The introduction of West Nile virus (WNV) into. To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. Growth curves of the two WNV strains were performed in African green monkey kidney (Vero; 37-42 6C) and duck embryonic fibroblast (DEF; 37-45 6C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 6C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 6C relative to levels of intracellular virus-specific RNA measured at 37 6C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 6C demonstrated only a 17-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important factor leading to the increased avian virulence and emergence of this strain of WNV. INTRODUCTIONIn North America, West Nile virus (WNV; family Flaviviridae, genus Flavivirus) has become the leading cause of arboviral encephalitis in humans and equines (O'Leary et al., 2004) and is associated with mortality in >200 avian species (Hayes et al., 2005). The North American WNV strain, NY99, is highly virulent for American crows (AMCRs; Corvus brachyrhynchos) (Komar et al., 2003;McLean et al., 2001). AMCRs inoculated with this virus develop peak viraemic titres in excess of 10 log 10 p.f.u. ml 21 and suffer 100 % mortality within 6 days post-infection (p.i.) (Brault et al., 2004;Komar et al., 2003).Mortality in migratory storks and domesticated geese was identified in Israel between 1997(Bin et al., 2001 Malkinson et al., 2002), where a strain almost identical to the NY99 genotype has circulated (Lanciotti et al., 1999). Other studies have demonstrated that WNV strains from Africa and Australia cause reduced viraemia and mortality in AMCRs compared with the NY99 strain (Brault et al., 2004). These experimental avian infection data, coupled with the genetic relatedness of avian virulent strains, indicate that viral genetic determinants are responsible for the emergence of WNV-associated avian mortality.The WNV genome is a single-stranded, positive-sense RNA of approximately 11 kb. The single open reading frame has 59-and 39-terminal non-coding regions (NCRs) and encodes a polyprotein, which is co-and post-translationally cleaved by viral and host proteases to yield three structural et al., 2005), as well as the construction of an infectious clone of the African KEN-3829 strain. We report phenotypic characterization of viruses generated from these constructs in temperature-sensitivity experiments in vitro and in the in vivo AMCR model. METHODSCells and viruses. Duck embryonic fibroblast (DEF; ATCC CCL-141) and African green monkey kidney (Vero) cells were utilized for temperature-sensitivity s...

West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis. While three of these substitutions had little effect on replication or mouse virulence phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype at 41 degrees C as well as attenuation of the neuroinvasive and neurovirulence phenotypes in mice.

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West Nile (WN) virus, a member of the Flavivirus genus, is a mosquito-borne virus of the Japanese encephalitis (JE) serocomplex. The JE serocomplex contains viruses that cause central nervous system infections, such as JE virus in Asia; St. Louis encephalitis virus in the Americas; Rocio virus in Brazil; Murray Valley encephalitis virus in Australia, New Guinea, and New Zealand; and Kunjin virus (reclassified as subtype WN recently) in Australia (44). Before the mid 1990s, WN virus caused sporadic outbreaks of illness, ranging from fever to occasional encephalitis, in Africa, the Middle East, and Western Asia. However, since 1996, WN encephalitis in humans has been reported more frequently in Europe, the Middle East, northern and western Africa, and Russia (33). In 1999, WN virus first emerged in the western hemisphere in New York City and surrounding areas, where the virus caused the deaths of seven humans and numerous birds and horses (15,33). Since then, WN virus has spread throughout most of the continental United States, with more than 4,156 reported human cases and 284 deaths in 2002 (37) and 9,862 cases with 264 deaths in 2003 (12). WN virus activity in humans, birds, and horses has been documented in Canada, the Caribbean, and Central America. The rapid spread of WN virus suggests it may pose a significant public health problem in future years (15). There is no licensed human WN vaccine available to protect at-risk populations from WN illness.Dengue (DEN) viruses are also human pathogens that are transmitted by mosquitoes. These viruses cause illness in millions of people every year throughout tropical regions of the world. Flaviviruses of the DEN serocomplex are classified into four serotypes, DEN 1 to DEN 4 (D1 to D4). Flaviviruses contain a single-stranded positive-sense genomic RNA of approximately 11 kb with the genomic organization 5ЈNCR-CprM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3ЈNCR (where NCR is noncoding region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural protein). One of the most promising D2 vaccine candidates, strain PDK-53, was derived by passage of the wild-type D2 16681 virus 53 times in primary dog kidney (PDK) cells (48). To study the attenuation loci of the candidate D2 PDK-53 vaccine virus, as well as to develop effective tetravalent DEN vaccines against all four DEN serotypes, we constructed infectious cDNA clones of the D2 viruses (9, 26) and used them to engineer chimeric DEN viruses containing the prM-E genes of D1, D3, or D4 virus in the D2 genetic backbones (18,19). The uncloned D2 PDK-53 vaccine virus contains a mixture of two genotypic variants (26), designated 19) in this report. The PDK53-V variant contains all nine PDK-53 virus vaccine-specific nucleotide mutations, including the Glu-to-Val mutation at amino acid position NS3-250 (26). The PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine, and the NS3-250-Glu of its parental D2 16681 virus. Our results showed that the phenotypic markers associated with the attenuation of PDK...

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41 °C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.

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