Friday, May 15, 2015

Hepatitis C Virus Mutation will be more deadly than Ebola and it's coming !!!

The Origin of Hepatitis C Virus
Peter Simmonds

Abstract  The origin of hepatitis C virus (HCV) can be conceptualised at several 

levels.  Firstly,  origins  might  refer  to  its  dramatic  spread  throughout  the Western 

world  and  developing  countries  throughout  the  twentieth  century.  As  a  blood-

borne  virus,  this  epidemic  was  fuelled  by  new  parenteral  transmission  routes 

associated  with  medical  treatments,  immunisation,  blood  transfusion  and  more 

recently injecting drug use. At another level, however, origins might refer to the 

immediate  sources  of  HCV  associated  with  its  pandemic  spread,  now  identified 

as areas in Central and West sub-Saharan Africa and South and South East Asia 

where  genetically  diverse  variants  of  HCV  appear  to  have  circulated  for  hun-

dreds of years. Going back a final step to the actual source of HCV infection in 

these  endemic  areas,  non-human  primates  have  been  long  suspected  as  harbour-

ing  viruses  related  to  HCV  with  potential  cross-species  transmission  of  variants 

corresponding to the 7 main genotypes into humans. Although there is tempting 

analogy between this and the clearly zoonotic origin of HIV-1 from chimpanzees 

in Central Africa, no published evidence to date has been obtained for infection of 

HCV-like viruses in either apes or Old World monkey species. Indeed, a radical re-

think of both the host range and host-specificity of hepaciviruses is now required 

following the very recent findings of a non-primate hepacivirus (NPHV) in horses 


1   HCV Genetic Diversity and Genotype Classification ..........................................................  2

2   The Recent Spread of HCV .................................................................................................  5

3   Endemic Circulation of HCV ..............................................................................................  6

4   Origins of Human Infections and HCV Homologues  

    in Other Mammals ...............................................................................................................  8

5   Concluding Thoughts ...........................................................................................................  11

References ..................................................................................................................................  12

P. Simmonds (*) 

Infection and Immunity Division, Roslin Institute, University of Edinburgh,  

Easter Bush, Edinburgh EH25 9RG, UK


R. Bartenschlager (ed.), Hepatitis C Virus: From Molecular Virology                                          1

to Antiviral Therapy, Current Topics in Microbiology and Immunology 369,  

DOI: 10.1007/978-3-642-27340-7_1, © Springer-Verlag Berlin Heidelberg 2013

and potentially in dogs. Further research on a much wider range of mammals is 

needed  to  better  understand  the  true  genetic  diversity  of  HCV-like  viruses  and 

their host ranges in the search for the ultimate origin of HCV in humans.

This  review  is  written  at  a  highly  significant  time  in  evolutionary  studies  of 

HCV and its origins. The discovery of closely related viruses to human HCV in 

horses  and  possibly  dogs  termed  non-primate  hepacivirus  (NPHV)  (Burbelo 

et al.  2012; Kapoor et al. 2011) throws an entirely new light on the species dis-

tribution  of  hepaciviruses  and  their  host  range.  Despite  the  significance  of  these 

very recent discoveries, however, in many ways it is a particularly difficult time 

to write a review of HCV origins and evolutionary history. Frequent infection of 

horses worldwide with a virus reasonably similar to HCV breaks a key assump-

tion of much previous research that the closest relative of HCV would be found in 

non-human primates. In its place we now have total uncertainty; domestic horses 

seem an incongruous host species and the suspicion must be that hepaciviruses are 

much more widely distributed in other mammals. At present, however, we simply 

do not know what these are. More importantly, we do not know whether viruses 

more similar to human HCV than NPHV exist and what species these may infect. 

Discovering a zoonotic source for the epidemic of HCV infection that has swept 

through the human population in the last century would be a truly important step 

in our understanding of host relationships, adaptation and pathogenicity.

This review of HCV origins therefore concentrates initially on the better character-

ised recent epidemic transmission of HCV in the twentieth century and the existence 

of suspected source areas for infection in sub-Saharan Africa and South-East Area. 

Some  aspects  of  the  much  less  well  understood  history  of  HCV  before  this  recent 

spread will be speculatively discussed, as will the existence of HCV-like viruses in 

non-human species. Inevitably any comments made about the latter will, through fur-

ther research, be revealed as either hopelessly cautious or naively overstated in a very 

short space of time, errors for which I apologise in advance. However, the findings 

cannot be simply omitted from a review with this title and I hope that they spur a 

greater interest in the wider group of hepaciviruses and whether the attributes of HCV 

that make it such an important human pathogen (persistence and hepatotropism) are 

shared with other members of the genus.

1   HCV Genetic Diversity and Genotype Classification

HCV is classified as the type member of the genus Hepacivirus within the virus 

family Flaviviridae (Fig.  1) (Bukh  1995; Simmonds et al.  1993, 2005). Although 

variants  of  HCV  show  substantial  genetic  diversity  from  each  other,  the  7  cur-

rently  classified  genotypes  are  all  classified  as  one  species  under  current  ICTV 

rules  notwithstanding  their  considerably  antigenic  variability  and  geographical 

differences in distribution (Simmonds et al.  2011). Until recently, the only other 

virus classified as a hepacivirus was GBV-B, a virus recovered from a laboratory 

The Origin of Hepatitis C Virus                                                                          3

Fig.  1   Phylogenetic  tree  of  members  of  the  family  Flaviviridae  showing  its  primary  division 

into four genera. The tree was based on comparison of conserved regions of the RNA polymer-

ase sequences (positions 7704–8550 numbered as in the HCV-1 genome, AF011751) from repre-

sentative variants within each genus and species. The unrooted phylogenetic tree was constructed 

by neighbour-joining of (uncorrected) amino acid p-distances. A divergence of 0.1 (10  % amino 

acid sequence divergence) is depicted on the scale bar. Variants variously described as GBV-A, 

GBV-C  and  hepatitis  G  virus  have  been  assigned  to  the  new  proposed  genus,  Pegivirus  as 

recently proposed (Stapleton et al. 2011)

housed tamarind [New World primate; (Simons et al.  1995; Muerhoff et al. 1995)]. 

Only the one isolate of GBV-B has been identified to date and its ultimate origin  

(primate or non-primate) remains unclear.

Members of the Hepacivirus genus are distinct genetically and in genome organ-

isation  from  members  of  the  recently  assigned  Pegivirus  genus  (Stapleton  et  al. 

2011) (Fig.  1). This group comprises a number of non-pathogenic viruses infecting 

humans apes (Adams et al.  1998; Birkenmeyer et al.  1998), non-human primates 

(Simons et al.  1995) and more recently, bats (Epstein et al. 2010). The recent pro-

posal to re-designate these viruses as human, simian and bat pegiviruses (Stapleton 

et  al.  2011)  was  designed  to  dispel  the  confusion  in  their  original  nomenclature 

(terms such as GB virus C and hepatitis G virus have both been applied to pegivi-

ruses  infecting  humans)  and  to  differentiate  these  viruses  clearly  from  GBV-B,  a 

member of the Hepacivirus genus.

HCV genotypes are substantially divergent in sequence from each other and fall 

into 7 phylogenetic clades, designated as genotypes (Fig.  2). Within these, a variable 

number of sub-groupings are apparent. HCV variants circulating in Western countries 

have been designated as subtypes, of which 1a, 1b, 2a, 2b, 3a, 4a and 6a are the most 

frequently identified. HCV subtypes are epidemiologically distinct, with differences 

in risk group targeting and geographical distributions that reflect their recent epidemic 

spread. As examples, genotype 3a (along with 1a) typically infects injecting drug users 

in Northern Europe and 4a in most frequently found in the Middle East. Genotypes 

Fig.  2   Evolutionary tree of NS5B sequences of HCV genotypes 1–7 (positions 8276–8615 as 

numbered  as  in  the  H77  reference  sequence).  High  diversity  areas  in  sub-Saharan Africa  and 

South East Asia contain a large number of variants additional to subtypes such as 1a, 1b and 3a 

found in Western countries, displaying an endemic pattern of diversity. The tree was constructed 

by  neighbour-joining  using  maximum  composite  likelihood  distances  as  implemented  in  the 

MEGA 4 program (Tamura et al. 2007). The scale bar depicts an evolutionary distance of 0.05

The Origin of Hepatitis C Virus                                                           

1b,  2a  and  2b  infections  are  in  contrast  most  prevalent  in  older  population  groups 

throughout Europe and Asia and are most frequently linked to past blood transfusions.

A  distinct  pattern  of  viral  diversity  is  observed  in  areas  such  as  sub-Saharan 

Africa and South East Asia, where infections with individual genotype predomi-

nate  over  large  geographical  areas  (such  as  genotype  1  in  Central Africa,  geno-

type 2 in West Africa and genotype 6 in South East Asia), within which there is 

substantial genetic diversity. The pattern of diversity observed within HCV is thus 

both  the  consequence  of  its  very  recent  epidemic  spread  into  new  risk  groups, 

overlaid on top of the much older “endemic” circulation of HCV in sub-Saharan 

Africa  and  South  East Asia.  These  different  ways  to  conceptualise  “origins”  of 

HCV are discussed in the next two sections.

2   The Recent Spread of HCV

The discovery of HCV in 1989 (Choo et al.  1989) was a remarkable achievement 

that heralded the use of molecular methods for virus aetiological studies refractory 

to previously used virus isolation methods. The very active research programme 

throughout the 1970s and 1980s that culminated in the discovery of HCV was pri-

marily driven by pressing concerns of clinicians and epidemiologists who increas-

ingly recognised chronic non-A, non-B hepatitis associated with blood transfusion 

and therapy with plasma-derived blood products (Prince et al.  1974; Feinstone et 

al.  1975; Alter  et  al.  1975).  Since  the  development  of  effective  diagnostic  tests 

for HCV, the full scale of the spread of HCV became rapidly apparent. It is cur-

rently thought that HCV chronically infects 170  million people worldwide, 3  % 

of the world’s population and creates a huge disease burden from chronic progres-

sive liver disease (Pawlotsky  2003; Hoofnagle 2002; Seeff 2002). In addition to 

recipients  of  blood  transfusion  and  medical  treatment  with  unsterilised  needles, 

diagnostic screening has identified the extensive spread of infection through nee-

dle-sharing  drug  abuse,  an  epidemic  starting  in  the  1960s  or  earlier  in  Western 

countries  and  the  primary  route  of  ongoing  transmission  of  infection  following 

the introduction of effective blood donor screening and blood product inactivation 

steps in the 1990s (Nelson et al. 2011).

Both the time of initial spread of HCV into Western countries and the popula-

tion dynamics of the epidemic can only be indirectly inferred. However, available 

evidence is consistent with relatively recent dates for its worldwide spread although 

it likely preceded the AIDS epidemic by some decades. A lack of samples available 

for screening collected before the Second World War has prevented a direct demon-

stration of this hypothesis and reconstruction of the HCV epidemic has been largely 

based  on  modelling  evolutionary  histories  of  currently  circulating  variants  and  by 

identifying historical factors such as widespread use of blood transfusion and other 

parenterally delivered treatments and vaccinations that facilitated HCV transmission.

In  epidemiological  terms,  transmission  of  HCV  through  sexual  contact  or  from 

mother to child is inefficient and infrequent (Wasley and Alter 2000; Pradat and Trepo 

2000; Thomas 2000). The restriction of HCV transmission through primarily paren-

teral routes therefore implicates medical treatment with unsterilised needles (including 

large-scale  vaccination  programmes),  blood  transfusion  and  more  recently  inject-

ing drug use as routes as the principal means of HCV spread and a relatively recent 

timescale (Drucker et al.  2001). None of these risk factors were common before the 

Second World War and supports the current model for the spread of genotypes 1b and 

type 2 subtypes from the 1940s–1950s, overlaid by more recent transmission among 

IDUs from the 1960s onwards (Pybus et al. 2001; Cochrane et al. 2002).

This scenario is strongly supported by genetic analysis of HCV genotypes and 

subtypes  most  frequently  detected  among  IDUs  and  those  infected  previously 

through  medical  treatment. A  recent  large-scale  coalescent  analysis  of  1a  and  1b 

subtypes demonstrated relatively small and constant population sizes for both sub-

types from the early twentieth century followed by an exponential period of popu-

lation growth between the 1940s and 1980s in the USA (Magiorkinis et al. 2009). 

The slowing of population growth thereafter is additionally consistent with reduc-

tions in blood transfusion risk through HIV-1 followed by HCV screening and the 

expansion of needle exchange programmes that have led to significant falls in HCV 

incidence among IDUs. Emphasising the global nature of the recent spread of HCV, 

parallel  phylogeographic  analyses  have  revealed  similar  demographic  histories 

of these subtypes in Brazil, Indonesia and Japan (Nakano et al.  2004). A detailed 

analysis of reconstructed population sizes of HCV and the emergence of parenteral 

routes of exposure in Japan Egypt and the USA further strengthens these conclu-

sions (Mizokami et al.  2006), including the close links between HCV emergence 

and parenteral antischistosomal therapy in Japan and subsequently in Egypt. In the 

latter, the extremely high population prevalence of HCV is dominated by genotype 

4a, whose spread can be reconstructed to have occurred between the 1930s–1950s, 

a  period  that  coincides  with  targeted  extensive  antischistosomal  injection  cam-

paigns using largely unsterilised injection equipment (Pybus et al. 2003).

   Collectively, these and several further combined phylogenetic and epidemiologi-

cal reconstructions provide a convincing narrative for the spread of HCV worldwide. 

Although earlier by some decades, its spread is paralleled by the explosive world-

wide spread of HIV-1 from Africa from the 1980s onwards leading to the current 

AIDS pandemic. In one sense, the question of the origins of HCV has likely already 

been answered. However, where HCV was before then and what factors led to its 

emergence are much less well understood and are discussed in the next section.

3   Endemic Circulation of HCV

While  the  epidemic  spread  of  HCV  is  associated  with  specific,  very  prevalent 

subtypes such as 1a, 1b, 3a and 4a, these represent a small part of the diversity 

existing with HCV. In sub-Saharan Africa and South East Asia, a quite distinct pat-

tern  of  genetic  diversity  exists  (Fig.  2).  Discounting  recent  introductions,  infec-

tions in large, geographically contiguous areas among several countries in Central 

Africa or the South East Asian peninsula are dominated by individual genotypes                                                        

(genotypes  1  and  6  respectively  in  these  examples).  Individual  variants  within 

these  genotypes  show  striking  genetic  diversity  from  each  other  matching  the 

genetic divergence observed between subtypes such as 1a and 1b found in Western 

countries. For example, sequence characterisation of genotype 2 variants infecting 

23 blood donors in Ghana (West Africa) revealed the presence of 20 highly diverse 

variants  that  would  merit  their  assignment  as  new  subtypes,  as  divergent  from 

each  other  as  2a  is  from  2b  (≈25  %  nucleotide  sequence  divergence)  (Candotti  

et al. 2003). Although far from fully mapped systematically, infections throughout 

Western Africa are predominantly by genotype 2 (Candotti et al.  2003; Jeannel et 

al.  1998; Mellor et al.   1995; WansbroughJones et al.  1998; Ruggieri et al.  1996), 

while  those  in  Central Africa,  such  as  the  Congo,  Cameroon  and  Gabon  are  by 

genotypes 1 and 4 (Mellor et al.  1995; Bukh et al. 1993; Fretz et al. 1995; Stuyver 

et al.  1993; Menendez et al.  1999; Xu et al.  1994; Ndjomou et al. 2003; Li et al. 

2009,  2012).  Genotype  3  and  6  are  typically  found  in  the  Indian  sub-continent 

and South East Asia (Mellor et al.  1995; Tokita et al.  1994,  1994,  1995; Lu et al. 

2008). It is further suspected, although with very limited data that genotypes 5 and 

7 are concentrated in Central/Southern Africa.

The extensive genetic heterogeneity of HCV in these regions has been described 

as  an  “endemic”  pattern  of  diversity  and  is  consistent  with  its  long-term  pres-

ence and diversification in these populations. As such, it is currently hypothesised 

that they represent source areas fuelling the worldwide spread of HCV in the last 

100–200  years. Indeed, the distinct subtypes that have been described in Western 

countries  such  as  1a,  1b  and  3a  might  simply  represent  the  explosive  expansion 

of certain variants within new risk groups for infection. Although we do not know 

and  may  never  be  able  to  reconstruct  their  ultimate  origins  and  initial  transmis-

sion pathways, 1a, 1b, 3a and others may simply happen to be the most success-

ful of variants that entered previously unexposed and highly susceptible individuals 

exposed  parenterally.  In  the  same  way  that  HIV-1  subtype  B  entered  and  spread 

within male homosexuals and IDUs in the USA and subsequently in Europe (Gao 

et al.  1999), our current collection of classified subtype might similarly represent 

founder viruses that were among the first to spread epidemically in the last century 

in Western countries where HCV was first genetically characterised.

Supporting  this  model  are  the  more  recently  described  examples  of  introduc-

tions  and  varying  degrees  of  local  spread  of  a  range  of  otherwise  undescribed 

“subtypes”  of  HCV.  As  examples,  substantial  diversity  and  restricted  distribu-

tions  of  genotype  2  variants  infecting  have  been  described  in  Europe  (Thomas  

et al. 2007), Indonesia (Utama et al. 2010) and throughout the Caribbean (Sulbaran 

et al. 2010; Martial et al. 2004), the latter examples in particular perhaps represent-

ing the shipment of infected West Africans through the slave trade in the eighteenth 

and nineteenth centuries (Markov et al. 2009). The more recent spread of genotype 

4 variants within Cameron and Egypt through medical treatment (Pybus et al. 2003; 

Pepin and Labbe 2012), into Mediterranean countries and the recent rapid spread of 

genotype 4 variants among IDUs in Southern Europe (Nicot et al. 2005; de Bruijne 

et al. 2009) provide further examples of this model (Ndjomou et al. 2003).

What  remains  unexplained  is  the  nature  of  the  “endemic”  circulation  of  HCV 

in these implicated source areas and in particular the transmission routes that have 

sustained  long-term  circulation  of  HCV  in  what  have  been  until  recently  relatively 

frequently highly isolated human communities. As discussed, transmission by either 

sexual contact or from mother to child is inefficient at least in areas where it has been 

studied (Wasley and Alter 2000; Pradat and Trepo 2000; Thomas 2000) and various 

factors that may enhance transmission have been proposed. Examples include sexu-

ally  transmitted  infections  (STIs),  circumcision,  excision  and  scarification  practices 

(Shepard et al.  2005) which at least in Central Africa show associations with HCV 

infection and more remote possibilities such as mosquito or other arthropod vectors 

(Pybus et al. 2007). These various hypotheses are yet to be resolved.

The time depth of “endemic” circulation of HCV remains similarly uncertain. 

Molecular  evolutionary  reconstructions  of  the  recent  spread  of  HCV  have  pro-

duced robust and reproducible estimates of its substitution rate (see previous sec-

tion).  Substitution  rates  extrapolated  to  the  much  larger  sequence  distances  that 

exist within genotypes (such as between subtypes 1a and 1b) have been used to 

provide  some  kind  of  estimate  of  the  minimum  period  over  which  the  observed 

“endemic”  diversity  developed  (Pybus  et  al.  2001;  Markov  et  al.  2009;  Smith  

et al.  1997; Pybus et al. 2009). Reconstructed dates for the common ancestor of 

different genotypes vary but are estimated to be several hundred years ago for gen-

otype 2 and even longer for genotype 6. In the opinion of the author of this review, 

such estimates should be treated with extreme caution and minimum estimates at 

best. Extrapolating substitution rates measured over short observation intervals to 

the much longer periods of subtype and genotype diversification makes assump-

tions  about  the  evolutionary  process  that  are  not  self-evidently  justified.  Factors 

such as extreme rate variation between sites, large-scale RNA secondary structure, 

greater  selective  constraints  and  fitness  optimisation  of  viruses  association  with 

large population sizes may create substantial underestimates of the real period of 

virus diversification [reviewed in (Sharp and Simmonds 2011)]. While it is beyond 

the scope of the current article to discuss this in detail, what can be said is that the 

subtype diversification in HCV and thus the likely period of endemic circulation 

in sub-Saharan Africa and Southern Asia is prolonged and likely long before long 

distance travel and interactions with colonial powers. These genotypes are there-

fore likely to be truly indigenous to areas where they currently endemically cir-

culate. This takes us a step further back to the question of the ultimate source of 

HCV. This much more speculative area will be reviewed in the next section.

4   Origins of Human Infections and HCV Homologues  

   in Other Mammals

A  compelling  scenario  which  has  driven  much  research  endeavour  in  the  last  

decade  and  a  half  is  the  hypothesis  for  a  non-human  primate  source  for  HCV 

infections in humans. The theory makes epidemiological sense in that high diver-

sity areas of endemic circulation in humans are those where human, ape and Old 

World  monkey  populations  overlap.  Before  long  range  travel  and  the  means  for 

wider  dissemination,  human  infections  acquired  zoonotically  from  non-human 

primates  may  have  remained  geographically  focussed  and  thus  account  for  the 

specific  association  of  each  of  the  genotypes  in  defined  areas  of  sub-Saharan 

Africa  and  Southern Asia. The  idea  of  a  non-human  primate  source  for  humans 

is additionally consistent with the observation of its poor transmissibility between 

humans, largely confined to parenteral routes and a reflection perhaps of its lack of 

host adaptation as might also be its severe, immune-mediated liver pathology.

This model is, of course, also driven by the tempting analogy with the origin 

of HIV-1, which similarly exploded worldwide out of Central Africa in the twen-

tieth  century  through  infections  directly  or  indirectly  from  chimpanzees  (Gao  et 

al.  1999). As might be imagined for HCV, HIV-1 infections acquired through con-

tact with Central African chimpanzees (Pan troglodytes) may have been occurring 

for centuries or millennia, but only in the last 50–70 years were demographic and 

societal changes suitable for its wider pandemic spread. Important differences from 

the  HIV-1  model  of  origins  would  be  the  earlier  spread  of  HCV  worldwide  and 

the existence of multiple potential source areas and possibly different primate spe-

cies. These  would  be  necessary  to  account  for  the  distinct  endemic  distributions 

of  HCV  genotypes  in  different  parts  of  sub-Saharan Africa  and  also  South  East 

Asia. Finally, into this model would come GBV-B, which might perhaps represent 

a much more divergent homologue of HCV in a New World primate species.

Despite   the   elegance,   plausibility   and   potential   medical   relevance   of   the  

primate  origin  hypothesis,  the  fundamental  problem  has  always  been  that  HCV 

or homologues cannot be found in ape or monkey species, at least to the author’s 

knowledge.   Extensive   screening   programmes   both   published   (Makuwa   et   al. 

2003, 2006) and unpublished have failed to document either seropositivity or viral 

sequences in literally hundreds or thousands of plasma samples collected from dif-

ferent ape and monkey species. As a possibly related problem, GBV-B has to date 

never been recovered nor serological evidence for past infections obtained from any 

tamarind or other New World primate among wild populations in South America.

Without an obvious primate source for infection and the genetic evidence for 

circulation of HCV in what would have been largely isolated human populations 

in distinct parts of the world for centuries or more likely millennia, studies of the 

ultimate origins of HCV have reached something of a frustrating impasse. As with 

many other virus discoveries, however, its resolution is likely to be considerably 

stranger than could have been imagined even as recently as last year. By pure ser-

endipity, attempts by Kapoor and colleagues to identify viral causes of respiratory 

disease  in  dog  held  in  kennels  by  deep  sequencing  revealed  the  existence  of  an 

RNA  virus  extraordinarily  similar  to  HCV  (Fig.  3)  but  with  suspected  biologi-

cal and epidemiological properties quite different from what had been previously 

described for both HCV and GBV-B (Kapoor et al. 2011).

The  virus,  initially  termed  canine  hepacivirus  (CHV)  showed  approximately 

50  %  nucleotide  sequence  divergence  from  HCV.  Data  presented  in  that  study 

demonstrated  high  viral  loads  in  respiratory  samples  and  an  implied  respiratory 

route of transmission and association with respiratory disease, none of which have 

been observed in HCV (or GBV-B) infections. Infections were found in dogs from 

Fig.  3   Amino  acid  sequence  divergence  scan  of  members  of  the  Hepacivirus  genus,  with 

genome diagram drawn to scale underneath plot. NPHV is more similar to HCV throughout the 

genome (red line) than GBV-B (dark green line). However, NPHV/HCV divergence is substan-

tially  greater  than  between  genotypes  (dark  and  light  blue  lines  respectively).  This  figure  has 

been adapted from Fig. 2 in (Kapoor et al. 2011). For details related to the HCV polyprotein and 

the  cleavage  products  see  chapter  “Hepatitis  C  virus  Proteins  From  Structure  to  Function”  by 

Moradpour and Penin, this volume

different regions of the USA but partial genome characterisation demonstrated a 

virtual absence of genetic diversity that would be expected for an RNA virus like 

HCV.  Whether  the  virus  spread  systemically  or  persisted  was  not  demonstrated 

although imaging of viral RNA in liver by in situ hybridisation was presented.

More recently, further studies of the host range of hepaciviruses in a range of 

mammalian species were performed by the same group using a serological assay 

for  antibodies  to  a  peptide  expressed  from  the  NS3  region  of  the  CHV  genome 

(Burbelo  et  al.  2012).  This  produced  further  unexpected  findings.  From  the  80 

dogs, 81 deer, 84 cows, 103 horses and 14 rabbits screened, only horses showed 

frequent seropositivity (35  %) with one weak positive sample from a cow while, 

remarkably, all 80 dogs were seronegative. Of the 103 horse samples, 8 were PCR-

positive (all seropositive) and from each of these near-complete genome sequences 

were  obtained.  Sequences  showed  moderate  sequence  diversity  from  each  other 

(6.4–17.2  %  nucleotide  sequence  divergence)  with  the  CHV  sequence  grouping 

with horse-derived variants. As viruses similar to CHV were frequently found in 

horses, the investigators coined the name NPHV to describe this group.

The diversity of NPHV variants was somewhere between inter-subtype and within 

subtype divergence of HCV, certainly not the equivalent of HCV genotypes (Fig. 3). 

The high degree of amino acid sequence conservation did, however, contrast mark-

edly with the degree of sequence variability at synonymous (non-coding) sites in the 

genome.  The  extraordinarily  low  ratio  between  synonymous  to  non-synonymous 

substitutions (0.03–0.06) indicates however that its evolution has been more severely 

constrained  and/or  less  subject  to  positive  selection  pressures  than  HCV.  These 

low  sequence  distances  are  therefore  not  necessarily  an  indication  of  their  recent 


There  was  no  information  available  on  the  clinical  features  of  infection  with 

NPHV in horses. To address this we have recently surveyed horses in Scotland by 

PCR and identified 3 viraemic horses from 136 screened (Lyons et al.  2012). Using 

veterinary records and further sampling, these have been evaluated for evidence of 

hepatitis  or  other  systemic  disease  manifestations.  Positive  horses  were  originally 

referred for reasons such as lameness, foot abscess or respiratory infections with no 

evidence  of  the  ill-health  that  might  be  associated  with  severe  systemic  infections. 

Although most liver indices were in the normal range, gamma glutamyl transferase 

(GGT)  levels,  a  sensitive  marker  of  liver  inflammation  were  marginally  or  signifi-

cantly elevated along with elevation in bile acids, perhaps providing some tentative 

evidence for an aetiological role of NPHV in hepatitis. Repeated sampling from one 

of the study horses demonstrated persistence over  at least a 6-month period and viral 
loads  comparable  to  those  observed  in  HCV  infections  (7  ×  10 −5  ×  10   RNA 

copies/ml).  Respiratory  samples  and  peripheral  blood  mononuclear  cells  from  the 

infected horse have proven uniformly negative although no opportunity to perform a 

liver biopsy of the horse has yet presented itself. Overall, these more recent findings 

provide some reassurance that hepacivirus infections in horses are both persistent and 

potentially associated with mild liver disease rather than the respiratory disease and 

viral secretion found originally in dogs. However, large-scale PCR-based screening of 

other mammalian species using primers conserved between NPHV and HCV failed to 

detect hepaciviruses in dogs (nearly 200 screened), cats, pigs and rodents (Lyons et al. 

2012), very much as found in the previous serology-based study (Burbelo et al. 2012).

This, to date, represents current published knowledge of non-human hepacivi-

ruses, a series of findings that present several conflicting interpretations and dif-

ficulties. This author believes that, despite the negative results from screening so 

far,  domestic  horses  are  most  unlikely  to  be  the  only  mammalian  species  (other 

than human or tamarins) infected with hepaciviruses and there is clearly much to 

be learned in short term from more extensive screening.

5   Concluding Thoughts

Our understanding of the ultimate origins of HCV infection in humans will doubt-

less be hugely enhanced once proper mammalian screening for other hepaciviruses 

has been performed and the genetic diversity and, more importantly, the specific-

ity of different hepaciviruses to individual host species is more clearly established. 

From  such  studies,  it  may  well  turn  out  that  hepaciviruses  are  highly  catholic  in 

their host range perhaps capable of jumping between horses and dogs as suggested 

by the published screening data (Burbelo et al.  2012; Kapoor et al. 2011) and per-

haps all species in-between. An ability of hepaciviruses to jump species is consistent 

with the observation that the NPHV protease is able to cleave human MAVS and 

TRIF (Parera et al. 2012); this ability to prevent interferon signalling is essential for 

HCV replication (Foy et al.  2005) and may therefore function across species bar-

riers and potentially favour zoonotic transmission. A wide mammalian host range 

is also characteristic of vector-borne flaviviruses and pestiviruses, the latter at least 

within ruminant species. In this scenario, HCV infections in humans may well have 

a zoonotic origin consistent with its relatively recent emergence (at least in Western 

countries). While being still relatively poorly adapted for infecting its new host, this 

may further account for its peculiar, inefficient transmission routes.

Alternatively,  it  may  be  that  each  hepacivirus  species  is  uniquely  adapted  to 

one  target  species,  HCV  in  humans,  NPHV  in  horses  and  perhaps  further  hepa-

civiruses  in  other  mammalian  species  waiting  to  be  discovered.  The  ability  of 

HCV to persist lifelong in humans, an attribute that greatly enhances its transmis-

sibility and evidence for subtle virus/host interactions such as the enhancing role 

of  human  micro  RNA,  miR-122  expressed  in  liver  on  virus  replication  (Jopling  

et al. 2005) certainly hints at long-term virus/host co-adaptation. HCV may always 

have infected humans throughout their evolution and it is only through greater life 

expectancy,  scope  for  epidemic  transmission  and  better  surveillance  and  under-

standing of causes of hepatitis that it has come to current medical attention. In that 

sense, HCV does not have an “origin”, it is just one of those viruses like herpes-

viruses that have always infected humans and before them hominoids, proto-apes 

and potentially right back to the ancestor of mammals themselves.

Future  research  will  be  truly  important  in  resolving  these  two  diametrically 

opposed possibilities.


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