Molecular Epidemiology And Biology Of Marek's Disease Virus Field Isolates In China

To investigate the pathogenicity and gene variety of epidemic field strains of isolatedMarek's disease virus (MDV) in China in last several years, some samples from differentflocks or provinces in China were collectted to isolate and identify MDV by cell culture,indirect immunofluorescrence assay( IFA), dot blot hybridization, histopathologicalexamination and polymerase chain reaction (PCR).Collecting the 63 bloods samplessuspected tumor and inoculating onto the monolayer chicken embryo fibroblasts(CEF), wehad isolated 19 MDV field strains. To further study weather these MDV field strains includereticuloendotheliosis virus (REV), we found that 7 MDV field strains infected CEF showednegative with REV monoclonal antibodies (Mabs) 11B154 and 11B158 and didn't amplify thelong terminal repeat (LTR) of REV by PCR, while another 7 MDV field strains infected CEFshowed positive both with REV Mab and PCR which mean these MDV strains contained freeREV. However, there were 5 MDV field strains that showed positive by PCR but didn't reactwith REV Mabs that indicated the REV fragments may be integrated into these MDV fieldstrains genomes. These results indicated that the co-infection phenomenon of REV and MDVwas common and maybe some MDV field strains had integrated the REV genome fragments. To study the relationship during the tumor development of MDV and REV coinfection onchicken, 1-day chickens were inoculated both REV and MDV strain. On 94 days postinoculation(PI), the mortality of chickens coinfected could reach 63.3% after a first deadchicken appeared while the mortality of chicken infected with MDV or REV alone were only33.3% and 36.7% and the first dead chicken appeared on 47 and 45 PI. The weight of chickencoinfected with REV and MDV were 153,397 and 920 gram on 21, 38 and 76 PI separately,while the weight of chicken infected with REV or MDV were 200, 494, 1197 and 173, 410,1107 respectively. It is obvious that affect coinfection of MDV and REV was more thaninfection with MDV or REV separately. By gross examination on dead chickens, there were 2 different tumors of sporadic, largetumors and diffusible, small tumors that induced and mixed on the aspect of liver, spleen,kidney and heart. These samples were embedded with paraffin and cut into a series ofsegments that were stained with hematoxylin and eosin, and examined for existence of two 6我国近年来马立克氏病病毒野毒株分子流行病学和生物学特性的研究different kinds of tumor cells by microscopic lesions. Some segments were reacted withmonoclonal antibody of MDV BA4 and REV 11B118 respectively. The IFA examinationsshown that both MDV and REV antibody could react with the same segment that mean REVand MDV could induce tumors separately in chickens. On 76 days PI, the average positiverate for MDV of white cell isolated from 3 chickens were 3.4% while the average positive ratefor MDV from chickens coinfected with REV and MDV was 7.67%. After passages for 5 times, the content of MDV in CEF infected with MDV alone were 5times than that coinfected with MDV and REV. It is included that the productivity of MDV inCEF was inhibited greatly if the CEF were infected with REV. Two Marek's disease virus (MDV) field strains were isolated from chickens with tumorsindependently from Guangdong and Guangxi provinces, and it was confirmed that there wereno co-infections with REV in CEF in IFA with REV-specific monoclonal antibodies. By dotblot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated thatLTR fragments of REV genome were integrated into genome of these two MDV field strains.To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primersfrom REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hotpoint were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDVfor each pair) were used in PCR while the genomic DNA of both strains were used a...