Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro . Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro , prior to transfection, provided that a 5′-cap structure was added to the 5′ end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

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 ...