Most acute infections with RNA viruses are transient and subsequently cleared from the host. Recent evidence, however, suggests that the RNA virus, West Nile virus (WNV), not only causes acute disease, but can persist long term in humans and animal models. Our goal in this study was to develop a mouse model of WNV persistence. We inoculated immunocompetent mice subcutaneously (s.c.) with WNV and examined their tissues for infectious virus and WNV RNA for 16 months (mo) post-inoculation (p.i.). Infectious WNV persisted for 1 mo p.i. in all mice and for 4 mo p.i. in 12% of mice, and WNV RNA persisted for up to 6 mo p.i. in 12% of mice. The frequency of persistence was tissue dependent and was in the following order: skin, spinal cord, brain, lymphoid tissues, kidney, and heart. Viral persistence occurred in the face of a robust antibody response and in the presence of inflammation in the brain. Furthermore, persistence in the central nervous system (CNS) and encephalitis were observed even in mice with subclinical infections. Mice were treated at 1 mo p.i. with cyclophosphamide, and active viral replication resulted, suggesting that lymphocytes are functional during viral persistence. In summary, WNV persisted in the CNS and periphery of mice for up to 6 mo p.i. in mice with subclinical infections. These results have implications for WNV-infected humans. In particular, immunosuppressed patients, organ transplantation, and long term sequelae may be impacted by WNV persistence.

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.West Nile virus (WNV) is a member of the genus Flavivirus, which includes many significant human pathogens of global epidemiological importance, including four serotypes of dengue (DEN) virus, yellow fever (YF) virus, Japanese encephalitis (JE) virus, St. Louis encephalitis (SLE) virus, and tickborne encephalitis (TBE) virus, as well as WNV (3). Among them, DEN virus, YF virus, TBE virus, JE virus, and WNV are listed by the National Institutes of Health as potential biodefense pathogens. Since its introduction into the United States in 1999, WNV has resulted in more than 4,156 known human cases, with 284 deaths (for updates, see http://www.cdc.gov /ncidod/dvbid/westnile/surv&controlCaseCount03.htm). Recent studies have shown that, besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion, organ transplantation (9), breast feeding (8), intrauterine exposure (6), and laboratory-acquired infection (7). These findings have underlined the importance of developing an accurate serologic assay for diagnosis of WNV infection.Flavivirus genomic RNA contains a single open reading frame encoding 10 viral proteins: three structural and seven nonstructural (NS) proteins (Fig. 1A). Viral envelope protein (E protein) (14), NS1 (18,25,26), and NS3 (24) are the most immunogenic proteins during flavivirus infection (15). The current serologic diagnosis of WNV infection is based on detection of antibodies against viral structural proteins, mainly the E protein (12, 21). Unfortunately, the high cross-reactivity of the E protein among flaviviruses limits the specificity of the assay. Positive sera or spinal fluids identified by the current assay must be verified by cross-species plaque reduction neutralization tests (PRNT) to exclude the possibility of infection with cross-reactive viruses such as SLE and DEN. These confirmatory tests have to be performed in level 3 biocontainment for many flaviviruses and substanti...