Molecular characterization of a novel Muscovy duck parvovirus isolate: evidence of recombination between classical MDPV and goose parvovirus strains

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Goose parvovirus; Inverted terminal repeats; Muscovy duck parvovirus; Recombination.

Fig. 1

The strategy for cloning the entire genome of strain ZW. a. Restriction endonuclease digestion of the extracted genome of strain ZW. 1. The annealed genomic dsDNA of about 5.1 kb; 2. NcoI digestion of the genome resulting in 3.1-, 1.3-, and 0.7-kb DNA bands; M: 1-kb DNA ladder. b. Schematic diagram of the experimental strategy for cloning three sub-genomic fragments of strain ZW

Fig. 2

Alignment of inverted terminal repeat (ITR) sequences at the 5′ terminus of the genome of strain FM, YY, and ZW of MDPV. Dashes denote nucleotide deletions. The bubble region of ITR comprises of 45 nucleotides and is indicated by open circles above the letters. The nucleotide differences between three strains are shaded grey. The nucleotides constituting the D region are italicized and underlined, which complement the D′ sequences at the 3′ ITR. The numbers above the alignment denote the nucleotide’s position

Fig. 3

Recombination analysis using Simplot 3.5.1 and RDP 3.4.1. a. Similarity plot analysis of full genome sequences of strain ZW, B, SYG61v, YZ99–6, and YY using Simplot. Bootscan analysis was executed with the following parameters: 1000 bootstrap replicates, sliding window of 200 bp, and step size of 20 bp. Strain ZW was used as query. b. Recombinant events that occurred in strain ZW were detected by RDP. The first recombination breakpoint began at nucleotide position 424 and ended at nt position 615. The second recombination breakpoint began at nucleotide position 3120 and ended at nt position 4246. In these recombinant events, strain YY is the major parent. SYG61v is the sole minor parent in the first recombinant site

Fig. 4

Alignment of partial nucleotide sequences spanning from downstream of ITR to the P9 promoter. Identical nucleotides relative to the SYG61v strain are denoted by dots. The recombinant strain ZW shares 100% nucleotide homology with the vaccine strain SYG61v

Fig. 5

Phylogenetic analysis of strain ZW and other 11 MDPV and GPV isolates. These isolates have complete Rep1 and VP1 gene sequences deposited in GenBank. The neighbor-joining method in MEGA 6.0.6 with 1000 bootstrap replications was used for construction of phylogenetic trees. Phylogenetic trees was constructed based on the C-terminal 1774-bp fragment of the Rep1 gene (a), the N terminal 694-bp (b) and the C-terminal 405-bp (c) fragments of the VP1 gene, and the middle 1100-bp fragment of the VP3 gene (d)

Fig. 6

Antigenic comparison between the recombinant strain ZW and the classical GPV and MDPV strains by agar gel precipitation (AGP) tests. A monoclonal antibody (mAb) against GPV was used in the AGP tests. A 1:20 dilution of this mAb was further diluted by two-fold series in phosphate buffered saline (pH 7.4) and placed in the outer wells surrounding the central well containing antigen. The classical GPV strain LH (a), the recombinant strain ZW (b), and the classical MDPV strain YY (c) were used as the AGP antigens, respectively