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2020-02-23 12:07:39Beijing News reporter:Li Yukuneditor: Zhou Bohua
Is pangolin a potential intermediate host for a new coronavirus? Expert opinions differ
The source initially pointed to the South China seafood market, but recent research results increasingly do not support this conclusion.
Beijing News (Reporter Li Yukun) Since February 7, researchers at South China Agricultural University have declared that pangolins are potential intermediate hosts for a new type of coronavirus. Concerns about "intermediate host theory of pangolins" have continued to rise. Wu Yuanbin, director of the Department of Social Development and Science and Technology of the Ministry of Science and Technology, also said on the 15th that relevant scientific research teams are being organized to demonstrate that the transmission of the new crown virus from pangolin to humans is also being further studied.
Recently, 4 manuscripts related to pangolin coronavirus were published on bioRxiv, a pre-release platform for biological thesis. These are the thesis identification of 2019-nCoV related coronaviruses in Malayan pangolins in southern China. Identification of 2019-nCoV-associated coronaviruses in Laiya pangolins, Shen Yongyi, professor of South China Agricultural University, et al. Isolation and Characterization of 2019-nCoV-like Coronavirus from Malayan Pangolins And the paper by Chen Jinping, from the Institute of Applied Research of Biological Resources of Guangdong Province, Are pangolins the intermediate host of the 2019 novel coronavirus (2019-nCoV)? Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019 submitted on February 7 (Evidence of coronavirus recombination indicates that the new coronavirus originated from pangolin).
Three papers in China have analyzed the similarity between the genetic sequences of coronaviruses found in the pangolin body and the human prevalent neocoronavirus. The papers by Guan Yi and Shen Yongyi believe that pangolins are likely to be intermediate hosts of the new coronavirus, Chen Jinping's paper Some doubts were raised. In addition, some experts have also posted articles online and put forward their views.
Evidence 1:
Pangolin coronavirus and neocoronavirus 90% similar
South China Agricultural University said at a press conference on February 7 that, by analyzing the virus's genome, it was found that the coronavirus strain isolated from pangolins had a sequence similarity of 99% with the currently infected human strain.
However, according to the paper submitted recently, the researchers obtained a near-complete coronavirus genome (29,578 bp) from multiple samples, of which the E, M, N, and S genes were expressed in 2019-nCoV (new crown virus), respectively. Gives 100%, 98.2%, 96.7% and 90.4% amino acid identity. S, E, M, and N4 genes are 3798, 228, 669, and 1260 bp (base pairs) in length. From this point of view, it is not that the entire gene sequence has reached 99%.
The South China Agricultural University paper pointed out that in the whole genome sequence analysis, the pangolin coronavirus genome is very similar to the human new coronavirus and a bat SARS-associated coronavirus (Bat SARSr-CoV RaTG13) in the entire genome. Sex is between 80% and 98%.
In Guan Yi's paper, it is speculated that the pangolin coronavirus genome has 85.5% to 92.4% similarity to the new coronavirus. Chen Jinping's research showed that the nucleotide sequence identity between pangolin coronavirus and neocoronavirus is 90.23%, and the sequence identity of a single protein can reach 100%. The nucleotide sequence similarity between pangolin coronavirus and bat SARS-associated coronavirus (Bat SARSr-CoV RaTG13) was 90.5%.
The conclusions of all three groups of studies can prove that pangolin coronavirus is genetically related to neocoronavirus and a group of bat coronaviruses.
According to the paper, the reporter found that the pangolins used by the three groups of research institutes were not exactly the same batch, but part of Guangdong came from the same batch of customs anti-smuggling operations. Lung tissue was obtained from four Chinese pangolins (Manis pentadactyla) and 25 Malayan pangolins (Manis javanica) collected from the Wildlife Rescue Center from March to December 2019. Amplification. Most of the pangolin samples studied by Guan Yi were obtained by Guangxi Customs in an anti-smuggling operation from August 2017 to January 2018.
Evidence 2
The receptor binding domains are very similar
The paper from South China Agricultural University specifically pointed out that the receptor binding domain of the S protein of pangolin coronavirus is actually the same as the S protein receptor domain of human neocoronavirus, but with one amino acid difference. Chen Jinping's paper explained that the coronavirus S protein binds to the host receptor through the receptor binding domain (RBD) and plays an important role in triggering viral infection and determining host tropism.
Previous studies have shown that coronaviruses found in neocoronavirus, SARS virus, and bats have similar receptor-binding domains, suggesting that they all use the same receptor ACE2, which is different from the receptor domain of the MERS virus. "Our analysis shows that the pangolin coronavirus is more consistent with the receptor binding domains of these three viruses, which indicates that pangolin CoV is likely to use ACE2 as a receptor. On the other hand, ACE2 receptors are present in pangolin and interact with humans. The sequences of the gene homologs are highly consistent. However, the zoonotic situation of this pangolin coronavirus is unknown. "Chen Jinping and others analyzed in the paper.
The paper specifically analyzes that by comparing and analyzing the S protein of SARS-associated coronavirus found in four animals: human, pangolin, civet, and bat, the receptor binding domain of the S protein of SARS virus can effectively bind human and fruit. The raccoon's ACE2, in addition, it seems to be able to bind the pangolin's ACE2, as well as the bat strain (Bat-CoV-RaTG13). In contrast, the S protein of neocrown virus and pangolin coronavirus may only recognize human and pangolin ACE2. Therefore, ACE2 of humans and pangolins may recognize the S proteins of all four SARS-associated coronaviruses, while ACE2 of civet cats can only reliably recognize the S proteins of SARS and bat coronavirus (Bat-CoV-RaTG13).
Guan Yi and others also analyzed in the paper that in terms of the receptor binding domain, the new coronavirus and the coronavirus found in Guangdong pangolin showed very high sequence similarity.
Matthew Wong, a member of Joe Petrosino's research group, posted on the website of virology 21 days ago that spike protein (or spike protein or ear protein) of Wuhan New Crown Virus strain is the "crown tip" of coronavirus. The receptor binding domain mentioned in the article is on the spike protein, which is responsible for binding with the receptor and letting the virus enter the cell .—— Reporter's Note) Generally closer to the bat virus strain (RaTG13), which may be due to the pangolin coronavirus Some subdomains of the S protein are very different, but starting from a certain part (S1-CTD), the Wuhan strain of new crown virus and pangolin coronavirus receptor binding domains are very similar, about 97%.
The paper by Matthew Wong et al. Shows that from the perspective of the composition of the receptor binding domain of the neocrown virus, the bat strain virus (RaTG13) genome shares only one of the five key amino acids involved in the receptor binding motif, while the pangolin corona virus Five key residues. It appears that the recombination of coronavirus strains and bat virus strains found in pangolins has resulted in the receptor binding domains of neocoronaviruses. However, Guan Yi and others did not tend to reorganize, but believed that convergent evolution occurred.
Evidence 3
Pangolin coronavirus has the potential to infect humans
Although the research data of Chen Jinping's team does not support the direct evolution from pangolin coronavirus to new coronavirus, this study shows that pangolin is a natural host of Beta coronavirus (SARS / new coronavirus belongs to this category-reporter's note) The potential to infect humans.
Studies have shown that the coronavirus genome sequences detected in two batches of pangolins intercepted from two different customs offices on different dates were related to bat coronavirus. Although it is not clear whether the two batches of smuggled pangolins came from the same area, it can be shown that pangolins are natural hosts of βeta coronavirus and may be symbiotic with pangolins. All three pangolins detected in Chen Jinping's research were infected with Beta coronavirus, which is very ill and suffers from severe respiratory diseases. It is unclear whether the coronavirus is a common virus in the pangolin respiratory tract flora. The pathogenesis of pangolins by a coronavirus remains to be studied.
Chen Jinping and others believe that their findings cannot prevent other coronaviruses from spreading in pangolins, and extensive monitoring of coronaviruses in pangolins can improve people's understanding of the pandemic coronavirus spectrum. Protecting wildlife and limiting human and wildlife contact can minimize the risk of coronavirus transmission from wildlife to humans.
Zhao Suwen also mentioned that pangolins are basically living alone in the wild, and even if they are sick, they are not likely to cause a pandemic. In the smuggling process, live pangolins are often smuggled together. Due to the harsh environment, it is easy to get lung disease and infect each other. Therefore, pangolin coronavirus has the conditions for long-term epidemic among the smuggled pangolin groups.
Doubt 1
Intermediate host similarity lower than native host?
However, the reporter found that about 90% of the similarities are far lower than the similarities between the new coronavirus and the coronavirus found in bats. Studies by the Wuhan Institute of Virology of the Chinese Academy of Sciences and Huazhong Agricultural University have confirmed that the similarity between the new coronavirus and the coronavirus found in a bat sample from Yunnan is as high as 96%. (Bat-CoV-RaTG13 mentioned above, the bat settles in Yunnan Province, nearly 2000 kilometers away from Wuhan)
Compared with the spread of SARS virus, this situation is not reasonable. In 2017, the Wuhan Virus Research Institute of the Chinese Academy of Sciences determined that the primary host of the SARS virus was a chrysanthemum, and the civet was previously thought to be only an intermediate host. Gene sequencing revealed that the SARS-associated coronavirus detected in civet was in agreement with the entire genome sequence of the SARS virus prevalent in the population of 99.8%, and the two were highly correlated. Sequencing of a bat coronavirus full-length genome proved that the genome sequence similarity to the SARS coronavirus capable of infecting humans and civet cats was 92%.
Zhao Suwen, a researcher at the iHuman Institute of the University of Shanghai for Science and Technology, pointed out that in terms of distantness and closeness with the new coronavirus, bat coronavirus RaTG13 ranks first, and pangolin coronavirus currently ranks second.
Chen Jinping and others directly pointed out in the paper that there is a high degree of sequence similarity between pangolin coronavirus and neocoronavirus. However, phylogenetic analysis does not support coronavirus generated directly from pangolin coronavirus.
Doubt 2
New crown virus-specific Furin protease cleavage site
Matthew Wong has previously mentioned that the receptor binding domains of pangolin coronavirus and neocoronavirus are very similar, but neocoronavirus has an additional Furin protease cleavage site.
Nankai University Takayama et al. Submitted a manuscript for the preprinted ChineseXiv website of the Chinese Academy of Sciences on February 2, 2020, “The Wuhan 2019 Coronavirus S protein may have a Furin protease cleavage site” for the first time in the world to report an important mutation. This mutation introduced A site for Furin digestion is not available in all previously discovered SARS and SARS-like coronaviruses. This mutation may enhance the ability of Wuhan 2019 coronavirus to spread.
According to the author, on the basis of previous work, it was inadvertently discovered that there may be Furin protease cleavage sites in Wuhan 2019 coronavirus genome. This finding suggests that Wuhan 2019 Coronavirus may be significantly different from SARS Coronavirus in the infection pathway. The virus may use packaging mechanisms of other viruses such as HIV and Murine Hepatitis Coronavirus, unlike most other Betas such as SARS. Coronavirus, due to changes in the packaging mechanism, Wuhan 2019 Coronavirus S protein has achieved a higher efficiency in infecting cells, which may be one of its reasons for its ability to spread more than SARS coronavirus. In addition, some avian influenza viruses can also obtain a Furin protease cleavage site through mutation to improve the efficiency of infecting cells.
Li Yanze, an associate researcher in the Department of Microbiology at the University of Pennsylvania School of Medicine, has commented on this. He believes that the new coronavirus has more Furin cleavage sites than other closely related viruses, and its receptor binding region is similar to the overall genome relative to bat coronavirus. The degree is not as good as that of the pangolin coronavirus, whose kinship is farther away. It would be incredible if these uniqueness were acquired through natural evolution.
Zhao Suwen also believes that the spike protein of pangolin coronavirus does not have the Furin digestion site like the new crown virus. From the pictures in the news report, we can see that Guangdong experts only wore gloves, no masks, no goggles, and no protective clothing when they participated in the pangolin treatment. We know that there is no outbreak in Guangzhou. Obviously this pangolin virus It is not as contagious as the new crown virus. From the point of being intercepted upon entry, it can be speculated that this batch of pangolins should have been carrying the coronavirus before entering (Vietnam). Guan Yi's team has also detected similar coronaviruses in multiple batches of pangolin samples intercepted by Guangxi Customs, which proves this.
Sorry: No animal sampling in South China market
Although the source of pneumonia of unknown origin in Wuhan was initially directed to the South China seafood market, recent research results increasingly do not support this conclusion.
Thesis of Yu Wenbin, associate researcher of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, et al. Decoded the evolution and transmission analysis of new coronaviruses based on whole-genome data. Patient samples associated with the South China seafood market were all haplotypes (and not the oldest haplotypes). And its derived haplotypes, only one from Wuhan appears in the sample, and there is no connection with the South China market. This type of haplotype appears in samples from other countries and regions, and even some countries have the new crown virus haplotype in Wuhan. No findings were found during sampling.
They analyzed that it could be as early as mid-to-late November last year. Infected people are likely to spread the new crown virus to workers or sellers on the market, and then quickly circulate on the market.
However, the positive samples of shops selling wild animals in the South China market are concentrated. Before and after the market was ordered to close on January 1, the shop owners also had multiple confirmed cases.
According to the information from the China Disease Control and Virus Disease Prevention Institute, they went to South China Seafood City at 8 am on January 1st, and collected 515 environmental samples for case-related merchants and related neighborhoods. On January 12th, experts from the Viral Diseases Institute visited South China Seafood again The market collected 70 specimens from wildlife dealers. Of these 585 environmental samples, 33 samples were detected to contain novel coronavirus nucleic acids, and the virus was successfully isolated from positive environmental samples, suggesting that the virus originated from wild animals sold in the South China Seafood Market.
The CDC report states that there are wildlife trades in the western area of the South China Seafood Market, especially in the areas near the 7th and 8th streets in the western area. There are many wildlife dealers in the area. 42.4% (14/33) of the sample. In summary, it is highly suspected that the outbreak is related to the wildlife trade.
Unfortunately, from the current reports, relevant organizations have only taken environmental samples, not animals, and the market has been disinfected several times. In this regard, Guan Yi said in an interview with the media that the South China Seafood Market was closed and washed, and the "crime scene" was gone, making it difficult to solve the case. He said that tracing animal sources is a complicated process, and it is impossible to find an animal with a virus and blame it as the culprit.
Chen Jinping and others also pointed out in the paper that the important point to control the spread of the disease is to determine the animal source of the new crown virus. In the wet market in Wuhan, environmental samples have reported high viral loads. However, a variety of animals, including some wild animals, are sold in this market, and the numbers and species are very dynamic. It is not clear which animal started the first infection.
Beijing News reporter Li Yukun
Editor Zhou Bohua proofreading He Yan
http://www.bjnews.com.cn/news/2020/02/23/693700.html
