Isolation,Identification And Whole Genome Sequence Analysis Of Two Tree Shrew-derived Mammalian Orthoreoviruses And Their Infective Characteristics In Vitro And In Vivo Of Tree Shrew

Mammalian reovirus is a double-stranded RNA virus which infects vertebrates via aerosol or fecal-oral route.The susceptible organs are intestinal epithelium,bile duct epithelium,pulmonary epithelium,white blood cells,central nervous system endothelium.The species known to be infected by reoviruses include humans,birds,cattle,monkeys,sheep,pigs,baboons and bats.Although MRV is a low pathogenic virus which in most cases made host has only mild symptoms,the virus may cause increased virulence because of virus reassignment,leading to a single host or animal population outbreak of upper respiratory tract disease,gastroenteritis,biliary tract Atresia,cerebral edema or encephalomyelitis and other diseases.Tree shrew shares considerable genetic homology with both humans and primates,and is considered to be well suited for use as a model for studies on viral infection and preclinical drug development.However,in the tree shrew breeding process,there will be bacteria or viruses and other pathogens infected health threat events and even lead to animal deaths.Tree shrews occasional diarrhea events,presumably may be caused by a virus infection.For the first time,two Mammalian reoviruses were isolated from tree shrew.Whether MRV/TS has a potential threat to tree shrew,whether it affects the study of other types of virus infection or pharmacological and toxicological studies.These issues need to be resolved.Through the isolation,identification and whole genome sequence analysis of two tree shrew-derived mammalian orthoreoviruses and their infective characteristics in vitro and in vivo of tree shrew the following results were obtained:1.Isolation and identification of MRV1/TS/2011 and MRV3/TS/2012,followed by whole genome sequence analysis.MRV/TS were isolated from Vero cell culture.The unknown pathogen was deduced into two reoviruses as the result of polyacrylamide gel electrophoresis and negative electron microscopy,and named MRV1/TS/2011 and MRV3/TS/2012,respectively.The whole genome sequence of MRV 1/TS/2011 were obtained by sanger sequencing,and MRV3/TS/2012 were obtained by high-throughput sequencing.MRV/TS have conserved sequences at the 5 'and 3' ends of gene,according to their genetic structural features.The genetic relationship of MRV/TS is close to stains isolated from human,pigs and bats from the phylogenetic trees.MRV1/TS/2011 is located in the Type ?,whereas MRV3/TS/2012 is located in the Type ?,MRV/TS is rearranged by fragments of different types of MRV.This study identified the viral typing and evolutionary relationship of tree shrew source reovirus strains.2.Infection characteristics of MRV1/TS/2011 and MRV3/TS/2012 in vitro.We cultivated primary intestinal epithelial cells of tree shrews,primary alveolar epithelial cells of tree shrews,primary neurons of tree shrews,and then use the means of immunofluorescence to identify the cell surface marker molecules.Electron microscopic ultrasonography showed that viral replication occurred in the cytoplasm,forming a lattice-like viral inclusion body.Plaques of MRV/TS are too small to be counted.Immunofluorescence shows MRV/TS replication in tIECs,tAECs,tNCs.Cross-neutralization tests showed that the antigens of the two viruses were not cross-linked.One step growth curves of MRV/TS in 5 kinds of cells,including tIECs,tAECs,tNCs,Vero and KMB17,show the relationship between viral load and time.The infectivity of MRV1/TS/2011 is stronger than MRV3/TS/2012.tAECs are more susceptible to MRV/TS than tIECs.MRV/TS has been limited to penetrate into KMB17 or Vero.The MRV/TS grew slowly at first,then became fast in KMB17;Nevertheless,they manifested a exponential growth immediately in Vero.The viral load in the supernatant was much lower than that in the cell,indicating that MRV/TS had a limited ability to penetrate the cell membrane in KMB 17.It was easier for MRV/TS to bind the cell or release itself in tNCs,but its replication has been limited.We studied JAM-A-mediated and NgR1-mediated infection of MRV/TS in vitro of tree shrew.The gene sequence of JAM-A and NgR1 was obtained by SMARTer RACE PCR.The expression and distribution of JAM-A and NgR1 in various tissues and organs of tree shrew could be tested by RT-qPCR.JAM-A mainly expressed in peripheral tissues;thereinto,the expression of intestinal tract is higher than other tissues.NgR1 mainly expressed in nerve tissue,and in brain,especially in temporal lobe it had a highest expression.The expression of JAM-A mRNA in tIECs and tAECs after infected of MRV/TS was measured.At the time of virus adsorption,the expression of JAM-A would not be up-regulated or down-regulated obviously compared to GAPDH even if time and concentration changed.The expression of JAM-A increased with time in the proliferative phase of virus replication.The mediated effect of JAM-A for infection of MRV/TS on tIECs,tAECs and HELA was found by anti-cell receptor method.Combination with NA of Human monoclonal antibody JAM-a 10.4 and rabbit polyclonal antibody JAM-A ab180821 could inhibit the infection of tAECS and tIECs by MRV1/TS/2011 and MRV3/TS/2012.The mediated effect in MRV/TS infection of NgR1 on tNCs was revealed by anti-cell receptor method and antagonist treatment.Sheep polyclonal antibody NgR1 ab32890,NEP1-40,PI-PLC and NA can block the binding site for MRV1/TS/2011 and MRV3/TS/2012 in tNCs.This study confirmed the susceptibility of MRV/TS to tree shrew cells and provided the infectivity characteristics of different species-derived cells.In addition,we found that MRV1/TS/2011 and MRV3/TS/2012 could infect neuronal cells.This is different from the previous study.JAM-A receptor and NgR1 receptor-mediated MRV virus adsorption protein VAP binding to host cells were confirmed on tree shrew cells.This part of the study laid the foundation for the study of the characteristics of living level infection.3.Infective characteristics of MRV1/TS/2011 and MRV3/TS/2012 in vivo of tree shrew.Adolescent tree shrews were supposed to infect with MRV1/TS/2011 and MRV3/TS/2012 by gastric perfusion.There are differences in tissue tropism and animal infection characteristics between MRV1/TS/2011 and MRV3/TS/2012.The viral load of MRV1/TS/2011 was about 103copies/?L in the blood,feces and main tissues on the 14th day after infection,then it would be cleared gradually.There is a small amount of virus has been detected in the blood and feces after infected of MRV3/TS/2012.Only lung tissue was detected 103copies/?L on the 1th day,8th day and 14th day,and there is no detection on the 21th day.Viral antigen IHC antigen localization showed the replication of MRV1/TS/2011 in the liver,and MRV3/TS/2012 in the lungs.There are differences in tissue tropism and animal infection characteristics between MRV 1/TS/2011 and MRV3/TS/2012.Transcriptions of tIECs infected with two strains of MRV/TS were analyzed respectively.There was a significant difference in the expression of host gene cluster of tIECs infected with MRV1/TS/2011 and MRV3/TS/2012.MRV/TS is age-dependent virus which has strong infectivity to newborn shrew,and it can cause severe mesenchymal pneumonia and encephalitis.MRV/TS can infect the adolescent tree shrew but does not lead to death or serious clinical lesion.This study revealed that MRV/TS was genotyped and age-dependent in tree shrews.To sum up,the new isolates of two reoviruses come from the new host-tree shrew,and is a recombinant strain of reoviruses,adding new hosts and new references for the evolution of reoviruses.In contrast to previous studies,this study selected the virus infection experiment on the primary cells of tree shrews in vitro,which was closer to the actual infection and provided the basic data for the selection of reovirus research materials.The study of JAM-A and NgR1-mediated MRV/TS infection in tree shrews enriches the contents of peripheral virus receptor and neurotoxin receptor research.Pathogenicity characteristics and infection characteristics of MRV/TS,benefits to understand the evolution of MRV virus in depth,providing a basic theoretical basis for the mechanism of mammalian tissue injury.MRV1/TS/2011 is more sensitive to JAM-A than MRV1/TS/2011,and MRV1/TS/2011 is more robust than MRV3/TS/2012 for tree shrew,suggesting that the mutation of key amino acid sites,pending further verification analysis.