Genome Analysis Of A Muscovy Duck Parvovirus Strain And Establishment Of IFA Method

The muscovy duck parvovirus(MDPV) is the causative agent of muscovy duck parvovirus diseases, which is characterized by panting, diarrheal, and markedly reduced body weights. In 2012, muscovy ducklings of about 19-day-old in a Shanghai suburb farm showed clinical symptoms of MDPV. However, the morbidity and mortality rate were much higher than previously reported.7 pairs of primers were designed according to the genomic sequences of MDPV and GPV deposited in GenBank, which were used to amplify the corresponding genes by polymerase chain reaction(PCR). DNAStar software was used to obtain the complete genomic sequence. The nucleotide sequences showed 93.7% homology with MDPV FM. The strain was named SAAS-SHNH. The full genome of the strain was 5061 nucleotides(nt), the inverted terminal repeat(ITR) was 381nt at the 5’and 3’ end of the genome; the left open reading frames(ORF) encodes for nonstructural proteins NS1(512bp-2395bp) and NS2(1040bp-2395bp); the right ORF encodes for structural proteins VP1 (12414bp-4612bp), VP2 (2849bp-4612bp) and VP3 (3008bp-4612bp). Two genetic recombination events have been found when SAAS-SHNH strain was compared with MDPV and GPV which were registered in the GenBank. The second recombination events covered nearly 70.0% of the full-length of VP3 gene.According to the full-length genome of MDPV strain SAAS-SHNH, a pair of primers were designed to amplify the VP3 gene by PCR. After being cloned and sequenced, the VP3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was induced with 1.00mmoL/L IPTG. SDS-PAGE and Western blotting analysis showed the MDPV VP3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with recombinant VP3 protein and MDPV vaccine strain.The detection of cultured MDPV on primary duck embryo fibroblasts(DEF) by IFA. The virus was inoculated to DEF after being passagged for 10 generations in the duck embryos.6-well plates were seeded by DEF cells with a seeding concentration 1×106/mL. The immunofluorescence method(IFA) was established by deciding titers of primary and secondary antibodies. Results:CPE could be observed with inoculated cells, which were longer and thinner. The immunofluorescence signal could be detected by IFA. By fluorescence microscope, the VP3 particles could be observed both in the cytoplasm and nucleus.