Development Of Loop-mediated Isothermal Amplification Assay And The Infectious Molecular Clone For Reticuloendotheliosis Virus

Reticuloendotheliosis is an important immunosuppressive and oncogenic disease in avian species. Recently, REV is prevailing in avian in the world. Co-infection of REV with subgroup J avian leukosis virus (ALV-J), Marek's disease virus (MDV), and chicken anemia virus (CAV) has become endemic in layer and breeder flocks, which induce immunosuppressive and immuno-failure of some important disease (AIV, NDV, et al) so that secondary infection. So it is very difficult to diagnose and control REV. In addition, REV genome can integrate into MDV and fowl poxvirus (FPV) genome and contaminate these commercial vaccines. Besides, the vaccines developed with non SPF embryonated egg are also easy to be contaminated with REV. Now, the threat posed by REV has been got more and more attention. However, the diagnosis, epidemiology and basic research are not consummate. In order to further understand and control RE in a new efficient level, the following experiments were performed in this study.1 Development of LAMP assay for REVThe convenient, specific and sensitive LAMP assay for detecting REV was developed, in which 4 primers aiming at six sites to amplify the pol gene. The REV LAMP assay did not cross react with common avian DNA viruses (MDV, CAV) and ALV-J. Additionally, the assay could detect different REV strains and has a detection limit of 5 copies, which has a 25 times sensitivity than traditional PCR methods. Furthermore, the efficiency of LAMP to detect REV in clinical samples was comparable to traditional PCR. The procedure of LAMP was straight-forward, not rely on any special equipment and performance was simple. The result of LAMP could be observed directly by the unaided eye.2 Expression of gp90 protein and development of anti-gp90 monoclonal antibodyThe gp90 gene was amplified by PCR and then cloned into pFastBacHTA, pET-28a (+) , respectively. The donator plamid pF-gp90 of eukaryotic expression and the prokaryotic expression plasmid pET28-gp90 were acquired. The recombinant donor plasmid pF-gp90 was transformed into E coli DH10Bac. The recombinant bacmid plasmid Bacmidgp90 was obtained by integrating gp90 gene into Bacmid vector in DH10Bac cells. The recombinant baculovirus rBacgp90 was obtained after transfecting Sf9 cells. Western blot and IFA showed that the gp90 protein was expressed successfully and its weight was about 45kDa. It has good reactionogenicity. The plasmid pET28-gp90 was transformed into E coli BL21 (DE3) and was induced by IPTG. Analysis by SDS-PAGE and Western blot showed that the gp90 protein was expressed successfully in the form of inclusion body. Then the gp90 protein was purified and immunized to 6-8 weeks Balb/c mice to prepare anti-gp90 serum. The protein has good immunogenicity and the mouse anti-gp90 serum with the ELISA titer of 1:12800 could recognize REV.3 Research of RE epidemiologySerological survey was performed in nine province. Serum samples were collected from thirty-five layer farms and REV antibody were detected by IDEXX ELISA kit. 89% farms were positive for REV antibody. The positive rate in each farms were 1%-98% and the average positive rate was 15%. These data suggested REV was popular in China and some farms were serious. Grinded and sterilized, the samples were inoculated CEF or DF1 and cultured in 37℃CO₂ incubator for 7 days. Virus was collected and blind passaged. Finally, REV was identificated by indirect immunofluorescence assay (IFA), PCR, electron micrograph. We isolated 10 REV strains from some chicken farm of Heilongjiang, jilin, liaoning, et al. The TCID₅₀ of HLJ0901 strain was tittered as 10^5.2/mL. With the replication kinetics curve, the regular pattern of HLJR0901 replication in CEF cells was researched: the highest titer of HLJR0901 was observed at 6 and 7 days post-infection. Gp90 gene of fourteen strains, encoding the most important protective protein, was amplification and cloned into pMD-18T vector, then were sequenced and submitted to GenBnak. Six overlapping fragments covering the full-genome of HLJR0901 were cloned and sequenced. The full-genome sequence of HLJR0901 (8284bp) was ascertained. The GenBank accession number is GQ415646. Sequence analysis showed that the LTR of HLJR0901 was completely homologous with FA, and has 99% homology with APC-566. Pol was the most stable gene. The phylogenetic trees of LTR nucleotide, gag amino acids, pol amino acids, gp90 amino acids, env amino acids and the genome displayed a common character:â‘ all the strains was diveided into three distinct branches, which represents three subtypes, respectively.â‘¡the northeast REV isolates formed a distinct cluster far from the early Chinese strain HA9901.(subtype I), which derive from different ancestors.â‘¢at least two subtypes of REV (subtype I and III) are popular in China, the northeast REV belong to subtype III.â‘£the northeast REV isolates have high similarity with some popular strains in American and Taiwan.4 Development and application of the infectious molecular clone of REVThe full-length genome of the HLJR0901 strain was jointed and cloned in the pBluescript II vector by SOE PCR and restriction enzyme, and the genetic tag C2250G (deleting the MscI site) was introduced. The infectious molecular clone pBlu-mHLJR0901 was constructed. pBlu-mHLJR0901 was transfected and then mind-blinded in CEF cells. With identification of IFA, PCR, sequencing, electron microscope assay, REV could be rescued efficiently. The rescued REV has similar characters of replication and infection. Based on the reverse genetics of REV,the construction of recombinant REV expressing EGFP was performed. EGFP was inserted into the 934 and 7713 nucleotide site of HLJR0901 genome, which was lied in the upper stream before gag gene promoter and the down stream after env gene terminator, respectively. After being transfected into CEF cells, EGFP was transient expressed transient with high performance in the former but not in the later. No recombinant virus was rescued. We also observed that the promoter activity of REV LTR was broad-spectrum not only in chicken cells but also in pig cell. The experiments provide some reference for the basic researches of REV.5 Animal experiment of REVThe REV-infection-animal model with the resued rHLJR0901 was studied preliminarily. Infected 1d SPF chicken, REV could influence the growth and induced a little lesion in liver, spleen, thymus and bursa. Seroconversion was observed at 3w post-infection, but the positive rate was 75% till 8w post-infection. Viremia existed during all experiment period. The experiments provide necessary data for REV-infection-animal model. Viremia will be a stable index for REV-infection-animal model.