The Research On Evolution And Pathogenicity Of Coronavirus Carried By Pangolins

Pangolin is a frontal mammal covered with scales,and is widely hunted and consumed by humans due to its scale and meat being considered to have medicinal value.In addition,its habitat has been severely damaged by human activities,posing a serious threat to the survival of the pangolin and being listed as an endangered animal.Its immunity is low,and it is easily infected by various pathogens,resulting in high mortality rate during rescue.After the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)outbreak,the beta-coronavirus(β-CoV)carried by the dead Malayan pangolin(Manis javanica)was discovered in our early reserch is highly similar to SARS-CoV-2,so pangolin is considered to be a potential intermediate host for SARS-CoV-2 transmission.Although subsequent studies have confirmed that pangolin is not the direct intermediate host of SARS-CoV-2,there is still no systematic research on the origin,transmission and pathogenesis of the coronavirus carried by the pangolin.Thus,for better rescue and protection of the pangolin,it is nessary to clarify the epidemiological situation,transmission route and pathogenic mechanism of coronavirus carried by pangolins based on the study of evolution of the coronavirus carried by pangolin and its influence on the host’s preference and immunity,which can provide an important reference for the protection of pangolin and preventing the overflow of zoonosis to humans.The present study forces on pangolin and its coronavirus,analyzing the evolution and epidemic variation of pangolin coronavirus.And further grasps the situation of coronavirus carried by pangolins through the investigation of rescued pangolin and analysis of phylogenetic relationships with coronavirus carried by bats.The infectivity and immune response mechanisms of pangolin coronavirus in human and bat lung cell lines were also evaluated herein.The main results are as follows:1.Beta-coronavirus were detected in both healthy and dead Malayan pangolins and shared 93.75-95.63%similarity with SARS-CoV-2 based on the N gene fragment.The coronavirus carried by pangolins was mainly detected in the feces of healthy individuals and the organs of dead individuals,indicating that β-CoV spread among pangolins through feces and attacked organs.Therefore,to protect the health of pangolin and human beings,it is necessary to regularly detect the pangolin epidemic and take corresponding preventive and control measures in time.2.Bats in Guangdong have not been found to carry pangolin coronavirus,but the whole genome sequence of two new severe acute respiratory syndrome related coronavirus(SARSr-CoV)strains from Guangdong bats was obtained.The nucleotide homology with pangolin coronavirus was 68.23-72.85%,lower than that with human SARS-CoV and SARS-CoV-2.The two new SARSr-CoVs did not recombine with pangolin coronavirus,but it happened with the two new SARSr-CoVs.At the same time,the two new bat SARSr-CoVs were far from the SARS-CoV-2 branch virus strain but close to the human SARS-CoV.According to the sequence analysis of receptor binding motif and the expression pattern of angiotensin-converting enzyme 2(ACE2),these bat viruses can not directly infect humans.3.Pseudovirus was constructed using the spike protein of coronavirus carried by pangolin and its infection ability to different host cells was studied.It was found that pangolin coronavirus may infect human and bat lung cells.Our result indicated that pangolin coronavirus might have the ability of cross-species transmission.4.The open reading frame 8(ORF8)of coronavirus carried by pangolin as a accessory protein suppressed the basic expression level of antiviral immune genes in human lung epithelial cells,including OAS3、IFITM1、MX1、MX2、DDX60 and DHX58,so which revealed that pangolin coronavirus might inhibit the expression of immune genes through accessory proteins,and subsequently avoiding the surveillance of innate immunity.In addition,the ORF8 of pangolin coronavirus was found containing a histone H3 mimic H3K27 motif(ARKSAP)as SARS-CoV-2 does.Therefore,it was inferred that pangolin coronavirus might be able to suppress the expression of host immune genes and effectively respond to virus infection for achieving immune escape strategy by simulating host histone.To sum up,through the molecular epidemiological investigation of the coronavirus carried by the rescued pangolins and wild bats in Guangdong,we have a comprehensive understanding of the background of these potential hosts of SARS-CoV-2.Our results provide important reference for the traceability of SARS-CoV-2 and pangolin coronavirus.By studying the susceptible host and immune escape mechanism of coronavirus carried by pangolins,we preliminarily reveal the cross-species transmission and immune escape mechanism of pangolin coronavirus,clarify the pathogenesis of pangolin coronavirus,and provide theoretical basis for the protection of pangolins and prevention of public health events.