Abstract

INTRODUCTION

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Currently, about 160 million individuals are persistently infected with HCV.¹ Acute HCV infection is asymptomatic in many cases, but 50%–80% of infected individuals are unable to clear the virus and a state of persistent viral replication and hepatic inflammation, i.e., chronic hepatitis C, follows. Some patients remain asymptomatic, but over years or decades others develop cirrhosis, portal hypertension, deteriorating liver function and hepatocellular carcinoma. It is due to these late complications that chronic hepatitis C is a leading cause of liver-related death and the prime indication for liver transplantation worldwide.² Combination treatment with pegylated interferon-α and ribavirin has been the standard of care for more than a decade.³ Recently, the first directly acting antivirals targeting the viral NS3-4A protease were licensed and triple therapy has further improved treatment options for genotype 1.⁴ However, resistance development, side effects and viral genotype-specific efficacy of these drugs require alternative antiviral treatment options.Hepatitis C Virus Homologs in Nature Essay.  The HCV genome is 9.6 kb in size, encodes for a single polyprotein that is cleaved by cellular and viral proteases into at least 10 different proteins: the structural proteins core, E1, E2, the ion channel p7 and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B.⁵ These viral factors act in concert with host proteins to mediate virus entry and to coordinate RNA replication and virus production which is coupled to lipoprotein biogenesis.⁶

Ongoing transmission of productive HCV infection is limited to human populations, although higher primates are susceptible to experimental infection.⁷ Due to this narrow host range, a robust immunocompetent small animal model is still lacking. However, a recent study demonstrated infection, replication and de novo virus production in an inbred mouse strain engineered to express human HCV entry factors in the liver and carrying a targeted lesion within innate immune signalling genes (Stat1, Irf7, Irf9, IFN-αβR).⁸ Although not fully immunocompetent, this landmark study shows that HCV can be propagated in mice and raises the hope that more robust models ultimately with fully functional innate and adaptive immune system can be developed. Despite the rapid advancements in HCV research over the past decade, the origin of HCV still remains elusive, with no evidence of an animal population that might have transmitted the virus to humans.⁹ Nevertheless, the idea that there might be a non-human primate source for HCV infections, as described for HIV,¹⁰ has engaged researchers since the discovery of HCV in 1989.¹¹ The recent discovery of novel hepaciviruses in different animals could now reveal new insights in the origin of HCV. Several reports identified new homologs of HCV in mammals that cluster in the genus hepacivirus or the newly proposed genus pegivirus. Non-primate hepaciviruses (NPHV) were initially discovered in domestic dogs and subsequently in horses^12,13 and other diverse and widespread HCV-like viruses have been reported in wild populations of rodents and bats.^14,15,16

HCV GENETIC DIVERSITY AND CLASSIFICATION

HCV is an enveloped, positive-stranded RNA virus belonging to the genus hepacivirus in the family of Flaviviridae. A second species tentatively assigned to this genus is GB virus B (GBV-B), a virus distantly related to HCV identified in a laboratory-housed tamarin, a New World monkey and causing hepatitis.¹⁷ However, GBV-B infection has not been reported in any other tamarin or other New World monkey species and its origin, as well as its natural host, remains unknown. Hepatitis C Virus Homologs in Nature Essay. Besides hepaciviruses, the Flavivridae family consists of three other genera: flaviviruses including yellow fever and dengue virus; pestiviruses including bovine viral diarrhea virus and classical swine fever virus and the recently assigned pegivirus genus. Pegiviruses encompass the previously unclassified GB virus A (GBV-A) which was found in primates,¹⁸ GB virus C (GBV-C) or hepatitis G virus (HGV) which infects humans and chimpanzees^19,20 and GB virus D (GBV-D) identified in bats.²¹ Of note is that in contrast to the hepaciviruses, GBV-A and some GBV-C/HGV isolates do not appear to encode a core protein.^22,23,24On the basis of phylogenetic relationships, observed similarities in genome organization and pathogenic features Stapleton and colleagues proposed this new genus within the family of Flaviviridae.²⁵

HCV is genetically highly variable and, based on phylogenetic analyses, viral isolates are grouped into seven genotypes (1–7) (Figure 1), which are associated with specific global regions and modes of transmission.²⁶ The high observed genetic diversity is a result of the error-prone replication of the virally encoded RNA dependent RNA polymerase, coupled with the high replication rate in vivo.²⁷ This genetic variability exhibited by HCV facilitates immune evasion and contributes to viral persistence. HCV genotypes differ from each other by 30%–35% at the nucleotide level. However, this genetic heterogeneity is not evenly distributed throughout the viral genome, and is concentrated in regional hot-spots. The greatest levels of diversity are observed in the E1E2 genes encoding the envelope glycoproteins E1 and E2, while other genome regions, such as those encoding the core protein or the helicase domain of the NS3 protein, exhibit higher levels of conservation.²⁸ Despite substantial sequence variation, all genotypes share the same genome structure, encoding a single polyprotein which is postranslationally cleaved by viral and host proteases into 10 mature proteins. The single open reading frame (ORF) is flanked by two highly conserved untranslated regions (UTRs). HCV genotypes can be further divided into multiple distinct subtypes (a, b, c, etc.) differing by 20%–25% at the nucleotide level.²⁹ Even within an infected patient, HCV exists as a heterogeneous population of genetically distinct yet related variants.³⁰The prevalence of HCV genotypes differs significantly in different parts of the world. Seventy percent of patients in North America and Europe are infected with genotype 1, while HCV genotypes 2 and 3 account for about 25% of cases, but are overrepresented among patients who inject drugs.³¹ In contrast, HCV genotype 3 can be detected in about 65% of patients in South East Asia and genotype 4 is the major genotype in the Middle East and northern Africa. Genotypes 5 and 7 have been detected mainly in Africa, while genotype 6 has been found in China and Southeast Asia.²⁹ Phylogenetic analyses, coupled with global distribution patterns of HCV genotypes, indicate that genotypes 4 and 6 emerged CA 350–700 years ago.³² The worldwide dissemination of genotype 2 began later some 90–150 years ago, with the restricted diversity of sequences among genotype 1b indicating emergence around 60–70 years ago.³³ One has to keep in mind that this molecular clock analysis might be under- or overestimated due to mutational masking and substitutional saturation. Hepatitis C Virus Homologs in Nature Essay.

NOVEL HEPACIVIRUSES IN DIFFERENT ANIMALS

NOVEL PEGIVIRUSES IN NEW HOSTS

As a newly proposed genus of the family of the Flaviviridae, the genus pegivirus has been proposed to encompass the so far unclassified viruses GBV-A, GBV-C/HGV and GBV-D.²⁵ Based on this new classification, different groups also identified multiple viruses falling into the pegivirus genus, which are emerging in new hosts. Chandriani and colleagues⁴² identified a highly divergent member of the Flaviviridae family, which is likely the causative agent for an outbreak of acute hepatic disease occurring on a horse farm. The virus was present in equine serum from two overtly clinical index cases of hepatitis and from an equine plasma product which was administered to the horses before the outbreak. This new pathogen was designated Theiler’s disease-associated virus (TDAV), presumably the causative agent of Theiler’s disease, an acute hepatitis in horses. Viral titers in most of the infected horses ranged from 10⁷ to 10⁸ genomes/mL. The viral genome contained a single ORF encoding a polyprotein of 3189 aa flanked by a 5′ and 3′ UTR. Based on comparison with related members of the Flaviviridae, the virus is predicted to encode three structural proteins (core, E1 and E2) and seven putative non-structural proteins (a protein between E2 and NS2 as well as NS2, NS3, NS4A, NS4B, NS5A and NS5B). Phylogenetic analyses grouped TDAV as belonging to the newly proposed pegivirus genus (Figure 1). Over the length of the entire polyprotein, the virus shares 35.5% aa identity with GBV-D, 20.5% identity with HCV (genotype 1) and 20.4% identity with NPHV. The most conserved regions can be found in NS3 and NS5B, with regions exceeding 75% aa identity with GBV-D. Interestingly, even though TDAV is linked to an acute hepatitis, no miR-122 binding site could be identified, suggesting that, unlike HCV, the virus can replicate independently of miR-122. Transmission between horses appears to be low as the virus was undetectable in horses which had contact with other infected animals.Hepatitis C Virus Homologs in Nature Essay.  The TDAV-positive horses were further ranked as clinical or subclinical, with the clinical cases displaying significantly elevated liver enzymes in serum. The subclinical cases displayed varying degrees of elevated liver enzymes in the serum, but without overt clinical manifestation of liver disease in most cases. In general, a slightly higher proportion of asymptomatic TDAV-positive animals were observed. Furthermore, a clear relationship between viral load and clinical hepatitis could not be observed, indicating that the viral load was not predictive of the extent of hepatic injury. Further analyses of the infected horses 1 year after the outbreak provided evidence that TDAV can establish a chronic infection. To further investigate the route of transmission for TDAV infections, the authors performed an inoculation study in which they experimentally injected four healthy animals with a TDAV-positive plasma product. Even though the viral genome could be detected in all inoculated horses, only one horse showed a clear elevation of liver enzymes over the course of the study. In summary, this study provides evidence for the association of TDAV with Theiler’s disease, identifying TDAV as the sole member of the newly described genus pegivirus for which disease association has been demonstrated.

Kapoor and colleagues⁴³ independently identified another equine pegivirus (EPgV) which is genetically distinct to TDAV. The group screened serum samples from horses with elevated liver enzyme levels and detected viral RNA in two samples, which were highly divergent from all previously known pegiviruses (Table 3). The virus encodes a 5′ UTR, a single ORF encoding a putative polyprotein of 3305 aa and a 3′ UTR. Sequence alignments revealed the divergence of EPgV from other pegiviruses, ranging from 62% to 77% in the structural genes and from 49% to 59% in the non-structural region. The most conserved regions were the NS3 and NS5B genes, as described for the hepaciviruses genus, whereas the highest sequence diversity can be found in the glycoproteins E1/E2 and NS4B. To analyze the prevalence and persistence of EPgV, two horse cohorts were studied. In one cohort, the viremia frequencies were 25% (3/12) among horses with elevated liver enzymes and 6.4% (4/62) among healthy animals. In another herd analyzed at different time points over four years, 15%–32% of the horses were found to be positive, with two horses remaining viremic for at least three and a half years and two horses being able to clear the infection. Viral genome copy numbers ranged from 10^4.5 to 10^6.5 genome equivalents/mL in the serum and viral RNA could also be detected in liver and lymph node biopsy samples, as well as in peripheral blood mononuclear cells, with no major differences in viral RNA between the tissues, suggesting that the virus is not strictly hepatotropic.⁴³ Further studies are necessary to determine the prevalence, tissue tropism and possible disease association of this newly identified virus. Hepatitis C Virus Homologs in Nature Essay.

The same group additionally identified two new species of pegiviruses in rodents (see the section on ‘Rodents/bats’), one from white-throated wood rats (RPgV-cc61) from which the whole genome was acquired (Table 3) and the other from deer mice.¹⁴ The viral genome displayed the typical Flaviviridae genome organisation including a 5′ UTR, a polyprotein coding region (3484 aa) as well as a 3′ UTR. Genetic analyses revealed that RPgV was substantially divergent from GBV-A, GBV-C, GBV-D and from the EPgV (Figure 1). Amino-acid divergence ranged from 78% to 81% in the structural proteins and 54% to 56% in the non-structural region.

Apart from rodents, bats comprise the most diverse group of mammals. The Pteropus giganteus bats have already been identified as carriers of GBV-D.²¹ The group of Quan and colleagues further enhanced our understanding of the viral diversity in bats, with the identification of bat-derived hepaciviruses and pegiviruses¹⁵ (see the section on ‘Rodents/bats’). Among the total of 83 bat-derived viruses uncovered, 19 potential novel viral species within the pegivirus genus were identified, which formed three distinct lineages. Hepatitis C Virus Homologs in Nature Essay. Clade H viruses clustered with the previously identified GBV-D, whereas the clade G and K viruses formed distinct phylogenetic clusters within the genus pegivirus. Interestingly, in some of the animals, coinfections of clade G and K viruses and in one case even coinfection with a hepacivirus (clade C) and a pegivirus (clade K) were observed. The genomic organization of these pegiviruses is similar to that observed for other flaviviruses. Amino acid sequence identities were greatest in non-structural proteins NS3 and NS5B, whereas the envelope proteins as well as NS2 and NS5A displayed the highest variability. In summary, these studies show that also pegiviruses are widely distributed among different mammalian species and future studies are necessary to investigate disease association, transmission and tissue tropism.

THEORIES FOR THE ORIGIN OF HCV

Viruses are usually well adapted to their hosts, resulting in multiple barriers to cross-species transmission.⁴⁴ Nonetheless, the majority of recent emerging infections in human populations represent zoonoses from wild animal species.⁴⁵ A recent survey, using fruit bats as a model organism, uncovered 55 novel viruses from seven viral families.⁴⁶ Extrapolating this number to all mammalian species, the authors estimate a minimum of 3.2×10⁵ mammalian viruses awaiting discovery. Although this number is likely to be an overestimation due to the tendency of bats to act as reservoirs for a broad spectrum of viruses, it is clear that many as yet undiscovered viruses circulate in wild animals, all with the potential to jump the species barrier to humans. Cumulatively, these findings would support a scenario where HCV in humans is likely the result of a cross-species transmission, probably from an as yet unidentified source.

Current patterns of global HCV diversity could be due to a single ancestral zoonotic transmission to archaic humans prior to their global dispersal, with subsequent viral diversification in isolated populations. Identification of a more closely related hepaciviral progenitor closer to the root of extant circulating HCV isolates than NPHV, the current closest relative, would provide support for this scenario.⁴⁷ However, the geographical associations of different HCV genotypes, which appear to have been evolving and diverging in geographically discrete human populations over an extended time scale, could also be the result of multiple, more recent, independent cross-species transmissions to geographically separated human populations.Hepatitis C Virus Homologs in Nature Essay.  Identification of novel hepaciviruses, isolated from animal hosts, which fall within the global HCV radiation and are positioned basally to distinct HCV clades, would ultimately provide strong evidence for this alternative.⁴⁷ 

    ^{ORDER A PLAGIARISM-FREE PAPER NOW}

The >1200 bat species are known to harbour a diverse array of viruses from multiple families, many of which have crossed the species barrier to cause a variety of diseases in humans, often via an intermediate host.⁴⁸ For example, the Hendra virus, an RNA virus from the family Paramyxoviridae, has caused multiple outbreaks in horses and four reported human fatalities.⁴⁹ Interestingly, Hendra virus was subsequently demonstrated to originate in fruit bats and was transmitted to humans via close contact with horses. Of note, the most closely related animal hepaciviruses to HCV described to date are found in horses (NPHV)¹², and Kenyan fruit bats (BHV)¹⁵ (Figure 1). Thus, if continued wild animal sampling fails to identify more likely candidates for the ancestor(s) of HCV, one could envisage an historical scenario whereby the progenitor of HCV was transmitted to humans from bats via horses, as all three species are susceptible to hepaciviral infection. However, viruses are dependent on a multitude of host factors to complete their life cycle in susceptible cells. Thus, species-specific barriers to viral cross-species transmissions are likely to be proportional to the genetic relatedness of host species: one would expect viruses to jump the species barrier more easily between closely related host species. In addition to HCV infection of humans, GBV-B infection of New World monkeys and the recent discovery of distantly related hepaciviral homologs to HCV in Old World monkeys³⁷ indicates the susceptibility of a broad array of primates to hepaciviral infection. Two of the biggest infectious disease burdens afflicting humanity presently are HIV and Malaria, zoonoses from chimpanzees and gorillas, respectively,⁵⁰our closest great-ape relatives. Thus, it might be also possible that great apes, or other primate species, harbor as yet undescribed hepaciviruses, which may ultimately have given rise to the current HCV pandemic. Hepatitis C Virus Homologs in Nature Essay.

CONCLUDING REMARKS

Despite successful development of cell culture systems to study the complete HCV replication cycle,⁵¹ analysis of mechanisms of virus pathogenesis and immune control as well as vaccine development are severely hampered by the lack of robust immunocompetent small animal models. Furthermore, the origin of HCV has remained elusive. Recently, studies combining powerful next-generation sequencing technologies with coordinated sample collection from multiple geographical localities have uncovered related hepaciviral and pegiviral homologs in diverse animal species. These research efforts have resulted in a rapid expansion of the known viral diversity in hepacivirus and pegivirus genera, and identified an increasing number of animal host species susceptible to hepacivirus and pegivirus infection. Differences and similarities between these newly discovered viruses and HCV may ultimately advance our understanding of hepacivirus biology with respect to mechanisms of hepaciviral replication, permissiveness of small animal models to productive infection, epidemiology and vaccine development, in addition to further elucidating HCV origins. Future studies should address transmission and ecology of these new viruses in their natural hosts to evaluate any potential risk of trans-species transmission to humans.Hepatitis C Virus Homologs in Nature Essay.  The generation of functional cDNA clones will further advance our knowledge on hepaciviral replication strategies and could be used for the development of recombinant HCV vaccines. In conclusion, the recent discoveries of multiple novel hepaciviruses in diverse mammalian species have undoubtedly intensified research efforts to identify the true origins of HCV. Ultimately, these studies will expand our knowledge of circulating mammalian viruses, in addition to further illuminating barriers/pathways to viral cross-species adaptation.

Acknowledgments