RESUMO
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.
Assuntos
Flaviviridae/genética , Expressão Gênica , Genes Reporter , Recombinação Genética , Animais , Antivirais/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Flaviviridae/efeitos dos fármacos , Genoma Viral , Hepacivirus/genética , Humanos , Camundongos , Mutagênese InsercionalRESUMO
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.
Assuntos
Doadores de Sangue , Flaviviridae , Anticorpos Anti-Hepatite/sangue , Hepatite Viral Humana/epidemiologia , RNA Viral/sangue , Reação Transfusional , Proteínas do Envelope Viral/imunologia , Adulto , Alanina Transaminase/sangue , Sequência de Bases , Transfusão de Sangue Autóloga , Feminino , Flaviviridae/genética , Flaviviridae/isolamento & purificação , Alemanha/epidemiologia , Pessoal de Saúde , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/complicações , Hepatite Viral Humana/sangue , Hepatite Viral Humana/prevenção & controle , Hepatite Viral Humana/transmissão , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Prevalência , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
DNA Helicases/genética , Flaviviridae/genética , Hepatite Viral Humana/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cromatografia em Camada Fina , Clonagem Molecular , DNA/análise , DNA/metabolismo , DNA Helicases/metabolismo , DNA Complementar/análise , DNA Complementar/genética , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/metabolismo , Expressão Gênica , Hepacivirus/genética , Humanos , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
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.