Yasir Waheed

Interaction of Hepatitis C virus proteins with pattern recognition receptors

Hepatitis C virus (HCV) is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR) family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I) as the receptors for intracellular viral double stranded RNA (dsRNA), and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C), non structural 3/4 A (NS3/4A) and non structural 5A (NS5A) have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells) via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

Development of Global Consensus Sequence and Analysis of Highly Conserved Domains of the HCV NS5B Protein

Background Hepatitis C virus (HCV) is a plus stranded RNA virus which encodes 10 different genes. The HCV NS5B gene encodes a polymerase, which is responsible for the replication of the virus and is a potential target for the development of antiviral agents. HCV has a high mutation rate and is classified into six major genotypes. Objectives The aim of this study was to draw a representing consensus sequence of each HCV genotype, align all six consensus sequences to draw a global consensus sequence and also study the highly conserved residues. Materials and Methods 236 HCV NS5B sequences, belonging to all six genotypes, reported from all over the world were aligned then a representing phylogenetic tree wasdrawn. Results The active site residues D220, D225, D318 and D319, which bind the divalent cations, are highly conserved among all the HCV genotypes. The other catalytic pocket residues, R158, S367, R386, and T390 and R394, which interact with the triphosphate of NTPs, are also highly conserved while T390 is mutated to valine in the genotype 5. The motif B residues G283, T286, T287 and N291, which take part in sugar selection by RdRp, are also highly conserved except for T286 which is mutated to proline in the genotypes 3 and 6. The residues E18, Y191, C274, Y276 and H502, which take part in primer/template interaction, are also high conserved except for H502 which is mutated to serine in genotype 2. High variation in all the six consensus sequences was observed in a 12 amino acid beta hairpin loop, which interacts with the double stranded RNA. Nine different peptides from the highly conserved regions of HCV NS5B protein were drawn which can be used as a peptide vaccine. The HCV NS5B phylogenetic tree shows the clusters of different genotypes and their evolutionary association. Conclusions In spite of a high mutation rate in HCV, the residues which are present in the catalytic pocket, sugar selection and template/primer interaction are highly conserved. These are target sites for the development of antiviral agents or peptide vaccines. The phylogenetic analysis suggests that different HCV genotypes have been evolved from the genotype 1a.

Evaluation of prognostic factors for Peg Interferon alfa-2b plus ribavirin treatment on HCV infected patients in Pakistan

The effective standard therapeutic regimen for patients with chronic hepatitis C is pegylated interferon plus ribavirin. The efficacy of treatment in chronic hepatitis C is defined as absence of detectable virus at six months after treatment. Analysis of patient dependent and virus related factors that enable us to predict the response to antiviral treatment is very important. We prospectively studied 403 patients who received PEG-IFN alpha-2b 1.5 μg/kg/body weight plus ribavirin. Treatment was administrated for 24 weeks and 48 weeks for hepatitis C virus (HCV) genotypes 3 and 1, respectively. Out of 403 treated patients, 301 patients (74.7%) showed a sustained virologic response (SVR). Seven variables (age, sex, ethnic group, pretreatment viral load, HCV genotyping and pretreatment ALT) were chosen as possible predictors of SVR and were analysed by means of univariable and multivariable logistic regression analysis. Five variables were statistically significant (p<0.005) on univariable analysis: age, ethnic group, pretreatment viral load, response rate at week 4, and HCV genotype. In multivariable analysis independent factors associated with SVR were low pretreatment viral load (1.97; 95%CI, 1.06-3.66; p=0.03) and attainment of rapid virological response (RVR) (7.19; 95%CI, 4.15-12.45; p<0.001). Our findings support the association between viral load and SVR to PEG-IFN-alpha-2b plus ribavirin therapy. No achievement of RVR is an unfavorable marker for SVR. These findings suggest that all patients considered for treatment should have quantification of serum HCV RNA levels. The result can be used to counsel patients on the likelihood of achieving SVR and may influence the patients decision on treatment. Future studies should confirm and explore this observation in other ethnic groups and in relation to HCV genotypes 1 and 3.

Awareness and risk factors associated with barbers in transmission of Hepatitis B and C from Pakistani population: Barber's role in viral transmission

Abstract Background: In Pakistan, most patients with Hepatitis B and C have history of facial and armpit shaving from barbers. Objective: Evaluate the awareness and risk factors associated with barbers in transmission of Hepatitis B and C. Subjects and methods: A cross sectional survey was conducted in Rawalpindi and Islamabad, the twin capital cities of Pakistan between January and July 2009. Five hundred and eight barbershops were surveyed. Results: Out of 508 barber shops, 99.8% and 98.2% were washing their razor with water and water plus antiseptic solution, respectively, while 99.8% were using new blades. Only 39.6% knew that hepatitis B virus (HBV) and hepatitis C virus (HCV) were viral diseases, 26.6% knew that it can lead to cancer, 90.7% thought that hepatitis could be transferred by blade sharing, 47.8% knew that a vaccine for HBV was available, and 43.0% had education beyond the primary level. None of the barbers used a new or washed apron/towel on every customer. Conclusion: In Pakistan, a small number of barbers are shaving clients with an old style razor with a permanent blade. There is moderate awareness about the various modes of transmission of hepatitis among the barbers, and most of them don’t know about vaccination. A predominant number of them are considering interferon treatment as a vaccine for hepatitis B and C.

Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis.

BACKGROUND Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. In Pakistan, more than 10 million people are living with HCV. Very little is known about the genotype distribution in Punjab Province, the largest province of Pakistan. Pretreatment genotype identification is very important, as different HCV genotypes respond differently to interferon therapy. METHODS In this study we performed HCV genotyping for 1537 HCV-infected patients from different districts of Punjab Province, Pakistan. Sequencing of partial HCV NS5B sequences from 14 samples belonging to genotypes 3 and 1 was also done. A sequence comparison was made of our reported sequences with those reported in the National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed. RESULTS Our study showed that the most frequent HCV genotype was 3a (in 88.1% of infected individuals), followed by 1a (3.5%), 3b (3.0%), 1b (0.8%), and 2a (1.0%). A mixed genotype infection was found in 3.6% of infected individuals, with 0.3% living with 1a + 1b co-infection, 3.1% with 3a+3b, and 0.2% suffering from 3a+1b co-infection. The sequence comparison showed that HCV NS5B motif B residues G283, T287, and N291 were highly conserved in both genotype 1 and genotype 3 sequences, while the motif B residue T286 was mutated to proline in all the genotype 3 sequences. The GDD motif, which forms the catalytic pocket and binding site for the divalent cations, was highly conserved in all the reported sequences. The phylogenetic tree suggests clustering of genotype 1 sequences with sequences from the USA, UK, Germany, and France, while genotype 3 sequences are clustered with sequences from Japan and the UK. CONCLUSIONS The major prevalent genotype in Punjab Province of Pakistan was genotype 3a, followed by genotype 1a, and only 3.6% of infected individuals had a mixed genotype infection. Sequencing of the HCV NS5B gene suggested that the active site residues were highly conserved in all the reported sequences. Our sequences, which are clustered with sequences from the USA, UK, France, and Japan, show the diversity in origin of the different genotypes prevalent in Pakistan.

Phylogenetic analysis of torque teno virus genome from Pakistani isolate and incidence of co-infection among HBV/HCV infected patients

BackgroundTorque Teno Virus (TTV) was the first single stranded circular DNA virus to be discovered that infects humans. Although there have been numerous reports regarding the prevalence of TTV from other countries of South Asia, there is severe lack of information regarding its prevalence in Pakistan. Thus the present study compiles the first indigenous report to comprehensively illustrate the incidence of the virus in uninfected and hepatitis infected population from Pakistan. Another aim of the study was to present the sequence of full length TTV genome from a local isolate and compare it with the already reported genome sequences from other parts of the world.MethodsTTV DNA was screened in the serum of 116, 100 and 40 HBV infected, HCV infected and uninfected individuals respectively. Nearly full length genome of TTV was cloned from a HBV patient. The genome sequence was subjected to in-silico analysis using CLC Workbench, ClustalW, ClustalX and TreeView. Statistical analysis was carried out in SPSS v17.0.ResultsOur results report that 89.7%, 90.0% and 92.5% of HBV, HCV patients and healthy control population were positive for TTV infection. TTV genome of 3603 bp was also cloned from a local isolate and given the identity of TPK01. The TTV genome sequence mentioned in this paper is submitted in the GenBank/EMBL/DDBJ under the accession number JN980171. Phylogenetic analysis of TPK01 revealed that the Pakistani isolate has sequence similarities with genotype 23 and 22 (Genogroup 2).ConclusionThe results of the current study indicate that the high frequency of TTV viremia in Pakistan conforms to the reports from other areas of the world, wherever screening of TTV DNA was performed against 5′-UTR of the genome. The high sequence diversity among TTV genome sequences and the high frequency of prevalence makes it harder to study this virus in cellular systems.

Molecular study of HCV detection, genotypes and their routes of transmission in North West Frontier Province, Pakistan

OBJECTIVE To determine hepatitis C virus (HCV) genotypes and explore the associated risk factors in chronic HCV patients. METHODS A total of 116 patients with chronic hepatitis C were subjected to polymerase chain reaction (PCR) based detection out of which 112 chronic HCV patients [53 male (47.32%), 59 female (52.68%); mean age (43.76±16.40) years; mean BMI (23.8±3.9) kg] were enrolled in this study. The frequency of 6 HCV genotypes and associated risk factors were evaluated from five districts of North West Frontier Province (NWFP). RESULTS Genotype 3 was the most prevalent in 73 samples (65.17%) followed by genotype 1 in 24 (21.42%) and genotype 2 in 13 (11.60%) samples. Genotype 3 had significantly high prevalence (P=0.000 2). The results showed that 48 (42.85%) samples were infected with HCV 3a; 25 (22.32%) with 3b; 14 (12.50%) with 1a; 10 (8.92%) with 1b; 11 (9.82%) with 2a; 2 (1.78%) with 2b; and 2 were untypable. The distribution of HCV genotypes in Mardan, Charsadda, Peshawar, Sawabi and Nowshehra districts was different. Use of unsterile equipment for medication, barbers and previous history of hospitalization were the main risk factors for HCV transmission. CONCLUSIONS Genotype 3a and 3b, 1a, 1b and 2a are the common genotypes in NWFP. Genotype 4, 5, and 6 can not be found in a single sample. The level of awareness about various modes of transmission of HCV among the population is found to be very low.

Analysis of variables and interactions among variables associated with a sustained virological response to pegylated interferon alfa-2a plus ribavirin in hepatitis C virus genotype 3-infected patients

BACKGROUND The recommended standard therapeutic regimen for chronic hepatitis patients with hepatitis C virus (HCV) genotype 3 is pegylated interferon plus ribavirin for 24 weeks. The aim of the present study was to evaluate treatment efficacy and variables predictive of treatment success, interactions among variables contributing to a response to therapy, and the utility of the rapid virological response (RVR; week 4 virological response) to predict treatment outcomes in HCV genotype 3-infected patients in routine clinical practice. METHODS We prospectively studied baseline and during-treatment factors associated with a sustained virological response (SVR) in HCV genotype 3-infected patients who received pegylated interferon alfa-2a (PEG-IFN α2a) 180 μg/week plus ribavirin 800 mg daily for 24 weeks and who were followed for 24 weeks after the completion of treatment. RESULTS Four hundred and twenty-six treated patients were included in the analysis; 320 (75.1%) showed an SVR. The following factors were assessed for their ability to predict SVR by means of univariable and multivariable logistic regression analysis: patient age, sex, pre-treatment viral load, pre-treatment alanine aminotransferase (ALT), body mass index (BMI), and RVR. Four factors - age, pre-treatment viral load, pre-treatment ALT, and RVR - were statistically significant predictors of SVR (p<0.05) in the univariable analysis. Factors showing a significant association with SVR were assessed by multivariable logistic regression analysis. In the multivariable analysis, independent factors associated with SVR were the attainment of RVR (odds ratio (OR) 11, 95% confidence interval (CI) 6.15-20.69; p<0.0001), patient age ≤40 years (OR 4.2, 95% CI 2.30-7.96, p<0.0001), and a low pre-treatment viral load (≤8 × 10(5) IU/ml; OR 3.4, 95% CI 1.87-6.25; p<0.0001). The effect of RVR in patients aged >40 years was more pronounced than in those aged ≤40 years: 81.1% of patients aged >40 years who achieved an RVR had an SVR, whereas only 7.5% of patients aged >40 years who did not achieve an RVR had an SVR (p<0.05). CONCLUSIONS RVR is an independent variable that is predictive of SVR. Moreover older patients (>40 years) who achieve an RVR are likely to have an SVR, while patients who do not achieve an RVR and who have a high pre-treatment viral load (>8 × 10(5) IU/ml) are unlikely to have an SVR.

RNA dependent RNA polymerase of HCV: a potential target for the development of antiviral drugs.

Hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma, cirrhosis and end stage liver disease. More than 200million people are living with HCV worldwide with high morbidity and mortality. There is no vaccine available for this virus; the approved treatment option for the majority of HCV genotypes is the combination of pegylated (Peg) interferon and ribavirin. The therapy has a different response rate on different HCV genotypes and has a number of side effects. Recently, as well as Peg interferon and ribavirin, two protease inhibitors have been introduced to treat patients with HCV genotype 1 infection. The protease inhibitors have rapid onset of resistance and are not approved for use for infections with other HCV genotypes. The HCV NS5B gene encodes RNA dependent RNA polymerase (RdRp), which is the key player in viral replication and is a promising target for the development of antiviral drugs. HCV NS5B has been studied in various biochemical assays, cell based assays and animal model systems. So far, a number of nucleoside and non-nucleoside inhibitors have been screened for effects on viral replication. This review presents a deep insight into the structure and function of HCV polymerase and the effect of various nucleoside and non-nucleoside inhibitors on viral replication.

Distribution of hepatitis C virus genotypes, hepatic steatosis and their correlation with clinical and virological factors in Pakistan

Abstract Background: Due to the inherently unstable nature of HCV, various genotypes have been identified. Steatosis is a histological feature in the progression of HCV-associated liver disease and has been shown to alter the host lipid metabolism. Objective: Assess the distribution of HCV genotypes in the two provinces of Pakistan, and determine the association of hepatic steatosis with altered clinical and virological factors in chronic HCV patients. Methods: One hundred twenty six chronic HCV patients (steatosis in 49 patients) were enrolled for qualitative analysis by PCR. Out of 126 ELISA and PCR positive samples, 119 (48 with hepatic steatosis) chronic HCV patients (mean age 42.0±13.3 years, mean body mass index (BMI) 24.2±4.1) were proved positive after PCR-based detection. Biochemical and virological factors such as HCV genotype, or glucose, in 119 CHC patients were determined and compared between patients with and without hepatic steatosis. Results: Out of 126 samples, 119 were HCV positive, where 58 (48.7%) were genotype 3a, 24 (20.2%) were 3b, 12 (10.1%) were 1a, eight (6.7%) were 2a, six (5.0%) were 1b, and one (0.8%) was 4. Furthermore, seven (5.9%) had a co-infection and three (2.5%) were untypable. BMI (p=0.004), genotype 3a (p<0.001), and triglycerides (p=0.002) were significantly associated with steatosis. It is noteworthy that cholesterol (p=0.281), glucose (p=0.305), lowdensity lipoprotein (p=0.101), high-density lipoprotein (p=0.129), alanine amino transferase (p=0.099), aspartate transaminase (p=0.177), bilirubin (p= 0.882), and age (p=0.846) showed non-significant association. Conclusion: Genotype 3a is the predominant genotype in Pakistan. Hepatic steatosis is quite frequent feature in HCV patients and strongly correlates with BMI, genotype 3a, and triglyceride contents in patients infected with HCV.