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    Distance analysis of the West Nile virus envelope glycoprotein coding region. Numbers at nodes indicate bootstrap confidence based on 1,000 replicates. GenBank accession numbers are provided in Table 1, except for ITequine98 (Italian isolate -af404757), ROpipiens96 (Romanian isolate - af260969), VOhuman99 (Russian isolate - af317203), and Eg101 (Egyptian isolate - af260969).

  • View in gallery

    Unrooted distance analysis of the West Nile virus envelope glycoprotein coding region. Numbers at nodes indicate bootstrap confidence levels. Only values greater than 50 are shown.

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GENETIC AND PHENOTYPIC VARIATION OF WEST NILE VIRUS IN NEW YORK, 2000–2003

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  • 1 Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, Slingerlands, New York; Department of Biomedical Sciences, The University at Albany, State University of New York, Albany, New York

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.

INTRODUCTION

West Nile virus (WNV) was introduced into the New York City area in 1999, producing an equine and avian epizootic and 68 cases of human disease.1 Surveillance data from 1999 do not exist for much of New York State, but all evidence for intense transmission (the presence of human cases and/or detection of infection in mosquito pools) occurred in the downstate region of New York City, Long Island, and in Westchester and Rockland counties, immediately to the north of New York City. In 2000 and 2001, although transmission was documented broadly throughout the state, most evidence for intense transmission and all human cases occurred within the initial downstate epicenter2 of WNV activity. During the 2002 and 2003 transmission seasons, intense transmission again occurred in the downstate epicenter, but was also detected in upstate and western New York, where human cases occurred for the first time (http://www.health.state.ny.us/nysdoh/westnile/update/2002/today2002.gif and http://www.health.state.ny.us/nysdoh/westnile/update/2003/wnv_map.gif).

The RNA viruses are notable for their high mutation rates and consequent potential for rapid evolution.3 Nonetheless, arboviruses possess remarkable genetic and antigenic stability in nature.4–6 West Nile virus apparently has undergone limited evolution in the years during which it has been observed in the United States. Strains collected from New York in 2000 were relatively homogeneous with genetic distances of 0.003 or less.7 Strains collected in Texas during the year 2002 transmission season were similarly homogeneous, but two distinct genotypes were detected.8 Most studies of WNV evolution since its introduction into the Americas have analyzed a relatively small portion of the WNV genome, focusing on the envelope (E) glycoprotein due to its functional significance in viral attachment to host cell receptors and subsequent fusion with the host cell membrane, and because it contains major immunodominant epitopes.9–12 Sequencing studies of the entire WNV genome included strains collected worldwide, and included a relatively small number of strains collected relatively early in the course of the North American epidemic.13 No published study of WNV since its introduction into the Americas has included comparative phenotypic studies of viral replication in vitro or in vivo in mosquitoes.

Accordingly, we collected WNV strains from New York State during the years 2000–2003 and defined their genotype based on the complete coding region of the E protein to determine whether changes in viral genetics accompanied the observed changes in WNV epidemiology in 2002 and 2003 compared with 2000 and 2001. In addition, fragments of the NS5 and 3′ untranslated region (3′UTR) of strains collected during the 2002 and 2003 transmission seasons were analyzed to determine whether estimates of nucleotide and amino acid diversity may be dependent on the region of the genome studied. Finally, it was determined whether differences in viral genotype were associated with either in vitro or in vivo replicative efficiency. The resulting data allow us to more fully characterize WNV evolution since its introduction into North America, and to ascertain whether this agent is adapting to local transmission cycles.

MATERIALS AND METHODS

Virus strains.

Infectious virus or viral RNA was isolated from selected pools of infected mosquitoes or vertebrate tissues. Mosquito pools and vertebrate tissues were submitted to the New York State Arbovirus Laboratories during the years 2000–2003 and screened for infection as described.14 Viral RNA was isolated directly from infected materials using RNEasy spin columns (Qiagen, Inc., Valencia, CA) as described elsewhere7 whenever possible. One or two passages on African green monkey kidney (Vero) cells was required for mosquito-derived WNV and for lightly infected vertebrates because a reverse transcriptase-polymerase chain reaction performed directly on RNA extracts from these materials generally failed (see Table 1 for passage history information on specific WNV strains). Amplification of virus strains on Vero cells was performed as previously described.7 Additional nucleotide sequence data were obtained from GenBank.

Amplification of RNA and sequencing.

To obtain sequence data, RNA samples were reverse transcribed using specific primers. cDNA was amplified using primers designed to amplify the entire envelope region (see Ebel and others7 for primer sequences) and fragments of the nonstructural protein 5 (NS5) (608 basepairs [bp]) and 3′ nontranslated region (464 bp) (NS5F: 5′-tgaggagcgcgaggcacat-3′, NS5R: 5′-cggctgag-tctttcttccccattc-3′, 3′UTRF: 5′-cgccaccggaagttgagtagac-3′, and 3′UTRR: 5′-tcgaccaccagccaccattgt-3′). Reaction products were electrophoretically separated on a 2% agarose gel and sequenced on an ABI 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). The primers used in the sequencing of the envelope region have been described elsewhere,7 and the sequences of the primers used in NS5 and 3′UTR sequencing are available from the authors upon request. Raw sequence data were assembled and edited using the SeqMan module within the DNAStar software package (DNAStar, Inc., Madison, WI). A minimum of two-fold trace redundancy was required for sequence data to be considered complete.

Phylogenetic analysis.

To reconstruct the phylogenetic relationships among the strains studied, alignments were generated using the ClustalW algorithm as implemented in DNAStar, and analyzed by the distance method using MEGA.15 Rooted trees included lineage one WNV strains from Africa and Europe. Evolutionary distances were computed using the Kimura 2-parameter method including transitions and transversions, trees were constructed using the neighbor-joining method, and their robustness was estimated by performing 1,000 bootstrap replicates. To determine the overall nucleotide and amino acid diversity within the sample of strains, the total number of substitutions within each genomic region with respect to a 1999 reference strain was divided by the total number of nucleotides sequenced in each year of observation. Statistical analyses were performed using Stata software (Stata Corporation, College Station, TX).

Mosquito transmission assays.

To determine whether genotypically defined strains may differ in in vivo phenotype in mosquitoes, we fed colonized Culex pipiens mosquitoes on selected virus strains and determined the infection, dissemination, and transmission rates of each strain at five, seven, and nine days postfeeding. Culex pipiens mosquitoes were initially collected in Albany, New York in 2001 and have been maintained in our insectary since that time (>20 generations). Virus stocks were diluted in BA1 to achieve a standard titer of 108 plaque-forming units (PFU)/0.1 mL. Two hundred fifty microliters of virus stock were then added to 5 mL of defibrinated goose blood containing 2.5% sucrose to achieve a final virus titer of 106.7 PFU/0.1 mL. Mosquitoes were allowed to feed on gauze pledgets soaked with virus-blood-sucrose mixture for two hours during which time the pledget was re-soaked once. Mosquitoes that took a full blood meal were then removed to one-pint cardboard cups and held at 27°C, a relative humidity of 85%, and 16:8 light:dark photoperiod until transmission assays were performed. At five, seven, and nine days postfeeding, mosquitoes were removed and anaesthetized with triethylamine (Sigma, St. Louis, MO). Their legs were removed to a microcentrifuge tube containing 1 mL of mosquito diluent (20% heat-inactivated fetal bovine serum [FBS] in Dulbecco’s phosphate-buffered saline plus 50 μg/mL of penicillin/streptomycin, 50 μg/mL of gentamicin, and 2.5 μg/mL of fungizone). Salivary secretions were collected in an in vitro transmission assay as described.16 Briefly, mosquito mouthparts were inserted into a capillary tube charged with a 1:1 mixture of FBS and 50% sucrose. After approximately 30 minutes, the contents of the capillary tube were discharged into 0.3 mL of mosquito diluent and the mosquito body was placed into a tube containing 1 mL of mosquito diluent. Mosquito bodies and legs were homogenized in a mixer mill (Qiagen, Inc.) at 24 cycles/second for 30 seconds and clarified by centrifugation. Mosquito bodies, legs, and salivary secretions were assayed for infection by plaque assay on Vero cells.17,18 Infection, dissemination, and transmission are defined as the proportion of exposed mosquitoes with infectious virus present in bodies, legs, and salivary secretions, respectively.

RESULTS

Virus strains representing the first four years of WNV transmission in New York and strains from previous WNV epidemics were included in this study (Table 1). Strains were collected in each year of observation from mosquito, avian, and mammalian hosts, and from throughout the state of New York. The sample of WNV strains in this study was selected to represent the population of WNV that has been actively transmitted in NY since its introduction in 1999.

Distance analysis of the envelope region of all strains in this study confirms the close relationships of WNV circulating in NY since its introduction (Figure 1). The overall tree topology is unremarkable, and the bootstrap confidence in the branching pattern is generally weak. One poorly supported cluster contained strains isolated initially during the year 2002 transmission season. In contrast, the remainder of the tree is populated by a mixture of strains isolated in each of the years surveyed. The proportion of bases sequenced and their predicted amino acids that differed from the NY99 reference sequence increased in 2002 and 2003 compared with 2000 and 2001 (Table 2). Nucleotide diversity increased approximately two-fold, and amino acid diversity increased approximately four-fold. Although WNV remained relatively homogeneous in New York during the period of observation, strains collected starting in 2002 were more distant from the strain introduced in 1999 than had been observed previously.

Unrooted neighbor-joining analyses (Figure 2) demonstrated the presence of two relatively well-supported WNV clades in New York during the period of observation: one containing strains collected during 1999–2003 (designated NY99 in Figure 2), and another containing strains collected solely during 2002 and 2003 (designated WN02 in Figure 2). These two clades were still evident when strains from the year 2002 and 2003 were considered independently, but were in-apparent in separate analyses of fragments of the NS5 and 3′ untranslated regions. Two nucleotide substitutions defined membership in this clade. One, a U to C transition at nucleotide position 1442, results in a valine to alanine substitution at position 159 of the WNV E protein. The second, a C to U transition at position 2466, is silent. Analysis of functionally diverse regions of the WNV genome showed region-specific trends of nucleotide and amino acid diversity. Protein coding regions of the WNV genome (E and NS5) had approximately two-fold more nucleotide substitutions per base sequenced than the 3′UTR, and the E coding region had approximately three-fold greater amino acid diversity than the NS5 fragment analyzed (Table 3).

In vitro growth studies in Aedes albopictus (C6/36) and African green monkey kidney (Vero) cells at multiplicities of infection of 5 and 0.05 showed little variation in viral replicative efficiency (data not shown). In vivo phenotypic analysis was conducted for two pairs of strains, each pair consisting of one member of each clade. In the first comparison (Table 4) between WNV strains 32010157 (NY99) and 02002640 (WN02), strain 02002640 more efficiently infected mosquitoes at five days postfeeding. At seven days postfeeding, strain 02002640 was again more likely to infect mosquitoes, but was also more likely to have disseminated into the hemocoel, and was more likely to be transmitted than was strain 32010157. Strain 32010157 was not transmitted by Cx. pipiens mosquitoes until nine days postfeeding: by this timepoint, the rates of infection, dissemination, and transmission were similar. In the second comparison (Table 4) between WNV strains 31000352 (NY99) and 02002831 (WN02), strain 02002831 was more likely to have disseminated by five days postfeeding, and the first transmissions occurred with this strain at five days post-feeding. At nine days postfeeding, the proportion of strains infected with strain 02002831 was lower than the proportion observed for strain 31000352. Overall, the proportion of mosquitoes that became infected and developed disseminated infections following exposure to strains of the newly recognized clade (WN02), was greater at five days postfeeding (Table 4). At seven days postfeeding, the proportions of mosquitoes infected, with a disseminated infection, and transmitting virus were also significantly higher for strains belonging to the WN02 clade than the proportions that ingested strains more closely related to NY99. Mosquito transmission assays were performed four times and similar results were obtained in each trial. These results indicate that WNV strains belonging to the WN02 clade may be more efficiently transmitted at early timepoints following mosquito acquisition of an infectious blood meal.

DISCUSSION

Strains of WNV included in this study were collected in New York State during each year since its introduction, from a wide geographic area, and from taxonomically diverse hosts. During the years 2000 and 2001, WNV was sampled from both mosquito and avian hosts to determine whether host-related differences in the envelope coding region would be apparent. When no differences were observed in sequences obtained from mosquitoes compared with sequences from birds,7 we focused our sampling in 2002 on infected birds to avoid cell culture passage of virus, which was often necessary to sequence mosquito-derived isolates. A geographic bias is apparent in the strains selected for this study, with more strains being sampled from the initial epicenter of WNV transmission in New York. This bias is a necessary consequence of WNV transmission in our sampling area: during the first three years of transmission (1999–2001), WNV was more intensely transmitted in the original epicenter than elsewhere. The sample of strains included in this study is thus an accurate representation of the virus population that has been transmitted in New York since its 1999 introduction.

The majority of the analyses focused on the WNV E coding region due to the functional significance of the flavivirus E protein in host receptor binding and membrane fusion. The E protein also contains the immunodominant epitopes responsible for neutralization and serogroup, type, and subtype definition.19 During the period of observation, only three amino acid substitutions in the E protein were detected on more than one strain examined. All three of these occurred in the central domain (Domain I) of the predicted three-dimensional structure of WNV E protein.12 The first, a V to A substitution at E159, is a conservative change that has been reported by Beasley and others,8 and is mainly buried within the protein structure. The second and third, a Y to H change at E176 and a L to Q change at E178, are less conservative and more exposed to the external environment, suggesting that immune selection may drive limited diversification of selected amino acids on the WNV E protein.

Comparative analyses of nucleotide sequences from E, NS5, and 3′UTR suggest that nucleotide substitutions may be more common in E and NS5 than in the 3′UTR, and that amino acid substitutions may be more common in E than in NS5. The relative paucity of nucleotide substitutions in the 3′UTR may indicate constraints imposed on this region by its functional significance in RNA replication.20–22 The difference observed between the proportion of substituted amino acids in E and NS5 may be due either to functional constraints (i.e., purifying selection) imposed on the NS5 protein, which codes the viral RNA-dependent RNA polymerase,23,24 or to diversifying selection imposed on the E protein due to its antigenic importance. Resolving the relative contribution of these selective forces requires further study. Both, however, have been described for flaviviruses.25–27

Phylogenetic analyses of the WNV strains sampled in this study indicate that WNV in New York remains a relatively homogeneous virus population. This finding is consistent with our previous studies of WNV in New York State7 and of WNV sampled more broadly throughout North America.8 In contrast, the proportion of bases sequenced, and their predicted amino acids, that differed from NY99 was greater in 2002 and 2003 than in 2000 or 2001, suggesting that WNV was more diverse in later than in earlier years. These findings suggest either in situ drift of WNV away from the introduced strain, or the introduction and spread of a closely related strain producing a similar effect on our analyses. The abrupt appearance of a previously unrecognized genotype in NYS in 2002 favors the latter hypothesis. The documentation of strains with this genotype in Texas,8 in addition to the now-widespread enzootic transmission of WNV in surrounding states,28 further supports the observation that an additional WNV genotype may have been introduced into New York during 2002. This genotype represents a substantial proportion (55%) of strains collected during 2002, and the majority (85%) of strains collected in 2003. Indeed, it has become dominant throughout New York State.

Phenotypic studies were conducted to assess the possibility that this newly introduced genotype may displace the existing WNV strains in New York. Selected strains representing both clades present in New York during the course of this study were included in in vitro growth and mosquito transmission experiments. Growth studies in cell culture models of WNV infection were generally uninformative: replication was essentially equivalent, with less than one log10 difference between the growth of phylogenetically defined strains at each time-point. Because cell culture systems may not be adequate models of natural transmission cycles, we then focused on an in vivo mosquito transmission model to determine whether the near complete displacement of NY99 by WN02 may be due to differences in mosquito transmission efficiency.

The term vector competence refers to the inherent ability of a particular vector mosquito to become infected with a virus (or other infectious agent) following exposure to an infectious blood meal, support viral replication, and to deliver an infectious inoculum upon taking a second blood meal. Most research on vector competence has focused on the contribution of environmental or mosquito genetic factors to vector competence.29–38 It also has been recognized that viral genotype may impact vector competence,39–42 which may lead to the displacement of one viral genotype with another.43 In the experiments reported here, viral genotype did not seem to impact overall vector competence: after nine days extrinsic incubation, similar proportions of mosquitoes infected with both genotypes were capable of transmitting WNV. However, viral genotype appears to affect the basic reproductive rate of WNV (vectorial capacity) through impacts on the extrinsic incubation period (EIP, the number of days incubation required for a mosquito to transmit an infection): WN02 reduced EIP by approximately two days, providing a possible mechanism for displacement of one viral genotype by another in the absence of an overall increase in vector competence. These findings suggest that genotype-specific increases in replicative efficiency and/or cell-to-cell spread within the mosquito host may be critical determinants of the basic reproductive rate of WNV in nature.

Interestingly, at nine days postfeeding, one strain belonging to the WN02 clade was significantly less likely to infect mosquitoes than its NY99 counterpart. This decrease may be due to either virus clearance from the mosquito or to virus-induced mosquito mortality. Additional studies are required to determine whether either of these hypotheses explains the observed differences in the proportion of infected mosquitoes.

A small number of nucleotide and/or amino acid changes may dramatically impact viral replication, virulence and host specificity. A small number of positively charged amino acid substitutions, for example, determines enzootic versus epizootic/epidemic phenotype of Venezuelan equine encephalitis virus.44 A single amino acid change in the hinge region of the flavivirus envelope region impacts neuroinvasion in mice and viscerotropism in primates.45 The single amino acid change (E159) that defines the newly introduced genotype seems unlikely to be the determinant of altered EIP phenotype due to its conservative and unexposed nature. However, sequencing of the complete genomes of all strains used in mosquito transmission studies (Ebel GD and others, unpublished data) has failed to identify any other amino acid substitutions that WN02 strains have in common. Additional studies using reverse genetics systems are required to determine whether the V to A substitution at E159 may impact mosquito transmission of WNV.

We observed a dramatic increase in the intensity of WNV transmission in New York beginning in 2002 compared with previous years. Non-viral determinants are well known to impact arbovirus transmission and epidemiology,46–50 and it is highly likely that some of these contributed to the observed increase in cases of human disease. However, the observations reported here establish that this increase was accompanied by an increase in genetic diversity that was largely mediated by the introduction of a previously unrecognized WNV genotype during 2002. Studies are in progress to further assess the role of viral genotype in the WNV EIP, to define the molecular determinants of this phenotype, and to assess whether this newly recognized clade may be in the process of displacing the WNV clade that existed in New York State prior to 2002.

Table 1

West Nile virus strains included in the study

Accession no.‡
ID*StrainPassage history†SourceNew York countyCladeEnvNS53′UTRReference
* ID designations used in phylogenetic trees. The first two digits of the ID refer to the year in which the strain was collected (e.g., 03n was collected in 2003, 00a was collected in 2000, etc.).
† V1 = virus isolate following one vero cell passage; V2 = virus isolate following two vero cell passages; P = primary tissue RNA extract.
‡ Env = envelope; NS = nonstructural; UTR = untranslated region; NA = not applicable.
§ Strains used in mosquito transmission experiments.
NY99NY99 EQHSV1HorseSuffolkNY99af260967af260967af26096713
00a3000017V2Culex pipiens (CP)RichmondNY99af346309NANA7
00b3000259V2CP/Cx. restuans (PRE)SuffolkNY99af346316NANA7
00c3000548V2PREQueensNY99af346311NANA7
00d3000622V2PREWestchesterNY99af346313NANA7
00e3100271V2PRERocklandNY99af346312NANA7
00f3100352§V2Cx. salinariusRichmondNY99af346314NANA7
00g3100365V2CPRichmondNY99af346310NANA7
00h842V2American crow (AC)RichmondNY99af346317NANA7
00i2741V2ACAlbanyNY99af346315NANA7
00j3282PRuffed grouseOswegoNY99af346319NANA7
00k3356PACRichmondNY99af346318NANA7
01a01000431PACWestchesterNY99ay369403NANAThis paper (A)
01b01000658PACRocklandNY99ay369404NANA7
01c01001398PACSuffolkNY99ay369405NANAA
01d01001399PBlue jayRichmondNY99ay369406NANAA
01e01001779PACNassauNY99ay369407NANAA
01f01002053PACSuffolkNY99ay369408NANAA
01g01002363PHouse sparrowRichmondNY99ay369409NANAA
01h01002394PBlue jayWestchesterNY99ay369410NANAA
01i33010257V1PRENassauNY99ay369411NANAA
01j32010157§V1PRESuffolkNY99ay369412NANAA
01kNYC01006V1PRERichmondNY99ay369413NANAA
01lNYC01035V1PRERichmondNY99ay369415NANAA
01mNYC01038V1PRERichmondNY99ay369414NANAA
02a02003555PACOnondagaWN02ay369416NANAA
02b34020055V2PRENassauWN02ay369417NANAA
02c007365V2HorseSchenectadyWN02ay369418NANAA
02d02003688V2Gray squirrelSchenectadyWN02ay369419ay369453ay369401A
02e6302PHumanNassauNY99af533540af533540af53354051
02f02002395PACNiagaraWN02ay369420ay369438ay369386A
02g02002640§PACNiagaraWN02ay369421ay369440ay369388A
02h02002831§PACBroomeWN02ay369422ay369446ay369394A
02i02003204PACAlbanyWN02ay369428ay369449ay369397A
02j02002684PACClintonNY99ay369423ay369442ay369390A
02k02002681PACNassauNY99ay369424ay369441ay369389A
02l02002554PACManhattanNY99ay369425ay369439ay369387A
02m02003654PACSuffolkNY99ay369429ay369452ay369400A
02n02003557PACBroomeNY99ay369435ay369451ay369399A
02o02002306PACRichmondNY99ay369430ay369437ay369385A
02p02003535PACKingsNY99ay369426ay369450ay369398A
02q02002735PACRocklandWN02ay369427ay369443ay369391A
02r02003944PACQueensWN02ay369431ay369454ay369402A
02s02002868PACBronxNY99ay369432ay369447ay369395A
02t02003011PACQueensWN02ay369433ay369448ay369396A
02u02002771PACRichmondNY99ay369434ay369445ay369393A
02v02002758PACClintonWN02ay369436ay369444ay369392A
03a03000360PACWestchesterNY99ay590229ay590249ay590191A
03b03001087PACErieNY99ay590211ay590231ay590192A
03c03001426PACSt. LawrenceWN02ay590212ay590232ay590193A
03d03001516PACBroomeWN02ay590213ay590233ay590194A
03e03001543PACMonroeWN02ay590214ay590234ay590195A
03f03001619PACPutnamWN02ay590215ay590235ay590196A
03g03001700PACOnondagaWN02ay590216ay590236ay590197A
03h03001721PACQueensWN02ay590217ay590237ay590198A
03i03001734PACRichmondWN02ay590218ay590238ay590199A
03j03001816PACNassauWN02ay590219ay590239ay590200A
03k03001869PACBronxWN02ay590220ay590240ay590201A
03l03001895PACColumbiaWN02ay590221ay590241ay590202A
03m03001956PACNew YorkWN02ay590222ay590242ay590203A
03n03001986PACAlbanyWN02ay590223ay590243ay590204A
03o03002018PACSuffolkWN02ay590224ay590244ay590205A
03p03002031PACKingsWN02ay590225ay590245ay590206A
03q03002035PACSaratogaWN02ay590226ay590246ay590207A
03r03002066PACNiagaraWN02ay590227ay590247ay590208A
03s03002086PACChautauquaWN02ay590228ay590248ay590209A
03t03002094PACRocklandNY99ay590210ay590230ay590190A
Table 2

Proportion of bases and predicted amino acids of the West Nile virus envelope coding region that differ from strain NY99

NucleotidesPredicted amino acids
YearNo. of strainsNo. of substitutionsNo. sequencedProportion substituted*No. of substitutionsNo. sequencedProportion substituted*
* P < 0.01 by Fisher’s exact test.
2000111416,53310−3.0725,51110−3.44
2001131619,53910−3.0936,51710−3.34
2002225533,06610−2.781811,02210−2.79
2003206330,06010−2.682210,02010−2.66
Table 3

Proportion of nucleotides and predicted amino acids that differ from West Nile virus strain NY99 by genome region*

NucleotidesPredicted amino acids
RegionLength of fragment (nucleotides)No. of substitutionsNo. sequencedProportion substituted†No. of substitutionsNo. sequencedProportion substituted†
* E = envelope; NS = nonstructural; UTR = untranslated region; NA = not applicable.
P < 0.01 by Fisher’s exact test.
E150310958,61710−2.733619,53910−2.73
NS56085723,71210−2.6247,87810−3.29
3′UTR4641918,09610−2.98NANANA
Table 4

Impact of West Nile virus genotype on mosquito transmission phenotype

Days postfeeding
579
PercentPercentPercent
StrainInfectedDisseminatedTransmittedInfectedDisseminatedTransmittedInfectedDisseminatedTransmitted
* Cumulative totals from four trials.
† Determined by chi-square test.
32010157* (NY99)27 (n = 75)4032 (n = 123)5040 (n = 84)105
02002640* (WN02)51 (n = 73)5047 (n = 92)15538 (n = 68)134
P0.0040.7171.0000.0330.0160.0130.8680.6061.000
31000352* (NY99)28 (n = 112)4033 (n = 92)12429 (n = 49)144
02002831* (WN02)28 (n = 102)15338 (n = 98)13612 (n = 51)106
P0.9030.0100.0680.4580.7680.5840.0360.4910.680
Total NY9927 (n = 187)4032 (n = 215)8236 (n = 133)115
Total WN023 (n = 175)11242 (n = 190)14627 (n = 119)125
P0.0340.0170.0720.0370.0420.0370.1170.9040.843
Figure 1.
Figure 1.

Distance analysis of the West Nile virus envelope glycoprotein coding region. Numbers at nodes indicate bootstrap confidence based on 1,000 replicates. GenBank accession numbers are provided in Table 1, except for ITequine98 (Italian isolate -af404757), ROpipiens96 (Romanian isolate - af260969), VOhuman99 (Russian isolate - af317203), and Eg101 (Egyptian isolate - af260969).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 4; 10.4269/ajtmh.2004.71.493

Figure 2.
Figure 2.

Unrooted distance analysis of the West Nile virus envelope glycoprotein coding region. Numbers at nodes indicate bootstrap confidence levels. Only values greater than 50 are shown.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 4; 10.4269/ajtmh.2004.71.493

Authors’ addresses: Gregory D. Ebel, Kristen A. Bernard, and Laura D. Kramer, Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159 and Department of Biomedical Sciences, The University at Albany, State University of New York, Albany, NY 12208, Telephone: 518-852-5295, Fax: 518-869-4530, E-mail: ebel@wadsworth.org. Justin Carricaburu and David Young, Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159.

Acknowledgments: We thank Ilia Rochlin, Sarah Macinski, and Jennifer Longacker for generous entomologic assistance, Eric and Lauren Biesbroeck for technical assistance, Elizabeth Kauffman, Mary Franke, and Susan Jones for additional technical support, Jan Conn for suggestions regarding the manuscript, the New York State Department of Health’s Division of Epidemiology for supervising collection of specimens, and the Wadsworth Center Molecular Genetics Core facility for performing sequencing.

Financial support: This work was supported by National Institutes of Health grant N01 AI-25490.

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