HEPATITIS C VIRUS AND ITS GENE STRUCTURE AND FUNCTION

Hepatitis C virus (HCV) is a member of the family Flaviviridae, and the principal cause of parenteral non A non B hepatitis. HCV is a single stranded, positive sense RNA virus whose genome contains a single open reading frame of about 10 kb in length which encodes a polyprotein of about 3000 amino acids. HCV is responsible for majority of the transfusion associated cases of hepatitis and a significant proportion of community-acquired hepatitis which leads to a range of clinical manifestations from an apparent carrier state to severe chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Cell-free translation and cell culture transient expression studies revealed that HCV genome initially transcribed as a polyprotein is processed by cellular and viral proteases to produce the putative viral structural and nonstructural proteins. The preliminary map of the gene order for HCV was established: 5\u27 untranslated region (UTR)-core (C)-envelope 1 (E1)-E2-nonstructural 2(NS2)-NS3-NS4A-NS4B-NS5A-NS5B-UTR3\u27. Nonstructural proteins, NS2, NS3 and NS5, are believed to be a component of viral coded enzymes. Mutations in hypervariable region 1 (HVR1) of the E2 protein was considered to be a possible mechanism of chronicity of HCV infection. HCV infection can be monitored either by serodiagnosis or by genodiagnosis. Interferon seems to be considered as the sole drug for HCV treatment until now. In the history of virology, HCV was the first example that was identified by molecular cloning of its genome from infectious materials

An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses

We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection

Whole-genome analysis of a Vibrio cholerae O1 biotype classical strain isolated in 1946 in Sasebo city, Nagasaki prefecture, from a returnee from the northeast part of China

Background: Cholera is a water-borne disease caused by toxigenic Vibrio cholerae serogroups O1 and O139. Not a few studies on the whole-genome analyses of V. cholerae O1 biotype El Tor have been published; however, the number of analyses for biotype classical is limited. The whole-genome analysis was made on a V. cholerae biotype classical strain, Man9, isolated in 1946 in Sasebo city, Nagasaki prefecture, from a returnee from the northeast part of China.Methods: PacBio RSII was used to determine the whole-genome of Man9. De novo assemblies were made with CLC Genomics Workbench 8.5.1 and Canu. 2.0 and annotated by Prokka version 1.12. Upon determining the configuration of the CTX prophage region, combined procedures of PCR, RFLP with Southern blotting, and Sanger sequencing method were used. The phylogenetic tree was constructed by RaxML and visualized by Phandango. The identification of Cas genes and spacer sequences was made by CRISPR-finder and NCBI Blast search. These data were compared with those of V. cholerae serogroup O1 biotype classical O395.Results: The Man9 carried the 2.9 Mb (Chr1) and 1.1 Mb (Chr2) chromosomes with 2683 and 1198 CDSs, respectively. The genome similarity between Man9 and O395 was 97.0% when the total genomes were compared. Man9 carried a 380-kb inversion on the Chr1, and 95-kb and 35-kb fragments were not present on the Chr1 and on the Chr2, respectively. Man9 monophyletically clustered with 23 other biotype classical strains on the core gene phylogenetic tree analyses. Man9 carries “CTXcla” and a stretch of “truncated CTXcla-CTXcla” on the Chr1 and the Chr2, respectively, which is the opposite arrangement of O395. Man9 carries CRISPR–Cas system subtype I-E with 33 spacers, 64% of which were identical to those of O395.Conclusions: Man9 differs from O395 by 3% on the total genome comparison; however, genomic analysis of a strain having circulated in the interpandemic period between the 6th and the 7th cholera pandemic is valuable and contributes to understanding the evolution of pathogenic V. cholerae

Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein

AbstractThe NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3

Association of dengue virus type-specific IgG on platelets is specific for the acute phase in an imported Japanese patient with secondary dengue 2 virus infection

The mechanism of thrombocytopenia in dengue virus infection remains unknown. We report herein an imported case of a 21-year-old male Japanese with dengue fever caused by secondary dengue 2 virus infection. The thrombocytopenia detected around the day of defervescence was associated with an increased level of plateletassociated IgG (PAIgG). The eluate from the platelets during the acute phase of this case contained an increased activity of anti-dengue virus 2 IgG, while the eluate from platelets during the convalescent phase contained a low level of anti-dengue 2 IgG. These findings suggest the transient formation of PAIgG involving anti-dengue 2 virus IgG during the acute phase of secondary dengue 2 virus infection

Genotype Determination of Hepatitis C Virus Strains in Malaysia

Out of 12 patient sera obtained from Malaysia, seven sera (7/12) were found to contain hepatitis C virus (HCV) genome by nested RT-PCR with universal HCV specific primers which were derived from 5\u27 noncoding region (5\u27 NCR). Genotype determination of these 7 Malaysian HCV strains were carried out both by subtype-specific core amplification system (Okamoto et al., 1992 and 1993) and sequencing in the 5\u27 NCR. Four Malaysian strains were major genotype 1 (two of subtype II/1b, one of subtype I/1a and one of co-infection with I/1a + II/1b). The genotypes of 3 remaining strains were determined as major genotype 3 by nucleotide sequences in the 5\u27 NCR. The results showed that HCV major genotype 1, 2 and 3 are common in the world, especially in Asian countries including Malaysia

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection

Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity

Isolation of dengue serotype 3 virus from the cerebrospinal fluid of an encephalitis patient in Hai Phong, Vietnam in 2013

Dengue encephalitis (DE) is characterized as unusual presentation of dengue infection. Despite the reports that DE accounts for only 1-5% of dengue cases, this disease tends to be increasingly reported to threaten global human health throughout dengue endemic areas particularly in Southeast Asia. The molecular information of clinically characterized, neurotropic dengue virus (DENV) in human beings is extremely scarce despite it playing an important role in deciphering the pathogenesis of dengue-related neurological cases. Here we report a case of DE caused by DENV3 genotype III in a male patient with atypical symptoms of DENV infection in Hai Phong, Vietnam in 2013. The virus isolated from the cerebrospinal fluid of this case-patient was closely related to DENV3 genotype III strains isolated from serum of two other patients, who manifested classical dengue in the same year and residing in the same area as the case-patient. It is noteworthy to mention that in 2013, DENV3 genotype III was detected for the first time in Vietnam