Isolation And Identification Of Orf Virus And Goatpox Virus And Preparation Of Monoclonal Antibody Against B2L Protein Of Orf Virus

Orf virus (ORFV), that causes recurring contagious ecthyma or orf disease in goat, sheep and other wild and domestic ruminants. Although the infection is usually cleared within 1-2 months, delayed growth and associated secondary infections could still impact the herds. Goatpox virus (GTPV) can infect sheep or goat cause Capripox, the most serious disease of all kinds animal pox disease, classified as notifiable animal diseases by the World Organization for Animal Health (OIE). It can cause significant economic losses of domestic ruminants in endemic regions and threaten the stockbreeding.Clinical symptoms similar to parapoxvirus infection are caused in animals by sheeppox and goatpox viruses infection, along with cross reaction in serological tests, which lead to the indistinguishability in clinical and serological. As well as the mixed infections of capripox with orf or other disease/s can increase the severity of diagnosis. In this study, ORFV and GTPV separated and identified by cell culture, PCR and genetic evolution analysis. B2L protein of orf virus was expressed and preparation of monoclonal antibody. The following results were obtained:1. Isolation and Identification of ORFV and GTPV from 2013 to 2015 in the partial area of Jiangsu provinceA total of 121 tissue samples were collected in some farms (households) and subjected to PCR detection, bacterial isolation and viral isolation in the laboratory.4 strains were ORFV positive by PCR, named ORFV/Ovis/XZ/Jiangsu/2015/China, ORFV1/Ovis/DT/Jiangsu/2015/China, ORFV2/Ovis/DT/Jiangsu/2015/China, and ORFV/Ovis/SL/Jiangsu/2015/China, in which has one mixed infection with GTPV. The B2L gene was amplified for the phylogenetic study. The nucleic acid homology was 100.0%、98.6%、 98.6% of XZ and DT isolates, gathered into a cluster with SC-JY, GX-YB, JS-FX isolates. SL gathered into a cluster with LiaoNing, HuB, Gansu isolates, the nucleic acid homology was 98.8%、 98.9%、 98.9%, respectively. The nucleic acid homology of 4 strains and orf virus strain China vaccine was 96.8%-98.8%, and 2 strains were isolated with ovine fetal turbinate cells (OFTu) and MDBK.2 strains were GTPV positive, named Goatpox/WX/Jiangsu/2014/China and Goatpox/XZ/Jiangsu/2014/China, and 1 strain goatpox virus was isolated successfully by cell-culturing of lamb testicular cells (LT) and BHK-21.2. The establishment of a multiplex PCR method for the simultaneous detection of ORFV and GTPVA multiplex PCR (mPCR) method was established, in which Four primers were designed to amplify a part of the conservative gene P32 and VIR, the unique gene of GTPV and ORFV, respectively. The result of sensitivity of the target DNA showed that the minimum detectable limitation of goatpox virus and orf virus were 0.098ng/μL and 0.46ng/μL. Specificity and broad spectrum also is good. There can quickly identify ORFV and GTPV or both mixed infection in a same PCR reaction system.3. Prokaryotic expression of orf virus B2L geneB2L gene of orf virus was amplified and inserted into prokaryotic expression vector pET-32a, then transferred to BL21. The result of SDS-PAGE showed that a great quantity of B2L proteins could be expressed in soluable form under the condition of 37℃,200rpm, and 0.6M IPTG. After purification, the concentration of recombinant protein is 4.183 mg/mL.4. Development of monoclonal antibodies against B2L protein of orf virusBalb/c mice were immunized with purified recombinant His-B2L fusion protein, and spleen cells of immunized mice were fused with myeloma cell (SP2/0) after forth immunization. The cells, which have the positive reaction with B2L and the negative reaction with His protein by indirect ELISA, were be cub-cloned. Finally, one monoclonal antibody (McAb) was screened, named 6D5. The result of IFA revealed that specific fluorescence was observed when MDBK cells were infected with orf virus and stained with McAb. The result of Western-blot confirmed that the McAb has a specific band with B2L protein, but not with His protein.In summary, there is potentially dangerous of diseases outbreak, since ORFVs and GTPVs are still popular in Jiangsu province. The molecular epidemiological surveillance of ORFV and GTPV and development of detection method based on monoclonal antibody technology was very important for prevention and control of orf and capripox.