RESUMEN
Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.
Asunto(s)
Flaviviridae/genética , Flaviviridae/metabolismo , Ingeniería de Proteínas/métodos , Animales , Línea Celular , Flaviviridae/patogenicidad , Infecciones por Flaviviridae/metabolismo , Genes Reporteros/genética , Genes Virales/genética , Células HEK293 , Humanos , Ratones/virología , Proteómica/métodos , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Porcinos/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Classical swine fever (CSF), a highly contagious viral disease affecting domestic and wild pigs in many developing countries, is now considered endemic in Mongolia, with 14 recent outbreaks in 2007, 2008, 2011, 2012, 2014, and 2015. For the first time, CSF viruses isolated from these 14 outbreaks were analyzed to assess their molecular epidemiology and pathogenicity in pigs. Based on the nucleotide sequences of their 5'-untranslated region, isolates were phylogenetically classified as either sub-genotypes 2.1b or 2.2, and the 2014 and 2015 isolates, which were classified as 2.1b, were closely related to isolates from China and Korea. In addition, at least three different viruses classified as 2.1b circulated in Mongolia. Experimental infection of the representative isolate in 2014 demonstrated moderate pathogenicity in 4-week-old pigs, with relatively mild clinical signs. Understanding the diversity of circulating CSF viruses gleans insight into disease dynamics and evolution, and may inform the design of effective CSF control strategies in Mongolia.
Asunto(s)
Virus de la Fiebre Porcina Clásica/clasificación , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Peste Porcina Clásica/epidemiología , Peste Porcina Clásica/virología , Epidemiología Molecular , Enfermedades de los Porcinos/virología , Virulencia/genética , Animales , Secuencia de Bases , Línea Celular , China , Peste Porcina Clásica/patología , Peste Porcina Clásica/fisiopatología , Modelos Animales de Enfermedad , Brotes de Enfermedades , Genotipo , Mongolia/epidemiología , Filogenia , República de Corea , Sus scrofa/virología , PorcinosRESUMEN
Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.
Asunto(s)
Antígenos Virales/análisis , Antígenos Virales/genética , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Animales , Aves , Análisis por Conglomerados , Heces/virología , Variación Genética , Japón , Datos de Secuencia Molecular , Mongolia , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.