hepatitis c virus ( hcv ) infects 175,000,000 people worldwide and is a major cause of morbidity and mortality ( 1 , 2 ) . in the united states ,
recent advances in the development of interferon sparing , direct - acting antiviral agents ( daa ) suggest that most treated patients can be cured , but given the limited access and high cost of daa on a global scale , prevention of infection by effective vaccination remains the best hope for virus eradication .
however , in contrast to drug development , progress in vaccine development has been slow , in large part due to the extraordinary genetic diversity and rapid sequence evolution of the virus ( 37 ) .
globally , hcv is represented by seven major genotypes ( 1 to 7 ) that exhibit nucleotide sequence diversity of as much as 30% ( 8) . within individual infected subjects
it is estimated that up to 10 virions are produced daily in a typical infected individual ( 9 ) , and based on an rna - dependent rna polymerase error rate of 2.5  10 per nucleotide per replication cycle ( 10 ) , most of these are expected to be unique .
in fact , every possible single point mutation , as well as every possible combination of two mutations , across the 10-kb viral genome are predicted to be generated every day ( 11 ) .
this explains the rapid appearance of resistance mutations to daas and the virus 's capacity to evade host adaptive immune responses . against this backdrop of nearly unfathomable viral diversity
is substantial evidence indicating that hcv exhibits a relatively stringent population bottleneck at the moment of transmission from one individual to the next ( 1214 ) .
such a transmission bottleneck is of clinical importance because at this point in viral natural history the infection is most vulnerable to treatment and prevention measures , including , potentially , vaccination ( 5 , 1517 ) .
a virus bottleneck at transmission was initially inferred from clinical epidemiological studies that correlated risk of infection to the volume of blood exposure and the route of exposure ( e.g. , blood transfusion , injection drug use , needlestick injury , or mucosal inoculation , especially in hiv-1-positive men who have sex with men [ 1 , 1823 ] ) .
more refined estimates of the transmission bottleneck came from studies using a variety of increasingly sensitive and specific molecular techniques to characterize viral diversity , including oligonucleotide heteroduplex gel shift , vrna / cdna population sequencing , and molecular cloning , followed by sanger sequencing and next - generation deep sequencing ( 12 , 13 , 2432 ) .
these reports described the hcv transmission bottleneck in both qualitative and quantitative terms , but none allowed for a precise identification of transmitted or founder virus genomes .
the most exacting quantitative estimates of the transmission bottleneck thus far came from studies based on single - genome sequencing ( sgs ) , mathematical modeling , and phylogenetic inference ( 10 , 14 , 3335 ) .
this approach , which was adapted from hiv-1 infection studies ( 3639 ) , theoretically allows for an unambiguous inference and quantitative estimation of transmitted / founder ( t / f ) viral genomes that are responsible for establishing productive clinical infection .
the conceptual and mathematical models underlying this strategy assume a virus population emanating from each t / f genome that expands exponentially with no recombination , no selection pressure on variable sites , and a constant mutation rate across lineages ( 36 , 40 , 41 ) .
under these conditions , the frequency distribution of the hamming distances ( i.e. , the pairwise distance between genetic strains ) is given by a poisson distribution whose mean depends linearly on the number of generations since the founder strain ( 36 , 40 , 41 ) .
if the expansion is sufficiently rapid , small samples of sequences exhibit a star - like phylogeny whose coalescent corresponds to the t / f genome .
for this situation , an analysis by forward simulation suffices , and a bayesian inference ( e.g. , beast [ 42 , 43 ] ) , which is a backward simulation of genealogies , provides no advantages .
previously , we used the forward simulation model to analyze hcv sequences from 17 subjects in the very earliest stages of acute infection ( weeks before hcv antibody seroconversion ) .
sequences from 13 subjects conformed well to model predictions , showing a star - like phylogeny and a poisson distribution of mutations within one or more distinct and well - separated lineages , each with low diversity ( 14 ) .
we interpreted these low - diversity sequence lineages to represent the progeny of discrete t / f genomes , thereby allowing for the identification and enumeration of the t / f genomes that resulted in productive clinical infection . in these 13 study subjects , the estimates of numbers of t / f genomes ranged from 1 to 13 per individual with a mean of 4 and a median of 3 ( 14 ) .
in the same study , however , viral sequences from four other acutely infected subjects ( subjects 10003 , 10016 , 10020 , and 106889 ) appeared to violate this forward simulation model by exhibiting multiple closely related lineages that failed to conform to a poisson distribution of mutations or star - like phylogeny . some lineages differed from others by as few as one to three informative sites ( shared mutations ) out of the 5,000 bases sequenced . for these patients , our estimates for the numbers of t / f genomes were as high as 30 or more .
we considered two possible explanations for these outlier results : either early hcv diversification in these subjects failed to follow a simple pattern of random diversification , creating multiple distinct but closely related lineages early on because of features unique to the hcv replication schema confounded by other unknown virus - host factors , or viral sequences in the donors to these four cases had undergone a population diversity bottleneck event prior to the transmission of multiple closely related variants , which then evolved as expected by the forward simulation model .
such a bottlenecking event in the donors could occur as a result of antiviral treatment , immunological selection or very recent acquisition of virus ( i.e. , the donors were themselves acutely infected ) . to explore these possibilities
, we developed a different forward simulation model of early virus diversification that accounts for essential differences in replication dynamics between hcv and hiv-1 .
based on the patterns of shared mutations observed in these forward simulations , we developed a phylogenetically based clustering method that counts the number of putative t / f genomes ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) .
this approach thus incorporates the important features of hcv 's unique cytosolic life cycle , where many replication complexes continuously produce virus from long - lived hepatocytes ( 10 , 44 , 45 ) . because of the sequential creation of as many as 40 replication complexes per cell ( 46 ) , those at the same generation depth have widely varying numbers of descendants , unlike the situation in hiv-1 infection
this leads to two predictions that differ substantially from the hiv-1 case : ( i ) sequence diversity of hcv saturates around the same time during primary infection that viral load stabilizes , instead of growing linearly in time as is the case for hiv-1 , and ( ii ) there is an expectation of about three times as many stochastically shared mutations in hcv compared to hiv-1 infection when sampled in this stable phase , leading to more common deviations from a star - like phylogeny and poisson distribution of mutations ( 10 , 14 ) .
when the second model was applied to sequences from subjects 10003 , 10016 , 10020 , and 106889 , it still failed to account fully for the atypical sequence diversification patterns and large numbers of potential t / f lineages observed in these subjects ( 14 ) .
we thus sought alternative explanations for the atypical early viral diversity , including the possibility of a pretransmission population bottleneck , followed by high - multiplicity virus transmission . in one case ( subject 106889 ) , we found evidence for such a bottleneck in the donor virus population due to prior treatment with a protease inhibitor ( 14 ) . for subjects
10003 , 10016 , and 10020 , however , there was no evidence of a drug - induced bottleneck , so we instead hypothesized that the donors for these subjects might themselves have been acutely infected or their viruses subjected to stringent immunological selection ( 32 ; unpublished data ) . in this scenario
, the transmission of multiple viruses would be expected to yield a diversity pattern in the recipient comprised of very closely related viral lineages in addition to more distantly related lineages . in africa
, it is estimated that  acute - to - acute  infection is responsible for as many as 25% of incident hiv-1 cases ( 4749 ) .
this can result in a pretransmission virus population bottleneck in the donor and acquisition of closely related viruses by the recipient ( 36 , 38 ) .
unfortunately , in our previous hcv studies ( 14 ) , we had no documented cases of  acute - to - acute  virus transmission to evaluate this scenario .
so , in the present study , we sought new examples of epidemiologically linked , sequence - confirmed , acute - to - acute hcv transmission .
here , we analyze early patterns of hcv sequence diversity in epidemiologically linked , viral sequence confirmed , donor - recipient , acute - to - acute transmission pairs , including human - to - human and human - to - chimpanzee infections .
we tested two hypotheses : first , that hcv genomes responsible for transmission to a naive host can be identified unambiguously in paired donor - recipient plasma samples and that these sequences will be identical or nearly so , and second , that high - multiplicity infection of a subject with plasma from a donor who is acutely infected ( and thus harbors sequences exhibiting a pretransmission population bottleneck ) will recapitulate patterns of virus diversity observed in subjects 10003 , 10016 , and 10020 .
in so doing , they validate the strategy of t / f genome identification by sgs , mathematical modeling , and phylogenetic inference for hcv , and they provide a plausible explanation for what had previously been a confusing set of hcv sequence data in a subset of acutely infected subjects .
our findings indicate that simple models of random virus diversification can generally explain early hcv evolution , although models that account for unique features of the hcv replication strategy offer a more conservative estimate of t / f genome numbers in samples that fail to conform to a star - like phylogeny and a poisson distribution of mutations .
our results illustrate how t / f analysis in hcv infection , much like t / f analysis in hiv-1 infection , represents a novel experimental strategy that can be used to molecularly anchor and uniquely inform studies of hcv natural history , immunopathogenesis , treatment , and prevention .
the present study was conducted according to the principles expressed in the declaration of helsinki .
it was approved by the institutional review boards of the university of pennsylvania and mt .
sinai medical center , new york , ny . subjects provided written informed consent for the collection of blood samples and subsequent analyses .
chimpanzee specimens were collected and stored as part of a previously conducted study ( 50 ) , and thus the present research is in full compliance with national institutes of health ( nih ) policies ( not - od-12 - 025 ) concerning biomedical research involving chimpanzees .
the subjects either were regular source plasma donors ( zeptometrix , inc . ; seracare , inc . ) who were hcv and hiv-1 antibody negative and became hcv infected sometime in the course of their twice - weekly plasma donations , as evidenced by the development of hcv viremia on sequential viral rna testing , or they were hcv antibody - negative patients who became acutely infected with hcv and sought medical consultation .
chimpanzee inoculations with hcv - infected human plasma , subsequent specimen collections , and analysis of plasma virus load and liver specific transaminase levels were previously reported ( 50 ) .
plasma samples were tested for hcv rna and antibodies by a battery of commercial tests .
these included the roche molecular systems cobas amplicor hcv monitor ( v2.0 ) assay , the abbott anti - hcv 3.0 assay , and the ortho anti - hcv 3.0 enzyme - linked immunosorbent assay .
additional vrna analyses using the transcription - mediated amplification ( tma ) method were performed as described previously ( 50 ) . for each plasma sample
, approximately 100,000 viral rna copies were extracted using the qiagen biorobot ez1 workstation with an ez1 virus minikit v2.0 ( qiagen , valencia , ca ) .
reverse transcription of rna to single - stranded cdna was performed using mulv ( superscript iii ) reverse transcriptase ( rt ; invitrogen life technologies , carlsbad , ca ) according to the manufacturer 's instructions .
briefly , each cdna reaction included 1 rt buffer , 0.5 mm concentrations of each deoxynucleoside triphosphate , 5 mm dithiothreitol , 2 u of rnaseout ( an rnase inhibitor)/l , 10 u of superscript iii reverse transcriptase/l , and 0.25 m antisense primer .
the cdna primer used for amplifying each genome fragment corresponded to the first - round antisense primer listed in the single - genome amplification section below .
the reverse transcription reaction was carried out at 50c for 60 min , followed by an increase in temperature to 55c for an additional 60 min .
the reaction was then heat inactivated at 70c for 15 min and then treated with 0.1 u of rnase h/l at 37c for 20 min .
the sequences from all of the subjects were generated using the sgs method previously described ( 14 ) .
nearly full length , 5 half , 3 half , or partial ns2 , ns3 , and ns4a genomes were amplified for each subject by nested or seminested pcr , respectively .
cdna was serially diluted and distributed among wells of replicate 96-well plates ( applied biosystems , foster city , ca ) so as to identify a dilution where pcr - positive wells constituted less than 30% of the total number of reactions . at this dilution ,
this was confirmed in every positive well by direct sequencing of the amplicon and inspection of the sequence for mixed bases ( double peaks ) , which would be evidence of priming from more than one original template or the introduction of pcr error in early cycles .
pcr amplification was carried out in the presence of 1 high - fidelity platinum pcr buffer , 2 mm mgso4 , 0.2 mm concentrations of each deoxynucleoside triphosphate , 0.2 m concentrations of each primer , and 0.025 u of platinum taq high - fidelity polymerase/l in a 20-l reaction ( invitrogen , carlsbad , ca ) .
nearly full length , 5 half or partial ns2 , ns3 , and ns4a genomes were amplified .
the primers used were as follows : ( i ) subject 110069 , first - round sense primer 1a.core.f1 ( 5-gcacgaatcctaaacctcaaagaaaaa-3 ; nucleotides [ nt ] 346 to 372 , h77 ) , first - round antisense primer 1ans5bend.r1a ( 5-ccggagtgtttatcccaaccttcat-3 ; nt 9374 to 9398 , h77 ) , second - round sense primer 1a.core.f2 ( 5-cgggtggcggtcagatcgttggtggagttta-3 ; nt 415 to 445 , h77 ) , and second - round antisense primer 110069.ns5b.r2a ( 5-gcggccgctattggagtgagtttgag-3 ; nt 9201 to 9226 , h77 ) ; ( ii ) bgi and cci , first - round sense primer 1b.core.f1 ( 5-atgagcacgaatcctaaacctcaaaga-3 ; nt 342 to 368 , h77 ) , first - round antisense primer ns4a_r1 ( 5-gcactcttccatctcatcgaactc-3 ; nt 5451 to 5474 , h77 ) , second - round sense primer 1b.core.f2 ( 5-tcaaagaaaaaccaaacgtaacaccaaccg-3 ; nt 362 to 391 , h77 ) and second - round antisense primer ns4a_r2 ( 5-aggtgctcgtgacgacctccagg-3 ; nt 5297 to 5319 , h77 ) ; ( iii ) subjects 10081 and x355 , first - round sense primer 10081ns2_f2 ( 5-acccgaccctgatatttgatatcacc-3 ; nt 2983 to 3008 , h77 ) , first - round antisense primer 1ans4b.r3 ( 5-tattgtatcccactgatgaagttccacat-3 ; nt 5634 to 5662 , h77 ) , second - round sense primer 10081ns2_f3 ( 5-aaagtaccctactttgtgcgcgt-3 ; nt 3063 to 3085 , h77 ) , and second - round antisense primer 1ans4b.r4 ( 5-agggccttctgcttgaactgctc-3 ; nt 5517 to 5539 , h77 ) ; and ( iv ) subjects 10083 and x331 , first - round sense primer 10083stf1 ( 5-gacatcactaagctgctgatag-3 , first - round antisense primer 3av2r2 ( 5-ttacttccagatcagctgaca-3 ) , second - round sense primer new10083stf2 ( 5-cccgttatatttaatacaggcta-3 ) , and second - round antisense primer 3av2r2 ( 5-ttacttccagatcagctgaca-3 ) .
pcr amplicons were directly sequenced by cycle - sequencing using bigdye terminator chemistry and protocols recommended by the manufacturer ( applied biosystems ) .
sequencing reaction products were analyzed with an abi 3730xl genetic analyzer ( applied biosystems ) .
individual sequence fragments for each amplicon were assembled and edited using the sequencher program 5.0 ( gene codes ; ann arbor , mi ) .
inspection of individual chromatograms allowed for the identification of amplicons derived from single versus multiple templates .
the absence of mixed bases at each nucleotide position was taken as evidence of amplification from a single viral rna / cdna template .
this quality control measure enabled us to exclude from the analysis amplicons that resulted from pcr - generated in vitro recombination events or taq polymerase errors and to obtain multiple individual sequences that proportionately represented those circulating hcv virions .
all of the sequences alignments were initially made with clustal w and then hand - checked using geneious 7.1.9 to improve the alignments according to the codon translation .
eight hundred three quarter genomes , 103 5 half genomes and 114 near full - length genomes were generated by sgs from seven human and chimpanzee subjects for the diversity analysis .
among the 1020 amplicons generated , the sequences of 928 were unambiguous at every position .
nine near - full length genomes had mixed bases at 1 to 2 positions per sequence .
we inferred that these mixed bases resulted from taq polymerase errors in the early pcr cycles because the mixed bases represented only a subset of the polymorphisms in any one sequence .
these sequences were retained in the analysis and mixed bases interpreted as lineage consensus nucleotides .
the other 83 amplicons contained  double peaks ,  clearly resulting from amplification from more than one template , and these sequences were excluded from the analysis .
sequences were analyzed using phylogenetic tree analysis together with a sequence visualization tool , highlighter ( www.hiv.lanl.gov ) , that allows tracing of common ancestry between sequences based on individual nucleotide polymorphisms .
phylogenetic trees were generated by maximum - likelihood methods using phyml ( 51 ) . for subjects productively infected by more than one t / f virus
, lineages containing 3 or more closely related sequences were included in the lineage diversity analyses .
one was a forward simulation of early viral expansion ( 36 , 40 , 41 ) , and the second , a phylogenetic - based clustering method that accounts for essential differences in replication dynamics and replication mechanisms hcv and hiv-1 ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) .
lineage - specific sequences were analyzed by the poisson fitter program ( www.hiv.lanl.gov ) , which computes the best fitting poisson distribution through maximum likelihood , performs a goodness of fit test , and tests for star - phylogeny .
power calculations to estimate the likelihood of detecting rare sequence variants based on sampling depth were performed using methods previously described ( see fig . s9 and the associated description in reference 36 ) .
acute hcv infection has historically been defined as the first 6 months of infection , which reflects the natural history and immunopathogenesis of disease where a minority of individuals spontaneously resolves the infection by this point , while most patients , if left untreated , exhibit persistent viremia ( 1 , 18 , 52 ) . whether or not an individual clears virus depends on host immunogenetics , innate and adaptive immune responses , and viral genotype , as well as other factors ( 1 , 5355 ) .
figure 1 illustrates viral dynamics and diversity of plasma vrna / cdna sequences amplified as nearly full length ( 8.8 kb ) genomic amplicons in a typical subject ( e.g. , 110069 ) experiencing acute ( primary ) hcv infection . in this case ,
overall viral diversity during the acute infection period was dictated far more by the number of transmitted viruses and the genetic distances among them ( 0.15 to 0.47% ) than by early sequence evolution , which can undergo positive or negative selection , as well as population diversity bottlenecking ( 13 , 14 ) . very early in acute infection
prior to antibody seroconversion , sequences appeared as four readily distinguishable , low diversity lineages ( t / f lineages 1 to 4 ) . within each lineage , sequences differed by no more than 3 nucleotides ( nt ) out of nearly 9,000 nt ( 0.03% ) , and exhibited a star - like phylogeny and a poisson distribution of mutations ( table 1 ) .
furthermore , the diversity within each lineage was roughly the same , as would be expected if all of the lineages had been diverging at about the same rate and for the same time .
the pattern within each lineage conformed to a simple model of random virus evolution and is typical of most acute hcv infections ( 14 ) .
we inferred from these data that the coalescent of each low - diversity lineage corresponded to a distinct t / f genome in total numbering four .
this is a minimum estimate because deeper sequencing could potentially detect variants present at lower abundance . in this case , 48 sequences from days 2 and 17 following the first plasma vrna positive time point were initially analyzed .
power calculations ( 36 ) predict that at this level of sampling , there is greater than a 90% likelihood of detecting minor variants present at a frequency of 5% .
however , in data not shown or reported elsewhere ( 33 ) , we used other primer sets and plasma rna samples from early time points to generate 228 additional overlapping 5 and 3 half genomes from this subject , giving a total of 276 sequences evaluated for diversity and phylogeny .
at this level of sampling , power calculations indicate a > 90% likelihood of detecting minor variant lineages present at a frequency of 1% .
still , these are minimum estimates for the number of t / f genomes , and we can not exclude the possibility that a sequence such as 110069_2f27 ( situated between variants 3 and 4 in panel a ) represents a distinct t / f genome or is an example of homoplasy . nor can we exclude the possibility that next - generation deep sequencing could detect additional t / f lineages present at exceedingly low frequencies , although this has generally not been seen in acute hiv-1 infection ( 56 , 57 ) .
hcv sequence diversity in acutely infected human subject 110069 . a maximum - likelihood ( ml ) mid - point rooted phylogenetic tree ( a ) and highlighter plots ( c and d ) of nearly full length plasma vrna sequences spanning the initial 6-month infection period are depicted .
plasma viral load kinetics ( b ) are also depicted , and the sequences are color coded to correspond to sampling time points determined from the first vrna - positive / antibody - negative time point ( blue , day 2 ; red , day 17 ; green , day 142 ) .
hcv antibody tests were negative through day 17 but were positive at the next sampled time point on day 146 .
the tree and the highlighter plot reveal productive clinical infection by four t / f viruses ( t / f lineages 1 to 4 ) .
viral diversity is indicated by the scale bar and bootstrap values are shown . in the highlighter ( c ) , tick marks are color coded to indicate nucleotide substitutions by a ( green ) , t ( red ) , c ( blue ) , or g ( orange ) compared to the consensus sequence of the first time point ( tp1 ) . in panel d , tick marks denote synonymous ( green ) or nonsynonymous ( red ) substitutions .
statistical analysis of hamming distance frequency distribution and star - like phylogeny poisson - fitter ( www.hiv.lanl.gov ) computes the poisson distribution by maximum - likelihood and chi - square goodness - of - fit analyses and tests for star - like phylogeny .
the sequences from subject 10081 exceeded the diversity limits of the poisson fitter and thus did not conform to a poisson distribution or a star - like phylogeny .
pt1 , patient 1 ; pt2 , patient 2 . a goodness - of - fit ( gof ) p value of > 0.05 indicates a nonsignificant divergence from a poisson distribution .
approximately 5 months into infection , the pattern of sequence diversity in subject 110069 plasma was much different than at earlier time points ( fig .
there was evidence of a stringent population genetic bottleneck between the second and third sampling time points ( days 17 and 142 in fig .
the closer similarity of the 5-month sequences to t / f lineage 4 sequences ( 0.1% mean diversity ) compared to t / f lineages 1 ( 0.5% mean diversity ) , 2 ( 0.6% mean diversity ) , or 3 ( 0.3% mean diversity ) suggested that the 5 month sequences had all evolved from the t / f 4 lineage , with the descendants of t / f lineages 1 to 3 lost in a population / diversity bottleneck .
later sequences emanating from the t / f 4 lineage contained many shared mutations violating both a star - like phylogeny and a poisson distribution ( table 1 ) .
figure 1 ( panel d ) suggests that the population bottleneck likely resulted from immune selection since the highly selected nonsynonymous mutations in e2 ( see vertical red stripe in fig .
1d ) map to a previously identified cytotoxic t lymphocyte ( ctl ) epitope ( 58 ) and are adjacent to a contact residue of the broadly neutralizing antibody ar3c ( 59 ) . in addition
, the three strongly selected nonsynonymous mutations in ns3 map to previously identified ctl epitopes ( 58 , 60 , 61 ) . unfortunately ,
peripheral blood mononuclear cells were not available from this subject for human leukocyte antigen analysis or phenotypic testing to confirm phenotypic t cell recognition and escape of these and other potential epitopes .
the patterns of sequence evolution observed here for subject 110069 illustrate how high - multiplicity transmission of very early acute infection viruses or viruses arising following an immune - mediated population bottleneck could lead to sequence patterns in a recipient that resemble what was seen in the earlier study 's acutely infected outlier subjects 10003 , 10016 , and 10020 ( 14 ) . to further test the validity of hcv t / f genome inferences , we next studied epidemiologically linked human - to - human and human - to - chimpanzee donor - recipient hcv transmission pairs in an attempt to document transmission of actual virus genomes and to study their subsequent evolution .
figure 2 illustrates plasma vrna kinetics and 80 5 half genome sequences from two human subjects ( patient 1 [ bgi ] , donor ; patient 2 [ cci ] , recipient ) , who became acutely infected with hcv at about the same time .
these two individuals were hiv-1-positive men who have sex with men , who were regular sexual partners , who denied injection drug use ( idu ) , and who were hcv antibody negative at the time of first vrna detection .
the clinical history indicated that patient 1 first became symptomatic with acute hcv infection ( fatigue , jaundice , elevated liver transaminases ) leading to hcv screening of himself and his partner patient 2 by viral rna assay .
both individuals were confirmed to be positive for hcv rna and negative for hcv antibody .
figure 2 shows that patient 1 and patient 2 each harbored single low - diversity hcv sequence lineages that exhibited a near star - like phylogeny and a poisson distribution of mutations ( table 1 ) , indicating that each had been productively infected by a single t / f virus .
surprisingly , when the viral sequences from these individuals were analyzed together , they were found to be identical or nearly so . whether the sequences were analyzed separately or together , they coalesced phylogenetically to the same t / f virus genome ( fig .
2 ) . to be certain that a sample mix - up had not occurred , which could explain the identity or near identity of their sequences , five additional plasma samples taken from these subjects in subsequent weeks were analyzed .
a total of 162 sequences from patients 1 and 2 spanning a 15-week period all coalesced to the same single t / f genome .
this confirmed that donor and recipient had both been productively infected by viruses containing an identical 5 half - genome sequence .
given the clinical history , the most plausible explanation for this result is that patient 1 became acutely infected by a single t / f virus , and very early in his acute infection period , when most of his circulating virus still contained no mutations , he transmitted a single virus to patient 2 .
a less likely scenario is that patient 1 and patient 2 each acquired their virus from a third individual .
this interpretation was not consistent with the clinical history and would require that this third individual also be acutely infected so as to transmit identical viruses to two partners .
either way , acquisition of viruses containing identical genomes by patient 1 and patient 2 corroborates the t / f genome strategy and the conclusion that early virus evolution generally follows a simple model of random sequence diversification .
hcv plasma vrna kinetics and 5 half genome hcv sequences from an epidemiologically linked human hcv transmission pair .
( a ) viral load profiles in the presumed donor ( patient 1 [ bgi ] ) and recipient ( patient 2 [ cci ] ) are illustrated .
the sampling time points are circled and color coded red ( donor ) and blue ( recipient ) .
( b ) ml phylogenetic trees and highlighter plots show that each subject was productively infected by a single virus .
( c ) the combined tree and plot show that the t / f viral genomes infecting both subjects were identical in sequence .
we next sought to evaluate the t / f strategy in situations of low and high multiplicity infection from a transmitting donor who was acutely infected .
this was done to determine whether hcv sequence patterns similar to those found in subjects 10003 , 10016 , and 10020 could be recapitulated and explained on the basis of a pretransmission virus population bottleneck . because we had no access to additional epidemiologically linked human - to - human transmission pairs , we studied hcv transmission in the closely related human - to - chimpanzee infection model . biomedical research in chimpanzees is now restricted , so we analyzed stored plasma samples from a previous study by busch et al .
( 50 ) that was designed to evaluate the clinical infectivity of human plasma sampled from the earliest phases of infection before plasma hcv vrna could be detected by u.s .
food and drug administration ( fda)-licensed diagnostic assays . in those experiments , closely spaced sequential plasma samples from individuals who subsequently became acutely infected by hcv were inoculated into virus - naive chimpanzees to determine whether they contained infectious hcv .
this study plan provided an ideal experimental design and sets of well - pedigreed frozen plasma samples from linked 
donors  and  recipients  to test the concept of t / f genome identification under conditions of low- and high - multiplicity infections .
the human donors for these studies were hcv antibody - negative source plasma donors who had donated plasma once or twice weekly for many months until , unexpectedly , they were found to have become positive for plasma hcv rna . in both human subjects whom we studied ,
plasma hcv rna increased rapidly and plateaued at high levels , followed weeks later by hcv antibody seroconversion in patterns typical of acute ( primary ) hcv infection .
figure 3a depicts plasma vrna kinetics of the acutely infected human donor 10081 ( 50 ) and shows the time of sampling for our hcv sequence determinations . the first plasma sample positive for hcv rna by an fda - licensed assay ( roche molecular systems cobas amplicor hcv monitor pcr assay , v2.0 ; detection limit of 600 iu / ml or 2,040 vrna copies / ml ) is designated as day 0 so as to maintain consistency with the previous report ( 50 ) .
samples for day 21 through day 4 all tested negative for hcv rna by this fda - licensed viral load assay . a more sensitive transcription - mediated amplification ( tma ) assay ( gen - probe / hologic , inc . ;
detection threshold of 12 vrna copies / ml ) was then used to test replicate plasma samples .
samples from days 21 , 18 , 14 , and 11 were determined to be negative for vrna by this ultrasensitive test , but a subset of the replicates from days 7 and 4 was positive .
samples ( 50 ml ) of plasma from day 18 through day 7 were infused into a virus - naive chimpanzee but did not lead to productive hcv infection . however , intravenous infusion of 50 ml of subject 10081 plasma from day 4 , which was estimated to contain 60 ( 95% confidence interval , 25 to 95 ) vrna copies based on the tma assay , led to productive infection of chimpanzee x355 .
plasma viremia in chimpanzee x355 was first detected 2 weeks after inoculation and remained variably positive through week 8 ( fig .
this pattern of early viremia , transient elevation of alanine aminotransferase ( alt ) , followed by resolution of viremia is typical of chimpanzee hcv infection .
hcv viral load kinetics and sequences from a human - to - chimpanzee transmission pair .
( b ) fifty milliliters of 10081 plasma from the day 4 time point was infused intravenously into chimpanzee x355 at week 0 .
plasma vrna kinetics are indicated by filled black dots , with blue and green circles indicating time points subjected to sequence analysis .
alanine aminotransferase ( alt ) is a hepatic enzyme that when elevated above baseline indicates liver inflammation or injury .
an ml phylogenetic tree ( with sequences color coded red , black , blue , and green to correspond to time points indicated in panels a and b ) and a highlighter plot show that the human subject 10081 was acutely infected by multiple viruses , one of which , h1 ( top of panel c ) , was transmitted to chimpanzee x355 giving rise to the c1 t / f lineage .
note that the t / f genome corresponding to h1 is identical in sequence to the t / f genome corresponding to the c1 lineage .
one hundred eighty - four human hcv sequences and 140 chimpanzee hcv sequences are shown ( fig .
this sequencing depth provided over 90% likelihood of detecting minor variants present at frequencies of 2% .
phylogenetic analysis suggested infection of the human subject 10081 by a large number of viruses . in 10081 , widely divergent lineages were evident but
so too were many additional lineages comprised of sequences that contained one , two , three , or more shared nucleotide polymorphisms .
these multiple sets of very closely related sequences failed to conform to a simple model of random virus diversification and resembled those from subjects 10003 , 10016 , and 10020 ( 14 ) . using the revised models to account for differences in replication strategies of hcv and hiv-1 ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) , the sequence pattern in subject 10081 ( fig .
3c ) could be explained by the acquisition of between 9 and 14 t / f viruses .
these sequences exhibited a star - like phylogeny and a poisson distribution of random mutations ( table 1 ) .
importantly , the inferred t / f hcv genome in the acutely infected chimpanzee ( t / f lineage c1 ) was found to be identical to one of the human t / f sequence lineages ( h1 ) .
this result documents the transmission of an hcv genome unchanged from donor to recipient , again corroborating the t / f concept and the observation that early virus evolution generally follows a simple pattern of random sequence diversification .
figure 4 illustrates a second case of human - to - chimpanzee hcv transmission but at a much higher multiplicity of infection .
3 , except that in this case , plasma samples from human donor 10083 from days 49 , 7 , and 5 ( the sample immediately preceding frank viremia ) failed to infect chimpanzee x331 despite containing 784 hcv vrna copies ( 50 ) . to show that x331 was infectible by hcv , busch et al . next inoculated this chimpanzee intravenously with 50 ml of the day 0 human plasma , estimated to contain 3.4  10 hcv vrna copies ( 50 ) . productive infection of chimpanzee x331 was detected 1 week later and persisted through week 16 ( fig .
we sequenced by sgs the human 10083 plasma samples from days 0 and 2 ( fig .
4a ) and chimpanzee x331 plasma samples from weeks 1 , 8 , and 10 ( fig .
4d ) were analyzed . as for human donor 10081 , phylogenetic analysis of human donor 10083 sequences revealed multivariant hcv infection resulting in multiple sets of very closely and more distantly related sequence lineages . using our previously described clustering model ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) , we estimated that subject 10083 had been productively infected by at least 12 t / f lineages ( h1 to h12 ) .
this is a minimum estimate because of sampling limitations and because lineages differing by as few as one or two shared mutations could represent distinct transmitted variants .
the diversity among the 12 inferred t / f genomes ranged from as little as 0.1% between h1 and h2 to as much as 0.6% between h11 and h12 ( fig .
4d ) , reflecting the sequence diversity in the individual from whom 10083 acquired hcv infection .
the phylogeny of sequences from chimpanzee x331 was quite different from that observed in chimpanzee x335 where only a single t / f lineage was identified . instead , in chimpanzee x331
, we found evidence by the clustering model ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) of a minimum of 10 t / f lineages ( c1 to c10 ) , with an overall pattern of diversity closely resembling that of subject 10083 hcv sequences ( compare fig . 4c and d ) .
when 10083 and x331 sequences were combined , phylogenetic analysis confirmed their close similarity ( fig .
4f ) . in order to analyze this combined sequence set for unequivocal evidence of transmission of sequences from donor to recipient
, we selected all sequences that were identical between 10083 and x331 and analyzed them in a combined phylogenetic tree ( fig .
we identified 13 discrete t / f lineages in chimpanzee x331 that had matching identical sequences in the human donor 10083 .
this was three more t / f genomes ( c11 , c12 , and c13 ) than were inferred by the clustering model ( fig .
the 13 t / f genome lineages thus correspond to viral genomes that were transmitted unaltered from the human donor 10083 to chimpanzee x331 .
this is likely an underestimate of the numbers of t / f viruses giving rise to productive infection in chimpanzee x331 , given the limitations of sampling and the very high virus inoculum .
nonetheless , the unequivocal identification of 13 t / f genomes that were identical in donor and recipient again corroborates the t / f concept for hcv and recapitulates the patterns of sequence diversity that we observed previously in subjects 10003 , 10016 , and 10020 ( 14 ) .
hcv viral load kinetics and sequences in a human - to - chimpanzee transmission pair .
( b ) fifty milliliters of 10083 plasma from the day zero time point was infused intravenously into chimpanzee x331 at week 0 .
plasma vrna kinetics are indicated by filled dots , with blue , purple , and green circles indicating time points subjected to sequence analysis .
( c ) an ml phylogenetic tree ( with sequences color - coded red and black to correspond to time points indicated in panel a ) and a highlighter plot show that the human subject 10083 was acutely infected by multiple viruses .
the model described by bhattacharya and coworkers ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) suggests a minimum of 12 t / f genomes ( h1-h12 ) .
panel d depicts sequences ( color coded blue , purple , and green to correspond to time points indicated in panel b ) from chimpanzee x331 , and the bhattacharya model suggests a minimum of 10 t / f viruses ( c1 to c10 ) .
( e ) human ( red and black ) and chimpanzee ( blue , purple , and green ) sequences are combined .
( f ) an ml tree of sequences found to be identical between the human donor and the chimpanzee recipient .
this tree reveals 13 t / f genome lineages ( c1 to c13 ) that were transmitted unaltered from humans to chimpanzees .
these 13 transmitted genomes are enclosed in black ovals in panel d and include three lineages ( c11 , c12 , and c13 ) that were identified empirically ( f ) but not by the bhattacharya clustering analysis ( d ) .
the findings of the present study provide an essential validation of the t / f strategy , originally developed to identify transmitted hiv-1 genomes ( 36 , 38 , 39 , 62 , 63 ) , for hcv .
the results distinguish modes and multiplicities of virus transmission ( 14 , 35 ) and mathematical models of early virus diversification and evolution ( 10 , 14 , 36 , 40 , 41 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) .
1 ) ( 14 ) and cases of human - to - human ( fig .
transmission where hcv sequences in donors and recipients were found to be identical , thus proving that the inferred t / f viral genomes in these subjects corresponded to actual transmitted viruses . these findings
further support the conclusion that hcv sequence evolution in the initial weeks following virus transmission generally conforms to a simple model of random virus evolution with sequences characterized by a within - lineage star - like phylogeny and a poisson distribution of mutations .
this allows for precise estimates to be made of numbers of t / f genomes associated with different clinicoepidemiological circumstances or risks of hcv transmission such as needlestick injury , blood transfusion , injection drug use , mucosal exposure , and acute - to - acute infection outbreaks ( 1 , 14 , 1823 , 28 , 35 , 50 , 64 ) , where the numbers of t / f viruses responsible for productive clinical infection may vary widely ( 14 , 35 ) .
these observations are important for future studies aimed at molecularly characterizing viruses responsible for infection , coinfection , superinfection and reinfection ( 64 ) , for analyzing the selective or sieving effects of neutralizing antibodies and virus specific t cells that emerge postinfection or postvaccination ( 6569 ) , and for analyzing the transmission or emergence of drug - resistant mutants ( 14 ) .
but if identification of t / f genomes is straightforward in cases where sequences conform to a simple model of random virus diversification , how can the present results explain the atypical examples previously described where acute infection sequences show very closely related lineages that appear to violate neutral evolution ( i.e. , subjects 10003 , 10016 , and 10020 in reference 14 ) and subjects 10081 and 10083 in the present study ?
the current data show that in cases where a pretransmission virus population diversity bottleneck is present in a donor ( e.g. , subject 10083 in fig .
4c ) and the multiplicity of infection is high , the resulting pattern of virus diversity in the recipient ( chimpanzee x331 in fig .
4d ) can mirror that in subjects 10003 , 10016 , and 10020 ( 14 ) .
a pretransmission bottleneck can result from acute infection of the donor , treatment of the donor with antiviral drugs , or , in some instances , potent immune selection by neutralizing antibodies and/or ctls . if instead , a low - multiplicity infection occurs from a similarly bottlenecked donor ( e.g. , subject 10081 in fig .
3c ) , sequence diversity in the recipient ( chimpanzee x335 ) follows the predicted random pattern with star - like phylogeny and a poisson distribution of mutations ( fig .
these findings suggest that during acute infection , the majority of distinct sequence lineages reflect diversification from discrete , and in some cases , very closely related t / f genomes .
a star - like topology is expected to persist , while the majority of the replication complexes present result from a rapid burst of diversification , probably up to 6 weeks or so postinfection when an acquired immune response is typically first detected .
the 13 t / f genome lineages in chimpanzee x331 support this conclusion ( fig .
however , in the absence of sgs data from linked donors and recipients , the complex patterns of closely related , low diversity sequence lineages such as shown for 10081 ( fig .
4d ) , can not all be assumed to represent distinct t / f genomes , and it is impossible to determine with certainty which were transmitted and which evolved posttransmission as a consequence of early stochastic mutations that then persisted in the population .
such complex data sets can be evaluated more conservatively by a relaxed deterministic model of hcv diversification ( 14 ; http://www.santafe.edu/~tanmoy/programs/hcv/ ) .
in the case of chimpanzee x331 , we could evaluate how closely the approximations of t / f lineages based on this mathematical model corresponded to t / f lineages documented empirically : 10 by the former , 13 by the latter ( fig .
an interesting question is why , given the complex replication strategy of hcv , early evolution of individual t / f genome lineages does not violate the predictions of a simple model of random diversification more frequently .
the likely explanation is that in acute hcv infection , plasma virus titers , the numbers of infected hepatocytes , and the numbers of intracellular replication complexes all increase exponentially and proportionately until plateau viremia is reached . for a fast enough exponential expansion rate , at each generation the progeny of new replication complexes dominate the virus population and , as a result , the phylogenetic tree of a small sample of viruses shows few coalescent events at generations much after the common ancestor .
in this situation , hcv diversification resembles that of a fast - growing hiv-1 viral population , and the long - lived nature of hcv replication complexes in untreated infection ( 10 ) does not modify the essentially random mutational patterns observed in early samples . during the plateau phase of viremia , the linear  stamping press  replication process from a relatively fixed number of infected hepatocytes ( 10 ) dominates and most replication complexes formed show evidence of this shared viral ancestry .
consistent with this picture , it was observed that the average sequence diversity in a sample grows almost linearly at very early times during the virus titer exponential growth phase and saturates later when the virus reaches the plateau phase ( 10 , 14 ) . once adaptive immune responses , including neutralizing antibodies and cytotoxic t cells , emerge , the viral load decreases and nonrandom virus diversity becomes evident ( fig .
we presented examples of nearly full length ( 8.8 kb ) , 5 half ( 4.9 kb ) and 5 quarter ( 2.4 ) genomic analyses .
nearly full length single - genome amplifications are not always possible because of priming and extension inefficiencies , time and cost considerations , and other factors .
however , because hcv contains a nonsegmented rna genome , analyses of sequences of a reasonable length ( 2 to 5 kb ) that contain sufficient numbers of informative sites ( shared nucleotide polymorphisms ) are sufficient to identify and distinguish distinct t / f genomes .
in addition , for applications where the intent is to characterize particular gene products such as env ( e1e2 ) , a 2-kb 5 amplification product may be sufficient to identify t / f genomes , characterize population diversity bottlenecks , and identify mutational escape patterns . where the intent is to map on a proteome - wide basis all t cell epitopes recognized or escaped ,
nearly full length amplification is required in order to maintain genetic linkage across all alleles , as we have described previously for hiv-1 ( 37 , 70 ) .
there are additional caveats to the extrapolation of the findings reported here to other cases of acute hcv infection and to different risk groups .
first , we studied relatively few subjects , and for most of these individuals , risk behaviors leading to hcv infection were unknown .
future studies could benefit from an analysis of larger numbers of human subjects with more clearly defined risk factors for hcv acquisition ( e.g. , needlestick , blood products , idu , sexual , and other ) .
second , our models of early virus replication and diversification of hcv ( 14 ) , as for hiv-1 ( 36 ) , can not fully account for the possibility of extremely early mutations that occur during one or more of the initial replication cycles of the incoming virus ( for hcv , plus - sense rna to minus - sense rna to plus - sense rna , etc . ) .
this can result in the early occurrence and persistence of shared mutations , deviations from a star - like phylogeny , and inference of a t / f genome one or a few virus generations subsequent to the actual transmission event . in practice ,
very early mutations resulting in shared polymorphisms are uncommon in acute hcv vrna sequence data sets ( 14 , 33 ) , and when they do occur , they do not confound the inference of founder genomes . the paucity of very early mutations persisting to later virus generations may be due in part to a lower in vivo error rate of the hcv polymerase than that estimated from in vitro analyses ( 10 ) and because random mutations are generally neutral or deleterious and subject to early purifying selection ( 71 ) .
thus , while it will not always be the case that the consensus of a low - diversity sequence lineage that conforms to model predictions represents the transmitted genome , such a sequence is nonetheless a founder sequence likely to have arisen within one or few virus generations of the transmitted genome .
the phenotypic properties of such founder viruses , including their antigenicity , is likely to be equivalent to that of the corresponding transmitted viruses because of the short time interval and small numbers of virus generations that separate the two .
what are the implications of the present findings for future hcv research ? because of the exceedingly short half - life of hcv in human plasma ( t1/2  45 min ) ( 72 ) , plasma virus titers and genetic composition closely reflect the spectrum of viruses replicating in hepatocytes , along with the composite effects of biological , immunological and antiviral drug pressures influencing virus replication and persistence . as a consequence , quantitative analyses of plasma virus titers and genetic composition provide an extremely sensitive and precise real - time indicator of biologically relevant , and oftentimes clinically relevant , virus - host interactions .
the current findings show how sgs of hcv genes and nearly full length genomes can inform important areas of hcv research .
for example , with the recent development and clinical introduction of new curative treatment strategies , elucidation of treatment  failures  due to antiviral drug resistance , inadequate dosing or compliance , or virus reinfection becomes paramount .
sgs , which maintains genetic linkage within genes ( e.g. , ns5b in patients receiving polymerase inhibitors ) and across genomes ( e.g. , ns3-ns5b in patients receiving protease , ns5a , and polymerase inhibitors ) , can contribute uniquely to the elucidation of mechanisms of treatment failure , as described previously for hiv-1 infection ( 73 ) .
similarly , distinguishing hcv coinfection from superinfection or reinfection can be important in explaining apparent cases of treatment failure .
this can best be done by sgs , as suggested by irving and brown ( 28 , 64 ) and by the findings reported here .
in the area of vaccine development , sgs has been uniquely informative and represents a major tool for assessing virus responses to host adaptive immunity in natural hiv-1 infection and following vaccination .
this has included proteome - wide analyses of t - cell responses ( 37 , 70 , 74 ) , env gp160-wide analyses of neutralizing antibody responses ( 36 , 75 , 76 ) , and sieve analyses of vaccine breakthrough cases ( 6568 , 77 ) .
current and future hcv vaccine trials ( 3 , 4 ) are primed to adopt this methodology and the associated bioinformatic and statistical platforms to accelerate hcv vaccine development and testing .
comparable studies in other flavivirus or alphavirus systems may similarly benefit from a precise molecular identification of t / f genomes and their early molecular pathways of diversification .
 abstractdespite the recent development of highly effective anti - hepatitis c virus ( hcv ) drugs , the global burden of this pathogen remains immense . 
 control or eradication of hcv will likely require the broad application of antiviral drugs and development of an effective vaccine . a precise molecular identification of transmitted / founder ( t / f ) hcv genomes that lead to productive clinical infection could play a critical role in vaccine research , as it has for hiv-1 . 
 however , the replication schema of these two rna viruses differ substantially , as do viral responses to innate and adaptive host defenses . 
 these differences raise questions as to the certainty of t / f hcv genome inferences , particularly in cases where multiple closely related sequence lineages have been observed . 
 to clarify these issues and distinguish between competing models of early hcv diversification , we examined seven cases of acute hcv infection in humans and chimpanzees , including three examples of virus transmission between linked donors and recipients . using single - genome sequencing ( sgs ) of plasma vrna 
 , we found that inferred t / f sequences in recipients were identical to viral sequences in their respective donors . 
 early in infection , hcv genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the t / f sequence . 
 closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment , immune selection , or recent infection . 
 these findings validate sgs , together with mathematical modeling and phylogenetic analysis , as a novel strategy to infer t / f hcv genome sequences . 
 
 importance despite the recent development of highly effective , interferon - sparing anti - hepatitis c virus ( hcv ) drugs , the global burden of this pathogen remains immense . 
 control or eradication of hcv will likely require the broad application of antiviral drugs and the development of an effective vaccine , which could be facilitated by a precise molecular identification of transmitted / founder ( t / f ) viral genomes and their progeny . 
 we used single - genome sequencing to show that inferred hcv t / f sequences in recipients were identical to viral sequences in their respective donors and that viral genomes generally evolved early in infection according to a simple model of random sequence evolution . 
 altogether , the findings validate t / f genome inferences and illustrate how t / f sequence identification can illuminate studies of hcv transmission , immunopathogenesis , drug resistance development , and vaccine protection , including sieving effects on breakthrough virus strains . 