Year-round West Nile Virus activity, Gulf Coast region, Texas and Louisiana.

West Nile virus (WNV) was detected in 11 dead birds and two mosquito pools collected in east Texas and southern Louisiana during surveillance studies in the winter of 2003 to 2004. These findings suggest that WNV is active throughout the year in this region of the United States.

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Since the initial recognition of West Nile virus (WNV) in North America in 1999 (1), one question that has perplexed epidemiologists and public health officials is bow the virus persists during the winter in temperate regions. Arbovirologists and vector biologists have long pondered how arboviruses are maintained during periods when their vectors are absent or inactive (2-5).

For WNV, little information is available on how the virus is maintained in North America during cold periods, when little or no adult mosquito activity occurs. In the winter after the initial 1999 West Nile outbreak in the northeastern United States, Nasci et al. (6) reported detecting West Nile viral RNA and infectious virus in hibernating adult Culex mosquitoes collected from underground sanitation tunnels, vacant buildings, and other protective structures in New York City. During the same month (February 2000), another group (7) reported isolating WNV from tissues of a freshly dead hawk found in Westchester County just north of the city. These two observations suggested that, in northern latitudes, WNV may be maintained locally in hibernating Culex mosquitoes, as demonstrated earlier for St. Louis encephalitis virus (8), but that low-level virus transmission may also occur during winter.

The Study

WNV was initially detected in the Houston metropolitan area (Harris County) in the summer of 2002; 105 confirmed human infections with WNV were reported in Houston during the first year (9). In addition, 307 WNV isolates were obtained from dead birds, and 851 WNV-positive pools of ex. pipiens quinquefasciatus were collected during our surveillance studies in the summer and fall of 2002 (9). In 2003, we began a long-term study on the ecology of WNV in Harris County. As part of this program, mosquito and dead bird collections were made by Harris County mosquito control personnel throughout the year. Mosquito collections were made at selected sites throughout the county, with CDC-type light traps and gravid traps (9). Our trapping methods were designed to sample mainly Cx. p. quinquefasciatus; this species represented >95% of the mosquitoes collected. After collection, mosquitoes were sorted into pools of <50 females (mean pool size 28.8) and assayed for WNV by an antigen-capture enzyme immunoassay (EIA) (9). Selected EIA-positive mosquito pools (including all winter positives) were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), as described previously (10).

After the initial detection of WNV in Houston in 2002, the Harris County mosquito control personnel established a dead bird surveillance system (9); media reports and public messages instructed county residents to report dead birds, most of which were picked up by the county mosquito control personnel. After collection and species identification, the bird carcasses were frozen at -75[degrees]C for subsequent transport to the University of Texas Medical Branch in Galveston, where a sample of brain from each dead bird was cultured for WNV. Culture methods and tests used for virus confirmation were described earlier (9,10). During 2003 and January 2004, the University of Texas Medical Branch group also received a few dead birds from mosquito control districts in Galveston County, Texas, and Louisiana. These avian samples were processed as described above, but are listed separately from the Harris County collections.

Conclusions

Table 1 summarizes the monthly WNV surveillance results for dead birds and mosquitoes tested from Harris County from January 2003 to March 2004. During this 15-month period, 1,623 dead birds, representing 83 avian species, were examined. The number and species of dead birds examined each month varied, reflecting seasonal changes in local avian abundance and deaths, interest of the local citizens in reporting dead birds, winter bird migration into the region, and limits in our ability to process samples. For example, in June, July, and August 2003, a total of 3,352 dead birds were reported by local residents to the county mosquito control staff. During this period, we limited the number of birds tested for WNV to approximately 50 per week, and an attempt was made to sample birds from a variety of different sites within the county. Thus in these 3 months, only 588 dead birds (approximately 17% of the total reported for the period) were actually tested. Also during this period, corvids (Blue Jays and crows) were preferentially selected to be tested, since our experience indicated that these species were most likely to yield virus (9). In contrast, during the winter months (November, December, January, February, and March) fewer dead birds were reported; most of these birds were tested, regardless of species or collection locality. For this reason, Blue Jays (the most common species sampled) represented 35.9% of the birds tested during June, July, mid August 2003 but only 7.7% of the birds tested during the months of November, December, January, February, and March.

A similar seasonal bias occurred in our mosquito sampling. During the hottest months of the year (June, July, and August) in Harris County, large numbers of mosquitoes were collected, exceeding our capacity to test them. Thus only a subsample of the mosquitoes collected during this period were tested for WNV. In contrast, during the winter months, adult Cx. p. quinquesfasciatus abundance and activity were markedly reduced. During this period, most of the mosquitoes that were collected in traps were assayed for WNV. Our results provide information about the seasonal pattern of WNV activity in the western Gulf region, despite the sampling bias.

Table 2 shows the species composition and WNV infection rates of dead birds collected in Harris County from January 2003 to March 2004. Overall, Blue Jays were the most common dead birds submitted for testing and represented 23.2% of the total; 48.9% of the dead Blue Jays yielded virus. Only 23 American Crows were submitted for WNV testing, but 16 (69.6%) of them were virus-positive upon culture. Crows are much less abundant than Blue Jays in urban areas of the county. Mourning Doves were another commonly submitted dead bird (17.1% of total), but only 2.2% of this species yielded WNV after culture.

As indicated in Table 1, most of the WNV-positive dead birds and mosquitoes from the county were collected during the summer months of June, July, and August. These months are also the three warmest in Harris County (11). However, WNV was also detected in birds and mosquitoes during most other months. Table 3 summarizes the WNV-positive samples identified in our laboratories from November 2003 to March 2004. Ten WNV-positive bird or mosquito samples were from Harris County; the other three positive dead bird samples were submitted from Galveston County, Texas, and Iberia Parish, Louisiana. These isolations of WNV from dead birds and the identification of viral RNA in physiologically active adult mosquitoes collected during the winter season (November-March) imply that the virus is active year-round in Harris County (Table 1) and the western Gulf region. The wide geographic distribution of localities yielding infected birds and mosquitoes also suggests that virus was not restricted to a single community or site but was widespread. Our data from Harris County indicate that peak virus activity occurred mainly during the warm months of the year (June-September), as observed elsewhere in North America (12), but that low-level virus activity continued during the rest of the year in this region. Prolonged periods with temperatures <0[degrees]C are uncommon in Harris County and the western Gulf Coast, so Cx. p. quinquefasciatus (the presumed vector of WNV in the region) does not enter a true diapause, as does its northern counterpart, (2. pipiens (13). Our field observations in Harris County indicate that Cx. p. quinquefaciatus adults become relatively inactive during cold periods, resting under buildings and in storm drains and sewers; however, these mosquitoes become active again during warm periods in the winter months. The fact that adults can be captured in light traps and eggs laid in gravid traps throughout the year in the county (Table 1) is evidence of their continual activity. This intermittent host-seeking activity throughout the winter probably accounts for continued low-level WNV transmission and infection in the resident avian population. On the basis of these observations, we believe that this mechanism is probably the principal one by which WNV overwinters and persists in the western Gulf region of the United States.

The results of our study also confirm observations by others (14) that surveillance of dead birds is a sensitive method for detecting early WNV activity. The use of sentinel animals (in this case dead birds) is a well-established method of arbovirus surveillance and sometimes detects virus activity during periods when none can be detected in mosquitoes (15). The presumed increased sensitivity of dead bird surveillance may explain why a few bird isolates of WNV were obtained each month during winter, but no virus activity was detected in Cx. p. quinquefaciatus during the same period.

Table 1. Monthly summary of dead birds and mosquitoes tested for West
Nile virus (WNV) in Harris County, Texas

                                               Total       No. WNV-
                           Mean                birds       positive
Year/month     temperature ([degrees]C) (a)    tested    (% positive)

2003
  January                  10.6                    46         0
  February                 12.5                    62         0
  March                    16.7                    80         0
  April                    21.4                    98      1 (1.0)
  May                      27.1                   213      4 (1.9)
  June                     27.9                   219     33 (15.1)
  July                     26.2                   205     72 (35.1)
  August                   28.9                   164     83 (50.6)
  September                25.5                   127     38 (29.9)
  October                  22.2                    73     11 (15.1)
  November                 18.7                    20       2 (10)
  December                 12.6                     7      1 (14.3)
2004
  January                  13.2                    26      1 (3.8)
  February                 12.3                   146      3 (2.1)
  March                    19.5                   137      1 (0.7)

Total                       --                  1,623     250 (15.4)

                 Total            No. WNV-
               mosquitoes         positive          WNV minimum
Year/month       tested      mosquito pools (b)    infection rate

2003
  January         2,164               0                 0.00
  February        1,146               0                 0.00
  March           5,304               0                 0.00
  April          39,000               0                 0.00
  May            58,698               2                 0.03
  June           42,041              40                 0.95
  July           54,582             203                 3.72
  August         40,184             128                 3.19
  September      34,691              21                 0.61
  October        41,465               4                 0.10
  November        7,562               2                 0.13
  December        8,411               0                 0.00
2004
  January        12,816               0                 0.00
  February        9,790               0                 0.00
  March          14,714               0                 0.00

Total           372,568             400                 1.07

(a) Mean monthly temperature in Houston (Harris County) (11).

(b) Mean pool size was 28.2 mosquitoes/pool.

Table 2. West Nile virus-species infection rates among 248
culture-positive dead birds collected in Harris County, Texas,
January 2003-March 2004

Common name                    Scientific name       Total tested

Blue Jay                     Cyanocitta cristata         376
American Crow               Corvus brachyrhynchos         23
Loggerhead Shrike            Lanius ludovicianus          14
House Sparrow                 Passer domesticus          119
Northern Mockingbird          Mimus polyglottus           99
Mourning Dove                 Zenaida macroura           278
Rock Dove                       Columba livia             48
Inca Dove                      Columbina inca             38
Great-crested Flycatcher     Myiarchus crinitus            1
Carolina Chickadee           Parus carolinensis            3
Tufted Titmouse              Baeolophus bicolor            2
Common Grackle               Quiscalus quiscula           84
Orchard Oriole                 Icterus spurius             3
American Goldfinch            Carduelis tristis            1

Common name                 No. infected (%)

Blue Jay                       184 (48.9)
American Crow                   16 (69.6)
Loggerhead Shrike                7 (50)
House Sparrow                   19 (16.0)
Northern Mockingbird             8 (8.1)
Mourning Dove                    6 (2.2)
Rock Dove                        1 (2.1)
Inca Dove                        1 (2.6)
Great-crested Flycatcher         1 (100)
Carolina Chickadee               1 (33.3)
Tufted Titmouse                  1 (50)
Common Grackle                   3 (3.6)
Orchard Oriole                   1 (33.3)
American Goldfinch               1 (100)

Table 3. Confirmed West Nile virus (WNV) activity in birds and
mosquitoes collected in east Texas and southern Louisiana

Date collected         WNV-positive sample

Nov. 3, 2003                 Blue Jay
Nov. 7, 2003                 Blue Jay
Nov. 14, 2003                Blue Jay
Nov. 18, 2003                Blue Jay
Nov. 20, 2003     Culex p. quinquefasciatus pool
Nov. 20, 2003      Cx. p. quinquefasciatus pool
Dec. 16, 2003             American Crow
Jan. 2, 2004            Northern Cardinal
Jan. 22, 2004                Blue Jay
Feb. 12, 2004           American Goldfinch
Feb. 12, 2004             American Crow
Feb. 19, 2004           Loggerhead Shrike
Mar. 4, 2004              American Crow

Date collected               Locality

Nov. 3, 2003       Santa Fe, Galveston Co., TX
Nov. 7, 2003       Dickinson, Galveston Co., TX
Nov. 14, 2003        Houston, Harris Co., TX
Nov. 18, 2003        Pasadena, Harris Co., TX
Nov. 20, 2003        Houston, Harris Co., TX
Nov. 20, 2003        Houston, Harris Co., TX
Dec. 16, 2003         Spring, Harris Co., TX
Jan. 2, 2004      New Iberia, Iberia Parish, LA
Jan. 22, 2004        Tomball, Harris Co., TX
Feb. 12, 2004        Houston, Harris Co., TX
Feb. 12, 2004        Kingwood, Harris Co., TX
Feb. 19, 2004        Houston, Harris Co., TX
Mar. 4, 2004         Kingwood, Harris Co., TX

This work was supported in part by contracts from the National Institutes of Health (NO I-AI25489 and NO I-AI30027) and the Centers for Disease Prevention and Control (U90/CCU 620539 and U50/CCU620539).

References

(1.) Nash D, Mostashari F, Fine A, Miller J, O'Leary D, Murray K, et al. The outbreak of West Nile infection in the New York City area in 1999. N Engl J Med. 2001;344:1807-14.

(2.) Reeves WC. Overwintering of arthropod-borne viruses. Progr Med Virol. 1961;3:59-78.

(3.) Tesh RB. Transovarial transmission of arboviruses in their invertebrate vectors. In: Harris KG, editor. Current topics in vector research, Vol. 2. New York: Praeger Scientific; 1984. p. 57-76.

(4.) Reeves WC. Overwintering of arboviruses. In: Reeves WC, Asman SM, Hardy JL, Milby MM, Reisen WK, editors. Epidemiology and control of mosquito-borne arboviruses in California, 1943-1987. Sacramento: California Mosquito and Vector Control Association, Inc; 1990. p. 357 82.

(5.) Reiter P. Weather, vector biology and arboviral recrudescence. In: Monath TP, editor. The arboviruses: epidemiology and ecology, Vol. 1. Boca Raton (FL): CRC Press: 1988, p. 245-55.

(6.) Nasci RS, Savage HM, White DJ, Miller J, Cropp BC, Godsey MS, et al. West Nile in overwintering Culex mosquitoes, New York City, 2000. Emerg Infect Dis. 2001;7:742-4.

(7.) Garmendia AE, Van Kruiningen HJ, Franch RA, Anderson JF, Andreadis TG, Kumar A, et al. Recovery and identification of West Nile virus from a hawk in winter. J Clin Microbiol. 2000;38:3110-1.

(8.) Bailey CL, Eldridge BF, Hayes DE, Watts DM, Tammariello RF, Dalrymple JM. Isolation of St. Louis encephalitis virus from over-wintering Culex pipiens mosquitoes. Science. 1978;199:1346-9.

(9.) Lillibridge KM, Parsons R, Randle Y, Travassos da Rosa APA, Guzman H, Siirin M, et al. The 2002 introduction of West Nile virus into Harris County, Texas, an area historically endemic for St. Louis encephalitis. Am J Trop Med Hyg. 2004;70:676-81.

(10.) Davis CT, Beasley DWC, Guzman H, Pushker R, D'Anton M, Novak RJ, et al. Genetic variation among temporally and geographically distinct West Nile virus isolates, United States, 2001, 2002. Emerg infect Dis. 2003;9:1423-9.

(11.) National Climatic Data Center. Climate at a glance: Houston, TX, 2004. [cited Jun 29, 2004]. Available from: http://www.ncdc. noaa.gov/oa/climate/research/caz/V1.html

(12.) Campbell GL, Martin AA, Lanciotti RS, Gubler DJ. West Nile virus. Lancet Infect Dis. 2002;2:519-29.

(13.) Spielman A. Structure and seasonality of nearctic Culex pipieux populations. Ann N Y Acad Sci. 2001;951:220-33.

(14.) Guptill SC, Julian KG. Campbell GL, Price SD, Marfin AA. Early-season avian deaths from West Nile virus as warnings of human infection. Emerg Infect Dis. 2003;9:483-4.

(15.) Milby MM, Reeves WC. Natural infection in vertebrate hosts other than man. In: Reeves WC, Asman SM, Hardy JL, Milby MM, Reisen WK, editors. Epidemiology and control of mosquito-borne arboviruses in California, 1943-1987. Sacramento: California Mosquito and Vector Control Association, Inc.; 1990. p. 26-65.

Robert B. Tesh, * Ray Parsons, ([dagger]) Marina Siirin, * Yvonne Randle, ([dagger]) Chris Sargent, ([dagger]) Hilda Guzman, * Taweesak Wuithiranyagool, ([dagger]) Stephen Higgs,* Dana L. Vanlandingham, * Adil A. Bala, ([dagger]) Keith Haas, ([double dagger]) and Brian Zerinque ([section])

* University of Texas Medical Branch, Galveston, Texas, USA: ([dagger]) Harris County Public Health and Environmental Services, Houston, Texas, USA; ([double dagger]) Galveston County Mosquito Control District, Dickinson, Texas, USA; and ([section]) Mosquito Control Contractors, Inc., New Iberia, Louisiana, USA

Dr. Tesh is professor of pathology at the University of Texas Medical Branch in Galveston. His research interests include the epidemiology and pathogenesis of arthropodborne and emerging zoonotic viral diseases.

Address for correspondence: Robert B. Tesh. Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA; fax: [phone]; email: [e-mail]