Epidemiological Studies On Reticuloendotheliosis Virus Infection In Ducks And Analyses Of Env Gene Sequences

Reticuloendotheliosis (RE) designates a group of pathologic syndromes including acute reticulum cell neoplasia, chronic neoplasia of lymphoid or other tissues, runting disease syndromes and immunodepression in various avian species, such as turkeys, chickens, ducks, geese, and quail. The diseases were caused by a group of retroviruses collectively called Reticuloendotheliosis virus (REV). Although this kind disease can't cause the chickens dead directly, it can decline the resistance of the chickens and take a great loss to the breed aquatics of chickens.REV can infect turkey, chicken, duck, goose, pheasant, partridge and other birds. Spleen necrosis virus(SNV) and Duck infectious anemia virus(DIAV) were isolated from ducks among all representative strains, and REV was isolated from tumors in ducks in foreign countries. But there is not any report on REV natural infection in ducks in China. DNA probe was labelled by digoxin after analysising conservative sequnces of env gene of REV. Tissue samples of the Bursa, spleens and livers were randomly collected in duck groups in Shandong province.To detect REV by dot-blot hybridization,PCR and nest-PCR with tissue-extracted DNA as the templates and by cell cultures and the detected by IFA to finish epidemiological studies on Reticuloendotheliosis Virus infection in ducks groups.The positive products of nest-PCR of env gene which were randomly selected were cloned and analysise.d in order to explain the resources of REV in ducks by analysising genetic relationship.1. The mark of REV nucleic acid probe and examination of sensitivity, specificityREV env gene fragment was amplified by specific primer with SNV viral genome cDNA infectious clone plasmid pB101 DNA as the template.The PCR product was purified , quotaed and labeled by digoxin to prepare DNA probe. This probe can specificity hybrid with DNA extracted from tissures and cells infected by REV. The nucleic acid probe sensitivity and the specificity test result showed: This probe can examine the 1pg specificity nucleic acid, having high sensitivity and the specificity. This research provided the good method for REV diagnosis and the epidemiology investigation .2. Epidemiological Studies on Reticuloendotheliosis Virus Infection in DucksThere were 220 tissue samples of the Bursa, spleens and livers randomly collected from 97 sick or dead ducks from 3 districts in Shandong province. REV was detected by cell cultures, direct dot blot hybridization, regular PCR and nest-PCR with tissue-extracted DNA as the templates. REV was detected 121 tissure DNA by Nest-PCR, the positive rate was 55%. The positive rate of bursal samples was significantly higher than spleen and liver samples (P<0.01). All samples demonstrated negative with the same samples in virus isolation, direct dot blot hybridization or regular PCR. The results indicated that many ducks infected or have infected REV in Shan Dong province. All samples demonstrated negative with the same samples in virus isolation, direct dot blot hybridization or regular PCR , absolutely different from REV infection in chickens which can be easily isolated and detected in. That illustrated that REV infective dose was very low in ducks, or the probability of integration in genome of ducks by cDNA was low than chickens. The positive rate of bursal samples was significantly higher than spleen and liver samples (P<0.01) when REV is detected, bursal should be paid more attention. Since REV was existing only at a very low level in infected samples and difficult to be detected.3. env gene of REV clone and sequential analysisThe REV gene is relatively conservative and there is not obvious high variation area,but the pouch membrane glycoprotein gene (env) as the major viral antigenicity gene is easy to mutate compared with other structural gene like gag, pol. In sequence comparisons of amplified and cloned env gene fragments from 3 representative samples, sequence Yinan-1 and Binzhou-1 had 99.8% of identity with SNV strain isolated from ducks in USA and Linqu-1 had 100% identity with two strains isolated from chickens in USA, higher than the homology with any strains isolated from chickens in China. It suggested REV infections in these ducks may relate to imported breeder ducklings by sequence homology comparisons and phylogenetic tree analysis.