Complete genome sequencing of 22 West Nile virus isolates suggested 2 independent introductions into Mexico. A previously identified mouse-attenuated glycosylation variant was introduced into southern Mexico through the southeastern United States, while a common US genotype appears to have been introduced incrementally into northern Mexico through the southwestern United States.

Epizootics of encephalitis in El Salvador killed 203 equines between November 2001 and April 2003. During an investigation of the outbreaks, 18 (25%) of 73 serum samples collected from stablemates of deceased animals in 2003 had antibodies to West Nile virus. Ten of these infections were confirmed by plaque reduction neutralization tests, suggesting West Nile virus has extended its range and spread to Central America.

Since the mid-1990s, West Nile virus (WNV) has emerged as a significant agent of arboviral encephalitis in several regions of the world. In 1999, WNV was introduced into the northeastern United States and was associated with an outbreak of encephalitis affecting humans, birds and horses. Subsequently, the virus has spread across the country, and across southern Canada, and in 2002 and 2003 was associated with the largest outbreaks of arboviral encephalitis recorded in the Western hemisphere. Interestingly, the more recent spread of WNV into Mexico, Central America and the Caribbean has not been associated with the high levels of clinical disease observed in North America. This review addresses the most recent results from studies investigating the molecular biology and evolution of WNV, as well as progress in the development of diagnostic and therapeutic reagents.

Neutralization of flaviviruses by antibody is primarily mediated via epitopes in the viral envelope (E) protein. Comparative studies using neutralizing monoclonal antibodies revealed differential expression of epitopes within the E protein domain III of ten naturally occurring West Nile virus strains representing major subtypes of genetic lineages 1 and 2. Residues that defined these subtype-specific determinants were identified by mutational studies and found to be surface exposed in the domain III structure. Mutations of residue 332 had the most significant effects on variation of domain III neutralizing epitopes among strains.

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). 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.

ABSTRACT Genetic characterization of a human cerebrospinal fluid West Nile virus isolate from Beaumont, Texas, revealed several nucleotide changes and amino acid substitutions that differentiated it from all other North American strains isolated to date, suggesting that isolates from the Texas Gulf Coast may form a unique genetic group among North American strains.

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

In order to understand how West Nile virus (WNV) has evolved since its introduction into North America, we have studied the genetic and phenotypic variation among WNV isolates collected in various areas during consecutive transmission seasons. The present report describes for the first time phenotypic changes occurring in the North American WNV population. Several isolates collected in Texas during 2003 display a small plaque (sp) and temperature sensitive (ts) phenotype, as well as reduced replication in cell culture, in comparison to isolates collected in 2002 and New York in 1999. Studies of mouse neuroinvasiveness/neurovirulence also indicate that several of these isolates were attenuated in neuroinvasiveness, but not for neurovirulence. The complete genome and deduced amino acid sequences of several of these isolates have been determined in order to map the mutations responsible for this phenotypic variation. These data indicate microevolution of WNV and the emergence of isolates exhibiting phenotypic variation.

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a beta-barrel structure formed from seven anti-parallel beta-strands in two beta-sheets. One anti-parallel beta-sheet consists of beta-strands beta1 (Phe(299)-Asp(307)), beta2 (Val(313)-Tyr(319)), beta4 (Arg(354)-Leu(355)), and beta5 (Lys(370)-Glu(376)) arranged so that beta2 is flanked on either side by beta1 and beta5. The short beta4 flanks the end of the remaining side of beta5. The remaining anti-parallel beta-sheet is formed from strands beta3 (Ile(340)-Val(343)), beta6 (Gly(380)-Arg(388)), and beta7 (Gln(391)-Lys(399)) arranged with beta6 at the center. Residues implicated in antigenic differences between different West Nile virus strains (and other flaviviruses) and neutralization are located on the outer surface of the protein. Characterization of the binding of monoclonal antibodies to WN-rED3 mutants, which were identified through neutralization escape experiments, indicate that antibody neutralization directly correlates with binding affinities. These studies provide an insight into theoretical virus-receptor interaction points, structure of immunogenic determinants, and potential targets for antiviral agents against West Nile virus and highlight differences between West Nile virus and other flavivirus structures that may represent critical determinants of virulence.

ABSTRACTSerological diagnosis of West Nile virus (WNV) infection is complicated by extensive antigenic cross-reactivity with other closely related flaviviruses, such as St. Louis encephalitis virus. Here we describe a recombinant, bacterially expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from those against other related flaviviruses in indirect enzyme-linked immunosorbent assays using standardized control antisera and field-collected samples.

Since its first recognition in North America in 1999, West Nile virus (WNV) has spread rapidly across the continent, but in many communities, rapid diagnostic tests for detection of WNV infection are not fully available. We describe a patient with extrapyramidal movement disorders and changes in the basal ganglia noted on magnetic resonance images that are characteristic of other flavivirus encephalitides and may help in the recognition of patients with West Nile encephalitis. Detailed molecular analysis suggested that, although our patient received a blood transfusion infected with WNV, the virus that caused his initial infection and encephalitis was probably acquired naturally from a mosquito.

ABSTRACT 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 Leu→Thr/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 Indonesia-Malaysia 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 Indonesia-Malaysia 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.

Analysis of partial nucleotide sequences of 22 West Nile virus (WNV) isolates collected during the summer and fall of 2001 and 2002 indicated genetic variation among strains circulating in geographically distinct regions of the United States and continued divergence from isolates collected in the northeastern United States during 1999 and 2000. Sequence analysis of a 2,004-nucleotide region showed that 14 isolates shared two nucleotide mutations and one amino acid substitution when they were compared with the prototype WN-NY99 strain, with 10 of these isolates sharing an additional nucleotide mutation. In comparison, isolates collected from coastal regions of southeast Texas shared the following differences from WN-NY99: five nucleotide mutations and one amino acid substitution. The maximum nucleotide divergence of the 22 isolates from WN-NY99 was 0.35% (mean = 0.18%). These results show the geographic clustering of genetically similar WNV isolates and the possible emergence of a dominant variant circulating across much of the United States during 2002.

Analysis of partial nucleotide sequences of nine West Nile virus strains isolated in southeast Texas during June–August 2002 revealed a maximum of 0.35% nucleotide variation from a New York 1999 strain. Two sequence subtypes were identified that differed from each other by approximately 0.5%, suggesting multiple introductions of virus to this area. Analysis of sequences from cloned PCR products for one strain revealed up to 0.6% divergence from the consensus sequence at the subpopulation level. The presence of unique patterns of small numbers of mutations in North American West Nile strains studied to date may suggest the absence of a strong selective pressure to drive the emergence of dominant variants.

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.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruı́, Pará state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

Annals of the New York Academy of SciencesVolume 951, Issue 1 p. 332-335 West Nile Virus Strains Differ in Mouse Neurovirulence and Binding to Mouse or Human Brain Membrane Receptor Preparations DAVID W. C. BEASLEY, Corresponding Author DAVID W. C. BEASLEY WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USAAddress for correspondence: David W. C. Beasley, Department of Pathology, University of Texas Medical Branch, Galveston, TX 777555-0609. Voice: 409-772- 2547; fax: 409-747-2415; dwbeasle@utmb.edu.Search for more papers by this authorLI LI, LI LI WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this authorMIGUEL T. SUDERMAN, MIGUEL T. SUDERMAN WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this authorALAN D. T. BARRETT, ALAN D. T. BARRETT WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this author DAVID W. C. BEASLEY, Corresponding Author DAVID W. C. BEASLEY WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USAAddress for correspondence: David W. C. Beasley, Department of Pathology, University of Texas Medical Branch, Galveston, TX 777555-0609. Voice: 409-772- 2547; fax: 409-747-2415; dwbeasle@utmb.edu.Search for more papers by this authorLI LI, LI LI WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this authorMIGUEL T. SUDERMAN, MIGUEL T. SUDERMAN WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this authorALAN D. T. BARRETT, ALAN D. T. BARRETT WHO Collaborating Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USASearch for more papers by this author First published: 25 January 2006 https://doi.org/10.1111/j.1749-6632.2001.tb02710.xCitations: 23Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume951, Issue1WEST NILE VIRUS: DETECTION, SURVEILLANCE, AND CONTROLDecember 2001Pages 332-335 RelatedInformation

Three of 41 IgM monoclonal antibodies derived from dengue 1 virus immunized mice neutralized dengue 1 infection in vitro. All three neutralizing monoclonal antibodies reacted with spatially related epitopes on the E protein of dengue 1 which were also recognized by antibodies in sera from dengue patients. Two neutralization-resistant populations of dengue 1 virus, D1-M10 and D1-M17, were selected by sequential passage of virus in C6/36 cells in the presence of neutralizing IgM monoclonal antibodies M10 and M17, respectively. Single nucleotide changes occurred in the E protein gene of each of these virus populations resulting in single amino acid substitutions at E279 (Phe-Ser) in D1-M10 and at E293 (Thr-Ile) in D1-M17. Both neutralization-resistant populations of virus were more sensitive to elevated temperature than was the wild-type dengue 1 virus and the infectivity and haemagglutinating ability of the neutralization-resistant populations decreased more slowly than that of wild-type virus when exposed to pH in the range 5.8 to 7.0. These are the first epitopes involved in neutralization to have been identified in dengue 1 virus and the first outside domain III of the E protein on any dengue virus.