Guan Yi is currently the director of the State Key Laboratory of Emerging Infectious Diseases and the director of the Influenza Research Center of the University of Hong Kong. During the "SARS" in 2003, the Guan Yi team took the lead in isolating the SARS virus, and confirmed that civet cats were the intermediate host of SARS and the direct source of human infection. According to the definition of the intermediate host and the report of Guan Yi and others, Guangdong Province subsequently cleared the civet cats in the market, thus effectively curbing the spread of SARS.
In this latest study, Guan Yi and others reported that COVID-19-related coronavirus was found in pangolins seized in anti-smuggling operations in southern China. They found that pangolin-associated coronavirus belongs to two subtypes of COVID-19-associated coronavirus, and the receptor binding domain of one subtype is very close to that of COVID-19.
The authors believe that the pedigrees of various pangolin coronaviruses found in the study and their similarities with COVID-19 indicate that pangolin should be considered as a potential intermediate host of this novel coronavirus and should be removed from the vegetable market to prevent the spread of human and animal diseases.
Of course, according to the current research, the research team believes that it is not enough to show that pangolin is an intermediate host directly involved in the epidemic. But so far, pangolin is the only mammal infected with COVID-19-associated coronavirus except bats. They may at least play an important role in coronavirus community ecology.
In this epidemic caused by COVID-19, the first scientific researchers who showed pangolin or played an important role were Professor Shen Yongyi and Professor Xiao Lihua from South China Agricultural University, Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology, and the teams from the Academy of Military Sciences of the Chinese People's Liberation Army and the Scientific Research Department of Guangzhou Zoo. On February 7th, they announced the research results that pangolin is a potential intermediate host in novel coronavirus.
It is worth mentioning that pangolin is the most trafficked of all mammals. They are used as food sources and scales are also used as medicines. Therefore, pangolin has attracted more and more attention in recent years. China pangolin and other pangolin species have been listed as critically endangered species in the Red List of Endangered Species by the World Conservation Union.
Many batches of pangolin samples tested positive for coronavirus.
The outbreak of COVID-19 epidemic is temporarily thought to be related to the South China seafood market in Wuhan, where the wild animals sold may be the source of human and animal diseases and infections. Although bats are probably the host of COVID-19, the intermediate host of the transferred virus is still unknown.
Guan Yi et al. had previously collected 18 samples of frozen tissue from August 20 17 to June 20 18. These pangolins were obtained in the anti-smuggling operation of Guangxi Customs.
Phylogenetic analysis described the evolutionary relationship between COVID-19, pangolin coronavirus sequences obtained in this study and other coronaviruses.
Notably, RNA high-throughput sequencing of these samples showed that coronavirus existed in 6 of 43 samples. The research team then obtained six full-length or nearly full-length genome sequences, which were labeled as GX/P 1E, GX/P2V, GX/P3B, GX/P4L, GX/P5E and GX/P5L respectively. These viruses also have a genome structure similar to that of COVID-19, with an open reading frame of 1 1. The research team also successfully isolated the virus.
According to the new genome sequence, the research team designed qPCR detection primers to confirm that the original sample was positive for coronavirus.
Next, the research team further tested another batch of pangolin samples collected from May to July in 20 18. 12 pangolin 19 samples, 3 lung tissue samples were positive for coronavirus.
In addition to these pangolins from Guangxi, after the outbreak, Guangzhou Customs Technology Center also re-examined five archived pangolins samples seized in the anti-smuggling operation in March, and coronavirus was also found in these samples.
Through high-throughput sequencing, the research team found that the large-scale samples contain coronavirus sequences, and used these data to assemble a partial genome sequence of 2 1505bp, which can represent 72% of the genome in COVID-19.
It is worth noting that another study on diseased pangolins conducted in Guangdong on 20 19 also found virus overlapping groups similar to those in COVID-19 from lung samples. Through different assembly methods and manual screening, some genome sequences accounting for about 86.3% of the full-length virus genome were obtained.
The genomes of these coronaviruses found in pangolin are 85.5%-92.4% similar to those of COVID-19, and they represent two subtypes of COVID-19-related viruses in the phylogenetic tree, among which GD/P 1L and GD/P2S are closely related to COVID-19.
Pangolin may be a long-term host of coronavirus, but it is not a close relative of COVID-19.
COVID-19 belongs to rotavirus subgenus of β coronavirus. Previous studies have noticed that members of coronavirus of Subgenus Sabovirus have undergone extensive gene recombination.
In order to verify the above viewpoint, the research team further conducted a restructuring analysis. The analysis showed that bat coronavirus ZC45 and ZC2 1 may be recombinant, containing several genome fragments of SARS-CoV related pedigree and COVID-19 related pedigree, including the genome fragment from pangolin.
More notably, however, the research team observed the recombination signals between pangolin coronavirus, bat coronavirus rat 13 and COVID-19. Especially, although COVID-19 and bat coronavirus RaTG 13 have the closest genetic relationship in the rest of the virus genome, the amino acid homology of receptor binding domain between COVID-19 and Guangdong pangolin is 97.4%, while the homology between RaTG and COVID-19 is only 89.2%.
In fact, Guangdong Pangolin coronavirus and COVID-19 have the same amino acid on five key residues of RBD, while RaTG 13 and COVID-19 have only one amino acid. However, the phylogenetic analysis of RBD synonymous sites only showed that the genetic relationship between Guangdong Pangolin coronavirus and COVID-19 was not recent.
Therefore, the research team speculated that the amino acid homology between Guangdong Pangolin coronavirus and COVID-19 RBD may be due to selective-mediated convergent evolution rather than recombination. Of course, it is still difficult to judge according to the existing data. This observation is consistent with the high similarity between human and pangolin ACE2 sequences and between human and bat ACE2 sequences.
It is mentioned that the occurrence of recombination or convergent evolution further emphasizes the role of intermediate animal hosts in the emergence of human viruses. But importantly, all the spiking proteins of pangolin coronavirus found so far have not inserted multi-base restriction sites at the S 1/S2 junction. This insertion feature can distinguish the current novel coronavirus and β coronavirus, which may also promote the rapid spread of COVID-19 in the population.
So far, pangolin is the only mammal infected with COVID-19-associated coronavirus except bats.
In this study, the research team found two related coronavirus lineages in pangolin, both of which are related to COVID-19. This indicates that pangolin may be the long-term host of these viruses.
Or COVID-19 virus from bats and other animal hosts.
Surprisingly, pangolin is a solitary animal with a relatively small number and is in an endangered state. However, it cannot be ruled out that pangolin has independently obtained COVID-19-related virus from bats or other animal hosts. Therefore, their role in the emergence of COVID-19 is still unclear.
It is worth noting that these two kinds of pangolin coronavirus are obtained from smuggled Malay pangolins, probably from Southeast Asia, and their local virus diversity is unknown at present.
The research team believes that there is no doubt that the spread of coronavirus in pangolin population needs further investigation, but the repeated infection of COVID-19-related coronavirus in Guangxi and Guangdong provinces indicates that pangolin may play an important role in the community ecology of coronavirus.
The research team mentioned that coronaviruses, including those associated with COVID-19, are obvious in many wild mammals in Asia. Although the epidemiology, pathogenicity, interspecific infectivity and transmission of pangolin coronavirus still need to be studied, the data provided by this study strongly show that these animals need to be handled very carefully and should be strictly prohibited from being sold in the vegetable market.
They also mentioned that it is obviously necessary to further monitor pangolins in the natural environment of China and Southeast Asia in order to understand their role in the emergence of COVID-19 and the risk of human and animal diseases spreading in the future.
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