Hepatitis C virus genotypes circulating in patients with chronic hepatitis C in Thailand and their responses to combined PEG-IFN and RBV therapy.

Different genotypes of hepatitis C virus (HCV) are circulating in different areas of the world. In Thailand, distribution of HCV genotypes has been investigated mostly in the central area while the information in other regions is limited. This study aimed to determine the HCV genotypes circulating in chronic hepatitis C patients in Chiang Mai, Thailand and to investigate the response of different HCV genotypes to pegylated interferon (PEG-IFN) and ribavirin (RBV) treatment. Patients infected chronically with HCV were treated with PEG-IFN/RBV based on the standard regimens for each HCV genotype and followed up the patients until the end of treatment and 6 months afterward. Out of 158 patients, three major HCV genotypes and eight subtypes were identified. Genotype 3 was the most predominant at 54.5%, followed by genotypes 1 (31%) and 6 (14.5%). Among subtypes, 3a was the most prevalent subtype (45%), followed by 1b (18.4%), 1a and 6f (each at 12.6%), 3b (9.5%), and 6a, 6i, 6n (each at 0.63%). Patients with genotype 3 showed higher rate of responding to the treatment at 80.2% compared to genotypes 1 (73.5%) and 6f (65%). Additionally, patients with genotype 6f showed higher rate of relapsing (25%) compared to genotypes 1 and 3 (14.3% and 16.3%, respectively). In conclusion, this study reported multiple HCV genotypes circulated in Thai patients and the response of different HCV genotypes to PEG-IFN/RBV treatment.

Hepatitis C virus NS5A disrupts STAT1 phosphorylation and suppresses type I interferon signaling.

Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.

Analysis of the VP6 gene of human and porcine group A rotavirus strains with unusual subgroup specificities.

Full-length VP6 amino acid sequences of human and porcine rotaviruses with subgroup (SG) (I + II) and SG non-(I + II) were analyzed in comparison with those of SG I and SG II. In human rotaviruses, the strains in the same SG shared a very high degree of amino acid identity, ranging from 97.4% to 99.4% for SG I, 95.9% to 100% for SG II, and 99.4% to 100% for SG non-(I + II), while viruses in different SGs shared somewhat lower sequence identity at 90.4-93.1%. Conserved amino acids that distinguished the strains of SG I from SG II were observed at 21 positions. The viruses with SG non-(I + II) shared sequence identity with SG II as high as 97.2-99.7%, suggesting that they belonged to genogroup II. Similarly, porcine rotaviruses in the same SG shared 96.4-99.7% for SG I, 98.2-100% for SG II, 97.4-100% for SG (I + II), and 96.2-99.7% for SG non-(I + II), while strains in different SGs shared sequence identity ranging from 91.9% to 94.4%. Interestingly, the strains with SG (I + II) and SG non-(I + II) shared a high degree of sequence identity with SG I, at 96.4-100% and 94.7-99.7% respectively, suggesting that they are related to porcine SG I strains. The conserved amino acids which distinguished SG I from SG II were observed at 13 positions. The strains with SG I, SG (I + II), and SG non-(I + II) showed identical amino acid residues at these positions. Phylogenetic analysis strongly supported the findings of the sequence analysis.

Genotypic distribution of hepatitis C virus in voluntary blood donors of northern Thailand.

The purpose of this study was to determine the prevalence and distribution of HCV genotypes among voluntary blood donors in northern Thailand. From 1998 to 2000, 167 serum samples which tested positive for anti-HCV antibodies in the screening of voluntary blood donors from 5 provinces in northern Thailand were selected for genotyping. Viral RNA was extracted from the sera. The core-E1 region of the HCV-RNA genome was amplified using a OneStep RT-PCR kit. The core-E1 amplicon was sequenced and the HCV genotype was assigned by comparing with the reference sequences available in the GenBank database. Of 167 anti-HCV positive serum samples, 126 (75.4%) contained HCV RNA as detected by PCR. HCV genotype 3 was the most predominant genotype (39.6%), of which 33.3% belonged to genotype 3a and 6.3% to 3b. Genotype 6 was detected in 31%, and genotype 1 was detected in 27.8%. Of the genotype 1 isolates, 14.3% were la, 12.7% were 1b, and 0.8% were 1c. Two HCV isolates detected in the present study were untypeable. About 75% of anti-HCV positive blood donors had chronic HCV infection. In northern Thailand, genotype 3a was the most predominant genotype, while genotype 6, 1a and 1b were also commonly found. The genotypic distribution of HCV isolates from various regions of Thailand were more or less similar. Nevertheless, in this study, the prevalence of HCV genotype 6 (31%) was higher than previously reported by others (8-18%). Phylogenetic analysis of the HCV isolates detected in the present study was also performed.

Multiple combinations of P[13]-like genotype with G3, G4, and G5 in porcine rotaviruses.

Epidemiological surveillance of porcine rotavirus (PoRV) strains was carried out in Chiang Mai Province, Thailand, from 2002 to 2003, and eight rotavirus isolates could not be completely typed by PCR. Of these, six were G3 and one was G4 and displayed a P-nontypeable genotype, while another isolate was both G and P nontypeable. Analysis of a partial VP4 gene of all eight P-nontypeable strains revealed a high degree of amino acid sequence identities (94.7% to 100%), suggesting that they belonged to the same P genotype. Comparison of the amino acid sequences of two representative strains (namely, strains CMP178 and CMP213) with those of 27 other known P genotypes revealed a high degree of amino acid sequence identity with those of P[13] porcine rotavirus reference strains HP113 and HP140, which were recently isolated in India. However, amino acid sequence comparison with non-P[13] rotavirus strains revealed relatively low identities, ranging from 58.2% to 84.8% for full-length VP4 sequences and 35.1% to 80.6% for VP8* sequences. Phylogenetic analysis revealed that CMP178 and CMP213 clustered together in a monophyletic branch with P[13]-like genotypes HP113 and HP140 which was clearly separated from the other lineages of P[13] or P[22] strains. Altogether, these findings indicate that PoRV strains CMP178 and CMP213 should be considered the P[13]-like VP4 genotype, a rare genotype that has been identified only in pigs. This study provides additional evidence of increasing genetic diversity among group A rotaviruses in nature.

Genetic diversity of norovirus, sapovirus, and astrovirus isolated from children hospitalized with acute gastroenteritis in Chiang Mai, Thailand.

Norovirus (NV), sapovirus (SV), and human astrovirus (HAstV) are important causes of acute gastroenteritis in infants and young children. This study investigated the prevalence of NV, SV, and HAstV infections in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand from May 2000 to March 2002. Fecal specimens were tested for NV, SV, and HAstV by reverse transcription polymerase chain reaction (RT-PCR) using degenerate specific primers. These viruses were characterized further by sequence and phylogenetic analyses of the partial capsid gene. From 296 fecal specimens tested, 13.5% (40 of 296) were positive for NV, SV, and HAstV. Of these, NV most predominant, with a prevalence of 60% (24 of 40), of which 17.5% were NVGI and 42.5% were NVGII. Of note, one specimen was positive for both NVGI and SV. SV was detected in 25%, while HAstV was detected in 17.5%. Analysis of nucleotide and amino acid sequences revealed that NVGI strains comprised GI/3, GI/4, GI/6, GI/7, and GI/13 genotypes. Among NVGII strains, approximately half of them belonged to genotype GII/4 (Lordsdale virus cluster), followed by GII/3, GII/10, GII/1, GII/6, GII/8, and GII/15. Analysis of SV sequences revealed that SVGI (Manchester virus) was more common than SVGII (London virus). The SV genotypes detected in this study belonged to SVGI/1, SVGI/4, SVGI/5, SVGII/1, and SVGII/2, whereas the HAstV belonged to genotypes HAstV-1, HAstV-2, HAstV-3, and HAstV-5. The findings suggest that NV, SV, and HAstV are important enteric viruses cocirculating among hospitalized children in Chiang Mai, Thailand.

WITHDRAWN: Molecular Characterization of VP4 and VP7 Genes of Nontypeable Strains Identifies a Novel P[28] Genotype in Porcine Rotaviruses.

This article has been retracted.

Analysis of mutations in the core and NS5A genes of hepatitis C virus in non-responder and relapser patients after treatment with Peg-IFN-α and ribavirin.

Mutations in several regions of HCV genome are shown to correlate with response to interferon (IFN) treatment. Persistence of HCV infection and poor susceptibility to treatment might be contributed by mutations arising within HCV genome which enable the virus to escape from host immune response/IFN treatment. This study investigated mutations in core and NS5A genes of HCV from non-responder and relapser patients after treatment with Peg-IFN-α and ribavirin. Viral RNA was extracted from patient sera and core and NS5A genes were amplified by RT-PCR. Nucleotide sequences of the core and NS5A genes were determined by direct sequencing, and converted to amino acid sequences. Nucleotide and amino acid sequences in the core region, ISDR, PKRBD, and V3 regions within NS5A after treatment were highly conserved when comparing to their corresponding sequences obtained before treatment. Interestingly, when comparing the virus from relapsers to those from non-responders, the number of mutations after treatment in N-terminal region of NS5A of virus from relapsers was significantly higher than those from non-responders (P < 0.05). Amino acid mutations at the N-terminus of NS5A of the virus in relapsers might help the virus to survive and somehow relapse after the cessation of the treatment.

Influence of amino acid variations in the NS3, NS4A and NS4B of HCV genotypes 1a, 1b, 3a, 3b and 6f on the response to pegylated interferon and ribavirin combination therapy.

BACKGROUND: It has been suggested that HCV proteins, core, NS3/4A, NS4B, and NS5A, contribute to the resistance of HCV to IFN and ribavirin (RBV) treatments. AIM: To assess the effects of HCV amino acid variations in NS3, NS4A and NS4B of HCV subtypes 1a, 1b, 3a, 3b and 6f on the response to pegylated interferon (Peg-IFN) and RBV therapy. METHODS: One hundred and thirty four HCV isolates of genotypes 1a, 1b, 3a, 3b and 6f obtained from HCV patients both before and at week 4 of treatments were evaluated. Amino acid sequences of NS3, NS4A and NS4B were analyzed and in compared to reference sequences of corresponding genotypes. RESULTS: The data revealed that amino acid variations within the full-length NS3, protease and helicase domains of NS3 of HCV 1a from responders were significantly higher than those from treatment failure groups as compared to reference sequences of each corresponding genotype. Similar results were observed in the full-length and helicase domain but not in the protease domain of HCV 1b. However, the number of amino acid variations in NS3 of HCV 3a, 3b and 6f as well as in NS4A and NS4B showed no difference between the viruses from responders and treatment failure group. Analysis of amino acid variations both before and at week 4 of treatment revealed that the mean number of amino acid variation in the full-length NS3 of HCV 3a and 3b from responders were also significantly higher than those from the treatment failure group. CONCLUSION: Our study suggests that the increase of amino acid variations within the NS3 protein of HCV 1a, 1b, 3a and 3b were associated with the response to Peg-IFN and RBV treatment in Thai patients.

Comparison of Xpert MTB/RIF Assay and the Conventional Sputum Microscopy in Detecting Mycobacterium tuberculosis in Northern Thailand.

Background. Despite low sensitivity in detection of Mycobacterium tuberculosis, sputum acid-fast smear remains the main diagnostic method. This study aimed to compare the diagnostic performance of Xpert MTB/RIF assay versus conventional sputum acid-fast smear. Materials and Methods. A cross-sectional study was conducted at Chiang Mai University Hospital, Thailand. Patients who were ≥15 years old and had clinically suspected pulmonary tuberculosis were included. Results. 109 specimens from 57 patients were included. Using MGIT sputum culture as a reference standard, the sensitivity (SEN) and specificity (SPEC) for Xpert were 95.3% (95% CI, 84.2%, 99.4%) and 86.4% (95% CI, 75.7%, 93.6%). The SEN and SPEC for sputum acid-fast smear were 60.5% (95% CI, 44.4%, 75.0%) and 98.5% (95% CI, 91.8%, 100%). Xpert had significantly higher sensitivity (p value < 0.001) and lower specificity (p value = 0.022) than sputum acid-fast smear. Among 43 culture-proven M. tuberculosis specimens, sensitivity of Xpert was 100% (95% CI, 86.7%, 100%) in acid-fast positive smears (n = 26) and 88.2% (95% CI, 63.5%, 98.5%) in acid-fast negative smears (n = 17). Conclusions. The good sensitivity and specificity of Xpert assay in detecting M. tuberculosis from sputum specimens may help in early diagnosis and treatment of pulmonary tuberculosis, particularly among patients who had acid-fast negative sputum smear.

A novel multiplex RT-PCR for identification of VP6 subgroups of human and porcine rotaviruses.

VP6 protein antigens allow classification of rotaviruses into at least four subgroups, depending on the presence or absence of SG-specific epitopes: SG I, SG II, SG (I+II), and SG non-(I+II). However, MAbs against epitopes on the VP6 protein of human and porcine rotaviruses, sometimes, do not recognize SG-specific epitopes or recognize irrelevant-SG epitopes and therefore result in the incorrect assignment of subgroups. In order to solve this problem, a novel multiplex RT-PCR was developed as an alternative tool to identify VP6 genogroups using newly designed primers which are specific for genogroup I or II. The sensitivity and specificity of the newly developed multiplex RT-PCR method was evaluated by testing with human and porcine rotaviruses of known SG I, SG II, SG (I+II), and SG non-(I+II) strains in comparison with monoplex RT-PCR and VP6 sequence analysis. The results show that the genogroups of both human and porcine rotaviruses as determined by the new multiplex RT-PCR method were in 100% agreement with those determined by monoplex RT-PCR and VP6 sequence analysis. The method was shown to be specific, sensitive, less-time consuming, and successful in genogrouping clinical isolates of rotaviruses circulating in children and piglets with acute diarrhea.

Occult hepatitis C virus infection during an outbreak in a hemodialysis unit in Thailand.

Control of hepatitis C virus (HCV) in hemodialysis populations is a major public health priority, but the preferred methods to prevent and rapidly detect HCV outbreaks in these populations remains subject to debate. We enrolled 231 hemodialysis patients at three dialysis centers in Chiang Mai, Thailand. Patients were followed every 6 months for 3 years and tested for the presence of serum HCV antibody and HCV RNA at each visit. We additionally isolated and tested peripheral blood mononuclear cells (PBMCs) for HCV RNA collected at the 30-month follow-up visit. Fifty-one study participants negative for anti-HCV at the baseline enrollment visit seroconverted over the course of the 3-year follow-up period. Of 11 individuals who transiently lost detectable serum HCV viremia, we were able to detect HCV RNA from the PBMCs of two individuals. Our results suggest that occult HCV infection may be common among hemodialysis patients, and serum HCV RNA testing may be supplemented with PBMC testing to maximize diagnostic sensitivity and aid in outbreak containment. Further work on the diagnostic implications of HCV compartmentalization in hemodialysis and other settings is urgently needed.