Abstract:
Hepatitis B viruses belong to a family of partially double stranded DNA viruses that infect a range of organisms with host responses that vary from mild to chronic infection and carcinogenesis. The white sucker hepatitis B virus (WSHBV) was first described in the freshwater teleost, white sucker (Catostomus commersonii) in 2015 and unofficially classified as a parahepadnavirus in 2017. At present, the significance of WSHBV infection to the health of white sucker is unknown. To date, no other parahepadnavirus genetic diversity or association with fish health has been studied. Here, we investigate patterns of genetic diversity and identify geographically associated variation among WSHBV genomes from white sucker inhabiting tributaries of Lake Michigan, Lake Superior, and Lake Erie, and Alberta, Canada. We previously identified 13 WSHBV positive white suckers via a Nanostring codeset targeting viral core RNA. Here we identify an additional 22 virus positive fish using a qPCR method that detects viral DNA extracted from plasma and liver tissue. Subsequently, 28 WSHBV genomes were sequenced using a circular long range amplicon sequencing (CLAS) method that consisted of long-range PCR followed by amplicon sequencing using the Illumina MiSeq. The CLAS method was effective for genome sequencing; however, we observed inconsistencies in sequencing success of the 627 bp atypical region within WSHBV. The WSHBV genome and ORFs isolated here clustered together by geography similar to that observed with geographically separated human hepatitis B virus genotypes. Although the WSHBV genomes do not meet the criteria used to define subgenotypes of other hepadnaviruses, we suggest the use of the ratio of nonsynonymous/synonymous mutations (Ka/Ks) in the surface protein and total Dxy to differentiate subgenotypes. Between the five viral subgenotypes, Alberta 1522, Alberta 1538, St. Louis, Swan Creek, and Lake Michigan the Ka/Ks was ≥ 0.572 and the Dxy was ≥ 0.00683. Similar to other hepadnaviruses, variability between subgenotypes was concentrated in the PreS and spacer domain that is associated with immune epitopes and host specificity. This study of WSHBV genetic diversity should facilitate the development of molecular markers for future identification of genotypes and provide evidence in future investigations of possible differential disease outcomes.
