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On the Origin of a virus

Skepticism plays a crucial role in science; different possibilities or speculations should be investigated, results obtained must be reviewed by experts (nowadays discussions happen in multiple platforms, journal websites, media/social media platforms), and evaluated based on scientific evidence presented. Science cannot accept any speculations blindly, no matter how attractive it is, or how big the name of the scientist who proposes it. Any new hypothesis should go through apparent rigorous review processes, and only with strong supporting evidence, science endorses it. A person in science knows the routine, but it is hard for someone outside of science to understand when scientists argue, or proposes alternative hypotheses, and seek for further evidence. Science cannot ignore any ‘extremely-unlikely’ scenario, until there are scientific proofs to conclude so. ´Questions asked´ by skeptics are understandable to the public- which may ignite their skepticism and easily jump into wrong conclusions without searching deep for evidence. Difficult part is understanding the answers to those questions! The listeners must need patience and basic knowledge on the subject, sometimes a bit more than the fundamentals to understand the language Scientists speak to clarify their genuine doubts. Let’s dive into the current speculations and ‘explosive’ evidence on various origin hypotheses.

Why the Origin of this pandemic virus matters?

This pandemic devastated world’s economy, affected lives of billions, killed millions, and still counting. It is very vital to fathom the origin of this killer virus ‘SARS CoV2’- the culprit of catastrophes. Pandemics are rare events that give us invaluable opportunities to figure out critical flaws and help us to develop precautionary steps to avoid such incidents in future. For that very reason, to understand the origin of this pandemic is extremely vital- where does this virus come fromNatural-origin or lab-origin, the answer will aid us to compose corrective steps. Suppose we could scientifically prove a natural spillover event from bats/intermediate host as the origin, that will benefit us in creating strict measures to prevent such spillovers in future (natural spillovers occurred even in recent pasts). Conversely, if we have the scientific evidence that this virus leaked from a research lab (accidental or deliberate), discovering that is very crucial to strengthen safety measures to avoid any such incidents. 

WHO phase-I report

A concrete answer on the origin question is always great, however, due to scientific and political reasons it is not going to be a cakewalk. A team of multidisciplinary experts (17 from China and 17 from other countries) visited and conducted a joint study over a 28-day period from Jan 14th to Feb10th 2021, in the city of Wuhan, China and created an elaborate 120-pages report (plus extensive annexure). First comprehend that WHO (World Health Organization) team was not there for any forensic investigations or to reanalyze samples or interrogating researchers. Phase-I of the joint study reviewed available epidemiological, environmental genomic data, molecular & bioinformatics data, and reached a consensus report and set the stage for follow up Phase-II study. WHO team enumerated different possible origin scenarios and graded them in ‘most likely to less likely’ scenarios. Further, WHO team visited Wuhan Institute of Virology (WIV) and discussed on their research activities, interviewed lab personnel and examined investigations conducted by Chinese authorities on lab-origin scenario. It is very naive to presume that WHO team should have barged in to inspect lab freezers and databases as a part of investigation.  WHO report proposed that future collaborative efforts and detailed investigations only will shed light to the events that led to this pandemic.

The possible origin scenarios

WHO report discusses the 4 possible scenarios (1-4). They argued in favor and against each of the scenarios and assessed the likelihood of each. 

  1. Natural origin– direct zoonotic transmission from a host animal reservoir to human population, like Influenza, Nipah, HIV etc. Natural precursor virus from an animal reservoir spilled and infected someone who ignited the pandemic fire. WHO team’s assessment: a possible-to-likely pathway.
  2. Natural origin through intermediate host- virus jumped from animal reservoir to another animal, adapted in the latter and finally jumped to human populationlike SARS or MERS. WHO team’s assessment: a likely to very likely pathway.
  3. Natural origin through frozen food– carcass of an infected animal was stored as foods which could be the vehicle for transmission from infected animal to human population. WHO team’s assessment: a possible pathway. 
  4. Laboratory origin– Virus introduced to human population through a lab incident. Accidental infection of a lab personnel or deliberate engineering/release of the virus. WHO team’s assessment: an extremely unlikely pathway.

Though the scenario 4 is ‘extremely unlikely pathway’ from their assessment, I would like to elaborate more on scenario 4 through possibilities sub-scenarios 4a-4d. Note there are no evidence so far on these sub-scenarios, my effort is to list possible lab-origin plots.

4a. Natural virus/Accidental leak– a person in a Corona virus research lab gets infected and accidently propagates the virus to human population.

4b. Natural virus/Deliberate release– a person in a Corona virus research lab deliberately releases virus to human population.

4c. Engineered virus/Accidental leak– a person in a research lab that studies engineered Corona viruses got infected with the virus and accidently propagates it to human population.

4d. Engineered virus/Deliberate release– a person in a research lab that studies engineered Corona viruses releases the virus to human population.

Why the natural origin is a ´possible-to-likely´ scenario while laboratory origin is an ´extremely-unlikely´ scenario?

A deliberate release of a deadly virus into own population seems illogical (WHO report did not consider such a plot), because a virus can mutate into more virulent variants as we see now and comprise havocs to the creators themselves. While spillovers of viruses from its natural reservoirs or through intermediate hosts, subsequently causing outbreaks are not at all bizarre. Such events happened several times in the past for many viral diseases, for e.g., Ebola, SARS, MERS, Influenza, Nipah, HIV etc. At the right circumstances, a ready-to-go precursor virus could easily ignite a local outbreak and evolve into a pandemic. Spillovers can happen from infected wild animals in a wet market, or from an animal farm, or from a bat cave. Patient zero could easily be a wet market employee, a buyer, a caretaker at wildlife farm, a farmer who collects bat guano or a researcher who collects research sample from a bat cave, from a zoonotic transfer scenario. Though accidental lab-origin (or lab-leak) hypothesis is based on conjectures with no scientific evidence so far and WHO graded it as an ‘extremely unlikely’ scenario, it is important to investigate further on lab-leak hypothesis also.

An argument against natural origin hypothesis is the delay in identifying the natural host of the precursor virus for SARS CoV2. Let’s discuss that point in detail, why is it difficult to figure out SARS CoV2 precursor virus? It is true that, after SARS and MERS outbreak, scientists could quickly identify the reservoir host. Having said that, a quick reservoir identification cannot be generalized for all the zoonotic outbreaks (Ebola, HIV for example are in conflict). During SARS1, sample collections from infected animals in markets were possible and scientists could figure out civets as intermediate hosts in 4 months. Though later it took more than a decade to identify some of the precursor bat viruses that spilled into civets. For MERS, identifying camels as reservoir host was also faster due to quick sample collections from infected animals. In both those earlier Corona virus outbreaks, yes, retrieving the samples from the suspected places happened quickly. But, situation was different in Wuhan. Due to initial speculations that Wuhan seafood & wildlife market was the epicenter of SARS CoV2 outbreak, authorities ordered to sanitize the market to prevent any further spread of the virus, as per protocolThis made it difficult to collect samples from remaining animals in Wuhan markets and lost a golden opportunity to rapidly discovering the natural reservoir link to SARS CoV2. Later, the presence of SARS CoV2 was detected from environmental samples from drainages of Huanan market. However, chances to identify any infected host reservoir were missed, due to sanitation efforts of Wuhan market places. WHO team reports the detection of virus in environmental samples and presence of susceptible hosts in the Huanan market. A direction now left is to trace legal/illegal animal farms that provided animals to Wuhan markets, collect samples from those wildlife farms for identifying any precursor viruses and perform thorough epidemiological analyses on the animal caretakers. Further, China made more strict regulations to curtail wildlife farms which may pose more difficulties to identify the intermediate host from farms that provided those exotic animals to markets. Golden chances for identifying the reservoir becomes feeble as time goes by. Rapid follow-up analyses are warranted to pinpoint the real epicenter of this spillover. WHO team proposed extensive and timely collaborative follow-up Phase-II investigation to solve this origin conundrum.

The tale of 2 lineages

According to WHO’s report, 2 lineages of SARS CoV2 were found in initial cases from Wuhan markets. What is a lineage? When virus multiplies mutations occur in its genome, such mutations can create a variant. Scientists track the mutations in viral genome as they are passed down through a ´lineage´ study, which can be compared to a branch of the viral family tree. The report says two lineages A and B circulated during November/December 2019 in Wuhan. Note, currently the predominant one that sweeps through the globe is the lineage B (yes, B in B1.1.7 or in B1.617 variants). There are 3 nucleotide differences between lineage A and B, and it is assumed that A is ancestral to B, when you compare with the bat CoVs. WHO team looked at the genomic data from earlier cases in Wuhan and reported that Huanan market cases are from lineage B. Interestingly, lineage A mediated cases were from other Wuhan markets. Based on the samples analyzed we can currently conclude that cases in Huanan seafood wet market are mediated by lineage B SARS CoV2 virus, which is down in viral family tree. While cases in other markets in Wuhan are caused by lineage A, which further up in the tree than B. More interestingly, genomic analyses of the cases in Washington, by WA1 variant belongs to the lineage A. Proposing that the first Washington case might have contracted lineage A virus that propagated from those other markets. 

Lineage tracing studies direct towards the possibility of natural origin scenario through infected market animals. Infected animal reservoirs (infected with different lineages) might have reached a hub in Wuhan and routed to different markets. One lineage becoming more prevalent in one market could be explained by this scenario. Lab-origin hypothesis does not fit very well with the lineage tracing data. An individual spreading lineage A at one place and same person spreading lineage B in another market seems an improbable plot. While another scenario, where an infected person spreading lineage A in one market, the virus propagates mildly/asymptomatically for a while and modifies further into lineage B, dominates in another location seems plausible. But how the infected person got the virus to begin this avalanche is the big question? Again, it could be a natural spillover or lab-accident, we don’t have the concrete answer yet.

How easy is designing a zombie virus?

Nowadays, we have cutting-edge technologies to manipulate the viral genome by introducing specific mutations and make genetically modified viruses for research and treatment purposes (to understand the virus, to develop cures and to make life-saving vaccines). Recently, molecular biology has advanced to an extent that we can introduce desired mutations to fragments of foreign genes in any viral genome without leaving any signs of genetic engineering (genetic manipulations). However, our approaches are very modest, compared to nature´s massive approach to create variants. Though we have the ´know-how´, functional prediction of engineered mutations is very hard and may not be very accurate. One must screen a ton of mutants created to identify the one with the expected traits. Hence compared to massive scale evolution and screening approaches by nature through animal reservoirs, our technology is meager. 

Let´s dig into SARS CoV2 genome now. Some regions of SARS CoV2´s spike protein, like the receptor binding domain (RBD) is peculiar when compared to bat CoVs. The RBD of SARS CoV2 is like the pangolin-CoV´s RBD, they both have good ACE2 binding character (the reason initially why we thought that the pangolins could be the intermediate reservoir of the SARS CoV2 virus). While looking at that the regions outside RBD the similarity declined, which lead to the hypothesis that some intelligent designer inserted RBD from pangolin to a bat-CoV precursor backbone to make it more humanized. 

To create a novel SARS CoV2 virus a researcher needs a precursor virus or the sequence of it, like a backbone or a scaffold. The most discussed closely related one from WIV lab was a bat CoV named RaTG13, which is only 96% sequence similar to the original SARS CoV2, first detected in Wuhan. However, one has to change nearly 1200 nucleotide letter changes in RaTg13 to match the SARS CoV2. Only an extraordinary researcher with so far unknown skills can predict, design, and modify the RaTG13 viral genome to create the current circulating version of SARS CoV2 genome. If there were any unpublished precursor viral genomes in WIV freezer stocks is unknown! However, all the features of SARS CoV2 can be found in other similar viruses, and this class of viruses possess peculiar mechanisms like template switching, recombination etc., that can clearly acquire bits and pieces from other similar viruses to create the perfect SARS CoV2 virus. One can speculate a plot that, a distant precursor of SARS CoV2 might have jumped from a bat to an intermediate host (e.g., a pangolin, or a cat or a mink or even a human) and while adapting in new hosts, the precursor could encounter related viruses. During this process of adaptation, precursor’s genome could randomly pick up gene fragments from its cousin viruses. These processes might have taken several years below the radar (i.e., only mild or asymptomatic infections) before it gained all the features that propelled its pursuit as the ´pandemic´ virus. 

Recently, Dr. Shi Zheng-Li´s group in WIV put nearly 8 more SARS-related CoVs obtained from bats. One of the bat viruses, RaTG15 shows 97.2% amino acid similarity with SARS-CoV2, more similar than the previously discussed precursor RaTG13 (interestingly both were from bats found in same location). Intriguingly, this virus binds to bat ACE2 receptor but not human ACE2 receptor! Which proposes, RaTG15 or any such viruses in the WIV lab freezer may not be the direct backbone for SARS CoV2 virus that uses human ACE2 efficiently. So far identified precursor viruses needs several years of evolution in bats or an intermediate host or humans to create current genome version of SARS CoV2.

Is FCS a smoking gun?

Nobel laureate virologist Prof. David Baltimore recently commented that the presence of Furine Cleavage site (FCS) is a smoking gun that hints SARS CoV2 is man-made!

What is FCS? It is a stretch of positively charged amino acids (the building blocks of protein*) where host cell’s protease (Furin- another protein) binds and cuts at this specific stretch in other proteins. The cutting at FCS by the furin plays a crucial role in the entry of SARS CoV2 to the host cell resulting in a successful infection. FCS is not detected in RaTG13 or other SARS related Corona viruses (see figure; green letters are present in RaTG13, red letters are extra sequences in SARS CoV2), which created genuine suspicion even in experts about the deliberate introduction of FCS for gain-of-function research. Portraying that FCS in SARS CoV2 as suspiciously ´unique´ feature is not accurate since the exact site can be seen in other related viruses. FCS is present in other related beta Coronaviruses like MERS, HKU1 and in feline CoVs. Presence of FCS was recently detected in other bat Corona viruses suggesting that acquiring such stretches probably through recombination is possible. Further, presence of O-linked glycan sites near FCS was another feature pointing towards intelligent design, under the notion that these sites are introduced to evade host´s immune system. Later we understood that such sites are present in other CoVs, the one present near the FCS regulate the furin mediated cleavage more efficiently. 

*Spike protein is a string of different amino acids, and the instruction for making that string is coded in the viral genome sequence. Each amino acid bead is coded by a group of 3 nucleotide codons (triplet codons) in the genome and each amino acid can be coded by such multiple triplet codons (redundancy). Nature exploits the opportunity to have random single nucleic acid mutation, sometimes changing the triplet codon of an amino acid and thereby changing the message of the protein. There are several examples where even single mutation results in wrong messages thereby bad proteins creating risk factors or causing detrimental genetic diseases (e.g. sickle cell anemia).

Figure legend: The gene sequence corresponding to FCS ‘RRAR’ is shown (blue box). Those 12 nucleotide letters are missing in RaTG13, an ancestral bat CoV. Note that the 12-nucleotide fragment is not in frame (arguing that an experienced molecular biologist may not design this). This resulted is an extra P in the insert, creating SPRRAR that becomes a furin cleavage site-FCS.

Furin cleavage site that consists of a stretch of positively charged amino acids (RRAR) in the SARS CoV2 spike protein, is very minimal. The FCS stretch in discussion can be created with various permutations and combinations of triplet codons. The exact sequence that is present as an extra stretch in the SARS CoV2 viral genome is ‘CTCCTCGGCGGG’. Such an insertion at this specific locus could be due to various naturally occurring phenomena previously known- like mutations, polymerase slippage, template switching or recombination events that happened between a potential SARS CoV2 precursor and another related virus that had this notorious stretch of nucleotides. Corona virus can undergo such recombination events, and one can speculate that an ancestral virus after attaining an FCS, could adapt further to become the perfect human respiratory virus SARS CoV2 to start the pandemic fire!

Out of frame FCS- an intelligent but bad designer?

If a researcher inserts such a gene fragment coding FCS, one of the basic rules in cloning is the insert should be in-frame with the message coding spike in the backbone viral genome. But here we can see the inserted sequence is not in-frame with the spike´s genetic code while comparing with the RaTG13 sequence. So, if RaTG13 is the backbone for SARS CoV2, the person who designed was so intelligent to change all those over 1kilo base nucleotide sequence, but forgot to put the most important FCS site in-frame! An experienced molecular biologist won’t do that, on the other hand, nature has the freedom to try all permutations and combinations of codes and perform brute force screening which might have resulted in the perfect villain SARS CoV2.

Unusual codons

Presence of unusual codons for aminoacids R and R (RR, the 2-consecutive arginine aminoacids) in ´RR´AR furin cleavage site also raised suspicions. The R (amino acid Arginine) coded by the triplet CGG, is unusual instruction for a viral genome. Such a sequence will invoke immune cell activation and evolutionarily not ideal for a virus getting adapted in a host reservoir. Presence of RR with unusual codons appears like the virus was propagated in a system without an immune system- i.e. dissociated cells in a plate- the argument goes. But if you look closely at the viral genomes, usage of CGG codon for R is not very rare, and is present in FCS of feline CoV genome, and present in other genes in SARS CoV2. Prof. Kristian Anderson explained in his Twitter thread that the CGG codon is rare in SARS CoV2 but that does not mean it is absent. There are other Rs in SARS CoV2 viral genome coded by CGG- nearly 3% of Rs are coded by CGG. So, portraying that presence of CGG as a signature of human intervention is not scientifically accurate. Until we find a precursor virus from an animal host with the FCS and those unusual codons, this issue will remain as smoking gun for conspiracy theorists.

Recently, Dr. Amy Maxmen from Nature journal contacted Prof. David Baltimore on his opinions on Prof. Kristian Anderson´s explanation on the FCS and unusual codons. Baltimore explained, ´´natural origin could well be the scenario, but one cannot completely rule out any other possibilities´´. Which is absolutely reasonable and nothing unusual if someone in science hears, but what public heard was a Nobel laureate endorsing a man-made lab-leak hypothesis.

Safety first

Recently, a Singapore based Australian virologist Dr. Danielle Anderson who was undertaking research at WIV shared her experiences. She was working at WIV while the SARS CoV2 infections were slowly taking off in China. Dr.Anderson was quite impressed by the safety regulations at WIV which were like any other high-containment lab. Revelations from Dr. Shi Zheng-Li her bat-CoV research happened in safety level 2 labs is unacceptable for many of the experts including Prof. Ralph Baric (they were scientific collaborators in CoV research). WIV did not do anything wrong here, bat CoVs can be handled in level 2 facility, according to Chinese safety regulations. This points towards a very important issue about the need of consensus on safety regulations while working with dangerous pathogens (regulations vary country wise!). Current case warrants a universal regulatory body, uniform protocols, recommendations and safety measures on such research areas. 

Early origin?

WHO Phase-I investigation also reports the possibility that SARS CoV2 could have been circulating in human population ‘under the radar’ for months before the first case was recorded. Intriguingly, molecular analyses on sewage water collected on March-12-2019, from Barcelona (famous tourist place in Spain), detected pieces of SARS CoV2 genome. Data is non-peer reviewed and only PCR data (not sequencing), hence take with a grain of salt. Similar early detections of SARS CoV2 were reported from Italy, France and also from the USA, before the first recorded case in those places. If these data are correct, this invisible villain was with us long before and slowly setting the stage for the show. WHO team proposes a retrospective surveillance of stored samples frozen in other countries to identify any clusters ahead of recorded first case of COVID19.

Obfuscating wildlife trade in markets

Illegal wildlife trade and sale happened in Wuhan seafood markets. Along with fish the market was selling and slaughtering wild animals in those markets. WHO-report says ´´no verified reports of live mammals being sold around 2019 were found´´, while a study came out recently that addresses wildlife trade in Wuhan markets with photographic evidence. Serendipitously, the team conducted this study in the context of other diseases and their links to seafood markets. Though there are strict regulations on selling those exotic species in China, implementation seems not very strict. The article mentions the poor welfare of the live animals in cages and some of them had gunshots or trap wounds suggesting wild harvesting. Data shows that nearly 38 terrestrial wild animal species were sold in the market, but importantly, no bat or pangolin species were for sale. However, it is interesting to see the potential SARS CoV2 reservoirs like mink, raccoons in the menu. The new report warrants tracing the suppliers of wild animals to Wuhan markets, genomic analyses of animal samples from the suspected wildlife farms and seroconversion analyses in caretakers, before we miss that boat.

Internet sleuths 

A group of scientific and non-scientific analysts formed a club DRASTIC, to dig out the data deep in several databases giving air to lab-origin hypothesis. In 2003, during SARS1 outbreak Chinese authorities got criticized for not being open to the world in a timely manner. Current pandemic also flared similar accusations on timely reporting, about live animals in markets, viral genomic data base that went offline in September 2019, research on Mojiang lab-miners etc. An Indian based anonymous origin hunter ´theseeker268´ brought up relevant theses from WIV on infected miner’s data. Bloom lab excavated a set of sequence data of early COVID19 cases in China, from a public database which were deleted upon publication. In current circumstances, even usual practices get shadows of doubt. Being transparent in science is important and helps to avoid casting shadows of doubts and trust issues. If someone else digs up those inaccessible data, it nurtures suspicions- even though it does not add anything new to the already known facts.

Intelligence reports

Science is not simple; you must spend time and energy to understand it. For public, a statement from their political hero, or a comment from a Nobel laureate is enough. But Science does not work like that. Prof. Richard Feynman, one of the greatest minds ever lived said ´´It doesn’t make a difference how beautiful your guess is, it doesn’t matter how smart you are who made the guess or what his name isIf it disagrees with experiment, it’s wrong’. If there is no scientific evidence to prove, it remains as conjecture! Former secretary of state, Mr. Mike Pompeo first claimed about an intelligence report that proves the lab-leak theory. According to him ‘’there is a pile of evidence 100 feet high’’, though he could not solve the case while he was in power. Mr. Pompeo should have given such evidence to WHO team to investigate further while they were in China. Recently, the intelligence report came again under spotlight on the matter of 3 WIV researchers who showed COVID or flu like symptoms in November 2019 and visited hospital. As per the information provided by WIV to WHO, the serological data on their employees shows no COVID19 infections before December 2019. Let’s assume that we may find that some of the WIV personnel have visited hospitals in November 2019, is that enough evidence to prove a lab-leak theory? We need more solid evidence like medical reports, left over blood samples or serological analyses, or further analyses of immune cells to prove the case. US President Mr. Joe Biden has ordered US intelligence officials almost a month ago to investigate and report back to him in 90 days. It will be scientifically very interesting to learn the supportive scientific evidence they provide if it is concluded as lab-leak. The big question is even if the Chinese authorities provide the evidence to liberate the Wuhan Institute of Virology from clutches of suspicions or to support natural origin of SARS CoV2, will the skeptic minds trust those evidence provided? Any explanations or proofs will not satisfy minds tuned to conspiracy theories- they may come up with more questions. Instead of accusations what we need is collaborative efforts to understand the real origin of this virus, which will help us to avoid such future incidents. Need to trace out earlier symptomatic cases, analyze frozen samples if available, trace back the suppliers of animals to Wuhan markets, analyze samples from the suspected wildlife farms- the clock is ticking!

Collaborative Phase-II investigation

Generally, we learn and fantasize on pandemics from history lessons and sci-fi movies. When we first heard about the 2019 outbreak in China, nobody contemplated the extend of global spread and damages. However, years ago experts were alarming us about such possible scenarios, which fell on deaf ears. Even months after COVID cases started piling up in China, we thought that it may not cross the border. Warnings on transmission possibilities of this respiratory virus were neglected and many ridiculed that the authorities are overreacting. Governments failed on timely preparations and downplayed it as a normal flu that will disappear in a few weeks’ time. Some declared early victory over the pandemic at the time when it´s unimmunized population was hugely susceptible to circulating variants. Political reasons aside, deducing the origin these chaos is essential to prevent any such foreseeable outbreaks. The circumstantial evidence of presence of Corona virus research labs at Wuhan is enough for the conspiracy theorists to cook up a lab-origin story. No doubt, we should investigate any possibilities for figuring out the origin. Real scientific evidence is warranted to prove any origin hypothesis- one cannot declare a verdict just based on media guesses, intuitions from experts, social media posts, or circumstantial evidence. Origin hypotheses; namely natural spillovers and laboratory incidents should be seriously investigated until we have sufficient data. Science needs concrete evidence to prove a hypothesis- so far, we don’t have such evidence to prove either a natural or a lab-origin hypothesis.

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Video of the blog

References

WHO report

https://www.who.int/health-topics/coronavirus/origins-of-the-virus

Twiv with WHO team members

Live animal trade in Wuhan market

https://www.nature.com/articles/s41598-021-91470-2

Furin cleavage site in the SARS-CoV-2 spike

https://www.nature.com/articles/s41564-021-00908-w

https://www.sciencedirect.com/science/article/pii/S1097276520302641?via%3Dihub

Lab-leak hypothesis

FCS

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872347/

https://pubmed.ncbi.nlm.nih.gov/32416074/

https://www.biorxiv.org/content/10.1101/2021.04.23.441209v1

2 Lineages

https://www.biorxiv.org/content/10.1101/2021.01.22.427830v3

https://virological.org/t/early-appearance-of-two-distinct-genomic-lineages-of-sars-cov-2-in-different-wuhan-wildlife-markets-suggests-sars-cov-2-has-a-natural-origin/691

Nicholas Wade´s article

https://nicholaswade.medium.com/origin-of-covid-following-the-clues-6f03564c038

Virologists letter in Science

https://science.sciencemag.org/content/372/6543/694.1

The seeker- virus origin hunter

https://www.theweek.in/theweek/cover/2021/07/01/theseeker268-worlds-foremost-virus-origins-hunter-on-how-he-began-combing-internet.html

Intelligence report

Dr. Shi Zheng-Li’s interview

8 SARS related CoVs

https://www.biorxiv.org/content/10.1101/2021.05.21.445091v1

Early detection

https://www.medrxiv.org/content/10.1101/2020.06.13.20129627v1

https://science.sciencemag.org/content/371/6525/120

Dr. Danielle Andersen

https://www.bloomberg.com/news/features/2021-06-27/did-covid-come-from-a-lab-scientist-at-wuhan-institute-speaks-out

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A tsunami of variants?

From a premature victory declaration over COVID-19, now India is under the ruthless grip of the enemy in new disguise. After curtailing to less than 10,000 daily cases towards the end of the first wave, now a staggering over 350,000 daily cases and nearly 3200 daily deaths! Neglect on mitigation measures, worse policies and inadequate preparations, false belief that India had beaten COVID-19, vaccine hesitancy, recommendations of pseudoscience, opening of theaters, no restrictions on mass religious gatherings, elections in several states, mammoth political rallies and protests, emergence of variants, all these factors played their role to induce this massive tsunami of Coronavirus, with record daily cases, overwhelming hospitals and crematoriums! India was in an advantageous position to plan in advance, we were seeing the merciless massive second wave sweeping through several western countries and instead of preparing for the inevitable second wave, we neglected the signs and assumed that pandemic is over for us. The comparatively less damage inflicted by first wave (against the predictions), also gave the false impression that we are done with the pandemic, but this virus was not done with us, yet. Propelled by early victory declaration, masses laxed strict mitigation measures which furnished a cozy incubator for emerging variants (viruses are obligatory parasites- they need a host to propagate, the reason why social distancing and mitigation measures are relevant) and an easy take off- now a tsunami is in front of us! Postmortem analysis on factors that fire-triggered this wave is crucial to avoid future mistakes, but amid this disastrous wave let´s focus on solutions to bring this big wave down, study the variant(s) under investigation and escalate the vaccination program.

B.1.617 aka wrongly named ´double mutant´

Variants or mutants always emerge, nothing surprising about it. Mutations are changes in genetic material (DNA or RNA) that may cause changes in the proteins. The process is not something special to SARS CoV2 virus, such coping errors happen when genetic materials (DNA or RNA) are copied. Some of the variants attain favourable features to propagate and flourish (survival and propagation of the fittest). To confirm that they are variants, genomic sequencing should be done in large scale, i.e., a high number of viral genome sequencing from the infected is the way to detect variants and take subsequent timely actions to contain it. The current massive surge in India could be due to a dominant variant or multiple variants or could be due to factors mentioned above like relaxation in mitigation measures. The variants B.1.1.7 (first reported from UK), B.1.351 (first reported from SA) and B.1.617 (first reported from India) are in direct competition in many places in India, and the one with evolutionary advantages will take over the other and could become the dominant (like the variants competed out the original virus first reported from Wuhan, China). B.1.617 is currently more prevalent in the genome sequenced samples and uploaded in global database Global Initiative for Sharing Avian Influenza Data (GISAID) recent weeks. Even though anecdotal reports on changes in clinical behaviours of the disease are appearing, thorough investigations are needed to make any conclusions.

The popular media named the variant B.1.617 as ´double mutant´ (as an easier alternative to the confusing strings of letters, numbers, and dots) since it bears two key mutations reported in two other notorious variants. L452R mutation-present in the dominant strain first detected in California, and E484Q mutation- like the one present in the variants first reported from South Africa and Brazil. Furthermore, the variant B.1.617 has 13 mutations in total (not just 2) compared to the original COVID19 strain first reported from Wuhan, China. For those who are interested in detail, B.1.617 accumulated more than 2 mutations, namely D111D, G142D, L452R, E484Q, D614G and P681R, even in the spike protein including within the receptor binding domain (RBD). The RBD mutations L452R and E484Q along with P681R in the furin cleavage site, may result in better transmissibility of this variant. The two RBD mutations may result in decreased binding to selected monoclonal antibodies and may affect their neutralization potential. Thorough investigations are warranted to determine the transmissibility, infectivity, and virulence of this variant under investigation B.1.617 and the effectiveness of the current vaccines against it. 

Earlier, it was reported that nearly 50% of the inhabitants in Delhi were seropositive, which means nearly half of the inhabitants had antibodies against the Corona virus due to symptomatic/asymptomatic infections. The resurgence of the second wave could be driven by waning immunity in those infected or evasion of immunity by the new strains or both, which is worrisome. That is, over time, as the immunity attained by first infection got waned, meanwhile more contagious variants popped up and ignited a surge that turned into a tsunami. Caution: If B.1.617 does increase the chance of reinfection or breakthrough infections in the vaccinated, and manage to cross borders; then probabilities are high B.1.617 could drive resurgence of COVID-19 in other parts of the world where a population’s immunity (obtained from natural infection or vaccine) is on the decline. Several countries learned from their experiences from devastating 1st and 2nd waves, and are now proactive by canceling flights from India. In a closely connected world, by the time we learn more about a variant of concern, it might have propagated across many borders- the B.1.617 is already in circulation in several countries- the horse is already out of the barn!

B.1.617 genome was detected back in 5 Oct 2020, appeared in the global database Global Initiative for Sharing Avian Influenza Data (GISAID) but during that time the first wave was in an indisputable descending trend, remarkable vaccine trial results were coming out, hence whether we gave sufficient attention to gear up our genomic surveillance and analyze the characteristics of this variant (infectivity, transmissibility etc.) is doubtful. Important to note that original SARS CoV2 virus was quite successful in propagation compared to any other viruses and managed to spread waves around the globe in a short time. A variant with ´more transmissibility´ is in a way more detrimental in choking the health system than one that causes ´more severe disease´. Suppose if B.1.617 is more transmissible or virulent, what could we do differently to treat this disease is a question? Whatever mitigation measures currently we follow, that includes social distancing, masks, hand hygiene, avoiding indoor gatherings etc. should be continued for longer time, in fact more strictly, until a significant percentage of population gets vaccinated. 

Very recently, a promising non-peer reviewed report came out suggesting that the sera from the natural COVID-19 infected and recipients of BBV152 (Covaxin) were able to neutralize B.1.617, emphasizing the seriousness in boosting the COVID-19 vaccination program in India. Though neutralizing efficiency of sera from other COVID-19 vaccines against B.1.617 weren’t reported, they should also protect us from critical illness during the current wave of virus. In a pandemic situation, though the mutation rate of Corona virus is comparatively lower than Influenza virus, it infects billions around the world and chances are high for nature to experiment various mutations (some of them can escape from the current vaccines). Many vaccine manufacturers are already preparing booster doses that may protect from emerging variants.

Blood-clots and the vaccine link

In a record time science came up with several efficient vaccines and the whole world is engaged in the biggest vaccination program in the history of mankind. More than a hundred vaccine-associated blood clots were officially reported in EU and US after jabbing millions of doses of COVID-19 vaccines, and those vaccines were on pause and public demand for those dropped in many countries. Two scientific studies showed possible links between the Oxford vaccine with the rare blood clots. Further investigations on vaccine associated blood-clot patients detected antibodies against platelet factor-4 (PF4) in them, proposing a mechanism through antibodies against PF4, inducing an autoimmune response and clots. To prove the causal link between these rare phenomena and the viral vector or other vaccines further scientific investigations are warranted. European Medicines Agency (EMA) concluded that unusual blood clots with low blood platelets should be listed as very rare side effects of Oxford/AstraZeneca/Covishield (1 in 100,000 vaccinated people), a viral vector based COVID-19 vaccine.

Based on similar rare episodes in the USA, another viral vector vaccine Johnson & Johnson/Janssen was also on a temporary pause. Recently, following a thorough safety review, FDA and CDC recommended to lift the pause regarding the use of the J&J COVID-19 vaccine and use of the vaccine may resume. Both the EMA and FDA resonated in their decisions that the vaccine’s known and potential benefits outweigh its known and potential risks in individuals 18 years of age and older. EMA reminded the possibility of very rare cases of blood clots combined with low levels of blood platelets occurring within 2 weeks of vaccination. So far, most of the cases reported have occurred in women under 60 years of age within 2 weeks of vaccination. 

In India, the mass vaccination program is now extending to younger ages from May onwards, and the health system would be vigilant and expect the occurrence of such rare events of blood-clots (if there is a causal link between vaccine and blood-clots) since Covishield is the major COVID-19 vaccine currently being rolled out. Such case reports may create panic and decelerate the vaccination program, hence public awareness on these rare side effects is vital. Though chances of this unusual blood-clot are rare, EMA recommends being aware of the symptoms within weeks after the injection, to seek medical attention. Patients should seek medical assistance immediately if they have the following symptoms in the weeks after the injection such as shortness of breath, chest pain, swelling in leg, persistent abdominal (belly) pain, neurological symptoms, including severe and persistent headaches or blurred vision, tiny blood spots under the skin beyond the site of injection. The pause, safety review and subsequent lift suggests that the surveillance system is functioning efficiently as they were designed (that too after identifying even rare cases).

How to get out of this mess?

Getting back to ‘pre-pandemic period’ will take time. With nearly half a dozen effective vaccines in our catalog, it would take lesser time than we first anticipated. Currently, two effective COVID-19 vaccines (including indigenous Covaxin) are spearheading the humungous vaccination program in India. An effective COVID-19 vaccine protects you from critical conditions and keeps you safe. Even if you are vaccinated, one can get mild or asymptomatic disease and can spread the bug. You must wear mask to protect the vulnerable from you, even if you are vaccinated, “masks are not theater”- as Fauci says. If you care, follow the mitigation measures like social distancing, masks, hand hygiene, avoiding indoor gatherings etc. until a significant population is vaccinated. Several vaccines against deadly diseases do give long-term immunity. This pandemic is only a year long, predicting what happens in 10 years’ time is not Science (Scientists are not fortunetellers with crystal ball!). Some variants may escape current vaccines (such variants may be out there already), and in that case we may need 3rd dose or dose every year as flu vaccine.

Yes, the fatality rate of the disease is low, the disease is mostly dangerous for vulnerable (though it may vary with some variants). But when it becomes a pandemic, it infects and kills in millions, suffocates the health system and eventually kills more vulnerable. With vaccines we can reduce the deaths significantly. Go for a COVID-19 vaccine that is offered by the health system. Herd immunity for infection may not be possible but herd immunity to prevent deaths is possible. Quoting Roberto Burioni here “the Earth is round, gasoline is flammable, and vaccines are safe and effective, all the rest are dangerous lies.”

References

https://outbreak.info/resources/2021.04.22.440932

https://www.gisaid.org/hcov19-variants//

https://scroll.in/video/993076/watch-prime-minister-narendra-modi-had-declared-indias-victory-over-covid-19-in-january

https://www.ema.europa.eu/en/news/astrazenecas-covid-19-vaccine-ema-finds-possible-link-very-rare-cases-unusual-blood-clots-low-blood

https://www.ema.europa.eu/en/news/astrazenecas-covid-19-vaccine-benefits-risks-context

https://www.fda.gov/news-events/press-announcements/fda-and-cdc-lift-recommended-pause-johnson-johnson-janssen-covid-19-vaccine-use-following-thorough

https://www.nejm.org/doi/full/10.1056/NEJMoa2104882

https://www.nejm.org/doi/full/10.1056/NEJMoa2104840?query=recirc_curatedRelated_article

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Variants Of Concern- a nightmare coming true?

Viruses mutate when they make more copies in host cells- there is nothing unusual about it. Mutations are changes in genetic material (DNA or RNA) that may cause changes in the coded proteins. These processes are not something bizarre happening to SARS CoV2 virus, such coping errors happen when genetic materials (DNA or RNA) are replicated. Mutations are keys to evolution, which happens in all species, from viruses to human cells. A mutated version of the virus is called a ‘variant’. If such mutations in genetic material result in changes in the protein it codes, which subsequently can transform the virus functionally (that may alter the features of the disease)- then it is termed as a ‘strain’ (there are different strains of flu virus). Mutations accumulate in viral genetic material during propagations that eventually may result is a new strain with significant genetic drift from an ancestor virus (see the diagram above) and occasionally becomes the dominant version. We are experiencing viral evolution in front of us! In a pandemic they can easily capture millions of hosts and produces numerous copies harboring several random mutations. If the mutations are suitable for viral propagation-that variant will flourish, if the mutations are detrimental for virus-that variant will perish. 

RNA dependent RNA polymerase, the enzyme that makes copies of RNA does not have very efficient error correcting mechanism like our cell’s DNA coping system. Flu virus, which is a fragmented RNA virus, notorious for frequent mutations, escapes our immune surveillance system and that is the reason for different flu variants and why every year we need different flu vaccine. Compared to flu virus the ´copier machine´ for SARS CoV2 is less error prone. RNA being the genetic material of SARS CoV2, while making copies they also will make several errors. This virus accumulates variations during the process of transmission from one infected individual to another. Transmissions across the population may accumulate mutations in viral genome, generally these random mutations won’t drastically alter the coded protein and hence won’t change the virus much. However, some of those random mutations can make the virus more efficient in terms of transmission, that may be the case we are observing in UK, South Africa, Denmark etc. 

The UK variant

The recently reported UK variant is termed as VUI 202012/01 which means Variant Under Investigation year 2020, month 12, variant 01 also known as B.1.1.7, is spreading to multiple countries. UK identified this variant because the genomic sequencing analyses on COVID19 cases in UK is very efficient than other countries. Preliminary data suggests that this variant shows an increase in R-value (which indicates the disease spread), decrease in Ct value (indicates the higher viral copies in the sample), increase in viral load (quantity of virus in the sample). UK scientific advisory body reported that they had ´moderate confidence´ that VUI-202012/01 was substantially more transmissible than other variants of SARS CoV2. Underlying mechanisms of increased transmissibility is still unknown. Even though there are indications that this variant might have altered features from the original SARS CoV2, to declare this ‘alleged-variant’ guilty needs more thorough biological experimental evidence, which warrants time (biological experimental data for higher transmissibility, virulence etc.).

The UK variant shows a dozen of changes in the spike protein of SARS CoV2 (several in receptor binding region). Current origin hypothesis is- a patient with weakened immune system might have harbored the virus longer, thereby transformed into a breeding ground for such a variant with several mutations. The spike proteins on the surface of this virus help the bug to latch specifically to the host cell receptors. More importantly, the spikes studded on the corona are very vital for our defense system. Because, neutralizing antibodies created by vaccines bind to those spikes preventing from receptor binding. 

Interestingly, another gene in the virus called ORF-8 (a gene supposed to help in replication of this virus) was also affected by a mutation. This was earlier reported from Singapore and thought to attenuate the viral disease. These copying errors made by the virus are useful for genetic ´detectives´ researchers, since they can easily trace the track the viral propagation. It is very important during an epidemic episode to understand more about the route-map of pathogen. The UK variant is suggested to have rapid transmission, but this feature of the variant could be due to several other factors rather than just mutations according to some experts. Such as disregarding social vaccination protocols (public health measures like masking, social distancing etc.), which may result in rapid spread of a variant. This is technically referred as chance or founder effect. 

South African variant

Genomic analyses of surging cases in South Africa revealed a variant named 501Y.V2. Though the UK and SA variant emerged independently and contains multiple mutations, they have some common changes in spike proteins. UK variant has 8 changes while SA mutant has 9 changes in the spike protein. Some of these mutations can make the virus flourish in the host´s respiratory system, may be a reason for higher transmissibility.

Other variants

D614G mutation in the spike protein was reported in early March 2020 and its spread to global dominance by a month, suggesting viral evolution and natural selection of D614G. Genomic investigation in Netherlands and Denmark demonstrated human to mink, mink to mink, and mink to human transmission. Sequences from those outbreaks had a Y453F mutation in the receptor binding region of spike in addition to other mutations, affecting its binding to receptor. To avoid such potential recurrent spillovers large scale cullings were implemented in mink farms. 

Studies on the effectiveness of the administered vaccines on these reported variants are ongoing and some initial data are coming out. A non-peer reviewed data suggests that the antibodies produced in Pfizer/BioNTech vaccine work against a mutation (N501Y) shared by both UK and SA variants. Information on vaccine efficiency on other worrisome mutations (like E484K) are expected soon.

Fighting a variant

Even if this variant is proven more transmissive or virulent, what could do to treat this disease is the question. Whatever mitigation measures currently we take that includes social distancing, masks, hand hygiene, avoiding indoor gatherings etc. should be continued for longer time, in fact more strictly! Important to note that original SARS CoV2 virus is quite successful in propagation compared to other viruses, that’s why SARS CoV2 managed to spread around the globe in a short time. A variant with ´more transmissibility´ is in a way more detrimental than one that causes ´more severe disease´. ‘’A 50% more transmissible SARS CoV2 variant would be a much bigger problem than a variant that’s 50% more deadly’’ says Prof. Adam Kucharski, at London School of Hygiene and Tropical Medicine, UK. If a variant becomes more contagious, it exponentially transmits to many including the vulnerable, thereby more quickly can choke the health system in the absence of various mitigation strategies. Considering that, the hasty decisions on travel bans seems appropriate. We may have to think again about flattening the curve, protecting the vulnerable and may have to introduce lockdown at outbreak epicenters, but remember that there are significant differences this time- we know more about the disease, more equipped with diagnostics and moreover we have several vaccines in our arsenal. This gives huge relevance to the faster rolling out of the vaccines that were developed at lightning-speed, to as many as possible. 

Making of an escaping virus?

Whether the new variant can evade the trained immune system of the naturally SARS CoV2-infected or vaccinated is a big worry. The initial data suggests that this variant cannot escape the immunity from the lead running vaccines that are already rolling out and further, this variant is not making the disease worse. A dilemma here is, most of the vaccines are targeting the vital spike protein of the virus (though a vaccine will elicit multiple kinds of antibodies and cell-mediated immune responses) but this may be the first time a virus is subjected to such a tremendous selection pressure from vaccine in the population. Under a selection pressure, the virus may mutate into a strain with variations in spike protein, thereby can evade the immune system of the immunized individual-which is a nightmare (a situation of ‘vaccine escape mutants’ is unlikely but does not mean that it can’t happen). Data from vaccine trials suggest that the vaccinated are certainly protected from serious illness and hospitalizations, but they are prone to asymptomatic infections and thereby can spread around the virus. Having inactivated virus vaccines (e.g., Covaxin (India), Sinopharm (China) that may prepare immune system to create defense weapons against different viral proteins (not only the spike), in our stock thus becomes more relevant. We should be vigilant and only rigorous scientific studies will reveal the true colors of this variant or any yet unidentified ones! This variant should be taken ´very seriously´ while travel bans are premature according to Dr. Antony Fauci.

Some countries are planning to vaccinate as many with just the first shot and delay the booster dose shot. One cannot rule out the possibility of emergence of some variants evading such incompletely trained immune system. ‘’We can generate difficult escape mutants if we subject a virus to low antibody pressure and then slowly move up’’ according to Prof. Florian Krammer, Mount Sinai, USA. Under a selection pressure from inadequate vaccine regimen (especially, in the context that vaccinated can undergo asymptomatic infections), the virus can mutate into a strain that can evade the immune system in an immunized individual-which may become a real concern!

There are variants out there circulating and current data suggests that they could be more transmissible than the preexisting forms, which is a serious situation according to experts. Constant surveillance of the cases with genomic and epidemiological investigations are very vital. We need more biological evidence about the features of these variants, before ruling them as ‘nastier’ than the original…right now, gear up with vaccinations and follow the instructions from the health authorities. 

Video of the blog

Mutations lead to evolution- watch the awesome experiment done on bacterial antibiotic resistance. Bacteria undergo random mutations while they divide and some of them at the antibiotic boundary attains antibiotic resistance, which then creates a resistant colony. Then it easily propagates through the antibiotic medium until it reaches the next higher-level antibiotic boundary. Again, it evolves to survive in the highest antibiotic concentration. Thus, random mutations help bacteria to grow eventually at the highest concentration in a short-time period!

References

Corona virus variants and mutations

https://www.bbc.com/news/science-environment-55404988

https://www.medrxiv.org/content/10.1101/2020.12.24.20248822v1

https://www.medrxiv.org/content/10.1101/2020.12.21.20248640v1

https://www.medrxiv.org/content/10.1101/2020.12.24.20248834v1

https://www.biorxiv.org/content/10.1101/2021.01.07.425740v1

https://www.gov.uk/government/groups/new-and-emerging-respiratory-virus-threats-advisory-group

https://www.gisaid.org/references/gisaid-in-the-news/uk-reports-new-variant-termed-vui-20201201/

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Beginning of the end!

It’s been more than a year now, that we came to know about the SARS CoV2 virus and the COVID19 disease. In the absence of an effective antiviral drug for this pandemic, vaccine is the way out. Several vaccine candidates came out with remarkably high efficiency from the clinical trials in a record time and got emergency authorization in many countries. Even experts were pessimistic about the rapid launch of any efficient vaccine (so far mumps vaccine was the fastest, which took 4 years). Now we are ready for the biggest vaccination program in the history of mankind. Several questions arise as the vaccines roll out. Whether vaccinated ones are free to travel around? Do the naturally infected ones need vaccine? How long does the vaccine protect us? Can the vaccinated transmit the disease if they are infected? Which vaccine should one take? Remember, this is a new disease, and those authorized vaccines are still on trials, and we are learning more about it and only time will answer most of those questions accurately. Here, I will share the current scientific opinions on several of such questions.

Novel platform vaccines

More than 100 vaccine candidates are undergoing different phases of trials, nearly 20 are in clinical phase 3. In 2020, we have seen tremendous development of vaccines from various vaccine platforms, among them 2 novel platforms viz. mRNA and Viral vectors stood out. These technologies are going to revolutionize the vaccine science of course, moreover, hopefully will evolve into the future medicines for many ailments. Reasons for rapid development of COVID19 vaccines is manifold, mainly several years of research on related viruses, R&D in various novel vaccine platforms, enormous funding, multiple multicentered trials and fast-track decisions from regulatory bodies.

What is an mRNA vaccine?

mRNA is messenger RiboNucleicAcid, which is a transient biological molecule that our cell uses to translate the DNA code to the protein molecule. It is an important class of biomolecule that helps in translating the genetic information (DNA) to the active players (proteins), in our cells. COVID19 mRNA-vaccines (by Pfizer and Moderna) are designed to produce the SARS CoV2 viral spike in the host cells at the injection site. Subsequently the spike proteins will trigger our immune system to create antibodies that recognize and neutralize the SARS CoV2, as well as to trigger cell-mediated immune responses. Thus, the vaccine equips our immune system by creating antibodies that neutralize the attacking pathogens and activate the cell mediated immune responses and keep the memory intact so that our body mobilizes these arsenals quickly upon a real attack by that specific pathogen.

Moderna´s (an US based biotech company) and Pfizer/BioNTech´s COVID-19 vaccines, mRNA-1273 and Tozinameran/BNT162b2 respectively, consists of pieces of ´transient´ genetic information of vital spike protein of SARS CoV2 (information in the form of RNA molecules) packed in oil droplets. In theory, these 2 lead runners are the same, except some differences in their formulations and dose regimens. One concern about such a vaccine is whether it integrates in our genome or stay in our cells! These are transient messages to produce the viral spike protein and are not messages that linger around forever (an analogy often helps is these mRNA vaccines are like ´snapchat´ messages).

What is a Viral vector vaccine?

By utilizing leading edge molecular biology techniques, we can modify even the deadly viruses to harmless tools for specific research and medical applications (like vaccines). Such modified viruses (viral vectors) are useful ‘cargo-vans‘ to deliver foreign genes into host cells. One can alter the genetic material of the cargo virus to remove viral proteins essential for its replication and instead, introduce a foreign gene of interest (e.g. spike protein of SARS CoV2). Non-replicative ´gutless´ viral vectors thus produced have the innate ability enter inside the host cells and produce proteins of interest (for e.g. spike protein of SARS CoV2) instead of viral proteins. Our immune system will take care the rest, as mentioned above.

Several such vector based COVID19 vaccines are undergoing clinical trials. The Jenner Institute’s (University of Oxford) Vector based COVID19 vaccine is the most promising one in the limelight. Their vaccine is ´ChAdOx1 nCoV19´ which is the abbreviation of Chimpanzee Adenovirus Oxford1 novel CoronaVirus 2019) also known as Covishield or AZD1222. The vaccine uses another mild virus (a crippled Adenovirus), that cannot replicate in host cells, but to deliver the code for spike protein of SARS CoV2 and subsequently to elicit the immune response. The genetically modified Adenovirus will infect the host cells but cannot replicate itself. Upon infection, it faithfully expresses the cargo it carries (here the spike protein of SARS CoV2). This foreign protein will evoke the defense system and produce the appropriate immune mechanisms in vaccinated host. Giant vaccine manufacturers like Serum Institute of India (world’s largest supplier of vaccines) is involved in the massive production to provide huge world-wide demands of this COVID-19 vaccine.

Emergency use authorization (EUA) approval

UK approved emergency authorization of the Pfizer vaccine first and kick started the biggest vaccine program in human history. Later, the USA approved the Pfizer and Moderna vaccines. Recently, UK granted approval for the country´s indigenous viral vector Oxford/AstraZeneca vaccine for emergency usage. After that, Europe also issued emergency authorizations for Pfizer vaccine and considering applications of other vaccines. UAE approved China´s Sinopharm and India approved Oxford/AstraZeneca and indigenous Covaxin vaccine for emergency use. Soon other countries may approve and start the marathon vaccination program. 

Vaccine efficiency- what do the data tell us?

There are several questions regarding the emergency authorized vaccines. Whether the vaccine will prevent the transmission of disease is a big question. The trial data shows that they are extremely efficient in protecting the vaccinated from getting severe illness- which is great. But whether the vaccines stop them from transmission to the vulnerable is not certain yet. Whether those vaccines work efficiently in diverse ethnic background and various age groups are other questions. Another question is how long the vaccine provided immunity will last. The vaccines are designed to elicit strong immune responses not only by creating neutralizing antibodies against different regions of spike protein (expressed from vaccines), but also through specialized memory cells- hence the defense system is supposed to be in action multiple ways if it encounters the real threat. Here are the gist of the different phase 3 results so far,

Moderna-mRNA-1273

Moderna vaccine aka the mRNA-1273 vaccine is a lipid encapsulated mRNA-based vaccine that encodes the full-length spike protein of SARS CoV2. Regimen consists of 2 intramuscular injections of mRNA-1273 (100 μg per dose) 28 days apart. The mRNA-1273 vaccine showed 94.1% efficacy at preventing COVID19, including severe disease in persons 18 years of age or above. Apart from transient side effects similar to other vaccines, no safety concerns were identified. Out of the 30,420 volunteers, 11 cases of symptomatic COVID19 were observed in vaccine group, while 185 cases were identified in placebo group.

Pfizer/BioNTech

Pfizer/BioNTech vaccine known as BNT162b2 is a lipid droplet enclosing a modified mRNA that codes for SARS CoV2 full-length spike protein. A two-dose regimen of BNT162b2 (30 μg per dose), 21 days apart provide 95% protection against COVID19 in persons 16 years of age or above. Adverse events observed were similar to those associated with other vaccines, such as mild-to-moderate pain at the injection site, fatigue, headache etc. Based on their data, from 43,548 participants- 8 cases of symptomatic COVID19 in vaccine group, while 162 cases were reported in placebo group. Whether this vaccine protects against asymptomatic infections and thereby spreading to unvaccinated vulnerable people is unanswered.

Oxford/AstraZeneca

Oxford/AstraZeneca viral vector vaccine is cheaper and easier with transportation & storage than the two mRNA-based vaccines which need extreme low temperatures. Oxford/AstraZeneca were the first among the vaccine candidates to publish the Phase-3 trial results in a peer-reviewed journal. During their interim results they announced about a ‘dosage error’, i.e. some of the participants received only half the dosage of the first jab (!). The standard plan of two doses of the same strength administered a month apart — had an efficacy of 62%, whereas the regimen with a lower first dose- had an efficacy of 90%. So, the data from different dosing regimens were pooled and they observed that the vaccine was 70% effective at preventing symptomatic infections. Intriguingly, those participants with lower first dose showed increase in efficacy compared to the standard-plan participants. Though the dose calculation error made the data interpretations complicated, it becomes ‘beneficial’ here, while it is difficult to explain the reason behind higher efficiency with current data. All together the interim data looks very promising that the vaccine has acceptable safety profile and is efficacious against symptomatic COVID19. But may need more data or a follow up study to come to a concrete conclusion (due to the dose complications). 

Further, they reported that the efficient low dose plan was 60% effective in reducing the asymptomatic infections, which means though the vaccinated are protected from serious illness and hospitalizations, they are prone to asymptomatic infections and thereby can spread around the virus. Suggesting the necessity of public health measurements for a long time!

Other vaccines in EUA list

Chinese-state owned Sinopharm vaccine (2 doses of inactivated vaccine) got approval in several Arab nations after claiming 86% efficiency. In a press release UAE stated that 99% of those vaccinated developed neutralizing antibodies against SARS CoV2 and that the vaccine prevented the vaccinated from severe disease. Interestingly, Sinopharm recently said that the vaccine is 79.34% effective and obtained approval in China. Similarly, Russia´s Sputnik V, the first registered viral vector vaccine from state-run Gamaleya institute claimed more than 91% effective, based on interim Phase 3 trial results. Along with Oxford/AstraZeneca’s Covishield (Serum Institute India is the production partner), another inactivated vaccine from Bharat Biotech (Covaxin) got restricted emergency authorization in India, though they are still recruiting volunteers for the clinical phase 3. Detailed efficiency data on any of these have n´t appeared in peer-reviewed journals yet. 

Side effects

Reactions like pain at the injection site, fatigue, mild fever, chills, joint aches are quite common and appears as your immune system kicks in upon vaccination. You may remember such unpleasant episodes after your childhood vaccines. If one gets such common mild-to-moderate side effects, it is reflected as vaccine is doing its proper job. The reported serious allergic reactions (less than 1 in 100,000) of emergency approved vaccines are less on comparison with some medications (e.g., approx. 10% are allergic to Penicillin therapy). Usually, the adjuvants in the vaccines can cause some adverse effects. In order to have an enhanced immune reaction and very efficient immunologic memory, vaccines are usually mixed with some adjuvants. An adjuvant is any substance that accelerates, prolongs, or enhances immune responses in combination with specific vaccine. The mRNA vaccines contain a compound called PEG which may be the culprit of some of the rare allergic reactions reported after the vaccination. People at high risk of an anaphylactic reaction should stay at the vaccination center for 30 minutes after their shot so they can be treated in case of any allergic reactions.

Which vaccine should I take?

Provided the vaccine is approved for safety and efficacy, if offered, one should take it. The faster you take the vaccine the better. Even the COVID19 survivors are recommended to take the vaccinations if offered, since it is challenging to determine whether they are protected from re-infections (especially asymptomatic). Based on the interim results, some of the vaccines (from mRNA platforms) came out with remarkably high efficiency (approx. 95%), while another one (from Viral vectors) showed nearly 70%. Main takeaway messages from these studies are, they are efficient in fighting against the disease, it gives nearly 100% protection to the vaccinated from getting a critical disease condition, they are safe and side effects are not significant. Though rapid development was highlighting feature for these authorized vaccines, the data show that they have not compromised any safety regulations during these processes, and the efficiency of these novel-platform vaccines is unprecedented. 

More vaccines in the pipeline

More than 100 vaccine candidates developed using various classical and novel platforms, are undergoing different stages of clinical phase trials (nearly 20 are in the final clinical phases). Among these currently 3 (with peer-reviewed data) got Emergency Use Authorization (EUA) in multiple countries and millions in the priority groups are taking it. To jab billions of people (at least two times, because of 2 dose regimen) around the world, we need different safe and efficient vaccines. Those authorized vaccines have completed only a few months of the 2-year clinical-trial period. So, anyone received the vaccine will be monitored for any-yet unobserved signs of concern. In case of any drawbacks reported on the authorized vaccines on some populations, it is better to be equipped with alternative vaccine options in our catalogue. As long as the vaccine is approved for safety and efficacy (lead runners have exceptionally high efficiency in protecting from severe illness)- if offered, it is wiser to take it.

References

Rapid vaccines

https://www.nature.com/articles/d41586-020-03626-1

https://www.who.int/immunization/research/vaccine_pipeline_tracker_spreadsheet/en/

Pfizer vaccine

https://www.nejm.org/doi/full/10.1056/NEJMoa2034577

Moderna Vaccine

https://www.nejm.org/doi/full/10.1056/NEJMoa2035389?query=recirc_mostViewed_railB_article

Oxford/AstraZeneca vaccine

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)32661-1/fulltext

Covaxin Bharat Biotech

Vaccine approval

https://www.nature.com/articles/d41586-020-03542-4

https://www.nature.com/articles/d41586-020-03593-7

Side effects

https://www.sciencemag.org/news/2020/12/suspicions-grow-nanoparticles-pfizer-s-covid-19-vaccine-trigger-rare-allergic-reactions

https://www.cdc.gov/mmwr/volumes/70/wr/mm7002e1.htm?s_cid=mm7002e1_w

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A virus that changed us!

SARS-CoV2, a tiny string of RNA made of mere 30 thousand molecules (ribonucleotides) wrapped in a membrane stolen from the host, changed the behavior of us humans! Today, we walk around wearing masks, do frequent hand washings, soak in sanitizers, we feel nervous if someone next coughs or sneezes, and are socially distanced from other human beings. We refrained from celebrations & festivals, our old-time customs of shaking hands, hugs & kisses became ´risky´ practices. We closed schools, factories, offices, tourist venues for months, while turned our dwellings into offices/kindergartens and many parts of the world are under second round of lock down. As you know, all these strict measures are to protect the vulnerable, to avoid choking the health system and buy time until an efficient treatment protocol or vaccine appears for COVID-19. A few years ago, during an official visit to Japan, I felt a bit suffocating while watching several people wearing face masks in public transport. Now, I am walking around wearing face masks in buses and carrying hand sanitizers everywhere, quite surreal situation!

It is difficult to conceive that a microbe could alter the behavior of mighty human beings. SARS CoV2 is not a kind of virus that directly messes up our brain and alters our behavior. However, there are microbes that can mess up host brain and make them behave weirdly, such parasites can transform even humans! Those parasites perturb host´s nervous system, and propagate more efficiently by taking advantage of behavior manipulation. Let’s talk about some of such zombie pathogens.  

Rabies virus – bullet shaped killer that make you aggressive

Rabies virus has a peculiar bullet shape (see the simplified diagram), and rabies infection is known to alter behavior of the host. Rabid ´´mad dogs´´ become more aggressive and less fearful, which increases the probability that the host will bite another animal, thereby spreading the virus to a new host before the rabid dog dies. The entire mechanism by which this deadly virus jumps from the bite site, the slow invasion & subsequent hijacking of host brain and the aggressive transformation of the host, still remains a mystery. The symptoms of rabies infection include anxiety, confusion, partial paralysis, agitation, hallucinations, and finally “hydrophobia´´. Hydrophobia is a behavioral alteration, the irrational ´fear of water´, caused by the difficulty in swallowing and painful muscle spasms. The infected person may panic at the sight of water, moreover, at a later stage even the thoughts of swallowing water may cause painful spasms. The patient will produce a lot of saliva and froth, which contains infectious rabies virus. Exact mechanisms of aggressive viral-manipulations of host brain are still unknown. Note, once the unfortunate host starts exhibiting clinical signs, it will be too late to rescue, however there are very effective vaccines against deadly Rabies virus.

A simplified diagram of bullet shaped Rabies virus. https://www.ntnu.edu/kavli/vvc/products-and-services

Toxoplasma gondii- the kind, that can make you a risk-taker

Toxoplasma gondii´s intermediate host is cat. It is intriguing to know how these microscopic single-celled organisms reaches our cats, from an infected rodent. Toxoplasma infected rodents become extroverted and less fearful of cats, increasing its chance of predation and the parasite’s chance of completing its lifecycle. We can also get infected on consuming under-cooked infected meats or sometimes by cleaning an infected cat´s litter box. This parasite influences the behavior of humans, Toxoplasma infections can cause some mental illness and risky behavior like what happens with those infected ´fearless´ rodents. There were observations that women infected with the parasite exhibit slightly more suicidal tendency (though a causal link between infection and suicide attempt is lacking). This parasite can affect a specific area in brain called prefrontal cortex and interfere with the capacity of that brain region to apply ´brakes´. A scientific study describes a correlation between career risk-taking entrepreneurs and Toxoplasma gondii infection. Some researchers have linked Toxoplasma infection to human behavioral changes that position them at higher risk for car accidents and drug abuse. Again- this is just a correlation! Are we influenced by tiny microorganisms on our decision makings, we are only beginning to understand?

Hairworms- hijackers of insect´s mind

Hairworms or Gordian worms infect crickets/grasshoppers. These worms need water for mating while crickets prefer dry land. This parasite controls host´s mind to make a dive into water (which usually crickets hate!), and then escape from infected body. Hair worms hijack host brain by producing proteins to mess up cricket´s navigational system. Parasite-manipulated cricket jumps around and ends in water bodies, bingo! The worms come out, mate, lay eggs, which will be eaten by tiny insects in water, and those insects eventually end up in another cricket’s body. A complicated lifecycle, indeed! (see the attached video).

Cordyceps-the enslaver fungus

This zombie parasitic fungus, Cordyceps host on jungle ants. Once the fungal spore gets into an ant´s body, it creates chaos in host brain, makes the ant behave weirdly. The baffled zombie-ant, driven by the parasite wanders around to the top of a tree where it performs the final bite on a leaf and dies. Thus, finding a suitable environment, the fungus comes out of dead ant´s body, flourish and disperse more spores to enslave more hosts (see the attached video). 

The above-mentioned cases are some classic examples of how the microbial/parasitic infections can affect the behavior of mighty hosts. Mechanisms underlying such behavior alteration by microorganisms are mostly poorly understood, will be very interesting research area indeed. Our genome is the ´graveyard´ of prior viral infections, which might have happened to our earlier ancestors. A significant portion (8.3%) of human genome is made of remnants of viral genomes. Several of those viral elements get repurposed or ‘exapted´ for several cellular functions including neuronal functions. We believe, we are in good control of our behavior, but who knows, how much we are influenced by tiny microbes or their exapted genes? We are only beginning to understand how such infections impact our behavior.

During this pandemic time, SARS CoV-2 modified us but taught us several lessons as well; it revealed us how poor we are in terms of pandemic preparations, reminded that we are as vulnerable as any other fellow creatures that shares this planet, it showed the values of social and family life, how vital the basic research is, the relevance of sharing knowledge & technologies and the significance of simple hygiene practices. We witnessed the rejuvenation of nature during lockdown, appealing us to stall our merciless destruction of nature. Though indirectly, this ´bat virus´ forced us to alter human behavior. How these pandemic-influenced behavioral changes will affect our future; whether such ´changes´ may remain permanent and transform into the new ´normal´ in our lives?… is an empirical question!

References

Rabies virus

https://www.nature.com/articles/s41598-017-12726-4

https://www.medicalnewstoday.com/articles/319735

Parasitic manipulation

https://www.sciencedirect.com/science/article/abs/pii/S0376635704002049?via%3Dihub

Hairworms

https://www.newscientist.com/article/dn7927-parasites-brainwash-grasshoppers-into-death-dive/

https://royalsocietypublishing.org/doi/10.1098/rspb.2005.3213

Toxoplasma gondii

https://www.npr.org/2012/07/03/156216620/common-parasite-may-influence-human-behavior?t=1603907258796

https://www.discovermagazine.com/mind/how-a-worldwide-parasitic-infection-might-shape-human-behavior

Domestication of retroviral gene-Syncytin and Placenta

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000028

https://viralzone.expasy.org/5957?outline=all_by_species

Featured

War against a virus!

When you hear the word ´virus´, unpleasant memories rush to your mind. It could be the painful memories of childhood chicken pox or about exhaustive viral-fever days or about scenes from Hollywood movies like Outbreak and Contagion. Not all viruses are bad though, some are beneficial to us (will discuss that topic later). These days everyone fears about the pandemic caused by a nanoscale virus, called Corona virus (CoV) that already killed several thousands and literally locked down the whole world.

This disease is named COVID-19 (COrona VIrus Disease 2019) and ´SARS CoV2´ is the virus that causes this disease. Like the ´version 2´ of earlier SARS outbreak in 2003. SARS stands for Severe Actute Respiratory Syndrome, CoV is for Corona Virus. First it was called ´Wuhan virus´, then ´novel Corona virus´ (nCoV) and still virologists are debating about its name! The similarity in the genetic sequence with the earlier SARS CoV (in year 2003) is the reason why it is called SARS CoV2. SARS CoV2 is an RNA virus, it has approximately 30000 bases making it the largest among the known RNA viruses. The genetic material of this virus is RNA, similar to the DNA genetic material we have in our cells. 

The economic loss due to COVID-19 is estimated in billions already. Only a mere percentage of that would have been sufficient to study about Corona viruses in general (note: this is a variant of SARS CoV´1´, that broke out in 2003), including basic research on broad anti-virals, detection methods, to some extent vaccine preparations etc. We would have learned a lot about the ´Corona viruses´ and have been easier to fight against it now! This is the situation for many diseases.

Not much about SAR2 CoV2 is known. We came to know about this around three months ago. The Scientific world is learning about its characteristics. Why is it more lethal to elderly than young, in general? Why a vast majority gets only a mild fever, still they are silent carriers? Why some countries are worst affected? What makes it very efficient than other Corona viruses in transmission? How long it can stay in surfaces and in aerosols? Will it genetically drift to a more virulent one? In a matter of 3 months from first reported case in China, it spread almost 200 countries around the world and worldwide death toll crossed 18500+ (24-3-2020).

A virus is an ´infectious, obligate intracellular parasite comprising a genetic material (DNA or RNA) surrounded by a protein coat, sometimes with a membrane´ (definition by Virology Guru Prof. Vincent Racaniello @profvrr). Which means it must be inside a host cell to be functional. It depends extensively on host cell´s machinery (hijack the cell machinery and make it into a virus reactor) to make more copies. It has a genetic material (either DNA or RNA), which carries the code to make more copies of it. It´s precious genetic material is protected by a protein shell and some of the viruses like SARS CoV2 has an extra protective layer of membrane that it steals from the host cell while budding off.

Whether virus is living organism or not, is still a debatable question. Consensus now is it has 2 phases. Phase 1, which is non-living phase outside the host cell. Phase 2, is the ´infected cell´-the virus once inside the cell overtakes the cell machineries, redirect the cell for viral replication hence the ´virus inside the infected cell´ can be considered as living.

Structure of the novel Corona virus (SARS CoV2)

Why is structure of a virus important to know? The protein structure is important to design drugs to fight against the viruses and also to make vaccines against it. The above diagram is an oversimplified version of SARS CoV2. The spikes (they are proteins) studded on the on the corona (in Latin corona means crown, also it resembles the corona of Sun) are very vital, since antibodies bind to these structures and eventually our immune system destroys these viruses. These days, once the genomic sequence of as novel virus is revealed, one can make pieces or complete viral proteins in its proper structural conformation. Such recombined fragments or full proteins can be synthesised and purified in a research lab to use for vaccine development and also for creating antibodies against the virus. These approaches have huge potential in clinical and diagnostic applications to save millions of lives.

Cell receptors, proteases and entry to the host cell

The spike proteins on the surface of this virus help to latch specifically to the host cell receptors. These receptors are identified as ACE2 receptors, note that these are cellular enzymes/proteins having dedicated functions in cell. SARS CoV2 make use of these receptors and other cellular proteases like TMPRSS2 to release its content into the host cell. The receptors are mainly found in the respiratory tract cells, but it can be found in other vital organs of our body suggesting that it can infect those organs also. The virus utilizes ACE2 receptor to attach and subsequently with the help of cellular proteases it gets access inside the host cell. Once inside they shed its genetic material (here RNA) which will then hijack the cells machinery to make more copies of the virus. Literally making the cell it infected into a ´virus making factory´. Sometimes it takes several days for the patient to show the symptoms, that time period is called incubation period. For COVID19 it is reported as 4-14 days, while it was 2-7 days for SARS (2003 version) and 6 days for MERS. Note, you can be an infectious ´silent carrier´ during this period.

After a week, the infected person make millions of viruses mainly in lungs (which he spreads to others), forcing cells to release alarm calls which are heard by our own immune system. Which eventually destroys infected cells and cleans the battle field, resulting in recovery of nearly 80% of the infected cases. In critical cases, the destruction of the protective lining of the respiratory system opens up way to bacteria causing pneumonia and later a cytokine storm (causing deaths in COVID-19).

We need to know where the fire is!

World health organization (WHO), with their the vast experiences in fighting against various outbreaks warned about the importance of ´testing´ to tame an outbreak. According to them, fighting the fire blindfolded is not efficient, we need to know where the fire is! Testing/detecting the infection is vital in an outbreak,

  • to provide timely care to those infected.
  • to contain the disease spreading, and protect the vulnerable.
  • to understand how the disease evolves in a population.
  • to lower the rate of infection and fatalities.

In the absence of timely testing, an asymptomatic ´infected person´ does not know about his/her infection hence might not quarantine, instead unknowingly go around and infect risky groups. To know the total number of people infected with COVID-19 widespread testing is warranted, unfortunately many countries did not prepare for that. Many countries face scarcity of protective gears, testing kits, facilities, personnel for testing in the midst of this outbreak. Some countries like South Korea, Singapore were exceptional, in many places they even had drive-thru testing centers- which resulted in harnessing this outbreak to a good extent. If testing is done on ´cherry-picked´ patients, we do not have a clear picture of what’s going on. The number of ´infected´ in many countries are not the exact numbers, it is only the tip of an iceberg.

Lack of detection facilities and personnel became a nightmare at the peak of an outbreak. Which forced some research labs in countries like the USA to make their own protocols to detect this virus. Demands for testing on suspected will rise at the peak of an outbreak, and thousands of tests/day will be warranted.

How to detect COVID19 viral infection?

As mentioned earlier, SARS CoV2 is an RNA virus, which means the genetic material of this class of virus is single string of ribonucleic acid (RNA). Note, we have double stranded deoxyribo nucleic acid (DNA) as our genetic material. The testing method is reverse transcription polymerase chain reaction (RT PCR), a powerful technique that can be done in authorized facilities. The instruments and reagents are expensive, needs trained personnel for testing (not like a simple strip that tells whether you are infected or not). In some countries such detection facilities are rare posing a delay in detection. An oversimplified version of detection strategy is shown above. Swab samples from respiratory canals of the suspected are taken for isolating the viral RNA genetic material. In simple terms, first we need to convert the RNA in to DNA and then run a reaction to amplify a specific fragment of our interest, that is found in the viral genetic material. It is so sensitive that a few copies of viral genetic material are enough for detection. We know the complete sequence of this virus; hence we can amplify specific regions of the virus’s genetic material first converting it into DNA. This test can be made very specific/stringent by designing a region that is very specific to the SARS CoV2.

How many are infected?

In many countries prescreened patients are subjected to testing, hence what we see is the tip of an iceberg. Once this fever season subsides, a follow up study is warranted on the population to understand how many actually got infected by SARS CoV2. RT PCR based fingerprinting is not useful there, but an antibody-based ELISA will be (such detection kits are reported already). Because an infected body retains antibodies against this virus as a memory for a longer time, even if there are no SARS CoV2 present in the body (a defence mechanism to fight against the same virus on a second attack). Many of the mild cases are unnoticed, or not considered for testing now in the middle of this pandemic season- but will be detected at a later time with this serological (blood analysis) ELISA test. It is important to know more about this virus outbreak and important to prepare more for a future pandemic. 

Comparison with other Corona viruses

SARS outbreak that happened in China (2003) and MERS in Middle East was caused by same class of virus (Corona). A comparison between SARS CoV2 and these corona viruses was made by Prof. Akiko Iwasaki (@VirusesImmunity). The case fatality ratio for SARS is 9.6-11%, while that of MERS is 34.4%. SARS CoV2 virus is novel to Science, hence we are in the learning curve. With the current studies, this virus is showing a range 2-8% case fatality ratio (based on the reported numbers). Note, this vary among different countries, among different age groups. It is difficult to predict the features of an ongoing novel viral infection (the number of the infected cases are not accurate).

No! it is not just a ´bit more than flu´!

For seasonal flu, the fatality rate is approx. 0.1%, and it is several folds higher than flu (atleast 10 times, we don’t know the real number yet). Vaccine is prepared every year for flu. COVID-19 is a respiratory illness leading to pneumonia. Flu can also cause secondary respiratory infections, in absence of proper care. We know much about the influenza virus that helps us to fight against it, while we know very little about this novel CoV.

In beginning of this outbreak, many were making case fatality ratio comparisons with 1918 flu pandemic, to state the less aggressiveness of COVID-19. This is like comparing watermelons with oranges. We are comparing a century old pandemic episode with a pandemic in 2020. In 1918 the world was crippled by the WW1. Science was not that advanced; it took another 10 more years to discover Penicillin (Alexander Fleming discovered Penicillin in 1928). We did not know much about the viruses at that time. In this current era, even with much Scientific advances, this string of RNA is bringing the whole world to it´s knees.

The horse is out of the barn!

WHO was giving alarm bells and instructions in the beginning of this year about the seriousness of this pandemic, importance of testing, travel restrictions, precise quarantine, preparing health system etc. Some countries took it more seriously than others and are now back on track. Locking down always causes economic turmoil and some countries don’t have the luxury to lockdown several weeks! WHO praised China’s efforts to contain the transmission in Wuhan, though many countries first considered lockdown as a Draconian measure, were later forced to endorse such measures.

Flattening the curve

This is the graph that was widely discussed in media which says protective measures will flatten the curve (note the dotted line is the healthcare capacity). Protective measures will buy us more time to take care seriously-infected patients without choking the health system. With no such protective measures, the peak will overwhelm the health systems and more deaths will be the result. It is intriguing, that why the authorities in many countries did not take strict protective actions first? Many countries had almost 2 months of ´golden time´ to study and prepare, after the outside world knew about the outbreak in China. The horse is out of the barn, this invisible enemy does not respect borders and almost all the countries are inoculated with this virus now. Many countries are implementing strict regulations that their citizens never experienced so far!

Locking down, social distancing and catch-22

A report from Ferguson, N., M., et al (Imperial college, London) changed the mind of many leaders and forced them to take the social distancing, lockdown model as the norm, which worked in many countries including the epicenter Wuhan province, China. Social distancing is necessary to flatten the curve, will give time to the health system to breath, but will result in slowing down natural immunity. Even if many of us escape from this ´first wave of SARS CoV2´, this is going to be around for several months- or may appear next season as a variant strain (like flu coming every year), though experts predicts less probability for such a mutant. Later there may be clusters of cases since complete isolation is literally impossible in this open world. We should still practice some travel restrictions, social distancing, hygiene measures, until there is a vaccine or an anti-viral drug.

Herd immunity

Herd immunity is the process by which a majority of the population gets immunity by natural infection or vaccination thereby preventing further transmission. This is usually heard in the context of vaccination- you don’t have to vaccinate every single person, but vaccination of significant population will check the disease from spreading (the percentage population to be immunised varies depending on the nature of virus). Note, the measles outbreak happened recently in some places where anti-vaxers refused to give vaccination to their kids.

With herd immunity strategy in mind, some countries initially prepared to gamble with this new virus but later dropped their plans. Herd immunity is not about the creation of naturally immunised ´leftover population´, when the vulnerable are dead. There will be unseen issues on experimenting with a novel virus; such as time to infect the majority of population, medical system can get overloaded during peak time of infections and moreover at the cost of many valuable lives. Also, if the virus mutates significantly, it can evade the immune system then the herd immunity will be a failure (though chances for such a mutation are rare).

Silent carriers

Virus spread is substantially undetected, and it is flying below the radar due to the silent carriers. These ´silent carriers´ can spread the illness, and they outnumber the detected cases, hence will be difficult to contain. It will also spread globally and may become the fifth endemic coronavirus within the human population according to a recent study.

How long SARS CoV2 virus can stay outside?

COVID-19 is a droplet disease, this virus is viable in the droplets that get out when an infected person sneezes or coughs. The droplets can travel a short distance before it falls down on surfaces. Once outside the host, viability of the viruses depends on environmental conditions. A study came out recently, analyzed stability of SARS CoV2 in aerosols and different everyday surfaces. In laboratory conditions this virus can stay stable for upto 3 days on plastic surfaces, upto 2 days on steel and a day on cardboard surfaces. Interestingly, SARS CoV2 was viable upto 3 hrs in aerosols (but infectivity depends on the inoculum shed).

Disinfectants

Soap is the best and cheapest way to get rid of this virus. This nanoscale virus has a greasy membrane coat that they take from the host cell while budding off. This lipid membrane will be easily destroyed by approx. 20 seconds washing with a good soap. Bleach is good for cleaning hard surfaces, while 70% alcohol containing disinfectant is very handy.

Origin and Conspiracy theories

With any new diseases, conspiracy theories also fly around. Most popular one was, the bioweapon theory. The coincidence presence of a high safety level virus research lab at the epicenter of this outbreak and the presence of some researchers with previous CoV research experiences at that lab fuelled these theories. Conversely, Chinese argued bioweapon testing on them by their enemies. A non-peer reviewed preprint on bioinformatic analyses on the SARS CoV2 sequence appeared (disappeared later) and claimed sequence similarities of bits and pieces of this virus with HIV and proposed the possibility of a genetically engineered virus. While these conspiracy theories were flourishing in social media, a scientific article came out very quickly in journal Nature Medicine that suggested the high sequence similarity of SARS CoV2 with bat CoV, proposing the possibility that this CoV may be transmitted from bat through an intermediate host. Their data supported the possibility that SARS CoV is a natural mutant from bats or through an intermediate host and debunked the ´designed virus´ hypothesis. Pangolins were the suspected intermediate species because of the high sequence similarity to the receptor binding region in spikes (comparison between SARS CoV2 and Pangolin CoV). Further analyses of the full virus sequence is not supportive to the hypothesis (active research is going on this field to find the real intermediate host, to prevent further interspecies transmission). Note, CoV for SARS and MERS were also originated from bats through Civet cats and Camels as intermediate hosts respectively. In the recent past, Ebola reported first from African continent, MERS from Middle East, Nipah outbreaks were reported first from Malaysia and later from Indian subcontinent. Nature is carrying out high-throughput mutation experiments on these viruses in different species and our technology to manipulate the genetics of viruses is very modest so far, compared to Nature´s approach. When other animals including us come in close contact with nature´s viral reactors, some of the mutated viruses may find a new host causing serious illness. Such zoonotic diseases (diseases that are transmitted from animals to humans) will happen in future also.

Recombinant viral vectors- viruses can be our friends too

We can create specific mutations on viruses’ genetic material or even introduce specific genes. Recently we have the technology do that for curing many genetic diseases (viruses as gene therapy vehicles). One can use molecular engineering tools to create safe recombinant viruses (viral vectors). Recombinant viral vectors are created from cells in a dish from viral genes. Cloning of essential genes into viruses and recovering safe viral tools is possible, which are utilized in research and currently in clinics too. Note, nowadays engineered safe viral vectors are used as ´cargo vans´ for delivering correct copies of a faulty gene to cure many genetic diseases. 

Desperate times, desperate measures!

During this fever season, anything that has a keyword COVID-19 or SARS CoV2 gets huge attention. Many non-peer reviewed data, nowadays submitted in repositories like BioRxiv, MedRxiv get immediate attention, social media takes it, interpret in different dimensions and influence the policies of states, even that of WHO.

WHO suggested the use of Paracetamol over Ibuprofen, in case of fever during this season. This is because of a non-peer reviewed piece in Lancet, that suggested an increase in the receptor ACE2 (that is the receptor for the spikes on Corona virus) upon Ibuprofen treatment. Note, there is no strong evidence to support this. This became a topic of discussion since a French minister tweeted about this.

Old weapons in our arsenal

Remdesivir, Chloroquine/Azithromycin, Vaccines.

We are desperately trying all the weapons in our arsenal. This is a fishing experiment in the context of a new disease, a reasonable approach to bypass many stringent tedious protocols since most of these drugs are clinically approved and side effects are well documented. 

One among these is a malaria drug Hydroxychloroquine/Chloroquine usage against COVID19. There are some ´preliminary data´ on its effect (with main criticism on less sample numbers) currently WHO is conducting a megatrial to confirm its effect. Chloroquine is shown to have effects on the RNA virus replication. Further, it blocks a pathway that helps the virus to enter and release its content into the infected cell. Another study suggests that this drug can act as a Zinc gate which will interfere with the RNA polymerase thereby inhibiting viral replication. Azithromycin (antibiotics) in combo with Chloroquine was administered against bacterial superinfection during Pneumonia. 

Remdesivir is another promising drug candidate. It was designed for the Ebola treatment and there are some promising studies in that context. Ebola virus also uses a similar mechanism to replicate inside the infected cell, this drug will act on the RNA polymerase (which is the key protein for the viral replication) and stall it. SARS CoV2 also has RNA polymerase, hence the drug might work effectively to inhibit its function. WHO is on megatrial with this drug, along with 3 other antiviral drug combos.

Vaccine takes time. It took almost 20 months to develop the vaccine against SARS 2003. You cannot simply inject a new vaccine bypassing the strict protocols (complications from an understudied vaccine may be more damaging than this disease). Nowadays creating a vaccine or new drug may not take that much time, but the clinical trials and getting these cures into the pharmacy shelves may still take months. The virus may not wait that long, may do the damage to the vulnerable by that time. Some companies claim discovery of fragment of viral RNA as vaccine for COVID-19, but experts say that it is too early as sharpening the scalpel before the surgery. They found a fragment of RNA which may be a potential vaccine candidate for this illness. Detecting a specific region (protein or RNA) in this virus as an antigen to produce antibody in our body, that specifically recognises the virus without interfering endogenous molecules is a humongous task. This requires rigorous testings, first in lab models and animals, and eventually in people. As mentioned earlier, Corona viruses like other RNA viruses, contain single strands of RNA which mutates, and vaccines may be warranted every season, like the flu vaccine (flu virus mutates extensively). Ebola vaccine development was expensive, now resting on shelves since outbreak ended, a reason why many companies are not interested in R&D and developing vaccines ahead.

Ever-evolving viruses

RNA being the genetic material of SARS CoV2, while making copies inside the host cell there will be several errors, the reason why there are many mutations during transmissions. Also SARS CoV2 is the largest RNA virus known, with nearly 30,000 bases. RNA dependent RNA polymerase, the enzyme that makes copies of RNA does not have efficient proofreading mechanism like DNA coping system, our cells hold. This virus mutates and accumulate variations during the process of transmission from one infected individual to another. Every transmission may accumulate mutations, in general, these random mutations won’t change the virus much. SARS CoV2 virus is already quite successful in propagation. Some of such random mutations can make it more efficient, though experts like Dr. Trevor Bedford (@trvrb) predicts it may take several years to see a major drift. Flu virus which is a fragmented RNA virus, notorious for frequent mutations, escapes our immune surveillance system and that is the reason why every year we need different flu vaccine. Compared to flu virus the ´copier machine´ for SARS CoV2 is less error prone, hence natural immunity that a survivor attains by infection may help us at least for couple of seasons.

These copying errors made by the virus (that makes a new strain) are useful for genetic ´Sherlock Holmes´ researchers, since they can trace the tracks of the viral propagation. It is very important during an epidemic episode to understand more about the route-map of pathogen. For example, in US they identified viral genome from the ‘patient zero’ in Washington state who travelled from China and created a virulent cluster of cases in King county. By genomic analyses multiple transmissions from this patient zero was traced even in different states.

Don’t underestimate the power of mutations- evolution works through mutations. To understand that, here is a great experiment done on antibiotic resistance by bacteria (see the video below). The resistance is achieved by some random mutations first in a bacterium at the antibiotic boundary, which then creates a resistant colony. Then it easily grows in antibiotic medium until it reaches the higher concentration antibiotic boundary. Again it mutates/evolves to survive in the highest antibiotic concentration. Thus random mutations help bacteria to grow eventually at the highest concentration in a short time period!

Now why is it relevant in this COVID19 scenario. SARS CoV2 also undergoes random mutation and get selected depending on the environment. SARS CoV2 is a result of variation that happened in a bat or an intermediate host sometime ago. Such mutations happen in each and every infected one. When it transmits from one host to another the virus carries some of the mutations, the reason why different new strains appear. There won’t be drastic variations because more likely those variations may make virus inactive. This virus is already so successful in its transmission, managed to spread nearly 200 countries in a short time. Under a selection pressure, the virus may mutate into a strain with variations in spike protein, thereby can evade the immune system of a survivor or immunised individual-which may be a concern for future.

Kindly go through the second blog ´Briefings on the hidden enemy´ where updated research on the disease are discussed.

References

SARS CoV2

https://asm.org/Articles/2020/January/2019-Novel-Coronavirus-2019-nCoV-Update-Uncoating

https://www.nature.com/articles/s41579-018-0118-9

ACE2 Receptor and TMPRSS2

Hoffman, M et al 2020. https://www.sciencedirect.com/science/article/pii/S0092867420302294

Stability of virus on aerosols and surfaces

https://www.nejm.org/doi/pdf/10.1056/NEJMc2004973?articleTools=true

Ibuprofen and ACE2 receptor

Fang, L. 2020. https://www.thelancet.com/pdfs/journals/lanres/PIIS2213-2600(20)30116-8.pdf

Remdesivir

https://www.nih.gov/news-events/news-releases/nih-clinical-trial-remdesivir-treat-covid-19-begins

Hydroxychloroquine

https://www.sciencedirect.com/science/article/pii/S0924857920300996

Solidarity Megatrial 

https://www.sciencemag.org/news/2020/03/who-launches-global-megatrial-four-most-promising-coronavirus-treatments

SARS CoV2 Genome sequencing

https://www.sciencemag.org/news/2020/01/mining-coronavirus-genomes-clues-outbreak-s-origins

https://www.nature.com/articles/s41591-020-0820-9

Blood test for COVID19

https://www.sciencemag.org/news/2020/03/new-blood-tests-antibodies-could-show-true-scale-coronavirus-pandemic

Gene therapy and viruses as delivery vehicles

https://www.novartis.com/news/media-releases/novartis-announces-landmark-eu-approval-one-time-gene-therapy-luxturna-restore-vision-people-rare-inherited-retinal-disease

https://www.nature.com/articles/d41587-019-00015-6

Origin and Conspiracy theories

https://www.foreignaffairs.com/articles/united-states/2020-03-05/us-chinese-distrust-inviting-dangerous-coronavirus-conspiracy

https://www.nature.com/articles/s41591-020-0820-9

Pangolins may not be the intermediate host

https://www.nature.com/articles/d41586-020-00548-w

Silent carriers

https://science.sciencemag.org/content/early/2020/03/13/science.abb3221

Updates on COVID-19 and a solution to control spreading

Corona wave and its resurgence

In spite of the predictions that, this viral fever may subside in later months from its start, COVID-19 is surging worldwide even after 7 months. Earlier it was more prominent in the Northern hemisphere now it is escalating in the South. While Europe is starting to breath after the destructive first wave, several states in US, South America are still suffocating from the extended first wave. COVID-19 is flaring up in the second most populated country-India and hope the wave won’t transform into a tsunami. Now, Europe is cautiously back to business. Tourist places are filled mostly with locals this year, masks are not mandatory in many places, but social distancing is practiced to an extend in public transport and restaurants. When you take a closer look at the daily cases these days, you can see a resurgence in many of these countries. Only time will say whether this are the early signs of a ´second wave´? Until a vaccine arrives, with our knowledge gathered so far about this virus and disease, we must be able to test and contain those potential hotspots to avoid vast outbreaks and a nation-wide lockdown.

Vaccine is the way out!

Since our fishing experiments on repurposed drugs is not giving very promising results, our hope is on an ´efficient vaccine´. Nearly a couple of hundreds of vaccine candidates are on different levels of pre-clinical and clinical trials. The process of vaccine approval takes long time (to find its immunological response, the dose, moreover the efficiency in the real battlefield). Due to the urgency, supported by modern technology, we are seeing fast-track processing of several vaccine candidates. Hopefully we may start distributing vaccine/s against this disease before the end of this year (may have to wait several months, until everyone gets their priming dose). Preliminary results from different vaccine candidate came out recently and are promising. Many vaccine candidates are exhibiting proper immune responses, which is really exciting. Oxford/Astra Zeneca/Serum Institute of India, Sinovac, Sinopharm and Moderna are the lead runners in this Marathon and entered into the crucial efficiency test (Clinical phase 3) in the field with thousands of participants in many countries. Fingers crossed!

Airborne route of transmission

A group of international Scientists recently made commentary that urges the medical community, public-health authorities to acknowledge the potential for airborne transmission through aerosols (tiny droplets that can linger in the air for several minutes). WHO acknowledges that in crowded, poorly ventilated spaces, short-range aerosol transmission cannot be neglected, but waiting for more evidences on the infectivity of these aerosols? SARS CoV-2 virus spreads mainly through droplets on contaminated surfaces generated by coughing, sneezing and talking. Droplets travel short distances and drop quickly from air to the surfaces due to its density. The viruses from surface droplets are transferred by people’s hands. Hence the relevance of washing hands and using sanitizer to control the transmission. In crowded, poorly ventilated indoor spaces aerosols with viruses may form. Whether these aerosols carry ´viable viruses´ that can cause illness is something we are still waiting to prove. Clear recommendations from health authorities for physical distancing, wearing masks, avoiding indoor gatherings may control aerosol transmission if it is proven.

Herd immunity by natural infection is attainable, but slow

A serological study from Spain on COVID-19 patients got much attention. Their data (only 5% of the population got infected) was misinterpreted to a large extend and social/media spread the wrong message that herd immunity is ´not attainable´ for COVID-19. The study looked at one arm of immune response to detect the prior infection (antibody against SARS CoV-2) while did not test the T-cell mediated immunity, which plays a vital role in fighting against this virus. Sweden, who took a different approach than its Nordic neighbors could not achieve immunity in large percentage of population (mere 6% of population developed antibodies to COVID-19). Nearly 10% of population in Stockholm (worst affected area in the country) had developed antibodies to COVID-19. Similarly, the worst hit New York city could infect nearly 20% during the disastrous first wave. The data shows with strong mitigation steps that countries adapted, we could contain the virus and resulted in lower percentage of infected cases during the first wave. Note, most of the studies are analyzing only the antibodies in the COVID-19 survivors, while recent studies suggest that crucial role of cell mediated immunity (another path that our body´s immune system uses to combat pathogens). These data can be correctly interpreted as that herd immunity (target estimated as over 70%) is achievable but slowly, only through multiple rounds of natural infections through the population, at the cost of more millions of lives.

HCQ, how many more nails we need in that coffin

An anti-malarial drug hydroxychloroquine/chloroquine (HCQ), effective in some chronic disease conditions also (like arthritis, lupus) became very controversial during this pandemic. Some ´preliminary observational data´ on its effect to treat COVID-19 was reported from China followed by another one from France (later retracted) and was quickly blown out of proportion as the ´magic bullet´ and US President became the brand ambassador for this ´gift from Heaven´. FDA issued a quick emergency use authorization based on the ´evidences´ at that time. Due to overhyped anecdotal evidences as ´the anti-COVID-19 drug´, people all over the world started stockpiling this drug, posing a huge problem for those patients relying on this drug for their chronic clinical conditions. Scientific community was cautious and suggested for the scientific evidences from clinical trials. It is hypothesized that HCQ blocks a pathway that helps this virus to enter and release its content into the infected cell. 

Several studies came out during this pandemic and now we can have a conclusion on this so called ´anti-Corona drug´. Verdict from many clinical trials was against the claims from this magical drug. A study from Spain did not find any significant benefit from HCQ on illness, Recovery trial in UK pointed out no clinical benefit on hospitalized COVID-19 patients, a combined study from the USA/Canada also showed that HCQ did not prevent illness in virus exposed individuals. Recently two articles in journal Nature, reported failures of HCQ as anti-viral drug, when investigated using lung cells in a culture-plates and in non-human primates. Though a plethora of evidences against using HCQ against COVID-19 are already present, many anecdotal and observational evidences are still coming. Based on interim trial results on HCQ and lopinavir/ritonavir that showed little or no reduction in the mortality of hospitalised COVID-19 patients compared to standard of care, WHO decided to discontinue the trials with immediate effect. Dr. Anthony Fauci recently said ´valid’ scientific data shows HCQ is not effective in treating coronavirus. Even with surplus of valid scientific evidences against HCQ, if one still claims a marginal benefit from HCQ administration based on anecdotal evidences, the risks associated with its administration could negate any such benefits (if at all!) in COVID-19 scenario.

Remdesivir, another potential repurposed drug is not giving exciting results. A clinical study from China on this wonder drug suggested no significant clinical benefits. Another study showed that this drug shortened the duration of illness. Currently, the results do not suggest it as ´the cure´ for COVID-19.

Relapse, is that possible?

One disturbing question that comes quite often is whether there is relapse of COVID-19? Whether one can get the COVID-19 illness twice? Earlier there were reports of reinfection of the SARS CoV-2, weeks after the first infection. These could be rare incidents on immunosuppressed individuals or false detection by tests. Note, some viral infections/vaccines give immunity for life long, some for years while some for weeks. So still we are on the learning curve about this virus’s ability to invade the system multiple times. A decline in levels of antibody weeks after an infection is quite normal that does not mean we are prone to same illness again, note that we have other defense mechanism to protect us like cell-mediated immunity. Which means that this arm of protective immunity may last longer and can prevent or ease the symptoms on a second infection (if it happens).

Cell-mediated immunity: our body will not forget SARS-CoV-2 infection

Antibodies are specific warrior proteins produced by our defense system (triggered by the presence of a pathogen or its proteins or a vaccine) to selectively destroy the invading pathogens. Our immune system is very efficient also in directing specific cells (like activated T-cells) to scavenge the pathogens or even kill the infected body cell. Once the virus enters our system (a cell), the resident guard cells recognizes the signals of the infection (viral proteins or chemical signal calls from an infected cell). The activated guard cells will initiate an immune response producing antibodies and cells to neutralize/destroy the invading pathogen. Within days after a viral infection our body will create tailored antibodies and immune cells, which eventually inactivate the pathogen and eventually these immune responses subside. But the memories of this attack retain in specialized memory immune cells. Even after several years, upon a second attack by the same pathogen, these memory immune cells get quickly activated, produce antibodies quickly and protect our body. We have a strong immune system, and have a repertoire of memory immune cells, a reason why we are rescued from several pathogens that we encounter daily. 

Recently some scientific studies came out which suggest the cell-mediated response will have a major role in our combat against SARS CoV-2. Most studies analyze the antibody profiling due to the availability of tests. Routine clinical analyses of cell-mediated immune response are not practical. On several weeks post-infection, the antibody level against that pathogen goes down, while memory immune cells remain. On testing serum antibody level on a COVID-19 survivor at a later stage may show less (depending on the sensitivity of the test) antibody titer. Current studies point to the necessity to understand the cell-mediated responses to SARS-CoV-2 infection, as well as in understanding the immune response of COVID-19 candidate vaccine clinical trials.

Experience matters! Pre-existing immunity to SARS CoV-2

Interestingly, now we have convincing evidences (from various scientific groups from different countries) that, a significant percentage of SARS CoV-2 unexposed population (20-50%) has T-cell immune response to SARS CoV-2! These analyses were done on blood samples (collected from 2015-2018, long before COVID-19). The speculation is, this could be a cross-reactivity to circulating ‘common cold’ coronaviruses in those un-exposed. Or whether it indicates this virus was circulating much earlier than we thought? This surprising, but significant pre-existing T cell immune response to SARS-CoV-2 may elicit quicker immune response to these immune privileged population when they encounter SARS-CoV-2. The observations are compelling us to hypothesize that this could be a reason for less severe cases in a large percentage of general population (due to preexisting immunity donated by previous coronavirus infection episode/s). Also, these reports caution us to screen whether the participants in various vaccine trials are already having the advantage of this preexisting immune response to this virus. Further studies are warranted to identify the source pathogen for this unprecedented T-cell response to SARS CoV-2 and clinical relevance of this important observation.

A solution for the spreading problem- daily testing to find the ´infectious´

As the primary wave is shifting towards the Southern hemisphere, and there is possibility of a subsequent wave in recovered countries (herd immunity is no way near) we should be prepared for worst case scenario in coming winter. It is impossible to live in a bubble with borders closed until we are ready with vaccine. Extending nationwide lock down for more months is not always the ideal solution, since repercussions are already visible even in developed world. Developing world don’t have the luxury to have longer lockdowns. Economic burden, mental health issues, absence of social life are some of the backlashes of stricter steps like lockdowns. Daily testing to find out the ´infectious´ and isolating them is the method to control spreading. ´Behavioral/social vaccine´ such as, using masks, keeping social distancing, avoiding gatherings, practicing hygiene measures and closing down hotspots (if necessary) are the proper ways to fight until we have the COVID-19 vaccine in our hands. Country wide lockdown is drastic and is not a feasible idea in a long run. 

Expensive-limited test is not a solution, but a cheap-rapid daily test is!

Test, Trace and Isolate (TETRIS) is the ideal strategy to fight this hidden enemy. Though it is only 7 months, we learned much about this disease and its causative agent. Recent studies point out that this disease spreads significantly through pre-symptomatic and asymptomatic patients. Moreover, the viral load peaks up and infected person transmits his virus through droplets to others even before any noticeable symptoms (up to 2-3 days before any symptoms!). Hence by the time one realizes by symptoms that he may be infected; he might have done the damage by spreading his virus to others. This is one of the main reasons why it is difficult to control spread of this ´successful´ virus (a virus from Wuhan could multiply in astronomical order and infect 18 million worldwide within a span of 7 months!). So, catching the infected who can transmit the virus (infectious), tracing their contacts and isolating them (TETRIS) is the best strategy to control the spread.

If there is a way to warn an infected person at an early ´infectious´ or ´transmitting´ stage, we can reduce the extend of spread. Currently there is long queue for the tests and long waiting time for the results (from days to weeks depending on the country), which is not effective at all if we consider the nature of this infection. We are obsessed with the sensitivity of the test and qPCR becomes the expensive gold standard (qPCR is very sensitive in detecting minute amounts of viral RNA fragments in a sample). The machine to run the test and reagents are expensive, and it requires expertise to conduct and interpret the results. Remnants of the viral RNA, at early stage of the infection or at the later phase when infection subsides, could be detected by the sensitive qPCR. In either scenario above, detecting few copies of viral RNA does not mean that the person is spreading the disease.

I will try to explain it in a bit more detail (in an oversimplified manner, for the general audience) about the issues with the current COVID-19 tests. Currently we use RT-PCR reaction and use the expensive qPCR machine to amplify and detect the viral genetic material in a swab sample (if interested, you can read the earlier blog for more details https://viralvector.design.blog/2020/03/25/war-against-a-virus/ ). Suppose by qPCR (the highly sensitive test currently we use) we can detect as low as 100 viral particles from a swab (note: qPCR detects RNA fragments, hence sometimes remnants of inactive viral particles are also detected). Detecting such a low number of viral particles in a sample using this sensitive method is fantastic, but the question is whether it is necessary? Let’s assume a threshold viral load (e.g. 10,000 viruses) is needed for a person to successfully spread the virus through the droplets to next person. A lower viral particle number from the test means the person may be initial phase of infection or had surpassed the peak infection (now at the later phase of illness) but he is not a risk for transmitting this viral disease. For those who care the details, the Ct value in qPCR is what matters here; it gives a relative measure of the viral RNA in the sample. Hence it may be irrelevant to detect the minute number of viral particles in an infected. On the other hand, it is more relevant to determine whether the person is in the range or above the threshold viral load, for transmitting the disease. For that purpose, a less sensitive test is more than enough, a test that can faithfully detect nearly 10,000 viruses (number just as an example) or above will serve the purpose.

The question here is do we need such a sensitive but tedious, expensive and less frequent testing solution to control this disease? Prof. Michael Mina at Harvard T. H. Chan School of Public Health has a cool strategy for this problem. He suggests a less expensive, less sensitive test but more frequent testing (daily test) as a solution to prevent viral spread in an efficient manner than expensive, time consuming, less frequent test that currently we are deploying to fight against this disease. A person who is giving swab for test on Monday may know his result only after several days, in between the wait he may get exposed to a virus infected person (see the cartoon below). Conversely, if the person is in infectious phase while giving swab for test, due to delays he may be distributing his virus to several. To avoid such scenarios through which extensive transmissions are happening, a cheap-daily testing becomes necessary. 

A lateral flow assay strip (similar to pregnancy detection strip) that detects viral antigens from one’s spit may be the one warranted. Here is the oversimplified version of the rapid antigen test for daily use (see the diagram below). Such paper strips use antibody against the SARS CoV-2 printed on paper strips to detect the presence of viruses in the sample. The subsequent reaction chemistry with another modified SARS CoV-2 antibody will exhibit the presence of virus as clear colored bands. One does not need any expertise in conducting this test or interpreting the results. Spit on the strip (no unpleasant nasal swabs or painful needles), add some solutions, wait a few minutes, if there are band/s- you are tested positive, isolate! If there are no bands- you are tested negative, and free to cautiously get back to work, but remember to test tomorrow. Moreover, such a test will help one to decide whether he needs to go out for his daily life activities or self-isolate himself and be a responsible person by not spreading the disease to vulnerable. TWiV lead by Prof. Vincent Racaniello and team, (https://www.microbe.tv/twiv/) supports this 1-dollar daily-rapid antigen test as an effective solution for this fight. Such a daily cheap test (compared to qPCR) will have huge impact on the current testing strategies in 3rd world and preparing all of us in case of probable subsequent waves. This test will help to get the workforce back to their jobs, kids back to their schools and ultimately save the vulnerable from getting exposed to the infected.

LAMP based testing

This is also an alternative testing approach called LAMP (Loop mediated isothermal AMPlification). Abbot ID Now machine that Mr. Trump endorsed, uses a similar isothermal reaction mechanism. The LAMP PCR reaction chemistry is complicated but quicker and cheaper than qPCR. But it is not as cheap as a 1-dollar antigen detecting strip assay that Prof. Michael Mina was proposing. As the logic above mentioned, such a test is more than enough to screen those infectious who are with viral load above the threshold to transmit the disease.

Children can transmit disease!

In many countries the schools reopening after the summer holidays. The million-dollar question is whether the kids can transmit COVID-19? Now there is overwhelming evidence that kids or youngsters don’t get seriously ill compared to adults. That does not mean this virus is not getting transmitted through them. A recent study from South Korea showed the school aged children aged 10-19 years can spread the disease at a rate similar to adults. COVID-19 transmission rate was 18.6% for household contacts of school-aged children (10-19 years) and 5.3% for household contacts of children 0–9 years. Note that they analyzed these transmissions in the middle of mitigation. Their data shows clearly that, kids also can transmit the disease and sufficient precautionary measures should be taken while reopening the schools. Another recent study conducted in the USA on a small cohort of patients shows that SARS CoV-2 viral RNA levels in infected children are similar to adults, reinforcing the common notion that children drive spread of respiratory and gastrointestinal illness in the population, and COVID-19 transmission may not be different.

Comparing Sweden´s approach to other countries

Extrapolating the controversial approach carried out by Sweden to other settings may be foolishness. Sweden has several advantages to begin with when they tried this bold strategy; such as low population, high standard of living, healthy population, efficient public health system, most households with one generation etc. Even with such favoring conditions, Sweden suffered a lot with their out-of-the box experiment. Nearly 5700 precious lives got sacrificed during the first wave of this hidden enemy, attaining a mere 6% percentage infected (10% showed serum antibodies in Stockholm- worst affected area in the country) had developed antibodies to COVID-19 and economical backlash as rest. Note, here analyses were on the antibodies against COVID-19 and not on the cell-mediated immunity arm (hence percentage will be more than reported, but no way near to attain the herd immunity). 570 deaths/million in Sweden, is similar to nearly 780,000 deaths in a country like India (would be even more if you consider the huge population density and other conditions that makes it a favorable playground for this bug). For a better ´apples to apples´ comparison, let’s take a look at the number of Scandinavian neighbors Denmark (106 deaths/million) and Norway (47 deaths/million), who took stricter lock down approach at the initial phase. 

Better be prepared 

First round of COVID-19 provided infection to only less than 5-10% of the population even in many worst affected countries. Unfortunately, this means herd immunity through natural infection is far away and may need to multiple waves of infection to attain it (if that is the way, this virus will take lives of more millions). Until an effective vaccine arrives (due to the absence of an effective drug treatment), we have to strictly follow behavioural and social mitigation measures to protect the vulnerable. Mitigation until an effective vaccine arrives may be the only possible way out. First wave of infection exposed our weakness in pandemic preparations. Learn from the mistakes, prepare the testing and hospital facilities, stockpile gloves, masks, PPEs for health workers, essential medicines, ventilators etc. In order to prevent viral spreading, we need to detect, isolate and trace contacts of the infected at the earliest. We need to catch those who are infectious at the right time.  Limited, less frequent and expensive current strategy of testing is targeting the ´detection´ of virus and not focusing on ´infectivity´ of the patients, hence not appropriate to harness this viral transmission. A cheap, rapid and daily testing method on the other hand is more efficient for controlling viral spread. Now, with the schools reopening, people getting back to work after holidays and to their normal life, things are going to be easy for this virus to have a second round of carnage. Those countries survived the first wave with less injuries should not be under the false impression that ´we are done with this virus´, understand that this virus is not done with us yet, better be prepared!

References

Updates on COVID-19

https://science.sciencemag.org/content/369/6503/510

Airborne transmission

https://academic.oup.com/cid/article/doi/10.1093/cid/ciaa939/5867798

HCQ results, do we need more evidences?

https://www.nature.com/articles/s41586-020-2575-3

https://www.nature.com/articles/s41586-020-2558-4

https://www.nejm.org/doi/full/10.1056/NEJMoa2016638

https://www.sciencemag.org/news/2020/06/three-big-studies-dim-hopes-hydroxychloroquine-can-treat-or-prevent-covid-19

Remdesivir results

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31022-9/fulltext

https://www.nejm.org/doi/full/10.1056/NEJMoa2007764?query=featured_home

LAMP testing

https://www.colorado.edu/today/2020/07/22/new-covid-19-test-returns-results-45-minutes-without-nasal-swab

Children can transmit COVID-19

https://wwwnc.cdc.gov/eid/article/26/10/20-1315_article

https://jamanetwork.com/journals/jamapediatrics/fullarticle/2768952

Relapse of COVID-19 is that possible?

https://www.bbc.com/news/health-52446965

TETRIS

https://asm.org/Podcasts/TWiV/Episodes/COVID-Tetris-Test-trace-isolate-TWiV-614

Frequent daily testing (Prof. Michael Mina)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314112/

https://www.medrxiv.org/content/10.1101/2020.05.01.20086801v1

https://www.medrxiv.org/content/10.1101/2020.06.22.20136309v2

T-cell mediated immunity and neutralizing antibody

https://www.nature.com/articles/s41586-020-2550-z

Pre-existing immunity

https://www.nature.com/articles/s41577-020-0389-z

https://www.cell.com/cell/fulltext/S0092-8674(20)30610-3?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420306103%3Fshowall%3Dtrue

Vaccine preliminary results

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31604-4/fulltext

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31208-3/fulltext

https://www.nature.com/articles/d41586-020-02154-2

https://www.nature.com/articles/d41586-020-02174-y

Herd immunity is attainable but slow!

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31483-5/fulltext

https://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2020/juni/forsta-resultaten-om-antikroppar-efter-genomgangen-covid-19-hos-blodgivare/

https://www.medrxiv.org/content/10.1101/2020.06.28.20142190v1

Timing of shedding and transmission of SARS CoV-2

https://www.nature.com/articles/s41591-020-0869-5

Viral vectors; our friends, not foes!

The word ‘virus’ recalls unpleasant thoughts of maladies like small pox, rabies, AIDS, Ebola, flu (no need to say, nowadays COVID-19) that killed millions of people in the history of mankind. Not all viruses are villains that cause deadly diseases, some even help us to cure illness (yes, seriously!). By utilizing cutting edge molecular biology techniques, we can modify even those deadly viruses to harmless tools for specific research and medical applications. Such modified viruses (here onwards will write as viral vectors) are apt ‘cargo-vans‘ to deliver foreign genes into host cells. Recombinant virus technology has unique features, establishing it as one of the most efficient techniques to address several scientific questions. It may become ´the therapeutic tool´ for fixing genetic disorders in future clinics.

Before going farther into the viral vectors, let’s talk about the less conveyed ´good side´ of viruses. Do you know a good portion of human genome consists of viral genomes? A type of viruses, especially retroviruses (for e.g. HIV, that causes AIDS belongs to the same family) while infecting the host cells, integrate their DNA into the host cell´s DNA and may remain silent for long time. When appropriate condition arises (like cases of immune suppression in host) viral DNA wakes up from the dormant stage and start forming infectious viral particles which can spread the infection further. These DNA remnants of viral attacks sometimes get into germ cells (sperm and eggs) and propagate further down the generations. Human genome consists of 3.2 billion base pairs. Only 1.5 % of our genome codes for proteins (genes carry information to make proteins). Interestingly, the rest of the DNA was thought as ´junk´ a couple of decades ago, now we know that they are not junk but carries important instructions for gene production (to drive the protein synthesis from genes). Moreover, now we know that nearly 8.3 % of our genome consists of viral genetic material, contributed by the earlier viral infections. That means a good fraction of our genetic material is due to the viral infections and subsequent integrations of the viral genetic materials- that might have happened millions of years ago to our common ancestors, even before Homo Sapiens emerged. Human genome can be called as a ‘cemetery’ for such retroviral infections, that might have happened to our earlier ancestors.

Some of these viral genes may land on right locations in our DNA genome strand and may get repurposed into some cellular functions (a novel function even). Here is one example how we repurposed a viral protein for a very vital function in our body. A viral gene called ´Env´ which is on the surface of a retrovirus helps them to fuse to the host cells they attach. This process very important for the entry of the virus into the cells. Inside the cells, the viral genetic material will produce more of this Env protein along with other viral proteins, which is directed on the surface of the infected cell. The host cell that expresses this Env protein will fuse with the neighboring cells and form single big fused cells called ´Syncytium´ (see the diagram below). This is a technique employed by the virus to spread easily from one cell to another. In year 2000, a Scientific team in Boston, USA discovered a peculiar gene in the human genome that codes a protein (later they named ´syncytin´) made only by cells in the placenta. Syncytin helps to form a layer of fused cells, which is important for the proper functioning of the placenta. This layer of fused cells forms the outer lining of the umbilical cord and with the placenta, helps in nutrient exchange between mother and fetus. A protein, thus donated to mammals by one of the ancient viral infections, helped us to transform from our egg laying mammal ancestors to placental mammals.

Host cell that expresses Env protein will fuse with the neighboring cells and form single big fused cells called ´Syncytium´. Image from https://viralzone.expasy.org/5957?outline=all_by_species
Syncytin a protein we domesticated from virus, helps to form a layer of fused cells, which is important for the proper functioning of the placenta. This layer of fused cells forms the outer lining of the umbilical cord and with the placenta, helps in nutrient exchange between mother and fetus. Image from https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000028

Very recently there came some reports on repurposing (exaptation) of parts of a viral gene even in our brain. Retrovirus (mentioned earlier; the family of viruses that includes HIV) uses ´Gag´ gene to produce a protein that forms a protein shell called capsid to protect the genetic material of the virus. A protein in our brain called Arc (this protein is known to play an important role in memory formation) contains parts of the Gag which helps Arc to form functional capsid like structures, that may be vital for its function and thus memory process. This is another example of how a viral gene (donated by an ancient viral infection) is repurposed for functions like neuronal communication.

Viral vectors as gene delivery vehicles

Recent years have witnessed a tremendous development of molecular tools for editing the genome, gene regulation and visualising and manipulating cellular functions, allowing research that until recently was considered ‘science fiction’. There exist several ways such as chemical methods, physical systems and viral vectors, to introduce such tools to cells in a laboratory for research purposes or in the clinic to cure diseases.Viral vectors stand out as the most efficient foreign gene delivery system, especially for applications inside living organism (in vivo). Viruses that have evolved through millions of years can invade all kinds of cells (from tiny bacteria to human cells), hijack the host cell’s machinery to multiply themselves and spread. One can alter the viral genetic material to remove viral proteins essential for its replication and introduce foreign gene(s) of interest, instead. Non-replicative ´gutless´ viral vectors thus produced have the innate ability to target, enter inside the host cells and produce proteins of interest instead of viral proteins. Such viral vectors are highly efficient for difficult-to-transfect (transfection = scientific term for introducing foreign gene into host cells) cells, for studying biological mechanisms in tissue slices or in vivo systems, and recently for introducing genes in human patients in clinics. Viral vectors are clinically approved to deliver functional copies of faulty genes to fix human genetic disorders. No need to mention the tremendous application of viral vectors in research, even cell-type specificity can be achieved to an extent by using a combinatorial approach of conditional viral vectors with the ever-expanding list of transgenics models.

Common viral vectors

Various viruses evolved specific features, which we can exploit for particular research questions. Moloney murine leukaemia virus, for example, is only expressed in dividing cells, and hence suitable for studying cellular mechanisms of dividing cells alongside neighbouring mature cells in intact tissue (thereby a valuable tool for adult neurogenesis research). Rabies virus jumps across the synapses between neurons, but only in the backward direction (retrograde) and is therefore efficient for tracing neuronal connections in the intact brain (mapping connections in the brain). Adeno-associated viruses are the most popular vectors. They are easy to synthesize, do not integrate into the host cell’s genome and can target many types of cells. Lenti virus is useful for long-term gene expression both in vitro and in vivo. Some viruses evolved coat proteins to target only particular kinds of cells, termed innate tropism. By leveraging the innate tropism of a virus, or using cell-specific regulatory elements (we can design viral vectors to target specific type cells in an organ), one can target and introduce therapeutic molecules to specific cells and thereby avoid side-effects in neighbouring cells, a major concern in modern medicine.

Production of viral vectors in a lab

Viral vectors are prepared using mammalian cells as ´production factories´ in a sterile, confined environment with strict safety regulations (biosafety level 2 is enough for gutless viral vectors). Vital replication genes from a viral genome are replaced with foreign genes of interests using molecular biology techniques. Along with the genes/instructions to make our desired protein, additional essential genes are provided separately (in-trans) to cells that make the viral vectors. The gutless viral vectors thus created are infectious, produce exclusively the foreign gene in host cells and since it lacks the replication genes in its genome, they cannot make active, harmful virus particles in any infected cells. Recombinant viral vectors hence work as safe ‘cargo vans’ for delivering our cargo genes. These delivery vehicles with foreign cargo genes are further purified and administered to specific regions (e.g. retinal injections to fix blindness). Please see the simplified diagram below. Cargo genes will be expressed in infected cells and viral vectors being non-replicative, it cannot further infect neighbouring cells like a disease-causing natural virus.

Make a cocktail of all the genetic instructions (DNA) to make virus (viral vector) and introduce into cells in a culture plate. The cells will act as a virus factory. After several days purify the viral vector which can be used for downstream applications.
The top image is a culture plate with cells producing viral vector with the code of a red fluorescent protein . Upon shining those cells with a green light you can see those virus producing cells as fiery red spots. The below image is the microscopic view of the cells (red spots) producing virus.

Clinical applications (gene therapy)

We can alter the viral genomes by taking away the viral proteins and replacing them with the genes of our interest and use the ability of viruses to infect cells and produce the molecules of our interest instead of the viral proteins. Such genetically modified viruses (viral vectors) are used as delivery vehicles/vectors for genes, as research tools and recently to fix genetic diseases. It is not science-fiction anymore, such gene therapies are already in clinics. Here are a couple of examples how viral vectors are used for curing genetic diseases. Recently, FDA approved one-time viral based gene therapy for curing the genetic disorder causing Retinal dystrophy that lead to blindness. Some unfortunate kids are born with mutations in both copies of the RPE65gene, they can experience sight loss at the early age which will progress to total blindness. A functional copy of the RPE65 gene provided by Luxturna can restore vision in children and adults.

Another viral based gene therapy restores functioning of immune systems in young children with a severe disorder caused by mutations in a gene that is crucial for immune-system development, causing severe combined immunodeficiency disorder (SCID-X1). The disease is called ‘bubble-boy’ disease because of the plastic enclosures in which they live, to protect affected children from possible infection. For them, even a common cold can be fatal. The gene-therapy treatment introduces the functional copy of gene IL2RGrestoring the proper immune system. More recently a tool for fixing DNA errors (CRISPR) is entering into the clinics to fix the genetic disorders.

Viral vector-based vaccines (a COVID-19 story)

Beijing Institute of Biotechnology (China) and Oxford University (UK) are conducting clinical trials for vector based COVID-19 vaccine. The Jenner Institute’s (University of Oxford) Vector based COVID-19 vaccine a promising vaccine candidate for COVID-19. It is called ´ChAdOx1 nCoV19´ which is the abbreviation of Chimpanzee Adenovirus Oxford1 novel CoronaVirus 2019). They use another crippled Adenovirus, that cannot replicate in host cells, to deliver the code for spike protein of SARS CoV2 and subsequently to elicit the immune response. The genetically modified Adenovirus will infect the host cells but cannot replicate itself. Upon infection, it faithfully expresses the cargo it carries (here the spike protein of SARS CoV2). This foreign protein will evoke the defense system and produce the appropriate immune mechanisms in vaccinated host. Another example for viral vector-based vaccine is one with brand name ´Ervebo´, that uses another genetically modified virus to produce an important protein of ebola virus as antigen. More such vaccines are in the pipeline using these platforms.

Viral vectors, the future technology

There is one thing that is in common, that made all these applications possible-the usage of viral vectors. In all these cases viral vectors are used as delivery vehicles for introducing the genes. Genetically modified gutless Adeno virus, Adeno-associated virus (various serotypes), Moloney murine leukaemia virus, Lenti virus and Rabies virus are common ‘delivery vehicles’ in research nowadays, each having distinct characteristics making them suitable for different applications. Viral vectors became an inevitable cutting-edge tool for life science research, we deploy them to manipulate mechanisms in cells in a culture plate to behaviours of intact animal. They are used as vaccines against deadly diseases. ´Ervebo´ (brand name), uses another genetically modified virus to introduce an Ebola virus protein as ´antigen´ into a host. In clinics, we already started using viral vectors carrying the right copies of the faulty genes, to fix genetic disorders (gene therapy). One of the main drawbacks of several lifesaving medicines is their ´side-effects´- while it does the ´right job´ at the right place besides, it can create complications at the wrong places. In future, viral vectors may be able to deliver therapeutic molecules to specific regions in an organ. Researchers (including us) had already shown in lab animals that we can target specific cells even in a complex organ like brain (like accurately addressing someone and delivering a package in a huge crowd). Active research is happening to polish these valuable tools further both for research and clinical purposes. In recent future, viral vector mediated gene therapy will be playing a vital role in clinics. 

Visit our webpage (www.ntnu.edu/kavli/viral-vector-core).

Video of this blog: Viral vector carrying gene works in a cell

References

Viral elements in our genome

https://www.nature.com/articles/nrg1674#Sec4

Domestication of retroviral gene-Syncytin and Placenta

https://www.nature.com/articles/35001608

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000028

https://viralzone.expasy.org/5957?outline=all_by_species

Memory related Arc protein and it´s viral protein connection

https://www.sciencedirect.com/science/article/pii/S0092867417315040?via%3Dihub

Luxturna for curing a retinal blindness disorder 

https://luxturna.com/about-luxturna/

https://www.nature.com/articles/nbt0118-6a

https://sparktx.com/press_releases/fda-approves-spark-therapeutics-luxturna-voretigene-neparvovec-rzyl-a-one-time-gene-therapy-for-patients-with-confirmed-biallelic-rpe65-mutation-associated-retinal-dystrophy/

Gene therapy for Bubble-boy disease

https://www.nature.com/articles/d41586-019-01257-9

https://www.nejm.org/doi/full/10.1056/NEJMoa1815408

Viral vectors as research tools

https://www.frontiersin.org/articles/10.3389/fnana.2015.00080/full

Cell specific expression of viral vectors

https://www.nature.com/articles/nn.4430

https://www.sciencedirect.com/science/article/pii/S2589004220300729

Saviours of our life from deadly pathogens

Vaccines are powerful weapons in our arsenal against many fatal viral diseases. Eventually they became vital for our survival against several deadly pathogens. Vaccines saved millions of lives from deadly diseases like polio, measles, mumps, rubella, rabies and we globally eradicated disease like smallpox (almost eradicated polio by last year).

Once a virus enters our system (a cell), the resident guard cells recognizes the signs of the infection (viral proteins or chemical signal calls from an infected cell). Within weeks after a viral infection our body will create tailored antibodies and immune cells which will inactivate the intruder and eventually these immune responses subside. But the memory to create those weapons retains and even after several years our body will initiate a quick immune response on a second attack by the same pathogen. Antibodies are specific proteins produced by our defense system (triggered by the presence of a pathogen or its proteins) to selectively destroy the invading pathogens. Our immune system is very efficient in directing specific immune cells (like activated T-cells) also to scavenge the pathogens or even kill the infected body cell (altruism). We have a strong immune response, a reason why we are protected from several pathogens that we encounter daily. Note that the viruses are masters in evading several antiviral mechanisms of the host. There exists a constantly evolving battle between these intracellular parasites and host defense system. Both the invader and the host emerge with newer strategies to conquer each other, the fight continues.

Vaccines activate and teach our defense system by creating antibodies that neutralize the attacking pathogens and activate the cell mediated immune responses and keep the memory of the attack so that our body mobilizes the arsenal quickly upon a real attack by that pathogen. Nowadays, making a vaccine is much faster due to technological advances but the clinical trials to approve them for efficient & safe use takes times in months. We are hearing about frustrating delay in COVID-19 vaccine development and hopefully you may learn the reasons for that scenario, after reading this blog.

History of vaccines

Variolation (image source Wikipedia)

During 11th century A.D., China practiced primitive form of vaccination called ´variolation´ for smallpox. They took the pustules from smallpox patients, dried them up and blow the pustule contents into the nose of healthy individuals to protect them from deadly smallpox infection. Later in 17th century, Lady Montagu the wife of the British ambassador in Constantinople, brought this practice of variolation to Europe.

  • Smallpox vaccine
Edward Jenner (image source Wikipedia)

Smallpox, a dreadful viral disease that killed millions all over world since medieval times. During 1796, Edward Jenner in England, noted that milkmaids don’t get smallpox since they get a milder version called cowpox (Variolae vaccinae). He took pustules from a cowpox virus-infected milkmaid and inoculated a boy named James Phipps with the pustule contents. Later he challenged that inoculated boy with smallpox pustule and noticed that the boy was protected from smallpox (such a challenge experiment is difficult to imagine these days!). This became a practice which saved many lives from this deadly disease. Very interestingly, Jenner did not know anything about viruses at that time. You can watch very distressing images of smallpox infections to realize from ‘what‘ the vaccine saved us (see references, painful to watch those images!). Thanks to vaccines, in 1980, WHO announced the eradication of smallpox one of the greatest achievements in history of mankind.

  • Rabies vaccine
Louis Pasteur (image source Wikipedia)

Almost a century later, Louis Pasteur in France developed the Rabies vaccine and coined the word ´vaccine´ for all protective inoculations (vacca means cow in Latin) in honor of Edward Jenner´s work on cowpox. Rabies is a very deadly zoonotic disease which kills several thousand even these days. Louis Pasteur developed the rabies vaccine by weakening rabies virus in rabbits and subsequently harvesting it from infected rabbits´ spinal cords (not knowing the disease-causing agent is a virus). By serially growing the virus in a different species (here rabbit), the virus gets adapted to that species with mutations. This may result in less adaptation to its original host, along with decrease in virulence of the virus (attenuated). Such live but attenuated virus still retain the features that provide the immune system enough information to develop immunity to the original virus. Pasteur is also known for his works on chicken cholera vaccine and anthrax vaccine for cattle.

A bit more on vaccines; Passive and active forms of immunization

In ´active form´ of immunization, inactive forms of the pathogen or pieces of pathogen are applied as vaccines to trigger the immune system to produce the antibodies or the cells to fight against the pathogen. This provides long-term protection against that pathogen.

The ´passive form´ of immunization provides short-term protection where antibodies or immune cells from recovered person is applied to another person who is at the risk of infection. Recently, you might have read about the convalescent serum therapy given to many COVID-19 patients at risk, where antibodies from blood donated by COVID-19 survivors were applied to risk group patients (which provides a temporary protection).

Rules for an effective vaccine

  • It should trigger the immune response in vaccinated person
  • It should create the appropriate protective response (either antibodies, activated immune cells or both).
  • It should protect the vaccinated person from that specific pathogen
  • It should provide long-term protection
  • It should be safe (no side effects), easy to use, long-term storage

Making different types of active vaccines

  • Live-attenuated vaccine

Live-attenuated vaccine are weakened (attenuated) form of the pathogen. E.g. Salk´s polio vaccine (read below).

  • Inactivated vaccine

Chemically inactivated form of the pathogen. E.g. Sabin´s polio vaccine (read below).

  • Subunit vaccine

Here pieces of pathogen (antigens-proteins or genetic material) are used to evoke the immune response. E.g. SARS CoV2´s mRNA based Moderna vaccine against COVID-19 under testing (read below).

  • Vector-based recombinant vaccine

With the modern genetic engineering methods, we can use another crippled virus (viral vector) as the delivery vehicle. This ´cargo van´ virus will introduce the genetic message of another pathogenic protein into our cells. The pathogenic protein produced will elicit the appropriate immune response in host. E.g. Ervebo against ebola virus (read below).

  • Toxoid vaccine

Immune response can be targeted to the toxin created by the pathogen.

Vaccination mediated herd immunity

To stop a viral transmission, enough number of people in the population should be immunized (note: we don’t have to immunize 100% of the population to break the chain of viral transmission). The percentage of the population to be immunized to attain the herd immunity vary for different viruses. A highly transmissible diseases like measles need around 95%, while for COVID-19 somewhere between 50-70% of the population should be immunized either by natural infection or vaccination to attain the herd immunity.

Delay in approval; several phases in clinical trials

With any new medical intervention, Scientists should conduct careful experiments for attaining necessary evidences to obtain its approval as a medical treatment. It involves several phases; experiments start with animal testing after the discovery. After successful animal experiments with the novel treatment protocol, the next stages are the clinical phases in human subjects. Early phase involves dose determination and safety tests in a few healthy human volunteers. Later phases test many human participants to determine the efficacy of the treatment.

Vaccine trials takes months to years to complete and involve 2 experimental groups. One is control group treated with ´placebo´ (mostly an established vaccine against a different pathogen) and other group is treated with ´vaccine candidate´. These studies are ´double-blinded´ which means both the parties, the administrator and the recipient do not know if the recipient is receiving the vaccine, to avoid any bias. In some studies, on deadly diseases, in place of placebo control group, there will be ´immediate´ and ´delay´ vaccination groups due to ethical considerations (to provide all the volunteers a promising treatment for a deadly disease as quickly as possible).

Phase 1 trial; involve safety and immunogenicity studies in a small number of closely monitored human subjects.

Phase 2 trial; involve dose studies and hundreds of human subjects are needed.

Phase 3 trial; studies effectiveness and additional safety data required for approval as medical treatment, thousands of human subjects are needed.

Phase 4 trialstudies on many vaccines on side effects and long-term immunity, once they are on the market.

Some modern-day vaccines

There are several life-saving vaccines in the list. WHO ensures that the vaccines used in immunization programmes are safe and effective (list of the prequalified vaccines are in the references). Below, I will be discussing some of the cherry-picked ones.

  • Polio vaccine
Jonas Salk (image source Wikipedia)

Poliomyelitis (polio) is a highly infectious viral disease, mostly children under 5 years of age are main victims. Polio virus spread through fecal-oral route (contaminated water/food) and multiplies in the intestine. The virus can subsequently invade the nervous system and cause paralysis.

Jonas Salk developed injectable polio vaccine and became the saviour of several kids from paralysis and iron lungs (many ended up in tank ventilator called iron lungs to assist breathing). Jonas Salk was quite unconventional and used inactivated polio virus (chemically inactivated using formalin) to mobilize the immune system to fight against the virus. More than one million school kids participated in the polio trial in 1954. In 1955, the vaccine was announced safe and he became a hero after the vaccine development. Interestingly, in an interview he was asked about who owns the patent of the polio vaccine, his said ´there is no patent, could you patent the sun? ´ Prevention of polio is one of the greatest achievements of medical science. Later in 1962, Albert Sabin introduced the oral poliovirus vaccine which is a live-attenuated vaccine. This vaccine was simple to use but with a rare risk of reversion of attenuated virus to virulence.

  • Influenza vaccine

Influenza virus, that causes flu is a segmented RNA virus (not a single string), notorious for frequent mutations, escapes our immune surveillance system and that is the reason why every year we need different flu vaccine.A commonly used influenza vaccine is developed by chemically inactivating the influenza virus grown (after finding the current seasonal strain and guided by WHO) in chicken egg embryo (watch the video at the end).

  • Ebola vaccine

Another deadly zoonotic disease caused by ebola virus, that result in hemorrhagic fever which leads to internal bleeding and death. Several types of vaccines are being developed for this disease, making use of the modern technology. Some of the vaccines are ready to use and some are in clinical trials. Two interesting ones are discussed here. One is viral vector-based active vaccine with brand name ´Ervebo´, it uses another genetically modified another virus to produce an important protein of ebola virus as antigen. Another one, is a passive therapy, i.e. making antibodies against ebola virus a genetically modified in tobacco plants (a cousin of tobacco, the one that people uses for smoking), which is then harvested for making antibody cocktail against ebola named ´Zmapp´ (see video below).

All eyes are on SARS CoV2 vaccine now

According to WHO´s list, there are more than 100 vaccine candidates under different stages of testings. Some are undergoing clinical trials, and some are on preclinical trails. Here I am discussing 2 main vaccine candidates which got wide attention which may become the saviour/s of millions of lives in future.

RNA vaccine

Moderna´s (an US based biotech company) COVID-19 vaccine consists of a piece of genetic information of spike protein of SARS CoV2 (information in the form of RNA molecule). It is designed to produce a part of viral spike and trigger the immune system to make antibodies that recognize and neutralize the spike protein that SARS CoV2 uses to enter host cells. It is under clinical trials now (into Phase 2 trials, very promising).

Vector-based vaccine

Beijing Institute of Biotechnology (China) and Oxford University (UK) are ahead with clinical trials for vector based COVID-19 vaccine. The Jenner Institute’s (University of Oxford) Vector based COVID-19 vaccine a promising vaccine candidate for multiple reasons. They named it ´ChAdOx1 nCoV19´ which is the abbreviation of Chimpanzee Adenovirus Oxford1 novel CoronaVirus 2019). Here they use another mild virus (a crippled Adenovirus), that cannot replicate in host cells, but to deliver the code for spike protein of SARS CoV2 and subsequently to elicit the immune response. The genetically modified Adenovirus will infect the host cells but cannot replicate itself. Upon infection, it faithfully expresses the cargo it carries (here the spike protein of SARS CoV2). This foreign protein will evoke the defense system and produce the appropriate immune mechanisms in vaccinated host.

They had an advantage compared to other vaccine candidates, the platform had undergone and proven safety clinical trials with regard to MERS, another closely related Coronavirus from the same family. Further, recently they reported in BioRxiv that a single dose of this vaccine prevented SARS CoV2 pneumonia in monkeys. Giant vaccine manufacturers like Serum Institute of India (world’s largest supplier of vaccines) is involved in the massive production to provide huge world-wide demands of the vaccine if this promising vaccine candidate turns into ´the COVID-19 vaccine´. Fingers crossed!

Several other promising and interesting vaccine candidates undergoing trials these days. British American Tobacco company is going forward with clinical trials of a recombinant COVID-19 vaccine, where they produce SARS CoV2 protein in tobacco plants (similar engineering approach to ebola vaccine Zmapp, mentioned above). 

Anti-vaxxers

Opposition to this life-saving approach (vaccination) is not new. Soon after Jenner´s smallpox vaccination, there was anti-vaccination movement (below, see the interesting cartoon from 1802, inspired by the controversy related to Jenner´s vaccine; that parts of cow may grow after the cowpox pustule inoculation). This hesitancy towards vaccination, unfortunately observed even present days, could be due to several reasons like religious, political, sanitary, fear and pseudoscientific. A very recent study shows that though these anti-vaccination protests are small, but worryingly growing amid this COVID-19 pandemic time (to date, more than 300,000 COVID-19 deaths worldwide). Anti-vaxxers spread emotional messages on children’s health, advocate alternative medicines and connect vaccinations to conspiracy theories. On the other hand, pro-groups have a direct message that ´vaccines work and save lives´. By analyzing the social media activities, the study warns that within a matter of 10 years the anti-vaccination campaigns may dominate over the pro-vaccination views, which is alarming. A better way of communicating with concerned parents about the real risks and benefits of vaccination is warranted. Once the intensity of this novel emerging disease subsides, the anti-vaxxers may gain momentum.

Cartoon from 1802, inspired by the controversy related to Jenner´s vaccine.

Conclude

            In the absence of an efficient anti-viral drug against COVID-19, a vaccine is essential to stop the spread of SARS CoV2 virus. Attaining herd immunity through natural infection of this virus takes long time, moreover at the expense of millions of lives. Vaccination, in a way is teaching our body how to fight against a killer virus, when it encounters a real one. Nowadays we have technologies to quickly make vaccines against any novel virus, approval of a safe and efficient vaccine demands rigorous testing and hence takes time. Some of the vaccine candidates are showing promising preliminary results. We may have to continue practising social distancing measures, contact tracing and quarantine measures until there is an effective treatment protocol, to avoid choking the health system. Even though vaccine trials take time, it is important to ascertain that COVID-19 vaccines that eventually appear in market are safe and effective, or else this may cause more damages than the disease itself. Once approved, another challenge in the future may be bulk production and vaccinating billions in a short period of time.

Videos of this blog

Influenza vaccine production using egg embryos

Ebola vaccine development using Tobacco plants

References

Clinical trials

https://www.fda.gov/vaccines-blood-biologics/development-approval-process-cber/vaccine-product-approval-process

List of WHO-prequalified vaccines

https://extranet.who.int/gavi/PQ_Web/

Anti-vaccination campaign

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140172/

https://www.bbc.com/news/uk-england-leicestershire-50713991

https://www.nature.com/articles/s41586-020-2281-1

Vector based vaccine for COVID-19; ChAdOX1 nCoV19

https://www.biorxiv.org/content/10.1101/2020.05.13.093195v1

WHO’s COVID-19 vaccine candidates list

Convalescent serum for COVID-19 treatment

https://www.nature.com/articles/d41587-020-00011-1

Moderna’s mRNA vaccine to fast-track clinical trials

https://time.com/5835785/moderna-coronavirus-vaccine-phase-2/

Smallpox infection images (graphic images!)

https://www.bbc.com/news/uk-england-birmingham-45101091

COVID-19 origin conundrum

Along with a viral outbreak, many conspiracy theories emerge-it is unavoidable. Conspiracy theories flourish and spread even more virally than the viral disease. More interestingly, such conspiracy thinking may not disappear even after the outbreak subsides. If you look at the history of AIDS, vaccines, or any novel therapeutic interventions in the past, you may find several such theories which are still floating around. It is human tendency to easily distrust other groups, which may be the heart of conspiracy thinking. My efforts here is to present what the Science says so far about the origin (keep in mind, this is an area where serious investigations are happening now- our views may change accordingly). We are seeing thousands of deaths daily, crippled economy and health system throughout the world by this tiny string of RNA coated in a lipid layer. We need to dig into the available Scientific data to develop an opinion about the origin of this virus. Researchers are working round the clock to solve many questions with regard to the nature of this virus, the features of the disease, cure for COVID19 and revealing more about this virus every day.

With regard to the Origin of SARS CoV2, there are three main possible scenarios, 

a) Bioengineered origin– this virus is designed, cloned and prepared by molecular virologists somewhere in a secret lab and deliberately released to a population (as bioweapon) to eradicate the human race? (this virus is not selective for a specific regional population).

b) Natural origin– this virus, is natural origin and got transmitted to humans from wildlife (a zoonotic transfer happened from a bat or through an intermediate host).

c) Accidental lab-escape– this virus got leaked from a lab conducting research on bat Coronaviruses. Either through improper disposal of viral samples or through researcher accidentally got infected and became the patient zero for this worldwide mess.

Origin: Bioengineered or Natural?

I would take the two scenarios a) Bioengineered origin and b) Natural origin together since they are addressing same kind of question. To know more about a virus, uncovering its genomic sequence is very essential.

SARS CoV2 sequence in public database

In December 2019, an outbreak of acute respiratory syndrome was first reported in humans (Wuhan, China). The exact date of the first report is still under investigation and being updated according to the evolving events- so far, we know that the first hospitalized case was on December 12th. According to WHO, they were informed about the cases of pneumonia on December 31st, 2019. Initially it was thought human to human transmission may not occur. While on 24th January 2020, in scientific journal Lancet published a study from China on familial cluster of pneumonia suggesting the person-to-person transmission. It is quite impressive that after the first report the genomic sequence of SARS CoV2 was published within weeks by a team lead by Chinese researchers and deposited in GenBank open access public database on first week of January. They published their work in in scientific journal Nature, where they submitted their data to that journal on January 7th 2020.

One of the main criticisms was the delay in the publishing SARS CoV2 sequence. Noticing that first report was on December 12th, publishing sequence in public database within weeks’ time is impressive. Naming a disease and knowing this vital sequence information helped us all around the world to understand more about the causative agent of this disease, to create tests and to design vaccines. Thanks to the modern technology, it took several months after the first report to release the sequence of past outbreak SARS-1 (2002-2003).

Bat coronavirus research labs under suspicion

Though several viral labs have the capability to do world class research on viruses, due to its location at the epicenter of COVID-19 outbreak (Wuhan) and the presence of a bat coronavirus lab, Wuhan virology Institute (WIV) came under suspicion. Prof. Shi Zhengli in Wuhan Institute of Virology (WIV) is a renowned bat Corona virologist, known as the ´bat woman´ in the field. She is well known in the scientific world for her extensive contributions in understanding the SARS coronaviruses. Once applauded as hero, now fiercely criticized by the conspiracy theorists around the world for her research on bat coronaviruses. Let’s dig into the relevant works from Prof. Shi Zhengli and her collaborators.

Shi Zhengli´s research lab in Wuhan Institute of Virology (WIV) also sequenced this virus and published in scientific journal Nature on 3-February-2020. This scientific article provided vital information regarding the contagion. They found that, this virus belongs to SARS family (before that it was an ´unknown ´disease in Wuhan), they also sequenced this novel virus (which helped to design the test for detecting the virus), the entry mechanism of this virus into the host cell (cell surface protein ACE2 receptor helps the virus to provide the entrance). They took the samples from the patients and analysed complete genetic sequence (RNA) of this virus. They noticed that novel Corona virus is 96% identical to a bat coronavirus (bat CoV RaTG13). Thevirus shared 79.6% sequence identity to SARS-CoV (the lab was conducting researches on both viruses). From an evolutionary point of view this 96% similarity is not very close (note we share nearly 96% genomic similarity with our cousin chimpanzee). According to Prof. Edward Holmes (whose was in a different team that sequenced SARS CoV2) points out that the sequence divergence (is equivalent to almost 20-50 years apart). Considering the slow pace at which this virus is mutating, it may take several years for those bat viruses from WIV to attain the present form, as SARS CoV2.

She was in the scientific team that found the connection between the bat and the SARS CoV1 outbreak in 2002 and warned such spillovers from natural bat reservoir may happen in future also. Her team found the ´origin´ cave (Yunnan, China) and a population of bats (smoking gun) from which 2002 SARS Coronavirus might have jumped and ultimately reached humans through civet cats as intermediate hosts (2002-2003 SARS killed nearly 800 people). They predicted the possibility of similar spillovers from bat to humans or through an intermediate host in future.

She was also involved in a controversial study on a chimeric coronavirus (word chimera is from Greek mythology; a creature with lion´s head, goat´s body and dragon´s tail).  The chimeric virus was made with bits and pieces from different viruses, and the study was designed and conducted by US lab (Dr. Vineet Menachery and team at Prof. Ralph Baric´s lab in University of North Carolina), in which Prof. Shi Zhengli was a collborator. They created a lab-engineered virus with SARS backbone with a spike protein from a horseshoe bat coronavirus (SHC014). Contrary to predictions from sequences, their experiments proved that the spike protein in chimera can bind to ACE2 receptor of host cells and is vital for infecting and grow in mice and human airway cells in petri dishes (caused illness in mice). This controversial but crucial study showed that SHC014 virus can infect human cells and could be a potential threat for future outbreak. What was the rationale for making a chimeric virus? They claimed, such a chimeric virus-based approach may help us to find the potential pre-pandemic viruses. This designed/engineered virus got fierce criticisms from some members of scientific community.

Recently, Prof. Ralph Baric´s lab proposed the possibility of using stable, engineered SARS CoV as replacement of live-attenuated virus vaccine. Live-attenuated virus vaccine is a savior and used successfully for fighting against deadly diseases like polio, measles, mumps, chickenpox etc, but poses a risk of reverting back to virulent form by mutation or recombination events (rare chances though). Baric´s lab deleted the gene tools from the SARS viral genome that help the virus to recombine, thereby handicapped it from reverting back to its virulent form. Their strategy is advantageous to create attenuated vaccine for CoVs in general, as several zoonotic CoV outbreaks are happening recently (several outbreaks even before COVID19).

The above mentioned are the most relevant scientific research articles from Shi Zhengli and her US collaborators that links them to coronavirus. None of these labs under suspicion were doing any secret researches. While, they were publishing their research works in several journals in the past as well as during this present outbreak. In fact, their research helped us to understand more about the SARS CoV in general otherwise it would have been difficult to fight against this virus even to this extent. Dr. Peter Daszak (a member of the team that found the link between bat and SARS) says ´´she should be lauded as hero, not vilified”. 

´Errors make us to evolve´- Mutations lead to natural evolution

Mutations are the keys for evolution, this happens in all species. Viruses produces numerous progenies harbouring mutations that too very fast, we are experiencing evolution in front of us. Such mutations accumulate during viral propagations which sometimes may result is a new strain with significant genetic drift from an ancestor virus (see the diagram below).

Several random copying errors occur during replications of genetic material of SARS CoV2 inside a host cell, the reason why there are many mutations during transmissions. SARS CoV-2 is the largest RNA virus known, with nearly 30,000 bases giving more chances for copying errors. In general, RNA dependent RNA polymerase (RdRp); the protein machine that makes copies of RNA does not have efficient proofreading mechanism like our DNA coping system. Such viruses ´always´ mutate and accumulate mutations during the process of transmission from one infected individual to another. Due to frequent mutations, viruses that causes flu escapes our immune surveillance system and that is the reason why every year we need different flu vaccine. Very interestingly, RdRp of SARS CoV2 has a proof-reading mechanism (if error happens while copying, a part of machine will correct it). Hence experts predict that it may take several years to see a major genetic/antigenic drift when compared to a flu virus which is a fragmented RNA virus. Compared to flu virus the ´copier machine´ for SARS CoV2 is less error prone, and the genetic material is a single RNA-string hence natural immunity that survivors attain by infection may defend us for longer time period (though predictions on a novel virus is complicated). These copying errors made by the virus (that makes a new strain) are useful for researchers to trace the tracks of the viral propagation which is very important during an epidemic episode to understand more about the route-map of pathogen.

Reports on SARS CoV2 mutations

A natural mutant of SARS CoV1 was reported recently, where a 29-nucleotides deletion happened in its genome during its early transmission, that made it less virulent. Sometimes a deletion of a large chunk from the genome may result inactivation of essential genes (change in a couple of alphabets (nucleotides) may not affect a virus, but several changes may change the nature of the virus). The gene that was affected by this deletion is called ORF-8 (a gene supposed to help in replication of this virus). More interestingly a deletion of 382-nucleotide deletion was already noticed from Singapore in a BioRxiv manuscript (whether this strain of virus is the reason for the relatively less damage caused by COVID-19 in Singapore, will be interesting to study). 

SARS CoV2 sequence comparisons

Comparison of the published genomic sequence of SARS CoV2 with that of the other viruses will help us to identify where this virus belongs to. Several scientific articles came out suggested the high sequence similarity of SARS CoV-2 with bat CoVs (RatG13), proposing the possibility that this CoV may be transmitted from bat mostly through an intermediate host. Their data supported the possibility that SARS CoV-2 is a natural mutant and debunked the ´designed/engineered virus´ hypothesis. One research group compared sequences of an important part of the key viral protein (spike) and noticed that it matches with that of the pangolin CoV. The reason pangolins as the intermediate host thus came into picture. Also, SARS CoV2 virus optimized itself during initial propagation (possibly in intermediate host) by acquiring furin cleavage site in on the spike protein, that helps in fine tuning the host range. This virus is notorious for stealing genetic materials from similar CoV from the host (the mechanism is called recombination), and current thinking is that the ancestor SARS CoV from a bat from China leaked into intermediate host and acquired some gene sequences by recombination event from host CoV, before jumping into humans as SARS CoV2 causing this outbreak (see the diagram below). Note, millions of inefficient viruses fail during these processes, while a very few viruses become successful in adapting to new host. 

Early spillover?

It should be perceived very clearly that we noticed COVID19 by the end of 2019. That does not necessarily mean that this virus jumped from bats towards the end of 2019, it only means we detected it then. It is difficult to predict how long we had this virus among our population. SARS CoV2 might have infected the ´patient zero´ several months or even years before we detected it. Prof. Robert Garry proposes (article in journal Nature medicine) such a scenario that the virus might have spilled into human population long back and was silently optimizing itself to its present form (check interesting history of origin of AIDS pandemic). Virus adapts to a new species by undergoing mutations and sometimes recombination while propagating. On this road, the chances for a novel virus to fail is high. Hence, both natural events like recombination as well as genomic mutations helped in the making of this ´perfect virus´ named SARS CoV2.

Lets come to another pandemic, AIDS and tracing the history of HIV origin. We came to know about this virus HIV and the deadly disease AIDS in 1981, but analyses of older blood samples and genetic sequences of HIV changed our views about the origin of AIDS. Current understanding is human population was having HIV infections long back even first decades of 19th century. The spillover might have happened from monkeys to a bush-hunter in Congo, Africa around 1920s. Similarly, a retrograde analysis of the patient samples stored from hospitals may change the history of COVID19 too (it is tough when compared to HIV, to retrogradely analyse the long history of this virus since SARS CoVs don’t integrate into host genome like HIV). Isolating viral genomes from already collected patient samples (with regard to other ailments with similar symptoms) and analyses may give some insights into COVID19 history.

Zoonotic diseases

SARS and MERS were also originated from bats through civet cats and camels as intermediate hosts respectively. In the recent past, Ebola was reported first from African continent, MERS from Middle East, Nipah outbreaks were reported from Malaysia and later from Indian subcontinent. HIV is another zoonotic disease that spilled over from monkeys, in beginning of 19th century. Nature carries out high-throughput mutation experiments on these viruses (mainly by copying errors and by recombination) in different species.When other animals including us come in close contact with such nature´s viral reactors, some of the mutated viruses may find a new host causing serious illness. Such zoonotic diseases (diseases that are transmitted from animals to humans) will happen in future also. Moreover, we already know about hundreds of different viruses from bats, and thousands of unknown viruses are waiting in bat reservoirs that may pose future outbreaks (works from Dr. Peter Daszak).

Nature has several high-throughput viral labs

Nowadays, we have the technology to manipulate the viral genome by introducing specific mutations in genomes and make engineered viruses for research purposes (to understand the basics of viruses and preparing preventive & curative measures in case of potential outbreaks). But our technology is very modest so far, compared to nature´s massive approach. Though we have the ´know-how´, so far in most of the cases it is hard, and the functional prediction of engineered mutations may not be very accurate. One has to screen a ton of mutants to pick up the right one (remembering Yogi Berra, ´´in theory there is no difference between theory and practice; in practice there is´´). 

Just after the SARS CoV2 sequence in public domain, a non-peer reviewed manuscript appeared (later disappeared) in BioRxiv that claimed sequence similarity between regions of SARS CoV2 and HIV. Recently, Luc Montagnier (2008 Nobel prize winner in medicine and physiology, for his discovery of HIV) argued that SARS-CoV2 contains some genes of HIV hence could be an engineered one. It is easier to find some degree of similarity in bits and pieces of genetic sequences between any 2 species (if you check for sequences of two different types of viruses you may find some similarities in a certain parts of their genetic sequence), which is not a big deal since most of these similar sequences may not be functionally relevant. Further, in case a deliberate genetic manipulation was performed (designed or engineered virus) it is easy to identify ´the genetic markers´ on such reverse genetic backbone of coronavirus genomes. A group of expert scientists all over the world carefully analyzed the sequence of SARS CoV2 and published their verdict in scientific journal Lancet, that this virus is of wildlife origin.

Based on several research articles from multiple labs around the world on SARS CoV2, a) the designed/engineered SARS CoV2 virus theory can be debunked. There is strong consensus among the experts in the field on b) the wildlife origin of this virus (they made several collective statements and reviews in scientific journals with regard to its natural origin).

Accidental release from a high safety viral lab?

Now the question remains whether this virus was leaked out from a bat coronavirus research lab. Accidental releases of smallpox and anthrax samples (though inactive) from high containment lab were reported in the past from US facilities. Intriguingly, Virology institute in Wuhan (WIV) at the epicenter of this outbreak has bat coronavirus research lab, which led to the speculation of a possible lab-escape from their facility. Also, several ´patient zero´ (the first patient or the carrier of an infectious disease) stories were circulating in media and one among them is, a researcher from WIV as the patient zero. For a novel infectious disease, it may take months to years to find the patient zero. We may not even find that patient, especially this disease with enormous asymptomatic transmission. Chinese authorities stated that none of the employees associated with WIV were known to be infected. Currently we don’t have any evidences to prove that a leak happened from a high-safety lab and that caused this pandemic (we may know the truth one day, but takes time). Based on the data so far, the genetic sequence of the bat coronavirus on which the WIV lab was working does not show similarity with that of SARS CoV2 (RatG13 Vs SARS CoV2). Concerns were raised by leaders of many countries on this issue and investigations are being done currently. WHO recently expressed its interest to work with investigations by Chinese government on the origin of this novel coronavirus.

BSL4 lab-Safety first

Coming to the level-4 lab in Wuhan Institute of Virology (WIV), it is a modern facility completed in 2015 (first in the Chinese mainland), in collaboration with French Institute CIRI (a total cost was 44 million USD). WIV has many international collaborations and publishes frequently in top scientific journals about their research suggesting transparency (WIV is a pioneer on bat coronavirus research). 

Research on microbes are usually conducted under very strict guidelines all over the world (to avoid any accidental release into nature from the lab). A biosafety level (BSL) facility is an enclosed laboratory facility, with very strict set of biocontainment regulations (the facility deals with dangerous biological agents). The levels of biocontainment range from the lowest biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4). There are less than 50 BSL-4 facilities around the world. BSL-4 is the highest level of biocontainment and is highly expensive to create one and to maintain it. Such labs are often very controversial. In a BSL-4 lab, the operating rules are extreme (it should be). Access is very limited, change of clothing before entering the facility, should use positive pressure suits, shower on exit and use of filtered air are some of the requirements. Decontamination of all wastes before removal from facility (please watch the video links attached on routines in a BSL-4 facility). Chances are very rare for a biological agent to escape from such a high containment facility.

To conclude

There is no doubt about the wildlife origin of SARS CoV2. With the genomic sequence analyses, it is assumed that the spillover happened from a bat to human, possibly through an intermediate host. During early propagations before jumping into humans, it got optimized and made a ´perfect human virus´ that led to this pandemic. The probabilities are high that SARS CoV2 spilled from a wild creature to humans (based on previous SARS1 and MERS spillovers) from the Wuhan wet market. Accidental release from a high biocontainment lab is in general a rare possibility. The genome sequence of SARS CoV2 is not similar to the bat coronaviruses in WIV. Further investigations are warranted to find out whether there was any accidental leakage of an ancestral virus from the BSL-4 facility from WIV lab, to disprove the ´lab-escape´ hypothesis. It is interesting to see what new evidences that the US President have regarding his claims that COVID19 originated from Wuhan lab. This disease became a crisis for humanity now, it’s time to help each other to fight against this invisible enemy.

Even if there are plenty of evidences to substantiate the natural origin and transmission of this virus, those evidences may never satisfy conspiracy thinkers. Please don´t glance through the media headlines and conclude, in most cases the sensational headlines do not reflect the contents. Read through it and check the evidences they present to support their claims. Soon, we may hear other conspiracy theories like COVID19 is a pharma-conspiracy, or there is no SARS CoV2 virus (virus deniers, there are HIV deniers), or ´alien origin´ of this virus (why not, why should there be a limit for imagination?). Psychology behind, adhering to conspiracy theories will be a very interesting area to study. One may cling hard illogically to his/her favorite conspiracy theory; despite how much scientific evidences you display in front. Perhaps, blindly believing such conspiracies is easier than spending time and energy to understand the facts.

Videos of this blog

Biosafety level lab routines 

References

The proximal Origin of SARS CoV2

https://www.nature.com/articles/s41591-020-0820-9

SARS CoV1

https://science.sciencemag.org/content/310/5748/676

Chimpanzee and Human genome

https://www.nature.com/articles/nature04072

Prof. Shi Zhengli´s work

https://www.nature.com/articles/s41586-020-2012-7

https://www.nature.com/articles/nm.3985

https://www.nature.com/articles/nature12711

https://www.nature.com/articles/s41586-020-2012-7

Prof. Ralph Baric´s work

https://www.nature.com/articles/nm.3985

https://www.nature.com/articles/s42003-018-0175-7

Other labs on Origin of SARS CoV2

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30251-8/fulltext

https://onlinelibrary.wiley.com/doi/full/10.1002/jmv.25688

https://www.sciencedirect.com/science/article/pii/S1567134820300447?via%3Dihub

Pangolin Origin

https://www.sciencedirect.com/science/article/pii/S0960982220303602

SARS CoV-2 and HIV (BioRxiv)

http://dx.doi.org/10.1101/2020.01.30.927871

Lancet; statement of experts

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30418-9/fulltext

WHO guidelines for BSL-4

Accidental release of anthrax and smallpox samples

https://www.bbc.com/news/world-us-canada-28221185

https://www.bbc.com/news/world-us-canada-32910020

China lab WIV

https://www.nature.com/news/inside-the-chinese-lab-poised-to-study-world-s-most-dangerous-pathogens-1.21487

https://time.com/5824061/wuhan-lab-denies-link-coronavirus-outbreak/

SARS CoV-2 sequence publishing

https://www.sciencemag.org/news/2020/01/chinese-researchers-reveal-draft-genome-virus-implicated-wuhan-pneumonia-outbreak

https://www.ncbi.nlm.nih.gov/nuccore/MN908947

https://www.nature.com/articles/s41586-020-2008-3

https://www.sciencedirect.com/science/article/pii/S0092867420303287?via%3Dihub

Familial cluster in China (first report)

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30154-9/fulltext

29 nucleotide deletion of SARs CoV1 (2002-2003)

https://www.nature.com/articles/s41598-018-33487-8

Recombination of SARS CoV2

https://www.biorxiv.org/content/10.1101/2020.02.10.942748v2

382-nt deletion of SARS CoV2

https://www.biorxiv.org/content/10.1101/2020.03.11.987222v1

Briefings on the hidden enemy

Previous blog ´War against a virus´ was on the general features of the virus. Here I try to feed the information regarding the virus (SARS CoV-2) and the disease it causes (COVID-19), which may help you to understand better this hidden enemy. Science is on the learning curve for this novel virus. For that reason, journals, databases, media are flooding with new information regarding this disease (take them with a grain of salt, since most of them are non-peer reviewed; that means not well scrutinised by experts in the field). Please check the ´references´ section for original papers. Note, as we learn more about this virus, we have to update, change our thoughts and strategies to fight against this disease.

The top on the list is ´Hydroxychloroquine´ treatment

Though US President and other political leaders are pushing on this anti-malarial drug hydroxychloroquine-HCQ (´´a gift from heaven´´ according to Mr. Trump) for COVID-19, the Scientific community is cautiously waiting for the scientific evidences from clinical trials. The demand for this ´game-changer´ drug is so high that US President Trump was literally threatening India, the biggest manufacturer of HCQ, for lifting its export-ban.

Some hospitals in Sweden and France stopped using HCQ for COVID-19, due to severe cardiac and vision side effects. Further, the controversial original scientific article (that claimed the antiviral activity of HCQ for SARS CoV-2) is facing fierce criticisms from scientific community and investigation from the journal/publisher. Researchers are coming with conflicting results on this ´Corona drug´, the main drawback in common about these studies is very low sample numbers hence are considered as pilot studies. This drug is effective in some chronic disease conditions (like arthritis, lupus). Due to overhyped anecdotal evidences as anti-COVID-19 drug, people all over the world are stockpiling this drug, posing a huge problem for those patients relying on this drug for their chronic clinical conditions. Please wait some more weeks/months for the verdict from WHO´s solidarity megatrial, to understand whether HCQ will be a safe ´silver-bullet´ or is just a ´false hope´?

Remdesivir- the magic bullet?

Ebola virus is also an RNA virus and uses a similar mechanism to replicate inside the infected cell, remdesivir will act on the copier machine of virus-RNA dependent RNA polymerase (the key protein for the viral replication) and inhibit it. Remdesivir was designed for Ebola treatment. There are promising preliminary data that this drug might work effectively to inhibit RNA polymerase of SARS CoV-2 too. Recently, a study published in a scientific journal (NEJM) showed promising results from this drug. This study was carried on a small group of nearly 50 patients. Intriguingly, the outcome was judged based on improvement of clinical symptoms while no analyses were on viral load! Any concrete conclusion from this study is difficult to make. A randomized control study is warranted and hence here also we need to wait for the judgment from WHO´s megatrial, on remdesivir.

Ivermectin- from anti-parasitic drug to antiviral drug?

Interestingly, a known FDA-approved anti-parasitic drug Ivermectin showed antiviral activities on SARS CoV-2. This drug previously showed some effects for other viral diseases had antiviral effects in SARS CoV-2 (mechanism is unknown), but in cells in a dish, that too at a higher concentration than recommended. Extending these results from the cell-culture to humans is not very easy though. Need a long way to go!

Desperate attempts- trying everything in the pharmacy shelf

Japan is trying themselves and donating Favipiravir to other countries (an effective influenza drug). Mechanism of action is by inhibiting the copier machine of a virus (a common mode of action of many antivirals). Trials are currently going on to get approval for this drug for treating COVID-19. In Denmark, a trial is going on another drug called Camostat mesylate– that acts on the cellular protease TMPRSS2 (remember the diagram from previous blog- a protein that helps the virus to get inside the host cell). Note, here the strategy is different the drug targets a cell protein hijacked by the virus to do its job.

A simple mask to prevent the spread.

Recent studies suggest that SARS CoV-2 may be viable in the droplets an infected person sheds while he talks, sneezes or coughs. These droplets can travel a short distance before it falls down on the surfaces. Once outside the host, viability of the viruses depends on environmental conditions. Under laboratory conditions this virus can stay stable for upto 3 days on plastic surfaces, upto 2 days on steel and a day on cardboard surfaces. Interestingly, SARS CoV-2 was viable upto 3 hrs in aerosols (but infectivity depends on the inoculum shed). Prof. George Gao, a top virologist and director-general of Chinese Center for Disease Control and Prevention (CDC version in China), expressed his concern that it is a ´big mistake´ not wearing masks indicating micro-droplet mediated transmission should not be ignored. The evidences forced many countries to change their mind and recommended the usage of mask while going out. Assuming that asymptomatic spreaders are in significant numbers and since they have similar viral load as symptomatic patients, it is wiser to use some kind of covering to your face. One concern was this will take away the masks for health workers, but experts from CDC now recommend the usage of atleast a home-made mask while going out. 

Serological test- antibodies 

RT-PCR based testing can detect the ongoing infection but cannot detect the survivors who were asymptomatic or had mild symptoms. This is because RT-PCR detects the genetic material of the virus, once our body recovers from the viral infection its genetic material will not be present. On recovery, even though no SARS CoV-2 viruses are present in the body, our body gains antibodies for a longer time against this virus (assumption based on similar Corona viruses). Antibodies/immunity created against this virus by our body act as warriors against the same virus in case of second attack. There is another way that we can detect the survivors- using an antibody-based serological test. A drop of blood from your fingertip on a testing strip will tell whether you already had a silent infection and if you are immune to SARS CoV-2 virus. The strip holds pieces of viral protein (usually the viral spike protein), which will recognise and bind by the anti-SARS CoV-2 antibodies present in the survivor’s blood. This specific binding between the viral protein and the antibody can be detected as colored bands by the reaction chemistry, on the strip (like the pregnancy detection strip). With such a simple test one can roughly detect whether you had a recent infection or an ongoing silent detection. An oversimplified version of the serological test for COVID-19 is shown below (to understand the logic behind the test).

Serological test

If we assume that the immunity gained by COVID-19 survivors sustain for couple of seasons (we are learning about it), then with precise immunological tests we can determine the residents who are immune to a subsequent infection. Such a SARS CoV-2-immune population can get back to work, volunteer helping the vulnerable and bring the economy back on track. One major issue that we are facing with regard to this test is the sensitivity and accuracy (note, the sensitivity of RT-PCR is high in detecting an ongoing infection). Many vendors are in the market with such COVID-19 kits, whether all of them meet the quality criteria is a big question.

Bouncing back of SARS CoV-2; a second wave?

Now many countries have flattened the curve, and it’s time to get back on our duties. Even if many of us escape from this ´first wave of SARS CoV-2´, this bug is going to be around for several months. Since complete isolation for a longer period is literally impossible, we may expect bouncing back of virus mostly by importing virus from other countries with ongoing cases. Wuhan, the epicenter of this outbreak is open now. China harnessed this highly transmitting disease by Draconian measures of strict lockdown and social distancing measures. This means the population might have failed to attain the herd immunity, hence there is greater chance of second wave of COVID-19 in China in coming days.

Antibody levels are not same in survivors: A recent non-peer reviewed study analyzed that neutralizing antibody levels (antibody that binds the virus and thereby inhibiting the virus from entry and replication) from several recovered patients (they had only mild symptoms!). They observed difference in the level of neutralizing antibodies in young, middle-aged and elderly (surprisingly, young recovered showed lesser antibody levels than others). This study got attention, based on this study many warned that if real, this will lead to second infection in the recovered patients (rebound). Moreover, this study warrants careful titration of serum from recovered patients, for convalescence therapy. The caveats of this preliminary non-peer reviewed study are many folds. The antibody they analyzed was only one kind-a neutralizing antibody against spike protein, while our body makes antibodies against several other viral proteins. Further, our body has other mechanisms to fight against a virus other than with neutralizing antibodies. Most important is they don´t know the initial viral load of these patients- which will have an impact on their antibody levels.

Sweden-the black sheep in the flock

While almost all the countries were following a similar approach of strict social distancing measures and lockdown to tackle COVID-19, Sweden to a great extend is not following the WHO guidelines in this COVID-19 fight. Though not very explicit, they are looking towards the herd immunity- an unknown approach for a novel virus. Already they are facing fierce criticisms on this matter not only from the scientific community but also from leaders outside. It may be too early to say whether their approach is a blunder, but the preliminary data is not very promising to their bold approach (comparison to their Scandinavian neighbors).

BCG vaccination for tuberculosis and COVID-19- two birds with one stone?

Again, another non-peer reviewed manuscript came up with the correlation of BCG vaccination (protector of tuberculosis) and less prevalence of COVID-19 in countries with BCG still in their vaccination policy. BCG vaccination given at birth, in many countries may not provide life-long immunity though. The argument is that these vaccinations may boost up the immunity (trained immunity) in the vaccinated which will augment our body´s fight against COVID-19. While WHO does not recommend BCG vaccination for prevention of COVID-19, they are waiting for the outcome of clinical trials (BCG vaccination to healthcare workers treating COVID-19 patients). More data, as well as a molecular mechanism is needed for supporting this correlative observation. Quite ambitious to expect a specific vaccine to fight against multiple diseases by boosting the immune system (mechanism not well understood), but why not?

Strains-will that matter?

A recent article from Cambridge University researchers reported 3 types of the deadly coronavirus are spreading around the world. They analysed the genetic sequences of SARS CoV-2 virus isolated from the patients from different parts of the world and found three distinct variants- A, B, and C. A-type is the ancestral one but interestingly not the most common one in China. As you understood from my previous blog, this RNA-virus undergoes mutations (though to a lesser extent than flu virus). These mutations get accumulated on transmissions and forms different genetic types or strains (this does not necessarily mean that there will be a huge shift in its pathogenic features). Analysing these variations help researchers to keep track of the propagation of SARS CoV-2 from one place to another.

Seasonal pattern of Coronaviruses 

Researchers from University college of London studied the seasonality preferences of three other coronaviruses (same family as SARS CoV-2) and came up with the conclusion that these viruses show a preference towards winter. The study is under peer review, and is not specifically on SARS CoV-2 (on CoVs in general), but it gives hope to many countries that are suffocating with COVID-19, where the winter is almost ending. If true, this is good news to Northern hemisphere while it gives alarm bell to the South, for preparedness.

Pandemic endgame

If this virus lingers around several months in different regions, it may not be easy to extinguish this fire completely. Countries who successfully contained this virus now, but not attained herd immunity will have to tighten their borders to prevent influx of cases from abroad. Sporadic outbreaks will force countries to reinstall the lockdown measures in those regions. If the social distancing works in mitigating the rapid virus spread and deaths, we may need to extend it for longer time periods to take care of the vulnerable. In the absence of a quick silver bullet (safe drug or vaccine) a significant population will be infected, and the survivors may establish herd immunity. Eventually, the virus cannot encounter viable hosts and subsides, but this natural approach is at the cost of millions of deaths! Coronaviruses generally show seasonality, it may subside during summer in many countries (but it is hard to speculate on any novel virus). Conversely, places where the environmental factors become favorable for viral propagation in coming months should prepare proactively on personal protective equipments, medicines, ventilators etc. Assuming that the immunity attained by patients recovered from COVID-19 will sustain for couple of seasons, then with the help of precise immunological tests we can determine the residents who already got and are immune to COVID-19. Those who are immune can safely get back to work.

One thing is for sure, this tiny string of RNA exposed the incapability of the health systems of even many developed countries in managing a pandemic. We must learn from this episode and prepare for such pandemics which may happen in future.

Video of this blog

Physics of the sneeze

https://www.youtube.com/watch?v=bFxgVkslD-k

 

References:

Original article on HCQ and its antiviral activity

https://www.sciencedirect.com/science/article/pii/S0924857920300996?via%3Dihub

Concerns by the journal on the HCQ study

https://www.isac.world/news-and-publications/official-isac-statement

No evidence HCQ antiviral activity

https://www.ncbi.nlm.nih.gov/pubmed/32240719

Trump Threatens India

https://www.bbc.com/news/world-asia-india-52180660

Solidarity Megatrial

https://www.sciencemag.org/news/2020/03/who-launches-global-megatrial-four-most-promising-coronavirus-treatments

Masks are important

https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/diy-cloth-face-coverings.html

https://www.sciencemag.org/news/2020/03/not-wearing-masks-protect-against-coronavirus-big-mistake-top-chinese-scientist-says

Asymptomatic patients have similar viral load

https://www.nejm.org/doi/full/10.1056/NEJMc2001737

Remdesivir

https://www.nejm.org/doi/full/10.1056/NEJMoa2007016

Ivermectin

https://www.sciencedirect.com/science/article/pii/S0166354220302011

Camostat mesylate

https://www.en.auh.dk/press-room/news-archive/2020/the-first-patient-tests-new-drug-against-covid-19/

Neutralizing antibody responses in recovered patients

https://www.medrxiv.org/content/10.1101/2020.03.30.20047365v1

Silent carriers

https://science.sciencemag.org/content/early/2020/03/24/science.abb3221

Serological tests

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30788-1/fulltext

Second wave

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30845-X/fulltext

BCG vaccination and WHO

https://www.medrxiv.org/content/10.1101/2020.03.24.20042937v1

https://www.who.int/news-room/commentaries/detail/bacille-calmette-guérin-(bcg)-vaccination-and-covid-19

3 variants of SARS CoV-2

https://www.pnas.org/content/early/2020/04/07/2004999117

Seasonality of CoVs

https://wellcomeopenresearch.org/articles/5-52

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