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Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 39 non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus. INTRODUCTIONThe isolation of caliciviruses from diverse species has been reported by many investigators. These viruses have been classified into four genera: Vesivirus, e.g. vesicular exanthema of swine virus (VESV), feline calicivirus (FCV) and San Miguel sea lion virus (SMSV); Lagovirus, e.g. rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV); the human genera Norovirus and Sappovirus (Capucci et al., 1996; Green et al., 1994Green et al., , 2000Ohlinger et al., 1990;Schaffer et al., 1980, Smith et al., 1979Studdert, 1978;Wirblich et al., 1994). In addition, caliciviruses have been isolated from mink, dog, cattle and non-human primates (Dastjerdi et al., 1999(Dastjerdi et al., , 2000Guo et al., 2001; Liu et al., 1999a;Mochizuki et al., 1993;Smith et al., 1985).In humans, caliciviruses represent the main cause of non-bacterial gastroenteritis (Clarke & Lambden, 1997b), whereas animal caliciviruses can cause vesicular lesions in swine and sea lions, respiratory illness and conjunctivitis in cats, and severe haemorrhagic liver diseases in rabbits and hares (Neill et al., 1998;Smith et al., 1973). While VESV, SMSV or FCV can easily be propagated in cell culture and cause a cytopathogenic effect within a few hours (Studdert, 1978), there is no cell culture system available for lagoviruses or human caliciviruses (Konig et al., 1998; White et al., 1996). Caliciviruses consist of non-enveloped virions 32-40 nm in diameter. Electron microscopy has revealed that several, but not all species, display typical cup-shaped surface depressions, the characteristic 'calyx' morph...

HSV-1 amplicons can accommodate foreign DNA of any size up to 150 kbp. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, or multiple transgenes can be inserted in a modular fashion. HSV-1 amplicon vectors deliver DNA efficiently into the cell nucleus as an extrachromosomal, non-replicating circular concatenate, which is rapidly diluted, at least in dividing cells. Consequently, transgene expression is lost within days to weeks in dividing cells, but may be retained for months in non-dividing cells. In contrast, vectors based on Epstein-Barr virus, adeno-associated virus, or retroviruses can mediate long-term transgene expression, as vector DNA is retained by episomal replication or chromosomal integration. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, thereby conserving the large transgene capacity of HSV-1, and genetic elements from other viruses that confer genetic stability to the vector DNA within transduced cells. Additional strategies to sustain genetic material in infected cells include the incorporation of recombinases from different bacteriophages or transposable elements of the Tc1/mariner family in the amplicon vector. Moreover, modification of the HSV-1 virion itself offers a myriad of possibilities to improve gene delivery by targeting specific cell populations or transporting foreign proteins, such as Cre recombinase or the adeno-associated virus Rep protein, which can control the fate and expression of the therapeutic transgene.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.