Elucidation of Hepatitis C Virus Transmission and Early Diversification by Single Genome Sequencing
[["Hepatitis C virus ( HCV ) infects as many as 170 million people or nearly 3% of the world's population .", 'The virus causes a wide variety of pathologic outcomes , the most significant being chronic liver disease , cirrhosis and hepatocellular carcinoma , which is nearly always fatal .', 'HCV infection is the leading indication for liver transplantation in the United States [1] .', 'HCV is a positive strand , non-segmented , enveloped RNA virus of approximately 9 . 6 kb in length .', 'The virus is classified in the genus Hepacivirus within the larger family of Flavivirus , which includes the human pathogens West Nile virus , yellow fever virus and dengue fever virus among others [2] .', 'A common feature among the Flaviviridae is their dependence on a virally-encoded RNA-dependent RNA polymerase ( RdRp ) for replication [3] .', 'RdRp is error-prone , and HCV is notable for its extensive diversity within and among individuals .', 'Globally , there are seven major genotypes of HCV that differ by approximately 30% in nucleotide sequence [1] , [4] , [5] .', 'The extraordinary diversity of HCV complicates studies of virus biology , pathogenesis and susceptibility to novel therapeutics .', 'Clinically , the different HCV genotypes exhibit variable natural history and responsiveness to interferon , ribavirin and the newer direct acting antiviral ( DAA ) agents [2] , [6] , [7] .', 'HCV variation poses similar challenges to the development of effective vaccines and to the elucidation of viral immunopathogenesis [5] , [8]–[10] .', 'It is of interest then that the extraordinary diversity of HCV is similar to that of HIV-1 and that a novel experimental strategy to identify transmitted/founder ( T/F ) HIV-1 genomes has led to new insights into virus transmission and persistence [11]–[18] .', 'Acute HCV infection isconventionally defined as the initial 6 months of infection and sets into motion virus-host interactions that to a large extent dictate the natural history of the disease [9] , [10] , [19]–[30] .', 'Depending on viral genotype and host immunogenetic factors , most importantly IL28B alleles , a proportion of newly infected individuals spontaneously controls or eliminates virus [9] , [10] , [31]–[33] .', 'A greater number can be cured if the infection is treated with interferon and ribavirin alone or in combination with DAA drugs [6] , [34]–[36] .', 'Mechanistically , how this occurs is incompletely understood .', 'From a vaccine perspective , the acute infection period is critical , since transmitted viruses are the obvious targets of a vaccine and early stages of infection when viral diversity is lowest represent a period when the virus may be most vulnerable to elimination by vaccine-elicited immune responses [26] , [37] .', 'For all these reasons , there is considerable interest in the molecular features of the initial virus population ‘bottleneck’ associated with virus transmission and the subsequent pathways of virus evolution that lead to persistence [19]–[25] , [28] , [29] , [38] , [39] .', 'Previous reports have described different experimental approaches to the analysis of the HCV transmission bottleneck .', 'These include studies that employed a DNA heteroduplex gel shift method to estimate viral diversity [39] , [40] , conventional polymerase chain reaction ( PCR ) methods to bulk amplify , sequence , or clone and sequence fragments of HCV genomes [20] , [21] , [23] , [25] , [28] , [38] , [41] , [42] , and 454 pyrosequencing to interrogate early viral sequences more deeply but narrowly [19] , [43] .', 'These reports documented a restriction in viral diversity associated with virus transmission , but despite the use of increasingly sensitive methods , a precise quantitative , molecular description of HCV transmission and early diversification has remained elusive .', 'In the current study , we hypothesized that T/F HCV genomes could be identified unambiguously and their early pathways of diversification mapped precisely by single genome amplification ( SGA ) followed by direct amplicon sequencing , otherwise known as single genome sequencing [17] , an approach we used previously to gain insight into HIV-1 transmission [15] , [18] .', 'This strategy differs from previous methods applied to HCV by providing gene-wide or genome-wide viral sequences that are proportional to their representation in human plasma and are not confounded by template resampling or by Taqpolymerase errors of nucleotide misincorporation or recombination [15] , [17] , [18] , [44]–[47] .', 'We amplified and sequenced HCV core , E1 , E2 , P7 , NS2 and NS3 genes and analyzed them by adapting a model of random HIV-1 evolution [15] , [48] , [49] to account for differences in the biology of replication between the HIV-1 and HCV .', 'In an accompanying report , Ribeiro and colleagues [50] used these sequences together with plasma viral load kinetic data to develop an agent based stochastic model of acute HCV replication dynamics resulting in new estimates of the HCV mutation rate in humans .'], ['One hundred fifty-four plasma specimens from 17 subjects with acute HCV infection and 14 subjects with chronic HCV infection were analyzed for viral RNA ( vRNA ) load ( Figure 1; Table S1 ) .', 'Acute infection subjects were source plasma donors who had undergone once or twice weekly plasmapheresis for months , and in some cases years , with persistently negative testing for HCV , HBV and HIV antibody or RNA before becoming acutely infected by HCV .', 'Chronically infected subjects were patients at the University of Alabama at Birmingham who were known to have been HCV infected for approximately 3 to >20 years .', 'All subjects were untreated with anti-HCV medications .', 'A median of 8 ( range 5–11 ) sequential specimens per acutely infected subject was analyzed for vRNA load spanning the period of plasma vRNA negativity through exponential increase to an early plateau ( Figure 1 ) , and a subset of these was analyzed for sequence diversity .', 'The median peak plasma viral load in acute infection subjects was 2 , 850 , 000 IU/ml ( range\u200a=\u200a527 , 000–10 , 300 , 000 IU/ml ) .', 'Five of 17 acutely infected subjects developed HCV antibodies by the last sampling time point .', 'Chronic subjects had a median vRNA load of 2 , 081 , 138 IU/ml ( range\u200a=\u200a24 , 000–7 , 690 , 001 IU/ml ) , all were HCV antibody positive , and all were sampled once for vRNA sequence diversity .', 'A total of 2003 5′ half genomes and 919 5′ quarter genomes were amplified , sequenced , and recorded ( Genbank accession numbers JQ801756–JQ804520; JX178293–178443 ) .', 'Sequences corresponding to core antigen and envelope E1 and E2 genes ( 2 . 2 kb ) from the initial HCV RNA positive sample from acute and chronic subjects were subjected to maximum-likelihood ( ML ) phylogenetic analysis ( Figure 2 ) .', 'Sequences formed subject-specific clades ( bootstraps 93–100% ) and represented HCV genotypes 1a ( n\u200a=\u200a23 ) , 1b ( n\u200a=\u200a4 ) , 2b ( n\u200a=\u200a2 ) or 3a ( n\u200a=\u200a2 ) .', 'No subject was infected by more than one virus genotype and there was no intermixing of sequences between subjects in the phylogenetic tree .', 'Sequences from acute and chronic subjects revealedwidely varying degrees of maximum within-subject diversity ranging from 0 . 14% to 6 . 40% ( Tables 1 and S1 ) , which was not different between the two groups ( p>0 . 05 , Mann-Whitney test ) .', 'Acute sequences were , however , distinctly different from chronic sequences in having one or more discrete lineages characterized by extremely low diversity .', 'This was true for sequences from each acutely infected subject but from none of the chronically infected subjects .', "Maximum diversity of sequences within these discrete viral lineages from acute subjects ( mean 0 . 12%; median 0 . 12%; range 0 . 04–0 . 19% ) was significantly lower than the overall diversity observed within chronic subjects ( mean 2 . 27%; median 2 . 37%; range 0 . 56–3 . 83%; p<0 . 0001 , unpaired T-test with Welch's correction ) ( Figure S1 ) .", 'Because of the distinctive replication strategy of HCV , which is still not fully understood [3] , [5] , we could not predict a priori the patterns of early virus diversification that we might observe in acutely infected subjects .', 'Thus , we developed two mathematical models to analyze HCV sequence diversity ( Figure 3 ) .', 'The first model [15] , [48] , [49] , employed previously to analyze early HIV-1 sequence diversification , assumes a narrow genetic bottleneck associated with virus transmission , initial rapid exponential virus growth , constant lineage-independent mutation rates at all sites , no recombination between sequences or back mutations , and no differential selection .', 'When diversity is low , most base substitutions occur at distinct loci , pairwise differences between sequences ( i . e . , Hamming distances , HD ) follow a Poisson distribution , and sequences exhibit a star-like phylogeny and coalesce to distinct unambiguous T/F genomes [15] , [18] , [51] .', 'Early HIV-1 diversification conforms well to this model and it was suggested that other viruses including HCV might also [48] .', 'However , HCV replication differs from HIV-1 in that HCV RNA does not integrate into chromosomal DNA and it does not produce all of its daughter progeny in a large burst of viruses within a couple days after infecting a cell [52] .', 'Instead , it forms as many as 40 cytosolic replication complexes that continue to produce virions throughout the lifetime of the cell [50] , [53] .', 'In early infection prior to the onset of HCV specific cellular immune responses , the lifetime of infectedcells is likely to span our sampling period given that the lifetime of uninfected hepatocytes is estimated to be months to years [54] , [55] .', 'To account for these differences with HIV-1 , we developed an alternative simplified deterministic model of HCV diversification ( Figure 3 ) .', 'This model predicts occasional violations in star-like diversification and in the Poisson fit of mutations with increasing probability as time goes on , and it predicts greater numbers of shared stochastic mutations between HCV sequences compared with HIV-1 sequences .', 'The latter model provided us with a mathematical and statistical basis for distinguishing closely related T/F HCV lineages from sequences that evolved from a single genome but shared early stochastic mutations .', 'Importantly , it allowed us to vary key assumptions regarding the contributions of long-lived hepatocytes containing multiple generations of replication complexes and assess the effects on sample-based T/F virus enumeration .', 'From this analysis , we adopted a conservative operational cut-off of >2 shared mutations per quarter genome ( ∼2500 bp ) or >4 shared mutations per half genome ( ∼5000 bp ) to distinguish T/F genomes in most cases of HCV transmission from chronically infected individuals ( see Methods ) .', 'In more complicated transmission scenarios , where the index case was hypothesized to be either acutely infected or to have experienced a viral genetic bottleneck due to antiviral drug therapy , we applied both an empirical approach where a single shared polymorphism could represent a distinct T/F viral genome as well as the more conservative modeling approaches .', 'Importantly , both empirical and model-based analyses predicted that consensus sequences of low diversity lineages before the onset of immune-driven positive selection coalesced to founder viral genomes at or near the moment of transmission .', 'Figure 4 depicts ML phylogenetic trees and Highlighter plots of viral 5′ half genome sequences from two subjects , one typical of chronic infection ( WIMI4025 , panel A ) and one illustrating the reduced genetic diversity characteristic of a subject productively infected by a single viral genome ( 10051 , panel B ) .', 'Sequences from the chronic subject showed broad genotypic heterogeneity with a maximum inter-sequence diversity of 3 . 83% ( mean 1 . 2%; median 1 . 21%; range 0 . 12–3 . 83% ) typical of chronic infection ( Table S1 ) .', 'Sequences from the acute subject revealed a very different pattern of diversification ( Figure 4B ) .', 'These sequences , which were derived from the last sampled time point 21 days after the beginning of documented viremia ( see Figure 1 ) , were extremely homogeneous with a mean diversity of 0 . 03% , median diversity of 0 . 02% , and a range in diversity of 0–0 . 19% .', 'Nucleotide substitutions corresponded to a near star-like phylogeny , although unlike most early HIV-1 sequence sets they deviated from a Poisson distribution ( p<5×10−5 ) , a finding consistent with predictions of the HCV adapted model .', 'This deviation resulted from two sequences ( 2C3 and 2C2 ) that contained a single shared polymorphismat position 2197 and two other sequences ( 2A2 and 2B34 ) that contained a different shared polymorphism at position 4344 .', 'The shared polymorphism at position 2197 was a first position GAG to TAG transversion that resulted in the introduction of a stop codon .', 'Surprisingly , the same nonsense mutation was found in the identical position in two additional sequences ( 2B8 and 2B9 ) from this subject in a plasma sample taken 12 days earlier ( Genbank accession nos . JQ803586 , JQ803587 , JQ803590 and JQ803591 ) .', 'We could be assured that these persistent but defective genomes were authentic and did not result from cross-contamination of amplicon sequences since each of the four sequences had additional distinguishing nucleotide polymorphisms ( e . g . , compare sequences 2C3 and 2C2 in Figure 4B ) or were processed , PCR amplified and analyzed on different days ( e . g . , 2C3 and 2C2 versus 2B8 and 2B9 ) .', 'Occasional shared polymorphisms are commonly found in acute HIV-1 infection sequences and can be explained by polymerase errors early in infection being retained in the population [15] , [49] , but for neither HIV-1 nor HCV would nonsense mutations be expected to be retained unless they were complemented by competent genomes [50] , [56] , [57] .', 'Regardless , the 60 sequences depicted in Figure 4B coalesced to a single unambiguous consensus , which we inferredto represent a likely T/F virus in this subject .', 'To explore if additional T/F sequence lineages might have been overlooked due to inadequate sampling , we sampled 243 additional 5′ quarter genome sequences from this subject at three earlier time points spanning a 19 day period ( Figures 1 and S2 ) .', 'Power calculations indicate that a sample size of 60 sequences provides 95% likelihood of detecting variants present at 5% in the population [15] , whereas a sample size of 303 sequences provides 95% likelihood of detecting variants present at 1% prevalence .', '43 of 46 ( 93% ) of the sequences at the initial time point were identical , and diversity increased with time ( Figure S2 ) .', 'Whether the 303 5′ quarter genome sequences from the different time points were considered separately or together , they conformed to a near star-like phylogeny and coalesced to the same T/F genome .', 'Thus , we can conclude with a high level of confidence that subject 10051 was productively infected by a single virus whose sequence is represented by the consensus in Figure 4B and S2 .', 'Figure 5 extends the analysis of T/F HCV genomes to four acutely infected subjects where sequences from sequential time points revealed variable patterns of early viral diversity .', 'In each subject , sequences from the initial sample were more homogeneous than those from later time points , as expected in a model of random accumulation of mutations .', 'For subject 10021 ( panel A ) , 19 of 24 ( 79% ) sequences from the initial timepoint were identical .', 'For subject 10025 ( panel B ) , 32 of 43 ( 74% ) of initial sequences were identical .', 'The remaining sequences from this first time point in each subject differed from the respective consensus sequences by only 1 or 2 nucleotides .', 'At the second and third sampling time points 1–4 weeks later , an increasing proportion of sequences from each subject differed from the respective consensus sequences by as many as 3 or 4 nucleotides .', 'Interestingly , in both samples rare shared mutations became evident at later time points , again consistent with predictions of the HCV adapted model .', 'For subjects 10021 and 10025 , we thus concluded that the respective consensus sequences corresponded to single T/F HCV genomes .', 'Of the 17 acutely infected subjects , four had evidence of productive clinical infection by single viruses ( Tables 1 and S2 ) .', 'Panels C and D depict sequences from subjects 10012 and 10062 , each of whom had evidence of productive infection by more than one genetically distinct virus based on the presence of multiple discrete low diversity lineages whose consensus sequences differed from each other by far more than the 2 nucleotides per quarter genome cut-off .', 'Importantly , unlike HIV-1 where viral recombination in acute and early infection is extremely common [12] , [16] , we found no evidence of inter-lineage recombination in HCV sequences from these subjects or from any other subjects in this study . We thus interpreted the consensus sequences of each low diversity lineage in subjects 10012 and 10062 to correspond to a unique T/F HCV genome , three for each subject .', 'This represents a minimum estimate , since deeper sampling could conceivably identify additional T/F sequence lineages , although with 188–230 sequences analyzed there was a 95% likelihood of detecting variants present at 2% in the population ( Table S2 ) .', 'Figure 6 depicts sequences from subject 10029 where discrete low diversity sequence lineages indicated clinical acquisition of a minimum of 9 T/F viruses .', 'In 6 additional acutely infected subjects , clearly distinguishable T/F lineages ranged from 3 to 13 per subject ( Figures S3 , S4 , S5 , S6 , S7 , S8 ) .', 'Importantly , for all of the acutely infected subjects described above , the numbers of T/F sequence lineages identified by visual inspection using the cut-off of >2 per quarter genome and >4 per half genome were nearly identical to those inferred from the HCV adapted model of early virus diversification using standard ( maximum cut-off ) or stringent ( average cut-off ) assumptions ( Table S2 ) .', 'Virus diversity in three acutely infected subjects ( 10003 , Figure 7; 10020 , Figure S9A; 10016 , Figure S9B ) shared features that distinguished them from the other 14 acute subjects .', 'In each case , maximum sequence diversity was relatively low ( 0 . 27%–0 . 41% ) , consistent with recent infection .', 'However , unlike in other subjects , samples from 10003 , 10020 and 10016exhibited many closely related sets of sequences that shared unique polymorphisms , which appeared as multiple vertical ‘stripes’ in the Highlighter plots and shared nodes in the phylogenetic trees ( Figures 7 and S9A , B ) .', 'In subject 10003 ( Figure 7 ) , there were at least 37 distinctsequence sets , and in subjects 10020 ( Figure S9A ) and 10016 ( Figure S9B ) there were 10 and 15 , respectively .', 'Although we recognized that occasional shared mutations are predicted by the model and were found in samples from subjects infected by single or few viruses with well-defined T/F sequence lineages ( e . g . , see Figures 4B , 5 and 6 ) , the high frequency of unique shared mutations in subjects 10003 ( Figure 7 ) , 10020 ( Figure S9A ) and 10016 ( Figure S9B ) was quite unusual and led us to hypothesize that most of the 10–37 distinct virus lineages observed in these individuals resulted from discrete transmitted viruses from individuals who themselves were recently infected by a single virus or by closely related viruses .', 'An alternative hypothesis that we considered was that infection by single viruses had occurred in subjects 10003 , 10020 and 10016but was followed by atypical diversification patterns not seen in any of the other acutely infected subjects .', 'Our strategy to distinguish between these scenarios was both empirical and model-based .', 'First , we observed that the maximum Hamming distances , expressed as per cent diversity , between the consensus sequences of the discrete sequence sets ( i . e . , between potential T/F genomes ) from subjects 10016 , 10020 and 10003 , were 0 . 21% , 0 . 28% and 0 . 35% ( mean 0 . 28%; median 0 . 28% ) , respectively .', 'These values exceeded the maximum intra-lineage diversity ( mean 0 . 12%; median 0 . 12%; range 0 . 04–0 . 19%; p\u200a=\u200a0 . 0051 , Mann-Whitney test ) found in all lineages from all subjects in the initial ∼6 weeks of infection .', 'These findings suggested acquisition of multiple distinct variants from recently infected subjects , not evolution of viruses from single transmitted variants .', 'Secondly , we found that the multiple sequence sets in subjects 10016 , 10020 and 10003 were present from the initial sampling time points and did not accumulate over time , again suggesting acquisition not evolution of the variants .', 'Thirdly , we noted that the linear distribution of shared nonsynonymous nucleotide substitutions across the viral genomes in subjects 10016 , 10020 and 10003 was not random but instead was concentrated in regions of E1 and E2 ( Figure S10 ) previously associated with immune escape [41] , [42] , [58] .', 'Inspection of the amino acid substitutions in the E2 region of the putative T/F genomes revealed combinations of different nonsynonymous mutations in identical or neighboring positions in the primary sequence that were unlikely to have occurred by chance ( probability estimates <0 . 001 ) .', 'Since these subjects were sampled very early after infection , well before antibody seroconversion ( Figure 1 ) or the onset of cellular immune responses [9] , [10] , the data suggest that the patterns of viral diversity observed in the putative T/F viral genomes from subjects 10016 , 10020 and 10003 were the consequence of acquisition of multiple variants from transmitting individuals who themselves had become infected in the preceding ∼6 months and whose viral sequences had been subjected to early epitope-focused immune selection .', 'We next applied our HCV adapted model of early virus diversification to the sequences from the three subjects 10016 , 10020 and 10003 ( Figures 8; S14–15 ) .', 'Under standard model assumptions ( maximum cut-off , see methods ) for delineating T/F lineages , transmission by at least 6–19 genetically distinct viruses was necessary to explain the viral diversity observed in the three subjects .', 'Even under the most conservative model assumptions ( average cut-off ) , transmission of at least 2–9 distinct viruses was required .', 'Based on these findings , we conclude that the pattern of viral sequence variation in subjects 10003 , 10020 and 10016can be most plausibly explained by acute-to-acute virus transmission .', 'The phylogenetic pattern of sequences from subject 106889 ( Figure', '9 ) was still more complicated than that of any of the other 16 acutely infected subjects .', 'This subject exhibited a typical acute infection viral kinetic profile with four sequential plasma samples negative for HCV vRNA and antibody followed by rapid vRNA ramp-up to nearly 4×106 vRNA IU/ml ( Figure 9 insert ) .', 'These plasma samples from subject 106889 were obtained in June and July 2008 and were preceded by over 150 plasma collections from this individual in 2003–2008 , all of which were negative for HCV RNA or antibody , proving this individual had incident infection .', 'Eighty-seven 5′ half genome sequences were obtained from the initial plasma vRNA positive time point ( Figure 9 ) .', 'Maximum diversity of these sequences was 1 . 03% , indicating multi-variant transmission .', 'Similar to subjects 10003 , 10020 and 10016 , numerous discrete , low diversity sequence clades , many of which were closely related to each other , were apparent in the maximum-likelihood tree and Highlighter plot .', 'Unlike sequences from the other subjects , however , the distinct sequence sets in 106889 clustered with high bootstrap values into larger lineages ( color-coded in Figure 9 ) .', 'Thoseclades that contained sufficiently large numbers of sequences for analysis ( e . g . , clades identified by sequences 5 . B . F9 and 5 . 02C22 ) exhibited a star-like phylogeny and Poisson distribution of mutations ( Table 1 ) , indicating that they had evolved very recently from discrete T/F genomes . These findings suggested that subject 106889 had been infected by large numbers of virusesfrom a chronically infected individual whose HCV sequences had been subjected to a stringent genetic bottleneck .', 'A quite unexpected finding suggested a likely explanation: 86 of the 87 sequences from subject 106889 were found to contain two signature mutations in the NS3 protease gene ( V36M and R155K ) that confer high level drug resistance to the NS3 protease inhibitors Boceprevir and Telaprevir [7] .', 'One of 87 sequences ( sequence 02B11 in Figure', '9 ) contained one of these DAA resistance mutations ( V36M ) .', 'Since the combination of V36M and R155K mutations is uncommon in treatment-naïve individuals [59] , this result suggested that a transmitting partner to 106889 was chronically infected with HCV , was treated with an investigational NS3 protease inhibitor , experienced a DAA-induced viral population bottleneck followed by the emergence of NS3 protease resistant variants , and as these variants rebounded , transmitted multiple drug resistant variants directly to subject 106889 or indirectly through a second acutely infected individual .', 'Under this scenario , where closely related sequences differing by as few as one nucleotide would be expected to be transmitted , we estimated by visual inspection of the phylogenetic tree that as many as 30 or more T/F virus could have been responsible for productive clinical infection ( Table S2 ) .', 'By the standard model analysis , 28 distinct T/F viral genomes were necessary to account for the observed diversity .', 'By the more conservative model analysis , at least 16 distinct T/F genomes were needed ( Table S2 ) .', 'The identification of T/F viral sequencesin 17 subjects provided us with a unique opportunity for analyzing HCV sequence evolution in natural human infection beginning at or near the moment of virus transmission and extending through the establishment of early viral load setpoint , and in some subjects , antibody seroconversion .', 'Among the 17 subjects , we identified a total of 146 T/F genomes .', 'As expected , all 146T/F sequences had intact open reading frames for core , E1 , E2 , P7 , NS2 and NS3 .', 'Thirty T/F genomes had 5 or more identifiable progeny ( range 5–303; median 19 ) from which we could analyze molecular features of sequence diversification in vivo using phylogenetic tools and algorithms .', 'A summary of this analysis is presented in Table 1 .', "Maximum intra-lineage diversity for the 17 subjects ranged from 0 . 04% to 0 . 19% ( mean\u200a=\u200a0 . 12%; median\u200a=\u200a0 . 12% ) , which was significantly lower than the maximum , mean and median viral diversities observed in chronically infected subjects ( 3 . 83% , 2 . 27% and 2 . 37% , respectively; p<0 . 0001 , unpaired T-test with Welch's correction ) ( Tables 1 and S1; Figure S1 ) .", 'Evolved sequences compared to their respective T/F genomes revealed low frequencies of per nucleotide insertions ( 1×10−6 ) , deletions ( 3×10−6 ) and stop codons ( 1×10−6 ) .', 'Transitions outnumbered transversions by 8 . 8 to 1 , and when corrected for the number of available sites , by 18 to 1 .', 'The average dN/dS ratio was low at 0 . 39 .', 'The overall mutation frequencyamong all 17 acute subjects uncorrected for time from transmission or numbers of virus replication cycles was 1 . 4×10−4 .', 'When sequences were analyzed in the context of an agent-based stochastic model of virus diversification that incorporates estimated time from transmission and HCV specific parameters of virusreplication , the mutation rate of HCV in vivo was estimated to be 2 . 5×10−5 per nucleotide per genome replication [50] .', 'We confirmed this low value by an analysis of the nonsense codon frequency per nonsense mutation target site [50] .', 'Progeny of T/F viruses sampled at the earliest time points generally conformedto a star-like phylogeny and a Poisson distribution of randommutations ( Table 1 ) , but at later time points there were occasional deviations .', 'Deviations from the model were of three types:', '( i ) shared polymorphisms resulting from stochastic changes after the transmission event or from the transmission of multiple closely related viruses;', '( ii ) immune selection or reversion in later samples at the time of antibody seroconversion in a single subject 9055 ( Figures S16 , S17 ) ;', '( iii ) rare examples of short perfect inverted repeats , 3–20 nucleotides in length , that resulted from template switching between double-stranded RNA molecules in locations prone to RNA stem-loop secondary structure ( Figure S18 ) .', 'This latter finding , which was found in 7 sequences out of 2922 analyzed , was observed in samples from three different study subjects .', 'Four of these strand transfers occurred at the same location in the core gene .'], ['The present study provides new quantitative and qualitative insights into HCV transmission and early diversification in humans .', 'Previous reports documented a virus population bottleneck associated with HCV transmission , but none of those studies including ones based on 454 deep sequencing captured the broad range in multiplicity of infection or the full spectrum of genetic diversity that exists among transmitted viruses .', 'In our study of 17 acutely infected subjects , we could unambiguously identify and determine the exact nucleotide sequences of one or more T/F virus genomes in each subject .', 'This was true for all subjects whose HCV genomes were sequenced within the initial ∼6–8 weeks of infection; beyond that there were examples of immune selection that confounded the identification of T/F virus genomes ( Figures S16 and S17 ) .', 'We estimated the multiplicity of infection ( numbers of T/F viruses leading to productive clinical infection ) to range from 1 to as many as 37 or more with a median of 4 .', 'These are minimum estimates given our sampling limitations , although we note that our median sampling depth of 151 sequences ( Table 1 ) afforded us a 95% likelihood of detecting variants present at 2% prevalence [15] .', 'In subjects productively infected by lower numbers of viruses ( <10 ) , where the progeny of each transmitted virus is repeatedly sampled , our estimates ( Tables 1 and S1 ) are likely to be an accurate and precise measure of the number viruses that result in productive infection .', 'In subjects infected by higher numbers of viruses , especially in the setting of acute-to-acute transmission where transmitted viruses are expected to differ by as few as one nucleotide , the accuracy of our estimates are necessarily less .', 'This is because we could not sample deeply enough due to practical constraints of single genome sequencing of quarter and half genomes , and because we could not distinguish between transmitted viruses that differ by one or few nucleotides from single variant transmission followed by early stochastic mutations .', 'However , based on the striking differences in diversity patterns that we observed between subjects with chronic-to-acute versus apparent acute-to-acute transmission , we suspect that the actual numbers of T/F viruses in subjects 10016 , 10020 , 10003 and 106889 approximate or exceed our estimates of 15 , 10 , 37 and 30 T/F genomes , respectively ( Tables 1 and S2 ) .', 'The broad range in numbers of T/F viruses responsible for acute HCV infection in our cohort must reflect the different transmission routes and risk practices ofsource plasma donors .', 'Ostensibly , such individuals should be at low risk of acquiring HCV infection since they are qualified as regular source plasma donors only after extensive pre-enrollment screening that consists of medical histories , physical examinations and behavioral questionnaires designed specifically to eliminate from the donor pool individuals at risk for HCV , HBV or HIV infection ( http://www . fda . gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/default . htm ) .', 'However , self-reporting of risk behaviors among paid plasma donors is admitt
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['A precise molecular identification of transmitted hepatitis C virus ( HCV ) genomes could illuminate key aspects of transmission biology , immunopathogenesis and natural history .', 'We used single genome sequencing of 2 , 922 half or quarter genomes from plasma viral RNA to identify transmitted/founder ( T/F ) viruses in 17 subjects with acute community-acquired HCV infection .', 'Sequences from 13 of 17 acute subjects , but none of 14 chronic controls , exhibited one or more discrete low diversity viral lineages .', 'Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes .', 'Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more ( median\u200a=\u200a4 ) .', 'Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny .', 'In these cases , empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy .', 'These results provide new quantitative and qualitative insights into HCV transmission , revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome .', 'Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures .']
['Hepatitis C virus infects as many as 170 million people worldwide .', 'Globally , there are seven major genotypes of HCV that differ by approximately 30% in nucleotide sequence .', 'Importantly , the natural history of HCV infection is variable , ranging from spontaneous resolution to persistent viremia and chronic disease .', 'Factors responsible for this variability in clinical outcome are unknown but likely involve a combination of viral and host determinants .', 'To this end , a precise molecular identification of transmitted HCV genomes could illuminate key aspects of transmission biology , immunopathogenesis and natural history .', 'We used single genome sequencing of plasma viral RNA to identify transmitted viral genomes and their progeny in 17 subjects with acute infection .', 'Numbers of transmitted viruses leading to productive clinical infection ranged from 1 to 37 or more ( median\u200a=\u200a4 ) .', 'Surprisingly , we found evidence of high multiplicity acute-to-acute HCV transmission in 3 of 17 subjects , which suggests that clinical transmission of HCV , like that of HIV-1 , may be enhanced in early infection when virus titers are highest and neutralizing antibodies are absent .', 'These results provide novel insight into HCV transmission and early virus diversification key to our understanding of virus natural history and response to drug selection and immune pressure .']
['hepatitis c', 'medicine', 'infectious diseases', 'hepatitis', 'viral diseases']