Reverse Genetics System Of Bovine Parainlfuenza Virus3NM09Strain

Bovine respiratory disease complex (BRDC) is considered as the most significant illness affectedcattle industry,which can cause huge economic losses in cattle industry worldwide yearly It is mainlycaused by bovine parainfluenza virus3(BPIV3), bovine viral diarrhea virus (BVDV) and bovinerespiratory syncytial virus (BRSV).BPIV3is a long-recognized, currently underappreciated, endemicinfection in cattle populations. The clinical presentation of bovine infections with BPIV3can beconsiderably, ranging from asymptomatic infections to severe respiratory illness. In the majority ofcases, where BPIV3is implicated in disease, mild clinic signs characterized by coughing, fever andnasal discharge. In some instances infected with BPIV3, which can contribute to tissue damage andimmunosuppression, and result in severe bronchopneumonia from secondary bacterial infections.BPIV3is an enveloped, non-segmented, negative-sense RNA virus within the genus respirovirus.The genome of BPIV3encodes six proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein(M), Fusion protein(F), hemagglutinin-neuraminidase protein(HN),and large protein (L). The NP, P, Lproteins and the viral genomic RNA compose a ribonucleoprotein complex (RNP), which constitute thetemplate for viral gene expression and replication by the virus polymerase. Currently, reverse geneticshas become one of the most important means in molecular virology research. With the development ofreverse genetics techniques, it can be easy to understand the relationship between genome structure andfunction of RNA viruses, the interactions between viruses and hosts, the mechanism of virustranscription and replication. Reverse genetics system (RGS) of negative strand RNA virus make theBPIV3to be the most valuable lived virus expression vector. As a vaccine vector, BPIV3has a broadapplication space and great economic significance.In2009, one BPIV3strain, NM09, was isolated from a sick cattle tissue on MDBK cells in Chinaand then eleven pairs primer were designed for the amplication of the complete genome of BPIV3NM09strain. Eleven fragments covering the whole genome were obtained by RT-PCR, and they werecloned into Blunt vector and sequenced. Nucleotide phylogenetic analysis of partial matrix (M) gene,hemagglutinin-neuraminidase (HN) gene and the large protein (L) gene of the NM09isolate showedthat this viruse was a novel member of the genus respirovirus of the paramyxovirinae subfamily andmay be grouped into genotype A. Besides, Based on analysis of complete genome sequence, weconfirmed the distribution of restriction enzyme sites in the genome and designed seven pairs primer toamplicate the complete genome of NM09, seven cDNA fragments covering the whole genome wereobtained by RT-PCR, and cloned into Blunt vector and in turn linked together by restriction enzymedigestion and ligation. Eventually, a full-length cDNA clone, named pcDNA3.1-NM09, of BPIV3NM09strain was successfully constructed. The plasmid contains full-length viral cDNA flanked byhammerhead (HAM) ribozyme and hepatitis delta (HDV) ribozyme sequences, under regulation of thecytomegalovirus (CMV) promotor. BSR Cells were transfected with pcDNA3.1-NM09, pcDNA3.1-NP,pcDNA3.1-P, pcDNA3.1-L using liposome. Fourty eight hours after transfection, the transfected supernatants and BSR cells through freezing and thawing were passaged on MDBK cells for viruspropagation. The recombinant virus were carried out by CPE of MDBK cells, PCR and RT-PCR. Theresults suggested that a reverse genetic system of NM09strain of BPIV3was successfully established insome degree. This work provided the basis for the development of a novel live viral vector vaccine toprevent BRDC.