The clinical outcome of PEG-IFN/RBV therapy for HCV infection is influenced by a number of host and viral factors 
. It has recently been reported that host genetic polymorphisms near or within the IL28B gene on the chromosome 19 show a critical impact on the treatment outcome of patients infected with HCV-1a and -1b 
. Also, HCV genetic polymorphisms have been known to contribute to differences in the treatment outcome, as demonstrated by the observations that SVR rates for patients infected with HCV genotypes 2 and 3 are higher than those for patients infected with HCV genotype 1 
. Moreover, polymorphisms of NS5A and core regions of a given HCV genotype, in particular HCV-1b, have been linked to the difference in SVR rates 
. It should be noted that the significant link between polymorphisms of NS5A and core regions of HCV-1b and treatment outcome was inferred mostly from studies carried out on patients in Asian countries, in particular Japan, and that somewhat controversial results were obtained from studies carried out on patients infected with HCV-1a or -1b in non-Asian countries 
. However, we would like to point out that most of these publications focused mainly on ISDR and core mutations, but not on IRRDR. In addition, the impact of viral genetic variation on treatment outcome in non-HCV-1 infection, either in Asian or non-Asian countries, is still unclear.
In our previous study, we identified IRRDR in NS5A of HCV-1b as a significant determinant for PEG-IFN/RBV treatment outcome; EVR and, more importantly, SVR 
. Consistent with the previous observation, we have demonstrated in the present study that sequence heterogeneity within IRRDR is closely correlated with the treatment responses in HCV-2a and -2b infections. In HCV-2a infection, IRRDR[2a]≥4 was closely associated with RVR (Table S1
) and SVR (). In HCV-2b infection, the sequence heterogeneity within an N-terminal part of IRRDR (IRRDR/N[2b]) was significantly associated with RVR (Table S1
). Furthermore, both IRRDR[2a]≥4 and ISDR/+C[2a]≥1 showed remarkable positive predictive values (95%) for SVR prediction (Table S2
), suggesting the clinical usefulness of these markers to encourage those patients to receive PEG-IFN/RBV treatment. On the other hand, their negative predictive values for non-SVR were rather low (50% and 33%). This suggests the possible involvement of another factor(s) that determines non-SVR and may limit the clinical usefulness of these markers to accurately predict non-SVR.
The present results were dependent upon the small number of non-SVR patients due to the high response rates of HCV-2a and -2b. In spite of this, the parallels between the RVR/non-RVR and the SVR/non-SVR analyses, especially in HCV-2a infection, support the possibility that the sequences presented in this study are truly representative of the viruses in general circulation.
The clinical correlation between IRRDR sequence heterogeneity and virological responses of IFN-based therapy in HCV infection can be linked to a recent experimental observation by Tsai et al. 
that an HCV subgenomic RNA replicon containing NS5A of HCV-1b exerted more profound inhibitory effects on IFN activities than the original HCV-2a replicon, and that domain swapping between NS5A sequences of HCV-1b and -2a in the V3 and/or a C-terminal region including IRRDR resulted in a transfer of their anti-IFN activities. Also, it is worthy to note that IRRDR is among the most variable sequences across the different genotypes and subtypes of HCV 
whereas its upstream and downstream sequences show a higher degree of sequence conservation (). This may suggest that whereas the upstream and downstream sequences have a conserved function(s) across all the HCV genotypes, IRRDR sequences have a genotype-dependent or even a strain-dependent function(s). Indeed, the upstream sequences, especially a Pro-rich motif, play key roles in multiple stages of viral replication 
while the downstream sequence in viral particle assembly and production 
. Therefore, the sequence heterogeneity of IRRDR and its significant correlation with IFN-responsiveness imply the possibility that IRRDR is involved, at least partly, in the viral strategy to evade IFN-mediated antiviral host defense mechanisms. Its possible molecular mechanism, however, is yet to be elucidated. The IRRDR sequence heterogeneity also suggests genetic flexibility of this region and, indeed, the C-terminal portion of NS5A was shown to tolerate sequence insertions and deletions 
. This flexibility might play an important role in modulating the interaction with various host systems, including IFN-induced antiviral machineries. It is also possible that the genetic flexibility of IRRDR is accompanied by compensatory changes elsewhere in the viral genome and that these compensatory changes affect overall viral fitness and responses to IFN-based therapy 
Sequence alignment of IRRDR (interferon/ribavirin resistance-determining region) and its upstream and downstream sequences of different HCV genotypes.
The relapse rate was higher in HCV-2b infection than in HCV-2a (). It should be noted that while the sequence heterogeneity within IRRDR[2a] was significantly correlated with both RVR and SVR in HCV-2a infection, IRRDR/N[2b] was correlated only with RVR in HCV-2b infection. These observations might be linked to an intrinsic difference in IFN- and/or RBV-sensitivity between HCV-2a and -2b isolates 
. We assume that HCV-2b is considered between HCV-1b and HCV-2a in terms of resistance to PEG-IFN/RBV treatment and that an extended treatment for a total of 36~48 weeks would be needed to prevent relapse in HCV-2b infection, especially for patients who have risk factors that do not fit the SVR or RVR prediction criteria ( and Table S1
A mutation at position 70 of the core protein of HCV-1b has been reported to be correlated with PEG-IFN/RBV treatment outcome 
. In the present study, however, we found no significant correlation between core protein polymorphism and treatment outcome in HCV-2a or -2b infections. The residue at position 70 of the core protein of HCV-2a and -2b isolates was Arg, which is known to be associated with SVR in HCV-1b infection 
, and was well conserved in all the isolates tested in the present study (Figures S1
). The observed sequence conservation at position 70 might be the reason for the lack of significant correlation between core protein polymorphism and treatment outcome in HCV-2a or -2b infections. On the other hand, Thr at position 110 of the core protein of HCV-2a has recently been reported to be significantly associated with SVR 
. In the present study, Thr at position 110 was found in 35% (14/40) and 14% (1/6) of SVR and non-SVR cases, respectively (Figure S1
). Similarly, Thr at position 48 was found in 35% (14/40) of SVR cases, but not in non-SVR cases (0/6). The observed differences between SVR and non-SVR, however, were not statistically significant due possibly to the small number of samples tested. A larger-scale study would be needed to determine the possible importance of those residues.
We preliminarily analyzed a host genetic factor, the single nucleotide polymorphism (SNP) at rs8099917 near the IL28B gene 
, of a portion of the patients examined in the present study. The result showed that the minor genotypes (T/G and G/G) were found in 5.1% (2/39) and 15.4% (2/13) of RVR and non-RVR patients, respectively, and 2.8% (1/36) and 20.0% (2/10) of SVR and non-SVR patients, respectively (Kim et al., unpublished observation). Although the differences were not statistically significant due probably to the small number of the patients tested, the minor genotypes showed a trend toward being associated with non-SVR, and with non-RVR to a lesser extent, in HCV-2a and -2b infections, as has been reported for HCV-1a and -1b infections 
. The impact of the IL28B SNP, however, appeared to be weaker in HCV-2a and -2b infections than that seen in HCV-1a and -1b infections, and also weaker than that of the most powerful viral factor, IRRDR[2a]≥4, in HCV-2a infection. In this context, we found that, of the four patients with the minor IL28B genotypes, two patients (nos. 2 and 105), who underwent unfavorable treatment response (non-RVR and non-SVR), were infected with HCV isolates of IRRDR[2a]≤3 or IRRDR/N[2b]≤1 while the other two patients (no. 63 and 106), who achieved favorable treatment response (SVR and/or RVR), were infected with HCV isolates of IRRDR[2a]≥4. This might imply the possibility that, in HCV-2 infection, the combination of the minor IL28B genotypes and a low degree of IRRDR sequence heterogeneity has a strong power to predict unfavorable treatment responses whereas a high degree of IRRDR sequence heterogeneity has a dominant predictive power for favorable treatment responses regardless the IL28B genotype. Analysis in a large-scale multicenter study is needed to clarify this issue.
In conclusion, our data suggest that the sequence heterogeneity of NS5A, i.e., IRRDR[2a]≥4, and ISDR/+C[2a]≥1 to a lesser extent, would be a useful predictive marker for SVR in HCV-2a infection. Also, IRRDR/N[2b]≥2 is significantly associated with RVR in HCV-2b infection. These results further emphasize the importance of NS5A, a viral factor, in determining the responsiveness to PEG-IFN/RBV therapy.