RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5-and 3-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5-terminal 20 nucleotides (5End) or targeting the 3-terminal element involved in a potential genome cyclizing interaction (3CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5End or 3CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 M concentration without apparent cytotoxicity. The 3CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3CSI sequences of specific viruses. Modeof-action analyses showed that the 5End and 3CSI PMOs suppressed viral infection through two distinct mechanisms. The 5End PMO inhibited viral translation, whereas the 3CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3 untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.Many members of the Flavivirus genus, a group of arthropod-borne viruses in the family Flaviridae, cause significant human diseases; among these, West Nile (WN), dengue (DEN), Japanese encephalitis (JE), yellow fever (YF), Murray Valley encephalitis, and tick-borne encephalitis (TBE) viruses are emerging and reemerging pathogens (7). Approximately 50 to 100 million human cases of DEN virus infection occur annually (29). The recent epidemics of WN virus have caused significant morbidity and mortality in the United States (9). Vaccines for humans are available only for YF, JE, and TBE viruses (7). No drug therapy is currently available to treat flavivirus infections. It is therefore of great importance to public health to develop an efficacious drug therapy against flaviviruses.Flavivirus virions are spherical in shape, with a diameter of approximately 50 nm (21). The flavivirus genome is a singlestranded, plus-sense RNA of approximately 11 kb in length. The genomic RNA consists of a 5Ј untranslated region (UTR), a single long open reading frame (ORF), and a 3Ј UTR (43). The single ORF encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases...

SUMMARY Increases in tick-borne disease prevalence and transmission are important public health issues. Efforts to control these emerging diseases are frustrated by the struggle to control tick populations and to detect and treat infections caused by the pathogens that they transmit. This review covers tick-borne infectious diseases of nonrickettsial bacterial, parasitic, and viral origins. While tick surveillance and tracking inform our understanding of the importance of the spread and ecology of ticks and help identify areas of risk for disease transmission, the vectors are not the focus of this document. Here, we emphasize the most significant pathogens that infect humans as well as the epidemiology, clinical features, diagnosis, and treatment of diseases that they cause. Although detection via molecular or immunological methods has improved, tick-borne diseases continue to remain underdiagnosed, making the scope of the problem difficult to assess. Our current understanding of the incidence of tick-borne diseases is discussed in this review. An awareness of the diseases that can be transmitted by ticks in specific locations is key to detection and selection of appropriate treatment. As tick-transmitted pathogens are discovered and emerge in new geographic regions, our ability to detect, describe, and understand the growing public health threat must also grow to meet the challenge.

The recent outbreaks of West Nile virus (WNV) in the northeastern

West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter (67% positive, n=907). Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus (92%, n=48) and the highest number of human cases (n=10).

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West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus sigma 3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

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