Project description:Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E-like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.
Project description:The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related.
Project description:BackgroundHepatitis C virus (HCV) is one of the leading causes of chronic liver disease. Seven genotypes and more than 80 subtypes have been identified for HCV so far. To date, 10 subtypes (3a to 3i; and 3k) of HCV genotype 3 have been identified. In 2006, two HCV isolates were reported from Iran that belonged to a new subtype of genotype 3. However, considering the consensus proposal for HCV genotype nomenclature, the available sequences of the new subtype did not correspond to the regions that are required to be analyzed prior to subtype assignment. During a study on the molecular epidemiology of HCV in Iran, an HCV isolate (FSM165) which seemed to belong to a new subtype of genotype 3 was obtained from a patient residing in Tehran, Iran.ObjectivesThe aim of this study was to assess the relatedness of isolate FM165 together with several sequences retrieved from the database to the new HCV-3 subtype reported from Iran in 2006.Materials and methodsVarious parts of the genome including the core/E1 region and two segments of the NS5B region were amplified and sequenced for isolate FSM165. Furthermore, using the Basic Local Alignment Search Tool (BLAST), the HCV database was searched for sequences that had a high level of similarity with sequences of FSM165 isolate and such sequences were retrieved from the database. To investigate the relatedness of isolate FSM165 and also the retrieved sequences to a new HCV-3 subtype reported previously, phylogenetic analyses were performed using the Kimura two-parameter model and the neighbor joining method.ResultsPhylogenetic analysis of the partial NS5B region demonstrated the relatedness of isolate FSM165 to the new subtype reported from Iran in 2006. Moreover, some core/E1 and NS5B sequences that had a high level of similarity with FSM165 isolate were found through searching the HCV database. These sequences were previously either misclassified or could not be accurately classified. Phylogenetic analyses showed that all of the described sequences belonged to the new subtype of HCV genotype 3.ConclusionsData suggests that the new subtype has a vast geographical distribution in Iran. The core/E1 and the NS5B sequences described in this paper can be used as references for the new HCV-3 subtype in future studies.
Project description:Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.