The Isolation And Characterization Of Caprine Parainfluenza Virus 3 And Prokaryotic Expression Of The Nucleocapsid Protein

BPIV3 can cause severe respiratory disease in bovine.It also has a high rate of morbidity and mortality,causing huge losses to the cattle breeding industry.But the virus infection in goats were rarely studied and reported in the world.In this research,a Caprine parainfluenza virus 3(CPIV3)was isolated,identificated and purified from goats’ clinical material suffered severe respiratory diseases.Using the traditional cell culture,plaque assay methods,RT-PCR identification of viral nucleic acid,a PIV3 virus strain was isolated and identified in MDBK cells with cytopathotic effects(CPE).Through the pathogen study and the the genome sequencing,we found that the virus,characteristics was different from other PIV3,named it as JS2013.For further exploring about the pathogenic of CPIV3 strain JS2013,the guinea pigs as PIV3 susceptible animals were infected with the virus isolate to study on the virus replication and pathological injury in the experimental animal tissues.The noted clinical symptoms were sneezing,eye secretion and dyspnea in the infected guinea pigs at 2 days post-infection(PI).The virus replicated in the blood of the infected animals and could be positively detected from 3 days PI and kept for 7 days PI.Macroscopic appearances of the lungs from infected guinea pigs were almost complete consolidation and swelling.The histopathologic examinations showed that alveoli septa thickening,disappearance of alveolar architecture and inflammatory cell infiltration were seen in the lungs of the diseased.The infected guinea pigs produced the special HI antibodies against CPIV3 in 5 days PI.These results showed that CPIV3 strain JS2013 had a strong pathogenicity on guinea pigs,and guinea pig might be one good experimental animal model for CPIV3.The N gene segment of CPIV3 strain JS2013 was partially amplified by reverse transcription polymerase chain reaction(RT-PCR)with a pair of primers,which was designed according to the genome sequence of several different PIV3.The RT-PCR products were cloned into pET-32a(+)vectors and successfully constructed the expression plasmid(pET-32a-N1).After transformation into BL21,the expressed recombinant N protein with molecular weight of 35.6 ku was confirmed by SDS-PAGE and Western-blot with His MAb,and mainly expressed in the inclusion bodies.Balb/c mice immunized with the purified expressing N protein produced the strong antibody response,and the mouse polyclonal antibodies could specificly react with the protein in Western-blot.In addition,JS2013 infected MDBK cells were positively stained with the antibodies by indirect fluorescence assay(IFA).These results indicated that the expressed protein possessed good immunogenicity and reactionogenicity.The results lay a foundation for the development of diagnostic tests and vaccines in the future to establish a variety of CPIV3.