Study On Molecular Detection Techniques Of Caprine Arthritis Encephalitis Virus

Caprine arthritis and encephalitis (CAE) caused by Caprine arthritis encephalitis virus (CAEV) is one of the most destructive and economically important viral disease of the goat industry worldwide. The disease is commonly associated with polyarthritis, interstitial pneumonia, mastitis, and progressive weight loss in adults, while the newborn and young kids often suffer from CAE-induced encephalitis, accompanied with a high mortality rate.In this study, the whole genome of CAEV89-GB1026 strain was cloned and sequenced, and the molecular characterization was determined. Additionally, a rapid detection assay based on loop-mediate isothermal amplification (LAMP) has been developed for detecting CAEV proviral DNA. At last, the p25 gene was cloned into expression vectors and expressed in E.coli, and the indirect ELISA was developed for detection of antibody in serum to CAEV.Five DNA fragments covering the whole genome of CAEV89-GB1026 strain were amplified from total DNA extracted from CAEV infected GSM cell by employing polymerases chain reaction, and were cloned into pGEM-Teasy vector respectively. Then the whole genome sequence was determined by sequencing and assembling these fragments. CAEV89-GB1026 strain has a genome size of 9065nt, with a 453nt of 5'LTR, followed by three structural gene (gag, pol and env), two regular gene (rev and tat) and an accessory gene (vif), and a 308nt 3'LTR. Sequence comparison showed the complete nucleotide sequence of CAEV89-GB1026 strain shared 99.6% identity with gansu strain (AY900630), but only 73.0%~73.5% identity with Italy isolates (EU293537,GQ381130). The homology tree was generated based on gag gene, showed that CAEV89-GB1026 strain belongs to SRLV subtype B2. The secondary structure and epitopes of structural proteins were analyzed, several function domains were identified, CAEV and MVV shared several epitopes in gag protein.A rapid detection method was developed by employing LAMP assay, a set of five primers was designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs, had no cross reaction with the negative control. Amplification was monitored with the Loopamp real-time turbidmeter, several copies of CAEV proviral DNA can be detected in 36 min, which is 100 fold more sensitive than PCR. 68 clinical samples were tested using AGID, PCR and LAMP assay and the positive rates were 38.2%, 41.2% and 50%, respectively.The biochemical characteristics of the capsid protein (P25) and presumptive P25 fusion proteins were analyzed by bioinformatics analysis method, and the recombinant P25 was obtained by constructing different expression vector. The P25 protein was expressed as a fusion protein with glutathione-S-transferase (GST) in E.coli BL21 by using prokaryotic expression vector pGEX-6P-1, the GST-P25 was obtained by a single step of affinity chromatography using Glutathinione Sepharose 4B. The pET28-p25 expression vector was constructed using pET28a vector, the recombinant protein (His-P25) with his-tag located in N and C-terminal was expressed in E coli BL21(DE3), and was purified through denaturizing and refold of inclusion bodies. The recombinant proteins were analyzed by Western blot, results showed that the recombinant proteins could specifically react with sera from CAEV infected goats, indicating that the recombinant proteins shared good immunogenicity. An indirect ELISA was developed for detection of antibody in serum to CAEV, using purified His-P25 as coating antigen.