hepatitis c virus ( hcv ) is one of the main causes of liver cirrhosis and hepatocellular carcinoma , where approximately 350,000 deaths occur each year due to hcv - related liver diseases .
the latest estimation indicates that more than 185 million people around the world ( 2.8% of the world population ) have been infected with hcv . in china , approximately 2.2% of the population on average
were infected with hcv ( most of which were genotype 1b ) , with a distribution range from 2.1% in fujian province to 9.6% in henan province . to inhibit the global prevalence of hcv infection , various strategies with regard to therapeutic medication
the traditional standard of care contains pegylated interferon plus ribavirin ( rbv ) , which could help genotype 1b patients to achieve sustained virological response ( svr ) rates of 45% , further up to 6575% for genotype 2/3 patients .
although patients chronically infected with hepatitis c virus ( chc ) in asia carries more beneficial il28b genotypes , nearly 20% of them could not achieve svr . in this
regard , more than 30 newly developed direct - acting antivirals ( daas ) have readily been approved in the europe , north american , and japan or are currently being in phase 2/3 clinical trials .
daas mainly encompass ns3/4a protease inhibitors ( pis , such as telaprevir , boceprevir , simeprevir , mk-8742 , asunaprevir , etc . ) , ns5a inhibitors ( such as daclatasvir , ledipasvir [ ldv ] , mk-5172 , etc . ) , and ns5b polymerase inhibitors ( such as sofosbuvir [ sof ] , dasabuvir , etc . ) .
in general , they are effective toward pan - genotypic hcv by helping patients to achieve high svr rates up to 100% , especially genotype 1b chc patients
. the high replication rate of hcv , along with the low fidelity and poor proof - reading of its polymerase , generates a highly variable virus population denoted as  quasispecies .
 the creation of variants encoding amino acid substitutions may result in reduced susceptibility to antiviral agents .
compared with hiv , the rate of emergence of resistant variants is much higher for hcv .
it is estimated that the error rate of the hcv polymerase is 10-fold higher than that of hiv , and the rate of production of rna virus is 100-fold higher than that of hiv .
data from clinical trials have revealed that baseline resistance - associated variants ( ravs ) were correlated with treatment outcomes of daas .
for example , patients presented pis ravs , such as q80k , d168e / v , ns5a ravs l31m / v , y93h , and ns5b polymerase inhibitors ravs l159f , c316n , v321a achieved svr rates much lower than those who did not .
ravs could exist even as minor variants at baseline , which would rapidly become the main strain under selective pressure , subsequently leading to a treatment - failure .
however , compared with population sequencing , the expense for ultra - deep sequencing was relatively high .
since none of the daas has been approved in china , there have been seldom reports by far on baseline daas ravs in our country . in turn
, it remains unclear if it is necessary to detect baseline daas ravs before daas treatment . in this study , we perform both population sequencing and deep sequencing to detect baseline daas ravs in genotype 1b chc patients in china .
serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv .
population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients .
ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies .
the study was conducted in accordance with the international society for pharmacoepidemiology guidelines for good epidemiology practices and applicable regulatory requirements .
hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted .
hcv rna was extracted from 140 l ethylenediaminetetraacetic acid anticoagulated plasma using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) after centrifugation at 24,000  g for 1 h at 4c .
the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . the primers used are listed in table 1 .
cycling conditions included an initial cdna synthesis step at 55c for 30 min , followed by a denaturation step at 94c for 2 min , 40 cycles of pcr amplification ( 94c for 15 s , 58c for 30 s , 68c for 2 min ) , and a final 10 min extension step at 68c .
the pcr mix contained 25 l of 2 reaction mix , 1 l of each primer , 8 l of extracted rna as template , and nuclease - free h2o to a final volume of 50 l .
the pcr reaction was carried out with thermal cycler pcr machine ( thermo , ca , usa ) .
the pcr products were purified using the qiaquick pcr purification kit ( qiagen , hilden , germany ) . in the deep sequencing study ,
the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . in the population study ,
the amplified fragments were subjected to direct sequencing by abi 3730xl dna sequencer ( abi , usa ) .
primers used for amplifying the ns3 , ns5a and ns5b regions the bioedit 7.09 software ( borland company , usa ) was used for editing the sequences .
translation from the nucleic acids sequences into amino acids sequences was performed with revtrans 2.0 ( technical university of denmark , denmark ) .
the sequence alignment was performed with clustal x 2.0 ( university college dublin , irland ) .
the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . , for ns3/4a pis ; and l23f , l28 t , r30q , l31m / v , p32l , q54h , p58s , q62h , y93h , etc . , for ns5a protein inhibitors ; t19s , n142 t , s282 t , a442 t , s556 g , etc . , for ns5b ravs .
nonparameters mann  whitney u - test , chi - square test with yates correction , or fisher exact test was performed .
serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv .
population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients .
ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies .
the study was conducted in accordance with the international society for pharmacoepidemiology guidelines for good epidemiology practices and applicable regulatory requirements .
hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted .
hcv rna was extracted from 140 l ethylenediaminetetraacetic acid anticoagulated plasma using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) after centrifugation at 24,000  g for 1 h at 4c .
the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions .
cycling conditions included an initial cdna synthesis step at 55c for 30 min , followed by a denaturation step at 94c for 2 min , 40 cycles of pcr amplification ( 94c for 15 s , 58c for 30 s , 68c for 2 min ) , and a final 10 min extension step at 68c .
the pcr mix contained 25 l of 2 reaction mix , 1 l of each primer , 8 l of extracted rna as template , and nuclease - free h2o to a final volume of 50 l .
the pcr reaction was carried out with thermal cycler pcr machine ( thermo , ca , usa ) .
the pcr products were purified using the qiaquick pcr purification kit ( qiagen , hilden , germany ) . in the deep sequencing study ,
the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . in the population study ,
the amplified fragments were subjected to direct sequencing by abi 3730xl dna sequencer ( abi , usa ) .
the bioedit 7.09 software ( borland company , usa ) was used for editing the sequences .
translation from the nucleic acids sequences into amino acids sequences was performed with revtrans 2.0 ( technical university of denmark , denmark ) .
the sequence alignment was performed with clustal x 2.0 ( university college dublin , irland ) .
the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . , for ns3/4a pis ; and l23f , l28 t , r30q , l31m / v , p32l , q54h , p58s , q62h , y93h , etc . , for ns5a protein inhibitors ; t19s , n142 t , s282 t , a442 t , s556 g , etc . , for ns5b ravs .
nonparameters mann  whitney u - test , chi - square test with yates correction , or fisher exact test was performed .
in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study .
fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] .
baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus .
one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] .
nucleoside changes at codon position 36 , 55 , and 80 were detected but could cause only synonymous substitutions .
number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing ,
the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i .
all of the ravs detected by ultra - deep sequencing could be detected by population sequencing .
about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs .
about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant .
only synonymous substitutions were detected at amino acid sites 23 , 28 , and 32 .
nucleoside substitutions from a to g at 6347 site were detected in 42.6% ( 63/148 ) patients , which could only cause synonymous substitutions at amino acid site 31 [ table 3 ] . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h .
interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% .
the fragments of ns5b were successfully amplified in 85.6% ( 137/160 ) patients . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one .
when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical .
there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) .
, l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b ,
the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected .
other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study .
in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] .
number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants .
baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus .
it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t
( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc .
, regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors .
most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii .
as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g .
interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) .
in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study .
fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] .
baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus .
one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs .
about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] .
nucleoside changes at codon position 36 , 55 , and 80 were detected but could cause only synonymous substitutions .
number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing ,
the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i .
all of the ravs detected by ultra - deep sequencing could be detected by population sequencing .
about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs .
about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant .
only 3 patients presented p58s variant , and only 5 patients presented q62h variant . only synonymous substitutions were detected at amino acid sites 23 , 28 , and 32 .
nucleoside substitutions from a to g at 6347 site were detected in 42.6% ( 63/148 ) patients , which could only cause synonymous substitutions at amino acid site 31 [ table 3 ] . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h .
interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% .
the fragments of ns5b were successfully amplified in 85.6% ( 137/160 ) patients . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one .
when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical .
there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) .
, l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b ,
the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected .
other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study .
in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] .
number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants .
baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus .
it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t
( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc . , regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors .
most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii .
as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g .
interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) .
in our study , as many as 71% of genotype 1b treatment - nave patients presented at least one ns3/4a pis rav .
the most prevalent variant was s122 g ( 56.6% ) , which has been confirmed as one of the major ravs of simeprevir ( one of the first - generation second - wave pis ) . in concerto trial conducted in japan ,
the baseline prevalence of s122 g variant was relatively lower ( 34.9% ) than that of our findings , while the baseline prevalence of v170i variant was relatively higher ( 39.6% vs. 13.1% ) than our findings .
t54s variant detected in our study was also correlated with resistance to simeprevir , with relatively low prevalence ( 5.5% ) .
mutations associated with first - generation first - wave pis detected in this study were t54s ( telaprevir and boceprevir ) , s122 g ( telaprevir ) , and v170i ( telaprevir ) . however , these ravs only conferred low - level resistance to these two licensed daas . with respect to other reports
, none of other baseline polymorphisms correlated with treatment outcomes of pis were detected in our study .
for example , r155k and d168e / v variants were ravs of asunaprevir ( had licensed in combination with daclatasvir as interferon [ ifn]-free regimens in japan ) and mk-5172 ( one of the second generation pis ) , none of which were found .
this discrepancy might be due to the fact that virus of different quasispecies even within the same subtype has different mutations . since all of the ravs detected by ultra - deep sequencing could be detected by population sequencing
, we can conclude that population sequencing could qualified for baseline pis ravs screening in china .
as one of the most important ravs of most of the ns5a protein inhibitors , y93h variants were detected in 10.1% of patients included in our study .
l31 m variants , another mutation associated with treatment outcomes of ldv , daclatasvir , and abt-267 , were only detected by ultra - deep sequencing in three patients with low mutation frequencies .
several in vitro studies have reported that single l31 m variant could only lead to 3-fold change with ec50 of daclatasvir .
single y93h variant could lead to 24-fold change of it while combination mutations of l31 m plus y93h could lead to 7105-fold change with ec50 of daclatasvir . in addition , single y93h variant could lead to 77-fold change of ec50 of abt-267 while l31 m plus y93h could lead to 142-fold change of it .
what 's more , y93h variant could lead to extremely high - level ( 994-fold ) fold - change with ec50 of ldv .
although ns5a protein inhibitors were usually used in combination with pis ( such as asunaprevir ) , baseline polymorphisms of l31 m or y93h could make patients to experience virologic relapse more easily and achieved much lower svr rates .
data by ultra - deep sequencing revealed that most of the ns5a protein inhibitors ravs detected here were with relatively high mutation frequencies , except for l31 m variants .
nevertheless , l31 m variants occurred only in three patients with low mutation frequencies . in this way
, population sequencing might be qualified for baseline ns5a protein inhibitors ravs screening in china .
the detection of naturally occurring ravs of ns5b polymerase inhibitors at baseline has been reported in numerous studies . some of them may influence the treatment outcomes of daas - containing regimens . in the clinical trials of sof - containing regimens ,
the primary concern lies in the s282 t , an important variant associated with treatment - failure .
however , a representative study conducted by fda from the us suggested that ns5b - c316n was found to be associated with treatment failure when patients chc genotype 1b receiving sof plus rbv therapy , throughout re - systemizing all the data from sof - containing trials where ultra - deep sequencing was performed . in the clinical trial p7977 - 2025 , 16/61 posttransplantation patients failed therapy of sof plus rbv , where 6/16 patients harbored c316n at baseline , in other words , all of the six patients did not achieve svr . on the contrary , for the other 45 patients who achieved svr12 ,
eighty - two percent ( 45/55 ) of the patients who did not carry c316n pretreatment achieved svr12 .
however , 66.7% ( 4/6 ) of patients harbored l159f at the same time . in clinical trial neutrino , 4/66 of the patients harbored c316n variant at baseline , 50% of which ( 2/4 ) experienced virologic relapse after receiving treatment of sof plus rbv . in this trial ,
rav c316n is located in palm ii ( allosteric ) . despite the fact that the majority of ravs of sof reported are at the active sites ( such as s282 t and n142 t )
, position 316 was deemed as the only different residue within 8 a in the ns5b polymerase between hcv genotype 1a and 1b .
n316 in hcv gt1b polymerase structure is much larger than c316 , which might lead to the block of the active site of sof .
however , c316 has been reported to be relatively conserved in gt1a , but polymorphic in gt1b .
data from los alamos databank showed that the prevalence of c316n was much higher in asia than worldwide ( 91.6% vs. 36% ) as well as american continent ( from 0% to 18% ) . in our study , both the ultra - deep sequencing and population sequencing revealed that the prevalence of c316n variant in treatment - nave chinese gt1b chc patients was quite high , which is in good agreement with previous findings .
as part of ifn - free regimens , sof in combination with ldv with and without rbv has been one of the most important regimens nowadays . combination treatment could help patients to overcome antiviral resistance more easily .
, data showed that the patient presented ns5a - l31m - y93h joined variants as previously stated , simultaneously harbored ns5b - c316n . even in the combination regimens of sof plus ldv , baseline polymorphisms of y93h
results by ultra - deep sequencing showed that the mutation frequencies increased dramatically at the time of relapse . both rav
s282 t and n142 t were at the active site of ns5b polymerase , but none of the 137 patients exhibited the existence of these two variants .
since most of the patients presented ravs of one category , cross - resistance seems less likely to occur .
data by ultra - deep sequencing revealed that most of the ns5b polymerase inhibitors ravs detected here were with high mutation frequencies , especially for c316n variants .
nevertheless , l419 m variants occurred only in two patients with low mutation frequencies . in this way
, population sequencing might qualify for baseline ns5b polymerase inhibitors ravs screening in china . in conclusion , we have employed both the ultra - deep sequencing and population sequencing methods to detect daas ravs in genotype 1b treatment - nave patients , among which the prevalence of variants related with treatment - failure of ns3/4a pis ( s122 g ) , ns5b polymerase inhibitors ( c316n ) and ns5a inhibitors ( y93h ) were quite high , in particular , c316n . currently high prevalence and high mutation frequencies of rav c316n in china might impede the virologic response when distributing sof - containing regimens in the near future .
our findings suggest that pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china .
this work was supported by grants from the china national science and technology major project for infectious diseases control during the 11th 5-year plan period ( no . 2008zx10002 - 013 , 2008zx10002 - 012 ) and 12th 5-year plan period ( no .
this work was supported by grants from the china national science and technology major project for infectious diseases control during the 11th 5-year plan period ( no . 2008zx10002 - 013 , 2008zx10002 - 012 ) and 12th 5-year plan period ( no .
 background : it has been reported that several baseline polymorphisms of direct - acting antivirals ( daas ) agents resistance - associated variants ( ravs ) would affect the treatment outcomes of patients chronically infected with hepatitis c virus ( chc ) . 
 the aim of this study is to investigate the prevalence of daas ravs in treatment - nave gt1b chc patients.methods:direct sequencing and ultra - deep sequencing of the hcv ns3 , ns5a , and ns5b gene were performed in baseline serum samples of treatment - nave patients infected with genotype 1b hepatitis c virus ( hcvs).results : one hundred and sixty chc patients were studied . 
 complete sequence information was obtained for 145 patients ( ns3 ) , 148 patients ( ns5a ) , and 137 patients ( ns5b ) . 
 treatment - failure associated variants of daas were detected : 56.6% ( 82/145 ) of the patients presented s122 g for simeprevir ( ns3 protease inhibitor ) ; 10.1% ( 14/148 ) of the patients presented y93h for daclatasvir and ledipasvir ( ns5a protein inhibitors ) ; 94.2% ( 129/137 ) of the patients presented c316n for sofosbuvir ( ns5b polymerase inhibitor ) . 
 nearly , all of the daas ravs detected by ultra - deep sequencing could be detected by direct sequencing.conclusions:the majority of genotype 1b chc patients in china present a virus population carrying hcv daas ravs . 
 pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china . 
 population sequencing would be quite quantified for the work . 
I
M
Study design
RNA extraction, polymerase chain reaction, and sequencing
Sequence alignment and analysis
Statistical analysis
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Patient characteristics
Over 50% of patients presented PI resistance-associated variants NS3-S122G
NS5A PI resistance-associated variants Y93H were found, and L31M variants were detected only by deep sequencing
High percentage of patients presented NS5B polymerase inhibitors resistance-associated variants C316N
D
Financial support and sponsorship
Conflicts of interest