Identification of a Novel Hepacivirus in Domestic Cattle from Germany

Abstract

Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related sequences have been found in bats, rodents, horses, and dogs, indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a nearly full-length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic analysis confirmed that the virus represents a novel species within the genus Hepacivirus. Viral RNA screening determined that 1.6% (n = 5) of 320 individual animals and 3.2% (n = 5) of 158 investigated cattle herds in Germany were positive for bovine hepacivirus. Repeated reverse transcription-PCR (RT-PCR) analyses of animals from one dairy herd proved that a substantial percentage of cows were infected, with some of them being viremic for over 6 months. Clinical and postmortem examination revealed no signs of disease, including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues, together with the presence of an miR-122 binding site in the viral genome, strongly suggests a liver tropism for bovine hepacivirus, making this novel virus a promising animal model for HCV infections in humans.

Importance: Livestock animals act as important sources for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from the search for novel viruses, detailed characterization of these pathogens is indispensable in the context of risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. In addition, determination of the prevalence and of the course of infection in cattle herds provides valuable insights into the biology of this novel virus. The results presented here form a basis for future studies targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.

FIG 1

Summary of bovine serum samples taken during the study. (A) Pooling of serum samples and subsequent high-throughput sequencing led to the identification of bovine hepacivirus B1. (B) Serum samples included in BovHepV RT-PCR screening. (C) Schematic overview of sampling sites: 158 herds from six federal states of Germany were included. Red dots indicate the origin of BovHepV-positive herds. (D) Summary of serum sampling in two dairy herds.

FIG 2

Phylogenetic analysis of hepaciviruses from human and various animal species including cattle. A maximum-likelihood tree is presented based on the complete coding sequences of hepaciviruses. Bootstrap analysis was performed with 1,000 replicates (numbers next to the branches are percentages). Bootstrap values below 70% are not shown. The tree was rooted to hepatitis GB virus A. Sequences downloaded from GenBank are cited with their accession numbers. Sequences are identified as follows: blue triangles, HCV; green squares, NPHV; gray inverted triangles, bat hepacivirus; purple circles, rodent hepacivirus; teal diamond, GBV-B; red open circles, BovHepV.

FIG 3

Box-plots of serum concentrations of liver enzymes and of total bilirubin determined for BovHepV-positive (n = 12) and BovHepV-negative (n = 28) cows. AST, aspartate-aminotransferase; γ-GT, γ-glutamyl transferase; GLDH, glutamate dehydrogenase.

FIG 4

BovHepV genome equivalents per milligram of tissue and microliter of serum determined by quantitative RT-PCR. Mean values and standard deviations of three independent experiments are shown. Samples were taken during necropsy from BovHepV-positive animal 463.