Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States

Abstract

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5' end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.

Keywords: hepadnavirus; hepatitis B virus; immunosuppression; prevalence.

Figure 1

Gel electrophoresis products from cPCR in DNA isolated from a U.S. cat. DCH is identified by PCR from one cat from Ventura County, California. Lane 1 = 1 KB ladder; Lane 2 = PCR product from DNA extracted from the blood of a shelter cat from Ventura County, CA; Lane 3 = positive control provided from UC Davis; Lane 4 = Negative control (SPF cat DNA). Primers used are HgapF and HgapR with an expected size range of 200 BP. Arrow indicates approximately 200 bp.

Figure 2

Whole genome DCH from a U.S. domestic cat. Overlapping clones were amplified from a cat from Ventura County, CA, USA. Sequences were aligned to the Sydney DCH reference sequence MH307930. A 157 bp deletion was observed in the core protein open reading frame between nucleotides 1679 and 1836. Multiple primer pairs and clones overlapping this region consistently detected the deletion. Nucleotide 1 represents a conserved EcoR1 site. Conventional PCR binding sites are indicated.

Figure 3

Phylogram (A) and identity matrix (B) based on whole DNA genome of DCH isolates, human HBV, and tent-making bat HBV. LC685967.1 = Japan isolate 1 (Rara); NC_040719.1 = Sydney isolate; LC668427.1 = Japan isolate 2; MT506041.1 = Thailand isolate; MK902920.1 = Malaysia isolate; MK117078.1 = Italy isolate. Alignment and figure generated using Uniprot Align [23].

Figure 4

Phylogram (A) and identity matrix (B) based on the large surface protein sequence of DCH isolates, human HBV, and tent-making bat HBV. BDG15103.1 = Japan isolate 1 (Rara); YP_009553237.1 = Sydney isolate; BDD79976.1 = Japan isolate 2; QOW38044.1 = Thailand isolate; QFG73558.1 = Malaysia isolate; QDX15498.1 = Italy isolate. Analysis and figure generated using Uniprot Align [23].

Figure 5

Sequence alignment of the whole large surface protein sequence of DCH isolates, human HBV, and tent-making bat HBV. Red rectangle represents the highly conserved domain necessary for NTCP binding. BDG15103.1 = Japan isolate 1 (Rara); YP_009553237.1 = Sydney isolate; BDD79976.1 = Japan isolate 2; QOW38044.1 = Thailand isolate; QFG73558.1 = Malaysia isolate; QDX15498.1 = Italy isolate. Analysis and figure generated using Uniprot Align [23].