. To assess the importance of these topoisomerases in viral lytic replication, shRNA-mediated gene silencing was used. Depletion of Topo I and II severely inhibited viral lytic DNA replication as well as virion production, suggesting essential roles of these cellular proteins in viral DNA replication. The discovery of Topo I and II as enzymes indispensable for KSHV DNA replication raises a possibility that these cellular proteins could be new targets of therapeutic approaches to halt KSHV replication and treat KSHV-associated diseases. In this report, we examined one Topo I inhibitor and several Topo II inhibitors (inclusive of Topo II poison and catalytic inhibitors) as potential therapeutic agents for blocking KSHV replication. The Topo II catalytic inhibitors in general exhibited marked inhibition on KSHV replication and minimal cytotoxicity. In particular, novobiocin, with the best selectivity index (SI ‫؍‬ 31.62) among the inhibitors tested in this study, is effective in inhibiting KSHV DNA replication and virion production but shows little adverse effect on cell proliferation and cycle progression in its therapeutic concentration, suggesting its potential to become an effective and safe drug for the treatment of human diseases associated with KSHV infection.

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.

Noroviruses are an important cause of non-bacterial epidemic gastroenteritis, but no specific antiviral therapies are available. We investigated the inhibitory effect of phosphorodiamidiate morpholino oligomers (PMOs) targeted against norovirus sequences. A panel of peptide-conjugated PMOs (PPMOs) specific for the murine norovirus (MNV) genome was developed, and two PPMO compounds directed against the first AUG of the ORF1 coding sequence near the 5′-end of the genome proved effective in inhibiting MNV replication in cells. A consensus PPMO (designated Noro 1.1), designed to target the corresponding region of several diverse human norovirus genotypes, decreased the efficiency of protein translation in a cell-free luciferase reporter assay and inhibited Norwalk virus protein expression in replicon-bearing cells. Our data suggest that PPMOs directed against the relatively conserved 5′-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses.

The mechanisms of calicivirus attachment and internalization are not well understood, mainly due to the lack of a reliable cell-culture system for most of its members. In this study, rabbit vesivirus (RaV) virions were shown to bind annexin A2 (ANXA2) in a membrane protein fraction from HEK293T cells, using a virus overlay protein-binding assay and matrix-assisted laser desorption/ ionization time-of-flight analysis. A monoclonal anti-ANXA2 antibody and small interfering RNAmediated knockdown of ANXA2 expression in HEK293T cells reduced virus infection significantly, further supporting the role of ANXA2 in RaV attachment and/or internalization. INTRODUCTIONCaliciviruses are relevant pathogens in both veterinary and human medicine against which antiviral strategies are sorely lacking. These viruses cause a wide variety of diseases and symptoms, such as gastroenteritis, vesicular lesions, respiratory infections, reproductive failure and haemorrhagic disease (Green et al., 2000;Hoover & Kahn, 1975;Morens et al., 1979;Thiel & Konig, 1999). The family Caliciviridae comprises non-enveloped virions of 35 nm in diameter, with a single-stranded, positive-sense RNA genome of 7. 4-8.3 kb (Kapikian et al., 1996). This family has four accepted genera, Lagovirus, Norovirus, Sapovirus and Vesivirus (Green et al., 2000), and two putative novel genera, Becovirus (Oliver et al., 2006) and Recovirus (Farkas et al., 2008), which have recently been proposed.Studies on the replication of most caliciviruses have been hampered severely by the lack of a reliable cell-culture system. More recently, cell-culture systems have been developed for some caliciviruses such as porcine enteric calicivirus (Parwani et al., 1991), murine norovirus 1 (Wobus et al., 2004) and human noroviruses (Straub et al., 2007). Human noroviruses can also be recovered from cell cultures using reverse genetics approaches (Asanaka et al., 2005; Guix et al., 2007). In contrast, most of the members of the genus Vesivirus can be propagated readily in cell culture. Rabbit vesivirus (RaV) has been characterized in our laboratory as a putative novel member of the genus Vesivirus. RaV was isolated from young rabbits (Oryctolagus cuniculus) showing signs of diarrhoea and was grown in cell culture (Martín-Alonso et al., 2005).Viral infection usually starts with the binding of virus particles to specific receptor molecules on the host-cell surface. Several cellular factors have been shown to be critical in calicivirus entry, including a2,6-linked sialic acid present on N-linked glycoproteins (Stuart & Brown, 2007) and ABH histo-blood group antigens (Hutson et al., 2002;Marionneau et al., 2002). In addition, feline functional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for feline calicivirus (FCV) (Makino et al., 2006).In this study, we developed a virus overlay protein-binding assay (VOPBA) in an attempt to identify cell-surface proteins that interact with RaV and which might be involved in virus attachment and/or penetration. The VOPBA ...

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Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV.IMPORTANCE Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.