Calsyntenin-1 mediates hepatitis C virus replication.

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

Mutations in the E2-PePHD region of hepatitis C virus genotype-3a and correlation with response to interferon and ribavirin combination therapy in Pakistani patients.

Hepatitis C is a major health problem affecting more than 200 million individuals in the world. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed in eliminating the virus completely from patient's body. One of the mechanisms by which virus evades the antiviral effect of interferon alpha involves protein kinase (PKR) eukaryotic initiation factor 2 alpha (eIF2a) phosphorylation homology domain (PePHD). This domain in genotype 1 strains is reportedly homologous to PKR and its target eIF2a. By binding to PKR, PePHD inhibits its activity and therefore cause virus to evade antiviral activity of interferon (IFN). Many studies have correlated substitutions in this domain to the treatment response and lead to inconclusive results. Some studies suggested that substitutions favor response while others emphasized that no correlation exists. In the present study we therefore compared sequences of PePHD domain of thirty one variants of six hepatitis C virus patients of genotype 3. Three of our HCV 3a infected patients showed rapid virological response to interferon alpha and ribavirin combination therapy whereas the remaining three had breakthrough to the same combination therapy. It is found that PePHD domain is not entirely conserved and has substitutions in some isolates irrespective of the treatment response. However substitution of glutamine (Q) with Leucine (L) in one of the breakthrough responders made it more identical to HCV genotype 1a. These substitutions in the breakthrough responders also tended to increase average hydrophilic activity thus making binding of PePHD to PKR and inhibition of PKR more favorable.

Prevalence of hepatitis delta virus infection among hepatitis B virus surface antigen positive patients circulating in the largest province of Pakistan.

BACKGROUND: Hepatitis delta virus (HDV) and Hepatitis B virus (HBV) co-infection is well known to induce a spectrum of acute and chronic liver diseases which further advance to cirrhosis, fulminant hepatitis and hepatocellular carcinoma (HCC). AIM: The aim of the present study was to determine the prevalence of hepatitis D virus super-infection among hepatitis B surface antigen (HBsAg) positive individuals in the highly populated province of Pakistan which is not well known. METHODS: Sera samples were subjected to HBsAg and anti-HDV screening and finally anti-HDV and HBsAg positive coinfected samples were used for HDV active RNA confirmation using nested polymerase chain reaction (PCR). RESULTS: Out of total 200 HBsAg positive samples by rapid device, 96 (48%) were also found reactive for HBsAg using enzyme linked immunosorbant assay (ELISA). Out of these HBsAg ELISA positive samples, 80 (88.8%) were anti-HDV ELISA positive which were then subjected to PCR. The amplification results further confirmed 24 (30%) samples to be HDV RNA positive. HDV super-infection was more common in male patients than female patients (81% VS 19%). CONCLUSION: The current study shows a high prevalence rate of HDV-HBV co-infection in Pakistan that tends to increase over time.

Hepatitis C treatment: current and future perspectives.

Hepatitis C virus (HCV) is a member of Flaviviridae family and one of the major causes of liver disease. There are about 175 million HCV infected patients worldwide that constitute 3% of world's population. The main route of HCV transmission is parental however 90% intravenous drug users are at highest risk. Standard interferon and ribavirin remained a gold standard of chronic HCV treatment having 38-43% sustained virological response rates. Currently the standard therapy for HCV is pegylated interferon (PEG-INF) with ribavirin. This therapy achieves 50% sustained virological response (SVR) for genotype 1 and 80% for genotype 2 & 3. As pegylated interferon is expensive, standard interferon is still the main therapy for HCV treatment in under developed countries. On the other hand, studies showed that pegylated IFN and RBV therapy has severe side effects like hematological complications. Herbal medicines (laccase, proanthocyandin, Rhodiola kirilowii) are also being in use as a natural and alternative way for treatment of HCV but there is not a single significant report documented yet. Best SVR indicators are genotype 3 and 2, < 0.2 million IU/mL pretreatment viral load, rapid virological response (RVR) rate and age <40 years. New therapeutic approaches are under study like interferon related systems, modified forms of ribavirin, internal ribosome entry site (HCV IRES) inhibitors, NS3 and NS5a inhibitors, novel immunomodulators and specifically targeted anti-viral therapy for hepatitis C compounds. More remedial therapies include caspase inhibitors, anti-fibrotic agents, antibody treatment and vaccines.

Nucleotide identity and variability among different Pakistani hepatitis C virus isolates.

BACKGROUND: The variability within the hepatitis C virus (HCV) genome has formed the basis for several genotyping methods and used widely for HCV genotyping worldwide. AIM: The aim of the present study was to determine percent nucleotide identity and variability in HCV isolates prevalent in different geographical regions of Pakistan. METHODS: Sequencing analysis of the 5'noncoding region (5'-NCR) of 100 HCV RNA-positive patients representing all the four provinces of Pakistan were carried out using ABI PRISM 3100 Genetic Analyzer. RESULTS: The results showed that type 3 is the predominant genotypes circulating in Pakistan, with an overall prevalence of 50%. Types 1 and 4 viruses were 9% and 6% respectively. The overall nucleotide similarity among different Pakistani isolates was 92.50% +/- 0.50%. Pakistani isolates from different areas showed 7.5% +/- 0.50% nucleotide variability in 5'NCR region. The percent nucleotide identity (PNI) was 98.11% +/- 0.50% within Pakistani type 1 sequences, 98.10% +/- 0.60% for type 3 sequences, and 99.80% +/- 0.20% for type 4 sequences. The PNI between different genotypes was 93.90% +/- 0.20% for type 1 and type 3, 94.80% +/- 0.12% for type 1 and type 4, and 94.40% +/- 0.22% for type 3 and type 4. CONCLUSION: Genotype 3 is the most prevalent HCV genotype in Pakistan. Minimum and maximum percent nucleotide divergences were noted between genotype 1 and 4 and 1 and 3 respectively.

Association of hepatitis C virus with insulin resistance: evidences from animal studies and clinical studies.

CONTEXT: HCV infection is strongly associated with development of insulin resistance and type-2 diabetes, however molecular mechanism of these associations is not known. The aim of this review was to conduct a comprehensive literature search to understand the nature of the association between hepatitis C virus (HCV) infection and insulin resistance (IR). We also explored the role of HCV core protein and NS5a in modulating the course of the insulin-signaling pathway. EVIDENCE ACQUISITIONS: We searched Directory of Open Access Journals (DOAJ) Google Scholar, Pubmed (NLM), LISTA (EBSCO), Web of Science (TS and PakMediNet). RESULTS: Emerging evidence suggests an association between HCV infection and carotid/coronary vascular disease. IR appears to be a dominant underlying cause of accelerated atherosclerosis in patients with chronic hepatitis C (CHC). HCV can induce IR directly through the stimulation of SOCS3 and PPA2, and both of these molecules have been shown to inhibit interferon-α signaling. Improvement of insulin sensitivity may increase the response rate to antiviral treatment and prevent IR complications, including vascular diseases. The results of several clinical trials that have used insulin sensitizers (metformin and PPAR-γ agonists) have been inconclusive. CONCLUSIONS: Beside the association between HCV and IR, the published data also have showed the possible association of HCV core and NS5A protein with IR.

PCR could be a method of choice for identification of both pulmonary and extra-pulmonary tuberculosis.

BACKGROUND: Nucleic acid amplification assays including PCR have revolutionized the detection of Mycobacterium tuberculosis (MTB). Tuberculosis spread to almost every organ of the body and is characterized on the basis of localization of infection. Therefore, different types of body fluids and tissues can be used for the detection of MTB.From 2004 to 2010 total 766 different types of smear negative samples from patients, clinically suspected for tuberculosis were received and investigated at Division of Molecular Diagnostics, University of the Punjab Lahore for the diagnosis of tuberculosis. Mycobacterial DNA was extracted followed by PCR amplification. FINDINGS: A total of 356 (46.5%) samples were found positive by PCR for MTB. These included; serum (4.8%), blood (36.3%), urine (46.6%), cerebro spinal fluid (CSF) (42.1%), ascetic fluid (67.6%), pleural fluid (52%), pericardial fluid (30%), pus (38.6%), bone marrow (60%), sputum (38.8%) and bronchoalveolar lavage (BAL) (70%). Over all there was no significant difference in males and females neither in different age groups for the identification of MTB. CONCLUSION: We conclude that PCR is a useful and sensitive tool for the early diagnosis of MTB in variety of clinical samples.

High baseline interleukine-8 level is an independent risk factor for the achievement of sustained virological response in chronic HCV patients.

Hepatitis C virus (HCV), a major cause of liver disease throughout the world, is difficult to treat with interferon (IFN) (and various formulations and combinations thereof) being the only approved molecule available. It has been investigated recently that proinflammatory chemokine interleukin-8 (IL-8) induced by HCV partially inhibits the antiviral IFN-α therapy. Therefore, the current study was aimed to prospectively utilize the baseline IL-8 levels in the HCV infected serum and predicts its role in sustained virological response (SVR) to IFN-α+ribavirin therapy, in chronic HCV patients in Pakistan. One hundred and ten hepatitis C patients without any other infections underwent IFN-α+ribavirin combination treatment. Baseline IL-8 levels were determined before starting of the therapy for all these patients. Fifteen normal volunteers negative for HCV were kept as control. The baseline IL-8 levels were found significantly higher in all HCV positive patients as compared to normal healthy volunteers (1083.54 ± 85.72 pg/ml versus 6.99 ± 1.05 pg/ml [mean ± SEM], p<0.01) and were also significantly higher in non-responders than responders (p<0.05). Comparatively higher mean baseline IL-8 levels were observed in non-responders (2442.02 ± 159.92 pg/ml), than late (1009.31 ± 45.31) and rapid (540.91 ± 27.06 pg/ml) responders. Significant relation was observed between baseline IL-8 level and response to IFN therapy (p<0.01). Results of this study suggest that increased levels of IL-8 in HCV infection might be involved in pathogenesis, persistence and resistance to IFN-α+ribavirin combination therapy.

Pattern and molecular epidemiology of Hepatitis B virus genotypes circulating in Pakistan.

The continuously mutating nature of Hepatitis B virus (HBV) is responsible for the emergence of varying genotypes in different regions of the world affecting the disease outcome. The objective of the current study was to find out the pattern of HBV genotypes circulating in Pakistan. HBV genotypes were determined in HBV chronic patients of different age and gender from all the four different geographical regions (provinces) of Pakistan for a period of 2 years (2007-2009). Out of the total 3137 consecutive patients, 300 (175; 58.3% males and 125; 41.7% females) were randomly selected for HBV genotype A through H determination using molecular genotyping methods. Total 269 (89.6%) isolates were successfully genotyped where as 31 (10.3%) samples failed to generate a type-specific PCR band and were found untypable. Out of the successfully genotyped samples, 43 (14.3%) were with type A, 54 (18%) were with type B, 83 (27.6%) were with type C, 39 (13%) were with type D, 2 (0.6%) were with type E, 4 (1.3%) were with genotype F and total 44 (14.6%) were with mixed HBV infections. Of the mixed genotype infection cases, 16 were with genotypes A/D, 9 were B/C, six were A/D/F, five were with genotypes A/F, two were with A/B/D and B/E and one each for A/C as well as A/E genotypes. Four common genotypes of HBV found worldwide (A, B, C & D) were isolated from Pakistan along with uncommon genotypes E and F for the first time in Pakistan. Overall Genotype C is the most prevalent genotype. Genotypes B and C are predominant in Punjab & Balochistan and Khyber Pakhtoonkhwa, respectively whereas genotype A in Sindh.

The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan.

Information regarding the changing pattern in hepatitis C virus (HCV) genotypes/subtypes and resulting disease outcome is not well known. The specific objective of this study was to find out the frequency distribution of HCV genotypes and changing pattern of various HCV genotypes overtime in well-characterized Pakistani HCV isolates. The genotype distribution of HCV from all the four provinces of Pakistan was tracked for a period of 10 years (2000-2009) on total 20,552 consecutive anti-HCV and HCV RNA positive patients sample using type-specific genotyping assay. Of these, 16,891 (82.2%) samples were successfully genotyped. Of these 11,189 (54.4%) were males and 9363 (45.55%) were females. Of the successfully genotyped samples, 12,537 (74.2%) were with 3a, 1834 (10.9%) with 3b, 50 (0.24%) with 3c, 678 (3.3%) with 1a, 170 (0.83%) with 1b, 49 (0.24%) with 1c, 431 (2.1%) with 2a, 48 (0.23%) with 2b, 3 (0.01%) with 2c, 13 (0.06%) with 5a, 12 (0.06%) with 6a, 101 (0.49%) with 4, and 965 (4.7%) were with mixed-genotype infection. A changing pattern of HCV genotypes prevalence was observed in Pakistan overtime, with an increase in the relative proportion of genotype 3a and mixed genotypes and a decrease of genotypes 3b, 2b, 4, 5a and 2a. This changed HCV genotype pattern might have direct impact on HCV disease outcome and new therapeutic strategies may be needed.

Hepatitis C virus genotype 3a infection and hepatocellular carcinoma: Pakistan experience.

AIM: To assess the association between chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) in Pakistan, and the genotype distribution among these HCC patients. METHODS: One hundred and sixty-one subjects with HCC were included in this study. Liver biopsy was performed on 145 of the patients; sixteen were excluded because they failed to fulfill the inclusion criteria. Qualitative polymerase chain reaction (PCR) was performed for hepatitis B virus and HCV. Samples positive for HCV RNA were genotyped using genotype-specific PCR and confirmed by HCV 5' noncoding region sequencing analysis. RESULTS: Chronic HCV infection was identified a major risk factor (63.44% of tested HCC patients) for the development of HCC. The time from HCV infection to appearance of cancer was 10-50 years. In the HCC patient population, broader distributions of genotypes were present with genotype 3a as the predominant genotype. Using the type-specific genotyping method, we found HCV genotype 3a in 40.96%, 3b in 15.66%, 1a in 9.63%, and 1b in 2.40% of HCC tissue samples. About 28% of cases were found with mixed genotypes. Two cases were unable to be genotyped because of low viral load. Sixty-six percent of treated patients with cirrhosis had an end of treatment response, but unfortunately they relapsed quickly when the treatment was discontinued, and HCC developed during a median 3.8 years. CONCLUSION: There was a strong association between chronic HCV infection and HCC in Pakistan, and between HCV genotype 3a and HCC.