Differential Diagnosis And Genomic Characterization Of Waterfowl Parvoviruses

Waterfowl parvoviruses cause diseases with high mortality and morbidity to goslings and Muscovy ducklings,which can be divided into two groups:the Muscovy duck parvovirus(MDPV)and the goose parvovirus(GPV).Both MDPV and GPV were firstly identified in China.MDPV and GPV have single-stranded DNA genomes of about 5.1 kb in length and contain two major open reading frames(ORFs),the left ORF encodes the nonstructural(NS)proteins which is involved in viral replication and regulatory functions.However,the right ORF encodes the capsid proteins(VP).MDPV and GPV shared more than 80.0%nucleotide identity,and also related antigenic characteristic,which was evaluated by cross-neutralization test.Based on the present study about waterfowl parvoviruses researches,the variations in genotype,host pathogenicity and virulence were observed,which causes great losses to waterfowl breeding industry.In this study,alignment analysis for the NS gene of MDPV and GPV derived from the GenBank,only MDPV isolates have EcoR I restriction site(GAATTC)at position 973-978 of NS coding regions,while GPV have not.A primers-pair was designed and used to amplify the MDPV and GPV with the length of 641 nt.The MDPV-derived PCR products can be digested by EcoR I enzyme into two fragments of 463 nt and 178 nt in length,respectively.However,the GPV-derived PCR products cannot be digested by EcoRI enzyme and should potentially remain 641 nt in length.The method described in this study was shown to be specific,sensitive and repeatable and to be simple and low cost compared with conventional PCR method,when combined with tissue direct PCR kit and quickcut EcoRI enzymes.Then,a SYBR Green I dye based real time fluorescence quantitative PCR method was established to distinguish GPV and MDPV,using only one primers-pair.The primers were designed with Oligo 7 software targeting the NS gene differences,which have significant difference GC contents between GPV and MDPV genomic sequences,leading different temperature peak(Tm)at dissolution curve analysis after the real time PCR reaction.Compared with the PCR-RFLP results,all PCR-RFLP positive samples were tested positive by the establishe real time fluorescence quantitative PCR method,sharing 100.0%co-relationship.Twenty-two waterfowl parvoviruses were isolated with ten GPV isolates and twelve duck parvoviruses(four traditional MDPV and eight novel MDPV)by using the 10-day-old embryonated Muscovy duck eggs from the Muscovy farm had no previous history with MDPV or GPV infections.In order to recognize the waterfowl parvoviruses genomic characterization,eleven GPV isolates(including four goose-origin,five Muscovy duck-origin,one Anser cygnoides-origin and one GPV vaccine strain),five classical MDPV isolates(including four Muscovy duck-origin and one MDPV vaccine strain),eight novel duck parvovirus(N-MDPV)isolates were sequenced and analysis.These data found that the genome length of eleven sequenced GPV isolates among 5050 to 5106 nucleotides,sharing homology of nucleotide was 93.4%-99.4%.Polygenetic analysis based on the genome,NS and VP gene showed the GPV isolated with complex and diversity relationship,and there has virulent GPV isolates circulating in China.The genome length of five sequenced classical MDPV isolates among 5017-5133 nucleotides,sharing homology of nucleotide was 98.7%-99.3%with MDPV respective strains(FM),81.9%-82.2%with GPV respective strains(B),polygenetic analysis based on the genome found that novel Muscovy duck parvoviruses co-circulating in China.Then,eight novel duck parvoviruses were sequenced with the genome length 4977 to 5129 nucleotides,sharing homology of nucleotide more than 98.0%with each other,whereas sharing homology of nucleotide was 90.8%-95.0%with MDPV respective strains,82.2%-85.3%with GPV respective strains.Two breakpoints were found had recombinant event between MDPV and GPV using Simplot analysis software.Birds pathogenicity tested showed some resistant Muscovy ducks shared with short beak and dwarfism syndrome(SBDS).Combined with the characteristics of the genome,recombination analysis,genetic evolution analysis and pathogenicity difference,the newly identified N-MDPV shared different with waterfowl parvoviruses reported preciously,novel duck parvovirus Species should be add as a member of the parvovirus genus under Familiy Parvoviridae.In order to study the pathogenesis of N-MDPV,according the N-MDPV genomic stranded characterization—single genomic stranded DNA easily annealed form with double stranded,single-stranded were obtained and "A" was add at the terminal of the genome,then using TA cloning methodology cloned into the plasmids pBluescript ?KS(+),which constructed plasmids pBSK-N-MDPV containing the entire genome of N-MDPV.Site-direction mutation technique were used to mutated EcoR ? restriction site to BamH ? restriction site,which the mutations are nonsense mutation.Double genetic selection markers at the full length infectious clone plasmids pBSK-N-MDPV-BamH I of novel duck parvovirus were constructed,then co-transfection into Muscovy duck embryos to harvest the rescued virus r-N-MDPV,after double eyzmes(EcoR ? and BamH ?)digestion,immunofluorescence test and sequences analysis,the N-MDPV infectious plasmids clone were successed.These data lay good foundation for the next step transfection to the experimental animals and cells,and provide research platform to research the novel duck parvovirus recombination mechanism.