| In this study, an unknown virus designated DXMV was isolated from livers and spleens of two died pandas in a zoo using MDCK cell line. The virus could multifly on the MDCK cells.and induce regular cytopathic effect (CPE).Observed by negative staining electron microscope, the virus could be seen with coroniform spike .In the ultrathin sections, the viruses were found to multiply and induce inclusion body forming in the cytoplasm. Physicochemical assays showed that this viurs was a kind of RNA virus,and sensitive to choroform and ether. In trpsin tolerance assay.this virus could resist to 1% trpsis at 37℃ in an hour.In acid tolerance assay,this virus was resistant to pH3.0 and pH5.0 at 37 ℃ in 2 hours,and the average infection litre of the virus decreased little.In heat assay, at 50℃,the virus was processed from 5 minutes to 150 minutes and at each condition the viral virulence reduced to some certain degree.Among these conditions,when at 50℃ in 30 minutes.the average infection litre of this virus decreased over 2 tilre.and when al 50℃ in an hour,CPE of Ihis virus disappeared.When time was set for an hour.but with processed in different temperature as 50℃ 60℃ 70℃, 80℃,the virus losl the multiplication capacity complelely.In biological assay,we selected different cell lines to cultivate this virus by laking advantage of possesional cells at that time in our laboratory.Then we found that FCWF cell line was the most sensitive to DXMV and MDCK was the second.With F81 cell line,after passaged for 12 times continuously with low concentration of FCS.the virus could produce CPE.However,with Vero cell line.the virus could not procuce any CPE after many passages.The hemagglutination and lumadsorption reaction test proved that this virus had no any reaction to erythrocyte of pig, fowl and cavy.By neutrolizaion assay,DXMV could be identified as a kind of CCV.By RT-PCR method.we first selected a pair of primers named 2Bp and 4Bm which was designed in highly conserved 922 nucleotide region in open reading fram(ORF) Ib from coronavirus.This primer could amplify 11 kinds of coronaviruses.After synthesizing this pair of primers, we amplified the target fragement of 251bp from the panda's liver-tissue materials and other different passages of culture of this virus .However, the control cell showed negative result. Inorder to identifiy the virus further, a set of double nested primers for canine coronavirus was selected.The primers were designed in S gene region from CCV including two pairs of primers:CCVFl-CCVRl,CCVF2-CCVR2.The first is a pair of outer primer,and can amplify a fragement of 1086bp.The second is a pair of inner primer.and can amplify a fragement of 515bp.Using the nested primers,many CCV strains can be identificated including k378,Insave-l,CCV 1-71 etc.Synthesizing this set of primers,we selected the panda's liver-tissue materials and some different passages of viral culture to amplify by RT-PCR, and all of them respectively gained two target fragements of 1086bp and 515bp, but the control cell did not. Using a kind of restriction endonuclease named BST-X I (590,1110) for S sequence,we proved the amplified fragement was specific.Then the CCVF2-CCVR2 amplified fragements from panda's liver materials and the 2nd,the 3rd and the 29th passages of viral culture was collected, purifed and sequenced .With the help of biological software to analyze the nucleotide sequences of every passages of viral culture.all of them were proved to be the same virus.By collecting CCVF1-CCVR1 PCR product from DXMV3 and sequencing it.then a 1047bp sequence was gained.Using bio-software,we compared the sequence of DXMV with those of other CCV strains,such as NVSL,CCVl-71,K378,Insavc-l ,etc.From the results ,we learned that the nucleotide sequence homology respectively was 100%,99.5%,98.3% and 89.5% and the deduced amino acid sequence homology was respectively 100%,98.9%,96.6% and 90.5%.In this study.CCV was first isolated and identificated from pandas/Therefore pandas was proved to be a new host of CCV.The result of the s... |
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