West Nile Virus Infection in Birds and Mosquitoes, New York State, 2000.

West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter (67% positive, n=907). Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus (92%, n=48) and the highest number of human cases (n=10).

The emergence of West Nile (WN) virus in 1999 in four U.S. states (1) was followed by its spread to 12 states in 2000 (2). An enzootic cycle was established between birds and mosquitoes, and WN disease was observed in humans, horses, and birds in both years (2,3). Bird deaths due to WN virus are unusual outside North America, with the exception of deaths of geese in Israel (4) and pigeons in Egypt (5). In 1999 in North America, WN disease, characterized by meningoencephalitis and myocarditis, was observed in 14 species of wild and captive birds (6). WN virus has been detected in a number of mosquito genera in North America, including Culex and Aedes species (2,7). Vector competence has been confirmed experimentally for some North American species, including Cx. pipiens, Ae. vexans, and Ae. japonicus (8,9).

We have summarized surveillance data for WN virus in dead birds and mosquitoes for New York State in the 2000 transmission season. A quantitative and kinetic analysis of data within and outside the epicenter is shown for both the bird and mosquito samples. Vertebrate and invertebrate WN virus infections are compared for counties in the epicenter.

Materials and Methods

Bird and Mosquito Samples

Dead birds were collected and mosquitoes were trapped by local county health units and other agencies as part of the New York State WN virus surveillance effort. Dead birds were necropsied at the Wildlife Pathology Unit at the Department of Environmental Conservation. Kidney, brain, heart, liver, or spleen were harvested and stored at -70 [degrees] C. Additional avian tissue samples sent to the National Wildlife Health Center were not included in this analysis because selection criteria and testing procedures differed. Mosquitoes were trapped, speciated, grouped into pools of 5 to 50, and stored at -70 [degrees] C. For some pools, Cx. pipiens and Cx. restuans were not separated but were pooled together and denoted as Cx. pipiens-restuans. The Aedes genus is being reclassified into two genera, Aedes and Ochlerotatus (10), but is classified as Aedes in this manuscript. Avian tissue samples and mosquito pools were sent to the Arbovirus Laboratory at the Wadsworth Center for WN virus testing. The transmission season was defined as May 15 to October 31, 2000, with the first and last positive samples collected on May 22 and October 31, 2000, respectively. Two positive birds were found earlier in the year (a hawk on February 6 and a crow on April 1), but they were not followed by other positive specimens and did not therefore appear to represent the beginning of the mosquito-borne WN virus transmission season.

WN Virus Testing

Samples were processed and WN virus assays were performed as described by Shi et al. (11). Briefly, RNA was extracted from bird tissue or pools of [is less than or equal to] 50 mosquitoes, and real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed (TaqMan, ABI Prism 7700 Sequence Detector, Applied Biosystems, Foster City, CA). Confirmatory tests included a second TaqMan primer-probe set, standard RT-PCR, virus isolation in cell culture, and immunofluorescence assays (for avian tissues). A sample was confirmed as positive if at least two different test results were positive.

Other Arbovirus Testing

Virus isolation was attempted for all Aedes species (with the exception of some Ae. vexans pools), all Culiseta species, and most WN virus-positive mosquito pools (with the exception of some Cx. pipiens and Cx. pipiens-restuans pools). Samples were inoculated onto monolayers of Vero or C6/36 cells. Viruses from positive cultures were typed by using serogroup-specific polyclonal antisera for bunyaviruses (California and Bunyamwera serogroups), flaviviruses, alphaviruses, and rhabdoviruses (Hart Park serogroup). California serogroup isolates were further characterized by sequence analysis.

Definition of the Epicenter and Radial Regions

The epicenter of the New York State epizootic was defined as follows. The minimum infection rate (MIR) of each mosquito species was calculated for each county or New York City borough by the standard formula: (number of WN virus-positive mosquito pools/total number of mosquitoes tested) x 1000. The MIR was calculated only when at least 1,000 mosquitoes were tested per species per county or borough. When the MIR was at least 1.0 for any mosquito species in a region, the county or borough was included in the epicenter. In addition, any counties that bordered at least two other epicenter counties or boroughs were included in the definition. The epicenter included the five boroughs of New York City (the Bronx, Brooklyn, Manhattan, Queens, and Staten Island) and the four counties immediately east and north of New York City (Nassau, Suffolk, Rockland, and Westchester counties). For both 1999 and 2000, all human cases of WN virus in New York State were in one of these counties (2,7).

Counties outside the epicenter (the "non-epicenter") were divided into four radial regions, R1 to R4, with increasing distance from the epicenter (Figure 1). Radial regions were defined as follows: R1 = Putnam, Orange, Dutchess, Sullivan, and Ulster counties; R2 = Columbia, Delaware, Greene, Rensselaer, Montgomery, Albany, Otsego, Broome, Cortland, Schenectady, Schoharie, and Chenango counties; R3 = Fulton, Essex, Hamilton, Herkimer, Allegany, Lewis, Chemung, Madison, Cayuga, Schuyler, Yates, Washington, Warren, Tompkins, Tioga, Steuben, Onondaga, Seneca, Saratoga, Ontario, Oswego, and Oneida counties; and R4 = Monroe, Wyoming, Cattaraugus, Wayne, Chautauqua, Erie, Clinton, Genesee, Jefferson, Orleans, St. Lawrence, Niagara, Franklin, and Livingston counties.

[ILLUSTRATION OMITTED]

Results

During the 2000 transmission season, WN virus testing was performed on 3,403 dead birds, representing 153 species, 46 families, and 18 orders. The 1,201 WN virus-positive birds represented 63 species, 30 families, and 14 orders (Table 1). The percentage of WN virus-positive birds was 35% for all birds submitted for testing from throughout the state. Avian species that were [is greater than] 35% positive and for which at least 10 birds were tested included American Kestrel (57%, n=14), Cedar Waxwing (60%, n=10), Ovenbird (50%, n=18), American Crow (47%, n=1,687), Fish Crow (47%, n=45), and Red-tailed Hawk (43%, n=14). The discrepancies in number of birds tested make comparisons between species difficult.

Table 1. Birds positive for West Nile virus in New York State
during the 2000 season(a)

Order               Family                Common name

Anseriformes       Anatidae            Domestic Goose
                                       Canada Goose
                                       Mute Swan

Apodiformes        Trochilidae         Ruby-throated Hummingbird

Caprimulgiformes   Caprimulgidae       Common Nighthawk

Charadriiformes    Charadriidae        Killdeer
                   Laridae             Herring Gull
                                       Ring-billed Gull
                                       Greater Black-backed Gull
                   Rynchopidae         Black Skimmer
                   Scolopacidae        Ruddy Turnstone

Ciconiiformes      Ardeidae            Least Bittern
                                       Green Heron
                                       Great Blue Heron

Columbiformes      Columbidae          Mourning Dove
                                       Rock Dove

Coraciiformes      Alcedinidae         Belted Kingfisher

Falconiformes      Accipitridae        Red-tailed Hawk
                                       Sharp-shinned Hawk
                                       Cooper's Hawk
                                       Broad-winged Hawk
                   Falconidae          Merlin
                                       American Kestrel

Galliformes        Meleagrididae       Domestic Turkey
                                       Eastern Wild Turkey
                   Phasianidae         Peacock
                                       Ring-necked Pheasant
                   Tetraonidae         Chicken
                                       Ruffed Grouse

Gruiformes         Rallidae            Virginia Rail

Passeriformes      Bombycillidae       Cedar Waxwing
                   Corvidae            Fish Crow
                                       American Crow
                                       Blue Jay
                   Fringillidae        Zebra Finch
                                       Song Sparrow
                                       American Goldfinch
                                       House Finch
                                       Cardinal
                   Icteridae           Red-winged Blackbird
                                       Common Grackle
                   Mimidae             Gray Catbird
                                       Northern Mockingbird
                   Parulidae           Black-throated Blue Warbler
                                       Canada Warbler
                                       Warbler
                                       Yellow-rumped Warbler
                                       Ovenbird
                   Ploceidae           House Sparrow
                   Sturnidae           European Starling
                   Turdidae            Veery
                                       Eastern Bluebird
                                       American Robin
                                       Wood Thrush
                   Tyrannidae          Eastern Phoebe

Pelecaniformes     Phalacrocoracidae   Cormorant
                                       Double Crested Cormorant

Psittaciformes     Cacatuidae          Cockatoo
                                       Cockatiel
                   Psittacidae         Macaw
                                       Parakeet
Strigiformes       Strigidae           Snowy Owl
                                       Great Horned Owl

Order              No. tested   % positive

Anseriformes             2          50
                        15          33
                         3          33

Apodiformes              5          20

Caprimulgiformes         2          50

Charadriiformes          3          33
                         9          33
                        66          32
                         7          29
                         1         100
                         1         100

Ciconiiformes            1         100
                         3          33
                        29          10

Columbiformes           83          19
                        41          17

Coraciiformes            6          33

Falconiformes           14          43
                        17          35
                        30          30
                         7          14
                         5         100
                        14          57

Galliformes              1         100
                         3          67
                         8          25
                        16          25
                        14          29
                       131          21

Gruiformes               2          50

Passeriformes           10          60
                        45          47
                     1,687          47
                       500          29
                         1         100
                         5          60
                         4          50
                         8          38
                         3          33
                         6          17
                        53          13
                        22          23
                        10          20
                         1         100
                         1         100
                         1         100
                         1         100
                        18          50
                       127          13
                        23          17
                         3          33
                         4          25
                        74          22
                         5          20
                         2          50

Pelecaniformes           2         100
                         2          50

Psittaciformes           1         100
                         5          60
                         1         100
                         9          22

Strigiformes             2         100
                        16          19

(a) Season defined as May 15, 2000, through October 31, 2000.

The percentage of WN virus-positive birds was analyzed for the epicenter and non-epicenter regions. Data are included for avian species for which at least 10 birds were tested in one of the regions (Table 2). For all submitted birds, 51% and 23% WN virus-positive birds were found in the epicenter and non-epicenter regions, respectively. WN virus infection in dead birds was highest for American Crows (67%) in the epicenter. In the non-epicenter, WN virus infection for crows was lower, similar to infection in all birds in this region. High numbers of crows were tested in both regions, and the percentage positive differed by almost threefold.

Table 2. Summary of birds tested for West Nile virus in New York
during the 2000 season(a)

                          Epicenter         Non-epicenter

                                  % WN                % WN
                        No.      virus      No.      virus
Common name            tested   positive   tested   positive

American Crow            907        67       780       23
Fish Crow                 31        55        14       29
Blue Jay                 191        40       309       23
Cooper's Hawk             11        27        19       32
Sharp-shinned Hawk       <10     NA(b)        14       36
American Robin            11         9        59       22
House Sparrow            107         8        20       40
European Starling         15         7       <10       NA
Common Grackle            27         7        26       19
Gray Catbird             <10        NA        16       25
Ovenbird                 <10        NA        12       75
Common Yellow Throat      19         0       <10       NA
Mallard                  <10        NA        12        0
Ring-billed Gull         <10        NA        66       32
Great Blue Heron         <10        NA        28        7
Rock Dove                 16         0        25       28
Mourning Dove            <10        NA        77       19
Ring-necked Pheasant     <10        NA        15       27
Chicken                  <10        NA        10       30
Ruffed Grouse            <10        NA       130       21
Great Horned Owl         <10        NA        15       20
Total(c)               1,502        51     1,901       23

(a) Season defined as May 15, 2000, through October 31, 2000. Bird
species were included only if at least 10 birds were tested for one
of the regions throughout the season.

(b) NA = not applicable because of the low number of birds tested.

(c) All birds tested in each region throughout the season.

WN virus infection in dead birds was examined over time for American Crows and all other birds in the epicenter and four radial regions in New York State (Figure 2). Comparison of American Crows in the five regions (Figure 2A) revealed the highest peak in the epicenter during September. In addition, the peak for American Crows in the epicenter was much broader than for the other four regions. For September, the peaks for American Crows in R1 and R2 were greater than those in the more distant regions, R3 and R4, suggesting a minor extension of the epicenter during this month. Comparison of all birds except American Crows in the five regions (Figure 2B) revealed little difference between the regions, even for the epicenter. These data support the hypothesis that the susceptibility to WN disease was greatest in crows in the epicenter.

[GRAPHS OMITTED]

We tested 9,954 mosquito pools with 317,668 mosquitoes, representing 28 species and eight genera (Table 3). Of eight positive species representing four genera, most positive pools were Culex species (n=341), compared with only 22 positive pools in the other three genera. All but five of the positive pools were collected in the epicenter. The MIR was calculated for each species in the epicenter for which at least 1,000 mosquitoes were tested (Table 3). In the epicenter, the MIR ranged from 0.47 to 3.55. The MIR of Cx. pipiens was the highest for an individual species. All the pure Cx. restuans pools were negative, and the MIR for Cx. pipiens-restuans was almost half that of the pure Cx. pipiens pools; therefore, the positive mosquitoes in the mixed pools of Cx. pipiens-restuans were most likely Cx. pipiens. The MIRs for Cx. pipiens and Cx. pipiens-restuans were compared with the percentage of positive American Crows in the epicenter for each week of the transmission season (Figure 3). The MIRs for Cx. pipiens and Cx. pipiens-restuans peaked 3 and 2 weeks, respectively, before the peak for positive crows. These data support the hypothesis that Cx. pipiens is an important enzootic vector of WN virus in New York.

[GRAPH OMITTED]

Table 3. Summary of mosquitoes tested for West Nile virus in New
York State in 2000

                                        Epicenter

                                                   Total
                              Total       Total   positive
Mosquito species            mosquitoes    pools    pools     MIR(a)

Aedes abserratus-punctor          NC(b)      NC      NC          NC
Ae. canadensis                 8,018        167       0           0
Ae. cantator                   1,615         65       1        0.62
Ae. cinereus                     340         15       0          NA(c)
Ae. communis                      NC         NC      NC          NC
Ae. communis group                NC         NC      NC          NC
Ae. intrudens                     21          1       0          NA
Ae. japonicus                  3,342        257       2        0.60
Ae. sollicitans                5,003        131       0          NA
Ae. stimulans                     NC         NC      NC          NC
Ae. stimulans group              153          3       0          NA
Ae. taeniorhynchus               395         11       0          NA
Ae. trichuris                     NC         NC      NC          NC
Ae. triseriatus                2,956        203       3        1.01
Ae. trivittatus                4,289        184       0           0
Ae. vexans                    21,486        761      10        0.47
Aedes species                  1,340         72       1        0.75
Anopheles crucians                13          2       0          NA
An. punctipennis                 165         37       1          NA
An. quadrimaculatus               66         18       0          NA
An. walkeri                       NC         NC      NC          NC
Anopheles species                 16          2       0          NA
Coquillettidia perturbans      8,167        157       0           0
Culiseta melanura              8,189        211       0           0
Cs. morsitans                     NC         NC      NC          NC
Culiseta species                  98          3       0          NA
Culex pipiens                 22,120        831      78        3.53
Cx. pipiens-restuans(d)      114,517      3,208     211        1.84
Cx. restuans                   3,403        190       0           0
Cx. salinarius                19,541        483      31        1.59
Cx. territans                     76         15       0          NA
Culex species                  5,358        200      19        3.55
Orthopodomyia alba                NC         NC      NC          NC
Psorophora ferox                  63         10       1          NA
Uranotaenia sapphirina           208         19       0          NA
Unidentified                   1,173         26       0           0
TOTAL                        232,131      7,282     358

                                    Non-epicenter

                                                  Total
                              Total      Total   positive
Mosquito species            mosquitoes   pools    pools

Aedes abserratus-punctor         214         9       0
Ae. canadensis                 8,269       145       0
Ae. cantator                     993        22       0
Ae. cinereus                     247        10       0
Ae. communis                     335        12       0
Ae. communis group               235         8       0
Ae. intrudens                     NC        NC      NC
Ae. japonicus                  3,871       271       3
Ae. sollicitans                    1         1       0
Ae. stimulans                     42         1       0
Ae. stimulans group              540        17       0
Ae. taeniorhynchus                11         1       0
Ae. trichuris                     23         2       0
Ae. triseriatus                6,335       206       0
Ae. trivittatus                2,453        79       0
Ae. vexans                    13,490       422       0
Aedes species                     NC        NC      NC
Anopheles crucians                NC        NC      NC
An. punctipennis                 291        17       0
An. quadrimaculatus               59         3       0
An. walkeri                      833        22       0
Anopheles species                 NC        NC      NC
Coquillettidia perturbans     10,874       206       0
Culiseta melanura              6,281       178       0
Cs. morsitans                  1,821        85       0
Culiseta species                  NC        NC      NC
Culex pipiens                  8,698       288       1
Cx. pipiens-restuans(d)       16,228       537       1
Cx. restuans                     794        48       0
Cx. salinarius                   704        17       0
Cx. territans                     NC        NC      NC
Culex species                  1,108        32       0
Orthopodomyia alba               101         3       0
Psorophora ferox                 162         6       0
Uranotaenia sapphirina           419        18       0
Unidentified                     105         6       0
TOTAL                         85,537     2,672       5

(a) MIR = minimal infection rate per 1,000 mosquitoes. MIR calculated
as (number of WN virus-positive pools by RT-PCR/total mosquitoes
tested) x 1,000. MIR was calculated only if a minimum of 1,000
mosquitoes was tested from a defined region throughout the season.
None of the counties outside the epicenter met this criterion.

(b) NC = not collected.

(c) NA = not applicable because <1,000 mosquitoes were collected.

(d) Cx. pipiens and Cx. restuans were not separated and were
pooled together.

The epicenter was examined as individual boroughs and counties to compare vertebrate and invertebrate WN virus infections (Table 4). The percentage of positive American Crows was calculated, and human and equine cases were noted for each county over the entire season. The MIRs of mosquitoes from each county were calculated for species with at least 1000 mosquitoes tested. Six mosquito species or groups met this criterion. The highest number of vertebrates infected with WN virus was found in Staten Island and was associated with the highest mosquito MIRs. This borough had measurable MIRs for five mosquito groups: Cx. pipiens, Cx. species, Cx. pipiens-restuans, Cx. salinarius, and Ae. vexans.

Table 4. Comparison of infection in vertebrates and minimal infection
rate in mosquitoes for WN virus in the epicenter of the New York
epizootic of 2000

                                                       MIR(a)

              % positive     No.        No.                   Cx.
Borough         crows       human      equine     Culex     pipiens-
or county       (n)(b)     cases(c)   cases(a)   pipiens   restuans(e)

Staten
  Island      92% (48)       10          1        11.42        9.9
Brooklyn      73% (48)        2          0         3.12       1.42
Manhattan     85% (34)        1          0         2.91       3.86
Queens        64% (25)        1          0         0.16       0.24
Suffolk       70% (188)       0          8          NC        2.74
Bronx         44% (9)         0          2         2.38        NA
Rockland      76% (280)       0          0          NA        1.98
Westchester   44% (128)       0          0         0.51       0.73
Nassau        56% (147)       0          4          NC        0.28

                             MIR(a)

Borough        Culex    Cx. sali-   Aedes       Ae.
or county     species    narius     vexans   japonicus

Staten
  Island       6.92       2.61       0.79      NC(f)
Brooklyn        NA(g)     0.67        NA       NC
Manhattan       NA         NA         NA       NC
Queens           0        0.2         NA       NA
Suffolk         NC         NC        0.40      NA
Bronx           NA        0.87          0      NC
Rockland        NA         NA        0.44      NA
Westchester     NC         NC           0     0.43
Nassau          NC         NC        0.45      NC

(a) MIR = minimal infection rate per 1,000 mosquitoes. Mosquito
species were included only if a minimum of 1,000 total mosquitoes
was collected throughout the season for the county. MIR was
calculated as (number of WN virus-positive pools by RT-PCR/total
mosquitoes tested) x 1,000.

(b) Percentage WN virus-positive crows throughout the transmission
season with total number of crows tested in parentheses.

(c) Human cases reported by the Centers for Disease Control
and Prevention (2).

(d) Equine cases reported by S. Trock (personal communication).

(e) Cx. pipiens and Cx. restuans were not separated and were
pooled together.

(f) NC = not collected.

(g) NA = not applicable because <1,000 mosquitoes were collected.

Virus isolation was performed on mosquito samples by cell culture. WN virus was isolated from 110 samples, including Cx. pipiens-restuans (n=60), Cx. pipiens (n=25), Cx. salinarius (n=13), Culex species (n=9), Ae. triseriatus (n=1), Ae. vexans (n=1), and Psorophora ferox (n=1). No virus was isolated from any of the WN virus RNA-positive pools of Ae. japonicus, Ae. cantator, and An. punctipennis. Pools that were negative on Vero cell culture were passaged repeatedly in Aedes albopictus (C6/36) cells; these further attempts to isolate virus were unsuccessful. Other viruses isolated were trivittatus virus from Ae. trivittatus (n=4) and Ae. triseriatus (n=1), Cache Valley virus from Ae. trivittatus (n=2) and Ae. triseriatus (n=2), and Flanders virus from Cx. pipiens-restuans (n=7), Cx. pipiens (n=2), and Cs. melanura (n=11). Eastern equine encephalitis virus and California group viruses other than trivittatus were not isolated from any of the Culiseta or Aedes pools.

Conclusion

In the 2000 transmission season in New York State, we found 63 bird species infected with WN virus, compared with 14 species in 1999 (6). The percentage of WN virus-positive birds was higher in the epicenter than outside it. This high percentage almost entirely reflects infected crows in the epicenter; no increase in WN virus infection was noted in birds other than crows. In contrast, high WN virus infection of dead crows was not observed outside the epicenter, where the percentage of WN virus positivity was similar in crows and other birds over the entire season. High numbers of dead crows were also observed in 1999 (3,6). The cause of the increased sensitivity of crows to WN disease or infection is unknown, but may be due to virus-host interactions, mosquito-bird interactions, mosquito feeding preferences, crow population density, or crow behavior. The presence of WN virus in dead birds does not indicate a definitive diagnosis of WN virus as the cause of death. Many of the birds did not show gross pathologic lesions of WN disease (12). In addition, the rate of WN virus-positive birds in our surveillance samples is not equivalent to prevalence of infection, since we are sampling only dead birds.

The analysis of percentage positive birds over time revealed differences between various regions in New York State. The percentage of WN virus-positive crows was highest in the epicenter compared with other regions of New York State throughout the season, supporting the importance of crows as indicators in the epizootic. The percentage of WN virus-positive crows was higher than that for all other birds early in the season only in the epicenter. Outside the epicenter, the percentages of WN virus-positive crows and all other birds were similar until the peak month of September. At this time, the two radial regions closest to the epicenter showed higher infection in crows than in other birds, suggesting that the intense level of viral activity may have spread beyond the epicenter. An explanation for this apparent spread may be increased movement of crows during the fall. The surveillance data on avian deaths have implications for future surveillance activities. The similar percentages of positive crows and other birds outside the epicenter indicate the importance of testing all birds, not only crows, outside the epicenter.

Sampling errors are likely with the avian surveillance samples. The samples were from dead birds submitted to the Wildlife Pathology Unit, which relied on the cooperation of the general public, individual county health departments, and other agencies; therefore, surveillance samples do not represent a random sampling of dead birds throughout the state. The size and degree of urbanization of various bird species may have resulted in differences in submission. For example, large urban-dwelling species, such as crows, were more likely to have been submitted than small rural dwellers. In addition, more birds were likely sampled from areas with human cases, greater media coverage, and higher human population. The similarity in WN virus infection in birds other than crows from different regions suggests that the impact of sampling bias was not significant. Additionally, specimens submitted for testing to the Wadsworth Center from the Wildlife Pathology Unit represent a sample of those submitted by the public. Sampling at this level may introduce further bias into our surveillance sample. The similarity in WN virus infection in birds other than crows outside the epicenter, however, suggests that such bias was minimal.

The highest MIR for mosquitoes in the epicenter was for Cx. pipiens. Positive pools of Cx. pipiens also were identified in 1999 in New York (13). Cx. pipiens-pipiens mosquitoes feed almost exclusively on birds (14); thus, they are likely an important enzootic vector in the bird-mosquito cycle in North America. Other Cx. species have been implicated as enzootic vectors worldwide (15). High MIRs of Cx. pipiens and Cx. salinarius were associated with human and equine WN virus cases and high infection rates of crows in counties in the epicenter. Cx. salinarius feeds on both birds and mammals (16); therefore, it is a likely candidate as a "bridge vector," transmitting the virus from bird to mammal. Cx. salinarius has been proposed as a bridge vector for Eastern equine encephalitis virus (17). All MIR data were calculated by using RT-PCR-positive pools and therefore cannot be directly compared with MIRs calculated by using infectious virus-positive pools. No virus was isolated from five RNA-positive pools of Ae. japonicus, even after six serial passages through C6/36 cells. Much attention has been focused on this species because it has been reported to be a highly competent laboratory vector of WN virus (9), but current field data do not support this experimental observation. Different populations of Ae. japonicus have been described in the eastern United States (18), and differences in vector competence between the populations may explain the discrepancy between the field and experimental data.

The possibility of sampling error also exists for the mosquito surveillance samples. Individual counties collected mosquitoes in different numbers and set traps by different criteria. In addition, some counties used mosquito adulticides or larvicides during the season. These sources of bias are unlikely to have been uniformly introduced, and their impact on our analyses is unclear.

The results from the 2000 surveillance season for WN virus leave a number of unanswered questions. Many avian species can become infected with WN virus, but the prevalence of infection for each species is unknown without systematic serosurveys of the wild bird population. It is also unknown which birds have a high enough viremia for efficient transmission to the vector. The apparent mortality rate caused by WN virus is higher for crows than for other birds, but laboratory experiments are required to determine WN virus mortality rates and the pathogenic mechanisms in crows and other avian species. In addition to crows, many other birds, such as raptors and other corvids, also showed significant pathology. Some nonmigrating species (e.g., ruffed grouse) have potential use as an indicator species for WN virus infection. Vector competence, blood meal identification, and transovarial transmission studies for the potential mosquito vectors are also important research areas.

Acknowledgments

We thank the WN virus surveillance team, especially the New York City Department of Health and the local county health units, who collected the surveillance specimens. We also thank H. Taber for helpful discussions.

Support was provided by the Wadsworth Center and the Division of Epidemiology, New York State Department of Health, the Department of Environmental Conservation, and the Centers for Disease Control and Prevention.

Dr. Bernard is assistant director of the Arbovirus Laboratory at the Wadsworth Center. Her major research interests are viral pathogenesis and the maintenance of arboviruses in temperate climates.

(1) P. Bryon Backenson, Ivan Gotham, Yoichiro Hagiwara, Geraldine S. Johnson, Gary Lukacik, Kathryn Schmit, and Amy L. Willsey, Division of Epidemiology, New York State Department of Health

References

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(2.) Centers for Disease Control and Prevention. Update: West Nile Virus activity--Eastern United States, 2000. MMWR Morb Mortal Wkly Rep 2000;49:1044-7.

(3.) Centers for Disease Control and Prevention. Outbreak of West Nile-like viral encephalitis--New York, 1999. MMWR Morb Mortal Wkly Rep 1999;48:845-9.

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(6.) Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, Manduca RM, et al. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol 2000;37:208-24.

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(10.) Reinert JF. New classification for the composite genus Aedes (Diptera: Culicidae: Aedini), elevation of subgenus Ochlerotatus to generic rank, reclassification of other subgenera, and notes on certain subgenera and species. J Am Mosq Control Assoc 2000;16:175-88.

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(17.) Vaidyanathan R, Edman JD, Cooper LA, Scott TW. Vector competence of mosquitoes (Diptera: Culicidae) from Massachusetts for a sympatric isolate of eastern equine encephalomyelitis virus. J Med Entomol 1997;34:346-52.

(18.) Fonseca DM, Campbell SR, Crans WJ, Mogi M, Miyagi I, Toma T, et al. Aedes (Finlaya) japonicus (Diptera: Culicidae), a newly recognized mosquito in the United States: Analyses of genetic variation in the United States and putative source populations. J Med Entomol 2001;38:135-46.

Kristen A. Bernard,(*) Joseph G. Maffei,(*) Susan A. Jones,(*) Elizabeth B. Kauffman,(*) Gregory D. Ebel,(*) Alan P. Dupuis II,(*) Kiet A. Ngo,(*) David C. Nicholas,(*) Donna M. Young,(*) Pei-Yong Shi,(*) Varuni L. Kulasekera,([dagger]) Millicent Eidson,(*) Dennis J. White,(*) Ward B. Stone,([double dagger]) NY State West Nile Virus Surveillance Team,(1) and Laura D. Kramer(*)

(*) New York State Department of Health, Albany, New York, USA; ([dagger]) New York City Department of Health, New York, New York, USA; and ([double dagger]) Department of Environmental Conservation, Albany, New York, USA

Address for correspondence: Kristen A. Bernard, Arbovirus Laboratories, Wadsworth Center, 5668 State Farm Road, Slingerlands, NY 12159; fax: [phone]; e-mail: [e-mail]