Isolation Of Tupaia Belangeri Orthoreovirus, Establishment Of Nucleic Acid Detection Methods And Study On Pathogenicity Of The Virus

Reovirus is a double-stranded RNA virus exhibiting a largr host range. Tree shrews breeding colony work was carried out by Tree Shrews Germplasm Resources Centre, Institute of Medical Biology. Infected clinical symptoms were observated from wild tree shrews in different time. Three reovirus were named TRV1, TRV2and TRV3,and were isolated by cell culture method and identified by polyacrylamide gel electrophoresis. To confirm tree shrews was the infected host of reovirus, the qualitative and quantitative nucleic acid detection methods counter the newly isolated TRV were established, meanwhile, the pathogenicity of the virus was studied.Firstly, the RT-nPCR method was established in order to explore the infection rates of TRV in tree shrews populations. According to the conserved regions of L1gene which were published in GenBank, two pairs of nested primers were designed and synthesized. The RNA isolated from TRV1, TRV2and TRV3was amplified by RT-nPCR and the specificity and sensitivity tests were developed via optimizing the reaction conditions. It showed that the method which can be applied to the rapid qualitative detection of TRV is specific, sensitive and stable. The infection rate of adult-onset group, weaning group and sucking tree shrews was detected by using RT-nPCR method. The results showed that the TRV detection rate was76%,10%,20%respectively,which is considered high in tree shrews populations.It was found that Cytopathic Effects induced by virus were weakened with generation increase, and the virus titer was only1025TCID50/mL. In order to obtain high titer virus as the infection source, chymotrypsin was added to the cell culture medium with final concentration of10ug/mL and the results showed that CHT can facilitated TRV infection on Vero cells. The virus titer was increased to10TCID50/mL, which provides high title virus for TRV research.Secondly, the TaqMan fluorescent quantitative RT-PCR method of TRV was established for detecting virus levels in the organs quantitatively. It presents good linear relationship between the Ct values and the log value of initial template concentration and good reproducibility, which contributes to the application of this assay for quantitative detection of the TRV.Finally, the experimental infection of tree shrows with TRV2were performed for evoluating the pathogenicity. Three infection groups include suckling mice of inoculations intracerebrally (i.c) group, suckling tree shrews of inoculations intracerebrally (i.c) group and sucking tree shrews of peroral (p.o) group. Animals were euthanized at different day after inoculation. Anatomical change and histopathological changes were observed by HE staining and the number of tissue viral copies was detected by TaqMan fluorescent quantitative RT-PCR.The results are shown as follows:(1)The viral nucleic acid were detected in the brain, liver, lung and intestinal at1,3,6,8days after the injection of TRV2in suckling mice of i.c group. The virus copy number increased along with the infection days in brain tissue, and the highest copy number is2.55×109copies/mg on the eighth day. Pathological observation showed the encephalitis and interstitial pneumonia.(2)The viral nucleic acid was detected in the brain, heart, lung, liver, spleen, intestinal and feces at3,7,14,28days after injection of TRV2in suckling mice of p.o group. Pathological observation showed severe interstitial pneumonia.(3)The infection of suckling mice after intracerebral injection of TRV2is in close agreement with sucking tree shrew’s results and the copy number of TRV is up to4×107copies/mg on the sixth day. The results further suggest that reovirus could infect immunocompromised sucking tree shrews. The viral copies of108.3TCID50/ml did not cause sucking tree shrews to death but it can replicate in the brain, heart, liver, spleen, lung, kidney tissues, leading to the damage to the organs among which the brain and lung’s injury is more serious. Above all, tree shrew reovirus has certain pathogenicity.The results of the research lay the foundation for developing animal model of reovirus infection. At the same time, several problems will be explored in the future study, such as the pathogenic mechanism of tree shrews reovirus and if it coinfect with other virus and so on.