Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30

Abstract

Background: Muscovy 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.

Methods: The 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.

Results: The 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.

Conclusions: The 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.

Keywords: Attenuation; Infectious clone; Muscovy duck parvovirus; Rescue; Transfection.

Fig. 1

FZ91-30 5′ inverted terminal repeat (ITR) sequences. The numbers above the sequences denote the nucleotide position. The nucleotide differences relative to the FM strain are shaded with grey. The D sequence is composed of 42 nucleotides, which base pairs with the D′ sequence at the 3′ ITR. The sequences in the bubble regions can exist as two different sequences, termed flip and flop, which reversely complement with each other

Fig. 2

Amino acid sequence alignments of the VP1 proteins (a) and the Rep1 proteins (b) between the FZ91-30 and other pathogenic isolates. Amino acids in common are indicated by dots. Different amino acids are denoted by the numbers above the alignments and shaded in grey

Fig. 3

Experimental strategy used to construct the infectious FZ91-30 MDPV plasmid clone. a The digested MDPV subgenomic fragments were cloned into the pBSKN plasmid respectively and further combined to form the recombinant plasmid pFZ that contained the whole genome of FZ91-30. b Restriction enzyme digestion of pFZ. M: DNA markers. 1: Digestion with SphI. 2: Double digestion with XhoI and BamHI

Fig. 4

Pathogenic changes of the embryo died at day 7 post-transfection of pFZ (a), and haemorrhagical lesions in the head, neck, embryonic body, and legs were observed. In the control group (b), no pathogenic change was observed in the embryo which survived in transfection of the vector plasmid pBSKN till the 12th day

Fig. 5

Discrimination of the rescued virus from the parental strain FZ91-30 using NdeI digestion