by week 3 p.i. (fig. S2, A to C). We consistently
detected high viral titers in liver tissue (106 to 108
NrHV GE per gram of tissue) but not in spleen,
kidney, and lung (Fig. 1D). NrHV replication was
dependent on miR- 122, a liver-specific microRNA
required for HCV replication (7, 8) (Fig. 1E).
Like HCV in humans, NrHV infection was not
associated with signs of acute disease or mortality. Chronically infected NRG and AG129 mice
showed minimal to mild liver inflammation at
week 35 p.i. (fig. S3).
To identify the cellular mediators of NrHV
clearance, we characterized the immune response
during acute resolving NrHV infection in 8-week-
old C57BL/6J mice. High-titer viremia was detect-
able as early as 24 hours p.i.; titers started to
decline at day 15 p.i. and were undetectable at
day 21 p.i. (Fig. 2A). Early acute infection (days 3
to 9 p.i.) was associated with an expansion of
intrahepatic Ly6C+ monocytes and NKp46+ NK
cells (Fig. 2B). Starting at day 9 p.i., we observed a
substantial increase in proliferating (Ki67+) intra-
hepatic CD4+ and CD8+ T cells. These cells were
characterized by a CD44+ effector phenotype with
an antiviral type 1 differentiation signature (9)
as indicated by high T-bet expression and IFN-g
production (Fig. 2, B to E, and fig. S4, A and
B). CD8+ T cells also showed a significant up-
regulation of granzyme B (fig. S4C). The T cell
response was predominant in the liver and less
pronounced in peripheral blood and spleen (Fig.
2 and fig. S4). High levels of intrahepatic effector
T cells coincided with a decline in viremia starting
at day 15 p.i. and were associated with elevated
alanine transaminase (ALT) levels (Fig. 2F), in-
dicating T cell–mediated liver injury. Hepatic
leukocyte infiltration was confirmed by histology
(Fig. 2G). We detected NrHV-specific IFN-g pro-
duction by CD4+ and CD8+ T cells in response to
peptide pools covering the NrHV proteins NS3
and NS4 at multiple time points p.i., with a peak
response by intrahepatic CD8+ T cells against the
NS4 pool at day 21 pi. (fig. S5). These results are
consistent with a strong and broadly directed
virus-specific T cell response during acute resolv-
ing NrHV infection.
T cells play an important role in HCV and hepatitis B virus (HBV) infection in humans and in
the LCMV (lymphocytic choriomeningitis virus)
mouse model (10, 11). T cell depletion studies in
HCV-infected chimpanzees showed that these
cells are critical for viral clearance during primary and secondary infection (12, 13).
To analyze if T cells play an essential role in
NrHV clearance, we depleted them in mice (fig.
S6, A and B). Transient CD4+ T cell depletion
initiated before infection resulted in chronic
infection (analyzed until day 210 p.i.) in C57BL/6J
and BALB/c mice even after the CD4+ cell population recovered. Transient CD8+ T cell depletion
led to delayed viral clearance as compared to
controls (Fig. 3A and fig. S7, A and B). Mice that
were constantly (every 10 days p.i.) depleted of
CD8+ T cells failed to clear the virus (Fig. 3B).
We next analyzed NrHV infection during recovery of CD4+ or CD8+ T cells after transient
day 7 p.i.
liver spleen kidney lungserum
day 7 p.i.
Sequence development during mouse passage
68 29 88
Inoculum Long-term NRG Serial NRG
Putative non-synonymous mouse adaptive mutations
Position Inoculum Change
Serum pool, deep seq
190 T S 1% 73% 6/9 3/4
195 T N 49% 11% 3/9 1/4
353 V L 45% 89% 7/9 7/9 3/4 3/4
361 V A/I 19% 23% 3/9 0/4
369 F I/L 4% 62% 4/9 0/4
370 V A 11% 10% 1/9 0/4
371 S P 45% 32% 3/9 0/4
550* N S/D/Y 80% 82% 7/9 7/9 0/4 0/4
*Changing predicted glycosylation motif Nx(S/ T)
(NRG serum) LOQ
weeks post infection
weeks post infection
Fig. 1. NrHV establishes hepatotropic infection in common laboratory
mice. Four-week-old mice were infected intravenously (i.v.) with 104 GE
of NrHV. Viremia (NrHV RNA GE/ml) was analyzed by reverse
transcription–quantitative polymerase chain reaction (RT-qPCR). LOQ:
limit of quantification; n.d.: not detectable. (A) Viremia in NRG, A129,
AG129, and MAVS−/− mice infected with rat-serum–derived NrHV.
(B) Viremia in C57BL/6J and BALB/c mice infected with either rat-
or NRG-serum–derived virus. (C) NrHV sequence development during
mouse passage (see fig. S1). Percentages of two identified subpopula-
tions in the rat inoculum and long-term and serial pools are shown.
Below, putative mouse adaptive positions (all variants <1% in the
inoculum). Fraction of variants determined by deep sequencing of the
pools or consensus ORF sequences from the nine mice of the last
serial passage and four C57BL/6J mice challenged with the serial pool
(week 1 p.i.) are shown. (D) Viral loads in tissue and serum of C57BL/6J
at day 7 p.i. (E) Viremia in miR- 122−/− mice and controls at day 7 p.i.
Panels (A), (B), (D), and (E) show representative data from two to five
independent experiments with four or five mice per group (mean ±SEM).
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