The plus-strand RNA genome of flavivirus contains a 5 terminal cap 1 structure (m 7 GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA 3 m 7 GpppA 3 m 7 GpppAm. The 2-O methylation can be uncoupled from the N-7 methylation, since m 7 GpppA-RNA can be readily methylated to m 7 GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2-O cap methylations require distinct buffer conditions and different side chains within the K 61 -D 146 -K 182 -E 218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.Eukaryotic mRNAs possess a 5Ј cap structure that is cotranscriptionally formed in the nucleus. mRNA capping is essential for mRNA stability and efficient translation (13, 39). Most animal viruses that replicate in cytoplasm encode their own capping machinery to produce capped RNAs. RNA capping generally consists of three steps in which the 5Ј triphosphate end of nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (15). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase, to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (13). Both N-7 and 2Ј-O MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-L-homocysteine (SAH) as a by-product. The order of capping and methylation varies among cellular and viral RNAs (13).The genus Flavivirus comprises approximately 70 viruses, many of which are important human pathogens, including four serotypes of dengue virus (DENV), yellow fever virus (YFV), St. Louis encephalitis virus, and West Nile virus (WNV) (23).The flaviviru...

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

West Nile virus (WNV) strains circulating during the first five years of WNV transmission in New York were collected, partial nucleotide sequences were determined, and in vitro and in vivo phenotypic analyses of selected strains were undertaken to determine whether observed increases in the intensity of enzootic and epidemic transmission in New York State during 2002 and 2003 were associated with viral genetic changes. Functionally diverse regions of the WNV genome were also compared to determine whether some regions may be more or less variable than others. The complete envelope coding regions of 67 strains and fragments of the nonstructural protein 5 (NS5) and 3' noncoding regions of 39 strains collected during 2002 and 2003 were examined. West Nile virus in New York remains relatively genetically homogeneous. Viral genetic diversity was greater in 2002 and 2003 at both the nucleotide and amino acid levels than in previous years due to the emergence of a new WNV genotype in 2002. This genotype persisted and became dominant in 2003. Envelope and NS5 coding regions were approximately two-fold more likely than the 3' untranslated region to contain nucleotide substitutions, and the envelope region was approximately three-fold more likely to contain amino acid substitutions than the NS5 region. Variation was noted in in vivo mosquito transmission assays, but not in in vitro growth studies. Strains belonging to the epizootiologically dominant clade were transmitted after approximately two fewer days of extrinsic incubation, providing a possible mechanism for the dominance of this clade. The observed increase in the intensity of WNV transmission beginning in 2002 was associated with an increase in viral genetic diversity that was the result of the emergence of an additional phylogenetic clade. This genotype seems to possess an advantage over previously recognized WNV strains in mosquito transmission phenotype.

The arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20-31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin-agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0-50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 39 1159 nt of the WNV envelope (E) coding region and the 59 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0?016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19?5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra-and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations. INTRODUCTIONWest Nile virus (WNV) (family Flaviviridae, genus Flavivirus) was first detected in North America in 1999 in the New York City area, where it caused an avian and equine epizootic and 68 cases of human disease (CDC, 1999;Lanciotti et al., 1999). It subsequently spread throughout the United States and into Canada, Mexico and the Caribbean (Blitvich et al., 2003;Dupuis et al., 2003;Austin et al., 2004). Recently, several studies have examined the course of WNV evolution since its introduction and have concluded that WNV remains a relatively homogeneous virus population, with the most divergent strains containing only a few nucleotide and/or amino acid substitutions (Anderson et al., 2001;Ebel et al., 2001aEbel et al., , 2004Lanciotti et al., 2002;Beasley et al., 2003;Davis et al., 2003). However, a single WNV genotype that differs from the introduced strain has arisen since 1999 and has become dominant, largely displacing previously circulating strains throughout North America Ebel et al., 2004). Therefore, although WNV remains relatively genetically homogeneous, it appears to be undergoing a process of adaptation to local transmission cycles. To date, no published studies have quantitatively examined the role of positive and/or purifying selection in WNV since its introduction into North America.The mechanisms that lead to population-level genet...

We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 ؋ 10 9 to 5 ؋ 10 9 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

West Nile virus (WNV) belongs to the genus Flavivirus, which includes significant human pathogens such as dengue virus (DEN), yellow fever virus (YF), the tick-borne encephalitis virus complex, Japanese encephalitis virus (JE), Murray Valley encephalitis virus, and WNV. WNV is widely distributed throughout Africa, the Middle East, parts of Europe, Russia, India, Indonesia, and most recently, North America (6). Since the introduction of WNV into the United States in 1999, the virus has caused more than 4,156 known human cases and at least 284 deaths (for updates, see http://www.cdc.gov/ncidod /dvbid/westnile/surv&controlCaseCount03.htm). The WNV epidemic in the United States in 2002 represents the largest meningoencephalitis outbreak in the Western hemisphere and the largest WNV outbreak ever reported (7). Prevention and treatment of WNV infection have become public health priorities.Flavivirus virions contain a single plus-sense RNA genome of approximately 11 kb in length, which consists of a 5Ј untranslated region (UTR), a single long open reading frame (ORF), and a 3Ј UTR (24). The 5Ј and 3Ј UTRs are approximately 100 nt and 400 to 700 nt in length, respectively. The encoded polyprotein is cotranslationally and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (the glycoproteins NS1 and NS2a, the protease cofactor NS2b, the protease and helicase NS3, NS4a, and NS4b; and the methyltransferase and RNA-dependent RNA polymerase [RdRp] NS5) (8). During the flavivirus replication cycle, the synthesis of plus-and minus-sense RNAs is asymmetric; plus-sense RNA is produced in 10-to 100-fold excess over minus-sense RNA (11,23,28).The terminal nucleotides of the 3Ј UTR of flaviviruses can form highly conserved secondary and tertiary structures (4,5,24,26,31). Upstream of the 3Ј stem-loop structure, conserved sequences (CS) CS1, CS2, and repeated CS2 (RCS2) are retained among mosquito-borne flaviviruses. The JE subgroup and YF also have a distinct CS3 and repeated CS3 (RCS3). There is limited information available in regard to the functions of these CS elements. CS1 has the potential to base pair with a sequence within the conserved sequence in the 5Ј region of the capsid gene (5ЈCS) (13). Recent studies using DEN (33), Kunjin (16), and YF (10,22) replicons have shown that base pairing between 5ЈCS and CS1 is essential for flavivirus replication. For CS2, RCS2, CS3, and RCS3 elements, studies using