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We thank H. Osvath, I. Jacobs, A. Anikin, M. Lambert, L. Hall,
C. Balkenius, P. Johansson, and T. Persson for help with the
manuscript. This research was funded by the Swedish Research
Council grants 2012-1235 and 2014-6402, the latter conjoined with
Marie Sklodowska Curie Actions, Cofund, INCA 600398. The authors
declare no conflict of interest. Data are available in the main article
and supplementary materials.
Materials and Methods
Figs. S1 to S3
27 January 2017; accepted 8 June 2017
HEPATITIS C VIRUS
Mouse models of acute and chronic
Eva Billerbeck,1 Raphael Wolfisberg,2 Ulrik Fahnøe,2 Jing W. Xiao,1 Corrine Quirk,1
Joseph M. Luna,1 John M. Cullen,3 Alex S. Hartlage,4 Luis Chiriboga,5
Kalpana Ghoshal,6 W. Ian Lipkin,7 Jens Bukh,2 Troels K. H. Scheel,1,2
Amit Kapoor,4 Charles M. Rice1*
An estimated 71 million people worldwide are infected with hepatitis C virus (HCV).
The lack of small-animal models has impeded studies of antiviral immune mechanisms.
Here we show that an HCV-related hepacivirus discovered in Norway rats can establish
high-titer hepatotropic infections in laboratory mice with immunological features
resembling those seen in human viral hepatitis. Whereas immune-compromised mice
developed persistent infection, immune-competent mice cleared the virus within 3 to
5 weeks. Acute clearance was T cell dependent and associated with liver injury. Transient
depletion of CD4+ T cells before infection resulted in chronic infection, characterized by
high levels of intrahepatic regulatory T cells and expression of inhibitory molecules on
intrahepatic CD8+ T cells. Natural killer cells controlled early infection but were not
essential for viral clearance. This model may provide mechanistic insights into hepatic
antiviral immunity, a prerequisite for the development of HCV vaccines.
Hepatitis C virus (HCV), a major cause of human liver cirrhosis and cancer, is nar- rowlyrestricted to thehuman liver (1). Cur- rently, there are no immune-competent small-animal models for HCV, and this
limits the study of host-virus interactions and the
development of vaccine strategies (2). A prophylactic and protective vaccine against HCV, which
will likely be needed for global HCV eradication,
does not exist (3).
Several HCV-related hepaciviruses have been
discovered in horses, bats, and wild rodents (4).
In 2014, a hepacivirus was identified in Norway
rats from New York City (5) and named Norway
rat hepacivirus (NrHV) or rodent hepacivirus-nr-1
(RHV-nr-1). Similar to HCV in humans, NrHV can
establish a hepatotropic infection in rats (5). Rats
represent a natural context in which to study NrHV.
However, given numerous genetic variants and
tools available for mice that permit deep mechanistic studies, we aimed to develop a mouse model
of NrHV infection, speculating that NrHV might
infect laboratory mice, given their close phylogenetic relationship to rats.
We first explored whether NrHV could establish
infection in the immune-compromised mouse strains
NRG (NOD-Rag1−/−IL2Rg−/−), A129 (IFNRab−/−),
and AG129 (IFNRab−/−IFNRg−/−) that lack adaptive immunity, type I, and type I/II interferon
(IFN) signaling, respectively. We infected 4-week-
old mice intravenously with 104 genome equivalents (GE) of NrHV derived from the serum of an
infected laboratory rat. NrHV established a high-titer (106 to 108 GE per milliliter of serum) chronic
infection in these mice (Fig. 1A). Mice lacking MAVS
(mitochondrial antiviral signaling protein) cleared
the virus within 3 weeks postinfection (p.i.) (Fig. 1A).
Intravenous infection of the immune-competent
mouse strains C57BL/6J and BALB/c with 104 GE
resulted in a high-titer (106 to 108 GE/ml serum)
acute resolving infection (Fig. 1B). NrHV derived
from rat serum was cleared significantly faster
than NrHV passaged one time through NRG mice
(Fig. 1B), indicating that NrHV can adapt to the
mouse host. To test the extent of NrHV adapta-
tion in NRG mice, we performed either 12-week
long-term adaptation in one mouse or serial-
passage adaptation through five mice (4-week
infection of each mouse) (fig. S1A). We then chal-
lenged naïve NRG and C57BL/6J mice with 9 ×
104 GE of either the pooled adapted (long-term
pool or serial pool) or the parental virus. Adapted
viruses showed 0.5 to 1 log higher viral titers at
week 1 p.i. and persisted longer in C57BL/6J mice
than did the parental virus (fig. S1B), suggesting
increased viral fitness in the mouse host.
Comparing the consensus NrHV genome open
reading frame (ORF) sequences of the rat inoculum with those of the adapted viruses revealed
changes in 2 and 59 nucleotide positions in the
long-term and serial-passage pool, respectively
(fig. S1C). Phylogenetic analysis of full-ORF clones
revealed the presence of two subpopulations in
the inoculum; the minor one was selected during
the serial passage (Fig. 1C and fig. S1D). Single
coding mutations in viral envelope proteins E1
and E2 occurred in both pools and in individual
NRG mice from passage 5 of the serial adaptation
(Fig. 1C, fig. S1E, and tables S1 and S2). The E1
mutation V353L (Val353→Leu ), combined with
either T190S (Thr190→Ser ) (serial pool) or T195N
(Thr195→Asn) (long-term pool), represent putative mouse adaptive mutations, as they were maintained in challenged C57BL/6J mice. Mutations
at amino acid position 550, combined with at least
one mutation in the cluster 361/369/370/371, were
selected in NRG mice, but were immediately lost in
C57BL/6J mice. Changing position 550 would disrupt a predicted Nx(S/T) glycosylation site (where
x is any amino acid except proline), possibly de-shielding neutralization epitopes as observed for
HCV (6). For subsequent experiments we used serial pool virus as our source of NrHV.
Our results indicate that NrHV is both highly
infectious and hepatotropic in mice. Even a low-dose infection with 10 GE resulted in high-titer
viremia. The dose did not influence the outcome
of infection, as mice infected with 104, 103, 102, and
10 GE cleared the virus with similar kinetics. In
contrast, age influenced clearance: 4-week-old
mice typically cleared the virus by week 5 p.i.,
whereas 2- to 6-month-old mice cleared the virus
1Laboratory of Virology and Infectious Disease, The Rockefeller
University, New York, NY, USA. 2Copenhagen Hepatitis C
Program (CO-HEP), Department of Infectious Diseases and
Clinical Research Centre, Hvidovre Hospital and Department
of Immunology and Microbiology, Faculty of Health and
Medical Sciences, University of Copenhagen, Copenhagen,
Denmark. 3College of Veterinary Medicine, North Carolina State
University, Raleigh, NC, USA. 4Center for Vaccines and
Immunity, The Research Institute at Nationwide Children’s
Hospital and Department of Pediatrics, Ohio State University,
Columbus, OH, USA. 5Department of Pathology, New York
University Medical Center, New York, NY, USA. 6Department of
Pathology, Comprehensive Cancer Center, Ohio State
University, Columbus, OH, USA. 7Center for Infection and
Immunity, Mailman School of Public Health, Columbia
University, New York, NY, USA.
*Corresponding author. Email: firstname.lastname@example.org