Viral isolation and passage in embryonated goose eggs
Three organ tissue samples were collected from diseased cherry valley ducks showing typical SBDS between 2016 and 2019 in Shandong Province, China. The three samples were positive in PCR detection using specific GPV primers. Each sample was used to inoculate five susceptible 9-day-old embryonated goose eggs. The result showed that 1, 2, and 1 of five embryonated goose eggs died between 234 h and 288 h post-inoculation. The three viral isolates were successfully passaged in another five 9-day-old embryonated goose eggs and killed all the goose embryos in a shorter time (between 112 h and 190 h) post-inoculation. Hemorrhagic lesions in the head, neck, and legs typical of parvoviral infection were observed (Fig. 1). The three NGPV isolates were named SDHT16, SDJN19, and SDLC19.
NGPV propagation in cherry valley duck embryos and ELD50 calculation
Strain SDHT16 and SDJN19 were successfully passaged in 9-day-old embryonated cherry valley duck eggs (Fig. 1). Strain SDHT16 killed 16 duck embryos between 106 h and 197 h (averaging 150 h) post-infection, and strain SDJN19 killed 16 duck embryos between 69 h and 163 h (averaging 104.6 h). Statistical analysis indicated that the mean death time between the two NGPV strains was significantly different (P < 0.05) (Fig. 2).
Furthermore, the calculation of the medium lethal egg doses (ELD50) was performed in embryonated cherry valley duck eggs. The ELD50 values of strain SDHT16 and SDJN19 reached 5 × 102. 5/mL and 5 × 105.4/mL, respectively, demonstrating that strain SDJN19 has a better propagation capability in embryonated cherry valley duck eggs than strain SDHT16.
Genome sequencing and homology comparison
The whole genomes of three NGPV isolates were amplified by PCR and sequenced. The genome of strain SDHT16 is 5050 bases long, which is four bases shorter than that of strain SDJN19 and SDLC19. Nucleotide deletions or insertions occurred in ITR with no exception. The three NGPV strains shared 98.9%–99.7% similarities at the genomic level but displayed slightly lower nucleotide homologies (95.2%–96.1%) with the classical GPV strains (strain B, 82-0321, 06-0329, YZ99-6, and LH). The genome sequences of three NGPV strains were deposited in GenBank with accession numbers MN356043, MN356044, and MN356045.
Sequence comparison of the Rep1 proteins
The Rep1 gene of the three NGPV strains (SDHT16, SDJN19, and SDLC19) was composed of 1884 nucleotides, coding 627 amino acids. Homology comparison indicated that the Rep1 gene of three NGPV strains shared 99.3%–99.7% similarities between each other but displayed 96.0%–96.5% similarities with the Chinese GPV strains (82-0321, 06-0329, YZ99-6, and LH) and 96.8%~96.9% similarities with the European GPV strain B (Figure 3). Amino acid alignments among the six NGPV strains (SDHT16, SDJN19, SDLC19, SDLC01, JS1, and QH15) and the classical GPV strains indicated that 12 amino acid mutations were produced in the NGPV strains compared with the classical GPV strains (Table 3). The altered amino acid sites were I50T, K131R, A140S, A350V, V468I, E498R, R553K, N555T, E573Q/K, D594Y, N609D, and V617A.
Sequence comparison of the VP1 proteins
The VP1 gene of three isolates was composed of 2199 nucleotides, coding for 732 amino acids. Homology comparison indicated that the VP1 proteins of three NGPV strains shared 99.1%–99.7% nucleotide similarities between each other but showed 94.4%–95.1% similarities with the Chinese GPV strains (82-0321, 06-0329, YZ99-6, and LH) and 96.0%–96.6% similarities with the European GPV strain B (Figure 3).
Amino acid alignments of the VP1 proteins among the six NGPV strains (SDHT16, SDJN19, SDLC19, SDLC01, JS1, and QH15) and the classical GPV strains indicated that compared with the Chinese GPV strains, the six NGPV strains harbored 16 common amino acid alterations, among which nine mutated amino acid sites were identical with the European strain B (Table 4). The 16 altered amino acid sites were H28Q, T35S, A41D, Q89L, D114H, Q116H/Y, D142E, V144I, A180V, L207M, A/T210S, S450N, A524G, L537I, K575R, and D703N.
Sequence comparison of ITRs
The ITRs of three NGPV strains (SDHT16, SDJN19, and SDLC19) shared 98.1%–100.0% nucleotide sequence homologies but displayed 93.0%–94.0% homologies with the classical GPV strain LH. The alignments of ITR sequences revealed that strain SDHT16, SDJN19, and SDLC19 have additional two, four, and four nucleotide insertions in the stems of the palindrome region, respectively, compared with the classical GPV strain LH (Fig. 4).
Phylogenetic tree construction
Because only partial sequences of the European SBDS-related NGPV strains were sequenced, a 427-bp fragment at the N terminus of the VP1 gene was used to construct the phylogenetic tree. The results revealed that six Chinese SBDS-related NGPV, including SDHT16, SDJN19, SDLC19, SDLC01, JS1, and QH15, intimately clustered together and grouped with the European SBDS-related NGPV strains (D697/3/06 and D146/02), forming a separate branch (Fig. 5). The four Chinese GPV strains (LH, YZ99-6, 82-0321, and 06-0329) closely clustered together and grouped with two European GPV strains (VG32/1 and B), forming another separate branch. On the basis of the results of phylogenetic analysis, the Chinese SBDS-related NGPV probably evolved from a common ancestor with the European SBDS-related NGPV.