Amjad Ali

Background: Hepatitis C virus, a silent killer, has infected 71 million people globally. The recombinant viral antigenic proteins might be used in the early diagnosis of HCV infection. The NS3 and NS5A genes of HCV function in HCV replication and influence host cellular factors that are involved in HCV pathogenesis. The current study was designed to select NS3 and NS5A antigenic sites, amplified, cloned, and expressed in order to find out better assays for diagnosis or drug and vaccine development. The antigenic sites within NS3 and NS5A genes were selected and confirmed through sequencing and were cloned. The antigenic recombinant proteins were expressed in bacterial strain E. coli BL21ply*, and the expression was confirmed by western blotting by using gene-specific and vector-specific antibodies. Results: Specific antigenic regions within the NS3 and NS5A genes of the HCV 3A genotype were amplified. PCR results showed 328 bp and 747 bp antigenic regions, respectively. The regions were confirmed by DNA sequencing and cloned into a bacterial expression vector. Expression analysis showed 12 kDa and 28 kDa of NS3 and NS5A antigenic recombinant proteins, respectively. Taken together, these studies will help to analyze the genetic variability within the local HCV isolates as these antigenic recombinant proteins were quite important in the screening of HCV-infected patients. Conclusions: This study might help to enhance the progress in the treatment of HCV infection through the modeling of HCV non-structural genes (NS3 and NS5A) from local isolate, and it might also present the viral genes as potential therapeutic targets.

The exploration of genetically superior accessions is the key source of germplasm conservation and potential breeding material for the future. To meet the demand of better yielding chickpea cultivars in Pakistan the present study was organized to select more stable and resistant lines from indigenous as well as exotic chickpea germplasm obtained from Plant Genetic Resource Institute (PGRI), National Agricultural Research Centre, Islamabad, Pakistan. For the identification and evaluation of chickpea wilt resistant lines against Fusarium oxysporum f. sp. ciceris (Schlechtends), the germplasm was tested in the field for the selection of wilt resistant lines and the PCR based molecular markers were investigated to use Marker Assisted Selection (MAS) for selection of the desirable cultivars. In field trial, 70 % accessions were resistant to wilt disease, while the remaining 30 % have shown susceptibility to the disease. A total of 5 RAPD and 15 SSR markers were screened for molecular based characterization of wilt response. The data of molecular markers were scored by the presence (1) and absence (0) of allele and subjected to statistical analysis. The analysis was based on coefficient of molecular similarity using UPGMA and sorted the germplasm into two groups based on disease response. Among the total used RAPD/SSR primers, only TA194 SSR marker showed linkage to wilt resistant locus at 85 % probability. The linkage of a marker was reconfirmed by receiver operating characteristic curve. The use of the sorted wilt resistant genotypes through SSR marker TA194 can make available ample prospect in MAS breeding for yield improvement of the crop in Pakistan.

Background: Hepatitis C virus (HCV) is estimated to infect 200 million individuals in the globe, including approximately 10 million in Pakistan causing both acute and chronic hepatitis. The standard treatment against HCV is pegylated interferon therapy in combination with a nucleoside analogue ribavirin. In addition, several herbal extracts and phytochemicals derivatives are used traditionally in the treatment of liver diseases as well as HCV infection. The present study determines the inhibitory effect of kaolin minerals compound against hepatitis C virus in Huh-7 cell lines. Methods: Huh-7 cell lines were used for the in vitro HCV replication by using HCV positive sera from different patients with known HCV genotypes and viral titer/load. Total RNA was extracted from these infected cells and was quantified by real-time polymerase chain reaction (Real-time PCR). The viral titer was compared with the control samples to determine the anti-HCV activity of kaolin derived compounds. Kaolin is a group of clay minerals, with the chemical composition Al₂ Si₂O₅ (OH)₄. Results: The results showed promising effectiveness of local kaolin derived anti-HCV compounds by causing 28% to 77% decrease in the HCV titer, when applied to infected Huh-7 cell lines. This study provides the basis for future work on these compounds especially to determine the specific pathway and mechanism for inhibitory action in the replicon systems of viral hepatitis. Conclusions: Kaolin mineral derivatives show promising inhibitory effects against HCV genotypes 3a and 1a infection, which suggests its possible use as complementary and alternative medicine for HCV viral infection.

Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response was observed in both males and females patients and do not assemble the rapidly evolving HCV envelope glycoproteins. That is why in spite the presence of neutralizing antibodies in the blood they fail to resolve infections. Moreover E1 antibodies insignificantly (>0.05) inhibit HCVpp infectivity while E2 antibodies significantly (

Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1–E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag–Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.

Hepatitis C Virus (HCV) genotype and viral load are two significant predictive variables knowledge of which might persuade treatment decisions. The objective of the present study was to identify the distribution of different HCV genotypes circulating in the study area and to estimate viral load in chronically HCV infected patients. Out of total 305 HCV positive patients, 177 (58%) were males and 128 (42%) were females. Frequency breakup of the HCV positive patients was 169, 69, 38 and 29 from Abbottabad, Mansehra, Haripur and Battagram districts respectively. Out of the total 305 tested serum samples, 255 (83.06%) were successfully genotyped whereas 50 (16.4%) samples were found with unclassified genotypes. Among typable genotypes, 1a accounted for 21 (6.8%) 1b for 14 (4.6%), 2a for 4 (1.31%) 3a for 166 (54.42%) and genotype 3b for (8.19%). Twenty five (8.19%) patients were infected with mixed HCV genotypes. Viral load distribution was classified into three categories based on its viral load levels such as low (< 60, 0000 IU/mL), intermediate (60,0000-80,0000 IU/mL) and high (> 80,0000 IU/mL). The baseline HCV RNA Viral load in HCV genotype 3 infected patients was 50 (26.17%), 46 (24.08%) and 95 (49.73%) for low, intermediate and high categories respectively. For genotypes other than 3, these values for low, intermediate and high viral load categories were 50 (43.85), 35 (30.70) and 29 (25.43) respectively. Pre-treatment viral load in patients with untypable genotype was 19 (38.00%), 5 (20.00%) and 11 (44.00%) for low, intermediate and high viral load categories. Viral load distribution was also categorized sex wise; for males it was 58 (32.76%), 26 (14.68%) and 93 (52.54%) whereas for females it was 40 (31.25%), 34 (26.56%) and 54 (42.18%) for low, intermediate and high viral load respectively. In conclusion HCV genotype 3a is the most prevalent genotype circulating in Hazara Division like other parts of pakistan. Pre-treatment viral load is significantly high (p 0.014) in patients infected with HCV genotype 3 as compared to other genotypes.

Six major Hepatitis C virus (HCV) genotypes and hundreds of subtypes have been identified globally. All these genotypes are generally studied for epidemiology, their vaccine development and clinical management. This article comments the frequency distribution of various HCV genotypes circulate in different areas/districts of Khyber Pakhtoonkhaw Province of Pakistan. Sum of 415 HCV RNA PCR positive sera samples were tested by a molecular genotyping assay. Data analysis revealed that out of these 415 HCV RNA positive patients 243 were males and 172 were females. Distribution breakup of the patients was 135, 58, 51, 51, 36, 32, 6, 7and 9 patients come from the districts of Abbottabad, Mardan, Pehawar, Swat, Haripure, Swabi and Dera Ismail Khan, respectively. Out of the tested samples, genotype specific PCR fragments were observed in 299 (74.82%) patient serum samples. The distribution of genotypes of the typeable samples was as fallows: 3 patients (0.72%) each were infected with genotype 1a and genotype 1b; 240 patients (80.26%) of genotype 3a; 25 patients (6.00%) genotype 3b; and 28 patients (6.73%) were observed as with mixed genotypic infection. Sums of 116 serum samples (27.88%) were still found untypeable by the used molecular genotyping system. In conclusion, HCV genotypes 1a, 1b, 3a and 3b are distributed in various parts of KPK among which the genotype 3a is the most frequent genotype.