First Report of Canine Bufavirus and Its Whole Genome Sequence in India

Canine bufavirus (CaBuV), a novel protoparvovirus of dogs was reported only in Italy and China, till date. It was detected from dogs with enteric and respiratory symptoms and is distantly related to the human bufavirus. To explore the enteric prevalence of CaBuV in India, 186 diarrheic faecal samples were collected in Telangana State between 2019 and 2020. Among the samples, 4.3% (8/186) were positive for CaBuV by PCR. Co-infection with canine parvovirus CPV-2 was seen in 75% (6/8) of CaBuV positive samples. The near complete genome (4292 bp) of CaBuV was amplied and reconstructed for one isolate 407/PVNRTVU/2020. Sequence alignment indicated 93.42–98.81% homology with the other available CaBuV sequences; 70.88-73.39%, and 54.4-54.8% identity with human bufavirus, and CPV-2, respectively. Phylogenetic analysis showed that CaBuV 407/PVNRTVU/2020 was most closely related to CaBuV Chinese strains, together separated as Asian lineage. All Bufaviruses clustered together in one clade; however, the bat and sea otter parvoviruses also showed close relation with Bufaviruses. This rst report of prevalence of CaBuV in India provides a good reference, emphasizes the need for further epidemiological surveillance of CaBuV in India and its role in canine enteritis.

Information on the epidemiology and genetic features of CaBuVs is rather limited; the only reports of its existence were from Italy and China [11,[16][17][18]. To this end, in the current study, we investigated the prevalence of CaBuV in India and its role in canine gastroenteritis. For this, fecal samples from dogs presented with enteritis at veterinary hospitals in Hyderabad, Telangana State, India were collected. Also, the near full-length genome of one strain was determined and characterized, providing signi cant reference for further studies on the evolution and epidemiology of CaBuV.

Sample collection
In total, 186 fecal samples were collected from dogs (1 month -10 year old) presented with clinical gastroenteritis to Veterinary hospitals in Hyderabad, Telangana State, India during 2019-2020. Of the total number of samples, 138 were from puppies (<1-year-old), and 48 were from adult dogs (>1-year-old) (Table S1). Informed consents were obtained from all animal owners. The fecal samples were collected in sterile centrifuge tubes using rectal swabs and stored at − 20ºC until used.

DNA extraction and PCR ampli cation
The rectal swabs were homogenized in 3 ml of 0.1 M PBS (pH 7.4) containing antibiotics (100 IU/ml Benzyl Penicillin, 100 µg/ml Streptomycin sulphate), centrifuged at 6000 rpm for 10 min at 4 o C, and the supernatant ltered through 0.22µ syringe lter was used for further analysis. The viral DNA was extracted using phenol chloroform and isoamyl alcohol method as described by Sambrook and Russel [19]. The extracted DNA was stored at -20ºC until use.

Phylogenetic and recombination analysis
Sequence identity was analysed by multiple sequence BLAST analysis of nucleotide and amino acids.
Multiple sequence alignment of the whole genome was performed by CLUSTALW algorithm of MEGAX software followed by phylogenetic tree reconstruction [27]. The phylogenetic tree was generated using the Maximum Likelihood statistical method with the tamura-3-parameter model. The credibility of the phylogenetic tree was tested by applying a bootstrap test with 1000 replicates.
The recombination analysis tool (RAT), and recombination detection program (RDP) package Beta 4.101 were used for identi cation of recombinant sequences in default mode. A recombination event with a signi cance of p < 0.01 in at least four out of nine selected algorithms: RDP, GENECONV, Boot-Scan, Maxchi, Chimaera, SiScan, 3Seq, LARD, and PhylPro was considered to be reliable [28]. RAT is a distancebased method of recombination detection platform [29].

Detection of CaBuV in fecal samples
Of the 186 samples screened by real-time PCR, eight were found positive for CaBuV which accounts for a prevalence rate of 4.3%. Of these, six samples were from diarrheic puppies (<1 year old) and two samples were from diarrheic adult dogs (>1 year old). Seven of the eight CaBuV positive samples had co infections with other enteric viruses such as canine parvovirus CPV-2, canine adeno virus-2, and canine astrovirus; and negative for canine coronavirus, and canine distempervirus (Table 1).

Phylogenetic and recombination analysis
Phylogenetic analysis was performed with the near complete genome sequence in this study with other publicly available CaBuV sequences and other protoparvovirus sequences available in GenBank. It can be seen that the CaBuV from India was more closely related to Chinese isolates, together separated as Asian lineage (Fig. 2a). Further, it can be seen that CaBuVs show closest relationship with bat and sea otter parvoviruses in addition to all other Bufaviruses (human, porcine, rat and megabat), whereas, canine and avian parvoviruses together formed a separate branch (Fig. 2b).
To analyze whether recombination played a role in CaBuV evolution, CPVs, CaBuVs, HuBuVs, and PoBuVs, megabat BuV, rat BuV, bat BuV, avian BuV, human cutavirus, and sea otter parvovirus were analyzed by RAT and RDP software with multiple algorithms. Nine algorithms (RDP, GENECONV, Boot-Scan, Maxchi, Chimaera, SiScan, 3Seq, LARD, and PhylPro) were used to predict potential recombination events between the input sequences. Even though no recombination events could be found in the CaBuVs, it can be noted that major recombination events possibly drove evolution of other BuVs in the Protoparvovirus genus (Fig. 3).

Discussion
CaBuV is a novel protoparvovirus rst identi ed from an outbreak of CIRD [11]. Since then, only handful of studies from a few countries (Italy, Hungary, and China) reported its prevalence [11,[16][17][18]. We report CaBuV prevalence in India for the rst time. Varying prevalence rates of this virus have been reported so far ranging from 1.74-42.15% [11,[16][17][18]30]. Our study reports a prevalence rate of 4.3%. It has been suggested that geographical, temporal, and target canine population included in the studies can affect the prevalence rates [18]. A statistically non-signi cant correlation of CaBuV with enteritis was reported before; although non-signi cant the authors noticed higher loads of CaBuV in diarrheic samples than in controls [11,18]. Moreover, our study along with another study where prevalence of CaBuV was studied extensively in diarrheic dogs, stresses the role of CaBuV in causing enteritis [16]. We have noticed that seven out of eight CaBuV positive samples had co infections with other enteric viruses with CPV-2 being the predominant co infection (6/8) followed by CAstV (2/8), and CAdV-2 (2/8). Similar to our ndings few other studies also reported CPV-2 to be the major co-infecting agent with CaBuV with a prevalence of CPV-2 ranging from 87.5%-100% [17,18]. CPV-2 is a well-known viral agent involved in severe gastroenteritis in dogs, especially pups. Further experiments on the pathogenicity studies shall throw light on the role of co-infections in increased severity of gastroenteritis morbidity and mortality in dogs.
We have reconstructed near complete genome of one isolate of CaBuV detected in Indian dog population.
Comparison and analysis with the other publicly available CaBuV sequences revealed NS1 to be the most conserved gene and VP2 to be the most variable, both at the level of nucleotide (NS1: 96.48%-98.13%; VP2: 88.19%-99.47%) and amino acids (NS1: 94.98%-97.81%; VP2: 94.19%-99.82%). Based on the phylogeny reconstructed with the whole genome, the current CaBuV isolate clustered with CaBuV strains identi ed in China. Our analysis differentiates all the available CaBuV sequences into two groups: Asia and Europe. Being a DNA virus, parvoviruses exhibit low mutation rate [17]. The genetic grouping observed in the current study indicates unique evolutionary forces driving evolution of CaBuVs. In summary, the rst report of prevalence and complete genomic sequence of CaBuV isolate from India was obtained and genetically analyzed in our study. Indian isolate was grouped with CaBuVs isolated from China. The current study forms a foundation to further studies on the epidemiology and genetic diversity of CaBuV in India.  2a Phylogenetic tree of CaBuVs based on whole genome sequence. A total of 15 whole genome of CaBuV isolates published earlier in GenBank were used as reference to reconstruct the phylogenetic tree using maximum likelihood as statistical method, test of phylogeny as bootstrap with value of 1000 replicates, model as tamura-3-parameter. The isolate from this study is marked with a dark triangle. 2b Phylogenetic tree of Parvoviridae family based on whole genome sequence. A total of 46 whole genome of Parvoviridae family isolates infecting different species published earlier in GenBank were used as reference to reconstruct the phylogenetic tree using maximum likelihood as statistical method, test of phylogeny as bootstrap with value of 1000 replicates, model as tamura-3-parameter. The isolate from this study is marked with a dark triangle.