Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV‐related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma‐glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3‐specific Abs and transient elevation of serum levels of liver‐specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3‐specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV‐positive horse using quantitative real‐time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs. Conclusion: Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver. (Hepatology 2015;61:448‐459)

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

Significance: Hepatitis C virus (HCV) displays a narrow species tropism severely hampering development of small animal models that are required for vaccine and pathogenesis studies in vivo. The recent discoveries of HCV-related hepaciviruses in diverse hosts offer new opportunities with respect to the development of an immunocompetent animal model for HCV research. Among the hepaciviruses, the equine nonprimate hepacivirus (NPHV) represents the closest homolog of HCV discovered to date. We defined key aspects of natural immunity to NPHV challenge in the cognate host and provide evidence for natural protection from NPHV infection. Further characterization of the immune signatures that confer protection against NPHV could provide important information that may facilitate the development of new prophylactic strategies including protective vaccines against HCV.

Background: Equine parvovirus‐hepatitis (EqPV‐H) research is in its infancy. Information regarding prevalence, geographical distribution, genetic diversity, pathogenesis and risk factors enhances understanding of this potentially fatal infection. Objectives: Determining the prevalence of EqPV‐H in Austrian equids. Investigating factors increasing probability of infection, liver‐associated biochemistry parameters, concurrent equine hepacivirus (EqHV) infection and phylogenetic analysis of Austrian EqPV‐H variants. Study design: Cross‐sectional study. Methods: Sera from 259 horses and 13 donkeys in Austria were analysed for anti‐EqPV‐H VP1‐specific antibodies by luciferase immunoprecipitation system (LIPS) and EqPV‐H DNA by nested polymerase chain reaction (PCR). Associations between infection status, sex and age were described. Glutamate dehydrogenase (GLDH), gamma‐glutamyl transferase (GGT), bile acids and albumin concentrations were compared between horses with active infection and PCR‐negative horses. PCR targeting partial EqPV‐H NS1 was performed and phylogenetic analysis of Austrian EqPV‐H variants was conducted. Complete coding sequences (CDS) of four Austrian variants were determined by next‐generation sequencing (NGS) and compared with published sequences. Results: Horses' EqPV‐H seroprevalence was 30.1% and DNA prevalence was 8.9%. One horse was co‐infected with EqHV. Significantly, higher probability of active EqPV‐H infection was identified in 16‐ to 31‐year‐old horses, compared with 1‐ to 8‐year‐old horses ( P = 0.002; OR = 8.19; 95% CI = 1.79 to 37.50) and 9‐ to 15‐year‐old horses ( P = 0.03; OR = 2.96; 95% CI = 1.08 to 8.17). Liver‐associated plasma parameters were not significantly different between horses with active infection and controls. Austrian EqPV‐H variants revealed high similarity to sequences worldwide. No evidence of EqPV‐H was detected in donkeys. Main limitations: Equids’ inclusion depended upon owner consent. There was only one sampling point per animal and the sample of donkeys was small. Conclusions: EqPV‐H antibodies and DNA are frequently detected in Austrian horses, without associated hepatitis in horses with active infection. The risk of active EqPV‐H infection increases with increasing age. Phylogenetic evidence supports close relation of EqPV‐H variants globally, including Austrian variants.

Non-primate hepacivirus (NPHV), a recently discovered hepatotropic virus infecting horses, is phylogenetically the closest known homologue of hepatitis C virus (HCV). The main route for acquiring HCV infection in childhood is vertical transmission. However, nothing is known about the natural mode of transmission for NPHV. To investigate the possibility of vertically transmitted NPHV infection in horses, 20 Thoroughbred broodmares and their foals were monitored during foaling season 2015 until 6 months post-partum. Prepartal serum was taken from the mares, and during foaling umbilical cord blood and colostrum samples were collected. Postnatal serum samples were taken from the foals after delivery. In addition, serum was taken at 3 and 6 months after foaling from all mares and foals. Samples were analysed for the presence of NPHV RNA by quantitative real-time PCR and for the presence of anti-NPHV NS3 antibodies by luciferase immunoprecipitation system. Identified NPHV isolates were sequenced and phylogenetic analysis of the viral glycoproteins was used to track the course of naturally occurring infections and the circulation of distinct isolates within the herd. At parturition, 16 mares were seropositive, including four viraemic mares. Vertical transmission occurred in one of these four mare–foal pairs. Interestingly, NPHV isolates of newly infected foals and mares after 3 and 6 months cluster in their respective pasture herds suggesting another horizontal route of transmission.