17. Many scientists who have analyzed the sequence of SARS-CoV-2 have concluded that it does not have the signatures of a lab-engineered virus. But even some of these researchers say it remains possible that SARS-CoV-2 existed in your lab and accidentally infected a lab worker. They note that several labs had accidental infections with the virus that causes SARS. So how can you rule out this possibility?
A: We have isolated three closely-related bat coronaviruses over the last 15 years (here an isolated virus is a live virus which can grow in cultured cells in the laboratory) and all of them are SARS-related coronaviruses. These bat viruses share 79.8% sequence identity and are distantly related to SARS-CoV-2. On February 3, we published a paper in Nature and reported that SARS-CoV-2 is 96.2% identical at the whole-genome level to a bat coronavirus named RaTG13 (I would like to emphasize that we have only the genome sequence and didn't isolate this virus). With about 30,000 nucleotides, coronaviruses have a larger genome size than most animal RNA viruses. The 3.8% difference in genome sequence is a significant difference for coronaviruses. Five renowned virologists from Scripps Research Translational Institute, Columbia University, Tulane University, the University of Edinburgh and the University of Sydney published a paper titled "The proximal origin of SARS-CoV-2" in Nature Medicine on March 18. The authors stated that "although RaTG13 is 96% identical overall to SARS-CoV-2, its spike diverges in the receptor binding domain." On April 23, the US news site "VOX" quoted opinions from Prof. Edward Holmes, an expert in virus evolution at the University of Sydney. "The level of genome sequence divergence between SARS-CoV-2 and RaTG13 is equivalent to an average of 50 years (and at least 20 years) of evolutionary change," said Professor Holmes. The genomes of RaTG13 carried by bats and SARS-CoV-2 differ in 1,177 nucleotide positions. It would have taken a very long time to accumulate sufficient numbers of mutations through natural evolution. The probability is extremely tiny that the mutations occurred exactly in these 1,100-plus positions to be identical to SARS-CoV-2. Therefore, RaTG13 evolving into SARS-CoV-2 in nature is only theoretically possible.
Meanwhile, the research and experiments in our institute are in strict accordance with the international and national management requirements of biosafety laboratories and experimental activities, which are conducted in the required biosafety laboratories. Both the facilities and management of P3 and P4 laboratories are very strict. For example, personal protective equipment must be worn by the research staff. The air in the laboratory can only be discharged after highly efficient filtration. Waste water and solid waste must be sterilized under high temperatures and high pressure. The entire process of the experimental activities is video-monitored by biosafety management personnel. Every year, the lab's facilities and equipment must be tested by a third-party institution authorized by the government. Only after passing the test can the lab continue to run. The high-level biosafety laboratories at our institute have been operated safely and stably. To date, no pathogen leaks or personnel infection accidents have occurred.
18. The people who have floated these theories have proposed several ways in which the virus could have escaped from the Wuhan Institute of Virology. I'd like to ask some detailed, factual questions about the work at your lab that could shed more light on those scenarios:
(1) Are bat coronaviruses grown at the institute?
A: We have only isolated three strains of live SARS-related coronaviruses (SARSr-CoV) from bats, which shared 95-96% genome sequence similarity with SARS-CoV and less than 80% similarity with SARS-CoV-2. These results were published in Nature [2013, 593(7477):535-538], the Journal of Virology [2016, 90(6), 3253-3256] and PLoS Pathogens [2017, 13(11):e1006698], respectively.
(2) Does your group extract viruses from biological samples and do the sequencing or does that take place elsewhere?
A: We isolated viruses or extracted virus RNA from biological samples in the lab. The sequencing was done mostly in Wuhan.
(3) Has your lab done any animal experiments with SARS-related viruses recently? If so, can you provide any details?
A: We performed in vivo experiments in transgenic (human ACE2 expressing) mice and civets in 2018 and 2019 in the Institute's biosafety laboratory. The viruses we used were bat SARSr-CoV close to SARS-CoV. Operation of this work was undertaken strictly following the regulations on biosafety management of pathogenic microbes in laboratories in China. The results suggested that bat SARSr-CoV can directly infect civets and can also infect mice with human ACE2 receptors. Yet it showed low pathogenicity in mice and no pathogenicity in civets.
These data are being sorted and will be published soon.
(4) Is it possible that someone associated with the institute became infected in some other way, for instance while collecting, sampling, or handling bats?
A: Such a possibility did not exist. Recently we tested the sera from all staff and students in the lab and nobody is infected by either bat SARSr-CoV or SARS-CoV-2. To date, there is "zero infection" of all staff and students in our institute.
(5) Is it possible that you have biological samples from bats in your lab that you have yet to test for viruses? If so, how many samples have you tested and how many remain untested? If some remain untested, how do you know for certain that none contain SARS-CoV-2 or a close relative?
A: We tested all bat samples that we collected, including bat anal swabs, oral swabs and fecal samples, and 2,007 samples were positive for coronavirus. We did not find any viruses whose gene sequence is more similar to SARS-CoV-2 than RaTG13.
(6) Your lab was one of the first to sequence and isolate the virus. When and where
did you first sequence it?
A: We received the first batch of samples from seven patients on December 30 2019. Using pan-coronavirus RT-PCR and quantitative RT-PCR, which can detect all SARS-related coronaviruses, we found samples from five patients were positive. On December 31, when analyzing the sequencing result of the RT-PCR product, we identified that it was a novel SARSrelated coronavirus. We then confirmed the result via different methods and performed fulllengthgenome sequencing as well as virus isolation. We released the genome sequence to the global public on January 12 via WHO.
(7) What about the cave at Mojiang in 2013? When did you first isolate RaTG13? When did you complete the full sequencing of it?
A: We detected the virus by pan-coronavirus RT-PCR in a bat fecal sample collected from Tongguan town, Mojiang county in Yunnan province in 2013, and obtained its partial RdRp sequence. Because the low similarity of this virus to SARS-CoV, we did not pay special attention to this sequence. In 2018, as the NGS sequencing technology and capability in our lab was improved, we did further sequencing of the virus using our remaining samples, and obtained the full-length genome sequence of RaTG13 except the 15 nucleotides at the 5' end. As the sample was used many times for the purpose of viral nucleic acid extraction, there was no more sample after we finished genome sequencing, and we did not do virus isolation and other studies on it. Among all the bat samples we collected, the RaTG13 virus was detected in only one single sample. In 2020, we compared the sequence of SARS-CoV-2 and our unpublished bat coronavirus sequences and found it shared a 96.2% identity with RaTG13. RaTG13 has never been isolated or cultured.
(8) Some people who suspect a lab accident occurred have suggested that BtCoV/4991, a bat virus you described in 2016, is SARS-CoV-2. When you published, you only had the sequence of one protein, RNA dependent RNA polymerase (RdRp). A blast analysis on GenBank shows that the RdRp of BtCoV/4991 and RaTG13 are 100% homologous. Is BtCoV/4991 actually RaTG13, which would be consistent with your 2020 report that described how you did the full sequence of a virus you only had done the RdRp sequence for earlier? If so, why did you rename the virus? What does "TG" stand for in RaTG13?
A: Ra4991 is the ID for a bat sample while RaTG13 is the ID for the coronavirus detected in the sample. We changed the name as we wanted it to reflect the time and location for the sample collection. 13 means it was collected in 2013, and TG is the abbreviation of Tongguan town, the location where the sample was collected.
(9) Why do you have RdRp sequences for some viruses and not their full sequences? How many full-length sequences are there of the samples you've tested and how many are just RdRp?
A: Due to financial and manpower constraints, it is impossible for us to do the whole genome sequencing of all samples. We hope to conduct further full-length coronavirus genome sequencing in some other samples within the next two years. However, for some samples, it is impossible to obtain the whole virus genome sequences because of the low quantity of the viral nucleic acids in them.
(10) Were you ever instructed to destroy any viruses after the outbreak surfaced?
A: No.
(11) Is it possible that there was an accidental release at another lab in Wuhan? The Wuhan Center for Disease Control has been mentioned. If you have ruled this out as a possibility, why?
A: Based on daily academic exchanges and discussion, I can rule out such a possibility.
