Umar Saeed

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.

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 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.

Identification of novel silent HIV propagation routes in Pakistan.

Human immunodeficiency virus (HIV) is rapidly increasing in both high risk groups and the general population. In this study, silent routes of propagation in teenaged Pakistanis are discussed. In order to promote sexual activity in youths, regular clients write contact details of sex workers on the doors of public washrooms. HIV prevalence is much higher among Hijra sex workers. Hijra sex workers have apparently stepped into the profession of begging at public places, where they earn money by both begging and distributing visiting cards offering unsafe sex. In many educational institutes, sex education is lacking or absent; if delivered via teachers, government agencies and nongovernmental organizations this could prevent a future epidemic of sexually transmitted infections in Pakistan.

Viral outbreaks and communicable health hazards due to devastating floods in Pakistan.

Pakistan is a developing country that has a population of 190 million people and faces a huge burden of viral diseases. Every year during monsoon season heavy rain fall and lack of disaster management skills potentially increase the transmission of waterborne diseases, vector borne diseases and viral outbreaks. Due to severe flooding, thousands of people lose their lives and millions are displaced each year. In most of the cases the children who lose their family members are forced into illegal professions of begging, child labor and prostitution which make them prone to sexually transmitted infections. Up to date, no scientific study has been conducted nationwide to illustrate epidemiological patterns of waterborne diseases, vector borne diseases and viral epidemics during flash flood. Mosquito sprays would not be a sufficient approach for dengue eradication; mass awareness, larvicide and biological control by Guppy fishes are also effective strategies to overcome dengue problem. International health bodies and non-governmental organizations must take note of this alerting situation and take adequate steps such as financial/medical aid in order to defeat the after-effects of flood.

Contemplating the Importance of Toll-like Receptors I and II Regarding Human Viral Pathogenesis

Background: Toll-like receptors (TLRs) play a major role in innate immunity, since they detect conserved pathogen-associated molecular patterns (PAMPs) on a range of microbes, including viruses, leading to innate immune activation and orchestration of the adaptive immune response. Objectives: The current study aimed to discuss earlier evidence implicating TLRs I and II in the innate immune response to viruses, in the light of more recent clinical data demonstrating that TLRs are important for anti-viral immunity in humans. Materials and Methods: A literature search was performed via accessing research articles from PakMediNet, Pubmed and Google Scholar with key words of Toll-like receptors I and II Regarding human viral pathogenesis. The valued information on the recent scientific horizons was subjected to critical analysis. Results: Comprehensive literature review illustrates important signaling pathways involved in TLR1/TLR2 mediated regulation of viral pathogenesis. TLRs mediated activation of apoptosis tends to contribute towards defense strategies utilized by innate immune response. Activation of antiviral TLR1-dependent signaling cascade would ultimately lead to activation of NF-kappa B which promotes antiviral responses via induction of specific genes. TLR1/TLR2 dimer generates intracellular signaling via IRAK4 mediated activation of IRAK1/2 which results in activation of NF-kappa B, p38 and JNK proteins in cytoplasm. NF- kappa B, p38 and JNK enter the nucleus thereby causing activation of various pro-inflammatory cytokines such as IL-1 beta, TNF-alpha, IL-6, IL-8 and IL-18. Among the chronic HCV infection, the HCV core protein induces TNF-α and IL-10 from the macrophages thereby causing reduction in release of interferon alpha. Abnormal TLR1/TLR2 signaling may contribute to the enhancement of infection-related morbidity and mortality. Conclusions: To date, a large number of viruses are proved to trigger innate immunity via TLRs, suggesting that these receptors are likely to be important in the outcome of viral infection. This suggestion is supported by the observation that many viruses have evolved mechanisms not only to evade the innate immune system, but also to subvert it for the benefit of the virus.

Estimation of Hepatitis B Virus, Hepatitis C Virus, and Different Clinical Parameters in the Thalassemic Population of Capital Twin Cities of Pakistan

Hepatitis B and C are serious public health problems worldwide. Thalassemia patients are dependent on blood transfusions throughout their life and are at high risk of viral infections. The aim of this study was to estimate the prevalence of hepatitis B/C infections and different clinical parameters in multitransfused thalassemia population. In this study, 262 multitransfused β-thalassemia patients were enrolled from the capital twin cities of Pakistan. The presence of hepatitis B virus (HBV)/hepatitis C virus (HCV), alanine aminotransferase (ALT) level, serum creatinine, serum ferritin, hepatomegaly, splenomegaly, and splenectomy were analyzed. The overall prevalence of HBV and HCV was 3.08% and 55.73%, respectively, with 100% of patients older than 20 years had HCV infection. The ALT levels among HBV- and HCV-positive thalassemia patients were 92.62 ± 41.57 U/L and 98 ± 63.65 U/L, respectively; creatinine values observed were 0.4 ± 0.35 mg/dL (for HBV) and 0.39 ± 0.24 mg/dL (for HCV), while serum ferritin levels were 6865.87 ± 1649.13 ng/dL (for HBV) and 5445.95 ± 3059.28 ng/dL (for HCV). A total of 74.8% and 82.20% of HBV- and HCV-positive patients had hepatomegaly with an average increase in liver size of 4.17 and 4.33 cm, respectively. Splenomegaly was observed in 64.9% and 67.12% of HBV- and HCV-positive patients with an average increase in spleen size of 4 and 4.46 cm, respectively. Splenectomy was observed among 14.50% and 15.75% of HBV- and HCV-infected thalassemia patients. There is a strong need to properly screen blood before transfusions to reduce the future load of viral hepatitis from Pakistan.