| 1. A nested-PCR typing assay was used for testing the presence of canine coronavirus (CCV) from 138 faecal samples of diarrhea and healthy dogs, collected from August 2003-January 2004. In healthy dog populations, 70 out of total 81 faecal samples were CCV II positive. The positive ratio were 80.0% (24/30) for Nanjing population, 87.2% (34/39) and 100% (12/12) for Shengyang and Kunming population respectively. In diarrhea dogs, 18 out of 43 samples collected from Nanjing and 7 out of 9 pet dogs feces from Shanghai City were CCV II positively, 3 out of 5 were CCV II positive in Kunming faecal samples. No type I CCV was detected. Sequence analysis of CCV M gene fragments showed the presence of the CCVs shared highest similarity with CCV reference strain 1-71 (96.5-99.5%) and giant panda CCV (95.8-99.1 % ) . One M gene fragment was found between the type I and type II CCV branches in the phylogenetic tree, suggesting that a novel strain had emerged. It confirmed that type II CCV infection is very common in domestic dog.2. Four CCV wild strains were isolated from CCV positive diarrhea dog feses by CCV susceptively A72cell line, and identified by RT-nPCR and immunofluorescence tests, namely CCV KM, CCV NJ2, CCV NJ17 and CCV SH3. By Electron microcopy, CCV KM virions had a distinct "corona" similar to reference strain CCV 1-71. The cell adaptability tests showed that the CPE caused by wild CCV strains were different from reference strains syncytia CPE. CCV KM could caused cell shrinkage and fall off, CCV NJ2 had caused obvious CPE at its 6-15 passages, but after 15 passages it could not caused visible CPE, NJ17, SH3 could not caused visible CPE on SA72 cell. CCV 1-71 and wild strain KM, NJ2 (No.8 passage) could replicated on MDCK, F81, CrFK cell lines and only KM could grow on Vero cells and cause visible CPE. Cross-prevented tests showed that the serum anti CCV 1-71 can neutralized CCV KM and NJ2 NJ17, could not neutralized CCV wild strains HC, DF, HF (isolated from other different areas and animals), but the serum anti CCV KM could only neutralized CCV KM but not to other CCV strains. This suggested that the different CCV strains cross-prevented antigen were restrictive.3. BALB/C mouse were immunized with CCV antigen purified by sucrose gradient centrifuged, and its immunized spleen cells were fused with murine myeloma cell lineSP2/0. One specific hybridoma colony secreting high titer antibodies and two hybridoma colonies secreting high titer neutralized antibodies were selected by ELISA and neutralization test respectively, and namely MAb El, MAb N1, MAb N2. The ELISA titer of hybridoma El secreting antibody in its cultural medium and ascites fluid were 1: 2048 and 1: 38768. While the neutralized monoclonal antibody titers of Nl and N2 were respectively 1: 40 and 1: 80 in hybtidoma cultural medium, and 1:20480, 1:40960 in ascites fluids. Speciality and characters of the MAbs were tested by immunofluorescence test and neutralization test. The results showed that MAbs Eland Nl could interact with CCV 1-71.USCV, Tn449, NJ2, NJ17, KM, DF, SH3, MAb N2 could interact with CCV 1-71, USCV and Tn449. Mab El could not neutralize any strains, MAbs Nl and N2 could only neutralized CCV 1-71, USCV and Tn449 those caused similar CPE on A72 cells.4. The release of CCV from infected cells were studied by monoclonal antibodies (MAb). The results showed that MAbs E1, N1 can display release of CCV 1-71, USCV, Tn449, KM, NJ2, NJ17, DF from infected A72 cell, MAb N2 could not display the viruses release. In the study of monoclonal antibodies interact with other CCV strains, we found MAb E1, N1, N2 can not prevent wild CCV strains infection, while MAb Nl and N2 even enhanced the viruses infection. The CPE would be appeared earlier and the progeny titer be higher the CCV KM were mixed with MAb N1, N2. In the immunofluorescence tests, stronger fluorescent staining of cells infected by CCV KM with MAb Nl or N2 than those of only infected by CCV KM. The results showed that the neutralized monoclonal antibodies of CCV 1-71 play a role of antib...
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