Quantifying the Diversification of Hepatitis C Virus (HCV)
during Primary Infection: Estimates of the In Vivo
Ruy M. Ribeiro1¤, Hui Li2, Shuyi Wang2, Mark B. Stoddard2, Gerald H. Learn2, Bette T. Korber1,
Tanmoy Bhattacharya1, Jeremie Guedj1, Erica H. Parrish2, Beatrice H. Hahn2, George M. Shaw2,
Alan S. Perelson1*
1Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America, 2Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania, United States of America
Hepatitis C virus (HCV) is present in the host with multiple variants generated by its error prone RNA-dependent RNA
polymerase. Little is known about the initial viral diversification and the viral life cycle processes that influence diversity. We
studied the diversification of HCV during acute infection in 17 plasma donors, with frequent sampling early in infection. To
analyze these data, we developed a new stochastic model of the HCV life cycle. We found that the accumulation of
mutations is surprisingly slow: at 30 days, the viral population on average is still 46% identical to its transmitted viral
genome. Fitting the model to the sequence data, we estimate the median in vivo viral mutation rate is 2.561025mutations
per nucleotide per genome replication (range 1.6–6.261025), about 5-fold lower than previous estimates. To confirm these
results we analyzed the frequency of stop codons (N=10) among all possible non-sense mutation targets (M=898,335), and
found a mutation rate of 2.8–3.261025, consistent with the estimate from the dynamical model. The slow accumulation of
mutations is consistent with slow turnover of infected cells and replication complexes within infected cells. This slow
turnover is also inferred from the viral load kinetics. Our estimated mutation rate, which is similar to that of other RNA
viruses (e.g., HIV and influenza), is also compatible with the accumulation of substitutions seen in HCV at the population
level. Our model identifies the relevant processes (long-lived cells and slow turnover of replication complexes) and
parameters involved in determining the rate of HCV diversification.
Citation: Ribeiro RM, Li H, Wang S, Stoddard MB, Learn GH, et al. (2012) Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection:
Estimates of the In Vivo Mutation Rate. PLoS Pathog 8(8): e1002881. doi:10.1371/journal.ppat.1002881
Editor: Claus O. Wilke, University of Texas at Austin, United States of America
Received April 12, 2012; Accepted July 12, 2012; Published August 23, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Portions of this work were done under the auspices of the U. S. Department of Energy under contract DE-AC52-06NA25396 and supported by the
National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) through Grant Number
8R01-OD011095-21, the NIH Center for HIV/AIDS Vaccine Immunology (AI67854) and through NIH grants AI028433, P20-RR018754, AI45008 and AI27767 and by a
developmental grant from the University of Pennsylvania Center for AIDS Research. RMR has received partial funding from the European Union 7th Framework
Programme under grant nu PCOFUND-GA-2009-246542 and from the Foundation for Science and Technology of Portugal. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
Hepatitis C virus (HCV) is a member of the hepacivirus genus
within the flaviviridae family of virus, and it has a single positive
stranded RNA molecule (,9500 nucleotides) as its genome [1–3].
After entering a cell this RNA is translated into a single large
polyprotein, which is cleaved to produce the viral structural and
non-structural (NS) proteins [1–3]. The NS5B protein is a viral-
specific polymerase, which is involved in replicating the HCV
RNA genome [1,4]. During genome replication the virion’s
positive strand RNA is copied into a complementary negative
strand, which then must be copied back to produce a new positive
strand. In the simplest replication model, this negative strand or a
complex of the original positive strand and the newly created
negative strand form an intermediate that acts as the template for
producing new positive strands. This template plus various non-
structural proteins form a structure called a replication complex
. If all new positive strands, and hence virions, are created from
the same replication complex, we say that replication occurs by a
‘‘stamping machine’’ mechanism [6–9]. However, HCV infected
cells often have more than one replication complex; indeed in vitro
and in situ studies suggest there are about 40 such complexes in one
infected cell [4,10].
The HCV polymerase is an RNA-dependent RNA polymerase
(RdRp) and hence does not possess error correcting mechanisms.
Thus HCV replication, like that of other RNA viruses, is highly
error prone [1–3]. Measuring the actual mutation rate, which
derives both from the (+)RNA to (2)RNA and the (2)RNA to
(+)RNA steps of replication, has been difficult [6,11,12]. A recent
study determined the intrinsic error rate of the HCV polymerase in
vitro using enzyme kinetic measurements . They found high
error rates, of ,1023per site, for transitions and about 100-fold
lower rates for transversions. Still, the in vivo mutation rate is likely
different. Mutation is difficult to estimate in vivo due to selection,
PLOS Pathogens | www.plospathogens.org1August 2012 | Volume 8 | Issue 8 | e1002881
multiple rounds of replication and incomplete sampling [6,11].
One proposed way to determine the in vivo mutation rate is to
estimate it based on the frequency of lethal mutants in the viral
population at any given time . In fact, classical genetics shows
that the frequency of a lethal mutation in a haploid population in
mutation-selection balance is m, the mutation rate. A recent study
used this method to estimate an upper limit for the in vivo mutation
rate of HCV as (1.1560.29)61024per nucleotide per replication
round , which is within the range of other RNA viruses .
This high mutation rate is consistent with the high degree of
HCV diversity found across the population of infected individuals
[14,15]. Indeed, HCV is highly variable, with multiple subtypes,
and a global diversity that is higher than that of HIV-1 .
Clearly, this population level diversity, which reflects the HCV
evolution rate, is in part prescribed by the mutation rate of the
virus in vivo . Moreover, in chronically infected individuals the
HCV viral population is also diverse . This diversity allows fast
evolution and escape from immune  or antiviral drug pressure
, and may contribute to HCV pathogenesis [18,20].
An important question is how HCV diversity is generated.
While it clearly depends on the mutation rate, we shall show using
a model of HCV replication that it also depends on other
parameters of the HCV life cycle [7–9], such as the long-lived
nature of infected cells, as compared to HIV infected cells [21,22],
the existence of multiple replication complexes within an infected
cell [4,10], and the turnover rate of these replication complexes. In
order to validate this model and obtain quantitative estimates of
the in vivo HCV mutation rate, we shall exploit our observations in
an accompanying report  and those of others [24,25] that
during the initial stages of primary infection the viral population is
comprised of discrete low diversity lineages of viral sequences
emanating from the transmitted/founder viral genomes .
Further, early on, diversity increases with time since infection. We
shall show that the rate of diversification is not constant but rather
slows as infection is established. Our model provides a quantitative
explanation for this phenomenon. Analyses of HIV evolution in
acute infection have been used to estimate the time since infection
[26,27]. Here, we know with reasonable accuracy the time of
infection, but use the same ideas to estimate the in vivo mutation
rate of HCV.
In Primary Infection HCV RNA Levels Expand Quickly and
then Plateau at a High Level
The early dynamics of viral increase in HCV infection is
different from that seen in other chronic infections, such as HIV
 and HBV . The HCV viral load in the subjects in this
study increases roughly exponentially until it reaches a plateau
(Figure 1A). This has also been observed in a prior study of
acute HCV infection  and observed in chimpanzees
experimentally infected with HCV . Quantitative charac-
teristics of this early increase are given in Table 1. The median
time between the last negative sample and the first HCV
positive sample in our dataset was 5 days, which is consistent
with a viral dynamics analysis of larger numbers of plasma
donors . Because of this short interval, we assumed that the
virus started expanding at the last negative sample. If the virus
started expanding after this, our estimated expansion rate would
be an underestimate. The median HCV RNA exponential
growth rate was 2.2/day, corresponding to a doubling time of
0.31 days (or 7.4 hours). The median peak viral load observed
was 36106HCV RNA IU/ml and it took a median of 21 days
to reach this level. The virus then stayed at approximately this
high viral load level for a median of at least 26 days. In two
subjects, we did not have enough follow-up to conclusively
affirm whether a plateau exists or not. These estimates are in
agreement with a previous study of 77 plasma donors with
longer follow-ups, which reported an estimate of ,6 days of
viral expansion before the first positive measurement (compared
to a median of 5 days in our dataset) and a mean plateau
duration of ,56 days .
The observation of the viral load plateau suggests that the
number of infected cells reaches a steady state level a couple of
weeks post infection. It is possible that this is a dynamic steady
state, with removal of infected cells in equilibrium with generation
of new infected cells. However, HCV is likely non-cytolytic ,
consistent with the normal levels of alanine aminotransferase
(ALT,40 IU/L is upper limit of normal [33,34]) in these
individuals early in infection (Figure 1B). In addition, prior work
has suggested that the cytolytic immune response takes weeks to
months to emerge [31,35,36] (consistent with an increase in ALT
to 106to 206the normal level late in acute infection ). Thus,
it is likely that the rate of infected cell death during this early
period is comparable to that of uninfected cells. The lifespan of
uninfected hepatocytes has been estimated as being on the scale of
months to years [38,39], and thus infected cell death is probably
negligible at these early times. In this case, the plateau in viral load
suggests an equilibrium where all cells that can be infected are
infected and producing virus. Assuming that there are 1011
hepatocytes in the liver , we estimate that a median of 6%
(with range 1.7%–22%) of these are infected across our subjects
(Table 1), consistent with experimental measurements in chronic
infection , including recent estimates by two-photon micros-
copy of frozen sections of liver tissue . Thus, primary HCV
infection is characterized by fast growth of viral load to a plateau
where only a minority of hepatocytes is infected.
Dynamics of Early HCV Diversification
To evaluate how HCV diversity changes during primary
infection, we performed single genome amplification (SGA)
Hepatitis C virus (HCV) is a RNA virus that infects over 170
million people across the world. It leads to a chronic
infection in the majority of people who are infected
(.70%). Most people only discover that they are infected
long after initial infection. Thus, it is difficult to study the
very early events in infection. Here we study 17 individuals
during the earliest possible stages of infection, from before
thevirus is detectable intheplasma to around35 dayspost-
infection. We focus on understanding the viral kinetics and
the diversification of HCV during this acute phase of
infection. During chronic infection HCV is present in the
host as a swarm of multiple variants generated by its error
prone copying. We studied the early diversification of HCV
during acute infection using a new mathematical model of
HCV replication. We found that after a phase of fast increase
in viral load, accompanied by viral diversification, there is a
stabilization of viral load and diversity levels. Using our
model, we were able to estimate for the first time the HCV
mutation rate during acute infection. We estimated the
nucleotide per genome replication (range 1.6–6.261025),
about 5-fold lower than previous estimates. We also used a
different approach, based on results of classical genetics, to
calculate HCV’s mutation rate and obtained consistent
Estimates of the In Vivo HCV Mutation Rate
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followed by direct amplicon sequencing [23,26], otherwise known
as single genome sequencing , at multiple time points in the
subjects shown in Figure 1. SGA is achieved through serial dilution
of the cDNA obtained by reverse transcription of HCV RNA from
plasma (see Methods and  for details). We amplified 59 half-
genome sequences, on average 4879 nucleotides, covering core,
E1, E2, p7, NS2 and most of the NS3 proteins of HCV. For early
samples, with low viral loads, we amplified the same region, but in
two separate assays of one quarter genome each to enhance
sensitivity of amplification. In this way, we obtained 84 sets of
sequences for the 9 subjects at multiple (between 3 and 5) time
points. On average, we had 44 sequences per time point. All of the
sequences were deposited in Genbank; see Li et al.  for further
details and accession numbers.
Figure 1. Profile of (A) viral load and (B) ALT in the subjects studied. The black symbols are the observed viral loads, the lines are the
simulated trajectories with the model described in methods, and the dashed lines correspond to 95% CI based on 100 simulations. The parameters
used for the simulations are given in Table 1. (The first week of increase in virus is very variable due to the stochastic nature of the process, and it is
not represented in the graphs.). The profiles of ALT in (B) are about normal (the upper limit of normal – ULN – is ,40 IU/ml [33,34]) and much less
than typical later in primary infection, where they can reach 106to 206the normal value .
Table 1. Kinetic and simulation parameters for each subject studied.
(/day)(days) (log10/ml) (days)(%) (Q1)(Q2) (5 h)
90552.4 0.295.93311.8 0.0190.75N/AN/A4.5
10012 2.6 0.277.0126 22 0.0220.732.63.5 2.2
100172.0 0.355.98 262.1 0.0140.89 5.3 3.83.9
100211.6 0.44 6.8226 140.0130.772.3 2.62.7
100241.2 0.59 6.47N/A 6.40.0080.8184.108.40.206
10025 220.127.116.11281.7 0.0520.418.104.22.168
100291.8 0.396.7723 13 0.0140.822.2 2.4 2.3
10051 4.60.15 6.66N/A 9.90.0330.84 1.83.0 2.1
100622.2 0.316.42 38 5.70.018 0.753.81.6 2.3
Median 2.20.316.47 266.4 0.0180.77 22.214.171.124
Mean 2.40.336.4428 8.50.0210.75 126.96.36.199
Std Err0.4 0.040.1422.3 0.0040.040.430.48 0.45
r – exponential growth rate; t2– doubling time; VLmax– maximum viral load; Plateau – time that the virus remains at the plateau; Iss– percentage of cells infected at viral
plateau, assuming that there are 1011hepatocytes ; Q1/Q2/5 h – quarter 1, quarter 2 and 59 half HCV genome, respectively. Other symbols described in text. The
mutation rate is m61025per nucleotide per replication cycle.
Estimates of the In Vivo HCV Mutation Rate
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We then aligned separately the set of sequences for each time
point and for each sequence region and used a sequence
visualization tool (Highlighter – www.HIV.lanl.gov), to analyze
the sequence diversity based on individual nucleotides. This tool
allowed us to identify low diversity monophyletic lineages
corresponding to the putative transmitted/founder (T/F) viruses
– the consensus at the earliest time point from SGA data . We
next confirmed that these lineages were maintained across the
times sampled, to guarantee that we were analyzing the
diversification of the same lineage over time. In cases where there
were two or more putative T/F viruses, we analyze only the
dominant lineage, as SGA sequence data was too limited to study
the minor lineages.
From these 84 sequence alignments, we were able to study the
evolution of the virus and the emergence of new mutations from
very early in infection (mean: 7 days, range 2 to 15 days since the
last negative sample across the 9 patients) until late in the plateau
phase of viral load (mean: 33 days, range 21 to 42 days). We found
that HCV sequence diversity increases quickly early on, but then
stabilizes in 7 patients, starting at about day 14; in subject 10051
there was not enough follow up to assess this issue, and in subject
10029 a clear stabilization of diversity was not observed. The
plateau of diversity occurred when an average of 46% of the
sequences were still identical to the inferred T/F viral genomes. In
three subjects (10029, 10062, 9055) there was an increase in
diversity at late times, ,35 days. Note that for 10062, this is
coincident with an increase in ALT levels suggesting turnover of
infected hepatocytes (Figure 1B).
We also found that in the vast majority of cases, HCV diversity
at each time point was consistent with a star-like phylogeny, i.e.
the viruses’ sequences coalesce at a single genome founder [27,44].
The only exception was the 59-half of 9055 at the last sampling
time point, day 38, when there was evidence for the onset of
immune selection . The mutations detected in the sequence
sets also conformed to a Poisson distribution in the inter-sequence
pairwise Hamming distances . The exceptions were the 59-half
of 10029 at day 13, the second 59 quarter of 10029 at day 34, the
second 59 quarter of 10051 at day 7, and the first quarter and 59-
half of 10051 at day 21. Due to the specifics of the HCV
replication life-cycle, one predicts occasional violations in star-like
diversification and in the fit to the Poisson distribution, because
there is a non-negligible probability of shared stochastic mutations
between HCV sequences. That is, shared mutations may occur
even in the absence of selective forces. See the accompanying
report  for a more detailed discussion of these issues.
Model of HCV Replication during Primary Infection
We next developed a model of HCV replication to study the
time course of accumulation of mutations and to estimate the in
vivo mutation rate of HCV needed to describe the observations
above. This stochastic model of HCV replication allowed us to
study the time course of viral load changes and the accumulation
of mutations in the study subjects (see Methods). In the model, we
assume cells are infected by a single virion, i.e., that superinfection
does not occur [45,46]. We further assume that in every infected
cell, on average, only a fraction k of newly synthesized viral (+)
strand RNA (vRNA) is exported in new virions, and the rest, 1-k,
forms new replication complexes (RC). We assume that vRNA
degradation can be neglected, i.e., that the newly synthesized
vRNA is either rapidly complexed with proteins and converted
into stable RC, or rapidly encapsidated and exported. (Note that
this is very different from analyses of HCV treatment, when
production of vRNA and/or virion assembly/release may be
blocked, and vRNA degradation becomes an important parameter
in the clearance of infection ). These processes are assumed to
continue until the cell generates a maximum number of replication
complexes (RCM). Note that if we set k=1, so that all synthesized
vRNAs are exported, we recover the ‘‘stamping machine’’ mode of
replication [7–9], where all virions result from the same replication
complex, i.e., the same negative strand of RNA. The existence of
multiple replication complexes within one cell corresponds to
‘‘geometric growth’’. In our model, after a virus is exported, a
fraction 1-h of the released virions is assumed to be cleared from
circulation , and the remaining fraction, h, is assumed to infect
new cells. We also assume infected cells are long lived, and thus,
we initially neglect death of infected cells during the first few weeks
of infection. This assumption is consistent with the viral load
profiles seen in the infected subjects, where viral load increases
rapidly to a maximum level and plateaus at this level for weeks.
We used our model to reproduce the viral load data (Figure 1A).
For each subject, the only free parameter available to determine
the trajectory of virus over time is the fraction of vRNA exported,
k, since all other parameters are fixed a priori or are calculated as a
function of k (see Methods). We found that the model could
describe the viral load data well with just this single adjustable
parameter. The values estimated for k indicate that most of the
synthesized vRNA is exported as virions (median k=0.77, range
0.42–0.89). Moreover, the estimated values of k are quite similar
among the different individuals, with the exception of 10025, who
has a lower estimated k (=0.42). However, this subject has only
one viral load measurement during the up-slope of the virus, which
strongly influences the value estimated for k. Indeed, for this
individual, choosing higher values for k lead to only slightly lower
quality fits (not shown).
The Mutation Rate of HCV In Vivo
Next, we used our model to analyze the diversification profiles
of HCV in these patients. As the viral RNA is copied, errors in the
incorporation of nucleotides are possible, i.e., mutations occur. If
we let m denote the probability that a base in the newly produced
virion differs from that in the infecting virion, then for the
stamping machine model the mutation rate, m, is simply twice the
rate at which bases are miscopied by the HCV RdRp, to account
for the cycle of (+)RNA strandR(2)RNA strandR(+)RNA strand
copying. With multiple replication complexes in a cell, opportu-
nities exist for additional copying errors to be made since a newly
synthesized (+)RNA strand needs to be copied again to make a
replication complex. Every time a RNA strand incorporating a
mutation is made, there is a probability that this mutation is lethal,
and the virus or replication complex made from such RNA is non-
functional. Prior experimental studies indicate that the fraction of
random mutations that are lethal is about 40% in RNA viruses
We incorporated mutation in our model to analyze the viral
diversification data and estimate the mutation rate needed to
match the observed accumulation of mutations. We assume that at
time zero the putative T/F virus starts replicating and mutating.
We then compute the decrease over time in the fraction of
sequences identical to the T/F virus (i.e., ‘‘the fraction of
unmutated viruses’’). We compare this model prediction to the
identical measurement in our subjects and varied the mutation
rate to obtain the best agreement between model and sequence
data obtained from plasma HCV RNA, which corresponds to
The best description of the data was obtained for a median
mutation rate (for the half-genomes) of m=2.561025
nucleotide per replication (Figure 2A–C). Moreover, this estimate
was consistent across subjects and across regions of the genome
Estimates of the In Vivo HCV Mutation Rate
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Our model exhibits a fast decrease in sequence identity early in
infection, as the viral load increases exponentially and more and
more cells are infected, followed by a stable viral diversity level as
the virus reaches and stays at its plateau. This stasis in viral
diversification is compatible with the assumption that the plateau
in viral load corresponds to a stable pool of infected cells. This
indeed seems to be the case for 5 of the patients (Figure 2A–C); for
1 case there is not enough data. If the plateau in viral load
per nucleotide per replication, corresponded to a dynamic steady state in which infected cells
were dying and being rapidly replaced, our model would predict a
continuous increase in diversification resulting from the continu-
ous replacement of replication complexes. In a few cases, we did
see an increase in diversity at times later than 30 days, and in three
patients (10029, 10062, and 9055) the observed long term
behavior (later than about day 35) deviates from that predicted
by our simulations. This difference between model and data could
be due to sampling error, for example the 95% CI for theory and
data at day 42 overlap for patient 10062. Alternatively, some
Figure 2. Fraction of sequences identical to the T/F virus over time. The symbols represent the SGA data and corresponding binomial 95%
CI; the solid line is the average from 100 simulations and the dashed line the 95% CI for the proportion . (A) data for the first quarter (Q1) of the 59
HCV genome (note that for subject 9055, there is no data for Q1 or Q2); (B) data for the second quarter (Q2) of the 59 HCV genome; (C) data for the 59
half of the HCV genome.
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processes not accounted for in the model may be operational at
these later time points, leading to increased diversity. For example,
for subject 9055 anti-HCV antibodies are detectable at this late
time point and there is strong evidence of CTL selection (escape or
reversion) ; and for 10062 there is a late increase in ALT
(Figure 1B), which suggests the initiation of a CTL response
consistent with renewed cycles of infection.
Our model also makes predictions about the distribution of
mutations across the population. Interestingly, our model not only
matches the fraction of unmutated viruses, but also the fraction of
viruses with 1, 2, 3, … mutations, even though this detailed data
was not used to parameterize the model (Figure 3A–C). We
obtained excellent agreement with the data, except when we
observed a late increase in diversity in the three patients discussed
above (10029, 10062, 9055). We tested this agreement for the 5 h
genomes by a Monte Carlo test , since the number of expected
mutations is low (,5) in several cases. The null hypothesis is that
the data follows the theoretical expected values, and with the
exception of those three patients, there was good agreement
between observed and predicted mutation counts (p.0.05).
Moreover, if we consider the distribution of mutations at the
previous time for which we have SGA data, this agreement was
also seen in 10029 and 10062 (p.0.05, and we cannot reject the
We next tested whether our results were dependent on the
particular values assigned to the parameters that we fixed in the
simulation (see Methods). We found that both the viral load time
course and the viral diversification were not sensitive to particular
values of these parameters (Figure S1 in Text S1). For example, we
assumed a maximum of RCM=40 replication complexes per
infected cell, as seen in vitro  and in situ . Clearly this number
could be different in vivo. However, our results were essentially the
same, when we varied RCMfrom 10 to 80 (Figure S1 in Text S1).
To further confirm the robustness of our results, we next used
the method suggested by Cuevas et al.  for estimating the
mutation rate of HCV by analyzing the frequency of lethal
mutations. Classical genetics shows that the frequency of lethal
mutations is equal to the mutation rate, since all such mutations
should be produced directly by mutation in the last replication
round. As in Cuevas et al. , we used non-sense (stop codon)
mutations as a proxy for lethal mutations. The concept is to count
all stop codons in the data set and to divide this by the number of
mutation targets (non-sense mutation targets – NSMT), i.e. codons
that by a single mutation could generate a stop codon (see Text S1
for details). For these analyses, we were able to use all 17 patients
in our cohort, thus expanding our data set.
In total we had 898,335 NSMTs and 13 stop codons in the over
16107bases sequenced  (Tables S1 and S2 in Text S1).
Surprisingly, 4 of the stop codons were identical and at the same
position in 10051 at two different time points (see Table S1 in Text
S1). This strongly indicates that this stop codon appeared only
once in this patient, and that stop codons may not be lethal in
HCV but instead complemented by intact genomes within the
same cell. Thus, we counted this stop codon only once, for a total
of 10 mutations leading to stop codons. A calculation identical to
that proposed in  then shows that m=3.261025per nucleotide
per replication, which is fully consistent with our estimate above.
We also propose an improved way to calculate this rate from the
same data (see Text S1), and with this method obtain
m=2.861025(binomial 95% CI: 1.4–5.261025).
Altogether, these data and analyses indicate that HCV
sequences diversify early in infection, during the exponential
increase of viral load, which is then followed by a plateau in
diversity for up to a few weeks. The mutation rate needed to
explain these observations (m<2.5–3.261025per nucleotide per
replication, Figure 3D) is 5 and 100 times smaller than previously
reported for HCV  and its purified RdRp , respectively.
We next investigated in detail why HCV diversification appears
to stop after a few weeks of infection, and what processes could
break this plateau in diversity, since in chronic HCV infection the
virus is much more diverse . In particular, we analyzed the
effect of turnover of replication complexes and the emergence of
the cytolytic immune response.
Turnover of Replication Complexes
In the baseline simulations of the model, we neglected the
turnover of replication complexes (RC). However, RC may
degrade. In this case, to sustain viral replication, the RC would
need to be continuously produced to balance their degradation.
Thus, we next analyzed the impact on our model predictions of
including RC degradation.
For fast RC turnover (e.g., half-life 1.5 d), most (median of
59%) of the simulated infections die out, and those that lead to
sustained infection show a slow growth of the virus that is not
compatible with the data (Figure 4A, left panel). It is possible to
recover fast viral growth rates, if one postulates that a larger
fraction of newly synthesized RNA is used to form new
replication complexes (i.e., if k is smaller). When the turnover
of replication complexes is not negligible (tK,5 days), on the
time scale of our simulations, the accumulation of mutations is
faster at later times as replacement of replication complexes
occurs (Figure 4A, right panel). In this case, to describe the data a
smaller mutation rate would be needed, at least in some patients.
Importantly, turnover of replication complexes also implies a
continued increase in diversity throughout the observation
period, since more (2) strand RNA needs to be made and hence
there is more opportunity for mutations to occur. However, such
a continued increase in diversity is not seen for subjects 10012,
10017, 10021 and 10025. On the other hand, this process could
help explain the marked increase in diversity seen at late time
points in subjects 10029, 10062 and 9055. Note however that
even these subjects seem to have a stabilization of diversity prior
to this marked increase, which is not compatible with fast
turnover of replication complexes. If the turnover of replication
complexes is much slower (eg., ,15-day half-life) then the profiles
do not differ from our baseline case where there is no turnover
over the 50 day period studied.
Here we studied RC turnover inside the cell, but it is also
possible that cells die due to the immune response against HCV,
thus forcing re-generation of RC. Thus, we next considered the
effects of cell turnover on the results of our model.
Effect of the Immune Response
An effect of immune processes is removal of infected cells.
Because there may be some limit to the number of infected cells in
the liver , the death of infected cells may allow new cells to be
infected, which in turn generates new RC and the opportunity for
mutation accumulation. For all subjects for whom there is enough
data, we find a stabilization of diversity, which in a few cases is
then followed by a ‘‘sudden’’ marked increase at a later time point
(10029, 10062, 9055). It could be that this latter pattern is an
artifact of sampling. For example for 10062, the observed fraction
of unmutated sequences at the three time points sampled have
confidence intervals that overlap, and those fractions are not
significantly different, p=0.07 (Figure 2C, overlap of vertical
bars). In our model this stabilization in diversity accumulation
occurs because a steady-state is attained for the numbers of
replication complexes and infected cells, without continued
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PLOS Pathogens | www.plospathogens.org6August 2012 | Volume 8 | Issue 8 | e1002881
turnover. Rather than a sampling issue, it is possible that the
observed increase in diversity is due to an immune response
emerging at late time points, which leads to an increase of the
infected cell death rate (d). Indeed, this is indicated in studies of
experimental infection of chimpanzees, where the immune
response is delayed several weeks [31,36]. In this context, an
alternative explanation for the increase in diversity in 10062 is the
appearance of such an immune response as suggested by the
increase in ALT in this subject (Figure 1B). To study the effect of a
late immune response that kills infected cells, we allowed for this
process starting at 30 days post infection (Figure 4B). As expected,
the emergence of an immune response lowers the viral load,
possibly leading to a new lower viral load steady state, as is
observed in some experimentally infected chimpanzees . With
the loss of infected cells, new cycles of infection occur along with
creation of new replication complexes, and the model predicts a
renewed increase in the accumulation of diversity, which mimics
the data in some subjects (eg., 10062, 9055). However, we do not
Figure 3. Spectrum of mutations in the data. The last sampling time (closed symbol) and the corresponding prediction by the model derived as
the average of 100 simulations (open symbol) and respective 95% binomial CI based on the estimated mutation rates indicated in Table 1 and
Figure 2 for each subject. (A) data for the first quarter (Q1) of the 59 HCV genome (note that for subject 9055, there is no data for Q1 or Q2); (B) data
for the second quarter (Q2) of the 59 HCV genome; (C) data for the 59 half of the HCV genome. (D) Summary box plot of estimated mutation rates for
the different genomic segments.
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PLOS Pathogens | www.plospathogens.org7 August 2012 | Volume 8 | Issue 8 | e1002881
have enough data to precisely estimate the timing and magnitude
of this immune response.
We analyzed the viral dynamics and viral diversification of
HCV very early in acute infection. The early diversity of HCV is
very low, and the inter-sequence Hamming distances follow a
Poisson distribution, as would be expected when the mutations
occur approximately at the same rate at all positions and the
sequences are not selected for diversity [27,44]. Given this
observation, the number of mutations at early times should
depend on the time since infection, the mutation rate and the
biology of viral replication. This idea has been used before in the
context of primary HIV infection to estimate the time of infection,
assuming a given mutation rate [26,27]. In the present study, the
time of infection is known to within a short time window, with the
first HCV positive sample within 5 days of the last negative
sample. With this information, we could use our data to estimate
the in vivo HCV mutation rate. By developing a model of HCV
Figure 4. Changes in viral load and mutation profile predicted by the model. (A) different values for the half-life of replication complexes
(ln 2/r), and (B) the emergence of a cytolytic immune response at 30 days post-infection. Note that if there were less than 5 runs leading to
establishment of infection, no line is plotted, because the noise is too large. Thus in (A) for short half-life of replication complexes (i.e., t1/2=0.74 d in
cyan) the line may not appear, because the infection was not established, or the line may disappear, because an initial infection was aborted.
Estimates of the In Vivo HCV Mutation Rate
PLOS Pathogens | www.plospathogens.org8 August 2012 | Volume 8 | Issue 8 | e1002881
replication that takes into account the details of the viral lifecycle,
we found the estimated mutation rate varied among subjects
replication cycle, with a median of 2.561025(Table 1, 5 h
genome). This estimate was very robust to different assumptions
about model parameter values (see Text S1). Moreover, we
systematically made conservative assumptions for the less well
known parameter values leading to higher estimates for the
mutation rate. To further confirm our results, we estimated the
mutation rate by a completely different approach based on the
frequency of stop codons (non-sense mutations), corrected by the
number of non-sense mutation targets, as proposed by Cuevas et al.
. With this calculation we obtained a mutation rate of
2.861025or 3.261025mutations per nucleotide per replication
cycle depending on the calculation method (see Text S1), which is
consistent with the estimate from our more complex dynamical
model and substantially less than the rate (,1024) estimated by
Cuevas et al. . A likely explanation for the difference between
the findings of our nonsense mutation analysis and that of Cuevas
et al. is that in our study Taq polymerase errors are eliminated from
the finished sequences by the SGA-direct amplicon sequencing
method and thus do not enter in the error rate calculations; this
was not the case for the previous analyses [6,13]. We further note
that estimates of the HCV mutation rate based on nonsense
mutations are likely to be overestimates since we found that stop
codons were not always lethal (see Text S1). One explanation for
this observation is that there are multiple HCV RNAs in an
infected cell and another RNA may complement nonsense
mutations. Indeed, we also found a case of a chronically infected
patient who has a strain with a large deletion replicating in plasma
at multiple time points . Moreover, for dengue virus (in the
same Flaviviridae family of HCV) there is a report of a viral strain
with a stop codon that spread and attained a high frequency in the
population, implying replication in both humans and mosquitoes
In addition, our analysis does not account for mutational errors
resulting from the cDNA synthesis step of the sequencing process,
which again may lead to an overestimation of the mutation rate.
However, we used Superscript IIITM Reverse Transcriptase (Cat.
No. 18080-093, 2000 units, Invitrogen Life Technologies, Carls-
bad, CA) that has been reported to have an error rate of ,261026
mutations/nucleotide/replication [23,51], which is at least 10-fold
lower than our HCV mutation rate estimates, and hence should
not significantly influence our estimates.
Our estimates of the mutation rate for the HCV RdRp of
,2.561025are notable because previous reports have suggested
that the in vivo mutation rate of HCV is of the order of 1024
mutations per nucleotide per replication ; and that the in vitro
rate of the isolated RdRp could be as high as 1023. One
possible explanation for the latter discrepancy is that the mutation
rates observed with purified RdRp enzymes are generally larger
than those seen in vivo, because in vitro analyses cannot recapitulate
the intracellular milieu of the replication or polymerase complex.
For example, in the case of HIV reverse transcriptase, the errors
measured with purified enzyme were found to be up to 20-fold
higher than those measured in infected cells . Another
possibility is that we may have missed some low prevalence
strains. However, a detailed power calculation shows that with the
number of sequences obtained per patient, we would only miss
strains that are present at very low levels, below 2% , which is
much better than was possible before [25,53] (see Li et al.  for a
detailed discussion). Moreover, for the dynamical model we follow
time courses and analyzed the fraction of virus identical to the T/F
virus; and for the stop codon analyses, we corrected for the
mutations per nucleotide per
mutational targets. Both of these lower the impact of missing
Given the low level of diversity observed in early infection and
the relatively low mutation rate, the enormous diversity of HCV
[14,15,18] and its high substitution rate (i.e., substitutions/site/
year) have to be understood in light of HCV’s replication
mechanism . Relatively long-lived infected cells, with multiple
replication complexes allow for the accumulation of diversity in
the virions produced. At the same time, the turnover of both
replication complexes and infected cells, which must surely ensue
as the immune response develops, allows for renewed generation
of diversity throughout the course of infection (compare 10062 in
Figure 1B and Figure 2C). Indeed, it could be that these details of
the life cycle are responsible for the large diversity of HCV. We
note that HIV and influenza, which are thought to have similar
mutation rates to the one estimated here [6,52], also have high
substitution rates . In this context, we see that accumulation of
diversity is not only dependent on mutation rate, but also to a
great extent on the particular processes of the viral life cycle
[7,8,16]. Clearly, the pressure of the immune response, once
established, will be important in determining relative fitness of
many of the mutations and in determining the spectrum of
mutations observed. That we see only scarce evidence of positive
selection in our dataset indicates that there is a window of several
weeks before the effects of the immune response can be detected.
Another important parameter that we estimated was the
fraction of infected cells during the early plateau in viral load,
which ranged between 1.7% and 22% of hepatocytes. This
fraction is in reasonable agreement with other studies of HCV
[41,42]. In our model, this fraction depends on the value assumed
for the maximum number of replication complexes (RCM). The
larger the number of replication complexes in an infected cell, the
more viruses this cell can produce per unit of time, and thus the
fewer the number of infected cells needed to maintain a given
steady state viral load. However, increasing RCMhas little effect on
our estimate of the mutation rate (see Text S1).
In this study, we constructed a simple model of HCV replication
that tried to capture the most salient features of the viral life cycle.
Moreover, we were careful to choose parameters consistent with
the literature a priori, so that only 2 parameters had to be adjusted
to fit the data on viral growth and diversity increase. We tested
variation in the model assumptions and found that the results were
quite robust. Still, it is clear that many complexities could be
added to the model. For example, instead of having a fixed RCM,
we could allow it to vary from cell to cell and possibly even from
time to time; or we could allow for a distribution of generation
times for RNA synthesis. These and other processes are easy to
include in the model, however we opted to keep to the essential
aspects of the replication process, so that we did not have to make
further assumptions, which would complicate the interpretation of
the results. In essence, this is akin to choosing a simple
experimental system that is amenable to easy manipulation and
interpretation of results, even if it does not represent fully all the
details of in vivo system.
Altogether, the unique dataset presented here, including HCV
viral kinetics and genomic diversification very early in infection,
revealed that the initial exponential expansion of HCV RNA is
followed by a plateau in viral load that lasts up to a few weeks .
The initial viral expansion is accompanied by a fast early increase
in sequence diversity, whereas during the viral plateau viral
diversity remains approximately constant. During the plateau viral
production continues but is simply balanced by the rate of viral
clearance. In order to understand why viral diversity did not
continue to increase during this period, we develop a novel
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stochastic model of HCV infection. The basic idea behind the
model is that during the early exponential expansion of the virus,
new cells are being infected and generating multiple replication
complexes in each infected cell. This involves multiple copying
events of (+)RNA to (2)RNA to (+)RNA, etc, with errors
potentially being generated at each stage. We postulate that once
the viral plateau is reached a stable population of long-lived
infected cells has been generated which then produce the plateau
virus without any need for new RC generation. If no new
replication templates are made then there is little opportunity for
mutations to accumulate, though each virus can still mutate in
relation to its parent RC due to the (2)RNA to (+)RNA copying
event. We found that our model, based on this idea, agreed with
both the viral load kinetic data and the sequence diversity data if
we assumed that the in vivo mutation rate of HCV is ,2.561025
per nucleotide per replication cycle. This is about 5-fold lower
than previously reported, but still high enough that coupled with
the long-lasting nature of HCV infection and the very high
turnover of virus in chronic infection leads to substantial HCV
diversity in an individual and in the population.
Materials and Methods
Plasma samples were obtained from seventeen regular source
plasma donors, who became HCV infected during periods of
twice-weekly plasma donations. The donors were untreated and
asymptomatic throughout the collection period. All subjects gave
written, informed consent and the study protocols were approved
by institutional review boards at the University of Pennsylvania,
the University of Alabama at Birmingham and Duke University.
HCV RNA and antibodies were analyzed as described elsewhere
Single Genome Sequencing
Single genome amplification (SGA) followed by direct amplicon
sequencing was performed on sequential plasma vRNA samples
(i.e., (+) RNA strands), as described in detail elsewhere .
For our dynamical analyses, we selected subjects who had at
least two time points sampled with single genome amplification
assays . Thus, three subjects were not included – 6213, 6222,
10004. Six subjects (10002, 10003, 10016, 10020, 10029, 106889)
had more than 7 putative T/F viruses, which makes a
diversification analysis impractical, both due to the complexity of
the viral species in the subjects and the small number of sequences
representing each lineage . The exception was 10029, who
had a dominant lineage with more than 38 sequences for each
time point, and we included this subject in our analyses. Thus,
there were 9 subjects who were sampled at multiple time points
and who had a clearly dominant putative T/F virus lineage .
Here we only analyzed these dominant lineages, for which we
have the most data (SGA sequences).
Sequence alignments were initially made with ClustalW and
then checked individually using JalView 2.6.1 (www.jalview.org).
We used ConsensusMaker (www.HIV.lanl.gov) to calculate the
consensus of the first set of sequences sampled by SGA, which is
the putative T/F virus . The set of sequences from each SGA
sample with the corresponding consensus was analyzed by
PoissonFitter (www.HIV.lanl.gov) to calculate for each sequence
the number of mutations away (i.e., Hamming distance) from the
T/F, and to test whether sequence diversification conforms to a
star-phylogeny and if the set of inter-sequence Hamming distances
follow a Poisson distribution .
Altogether we analyzed time courses of thousands of sequences
with over 11.9 million base pairs and 1887 mutations .
To analyze the process of replication of HCV and how it affects
the generation of diversity in primary infection, we developed an
agent based model of HCV infection and replication. We assumed
cells are infected by a single virion, and that in every infected cell,
on average a fraction k of newly synthesized viral RNA (vRNA) is
exported in new virions, and the rest, 1-k, form new replication
complexes in the cell. These processes continue until the cell
contains a maximum number of replication complexes (RCM). We
assume this maximum value is set by the availability of host
factors. After a virus is exported, a fraction 1-h of released virions
are cleared from circulation, and the rest, h, infect new cells.
As the vRNA is copied, errors in the incorporation of
nucleotides are possible. Every time a mutation occurs, there is a
probability that this mutation is lethal, implying a virus or
replication complex made using such mutant vRNA is non-viable.
Sanjuan  estimates that the fraction of random mutations that
are lethal is about 40% for RNA viruses.
We assumed HCV is noncytolytic . Thus, infected cells can
produce virus for long periods of time – until the infected cell dies,
either from natural death or immune attack. Early in acute
infection there is little evidence of cytotoxic T cell activity and
CD8+ T cells do not appear to enter the liver until many weeks
after infection . In addition, normal hepatocytes live for
months  to a year or more , thus, we either totally neglect
death of infected cells or allow death after the first few weeks of
infection. The assumption of no early death is consistent with the
normal levels of alanine aminotransferase (ALT) measured in these
individuals (Figure 1B) and the viral load profiles, where viral load
increases rapidly to a maximum level and then stays at that level
for some time. (This is in stark contrast for example with HIV, a
cytolytic virus, where a clear peak in viral load is seen during
primary infection followed by a decrease in viral load .)
Replication of the RNA and formation of a new virion or
replication complexes is not instantaneous, as it takes a certain
amount of time for synthesis of the different molecular compo-
nents and their assembly. Although this time is most likely variable
from replication cycle to replication cycle and from cell to cell, we
assume that it is similar for all replication events in our model,
fixing it at an average time to complete all the replication steps.
This time we call the ‘‘generation time’’. Most likely it will take
longer to produce the first copied RNA upon cell infection than
later ones, as various molecular events need to occur before virus
production begins (eg., uncoating, polyprotein synthesis and
cleavage, assembly of the replication complex, etc) .
In the simulation, based on the assumptions described above,
we follow the number, age (in the sense of generations) and
mutational burden of each virion and each replication complex
inside infected cells. The simulation was implemented in the R
language (www.r-project.org). Because these are stochastic simu-
lations, there is variability from one run to the next, even when all
parameters remain the same. Thus, for each patient and each set
of parameters (in Figures 1–4) we present results from 100 runs.
Including more runs (we tested some cases with 200 runs) does not
significantly alter the results presented.
The parameters of the stochastic model are as follows:
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production, we assume that it takes ,6 h for a cycle of replication
to produce new virions or replication complexes based on the
following argument. A study of HCV replication kinetics  found
that there are about 200 (+)RNA strands in a cell at steady state.
With 40 replication complexes per cell, if we assume at steady state
(+)RNA is being produced at rate a and degraded by a first-order
process at rate dR, then at steady state 200=a/dR. In treatment
experiments in the replicon system the half-life of (+)RNA was
found to be between 11 and 18 h [55–57], i.e. ,15 h, so that
dR=0.693/15=0.0462 h21and a=9.24 h21. Then with 40
replication complexes per cell, each one would be producing
(+)RNA at a rate of 9.24 h21/40=0.231 h21and it would take 1/
0.231 h21=4.33 h to produce one new (+)RNA. Allowing for
some extra time for assembly of a virion or a new replication
complex, we thus assume ,6 h for a cycle of replication.
RCM– maximum number of replication complexes in an
Experimental results show that about 40 replica-
tion complexes can exist in one infected cell [4,10]. Our baseline
results use this number, but we also vary this parameter.
r – turnover of replication complexes.
observed after introducing treatment in a replicon system, that
the half-life of (2)RNA was ,12 h , but this decay only
started after a 12 h delay. In our simulation, this would correspond
to a degradation probability for the replication complex of ,0.3
per generation. To see this, note that with a half-life of 12 h, a
(2)RNA, or we assume equivalently a RC, decays at an average
rate dRC=0.693/12 h=0.0577 h21. To convert a continuous rate
to a probability that an event occurs during a time interval Dt ,
note that by the exponential distribution the probability that a RC
that is decaying with an average rate dRCdegrades at or before a
time Dt has elapsed is given by 1- exp(-dRCDt) . Choosing
Dt=6 h, i.e. a generation and dRC=0.057 h21, the probability of
degrading in one generation is r,0.3. However, we expect this to
be an upper limit for r, the probability of degradation in the
absence of treatment, because replication complexes are protected
within vesicular membranous structures (VMS) adjacent to the ER
membrane . Indeed, the 12 h delay until the start of
degradation of (2)RNA, which is thought to be mainly localized
within the VMS, supports this idea. Initially, as a conservative
approach to estimate the maximum mutation rate, we will assume
that replication complexes are not degraded on the time-scales
involved in primary infection, i.e., r=0. We later allow RC
degradation (r.0) and ask what impact it has in the dynamics of
virus and viral diversity.
k – probability that a newly formed vRNA is exported as a
For each subject, we choose k to match the observed viral
load profile. We varied k between 0 and 1 in increments of 0.01 and
found the value that best describes the data by minimizing the sum
data. For each case, we ran 100 simulations and then chose the
value ofk that led tothe best matchoftheaverageoftheviral load in
the simulations with the observed viral load.
d – turnover of infected cells.
long lived cells, but infected cells may die faster due to viral effects.
However, HCV is thought to be non-cytolytic , thus, we
assume that the infected cell death rate is similar to that of
uninfected hepatocytes, and can be neglected (d=0) in the time
frame of our study. We also investigate the effect of the emergence
of the cytolytic response (d.0) sometime after infection .
Initial estimates, mostly based on interferon therapy of chronically
infected patients, found that the loss rate of infected cells was quite
variable with median half-lives of about 7 days [21,60,61],
corresponding to a probability of death per generation d=0.025
During stable cell infection and virus
It has been
Hepatocytes are in general
(by the same argument using the exponential distribution as above
to estimate r).
h – probability of a free virus infecting a target cell (if
these are available).
In this model, as well as in the standard
model of viral infection (i.e., the differential equation model that
has been widely used to analyze both primary infection and
antiviral treatment [21,62]), free virus can either be cleared or
infect a new cell. In the standard model these processes occur at
rates c and bT, respectively, where T is the available target cell
density. If we write the differential equations corresponding to the
infected cells, I, and free virus, V, we have
where p is the daily viral production rate per infected cell. If we
make the common assumption of quasi-steady state, then I<(c/p)
V [21,62]. This essentially means that the viral dynamics are much
faster than the infected cell dynamics. From the first equation
above, we can now write
with the initial exponential rate of increase of the virus, r, given by
r=pbT/c. Moreover, in this model the probability of infection is
given by h=bT/(c+bT), because infection (bT) is one of two
possible events, the other being virion clearance (c). We can write
this probability of infection in terms of r and p as h=r/(r+p). Here,
r can be measured directly from the rate of exponential increase in
viral load observed in the data of each individual. Indeed, the
initial rise in viral load is well described by a constant exponential
rate of increase, as has been suggested before . In our model, p,
the daily virus production rate varies over time, because the
number of replication complexes in each infected cell is increasing.
However, to be consistent with the observed constant rate of
increase, r, we assumed that p=ng6RCM6k; where ng is the
number of generations per day (converting the production of
viruses per generation of the simulation into the production rate
per day), RCMis the maximum number of replication complexes,
and k is the fraction of newly synthesized RNA that is exported as
virions. Because we are fitting k, this expression for p corresponds
to a constant effective production rate throughout primary
infection that matches the viral load. Substituting this expression
Iss–number of infected cells at steady state.
most individuals exhibits a viral plateau a couple of weeks after
infection (see also ). That is, the virus does not continue to
grow exponentially. With our assumption of negligible infected cell
death (d=0), the logical implication of the observation of this
steady state is that the number of infected cells reaches a
maximum and is then kept constant at this level, Iss. When the
virus reaches the plateau, Vss, we can calculate from the standard
model and the quasi steady-state assumption [21,62] that
for p into h, we have h~
The data of
where the daily production of virus per infected cell at the steady
state is p=ng6RCM. Note that at the steady state, each cell has the
Estimates of the In Vivo HCV Mutation Rate
PLOS Pathogens | www.plospathogens.org11 August 2012 | Volume 8 | Issue 8 | e1002881
maximum number of replication complexes, because if it did not
then the production per cell would increase further contradicting
the assumption of a steady state. Thus, at the steady-state all newly
synthesized RNA is exported as new virions, and k does not appear
in the formula for Iss. This number represents the number of
infected cells at the plateau. For those few cases where we did not
observe the viral plateau, because of loss to follow-up, we use for
Vssthe maximum viral load observed. To calculate Iss, we use
c=23 day21, estimated from the rate of decay of HCV RNA in
patients treated with an NS5A inhibitor .
D – fraction of lethal mutations.
mutations are lethal and that the rest are neutral. The fraction of
lethal mutations has been estimated in different viruses using site-
directed mutagenesis . For two eukaryotic RNA viruses this
fraction was 40% as used here, whereas for an RNA bacteriophage
it was 30% . The lethal phenotype can have different causes
(from improper folding of the RNA molecule to lack of protease
function). Here we will assume that lethal mutations lead to
vRNAs that do not contribute to viral load or to make new
replication complexes. An alternative view would be that some
lethal mutations still allow production of viral particles, but that
these are not infectious. In this case, they would be included in
viral load measurements, but they would not infect new cells. In
our model, this possibility is accounted for by the parameter h, the
fraction of virus that infects new cells.
t – time (in generations) that a cell takes to start
producing RNA upon first infection.
infection and virus production, we have assumed that it takes
,6 h for a cycle of replication to produce new virions or
replication complexes. However, upon initial virus infection, a cell
does not produce virus immediately. It goes through an eclipse
phase before the first RNAs are produced. Replication in cellular
cultures is readily detectable at 24 h, albeit at low levels . Thus,
for the baseline scenario, we assume that upon infection cells can
start producing vRNAs after a time corresponding to two
We assume that 40% of all
During stable cell
generations, i.e. 12 h. However, we also investigate the effect of
larger values for t.
m – mutation rate per base per replication (2 copying
The mutation rate for HCV has been estimated
maximally at m=1.261024per base per replication . In our
simulations, we choose m to match the observed profile in the
decrease over time of the fraction of sequences without mutations
in relation to the putative T/F virus. That is, at each time point for
which we have a SGA sample, we calculate for the data and in the
simulations the fraction of virus that has not mutated and hence
has a genome (segment) still identical to the T/F virus. For each
individual and genome segment (quarter or 59 half), we vary m in
increments of 0.01/Nb, where Nbis the number of nucleotides of
the SGA sequence, and find the mutation rate that provides the
best fit to the data (i.e., minimizes the sum of the squared
residuals). Again, we ran 100 simulations for each value of m and
used the average of those simulations to compare with the data.
that our results are robust regarding our choices for the maximum
number of replication complexes, the initial delay after a cell is
infected and before virus is produced and the fraction of lethal
mutations. We also present details of the estimation of mutation
rate using the frequency of stop codons.
In Text S1 in online supporting information, we show
Conceived and designed the experiments: RMR HL BHH GMS ASP.
Performed the experiments: HL SW MBS EHP. Analyzed the data: RMR
HL GHL BTK TB JG GMS ASP. Wrote the paper: RMR HL TB JG
GMS ASP. Critically read and approved manuscript: RMR HL SW MBS
GHL BTK TB JG EHP BHH GMS ASP.
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