Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag.

The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses.IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses.

Avaliação da Atividade Antiviral de Extratos Vegetais e de Fungos contra Dengue Virus

Produtos naturais são potenciais fontes alternativas para o desenvolvimento de antivirais para o tratamento da dengue, assim como de outras doenças causadas por vírus da família Flaviviridae ou mesmo para um amplo espectro de viroses. Neste estudo foi feita a triagem da atividade in vitro contra o Dengue virus 2 (DENV-2) de 3101 extratos, provenientes de plantas e de fungos da Coleção de Amostras para Bioensaios da Fiocruz. Para tal, células BHK-21 foram infectadas com DENV-2 e tratadas simultaneamente com 25 μg/mL de extrato sendo o resultado analisado por dois métodos: observação do grau de inibição do efeito citopático (ECP) por microscopia óptica e análise da viabilidade celular pelo ensaio colorimétrico do MTT. Dentre os 3101 extratos testados, 115 extratos apresentaram atividade antiviral contra DENV-2 e foram selecionados para a determinação da respectiva concentração efetiva 50 (CE50).

Cinquenta e cinco destes extratos foram obtidos de plantas pertencentes a 20 famílias distintas: Amaryllidaceae (3), Annonaceae (1), Asteraceae (5), Begoniaceae (1), Clusiaceae (1), Combretaceae (1), Erythroxylaceae (1), Fabaceae (4), Lythraceae (2), Malpighiaceae (8), Malvaceae(1), Melastomataceae (2), Melochia (1), Myrtaceae (3), Rubiaceae (8), Sapindaceae(9), Ochnaceae (1), Primulaceae (1) Vitaceae (1), Vochysiaceae (1). Os demais extratos (60) foram obtidos de culturas de fungos endofíticos coletados no Brasil, no continente Antártico e no Deserto do Atacama, ainda não identificados. Até o momento, os extratos vegetais mais promissores foram obtidos de plantas da família Amaryllidaceae (IS = 32,15) e da família Fabaceae (IS = 20,47) e (IS = 24,47). Vinte extratos fúngicos apresentaram valores de CE50 que variaram entre 3,1 a 12,5 μg/mL e sem citotoxicidade aparente até a concentração de 100 μg/mL. Nossos resultados mostram que tais plantas e fungos são fontes promissoras de substâncias com ação antiviral contra DENV.

Design, synthesis and anti flaviviridae activity of N(6)-, 5',3'-O- and 5',2'-O-substituted adenine nucleoside analogs.

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)>100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.

Epidemiological and clinical aspects of hepatitis G virus infection in blood donors and immunocompromised recipients of HGV-contaminated blood.

BACKGROUND AND OBJECTIVES: The infectiousness and clinical relevance of the newly discovered blood-borne Flaviviridae-like agent, termed hepatitis G virus (HGV), are not well understood. MATERIALS AND METHODS: Twenty-three transfusion recipients of two HGV-affected long-term blood donors were studied for HGV genome and antibodies to the putative envelope 2 glycoprotein (anti-E2) of HGV. Nine recipients had nonhematological disorders and 14 suffered from severe hematological diseases and 7 of them received allogeneic bone marrow or blood stem cell transplantation. The molecular epidemiology of the observed HGV infection was studied by direct sequencing of parts of the 5'-noncoding region, NS3, and NS5 region of HGV in the 2 long-term donors and in their 6 recipients who became HGV RNA positive. Additionally, 549 individuals-homologous (n = 254) and autologous blood donors (n = 202), and medical staff (n = 89)--were investigated for the presence of HGV RNA. RESULTS: HGV RNA in serum was found in 15 of the 23 (65%) transfusion recipients with known exposure of HGV-contaminated blood. Seven of the remaining 8 recipients showed only an anti-E2 response, indicating previous HGV infection with spontaneous clearance of the virus. In one recipient neither HGV RNA nor anti-E2 could be detected. Molecular evidence for HGV transmission by the 2 donors was found in 3 of the 6 recipients studied. The alanine aminotransferase levels were not significantly different in the HGV RNA positive and negative recipients, and none of the 23 recipients developed posttransfusion hepatitis. Persistent HGV infection was observed especially in recipients with severe hematological disorders or in those in whom intensive immunosuppressive treatment was necessary. Of the 549 individuals studied, 10 (1.8%) were healthy carriers of HGV RNA. CONCLUSION: The persistence of transfusion-acquired HGV infection is not associated with acute or chronic hepatitis, but may be influenced by the recipient's underlying disease.

Expression and characterization of the hepatitis G virus helicase.

The hepatitis G virus (HGV) is a new member of the Flaviviridae family and has a genomic organization similar to that of hepatitis C virus (HCV). Protein sequence motifs are present suggesting that HGV encodes a serine proteinase, an RNA-dependent RNA polymerase and a helicase. We have cloned and expressed the putative helicase of HGV and have shown that it contains a poly (U)-stimulated NTPase activity and is able to function as a DNA helicase. Preliminary characterization of the HGV helicase activity reveals similarities with other members of the Flaviviridae, but especially with HCV, raising the possibility that HGV could be used as a surrogate virus for the development of therapies against HCV.

Past and present hepatitis G virus infections in areas where hepatitis C is highly endemic and those where it is not endemic.

We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5' noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (P < 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (P < 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (P < 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.

GB virus C infection by blood transfusion during and following liver surgery.

BACKGROUND/AIMS: GB virus C has been associated with some cases of fulminant hepatitis and post-transfusion hepatitis. We examined transfusion-related infection with this virus. METHODOLOGY: Of 150 patients undergoing liver resection, 108 received a homologous blood transfusion. Serum samples before and after surgery were examined for GB virus C RNA by a nested polymerase chain reaction with reverse transcription. We also studied the post-operative course of patients infected with GB virus C by blood transfusion. RESULTS: Viral RNA was detected in sera taken after transfusion in 4 (4%) patients receiving homologous transfusions. Viral RNA was not detected after surgery in the 42 patients given autologous transfusions or not receiving homologous blood. Post-operative courses in the 4 infected patients were uneventful. CONCLUSIONS: As 4% of homologous transfusions resulted in GB virus C infection in our small surgical study, autologous transfusion is recommended when circumstances permit.

Prevalence and risk factor analysis of GBV-C/HGV infection in prostitutes.

GB virus-C (GBV-C) and Hepatitis G virus (HGV) are variants of a recently cloned virus transmitted parenterally. It is unclear if sexual contact also transmits this virus. In this study, we detected serum GBV-C/HGV RNA in 140 prostitutes by reverse transcription polymerase chain reaction (RT-PCR) using different primers. Thirty (21%) were found with GBV-C RNA by nested PCR although only 22 (73%) had HGV RNA by single round RT-PCR. Both assays had a nearly perfect agreement (kappa value, 0.812). The prevalence of GBV-C RNA in prostitutes was significantly higher than the control group (30/140 vs. 2/40, P < 0.02). Multivariate analysis revealed that a frequency of paid sex more than 120 times per month was the only factor significantly associated with positive GBV-C RNA in prostitutes (P < 0.003). In summary, prostitutes are a high risk group and reservoir of GBV-C/HGV infection due to high frequency of paid-sex.