Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe

Abstract

Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.

Isolation of a goose parvovirus from swan and its molecular characteristics

“…Of note, the ITR of the swan isolate showed 100% identity with that of 82-0321 isolate. Although the nt deletions in ITR were observed also in other GPV isolates (Shien et al, 2008), we found that the nt deletion regions were mainly related to the sequences "TCCGGT" and "ACCGGA" as indicated in Fig.2 with red and purple squares respectively. These short sequences might serve as cis-regulatory elements, however, little is known about the role of these nt deletions or short repeats of complementary sequences in gene transcription and viral replication for GPV.…”

“…When compared with isolate 82-0321, the swan isolate had two mutations P485T and K523E in VP1. Since positions 485 and 523 are near to the two potential receptor binding sites (Opie et al, 2003;Shien et al, 2008), P485T and K523E mutations might be involved in the virus receptor binding and host tropism. Phylogenetic analysis also showed that the VP1 of the swan isolate and the other Chinese mainland isolates were clustered into the different groups as described in Fig.…”

“…The parameters of the PCR were as follows: one cycle of 95°C for 5 min, followed by 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, and then one cycle of 72°C for 10 min. For sequencing the whole genome of the GPV, three primes P1 (5΄-TT CAG CTG CTC ATT GGA GGG TT-3΄), P2 (5΄-TT CTCGAG GCG TGG TCA ACC TAA CA-3΄) and P3 (5΄-GCA TGC CGC GCG GTC AGC CCA ATA-3΄) were synthesized according to Shien (Shien et al, 2008). P1 and P2 primers annealing to the ends of the whole genome amplified the two inverse identical 203 bp fragments (designated as PCR II) (Shien et al, 2008).…”

“…For sequencing the whole genome of the GPV, three primes P1 (5΄-TT CAG CTG CTC ATT GGA GGG TT-3΄), P2 (5΄-TT CTCGAG GCG TGG TCA ACC TAA CA-3΄) and P3 (5΄-GCA TGC CGC GCG GTC AGC CCA ATA-3΄) were synthesized according to Shien (Shien et al, 2008). P1 and P2 primers annealing to the ends of the whole genome amplified the two inverse identical 203 bp fragments (designated as PCR II) (Shien et al, 2008). P3 primer located in nt position 198 to 221 and 4,885 to 4,908, amplified a 4,711 bp fragment (designated as PCR III) (Shien et al, 2008).…”

“…P1 and P2 primers annealing to the ends of the whole genome amplified the two inverse identical 203 bp fragments (designated as PCR II) (Shien et al, 2008). P3 primer located in nt position 198 to 221 and 4,885 to 4,908, amplified a 4,711 bp fragment (designated as PCR III) (Shien et al, 2008). Fifty μl PCR II or PCR III mixtures contained water, 10x buffer, 2.5 mmol/l dNTP, 25 pmol primers, 2 μl of template DNA and 0.5 μl of LA Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd., China).…”