Generation of Immune Responses against Hepatitis C Virus by Dendritic Cells Containing NS5 Protein-Coated Microparticles

Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4⁺, IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.An estimated 170 million individuals (3% of the world''s population) are infected with hepatitis C virus (HCV), leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The current standard-of-care therapy for chronically infected HCV patients is the combined administration of pegylated alpha interferon (IFN-α) and ribavirin (28). A sustained response is seen in approximately 50 to 60% of individuals (33). Treatment is long term (6 to 12 months), costly, and associated with substantial toxicity (42). Clearly, more-effective regimens are needed (10).Clinical studies of the adaptive host immune response to acute HCV infection suggest a rationale for immunization approaches. Clearance of HCV is associated with early, multispecific, strong CD8⁺ T-cell immunity that is matched by vigorous and sustained CD4⁺ T-cell proliferation in response to multiple recombinant structural and nonstructural viral proteins (13, 18, 47). Activated T cells secrete proinflammatory cytokines (TH1 type), such as IFN-γ, coinciding with large reductions in viral load during acute infection (47). HCV infections that are successfully controlled result in durable memory populations (44). This observation is supported by a substantially lower rate of HCV persistence in reexposed humans with a history of acute resolving HCV (29). Rechallenge experiments with chimpanzees showed that antibody-mediated depletion of CD4⁺ T cells resulted in HCV persistence, which is in contrast to a marked reduction in duration and peak of viremia in nontreated animals (4, 20). The importance of CD4⁺ T cells is further emphasized by the loss of immune protection against reexposure to HCV and correlated with low CD4⁺ T-cell counts in intravenous drug users who had recovered from HCV but subsequently acquired human immunodeficiency virus infection (29). Whether recovery from acute HCV coincides ultimately with virus eradication is still a matter of debate (34). The disappearance of HCV-specific antibodies in some individuals 10 to 20 years after viral clearance indicates that a subgroup of patients achieves complete virus elimination (44).Persistent HCV infection can be attributed to a number of viral evasion strategies. First, the rapid spread of HCV in a host outpaces the immune response by several weeks (39, 46). Second, HCV is a strong inducer of type I IFN but appears to render hepatocytes resistant to antiviral activity (45, 46). Third, NK cell function can be inhibited by cross-linking tetraspanin CD81 on the cell surface with the major envelope protein of HCV (HCV E2), thus blocking NK cell activation, proliferation, and cytokine production (12). Finally, persistent HCV infections correlate with the permanent loss of HCV-specific T-cell proliferation during acute HCV infection, the functional exhaustion of an initially vigorous response, and the inability of effector T cells to migrate into the infected liver (10, 13, 18, 36, 39, 46). Insufficient CD4⁺ T-cell activity appears to be a key event leading or contributing to chronic HCV. Failure to sustain the CD4⁺ helper response renders virus-specific CD8⁺ T cells inadequate (e.g., loss of cytotoxicity and IFN-γ production), thus contributing to persistent viremia (39).Recapitulating successful immune responses and addressing HCV-specific defects in immunity are mandatory in efforts to improve current treatment options. The purported involvement of dendritic cells (DCs) in the impaired immune responses observed in patients with persistent HCV infection makes DCs a principal target for immunomodulatory approaches. Access to a sufficient quantity of mature DCs is a critical issue since only mature DCs are capable of targeting the antigen and inducing cellular immunity rather than tolerance. Previously, we outlined a novel method of generating large numbers of DCs in vivo (17). These, in turn, are enriched in vitro by phagocytosis of magnetic beads and separation in a magnetic field. Here we further demonstrate that, by immunizing mice with DCs that contain beads coated with the nonstructural HCV antigen NS5 and compounds known to induce DC maturation, significant levels of cellular immunity are elicited. The key elements of this approach reside in the combined enrichment, maturation, and antigen targeting of DCs in a single step and the generation of immune responses of the type known to promote viral clearance in humans.     

Hepatitis C Virus Serotype-Specific Core and NS4 Antibodies in Injecting Drug Users Participating in the Amsterdam Cohort Studies

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75.5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5′ untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.     

丙型肝炎病毒NS3蛋白单克隆杂交瘤细胞株的建立及初步鉴定

应用国产基因工程表达的丙型肝炎病毒（ＨＣＶ）ＮＳ３区抗原免疫小鼠，然后取其脾细胞与小鼠骨髓瘤细胞系ＳＰ２／０融合，筛选出４株稳定分泌抗ＨＣＶＮＳ３区蛋白单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为２Ｂ６，２Ｆ３，３Ｄ８，３Ｄ９，经初步研究表明这４株单抗与ＮＳ３抗原具有良好的反应性，与ＨＣＶ核心区多肽及乙型肝炎病毒表面抗原和ｅ抗原均无反应。抑制实验表明这４株抗体分别针对ＮＳ３抗原分子上的２个不同的抗原决定簇。     

肝细胞肝癌中丙型肝炎病毒C区、E区、NS3区、NS4区抗原的表达

本文报道研究丙型肝炎病毒抗原在肝细胞肝癌组织内的定位分布情况。以丙型肝炎病毒（ＨＣＶ）的Ｃ、Ｅ、ＮＳ３、ＮＳ４区四种单克隆抗体用免疫组化方法检测了１３９例肝细胞肝癌（ＨＣＣ）的肝脏标本，结果总的阳性率为１５．１％。２１例阳性标本中，Ｃ区单抗检测阳性占８０．９％（１７／２１），Ｅ区占３３．３％（７／２１），ＮＳ３、ＮＳ４区均占５７．１％（１２／２１），表明应用多区段单抗有助于提高ＨＣＶ抗原的检出率。阳性物质主要存在于胞浆中，呈细、粗颗粒及块状，３例出现膜及膜下型，１例核内有阳性反应。ＨＣＶ感染与ＨＣＣ的发生发展有一定的关系。     

Antigenic Heterogeneity of the Hepatitis C Virus NS5A Protein

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n = 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.     

Generation and Serological Characterization of Murine Monoclonal Antibodies against O Antigens from Acinetobacter Reference Strains

O-antigen-specific monoclonal antibodies were generated against Acinetobacter strains from international type culture collections and characterized by enzyme immunoassay and Western and colony blotting. The antibodies aid in the further completion of an O-serotyping scheme for Acinetobacter and, due to their high specificity, are especially useful to all working with these strains.     

抗人Ⅳ型胶原蛋白单克隆抗体的制备和鉴定

用人Ⅳ型胶原蛋白免疫ＢＡＬＢ／ｃ小鼠，克隆了一组分泌抗人Ⅳ型胶原蛋白的杂交瘤细胞株，并且对其中的Ａ２、Ａ４、Ａ５、Ｂ３和Ｅ２进行了鉴定。其抗体亚类分别为ＩｇＧ１、ＩｇＧ２、ＩｇＧ２ｂ、ＩｇＧ２和ＩｇＧ１。其亲和力常数Ｋａｆｆ＝６．６×１０８Ｍ（－１）～６．０×１０（１０）Ｍ（－１）。它们均能特异性地识别人Ⅳ型胶原蛋白。     

Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells

Although reasons for hepatitis C virus (HCV) persistence are still unknown, specific cellular immune responses appear to influence the pathogenesis and outcome of the infection. Apoptosis of cells infected by viruses may appear suicidal to the viruses that induce programmed cell death of its host. However, apoptosis has been suggested to be a response to virus infection as a mean of facilitating virus dissemination. Annexin V-propidium iodide staining and DNA fragmentation, were used to show that expression of the core, NS3, NS5A, or NS5B protein induces apoptosis in mature dendritic cells. In addition, immunoblotting was used to demonstrate that expression level of p21waf1/cip1 protein decreased in cells expressing one of these HCV proteins. No expression of p53 could be detected and expression of Akt was independent of HCV proteins expression. These results suggest that the effect of these HCV proteins on HCV associated pathogenesis may be linked (at least partially) to its ability to modulate apoptosis pathways in mature dendritic cells.     

Antibodies against Hepatitis A and Hepatitis B Virus in Intravenous Immunoglobulin Products

The worldwide seroprevalence of hepatitis A virus (HAV) and hepatitis B virus (HBV) has changed over the last two decades, indicating a declining incidence of HAV and HBV infections. Therefore, vaccinations against HAV and HBV are recommended for unimmunized people before traveling to an endemic area. Unfortunately, primary antibody deficiency (PAD) patients can only obtain humoral immunity through intravenous immunoglobulin G (IVIG) replacement and not from vaccination because of a defect in antibody production. However, few studies have analyzed the titers of antibodies against HAV or HBV in IVIG products. In this study, the titers of anti-HAV and anti-HBs antibodies were measured in nineteen lots of IVIG products from five manufacturers from three countries (A, B from Korea; C, D from Japan; and E from the USA), and trough titers in plasma were estimated. Concentrations of anti-HAV antibody ranged from 1,888–8,927 mIU/mL and estimated trough titers exceeded the minimal protective value in all evaluated IVIG products. Concentrations of anti-HBs antibody ranged from 438–965 mIU/mL in products A and B and were 157, 123, and 1,945 mIU/mL in products C, D, and E, respectively. Estimated trough titers in products A, B, and E exceeded the minimal protective value but those in products C and D did not reach this threshold. These data demonstrated that available IVIG products generally provide sufficient antibodies against HAV and HBV to protect patients with PAD, although the trough concentrations of anti-HBs antibody in two IVIG products did not reach the minimum protective value.     

Baculovirus Expression of Chimeric Hepatitis B Virus Core Particles with Hepatitis E Virus Epitopes and Their Use in a Hepatitis E Immunoassay

Two hepatitis B core proteins bearing the immunodominant region of the hepatitis E virus (HEV) capsid protein, one at the C terminus of hepatitis B virus core antigen (HBcAg) and the other within the HBcAg immunodominant loop, were constructed. Both chimeric proteins exhibited HEV reactivity, but only the first construct retained HBcAg reactivity. The second construct was used to develop an anti-HEV test which is equivalent to a commercial test for the detection of anti-HEV immunoglobulin G (IgG) but is more sensitive for the detection of anti-HEV IgM.     

丙肝病毒NS5A蛋白对NS5B的RdRP活性影响的研究

丙型肝炎病毒 (Hepatitis C virus,HCV )非结构蛋白 (Nonstructural,NS) 5 A在 HCV基因组复制中的作用目前尚不清楚。本文研究 His- NS5 A对 NS5 B的 RNA依赖性 RNA酶 (Rd RP)活性的影响 ,以了解 NS5 A在HCV RNA复制中的作用。采用变性 -复性方法 ,纯化大肠杆菌表达的重组组氨酸 NS5 A融合蛋白。 GST结合洗脱实验 (GST pull- down assay)研究 NS5 A和 NS5 B是否结合。以不同的摩尔浓度比 ,将纯化的 NS5 B和 NS5 A蛋白混合 ,检测 NS5 A对 NS5 B的 Rd RP活性的影响。获得高得率的纯化 His- NS5 A蛋白。重组 NS5 A蛋白可在体外与NS5 B结合并抑制后者的 Rd RP活性。本研究报道了纯化重组 His- NS5 A蛋白的变性 -复性方法 ,结果显示纯化的重组 NS5 A在体外可与 NS5 B相互结合 ,并明显抑制 NS5 B Rd RP活性。提示了 HCV NS5 A在病毒复制中的可能作用。     

Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV genome. The biochip contains 120 oligonucleotides that identify genotypes 1 to 6 and 36 (1a, 1b, 1c, 1d, 1e, 2a, 2b, 2c, 2d, 2i, 2j, 2k, 2l, 2m, 3a, 3b, 3k, 4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r, 4t, 5a, 6a, 6b, 6d, 6g, 6h, and 6k) subtypes. The procedure included amplification of a 380-nucleotide (nt) fragment of NS5B and its hybridization on the biochip. Tests on 345 HCV-positive samples showed that the assay agreed with NS5B sequencing 100% for the genotype and 99.7% for the subtype. The hybridization on the microarray and the NS5B sequence were in 100% agreement for identifying the most common subtypes, 1a, 1b, 4a, 4d, and 3a. This approach is a promising tool for HCV genotyping, especially for implementing the new anti-HCV drugs that require accurate identification of clinically relevant subtypes.The hepatitis C virus (HCV) is a leading cause of chronic liver disease and increased risk of cirrhosis and hepatocellular carcinoma (51). More than 170 million people are infected with HCV worldwide (42). This enveloped, single-stranded positive-sense RNA virus is a member of the Flaviviridae family. The RNA genome contains a single large open reading frame composed of over 9,000 nucleotides (nt) encoding structural and nonstructural proteins (5). One of these proteins is an RNA-dependent RNA polymerase encoded by the so-called NS5B region. This error-prone enzyme lacks proofreading activity, which makes it responsible for the great genetic variability of HCV. Sequencing studies of HCV strains have identified 6 genotypes and more than 70 subtypes (43, 45).The HCV genotype is considered to be the major baseline predictor of a sustained virological response (SVR) to antiviral therapy. Patients infected with HCV genotypes 2 and 3 are more sensitive to combination therapy with interferon and ribavirin than are those infected with genotype 1 (8, 11, 21). The available data on HCV genotype 4 suggest that its sensitivity to HCV treatment lies somewhere between those of genotypes 1 and 2/3 (17). The sensitivity of genotypes 5 and 6 could be similar to that of genotype 2 or 3 (1, 9, 19). The HCV subtype has recently been implicated as a potential predictor of SVR. One study of 597 difficult-to-treat patients found that subtypes 1b, 4a, and 4d were independently associated with SVR (16). The virological response to new anti-HCV agents could also be influenced by the HCV subtype (31, 42).Several methods has been proposed for HCV genotyping (50), including commercially available techniques based on real-time PCR: the HCV genotyping analyte-specific reagent (ASR) assay (Abbott Molecular Inc., Des Plaines, IL) (23), semiautomated sequencing (the TruGene HCV 5′NC genotyping kit; Bayer HealthCare, Berkeley, CA) (10), and automated reverse hybridization (the Inno-LiPA HCV II assay; Innogenetics, Ghent, Belgium) (46, 49). Most HCV genotyping methods are based on analysis of the 5′ noncoding (NC) region of the HCV genome because the 5′ NC region is regularly amplified for HCV molecular diagnosis and quantification of the viral load. However, this highly conserved region is not suitable for accurately discriminating between subtypes and can lead to genotyping or subtyping errors (2, 3, 15, 39, 43). Hence, alternative genomic regions have been proposed for genotyping HCV, including the core fragment (35, 49) and the NS5B region (39). Sequencing and phylogenetic analysis of the NS5B region are presently considered to be the gold standard for HCV genotyping since they accurately identify the subtype and can be used to establish an epidemiological picture of circulating virus strains (27, 30, 39, 47). However, this method includes steps of purification of the amplified product, sequencing, and phylogenetic analysis that require the skill of laboratory personnel, a factor that can be a limitation to the wide use of the technique in routine clinical laboratories.Therefore, an assay was developed that involves hybridization on an oligonucleotide microarray for identifying HCV genotypes and subtypes. It uses a low-density hydrogel-based microarray (biochip) that has been successfully used in many fields of molecular diagnostics (26, 28, 37). The microarray contains genotype- and subtype-specific oligonucleotides based on the corresponding sequences of the NS5B region. This report compares this approach to accurately identifying HCV genotype and subtype with direct NS5B sequencing.     

Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death

Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179-1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-alpha induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-alpha mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-alpha induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-alpha mediated apoptotic cell death.     

Generation and Characterization of Monoclonal Antibodies Specific to Surface Antigens of Human Trophoblast Cells

PROBLEM : To generate and utilize specific monoclonal antibodies for routine fetal cell isolation from the maternal circulation. METHODS : Monoclonal antibodies specific to human trophoblast cell surface antigens were generated and characterized. After cell fusion, antibodies secreted by hybridomas were screened by enzyme-linked immunosorbent assay and immunohistochemical assays. RESULTS : By using cultured BeWo choriocarcinoma cells or the membrane fraction of human placenta as the immunogen, seven (BW-108, 110, 123, 124, HP-15, 16 and 17) antibodies specific to the surface antigens of trophoblast were produced. They were shown to have little cross-reactivity to other human tissues. Among the antibodies raised against human sperm, HSA-10 was also found to cross-react with human trophoblast, but not detected in other tissues. When immobilized to magnetic beads, these antibodies were shown to react only with BeWo cells in suspension, but not blood cells and ovarian carcinoma cell line, OC-3-VGH. CONCLUSION : Therefore, these antibodies may have potential application in fetal trophoblast cell isolation from the maternal circulation for prenatal genetic diagnosis.     

GOR抗体与HCV感染的相关性研究

用工合成的ＧＯＥ肽与ＢＳＡ交联后包被反应板，作为捕获抗原，建立了检测ＧＯＲ抗体的间接ＥＬＩＳＡ。共检测临床各类病人２７８例，正常供血员４８名。结果慢性丙肝患者抗ＧＯＲ阳性检出率为６０．０％（３６／６０），血透患者为４３．６％（１７／３９），白血病患者为５０．０％（６／１２），甲亢患者为４０．４％（１９／４７）。对抗ＧＯＲ抗体阳性的意义进行了分析。     

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny,an Alternative to Current Methods

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.