Analysis Of REVs Genome Sequence In Mixed Infection Cases Of Avian Tumor Diseases And Identification Of LTR Recombinant MDV

Marek’s disease(MD) which is caused by Marek’s disease virus(MDV) is an infectious and neoplastic disease of chickens. MD, which is characterized by immunosuppression and lymphoproliferation, could induce tumor until the host chickens death. Reticuloendotheliosis(MD) whose agent is Reticuloendotheliosis virus(REV) is another neoplatic disease, which causes oncogenic, atrophy of immune organs and runting syndrome in multiple avian hosts including chickens, ducks, turkeys and other bird species. Recent years, the cases of mixed infection of MDV and REV, leading the disease earlier and clinical symptom more serious, are gradually increasing. Additionally, the MD vaccine contaminated by REV could also be an important resource. These problems make the prevention and control of MD and RE more difficult.The REV genome especially LTR region can integrated to MDV genome, while mixed infection of the herpesvirus MDV and retrovirus REV in vivo and in vitro. Due to the activity of promoter and enhancer of LTR, the integrated LTR could regulate the expression of MDV genes, resulting in alteration of property of MDV, including pathogenicity and capacity of horizon transmission, which contributes to the evolution of MDV. In this study, two clinical samples were collected from two flocks in Shandong province and Liaoning province respectively, where neoplastic disease happened. The result of agar diffusion assay and PCR test diagnosed with MD. Further testing show the two flocks were mixed infected with REV. To study the molecular characteristic of the REV in the mixed infection, we succeeded in isolated the REV in DF-1 cells, and were identified through PCR, indirect immunofluorescence assay and electron microscopy. The two REV isolates were named CY1111 and SY1209. Furthermore, the complete proviral genome sequence of the two isolates were determined, and aligned with the genome sequences of other strains. Phylogeny trees were constructed based on different region, including LTR region, gag gene, pol gene, env gene and the full genome sequence. The results of phylogeny trees depicted that our two REV isolates and others isolated in China, except GD1210 strain, were in the same clade, indicating that the strains isolated in our country were closely related. Notably, REV CY1111 and SY1209 shared 99.8% identity with MD-2 isolated from a commercial MD vaccine, and phylogeny relationship also revealed they were in the same clade, indicating that the REV in the two flocks might be from the contaminant of vaccine. If so, we should pay more attention to the quality of MD vaccine. What’s more, the phylogeny results revealed that REV isolates did not present host-specific or territory-specific, thus, REV, under the natural condition, might freely spread between various hosts and diverse territories.To study the events that REV integrated into MDV genome, we employed the method of Hot Spot Combined PCR(HS-cPCR), which was used to identify the integration of REV LTR into MDV genome at the hot spots. We tested the recombinant in MDV and REV mixed infection in vivo and in vitro with the HS-cPCR. MDV LCY-BAC strain, originated from purification by bacterial artificial chromosome(BAC) following the process of virus isolation from the sample mixed infected with REV, was tested and the results showed that there were two copies of LTR in the genome of LCY-BAC, which were at the junctions between Us/IRs and Us/TRs, and they were at the same direction, contrary to the MDV genome direction. To study the stability of the recombinant virus, LCY-BAC strain was passed for 15 passages in vitro. The location, direction and copy number of LTR integration did not change, when testing the second, fifth, eighth, twelfth and fifteenth passage, certifying the LTR recombinant MDV could be stably passed, which contributed the research of the recombinant between MDV and REV. None of LTR recombinant was detected when checking the 20 mixed infected samples from 8 flocks. Thus, the probability of LTR recombinant MDV in nature was likely to be relatively low.In this study, the REVs in mixed infected samples were isolated, identified and the phylogeny relationship was analyzed, of which the results showed most of the REV strains isolated in our country were closely related, and indicating that contaminant of vaccine might be the resource of REV. A novel model of recombinant was detected when we tested the LTR recombinant MDV, and recombinant virus could stably passed. Some samples naturally mixed infected with MDV and REV were tested, but none of natural recombinant virus was detected. This study provided important data and theory basis to the research of recombinant, evolution and the control of MDV and REV.