Discovery and characterization of feline and canine bocaviruses in Hong Kong

Abstract

Objectives: To identify and characterize bocaviruses in cats and dogs in Hong Kong. Methods: DNA extracted from feline and canine samples were screened for bocaviruses, using degenerated primers targeted to NS1 gene. Once potential novel bocavirus was found, specific primers were designed for subsequent detection. PCR products were sequenced and compared with available NS1 sequences of bocavirus in GenBank. DNA extracted from the positive specimens was amplified by degenerate primers designed from multiple alignment of bocaviruses genomes. The terminal sequences were confirmed by a modified protocol for RACE. Sequences were assembled and edited manually to produce final sequences of the viral genomes. A maximum-likelihood (ML) phylogenetic tree was constructed. Protein domain and family analysis was performed using InterProScan and/or multiple sequence alignment. Results: Novel feline bocavirus (FBoV), and canine bocavirus (CBoV) which is closely related to CBoV strain Con-161, was identified in cats and dogs in Hong Kong respectively. FBoV was detected by PCR among 7.2%, 0.4%, 1.6% and 1.1% of feline fecal, nasal, urine and blood specimens respectively and CBoV was detected among 3.6%, 3.1%, 6.2% and 0.2% of canine fecal, nasal, urine and blood specimens respectively. The three ORFs characteristic of bocaviruses can be found in both FBoV and CBoV. In addition, a fourth ORF downstream to NS1 was predicted in CBoV. This ORF was not reported in CBoV strain Con-161 and other known bocaviruses. Second exon that encodes the C-terminal of non-structural protein NS1 and conserved RNA-splicing sites was found in CBoV. Phylogenetic analysis of FBoV and CBoV genome sequences showed that recombination was not involved in the emergence of the bocaviruses. Genetic analysis of partial VP1/VP2 sequences in 23 FBoV and 25 CBoV strains revealed two potential genetic groups in FBoV and an additional group that was distinct from CBoV-A to -C found in USA, in CBoV. Several tissue specimens were collected for the study of tissue tropism of FBoV and CBoV. FBoV was detected among 4.4%, 2.2%, 15.6% and 3.3% of feline lung, liver, spleen and kidney specimens respectively and CBoV was detected among 3.6%, 6.0%, 6.0% and 8.4% of canine lung, liver, spleen and kidney specimens respectively. It suggested wide tissue tropism in FBoV and CBoV, and CBoV may cause more systemic infections. In this study, all samples were collected from healthy cats and dogs, the pathogenicity of FBoV and CBoV remains to be determined. Conclusion: A novel bocavirus was identified in domestic cats in Hong Kong. The three ORFs characteristics of bocaviruses was present in the FBoV genomes. A bocavirus was found in dogs in Hong Kong which is closely related to the CBoV identified in the US. A novel genetic group distinct from the three groups of the US CBoV strains was revealed.