We reported earlier that B95a, an Epstein-Barr virustransformed marmoset B lymphoblastoid cell line, is more susceptible to infection with measles virus than other cells. The cell line also was found to be susceptible to infection with the lapinized Nakamura III (L) strain of rinderpest virus and various strains derived from it. The B95a cell line was therefore the only host cell system available for the propagation and quantification of the L strain. In contrast to the adaptation of the L strain to Vero cells which results in a diminution of virulence in rabbits, the propagation of the virus in B95a cells preserved the virulence and some other properties in rabbits. Furthermore, when Vero cell-adapted variants of the L strain with diminished virulence were serially passaged in B95a cells, virulence in rabbits was gradually regained.

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors. Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

We have reported a novel bovine rotavirus, the AzuK-1 (G21P [29]) strain, isolated from an asymptomatic calf. We isolated another bovine rotavirus, the Dai-10 strain, bearing new G24P [33] genotypes, assigned by the Rotavirus Classification Working Group (RCWG), from an asymptomatic cow in Hyogo Prefecture, Japan in 2007. To gain an insight into the origins and evolution of these strains, we determined the complete ORF sequences of all 11 genes of the two strains. The NSP3 genes of both strains were confirmed to belong to a new NSP3 genotype, T9, by the RCWG. Genotype determination of AzuK-1 and Dai-10 strains revealed that eight gene segments of both strains possessed genotypes typically observed in bovine rotaviruses, with the exception of VP4, VP7 and NSP3 gene segments. Unexpectedly, phylogenetic analyses showed that VP6 and NSP2 gene segments of the AzuK-1 and Dai-10 strains were clustered with those of simian or canine/feline rotaviruses, rather than with those of bovine rotaviruses. These findings indicate the possibility that both strains originated by interspecies transmission and multiple reassortment events involving bovine, simian and canine/feline rotaviruses, resulting in the introduction of some genes into the genetic background of bovine rotaviruses. INTRODUCTIONGroup A rotaviruses are the major pathogens causing acute gastroenteritis in infants and a wide range of animals, including birds. Rotavirus-induced diarrhoea is a serious public health problem worldwide, responsible for more than 600 000 child deaths each year (Parashar et al., 2006). Likewise, in domestic animals, rotavirus-induced diarrhoea is a major problem causing significant economic losses (Dhama et al., 2009;Martella et al., 2010).Rotaviruses are members of the family Reoviridae. Rotaviruses possess a genome of 11 segments of dsRNA, which encode six viral structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6). Because of the segmented nature of the genome, a reassortment event can occur in cells co-infected with two or more different strains (Estes & Kapikian, 2007;Palombo, 2002;Ramig, 1997). The rotavirus virion is a triple-layered icosahedral particle. The outer capsid is composed of VP7 and VP4. They elicit neutralizing antibodies independently. In a dual classification system, rotaviruses are classified into 24 G genotypes and 32 P genotypes based on the nucleotide sequences of VP7 and VP4 genes, respectively (Collins et al., 2010; Esona et al., 2010;Matthijnssens et al., 2006Matthijnssens et al., , 2008a Schumann et al., 2009; Solberg et al., 2009;Ursu et al., 2009). Recently, a new classification system has been established using nucleotide sequences of all of the 11 genomic RNA segments by the Rotavirus Classification Working Group (RCWG) (Matthijnssens et al., 2008b). In this system, the The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB513836-AB513838 and AB573070-AB573086.Supplementary material is available with the online version of this paper. , 2...

The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.The host immune response to viral infection is a key factor in defining viral pathogenicity and the outcome of the infection. This depends not only on the capacity of the host to mount an innate and/or adaptive immune response against the virus but also on the ability of the virus to evade/subvert this response (22).The principal response of host cells to viral infection is the production of type I interferons (IFNs) (including alpha interferon [IFN-␣] and IFN-␤), which, on binding to IFN receptors on the cell surface, activate the JAK/STAT intracellular signaling pathway that culminates in the phosphorylation, heterodimerization, and nuclear translocation of the transcription factors signal transducer and activator of transcription 1 (STAT1) and STAT2. In the context of a complex called IFNstimulated gene factor 3 (ISGF3), the activated STATs bind to promoters in the DNA that contain an IFN-stimulated response element (ISRE) sequence, resulting in the transcription of a plethora of IFN-stimulated genes (ISGs) encoding antiviral proteins which act to establish the antiviral state in cells (reviewed in reference 22).To propagate efficiently in host cells, viruses have had to evolve multiple strategies to dampen the host IFN system, which appear to involve the expression of viral proteins with IFN antagonist functions. These IFN antagonists are reported to exert their effect by a variety of mechanisms, reflecting the diversity of host antiviral responses, but the STATs are known as common targ...

These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.