Genetic characterization and phylogenetic analysis of Novel Duck-Origin Goose Parvovirus in Anhui Province, Eastern China

Recently, a novel duck-origin goose parvovirus (N-GPV) was reported to cause short beak and dwarsm syndrome in ducks. In this study, we performed complete genome sequencing and analyzed three different duck-derived parvoviruses that infected different breeds of ducks. Phylogenetic trees based on gene sequences indicated that they were classical goose parvovirus (C-GPV), Muscovy duck parvovirus (MDPV), and N-GPV, respectively. Furthermore, potential recombination events were found. These results improve our understanding of the diversity of duck-derived parvoviruses in the Anhui province, eastern China, and provide a reference for the prevention of associated diseases.

China. Liver, kidney, and spleen samples were collected and homogenized in 1 mL of sterile phosphatebuffered saline (PBS; pH 7.2). After centrifugation at 10,000 g for 10 min, freeze-thawing was repeated three times and the supernatant was stored at −80°C until use. Viral DNA was extracted using the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China) according to the manufacturer's protocol. A pair of speci c detection primers for conventional polymerase chain reaction (cPCR) was designed using Primer Premier 5 software (DNASTAR, Madison, WI, USA) according to the reference strain in the GenBank (Accession Number: MH444513.1). The cPCR reaction system was composed of 12.5 μL high-delity enzyme 2× Premix Taq®2.0 (TaKaRa, Dalian, China), 2 μL DNA template, 0.4 μM each of forward and reverse primers, and RNase-free H 2 O up to 25 μL. The ampli ed product was cloned into the pMD19-T vector (TaKaRa), and the recombinant plasmid was sent to General Biological System Co., Ltd. (Chuzhou, Anhui, China), for sequencing. After BLAST analysis of the sequencing results, we designed primers to allow the ampli cation of the complete N-GPV genome sequence. The information regarding the primers and PCR conditions are shown in Table 1. The complete genome sequences of C-GPV and MDPV were ampli ed using the primers previously reported [22,23].
The previously reported complete genome sequences of C-GPVs, N-GPVs, and MDPVs were downloaded from GenBank and used to analyze the variation and phylogenetic relationships between the three detected strains in this study and other duck and goose-related parvoviruses. Sequences of the three detected strains and all referenced strains were aligned using the MAFFT software (http://mafft.cbrc.jp/alignment/software/) and analyzed using the MegAlign 6.0 program (DNASTAR) [24]. A phylogenetic tree based on the complete sequences, NS1, and VP1 was established in 1,000 bootstrap replications using the maximum-likelihood method in the MEGA vX software. The phylogenetic tree of the complete sequences showed that the waterfowl parvoviruses were divided into three branches, namely C-GPV, N-GPV, and MDPV. The three detected strains belonged to these three branches, respectively, which was consistent with the above sequence alignment results. Of note, the phylogenetic trees based on NS1 and VP1 and that based on the whole genome sequences showed different topological structures; however, they all established that the three strains belonged to the three different Anseriform dependoparvovirus 1 (Figure 1). The phylogenetic trees also indicated that the AHAU30 strain found in the Anhui province was most similar to the Sheldrake-origin N-GPV strain recently reported in the Linwu area, forming an independent sub-cluster with other N-GPVs. Next, to investigate whether there was recombination events, the waterfowl parvoviruses were analyzed using the RDP 4.50 software. The results showed that potential recombination events occurred in two strains, AHAU41 and AHAU30. Particularly, two recombination events were found in the strain AHAU41; the major and minor parents of the two events were C-GPV and N-GPV. Additionally, only one recombination event was found in the AHAU30 strain and the major and minor parents of the recombination event were also C-GPV and N-GPV. Simplot analysis showed that the recombinant region in the AHAU30 strain was located in the VP1 (2,681 bp to 3,293 bp) locus, and those in the AHAU41 strain were located in the NS1 (204 bp to 957 bp) and VP1 (3,938 bp to 4,327 bp) loci ( Figure 2). Importantly, the recombination events in the AHAU30 and AHAU41 strains were veri ed using the R, G, M, C, S, and Q methods, and the obtained p values were between 4.860 × 10 -5 and 1.420 × 10 -13 .
Waterfowl parvoviruses include GPV and MDPV, causing higher morbidity and mortality rates in geese and ducks, respectively [12]. In recent years, N-GPV has been found in ducks in some provinces of China, leading to duck SBDS, and causing losses in the duck breeding industry [8,26]. Until now, little is known about the prevalence and evolution of N-GPV in the Anhui province in eastern China. In this study, three types of waterfowl parvoviruses, namely MDPV, C-GPV, and N-GPV were detected in samples collected from three breeds of ducks in different areas of the Anhui province. Our study highlights the diversity of parvoviruses that can infect ducks and provides evidence of the spread of goose parvovirus among waterfowl species. Notably, N-GPV was only widely detected in Cherry Valley and mule ducks [9,26]. However, in the last year, N-GPV was also detected in Sheldrakes. In the current study, N-GPV was again detected in Sheldrakes, demonstrating again that this virus can infect this species [27].
In our study, to explore the phylogenetic relationships between the three detected waterfowl strains and other reference strains, phylogenetic analyses were performed based on the complete genome sequences and on the sequences of NS1 and VP1. The phylogenetic trees exhibited different topological structures, further verifying the results of the sequence alignment. In addition, we found that the N-GPV detected in Sheldrakes formed a separate branch; of note, the diversity of transmission in waterfowl needs further study.
Remarkably, two recombinant strains (AHAU30 and AHAU41) were found in this study with a total of three recombinant events; the parents were N-GPV and C-GPV in all of the events. Previous studies have also reported recombination events between N-GPV and C-GPV, suggesting that such recombination may be widespread. Of note, recombination requires co-infection of the same cell [28]. Therefore, the recombination events identi ed in this study suggested co-infection with C-GPV and N-GPV, which further supports the notion of cross-species transmission of GPV, as previously reported. Importantly, coinfection and cross-species transmission of different GPVs is a challenge to prevent parvovirus infection in ducks and is likely to pose a major threat to the duck industry. Nevertheless, the number of samples in this study and the sample collection location was limited. Therefore, further epidemiological investigation is needed to investigate the prevalence of waterfowl parvovirus in eastern China.
In summary, this study contributes to the better understanding of the diversity of duck-originated parvovirus in the Anhui and Jiangsu provinces and highlights the need for the implementation of parvovirus infections control measures to avoid losses in the duck industry.

Declarations Con ict of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Author contributions
All authors participated in this study. Wang Yong and Sun Jianfei participated in the conception of the experiment, Da Zhang, Xu Guo and Wenhao Shen performed data analysis, and Yongdong Li performed the experiment. The manuscript was completed by Wang Yong and Sun Jianfei.