Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), and varicella-zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91–52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV-6 and human dUTPases than controls (p = 0.0053 and p = 0.0036, respectively), while the ME/CFS cohort had higher anti-EBV-dUTPase antibodies than in both GWI patients (p = 0.0008) and controls (p < 0.0001) as well as significantly higher anti-human dUTPase antibodies than in controls (p = 0.0241). These results suggest that screening of patients’ sera for the presence of various combinations of anti-dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment.

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the fC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in $6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied. KEYWORDS RMCE; Drosophila; cell line; single-transgene expression M AMMALIAN cultured cells can be genetically modified by site-specific insertion of transgenes using homologous recombination and various recombinase systems (Branda and Dymecki 2004;Sorrell and Kolb 2005). In contrast, transgenes are introduced into Drosophila cultured cells either by transient transfection or by stable transformation following random integration of transgenes together with a selectable marker (Eschalier 1997;Cherbas and Cherbas 2007). These methods fail to provide control of transgene expression because the number of transgene copies in the cells varies greatly. Moreover, in stably transformed lines, the sites of insertion vary, and each will be subject to position effects on gene expression (Spradling and Rubin 1983). For these reasons, methods that involve site-specific introduction of single transgenes into Drosophila tissue culture cells would be a significant improvement.Homologous recombination has been attempted in Drosophila cell culture, but the levels of nonspecific recombination observed make the approach unsatisfactory (Cherbas and Cherbas 1997). Several site-specific recombination systems have been used successfully in whole flies, including uC31, which mediates recombination between two heterotypic target sites referred to as attP and attB (Groth et al. 2004;Venken et al. 2006;Bischof et al. 2007). Insertion results in two new sites (attL and attR) that are not targets of the integrase, thus producing an irreversible cha...