Genotype 1a Research Articles

HCV Genotype Distribution Among People Who Inject Drug in Turkey: Findings from Multicenter and Cross-Sectional Study.

Hepatitis C virus (HCV) infection is very common in people who inject drugs (PWID). Studies about the prevalence and genotype distribution of the HCV among PWID are very crucial for developing strategies to manage HCV infection. This study's objective is to map the distribution of HCV genotypes among PWID from various regions of Turkey. This prospective, multicenter, cross-sectional study involved 197 PWID who tested positive for anti-HCV antibodies from 4 different addiction treatment facilities in Turkey. Interviews were done with people who had anti-HCV antibodies, and blood samples were taken to check the HCV RNA viremia load and genotyping. This study was conducted on 197 individuals with a mean age of 30.3 ± 8.6 years. 9.1% (136/197 patients) had a detectable HCV-RNA viral load. Genotype 3 was the most commonly observed genotype by 44.1%, followed by genotype 1a by 41.9%, genotype 2 by 5.1%, genotype 4 by 4.4%, and genotype 1b by 4.4%. Whereas genotype 3 was dominant with 44.4% at the central Anatolia region of Turkey, the frequencies of genotypes 1a and 3, which were predominantly detected in the south and northwest regions of Turkey, were very close to each other. Although genotype 3 is the predominant genotype in the PWID population in Turkey, the prevalence of HCV genotype varied across the country. To eliminate HCV infection in the PWID, treatment and screening strategies that differ by genotype are essentially required. Especially identification of genotypes will be useful in developing individualized treatments and determining national prevention strategies.

Hepatitis C virus genotype diversity in Ningxia Hui autonomous region, Northwestern China

Hepatitis C virus (HCV) genotypes vary greatly in different regions. The aim of this study is to investigate the distribution of HCV genotypes in HCV infected patients, in Ningxia Hui Autonomous Region. Nucleic acid extraction and amplification were performed with test kits on 153 HCV infected patients serum samples. The HCV viral load was measured using reverse transcriptase PCR (RT-PCR) and HCV genotypes were determined. Among the 153 HCV-infected patients, 56 had genotype (GT)1b (36.60%), 45 had GT2a (29.40%), 23 had GT3a (15.00%), 14 had GT3b (9.20%),13 had GT6a (8.50%), 1 had GT1g (0.70%), 1 had GT6xa (0.70%). In GT1b, 21.40% were female and 78.60% were male; in GT2a, 42.20% were female and 57.80% were male;Males were most prevalent in genotypes 1b(39.30%), while female were most prevalent in genotype 2a(46.30%). Rare GT1g and GT6xa were also detected in males. The 41-50 year age group had the highest HCV prevalence of 32.00%. HCV GT1b is the predominant HCV genotype in Ningxia Hui Autonomous Region.

Comparison of pathogenicity of 4 porcine circovirus type 2 (PCV2) genotypes (2a, 2b, 2d, and 2e) in experimentally infected pigs.

The objective of the current study was to compare the virulence of four porcine circovirus type 2 (PCV2) genotypes (PCV2a, 2b, 2d, and 2e) in pigs. Pigs were inoculated at 42 days of age with one of four PCV2 genotypes, then necropsied at 63 days of age. PCV2 genotype groups were evaluated through a comparison of clinical outcomes, antibody titers, level of PCV2 loads in blood and lymph nodes, and lymphoid lesion severity. Statistical differences did not occur between the evaluated genotype groups. Pigs inoculated with PCV2a, PCV2b, or PCV2d had a significantly (P<0.05) higher levels of PCV2 loads in blood and lymph node compared to pigs inoculated with PCV2e. The results of this study indicated that the PCV2a, PCV2b, and PCV2d are more virulent than PCV2e based on blood and lymphoid viral load of PCV2.

Identifying mutations conferring resistance to Sofosbuvir in DAA naive Genotype 3a isolates from HCV-infected patients in Kolkata, West Bengal

Identifying mutations conferring resistance to Sofosbuvir in DAA naive Genotype 3a isolates from HCV-infected patients in Kolkata, West Bengal

NS5A Resistance Associated Mutations to Daclatasvir in Hepatitis C Virus Genotype 3a Treatment Naive and SOF/DCV Treatment Failure Patients

NS5A Resistance Associated Mutations to Daclatasvir in Hepatitis C Virus Genotype 3a Treatment Naive and SOF/DCV Treatment Failure Patients

Recurrence of hepatitis C virus after treatment with pegylated interferon and direct acting antivirals in Punjab Pakistan.

Although increased response rates concomitant in hepatitis C virus but relapse after treatment is threatened. Therefore, it is terrible requirement to evaluate the response of Pegylated interferon and direct acting antivirals in Punjab Pakistan. The study was conducted to find the rate of recurrence of HCV infection after treatment with Pegylated Interferon and Direct Acting Antivirals in Punjab Pakistan. This study was conducted at Department of Pathology, Nawaz Sharif Medical College Gujrat, while treatment effects monitored in different Government and Private Hospitals of Punjab, Pakistan. Total 973 patients who administered the recommended dose and divided in two groups (i) Interferon based therapy (ii) direct acting antivirals (DAAs).Other parameters like ALT and viral load studied. The rate of recurrence was higher in female infected with genotype 2b and in male with mixed genotype 3a/2b after six month of antiviral therapy. Genotype 3a showed significant response to therapy after three month. 32 among 374 (8.5%) were positive after 24 weeks of treatment with interferon, 29 (7.7%) patients have same genotype while 3 patients were re-infected with different HCV strains. With DAAs, only 27 (4.8%) patients were positive among 558 after 2 weeks and one patient re-infected with different genotype. Early and sustained virological response noted in DAAs. ALT and viral load decreased faster with DAAs that not achieved after 4 weeks with pegylated interferon. Sustained virological response appears in DAAs and recurrence rate is high in interferon therapy compared to DAAs. Therefore, reinfection has implications for correct treatment efficiency and to select strategies for retreatment cases.

Prevalence of active HCV infection and genotypic distribution among the general population of district Mardan, Pakistan.

Hepatitis C virus (HCV) is the serious global public health burden of liver disease. Approximately 170 million people in the world are infected with (HCV). In Pakistan, where the disease has high occurrence rate. The present study envisages an up-to-date prevalence of HCV and genotypic distribution in the general population of Mardan District, Khyber Pakhtunkhwa (KP), Pakistan. The blood samples from 6,538 individuals including 3,263 males and 3,275 females were analyzed for hepatitis C surface antigen by Immuno-chromatographic test (ICT), Enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (PCR). It was found that 396 (12.13%) out of 3263 individuals contained antibodies in their blood against HCV, while among the different age groups, the highest incidences of HCV antibodies were found in the 31-40 age group (11.01%). The ICT positive samples were further screened by nested PCR to determine the existence of active HCV-RNA. It was identified that 7.11% (3263) of the total population (6538) tested was positive, among which the 461 (14.07%) females possessed antibodies in their blood against HCV. Our data showed total HCV infection in the investigated population was 5.78%. Higher percentage of HCV prevalence was detected in males than females in the age group 31-40 and 41-50. To compare the prevalence of HCV genotypes age-wise in male and female genotype 3a was found most prevalent genotype followed by 1a, 2a and 3b, respectively.

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

Methanolic Extract of Neem Plant Inhibits NS3 and NS5A Nonstructural Proteins of HCV 3A Genotype

Methanolic Extract of Neem Plant Inhibits NS3 and NS5A Nonstructural Proteins of HCV 3A Genotype

Studies of the symmetric binding mode of daclatasvir and analogs using a new homology model of HCV NS5A GT-4a

ContextEgypt has a high prevalence of the hepatitis C virus (HCV) genotype 4a (GT-4a). Unfortunately, the high resistance it exhibited still was not given the deserved attention in the scientific community. There is currently no consensus on the NS5A binding site because the crystal structure of HCV NS5A has not been resolved. The prediction of the binding modes of direct-acting antivirals (DAA) with the NS5A is a point of controversy due to the fact that several research groups presented different interaction models to elucidate the NS5A binding site. Consequently, a 3D model of HCV NS5A GT-4a was constructed and evaluated using molecular dynamics (MD) simulations. The generated model implies an intriguing new orientation of the AH relative to domain I. Additionally, the probable binding modes of marketed NS5A inhibitors were explored. MD simulations validated the stability of the predicted protein–ligand complexes. The suggested model predicts that daclatasvir and similar drugs bind symmetrically to HCV NS5A GT-4a. This will allow for the development of new NS5A-directed drugs, which may result in reduced resistance and/or a wider range of effectiveness against HCV.MethodsThe 3D model of HCV NS5A GT-4a was constructed using the comparative modeling approach of the web-based application Robetta. Its stability was tested with 200-ns MD simulations using the Desmond package of Schrodinger. The OPLS2005 force field was assigned for minimization, and the RMSD, RMSF, and rGyr were tracked throughout the MD simulations. Fpocket was used to identify druggable protein pockets (cavities) over the simulation trajectories. The binding modes of marketed NS5A inhibitors were then generated and refined with the aid of docking predictions made by FRED and AutoDock Vina. The stability of these drugs in complex with GT-4a was investigated by using energetic and structural analyses over MD simulations. The Prime MM-GBSA (molecular mechanics/generalized Born surface area) method was used as a validation tool after the docking stage and for the averaged clusters after the MD simulation stage. We utilized PyMOL and VMD to visualize the data.

Transient expression and purification of S2 protein from porcine epidemic diarrhea virus in plants

Porcine epidemic diarrhea (PED), caused by the Porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious disease of pigs of all ages, especially piglets under one week old, with the mortality rate reaching 95–100%. Developing an effective vaccine against PEDV in Vietnam is urgent. Spike protein containing S1 and S2 subunits is considered the main target for vaccine development, and the S2 subunit contains immunodominant neutralizing epitopes of PEDV. To date, the expression of S2 protein in plants has not been reported and evaluated. In this study, the gene encoding the S2 subunit of a PEDV strain belonging to genotype 2a was amplified, sequenced, and inserted in a pRTRA vector containing the GCN4pII (pII) motif. The plant expression cassette containing S2-pII was then inserted into the pCB301 vector. The Agrobacterium tumefaciens harboring the pCB301-S2-pII vector was transformed into Nicotiana benthamiana leaves via agroinfiltration. The accumulation level of S2-pII protein in tobacco leaves was semi-quantified by Western blot, accounting for approximately 86.7 mg/kg of fresh leaves and 1.47% total soluble protein, which was 294-fold higher than the accumulation level of S1-pII protein in our previous publication. S2-pII protein was successfully purified by immobilized metal affinity chromatography (IMAC). The oligomeric state of S2-pII protein was characterized by size exclusion chromatography (SEC). The S2-pII protein was determined to be a multimer protein with a high molecular weight. These results are the basis for more extended studies to develop a plant-based S2 vaccine against PEDV infection.

Production and physicochemical characterization of genotypes of Eugenia uniflora L.

Pitanga (Eugenia uniflora L.) is an exotic fruit species of significant economic importance. However, due to genetic variability, its exploitation is hampered by the lack of homogeneous fruit production. In this scenario, this study aimed to select pitanga genotypes according to the physical and physicochemical parameters of fruits grown under semi-arid conditions. The study was developed at the Federal Rural University of the Semi-Arid Region with genotypes resulting from the open pollination of the pitanga variety ‘Tropicana”. Thirty-nine pitanga genotypes were evaluated for fruit mass, fruit length, fruit diameter, soluble solids (SS), titratable acidity (TA), ascorbic acid (AA), pH, and SS/TA ratio. The pitanga genotypes showed high variability. The clustering method separated the genotypes according to desirable traits. Genotype A12 showed the largest fruit sizes, whereas genotype A8 showed the highest SS and TA contents. Genotypes A2, A13, A34, and A39 showed fruits with the highest AT values. On the other hand, genotypes A11, A16, A45, A9, A26, and A44 showed the most significant contents of pH and SS/TA. Highlights The grouping of two genotypes depends on the environmental conditions, mainly on the effect of two genotypes per year. The analysis of principal components allows selecting the genotypes based on their desired characteristics. The physical and chemical composition of two pitanga fruits are affected by climatic conditions, genotypes and years of cultivation.

Second hepatitis C virus transmission by blood components since introduction of mandatory NAT screening in Germany.

Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999. When a repeat donor who had tested negative for anti-HCV tested positive for HCV RNA by NAT in a minipool (MP) of eight, a look-back procedure was initiated. Qualitative, quantitative and genotyping assays were used to investigate the titers of the quarantined fresh frozen plasma (FFP) from the donor and a serum sample from the recipient of the pooled platelet concentrate (PPC). Amplified products of 5'UTR and HVR1 were used for sequence comparison to characterize the HCV genomic identity of donor and recipient samples. All NAT tests utilized in this procedure were able to detect a low HCV RNA titer (~15 IU/ml) in the FFP from the donation. Dilution of FFP by factor 8 was performed to mimic an MP, and the detection rate correlated well with the claimed sensitivity of the tests. Analysis of donor and recipient samples revealed genotype 3a viral transmission confirmed by sequence analysis. This TT HCV case could have been prevented by individual donation (ID) NAT. However, a low titer blood donation in the window period (WP) is very rare. Residual risk calculation for TT HCV in the WP revealed that, compared to MP-NAT testing, ID-NAT would improve blood safety only marginally.

Diagnostic markers of hepatic steatosis in patients with chronic viral hepatitis C

Objective was to conduct a comparative analysis of routine clinical and laboratory parameters in two groups of patients with chronic viral hepatitis C (CHC) depending on the presence or absence of hepatic steatosis.Methods and materials. The observational retrospective cross-sectional study included 411 patients with CHC, of which 43.80 % were those with hepatic steatosis. The studied parameters were routine clinical and laboratory parameters, the viral genotype, as well as formalized results of a liver biopsy and ultrasound examination of the abdominal organs. To assess the differences between the two groups, the following were calculated: Pearson’s test, two-tailed Fisher’s test, Wilcoxon rank sum test, Spearman’s correlation coefficient, and partial correlation coefficient.Results. It was found that CHC patients with hepatic steatosis were older and had a higher body mass index (BMI) than patients without hepatic steatosis (p&lt;0.01). Among patients with hepatic steatosis, the 3a genotype of the virus (p&lt;0.001) and advanced stages of liver fibrosis (p&lt;0.01) were more often detected, higher concentrations of hepatic transaminase (p£0.001) and iron (p&lt;0 .01) in blood serum were observed, as well as higher liver (p&lt;0.001) and pancreas (p&lt;0.001) sizes on ultrasound than among patients without hepatic steatosis.Conclusion. Age, BMI, viral genotype, stage of liver fibrosis, ALT, AST, GGT, and iron levels, as well as liver and pancreas size are potential predictors of the presence of hepatic steatosis in CHC patients.

Chronic hepatitis E in an elderly immunocompetent patient who achieved a sustained virologic response with ribavirin treatment.

A woman in her late 70s was diagnosed with liver injury at a health examination. Despite treatment with ursodeoxycholic acid at a nearby hospital, her transaminase levels elevated in two peaks. She was transferred to our hospital 77days after the health examination. She weighed 42kg and had a low body mass index of 19.8kg/m2. Viral markers, including immunoglobulin A (IgA) against hepatitis E virus (anti-HEV IgA), were negative. Drug-induced liver injury was negligible. We suspected autoimmune hepatitis because of the patient's female gender and positive antinuclear antibody. However, prednisolone and azathioprine failed to completely improve her hepatitis. On day 643, anti-HEV IgA was re-evaluated and found to be positive. She was diagnosed with autochthonous chronic hepatitis E because the virus strains in the preserved serum on day 77 and the serum on day 643 had identical nucleotide sequences (genotype 3a). Following prednisolone and azathioprine discontinuation, ribavirin (RBV) was administered for 3months. HEV RNA disappeared and remained negative for more than 6months after the cessation of RBV. The HEV RNA titer of 6.2 log10 copies/mL on day 77 was unusually high 2.5months after the onset, suggesting that hepatitis E had already been chronic before immunosuppressive treatment for possible autoimmune hepatitis. After getting married at 23years old, she had been a housewife and had no comorbidities that might deteriorate her immunity. Chronicity should be kept in mind when encountering HEV infection in elderly and underweight patients.

Isolation of BVDV-1a, 1m, and 1v strains from diarrheal calf in china and identification of its genome sequence and cattle virulence.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5'-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization.

Hepatitis C virus (HCV) genotype 4 is highly prevalent in the Middle East and parts of Africa. Subtype 4d has recently spread among high-risk groups in Europe. However, 4d infectious culture systems are not available, hampering studies of drugs, as well as neutralizing antibodies relevant for HCV vaccine development. We determined the consensus 4d sequence from a chronic hepatitis C patient by next-generation sequencing, generated a full-length clone thereof (pDH13), and demonstrated that pDH13 RNA-transcripts were viable in the human-liver chimeric mouse model, but not in Huh7.5 cells. However, a JFH1-based DH13 Core-NS5A 4d clone encoding A1671S, T1785V, and D2411G was viable in Huh7.5 cells, with efficient growth after inclusion of 10 additional substitutions [4d(C5A)-13m]. The efficacies of NS3/4A protease- and NS5A- inhibitors against genotypes 4a and 4d were similar, except for ledipasvir, which is less potent against 4d. Compared to 4a, the 4d(C5A)-13m virus was more sensitive to neutralizing monoclonal antibodies AR3A and AR5A, as well as 4a and 4d patient plasma antibodies. In conclusion, we developed the first genotype 4d infectious culture system enabling DAA efficacy testing and antibody neutralization assessment critical to optimization of DAA treatments in the clinic and for vaccine design to combat the HCV epidemic.

Identification of NS5B resistance against SOFOSBUVIR in hepatitis C virus genotype 3a, naive and treated patients.

Pakistan has the second highest prevalence of HCV with genotype 3a (GT-3a) being the most frequently circulating genotype. Currently, resistance-associated substitutions (RASs) are a major challenge in HCV treatment with direct-acting antivirals (DAAs). Sofosbuvir (SOF) is an FDA-approved NS5B nucleotide inhibitor. The aim of this study was to identify these RASs in the NS5B gene in naive and treated Pakistani HCV 3a isolates against SOF. Blood samples were collected from anti-HCV-positive patients, followed by HCV RNA isolation and real-time PCR quantification. HCV-positive patients were processed for HCV RNA genotyping, patients with genotype 3a were processed for NS5B gene amplification and sequencing. GT-3a was the most prevalent genotype (62.2%). S282T was identified in 2 (8.7%) patients, C316Y/G/R in 3 (13%), V321A and L320P in 1 (4.3%) each in SOF/RBV-resistant patients. Variants of S282 were detected in 3 (13%) of SOF/RBV-treated patients. While INF/RBV-associated mutations were also analysed, D244N, A333R and A334E were identified in 2 (9.5%), 3 (14.2%) and 7 (33.3%) in treatment-naive and 15 (65.2%), 7 (30.4%) and 5 (21.7%) treated patients, respectively. Q309R was observed only in one treatment-experienced patients. Some substitutions were present at higher frequency in both groups like N307G, K304R, A272D and R345H, considered that they do not have any role in sofosbuvir resistance. It was concluded that sofosbuvir RASs are present in Pakistani HCV GT-3a isolates, and they should be monitored carefully, especially in treatment-experienced patients, for further selection of treatment regimens. HCV RASs have been studied very well across the world but there is scarcity of data regarding this topic in Pakistani population, this study provides data regarding the prevalence of these RASs in Pakistani HCV isolates emphasizing the fact that these RASs must be carefully monitored before starting HCV treatment, especially in treatment failure patients.

Virulence Comparison of Four Porcine Circovirus Type 2 (PCV2) Genotypes (2a, 2b, 2d and 2e) in Pigs Single-Infected with PCV2 and Pigs Dual-Infected with PCV2 and Mycoplasma hyopneumoniae

The objective of this study was to compare the virulence of four porcine circovirus type 2 (PCV2) genotypes (2a, 2b, 2d and 2e). Pigs were infected with one of these four genotypes. Pigs were also dually infected with Mycoplasma hyopneumoniae and one of the four PCV2 genotypes. Virulence was determined based on the amount of PCV2 loads in the blood and lymph nodes and the severity of lymphoid lesions. Marked differences in virulence were found among the four genotypes. Within the single infection model, PCV2a, PCV2b and PCV2d were more virulent than PCV2e, while significant differences in virulence were not found among the PCV2a, PCV2b and PCV2d groups. Within the dual infection model, PCV2d was more virulent than the other three PCV2 genotypes. Mycoplasma hyopneumoniae potentiated the severity of PCV2-associated lymphoid lesions and increased the amount of PCV2 loads in the blood and lymph nodes, regardless of the PCV2 genotype. By contrast, PCV2 was not able to potentiate the severity of mycoplasmal-induced lung lesions or the level of M. hyopneumoniae laryngeal load. The results of this study demonstrate that PCV2d is of major clinical importance, while PCV2e is of minor clinical importance.

Repurposing Novel Antagonists for Targeting p7 Viroporin of HCV Using In Silico Approach

Background: P7 viroporin in HCV is a cation-selective ion channel-forming protein, functional in the oligomeric form. It is considered to be a potential target for anti-HCV compounds due to its crucial role in viral entry, assembly, and release. Method: Conserved crucial residues present in HCV p7 protein were delineated from the available literature with a specific focus on the genotypes 3a and 1b prevalent in India. Using the Flex-X docking tool, a library of FDA-approved drugs was docked on the receptor sites prepared around crucial residues. In the present study, we proposed drug repurposing to target viroporin p7, which may help in the rapid development of effective anti-HCV therapies. Results: With our approach of poly-pharmacology, a variety of drugs currently identified as antibiotics, antiparasitic, antiemetic, anti-retroviral, and anti-neoplastic were found to dock successfully on the p7 viroporin. Noteworthy among these are general-purpose cephalosporin antibiotics, leucal, phthalylsulfathiazole, and granisetron, which may be useful in acute HCV infection, and anti-neoplastic sorafenib and nilotinib, which may be valuable in advanced HCV-HCC cases. Conclusion: This study could pave the way for quick repurposing of these compounds as anti-HCV therapeutics.