Molecular Epidemiology And Evolution Of Reticuloendotheliosis Virus Under The Host Immune Selective Pressure

In order to investigate the molecular epidemy of the envelope glycoprotein gene (env) of Chinese domestic field reticuloendotheliosis viruses (REV) in the past few years and to make sure their evolution principle over a span of time, several overseas strains and seven Chinese field isolates identified using tissue-culturing, indirect fluorescent assay (IFA) and polymerase chain reaction (PCR) from the samples of 1999—2004 were used to be amplified for the env fragment by PCR, and the products were cloned and sequenced separately. Then the sequences were compared with the reported ones using a software of DNAstar. The result indicated that except the obvious difference of turner-causing mutant T s associate strain A from the others, the env gene was speculated to be very conservative, i.e. whether in the level of nucleotide or amino acid, the homology was over 92%. But the difference would be more remarkable if several segmental regions were focused on, and it seemed to be a rule although they were originally isolated from different times in different regions and birds. In another aspect, the changes focused on several regions in the sequence of env, while the ratio of nonsynonymous to synonymous (NS/S) was calculated, the result was obviously higher than 2.5 which is popularly acceped as the marker to give a conclusion whether the changes in some gene of a pathogen are under the influence of a certain selective pressure. Thus it would be speculated that the antigen epitopes in env of the analyzed strains had overtaken a long period of dominantal selection from the host immune system and gone to the nearly same evolution from their ancient original parent viruses.As a field isolate is a complex mixture of different virions in pathogenicity, the analysis mde to it will not be expercise enough, and in order to do so, it is necessary to establish a modle, i.e.to get one infectious virion with known genetic background of the dominant virus particles purified from the original isolate. Therefore, the interference from the chanciness of random mutations will be negalected in forthcoming analysis, and herein the REV Chinese field isolate HA9901 was chosen as the sample, and 6 continuously overlapping pairs of primers were synthysized to amplify the proviral cDNA of the virus. After the PCR products were cloned and sequenced, a software of DNAstar was used to splice and analyze the data,and finally the complete proviral genome sequence of HA9901 was finished and it was the first one for REV free infectious particles isolated from chicken. Compared with the other related genomic sequences, this one showed high homology with them indicating the relatively quite conservative for the genome of REV, especially the sequence of pol gene encoding the functioning protein. But the sequeces of LTR in proviral cDNA playing important role in the life cycle differed in two regions with deletion according to the original host, and its influence on the virus still needs further works.Furthermore, in order to verify the sequence of HA9901, two pairs of primers were designed to amplify the gene of gp3 and env, then the products were cloned into vectors of pGEX-6P-l and pcDNA3.1/Zeo(+). The identified plasmids were transformed into E.coli. BL21 or transfect SPF CEF by Lipofectamine? Reagent, and then they were verified by running SDS-PAGE gel or IFA to be existent of REV elements.On the basis of the complete genome sequence of HA9901, its proviral cDNA was amplified as three continuous fregments by PCR, which were then cloned and simultaneously subcloned into the vector pcDNA3.1/Zeo(+) digested with Pst I and Xho I., thus the recombined plasmid carrying the whole cDNA of HA9901 was constructed being named pcDNA/HA9901. Following this, the mutant recombinant of pcDNA/Ae?vHA9901was got with a deletion which covered about 130 amino acids on env gene following the stop code of pol. Then the experiment of the two clones to be separately transfected into SPF CEF is still in improving progress.On the other hand, to survey the genetic variation of env gene under the host immune selective pressure during the procedure of continuous infection, transmision and fighting with the host, the molecular cloned SNV was challenged and continuously passed in three ways in this experiment: one was inoculated in CEF cultured under the media of Ml 99 without REV-specific antibodies and passed till 15th passages; one was challenged in 1 day SPF chicks untill 10 days and passed till 15th passages; the last was challenged in 14 days SPF chicken untill 21 days and passed till 15th passages. During each procedure, the viruses of 5th 10* and 15th passage were collected and the CEFs in which they were cultured were also saved The genomic DNA extracted from these CEF was used as template to amplify the env and 3'LTR of every kept virus with two corresponding pairs of primers. Then the products were cloned into pMD 18-T vector and sequenced respectively, and the data were analyzed with DNAstar. The comparison of the finished env gene sequences showed that although there were 6 changed parts or deletion in about 2/20 to 3/20 sequences, the variation was not stable and genetic on the basis of the way which SNV was inoculated or challenged, and the NS/S ratiowas unnecesary to cal-culate, i.e. the variation could not imply any influence of immune selective pressure. So did the sequences of 3'LTR. Thus it would be necessary to improve the method or time of passage, for instance, the CEF infected with SNV was cultured in such media with a certain concentration of REV-specific antibodies or monoclonal antibody, or altenervely the procedure of passages and the time of the interaction between SNV and the host immune system were not long enough for the obvious and reguler changes to emerge.Although the result of SNV passages did not meet with the expectation, it gave us a opportunity to selecte natural tumor cells from the challenged birds with SNV; meanwhile, the natural material of tumors in field flocks was also a beneficial samples, and they both suppor the possibility of establish a few tumor cell lines. The lymphocyte mixtures from the two samples were cultured and passaged for as long as 12 months until the several strains were stable in vitro besides they had not appeaed to reproduce very fast. Then several tests will be prepared to perform, for example, nuclear-counting and IFA with monoclonal antibodies against REV ALV or MDV, so as to selecte several cell line candidates transformed by REV ALV and MDV respectively and to found a basis for the research in the mechanism of their tumor-causing and immune suppresion-causing.