Isolation of a new bat virus in a studypublished in the Journal of Virology on Dec. 30, 2015, titled “Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus,” found that the virus, named SL-CoV-WIV1, was almost identical to Rs3367 with 99.9% genome sequence identity. The researchers identified that WIV1 can use human ACE2 as an entry receptor and has the potential to infect human cells in this study. Subsequently, the same research group isolated another bat virus that can use ACE2 and infect human cell lines in the lab in 2015.
In addition, Dr. Shi’s group conducted another study in 2018 to address the question of whether some bat viruses can infect humans via using human ACE2, without the need of an intermediate host. But to the date of their study, “no direct transmission of SARS-Like CoVs from bats to people has been reported”.
Zhengli Shi’s group at the Institute of Virology at Wuhan was successful in isolating two infectious clones of bat SARS-Like CoV: SL-CoV-WIV1 and WIV16 from bats. In their further studies, they found out that these SL-CoV Spike protein (S protein) “[were] unable to use any of the three ACE2 molecules as its receptor; Second, the SL-CoV failed to enter cells expressing the bat ACE2; Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs; Fourth, a minimal insert region ( Amino acids 310 to 518 ) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding.”
Therefore, Shi’s group found in a study published in the Journal of Virology in February 2008 that the natural bat coronavirus cannot use the human ACE2 receptor to infect humans. However, when inserted with some amino acids from position 310 to 518 for the bat CoV S protein sequence, the chimeric bat CoV can use the human ACE2 receptor.
Meanwhile, another research group led by Dr. Li published their finding in 2013 that 5 amino acid sites on CoV spike proteins are crucial in making the binding to human ACE2 on SARS virus (those positions are Y442, L472, N479, D480, T487). These 5 sites just lie in the region that the Shi group noted to be important above.
Later, Li and Shi jointly conducted a gain-of-function study published in the Journal of Virology in September 2015 on the MERS virus and a bat virus (strain HKU4) in 2015. Since MERS virus can enter human cells but HKU4 can not, they introduced 2 single mutations in the HKU4 spike protein and found that the new mutant S protein can enable HKU4 to enter human cells. If they mutated 2 sites in MERS spike, the resulting MERS pseudovirus (experimental virus) cannot enter human cells anymore.
To their surprise, the chimeric virus (SHC014-MA15) can use SHC014 spike to bind to human ACE2 receptor and enter human cells. SHC014-MA15 can also cause disease in mice and cause death as well. Existing vaccines to SARS cannot protect animals from SHC014-MA15 infection. Therefore, these chimeric virus studies can lead to the generation of more pathogenic, more deadly CoV strains in mammalian models.
Due to the U.S. government-mandated pause on the gain-of-function (GOF) studies, this international research did not proceed further at that time. However, there is no evidence that Shi’s group in China stopped any further study on the track of introducing GOF mutations on the CoV. And it is clear that Shi’s group already mastered the reverse-engineering technology that is sufficient to introduce mutation in current SARS-CoV or SARS-Like CoV to create mutant infectious coronavirus.
t has been two months since the outbreak of the coronavirus in Wuhan and its spread has shown no signs of slowing down in China. More than 35 Chinese cities have been put on lockdown by Chinese authorities in an attempt to isolate confirmed and suspected cases. The lives of millions of people are in danger as the virus shows signs of spreading further in China as well as internationally.
There are significant gaps in the official investigations into the origins of the novel Coronavirus. In order to contain the virus, one first needs to understand how a virus that allegedly originated in animals found its way to humans. For this to happen, the Chinese authorities need to release their animal testing data and samples. Testing results from animal samples collected at epicenters would give important insights into what animals might serve as intermediate hosts for the new coronavirus.
This is critical to the containment of the epidemic. For example, if rats are the intermediate hosts for this virus, it would be futile to shut down the cities to restrict people’s movements while infected rats are still moving freely. Results from animal samples could also guide policy decisions that would reduce the risk of another outbreak.
An Animal Origin of the Virus
Scientific studies based on phylogenetic analysis have researched the sequence of the novel coronavirus, compared it to other coronavirus sequences, and found it likely originated in bats. Researchers from the Wuhan Institute of Virology found the genome in the virus found in patients was 96 percent identical to that of an existing bat coronavirus, according to a study published in the journal Nature. But there have been other theories as well. One Chinese study suggested, for example, that snakes were the source of transmission to humans. However, many scientists believe that reptiles are a less likely source and that mammals like rats and pigs, and some birds, have been the primary reservoir for coronaviruses.
With this in mind, phylogenetic studies of viral genome sequences need to be supported by animal studies to confirm the origin of the infection, as well as to determine whether there is an intermediate host.
It is not an easy task for a virus to establish zoonotic transmission, and coronaviruses rarely leap from animal to human infection with high transmissibility. There is even less chance to see a coronavirus leap directly from bats to humans. To infect new hosts, mutations need to occur with the viral surface proteins and/or envelope and structural genes, so that the mutated viruses can bind and enter the cells of new species, and efficiently complete the replication cycles in the new hosts.
Some scientists have argued that coronaviruses can jump directly to humans, without mutating or passing through an intermediate species. However, an intermediate host was clearly needed to establish zoonotic transmission to humans in the previous outbreaks of coronaviruses. Many studies suggested that the bat coronavirus jumped from its natural host bats to civets and then to humans during the 2003 SARS outbreak, and it jumped from bats to camels and then to humans for the MERS outbreak. So, civets and camels would serve as intermediate hosts for zoonotic transmission.
Because bats were not sold at the Huanan market in Wuhan—the epicenter of the infection—at the time of the outbreak, this suggests the existence of another intermediate animal host that may have transferred the virus to humans.
What is the most puzzling is that there have been no reports on the testing of animal samples collected in any epicenters in Wuhan, especially at the Huanan seafood market, to identify what animals might be the host or intermediate hosts of this novel Wuhan coronavirus.
Chinese scientists published a report in Lancet recently which stated that “the majority of the earliest cases included reported exposure to the Huanan Seafood Wholesale Market” and that patients could have been infected through zoonotic or environmental exposures. Another report on Lancet by Chinese CDC scientists claimed that “on the basis of current data, it seems likely that the 2019-nCoV causing the Wuhan outbreak might also be initially hosted by bats, and might have been transmitted to humans via currently unknown wild animal(s) sold at the Huanan seafood market.”
However, so far, no information was released about the amount, and species, of wild animals present at the Huanan seafood market upon closure; nor about how the animals were managed or disposed of when the market was closed on Jan. 1, 2020. And no information was released about how many animal samples were tested for SARS-CoV or Wuhan Coronavirus via viral nucleic acid testing methods.
Official Chinese state news agency Xinhua reported on Jan. 26 that 33 samples out of 585 environmental samples collected at the Huanan Seafood market were positive for nucleic acids from new Coronavirus, suggesting the virus originated from wild animals or stocks sold there. However, these samples were from the environment—not from animals.
It would be an ultimate failure of the Wuhan public health commission and Chinese CDC if no animal samples were collected and tested prior to, or at the time of, the shutting-down of the Huanan seafood market, where many animals were sold at the time of the outbreak. It would be similar to conducting an investigation on a food-borne disease outbreak without taking restaurant food samples related to the outbreak, and instead taking dining table surface swabs to test.
Background of the Huanan Seafood Market Closure
The 2019-nCoV has caused rapid infection in China and spread to other countries outside China, which has led to a global health crisis.
The Huanan seafood market is known to be a major outlet for the collection and distribution of live and dead wild animals. These included live wolves, hedgehogs, deer, birds, snakes, goats, hares, and boars that were sold and available in the east section of the seafood market.
A Wuhan medical and health committee identified multiple pneumonia cases associated with Huanan seafood market, which were announced on Dec. 31, 2019. The seafood market was closed by the Wuhan government on Jan. 1.
Chinese medical reporters visited the market on Dec. 31, 2019, the evening before its closure on Jan.1 where they observed poor hygiene, and wild animal bodies and organs disposed of in an unorganized manner. This suggested that a relatively large quantity of wild animals were still present at the market upon the forced closure.
No Information on Wild Animals at the Seafood Market Was Disclosed
Yet, no information was released about the amount, and species, of animals present upon closure, how many animals were tested for Coronavirus, and how the animals were managed or disposed of upon the closure of the market on Jan. 1. A Chinese media outlet, Yicai, inquired about the outcome of the wild animals sold at the market and confirmed that there was no disclosure from the Wuhan government.
Dr. Guan Yi, the current director (China affairs) of the State Key Laboratory for Emerging Infectious Diseases at the University of Hong Kong, visited Wuhan on Jan. 21 with the goal of identifying the animal source. He mentioned in a media interview that locals refused to cooperate with him. He pointed out that with the market now closed, it would be difficult to investigate the origin of the virus. He said the “Huanan seafood market was cleaned after the closure, ‘the crime scene’ was gone, and how can you solve a case without evidence?”
Gao Fu, director of the Chinese Center for Disease Control and Prevention stated,“it is clear that the source of infection was from wild animals, but we don’t know which species due to closure of the seafood market.”
The Huge Risks of Not Identifying the Original or Intermediate Animal Hosts
The U.S. Center for Disease Control (CDC) stated that “much is unknown about how 2019-nCoV, a new coronavirus, spreads.” So far the understanding is that the major pathway of 2019-nCoV infection is respiratory droplet transmission and contact from humans to humans.
Guan Yi and Kwok-yung Yuen of the University of Hong Kong (HKU) et. al. identified severe acute respiratory syndrome coronavirus (SARS-CoV) from caged palm civets from live animal markets in China in 2003. Their studies lead to the subsequent ban on selling civets and the closing of all wild animal markets in Guangdong and helped to confine the SARS epidemic.
Typically, if an animal is identified as host or source of spread of disease, authorities and the CDC would initiate prevention and control measures such as an awareness campaign, proper quarantine of sick animals and disposal of carcasses as well as monitoring the potential route for how the disease spread zoonotically.
Rodents are known to infest seafood markets. For example, tens of thousands of rodents are expected to be unleashed in Japan as one big fish market is closed.
Huanan seafood market is also infested by rodents. If rodents were elucidated as being a potential host for coronavirus, the risk of rats infesting beyond the current quarantine zone still persists. Given the fact that coronavirus was detected from feces from patients from Shenzhen and that bat SARS-like virus strains were isolated from bat feces, the possible fecal-oral route of 2019-nCoV transmission in addition to respiratory droplet transmission would lead to a reasonable warning for people to avoid contact with animals like rats. Thus, if rodents are indeed a source or host of the 2019-NCoV infection, then, rodent contamination of food or water is a potential way for the disease to spread, which needs to be brought to the awareness of the international community.
Similarly, if birds or other species were the hosts of 2019-nCoV in the seafood market, the information pertaining to the species, amount, virus type, biological reactions, and potential routes of spreading of the virus also need to be identified or reported to the world so that appropriate prevention measures could be taken.
It would be serious incompetence and malfeasance if Chinese authorities did not attempt to collect nasal, fecal, and blood samples from animals and birds sold at the seafood market. Testing animal samples would reveal very important information regarding the zoonotic transmission routes, the trends of viral mutations in this outbreak, and the loopholes in the current countermeasures.
Were There Other Epicenters Besides the Huanan Seafood Market?
The Chinese CDC did release data from environmental samples from the seafood market and suggested that “it is originated from wild animals with species uncertain.”
A team which included Dr. Feng from the Chinese CDC published a report titled “Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia,” in the New England Journal of Medicine on Jan. 29, 2020. The paper stated that “Although the majority of the earliest cases were linked to the Huanan Seafood Wholesale Market and the patients could have been infected through zoonotic or environmental exposures…the majority of the earliest cases included reported exposure to the Huanan Seafood Wholesale Market, but there was an exponential increase in the number of nonlinked cases beginning in late December.”
The Possibility 2019-nCoV Originated From Bat SARS-Like Virus (Bat-SL-CoV)
One recent Lancet report on Jan. 29, 2020, titled “Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding,” stated that “A Blast search of the complete genomes of 2019-nCoV revealed that the most closely related viruses available on GenBank were bat-SL-CoV-ZC45 (sequence identity 87.99%; query coverage 99%) and another SARS-like betacoronavirus of bat origin, bat-SL-CoV-ZXC21 (accession number MG772934;23 87.23%;” “Notably, the 2019-nCoV strains were less genetically similar to SARS-CoV (about 79%) and MERS-CoV (about 50%).”
This message might be interpreted as 2019-nCoV being biologically closer related to SARS-like betacoronavirus of Bat origin and bats may be the original host of this virus. However, the authors did not claim that the only host to 2019-nCoV is a bat.
The paper stated that “However, despite the importance of bats, several facts suggest that another animal is acting as an intermediate host between bats and humans. First, the outbreak was first reported in late December, 2019, when most bat species in Wuhan are hibernating. Second, no bats were sold or found at the Huanan seafood market, whereas various non-aquatic animals (including mammals) were available for purchase. Third, the sequence identity between 2019-nCoV and its close relatives bat-SL-CoVZC45and bat-SL-CoVZXC21 was less than 90%, which is reflected in the relatively long branch between them. Hence, bat-SL-CoVZC45 and bat-SL-CoVZXC21 are not direct ancestors of 2019-nCoV. Fourth, in both SARS-CoV and MERS-CoV, bats acted as the natural reservoir, with another animal (masked palm civet for SARS-CoV35 and dromedary camels for MERS-CoV) acting as an intermediate host, with humans as terminal hosts. Therefore, on the basis of current data, it seems likely that the 2019-nCoV causing the Wuhan outbreak might also be initially hosted by bats, and might have been transmitted to humans via currently unknown wild animal(s) sold at the Huanan seafood market.”
They mentioned that most bats in Wuhan are hibernating and no bats are sold at the Huanan seafood market. Thus, the chance of physical contact from bats to spread the virus to humans or animals at Wuhan is highly unlikely.
Studies From Wuhan Institute of Virology on Bat SARS-Like CoV.
Zheng-Li Shi and several other researchers at the Wuhan Institute of Virology published an article in Nature in 2013 titled “Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.”
In that study, their team harvested from anal swabs or fecal samples from bats and found 2 strains of sequences from Bat SARS-Like CoV that termed as RsSHC014 and Rs3367. They process 95% nucleotide sequence identity with human SARS-CoV Tor2 strain.
Isolation of a new bat virus in a studypublished in the Journal of Virology on Dec. 30, 2015, titled “Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus,” found that the virus, named SL-CoV-WIV1, was almost identical to Rs3367 with 99.9% genome sequence identity. The researchers identified that WIV1 can use human ACE2 as an entry receptor and has the potential to infect human cells in this study. Subsequently, the same research group isolated another bat virus that can use ACE2 and infect human cell lines in the lab in 2015.
In addition, Dr. Shi’s group conducted another study in 2018 to address the question of whether some bat viruses can infect humans via using human ACE2, without the need of an intermediate host. But to the date of their study, “no direct transmission of SARS-Like CoVs from bats to people has been reported”.
They collected serum from 218 residents who live close to bat caves with bats carrying the viruses. Those caves were the places where the Shi group collected the virus samples. Then ELISA assays were conducted to detect antibodies to bat SARS-CoV, since antibody existence would suggest a prior exposure to the bat coronavirus. They found that only 6 out of 218 (2.7 %) subjects showed seropositivity, which suggested likely infections to bat SARS-CoVs or related viruses. No clinical symptoms have been manifested in the 6 positive persons in the past 12 months. As a control, they collected 240 samples from random blood donors in Wuhan, 1000 km away from Yunnan, none of the Wuhan blood samples showed any positivity to bat SARS-like CoV.
This data suggests that the chance of bat virus infecting humans is very low, <2.9% if possible, and with no obvious symptoms in human beings that live very close to the bat caves. No infection from a bat to a human has been reported in Wuhan as of 2018.
[ZH: February 2020, her team published a paper in Nature showing that remdesivir, an experimental drug owned by Gilead Sciences, had a positive effect in inhibiting the virus invitro, and applied for a patent for the drug in China on behalf of the WIV.]
Track Record of Wuhan Institute of Virology on Engineering ‘Gain-of-Function’ Bat SARS-Like CoV.
Therefore, Shi’s group found in a study published in the Journal of Virology in February 2008 that the natural bat coronavirus cannot use the human ACE2 receptor to infect humans. However, when inserted with some amino acids from position 310 to 518 for the bat CoV S protein sequence, the chimeric bat CoV can use the human ACE2 receptor.
Meanwhile, another research group led by Dr. Li published their finding in 2013 that 5 amino acid sites on CoV spike proteins are crucial in making the binding to human ACE2 on SARS virus (those positions are Y442, L472, N479, D480, T487). These 5 sites just lie in the region that the Shi group noted to be important above.
Later, Li and Shi jointly conducted a gain-of-function study published in the Journal of Virology in September 2015 on the MERS virus and a bat virus (strain HKU4) in 2015. Since MERS virus can enter human cells but HKU4 can not, they introduced 2 single mutations in the HKU4 spike protein and found that the new mutant S protein can enable HKU4 to enter human cells. If they mutated 2 sites in MERS spike, the resulting MERS pseudovirus (experimental virus) cannot enter human cells anymore.
Furthermore, Shi’s group joined an international group to generate a chimeric virus with the bat virus SHC014 they harvested in Yunnan. Since they know SHC014 is unlikely to bind to human ACE2, they “synthesized the SHC014 spike in the context of the replication competent, mouse-adapted SARS-CoV backbone”. So, that is a lab-engineered virus with SARS-CoV Mouse adapted backbone (MA15) but with SHC014 spike.
To their surprise, the chimeric virus (SHC014-MA15) can use SHC014 spike to bind to human ACE2 receptor and enter human cells. SHC014-MA15 can also cause disease in mice and cause death as well. Existing vaccines to SARS cannot protect animals from SHC014-MA15 infection. Therefore, these chimeric virus studies can lead to the generation of more pathogenic, more deadly CoV strains in mammalian models.
Due to the U.S. government-mandated pause on the gain-of-function (GOF) studies, this international research did not proceed further at that time. However, there is no evidence that Shi’s group in China stopped any further study on the track of introducing GOF mutations on the CoV. And it is clear that Shi’s group already mastered the reverse-engineering technology that is sufficient to introduce mutation in current SARS-CoV or SARS-Like CoV to create mutant infectious coronavirus.
[ZH: In February 2020, the South China Morning Post reported that Shi's decade-long work to build up one of the world's largest databases of bat-related viruses gave the scientific community a "head start" in understanding the virus. The SCMP also reported that Shi was the focus of personal attacks in Chinese social media who claimed the WIV was the sources of the virus, leading Shi to post: "I swear with my life, [the virus] has nothing to do with the lab", and when asked by the SCMP to comment on the attacks, Shi responded: "My time must be spent on more important matters."
Caixin reported Shi made further public statements against "perceived tinfoil-hat theories about the new virus's source", quoting her as saying:
"The novel 2019 coronavirus is nature punishing the human race for keeping uncivilized living habits. I, Shi Zhengli, swear on my life that it has nothing to do with our laboratory"]
Interestingly, Shi’s group published on bioRxiv on Jan. 23, 2020 that a new bat coronavirus that they detected in Yunnan, named BatCov RaTG13, shares 96.2 percent overall genome sequence identity with 2019-nCoV. However, this virus was never mentioned or published in their research before.
In the sequence information provided by them in the supplemental material and method section, 3 sequences are shared between the 2019-nCoV they collected and the RATG13 virus but not in any of the other SARS or Bat SARS-Like CoV families in the paper listed. The 3 sequences are located close to N terminus of the spike protein, they are GTNGTKR, NNKSWM, RSYLTPGD.
Possibilities of an Animal Host of 2019-nCoV at Huanan Seafood Market
One recent Lancet paper titled “Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding,” reported that “as a typical RNA virus, the average evolutionary rate for coronaviruses is roughly 10⁴ nucleotide substitutions per site per year, with mutations arising during every replication cycle. It is, therefore, striking that the sequences of 2019-nCoV from different patients described here were almost identical, with greater than 99.9% sequence identity. This finding suggests that 2019-nCoV originated from one source within a very short period and was detected relatively rapidly.”
With mutations in every cycle, it is highly unlikely for different bats to host viruses with the same sequence. If bats alone are not enough for virus transmission, another animal is needed as the intermediate host, and the chance of the virus being identical is even slimmer. Since the seafood market is not the only source for the outbreak, it is reasonable to postulate that if another animal is the intermediate host for the virus, that animal needs to have contact with bats, allow bat coronavirus to proliferate in them, and, eventually, the animal needs to have the capacity to transmit viruses to human beings who may or may not have contact with the Huanan seafood market.
Therefore, there have been serious questions on whether this Wuhan coronavirus outbreak was due to a leak or mishandling of laboratory animals used in coronavirus studies. This is a reasonable public inquiry regarding the source of the outbreak and it warrants a transparent investigation from the Chinese authorities and foreign disease control and laboratory operation experts. This is not just about the accountability of medical ethics or laboratory safety operations, it is directly related to the current endeavors to contain the virus outbreak.
While the animal host of 2019-nCoV is yet to be identified, the data and information from possible animal hosts and potential zoonic infection is imperative for prevention and controlling disease on an international scale.
The Huanan seafood market has a high potential of harboring the animal host. Animal data and profiling results from the Huanan seafood market need to be disclosed immediately by Chinese authorities even if they are negative results. It is imperative for U.S. CDC and WHO officers to demand that Chinese authorities release the information about animal testing data.
If Chinese authorities refuse to disclose testing data for animal samples, it could imply an intentional cover-up of the true origin of the 2019-nCoV outbreak.
