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Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses.

ABSTRACT: A restriction fragment length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses (GPV and MDPV) was developed based on comparison of the NS gene of GPV and MDPV. Both GPV and MDPV genomic DNA can be amplified with 641 bp using the specific PCR primers. The PCR fragments can be cut into 463 bp and 178 bp only in the case of MDPV-derived PCR products, whereas the GPV-derived PCR products cannot. The method established in this study can be used to differentiate GPV and MDPV with high specificity and precision, by using a direct PCR kit and QuickCut enzyme, as quickly as conventional PCR.

SUBMITTER: Wan CH

PROVIDER: S-EPMC4905843 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses.

Wan Chun-He CH Chen Hong-Mei HM Fu Qiu-Ling QL Shi Shao-Hua SH Fu Guang-Hua GH Cheng Long-Fei LF Chen Cui-Teng CT Huang Yu Y Hu Kai-Hui KH

The Journal of veterinary medical science 20160205 5

A restriction fragment length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses (GPV and MDPV) was developed based on comparison of the NS gene of GPV and MDPV. Both GPV and MDPV genomic DNA can be amplified with 641 bp using the specific PCR primers. The PCR fragments can be cut into 463 bp and 178 bp only in the case of MDPV-derived PCR products, whereas the GPV-derived PCR products cannot. The method established in this study can be used to d ...[more]

PMID: 26854108

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Project description:Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are important etiological agents for Muscovy duck parvoviral disease and Derzsy's disease, respectively; both of which can cause substantial economic losses in waterfowl industry. In contrast to GPV, the complete genomic sequence data of MDPV isolates are still limited and their phylogenetic relationships largely remain unknown. In this study, the entire genome of a pathogenic MDPV strain ZW, which was isolated from a deceased Muscovy duckling in 2006 in China, was cloned, sequenced, and compared with that of other classical MDPV and GPV strains.The genome of strain ZW comprises of 5071 nucleotides; this genome was shorter than that of the pathogenic MDPV strain YY (5075 nt). All the four deleted nucleotides produced in strain ZW are located at the base-pairing positions in the palindromic stem of inverted terminal repeats (ITR) without influencing the formation of a hairpin structure. Recombination analysis revealed that strain ZW originated from genetic recombination between the classical MDPV and GPV strain. The YY strain of MDPV acts as the major parent, whereas the virulent strains YZ99-6 and B and the vaccine strain SYG61v of GPV act as the minor parents in varying degrees. Two recombination sites were detected in strain ZW, with the small recombination site surrounding the P9 promoter, and the large recombination site situated in the middle of the VP3 gene. The SYG61V strain is a vaccine strain used for preventing goose parvoviral disease. This strain was found to be solely involved in the recombination event detected in the P9 promoter region. Phylogenetic analyses between strain ZW and other classical strains of MDPV and GPV were performed. The results supported the in silico recombination analysis conclusion.MDPV Strain ZW is a novel recombinant parvovirus, and the bulk of its genome originates from the classical MDPV strain. Two virulent strains and a vaccine strain of GPV were involved in the recombination process in varying degrees.

Project description:BackgroundMuscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood.MethodsThe FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus.ResultsThe genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain.ConclusionsThe amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

Dataset Information

Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses.

Publications

Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses.

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