| Bovine parainfluenza-3 virus(BPIV3) is assigned in the genus Respirovirus of the subfamily Paramyxovirinae. BPIV3 is an important pathogen of bovine respiratory syndrome(BRDC), which cattle with BPIV3 infection can represent acute clinical symptom and results in significantly economic losses to the global cattle industry annually. So far, three genotypes(A, B and C) of BPIV3 have been tentatively confirmed and these three genotypes have been isolated in many countries worldwide. The epidemiological investigations suggest that the BPIV3 be widespread in China. BPIV3 s were isolated in Heilongjiang, Shandong and Inner Mongolia,whereand the genotypes type A or C BPIV3 was reported.To sequence the complete genome of a BPIV3 genotype C strain isolated from Ningxia region in 2014 and to analyze the evolution, five pairs of primers specific to BPIV3 were designed based on the reference strain SD0835 isolated in China in GenBank. The total RNA of BPIV3 was extracted and used as a template for RT-PCR. The each of PCR products was cloned to pMD®18-T vector, followed that the constructed recombinant plasmid was transformed to DH5α E. coli competent cells, and the selected each of positive clones was subjected to nucleotide sequencing. The sequencing results were assembled using molecular biological software and compared with the reference sequences in GenBank, by which a phylogenetic tree was constructed based on complete genomic sequence. The evolutionary analysis result showed that a BPIV3 strain was successfully isolated and designated as the NX49, of which the complete genome consisted of 15474 nt. The sequence was submitted to GenBank, which the accession No.was designated as KT071671. The characterization of the NX49 demonstrated that it was sensitive to high temperature, low pH and chloroform or diethyl ether. The presence of Mg2+showed no protection against the treatment at high temperature. The Hemagglutination assay test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4℃ and the titer was 1:4.The TCID50 value of sixth passage virus was 108.0TCID50/mL. The NX49 strain shared 99.3%nucleotide identity with the SD0835 strain of genotype C isolated in Shandong province, 82.5%with the NM09 strain of genotype A isolated in Inner Mongolia, and 81.4% with the Q5592 strain of genotype B isolated in Australia. The NX49 strain was clustered with the SD0835 strain.The geographic distribution and circulating period of the NX49 indicated that genotype C BPIV3 widely circulates in China.In addtion, BPIV3 standard strain BN-1 was chosen to build up a BPIV3 plasmid reverse genetics system and RT-PCR was used to segmented amplify full-length genome of BPIV3,fragments were cloned intothe expression vector rpCI-neo using the selected restriction endonuclease. The hammer head shape ribozymes(HamRz) and hepatitis delta virus ribozymes(HdvRz) were introduced to the 3ˊ and 5ˊ flank of the viral genome, respectively. The plasmid harbouring full-length genomic sequencewas used as template to amplify N, P and L fragments, followed that each of fragments was cloned into expression vector pCI-neo, which Kozak sequence was introduced to the forward primer to enhance the transcription efficiency.Each of helper plasmids was transfected to MDBK cells and identified by RT-PCR and indirect immunofluorescence assay. The results showed that full-length of BN-1 cDNA and helper plasmids which under control of the CMV promoter were successfully constructed. The transcription or expression of each of helper plasmids in MDBK cells was confirmed. This study will contribute to the research of gene function and live carrier vaccine against PRDC due to BPIV3 infection. |
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