Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu3Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the IndonesiaMalaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the IndonesiaMalaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.

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.

A technique is described that allows unimodal function optimization methods to be extended to locate all optima of multimodal problems efficiently. We describe an algorithm based on a traditional genetic algorithm (GA). This technique involves iterating the GA but uses knowledge gained during one iteration to avoid re-searching, on subsequent iterations, regions of problem space where solutions have already been found. This gain is achieved by applying a fitness derating function to the raw fitness function, so that fitness values are depressed in the regions of the problem space where solutions have already been found. Consequently, the likelihood of discovering a new solution on each iteration is dramatically increased. The technique may be used with various styles of GAs or with other optimization methods, such as simulated annealing. The effectiveness of the algorithm is demonstrated on a number of multimodal test functions. The technique is at least as fast as fitness sharing methods. It provides an acceleration of between 1 and l0p on a problem with p optima, depending on the value of p and the convergence time complexity.

Using a panel of neutralizing monoclonal antibodies, we have mapped epitopes in domain III of the envelope protein of the New York strain of West Nile virus. The ability of monoclonal antibodies that recognize these epitopes to neutralize virus appeared to differ between lineage I and II West Nile virus strains, and epitopes were located on the upper surface of domain III at residues E307, E330, and E332.West Nile (WN) virus is a member of the Japanese encephalitis (JE) serocomplex of the family Flaviviridae, genus Flavivirus. WN virus is a mosquito-borne virus, primarily transmitted by Culex mosquitoes to a number of vertebrates including humans. Human infections with WN virus may result in presentations ranging from subclinical infection to a mild undifferentiated fever to potentially fatal encephalitis. More severe disease presentation generally occurs in older individuals (16). Phylogenetic analysis of WN virus strains has defined two major lineages, I and II (9, 10). With very few exceptions, lineage II WN virus strains have been isolated only in Africa and Madagascar, while lineage I strains have a wide distribution including Africa, Europe, and North America.The flavivirus envelope (E) protein is a major determinant of tropism and the primary target of virus-neutralizing antibody. In particular, structural domain III of E has been proposed elsewhere as a putative receptor-binding domain (13). Studies with several flaviviruses-including JE, yellow fever, dengue, and Murray Valley encephalitis viruses-have identified epitopes recognized by neutralizing antibodies within this domain (4,14,15,17). Although neutralizing epitopes have been identified in other domains of the E protein, antibodies binding to domain III are reported elsewhere to be the most efficient at blocking virus attachment to cells, supporting the proposed role of this domain in receptor binding (6). Specific mutation of residues within domain III has also been shown elsewhere to affect virulence and tropism of flaviviruses (11,12).We used a panel of domain III-reactive monoclonal antibodies (MAbs) and a mouse brain tissue membrane receptor preparation (MRP) to select variants of representative lineage I (strain 385-99) and II (strain H-442) WN virus strains to identify residues that are potentially involved in neutralizing epitopes and receptor binding interactions. In addition, we investigated the effects of these mutations on the virulence of these viruses in a mouse neuroinvasion model.Expression and purification of recombinant WN virus E protein domain III. Recombinant E protein structural domain III (r-EIII, comprising amino acids 296 to 415) from neuroinvasive lineage I WN virus strain 385-99 was expressed from Escherichia coli DH5␣ as a glutathione S-transferase fusion by using the pGEX-2T vector (Amersham Pharmacia Biotech, Piscataway, N.J.) by protocols similar to those described by Bhardwaj et al. (3). Homogeneity of cleaved, purified r-EIII was confirmed by mass spectroscopy (data not shown). Neutralization of WN virus by domain ...

Despite recent advances in the genetics of West Nile (WN) virus, relatively little is known about the molecular basis of virulence of this virus. In particular, although the genotype of the WN virus strain that was recently introduced into North America has been determined, there have been few experimental studies on the virulence phenotype of the virus. We compared genetic and neurovirulence properties of 19 strains of WN virus, including 2 from North America, and observed significant differences in their neuroinvasive phenotype in mice and hamsters that correlated with virus genotype. Virus isolated in North America was found to be highly neuroinvasive with a lack of age-related resistance to infection in mice normally associated with mosquito-borne flaviviruses.

The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.

West Nile virus (WNV) RNA was demonstrated in 5 of 25 (20%) urine samples collected from convalescent patients 573 to 2,452 days (1.6 to 6.7 years) after WNV infection. Four of the 5 amplicons sequenced showed >99% homology to the WNV NY99 strain. These findings show that individuals with chronic symptoms following WNV infection may have persistent renal infection over several years.

West Nile virus (WNV) antibodies were detected in horses from five Mexican states, and WNV was isolated from a Common Raven in the state of Tabasco. Phylogenetic studies indicate that this isolate, the first from Mexico, is related to strains from the central United States but has a relatively high degree of sequence divergence.