The Infection Status Survey Of Main Immunosuppressive Disease Pathogen In Northeast Wild Birds And Yeast Two-hybrid Screening For The Interactional Host Protein With REV PR

The co-infection of several avian immunosuppressive disease pathogen, such as Reticuloendotheliosis virus (REV), Avian Leukosis virus subgroup J (ALV-J), Chicken infectious anemia virus (CAV) and Marek’s disease virus (MDV), is common in chicken farms in recent years. They not only leade to low immunity of chicken flocks, the immune failure of Avian influenza and Newcastle disease vaccine, but also cause secondary infection easy, and increase the difficulty of the diagnosis, prevention and control of these disease.The avian leukemia (AL), Reticuloendotheliosis (RE) and marek’s disease (MD) are referred to as three viral oncosis,cause serious damage to poultry health. Previous molecular epidemiology studies focused primarily on poultry and waterfowl, and reports of infection in wild birds were rare. The research on pathogens carried by wild birds mainly focused on the studies of avian influenza,and there were no correlation epidemiological investigation reports on avian immunosuppressive pathogens related to wild birds so far. As many pathogen carriers and natural hosts, wild birds may spread many important infectious diseases of humans and animals potentially. Along with migratory, there may be such a possibility that migratory birds which carried viruses can spread the virus to other places, and even lead to the outbreak of this disease. Therefore, it has important instruction meaning to do molecular epidemiological surveillance on wild birds. In order to know the condition of avian immunosuppressive pathogens infected or carried by wild birds, we carried out the epidemiological investigation on wild birds collected from flagged wader birds stand and wildlife endemic disease monitoring stations of three provinces in northeast China from May2010to November2012.916samples were collected in all, including581samples from wild ducks, the other335samples were passeriformes birds. On the one hand, we investigated the prevalence of several pathogens; on the other hand, we cloned and analized the genetic characteristics of the gene sequences.1. PCR detection of avian immunosuppressive pathogens of wild birds in Northeast ChinaDNA and RNA of wild birds samples were prepared with conventional method. And REV、ALV-A、ALV-B、ALV-J、CAV、MDV and IBDV were detected by specific PCR(or RT-PCR). Experimental results showed that there existed the infection of REV,ALV-A,ALV-B,ALV-J and CAV in wild birds. In contrast, the specific nucleic acid of MDV,IBDV and ARV were undetected according to the testing results of part of the samples. The results showed that the PCR positive rate of REV was10.7%of all wild bird samples, with wild duck’s13.1%and small birds6.6%; the PCR positive rate of CAV, ALV-A, ALV-B and ALV-J were4.1%,4.35%,6.4%and6.8%respectively. There were no apparent seasonal characteristic on the viruses wild birds infected with, There has a certain difference in the positive detection rate of REV and ALV-J in different kinds of wild birds. Pintails had the highest REV detection rate in wild duck, and Long-tailed Rosefinch had the highest REVdetection rate in small passerine birds. Tufted Duck had the highest ALV-J detection rate in wild duck, and Red-flanked Bush Robin had the highest ALV-J detection rate in small passerine birds.2. Isolation and identification of REV from wild birds of Northeast China and analysis of genetic evolution of gp90gene.Virus isolation was performed by inoculating DF1cells with the tissue homogenates. The filtered liver-and spleen tissue homogenates with positive results in PCR were inoculated onto DF1cells and monitored daily for7days. With identification of specific PCR, indirect immunofluorescence assay (IFA), and electron microscopy test,10REV strains were confirmed.2strains from small passerine birds,8strains from wild ducks. The gp90gene from each of the10REV strains was amplified, cloned, and sequenced. Sequence analysis indicated that the gp90genes of the10REV strains isolated in this study were more similar at the nucleotide level with the northeast Chinese strains HLJR0901and HLJR0801and some REV strains found in the US and Taiwan than with the early Chinese REV isolate HA9901. Furthermore, phylogenetic analysis indicated that the gp90genes of the10REV strains were more similar to the REV subtype Ⅲ-representing strain (CSV) than to strains170A (subtype Ⅰ) or SNV (subtype Ⅱ). This is the first study to investigate the status of wild birds infected with REV. The results of this paper will not only provide necessary information for further understanding the evolution of REV, identify the potential role of wild birds in REV transmission and furthers our understanding of the ecology of REV in wild bird species.but they also lay the foundation for further research on the pathogenic mechanism and immune suppression mechanism of REV. Six overlapping fragments covering the full-genome of DBYR1102were cloned and sequenced. The full-genome sequence of DBYR1102was ascertained.3. Isolation and identification of ALV-J from wild birds of Northeast China and analysis of genetic evolution of gp85gene.With identification of specific PCR, inderict ELISA and indirect immunofluorescence assay (IFA), a total of six ALV-J strains were isolated,2strains from small passerine birds,8strains from wild ducks. The GenBank sequences of the ALV-J strains that were isolated from broilers and layer chickens were included in a multiple sequence alignment and phylogenetic analysis. The six wild-bird ALV-J sequences were phylogenetically analyzed using the maximum-likelihood method with other ALV-J sequences. The gp85genes of the six ALV-J wild-bird isolates were921to924nucleotides long. Sequence analysis indicated that the nucleotide sequence identities ranged from93.1%to99.7%, and the deduced amino acid sequence identities ranged from93.1%to99.7%compared with the ALV-J prototype strain HPRS-103. The gp85genes of DBYJ1102,DBYJ1103, DBYJ1004and DBYJ106displayed close similarity (over95.8%) to each other and had relatively close similarity at the amino acid level (94.4to98.3%) to some ALV-J layer strains isolated in recent years. These isolates belonged to one branch, which was designated group I. In addition. However, the gp85genes of DBYJ1101and DBYJ1105displayed close similarity to each other and were99.6and99.4%identical, respectively, to the prototype strain HPRS-103at the amino acid level. These isolates belonged to another branch, which was designated group II. American strain UD5and most of the broiler isolates were located in the same group. Our studies provide evidence of the coexistence of two different ALV-J subgroups prevalent in Chinese wild birds.4.Expression of REV p30protein and the preliminary establishment of REV antibody indirect ELISA detection methodThe REV p30gene was amplified by PCR and then cloned into pET-30a, and the prokaryotic expression plasmid pET30-p30was constructed. The plasmid pET30-p30was transformed into E coli BL21(DE3) and was induced by IPTG. SDS-PAGE and Western blot showed that the p30protein was soluble expressed successfully. Then the p30protein was purified with resin and immunized to rabbits to prepare anti-p30serum. The purified protein has good immunogenicity and the rabbit anti-p30serum could recognize REV with the ELISA titer of1:25600. Used the purified recombinant p30protein as coating antigen, with the quantity of package3.2μg/hole, serum250times dilution, the P/N reached the maximum, and REV antibody indirect ELISA detection method had been preliminary established. The research laid the foundation for the REV detection and research on p30gene of REV.5. Screening the host proteins interact with polymerase protein PR of Reticuloendotheliosis virus (REV) in chicken embryo fibroblasts(CEF) with Yeast two-hybrid systemIt’s of great significance to research the interaction between the host cell proteinwhich interact with REV and the function of PR protein, pathogenicity and infection mechanism of REV. The recombinant bait vector pGBKT7-PR was constructed for screening the proteins which interact with polymerase protein PR of Reticuloendotheliosis virus (REV) in chicken embryo fibroblasts(CEF). The self-activation and toxic action of the bait vectors were tested in the Matchmaker Gold Yeast Two-Hybrid System. Results indicated that the bait vector pGBK-PR was successfully constructed and proved to be neither self-activation nor toxic to yeast cells, and could be used as bait to capture the interacting protein in the further research with the yeast two-hybrid system. To co-transform Y2H yeast, it was confirmed the interaction between Snapin and pGBKT7-PR. By analyzing the sequence of the insertion element of positive library plasmid, host protein ATP1B1and Snapin interact with PR were identified. Snapin protein play role in variety of cell biological processes, and can play a role of antivirus. The Snapin genes were amplified from the RNA of DF1and cloned into eukaryotic expression plasmid pCAGGS with Flag tag (pCAF), and eukaryotic expression vector pCAF-Snapin was constructed. The eukaryotic expression vector pCAH-PR was constructed by cloned PR gene into plasmid pCAGGS with Ha tag(pCAH). The above research not only laied foundation for further validate the interaction between PR and Snapin by immune co-precipitation (coimmunoprecipitation, Co-IP), but also benefit the further investigation on the interaction between REV and its host cell.