We assembled the complete genome of FZ91-30 after sequencing the sub-genomic fragments. We found that several amino acid differences between FZ91-30 and virulent isolates may contribute to MDPV attenuation. Here we sequenced the FZ91-30 genome and generated an infectious plasmid clone. To discriminate the vaccine strain from its parental strain FZ91, the attenuated virus is referred to as FZ91-30. FZ91 was passaged 3 times in embryonated Muscovy eggs and in embryonated goose eggs 30 times, resulting in an attenuated virus that was safe and immunogenic to ducklings. FZ91 is a virulent isolate obtained from deceased ducklings that died from an outbreak of “three-week” disease in a farm from Fuzhou city, China.
These attenuated vaccines were prepared by serial passages of the parental derivatives in cell cultures or embryonated eggs. Live vaccines have been utilized in day-old Muscovy ducklings to prevent disease outbreaks. VP1, VP2, and VP3 comprise the viral capsid at a ratio of ~1:1:8. The right open reading frame encodes the capsid proteins VP1, VP2, and VP3 that are generated from the use of different initiation codons and through proteolytic cleavage. Rep proteins can bind to ITR elements and are involved in genome replication, and modulation of the downstream P41 promoter. The left open reading frame encodes the non-structural protein Rep1 as well as several smaller proteins generated after splicing. The ITR also contains cis-acting elements required for rescue, excision from cloning vectors, and packaging. The genome is flanked by inverted terminal repeats (ITR 457 nts in the FM strain) that can form a palindromic hairpin structure to serve as origin of replication. The MDPV genome is a linear, single-stranded DNA molecule of ~5.1 kb. The disease is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. MDPV is the etiological agent of Muscovy duckling parvoviral disease, which is also named “three-week disease” in China. Its closest relative is Goose parvovirus (GPV). Muscovy duck parvovirus (MDPV) belongs to the Dependoparvovirus genus in the Parvoviridae family. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV. The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. 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. 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. 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.
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. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. 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. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. 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 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. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. 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. 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. 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.