key: cord-297679-swmb19ty authors: Wang, Yong; Guo, Xu; Zhang, Da; Sun, Jianfei; Li, Wei; Fu, Ziteng; Liu, Guangqing; Li, Yongdong; Jiang, Shudong title: Genetic and phylogenetic analysis of canine bufavirus from Anhui Province, Eastern China date: 2020-10-20 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2020.104600 sha: doc_id: 297679 cord_uid: swmb19ty Bufavirus is a novel virus associated with canine gastroenteritis. Three strains of bufavirus were first detected in dog feces collected from Anhui province in Eastern China. The near-complete genome sequences were amplified. Sequence alignment showed 98.3–99.5% homology between the three bufavirus strains and reference strains. Phylogenetic analysis showed the distributed viruses forming a cluster of close relationships. Selective pressure analysis of the VP2 region indicated that the canine bufavirus (CBuV) was mainly subject to negative selection during evolution. The negative selection site was located on the residue of B-cell epitopes, indicating minimal change to the virus's immunogenicity. Since this is the first report of CBuV circulating in Anhui Province, this study will provide further understanding of the phylogenetic and molecular characteristics of CBuV and serve as a reference for prevention and vaccine development. Members of the family Parvoviridae are common pathogens, which cause a wide range of animal diseases (Lau et al., 2012) Bufaviruses (BuV) are part of the Protoparvovirus genus (Hargitai et al., 2016; Huang et al., 2020) . It is a small, non-enveloped, single-stranded DNA virus with a genome size of 4.5-4.8 kb with complex hairpin structures at the 5' and 3' ends. BuV also contains two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes non-structural protein, and ORF2 encodes capsid protein (Sun et al., 2019) . In 2012, it was discovered in the fecal samples of children with diarrhea in Burkina Faso (Phan et al., 2012) . Subsequently, BuV was found in wild shrews, megabats, wild rats, pigs, dogs, and cats (Diakoudi et al., 2019; Huang et al., 2020; Martella et al., 2018; Sasaki et al., 2016; Sasaki et al., 2015) . In 2018, a virus with a close genetic relationship to the human bufavirus (HuBuV) was detected in dogs with either gastroenteric or respiratory disease in Italy; it was named canine bufavirus (CBuV) (Martella et al., 2018) . In China, the CBuV was first detected in Shanghai, causing a high infection rate in dogs (Li et al., 2019) . J o u r n a l P r e -p r o o f Presently, the distribution of CBuV has only been reported in Italy and China (Di Martino et al., 2020; Li et al., 2019; Martella et al., 2018; Sun et al., 2019) , and its genetic characteristics and pathogenicity are poorly understood. The primary symptom caused by a member of Protoparvovirus is diarrhea in carnivore (Chaiyasak et al., 2020; Piegari et al., 2020) . Recent studies have shown a positive correlation between CBuV and diarrhea, and CBuV DNA was also detected in the serum sample of dogs with gastroenteritis (Li et al., 2019) . In terms of genetic and phylogenetic characteristics, a report has shown that the potential heterogeneity of CBuV and recombination may be a factor in its evolution (Di Martino et al., 2020) . As mentioned, the CBuV was found in Shanghai, China with a hige prevalence. The prevalence of the virus in Anhui province, which has a close relationship with Shanghai in terms of trade, is unknown. To this end, fecal samples from different cities in Anhui province, eastern China were collected in this study to explore the molecular and phylogenetic characteristics of CBuV. The study reveals the epidemic status in Anhui province and related molecular characteristics of CBuV, which provide significant reference for studies on the evolution and epidemiology of CBuV. dogs (>1 year old). Fifty-two fecal samples (31 adult dogs and 21 puppies) were from healthy dogs, and 68 were from puppies with diarrhea. The fecal samples were collected in sterile centrifuge tubes using rectal swabs and stored at −80°C until used. The samples were dissolved in 10% phosphate-buffered saline and mixed in an oscillating manner. The mixture was centrifuged at 10,000× g for 10 min, and the supernatant was collected. CBuV was detected through conventional PCR (cPCR), as previously described (Martella et al., 2018) . The primers used for detecting canine parvovirus (CPV) were (Table 1 ). The PCR products were visualized through agarose gel electrophoresis and then verified by sequencing. The specific primers used to amplify the near-complete genome were designed from other sequences in GenBank (accession number: MK404087.1) using the Primer Premier 5 software (DNASTAR, Inc.; Table 1 ). PCR products were purified using a DNA purification kit (TIANGEN Biotech) following the manufacturer's instructions. After ligation of the PCR products into the pMD19-T vector (TaKaRa Bio Inc.), the recombinant plasmids were sent to Sangon Biotech Co Ltd. (Shanghai, China) for sequencing. Each plasmid was sequenced three times. Near-complete genomes of the CBuV strain obtained for this study were splined together using SeqMan software (DNASTAR). ORFs were identified using the NCBI ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Sequence identity was analyzed with the MegAlign 6.0 program (DNASTAR Inc.) by aligning nucleotide and amino acid sequences via the MAFFT method (Katoh and Standley, 2013) . Phylogenetic analysis was performed using PhyloSuite software (Zhang et al., 2020) . Maximum likelihood phylogenies were inferred using IQ-TREE for 5,000 ultrafast bootstraps (Minh et al., 2013; Nguyen et al., 2015) and Shimodaira-Hasegawa-like approximate likelihood-ratio test (Guindon et al., 2010) . ModelFinder selected the best substitution model (Kalyaanamoorthy et al., 2017 (Table 3) . Interestingly, a comprehensive analysis of pressure selected B-cell epitopes revealed that codons 240, 313, and 380 were located on the predicted B-cell epitopes (Fig. 2 ). Bufavirus is a potential enteric pathogen that causes diarrhea in children (Altay et al., 2015; Chieochansin et al., 2015) . CBuV was first found in canines with gastrointestinal and respiratory diseases (Martella et al., 2018) . In Italy, the positive rate was 7.7% (16/207) (Di Martino et al., 2020) . In China, CBuV has been found in Shanghai, Guangxi province and Henan province, and the positive rates were 42.15% (51/121), 2.5% (5/200), and 1.74% (2/115), respectively (Li et al., 2019; Shao et al., 2020; Sun et al., 2019) . In this study, three positive samples were detected, and the positive rate was 2.5% (3/120) in Anhui province. Positive rates in other parts of China are significantly lower than those in Shanghai, indicating that CBuV prevalence may have regional differences.Further studies are needed to confirm the significance of this difference. In previous reports from China and Italy, the presence of CBuV was found in J o u r n a l P r e -p r o o f More pathogenicity studies are required to clarify whether CBuV plays a major role in diarrhea in dogs. CBuV homology was compared with the references, and the results showed a high homology. Phylogenetic analysis, based on the nearly complete sequencing of the VP2, indicated that CBuV formed a unique cluster, and the three isolated strains were closely related to other reference strains. Compared with HuBuV with three distinct branches, CBuV was conservative in evolution (Yahiro et al., 2014) . As a member of the genus Protoparvovirus, CBuV was not closely related to CPV in the phylogenetic tree, which is consistent with the homology comparison between CPV and CBuV mentioned above. In addition, genetic heterogeneity has been recently reported to be found in the region downstream, where there may be recombination. This finding suggests that recombination may play a role in the evolution of CBuV. In this study, no significant recombination event was found, which may be due to geographical differences or limited genetic information. Hence, the role of recombination in CBuV evolution needs more attention. In amino acid alignment, we found relatively higher amino acid mutations in VP2 than in NS1. To elucidate whether external selective pressure was associated with J o u r n a l P r e -p r o o f Journal Pre-proof these mutations, selection pressure analysis was performed by analyzing existing sequences. The results showed a lower rate of positive selection in the VP2 but higher rates of negative selection. Negative selection pressure means that the gene will not increase in variation under pressure from the external environment, and that there is a tendency for gene sequence conservation (Miller et al., 2009) . Therefore, external selection pressure may not be significantly associated with the presence of mutations. Due to the lack of available genetic information, further analysis is limited. Our concern was regarding whether external selection pressure affects the immune response to the virus. Interestingly, we found that the positively selected codon site did not coincide with the B-cell epitope. However, negative selected codon sites 240, 313, and 380 were located on the predicted B-cell epitopes (Fig. 2) . This suggests that selective pressure does not significantly change the B-cell epitopes of CBuV. In parvoviruses, the VP2 region contains major epitopes (Lopez de Turiso et al., 1991) . As is known to all, B-cell epitope is a group of residues on the surface of an antigen which recognized by either a particular B-cell receptor (BCR) or a particular antibody molecule of the immune system and determine the immunogenicity of viruse (Zhang et al., 2016) .Therefore, the conservatism of epitope residues means that the immunogenicity of the CBuV may not significantly alter, which indicates that new serotypes of the CBuV may not easily produced. This is beneficial for virus prevention and vaccine development. Herein, three of 120 fecal samples were positive for CBuV, providing molecular evidence for the presence of the CBuV in Anhui Province. Phylogenetic analysis and J o u r n a l P r e -p r o o f Journal Pre-proof sequence alignment showed a high homology with other reference strains, indicating that CBuV was relatively conservative. In addition, CBuVs were subject to negative selection, which helped maintain the conservation of viral genes. And the negative selection codons were located on B-cell epitopes, it did not affect the immunogenicity of CBuVs. This study provides a reference for further understanding of the epidemic and molecular characteristics of the virus in China. Table 3 The selected sites of the VP2 gene according to three methods. Table 4 The results of B-cell epitope prediction. 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This study was supported by the Ningbo Health Branding Subject Fund (No. Note ：The bold were predicted by more than one method J o u r n a l P r e -p r o o f