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Five-year surveillance of West Nile and Eastern equine encephalitis viruses in southeastern Virginia.


Since the appearance of West Nile virus (WNV) in New York during the 1999 season, communities throughout the United States have initiated or enhanced their arbovirus surveillance programs so that they can monitor and better understand the spread of this emerging infection (Andreadis, Anderson, & Vossbrink, 2001; Blackmore et al., 2003; Gubler et al., 2000; Kent, Lacer, & Meisch, 2003; Lanciotti et al., 1999; Nasci et al., 2001). WNV and Eastern equine encephalitis virus (EEE) are capable of causing significant neurological disease and death, especially in the elderly and in immuno-compromised individuals, and viral encephalitis is on the bioterrorism list of the Centers for Disease Control and Prevention (CDC) (CDC, 2004); consequently, monitoring the environment for these pathogens is a vital public health function. Identifying trends in the distribution of the viruses in their endemic and bridge-vector populations and understanding their seasonal cycles will allow for more timely and efficient mosquito control. The trends may also provide health care providers with early warning signs of these deadly arboviral illnesses.

Mosquito pools and chicken sera were collected to determine the occurrence of WNV and EEE in the environment. Chickens were well suited to serve as sentinel birds, because they exhibit a transient viremia that stimulates the production of IgM antibodies against both WNV and EEE (Komar, 2001).

Environmental conditions, especially landscape ecology, temperature, and precipitation, have an important effect on the distribution of the mosquitoes that harbor the arboviruses, thereby influencing seasonal virus activity (Andreadis et al., 2001; Hayes, 1981; Hunter, 2003; Turell, Sandelis, Dohm, & O'Guinn, 2001). Southeastern Virginia (Figure 1), which is characterized by both urban and wetland ecologies, is the home of the Great Dismal Swamp, migratory and permanent bird sanctuaries, and the largest U.S. naval base. The wetlands provide an ideal place for mosquito breeding and therefore arboviral activity, while the bird sanctuaries provide an abundance of hosts for the mosquitoes to feed upon. The large Navy installation may be an attractive target for bioterrorism, and EEE is classified as a select agent for bioterrorism (CDC, 2005). Therefore, monitoring of southeastern Virginia for arboviruses is crucial to public health. Although the mosquito surveillance program has been active in previous years, the enhanced program has been ongoing since 2000. The activity of EEE and WNV will be reported in this study for each mosquito season from 2000 through 2004.

Materials and Methods

Mosquito Pool Processing

From 2000 to 2004, adult mosquitoes were collected between May and November at selected sites in the health districts of Chesapeake, Hampton, Norfolk, and Western Tidewater (including only Suffolk and Isle of Wight). Standard CDC light traps with carbon dioxide (ice and gas) and gravid traps (Hausherr Machine Works, Toms River, New Jersey) were used. Each pool contained up to 50 mosquitoes from the same species, collection site, and date of collection.

Mosquito pools were frozen and processed by addition of four copper-clad BB pellets (Copperhead 6000, Grosman, East Bloomfield, New York) and 1.5 mL of cold Dulbecco's modified Eagle's medium (DMEM) with 2 percent fetal bovine serum (FBS, Sigma, St. Louis, Missouri), according to a standard method described by Lanciotti and co-authors (2000). The supernatants and pellets were stored at -80[degrees]C (Model 5416, Forma Scientific Division, VWR Scientific Products, New York, New York).

Virus Isolation and Cell Cultures

The standard strain of EEE (Laboratory #1048) was isolated from a Cs. melanura mosquito pool in tissue culture on August 1, 2000. The standard strain of WNV (Laboratory #321) was isolated from a bird on November 1, 2000. Both EEE and WNV strains were confirmed by CDC's Division of Vectorborne Infectious Diseases at the National Center for Infectious Diseases in Fort Collins, Colorado. In 2004, a standard strain of WNV NY99 was obtained from CDC. These virus strains and their RNA were used as positive controls for tissue culture, RNA extractions, and reverse transcription polymerase chain reaction (RT-PCR).


Supernatants were tested for viral activity in duplicate on a monolayer of Vero cells in a 48-well microtiter plate and incubated at 35[degrees]C. After 60 to 90 minutes, 500 [micro]L of DMEM with 2 percent FBS was added to each well. The plate was incubated for up to one week so that the cytopathic effects (CPE) could be observed. Supernatants from cultures with CPE were harvested to identify the virus by RT-PCR (Lanciotti et al., 2000).

RNA Extraction, Reverse Transcriptase Polymerase Chain Reaction, and TaqMan Assay

The QIAamp Viral RNA Kit Mini procedure (Qiagen, Inc., Valencia, California) was used to isolate viral RNA from mosquito or Vero culture supernatants according to the manufacturer's procedure. The authors screened mosquito pools for viral RNA in the TaqMan assay with a 96-well plate (Bio-Rad Laboratories, Hercules, California) using the iCycler iQ Multi-Color Real Time PCR Detection System (BIO-RAD Laboratories, Hercules, California). A TaqMan RT-PCR ready-mix kit (PE Applied Biosystems, Foster, City, California) was combined with forward (WNV1160, EEE 9391) and reverse (WNV1229C, EEE 9459C) primers and probes for WNV (1186, 10692) or EEE (EEE probe, 9414, Qiagen Operon, Alameda, California) as designed by Lanciotti and co-authors (2000). Infected mosquito pools were confirmed with a second set of forward and reverse primers, respectively, WNV (10668, 10770C) and EEE (9431, 9452) primers and FAM/TAMRA probes (WNV 10692, EEE 9501c) (Lambert, Martin, & Lanciotti, 2003; Lanciotti et al., 2000).

During the 2000 and 2001 mosquito seasons (May through November), the mosquito pools were screened for viral RNA with the Titan One-Tube RT-PCR kit (Roche Molecular Biochemicals, Mannheim, Germany) and 3.5 percent agarose (NuSieve 3:1, BMA, Rockland, Maine) gel electrophoresis. The WNV primers were WN212, WN619, WN9483, and WN9794, and the EEE primers were DB10 and DB14. The PCR products were stained with 5 x [10.sup.-5] ethidium bromide in Tris Borate EDTA buffer (Sigma) and visualized with the BIO-RAD Gel Documentation System. Positive pools were confirmed by CDC in Fort Collins, Colorado.

The minimal infection rate (MIR), equal to the ratio of virus-infected mosquito pools divided by the number of mosquitoes tested, was calculated for the combined health districts to assure >1,000 mosquitoes for each primary species, according to the method described by Bernard and co-authors (2001).

Sentinel-Chicken Blood Collection

The health districts of Norfolk, Chesapeake, and Hampton (including Langley Airforce Base) submitted chicken blood from 2000 to 2004; Suffolk submitted blood only in 2004. At the beginning of each season (late April or early May), baseline blood from chickens approximately eight weeks old was obtained by each participating locality before the chickens were placed at their respective surveillance sites. Subsequently, 0.5 to 1.5 mL of blood was collected every two weeks from a vein on the ventral side of the wing and refrigerated. The sera were separated from the blood by centrifugation in the laboratory.

Sentinel-Chicken Sera Enzyme Immunoassay

Mouse anti-chicken IgM (Southern Biotechnology Associates, Inc., Birmingham, Alabama) was used to capture the IgM in the chicken sera by a standard ELISA method (Monath, Nystrom, Bailey, Calisher, & Muth, 1984; Olson, Scott, Lorenz, & Hubbard, 1991; Tsai et al., 1987). The antigens were WNV recombinant COS-1 tissue culture antigen (CDC, Fort Collins, Colorado; Focus, Cypress, California) or EEE (Strain NJ/60) mouse brain antigen (CDC), and the antibodies were either the St. Louis encephalitis (SLE) monoclonal antibody 6B6C-1 (CDC, Fort Collins, Colorado; Focus, Cypress, California), which cross-reacts with WNV within the flavivirus family, or the EEE monoclonal antibody 1B5C-3 (CDC) conjugated to horseradish peroxidase. The color change was detected in a microtiter plate reader (ELX 808 Ultramicro Plate Reader, Bio-Tek Instruments, Inc., Winooski,

Vermont) with a 450-nm filter and a reference filter of 630 nm. The positive/negative (P/N) optical-density ratio was calculated, and a P/N [greater than or equal to]2.0 was considered IgM-reactive for EEE or WNV. The authors calculated the seroconversion rate by dividing the number of IgM-reactive chickens by the total number of chickens tested.

Environmental Conditions

The average monthly temperature and precipitation in Norfolk, Virginia, from May through October, 2001-2004, were obtained from Mr. Scott Schumann at the National Weather Service Forecast Office in Wakefield, Virginia.


Mosquito Species with Arboviral Infections

For five years, 14,150 mosquito pools were screened for WNV and 14,949 for EEE, respectively. These pools included 23 different species of mosquitoes. Overall the most common enzootic vectors of EEE and WNV in southeastern Virginia were Cs. melanura and Cx. pipiens, respectively. Cs. melanura pools greatly outnumbered the Cx. pipiens pools for all the seasons; the latter increased in number every year until 2003 and then declined in 2004. The lowest number of Cs. melanura pools were tested in 2002.

Table 1 lists the total number of pools submitted each year for the vector species of WNV and EEE. Except in 2002, Cs. melanura was the mosquito species most commonly submitted for yearly arboviral screening. In 2002, more pools of Ae. albopictus and Ochlerotatus taeniorhynchus were collected than were pools of Cs. melanura. The primary enzootic WNV vector was Cx. pipiens, and the primary enzootic EEE vector was Cs. melanura.

Overall, Cs. melanura and Cx. pipiens were the primary vectors for EEE and WNV. In 2000, 2003, and 2004, the bridge vectors Cx. salinarius, Ae. vexans, and Anopheles crucians contributed to the arboviral activity. The number of Cs. melanura pools submitted for testing decreased in 2001 and 2002.

The Yearly Occurrence of Arboviruses in Southeastern Virginia

The minimum infection rates (MIRs) of WNV in Cx. pipiens and EEE in Cs. melanura for each year are summarized in Table 2. The peak MIR for EEE (0.9, Cs. melanura) occurred in 2001, with a decline in 2002 and a decline in 2004. The peak MIR for WNV (2.7, Cx. pipiens) occurred in 2002, with a decline in 2003 (0.64) and an upsurge in 2004 (1.8). The 2.8-fold increase in the MIR of WNV in Cx. pipiens from 2003 to 2004 was balanced by the decrease in WNV-infected Cs. melanura observed in 2004, because the peak WNV infections in Cs. melanura (MIR = 0.24) occurred in 2003 and then declined threefold in 2004 (MIR = 0.07).

The MIR was dependent on the total number of mosquitoes tested for WNV, EEE, or both. The number of mosquitoes varied markedly during the five-year period. The total numbers of Cx. pipiens tested for WNV during the five-year period were 1,011 (2000), 1,814 (2001), 2,254 (2002), 12,492 (2003), and 8,200 (2004) respectively. The total numbers of Cs. melanura tested for both EEE and WNV were 60,707, 30,093, 3,919, and 73,848, from 2000 through 2003, while in 2004 the numbers were 140,226 for EEE and 100,832 for WNV. In 2002, very few Cs. melanura were available for testing, while the number of Cx. pipiens was comparable to that in the previous year, but still only one-sixth of that in 2003. The number of Cx. pipiens that were screened increased an average of eightfold from 2000 to 2004.

The occurrence of sentinel chickens infected with the arboviruses from 2000 through 2004 was determined by the IgM seroconversion percentages (Table 2). Unlike the MIR in the mosquito pools, both WNV and EEE activity in sentinel chickens peaked in 2003 and declined in 2004. The seroconversion percentage in 2004 was, however, greater than the percentages in 2000 through 2002.


The Time Course of Arboviral Activity

The number of mosquito pools and chicken seroconversions for each season were examined monthly to determine the trend of WNV and EEE infections. The numbers of WNV- and EEE-infected mosquito pools are shown in Figure 2a and Figure 2b, respectively. Peak WNV activity occurred in August in 2003 and 2004 and in October in 2002 (Figure 2a). Only one WNV-infected mosquito pool was detected in 2001, in September. By contrast, peak EEE activity was observed in July in three years--2001, 2003, and 2004 (Figure 2b). In 2004, the EEE activity extended into November.



Similar to the peak mosquito infections, the highest WNV activity in sentinel chickens occurred in August during the 2003 and 2004 seasons (Figure 3a). The activity in sentinel chickens extended into September, however, and in 2003 lingered into November. The peak level of seroconversions for EEE occurred in July in 2001-2003 and in August in 2004 (Figure 3b). This result is consistent with the earlier appearance of EEE than WNV in mosquito pools (Figure 2a, Figure 2b).

The variation in arboviral activity is reflected in the changes in the average monthly temperature and rainfall during 2001-2004 (Table 3). Very little rainfall was observed in the fall of 2001 (October rainfall was the lowest in four years) and again in May-August of 2002. Very wet summers were recorded in 2003 and 2004; in the fall of 2004, the precipitation dropped dramatically. The temperatures in May 2004 and October 2002 were higher than normally observed for these months for the period 2001-2004; in August 2004, the average temperature was 3[degrees]F to 4[degrees]F lower than in previous years. The cooler temperature in August 2004 coincided with the peak level of IgM seroconversions for EEE.


The longitudinal surveillance study reported here compared the occurrence rates and time courses of WNV and EEE infections in mosquitoes and sentinel chickens. This report is the first of its kind from southeastern Virginia. The results indicate that EEE was endemic in southeastern Virginia from 2000 and was transmitted primarily via the enzootic vector Cs. melanura. This finding could be attributed to the abundant wetland regions and bird sanctuaries located in southeastern Virginia and is consistent with previous reports from other eastern states (Chamberlain, 1958; Chamberlain, Sudia, Coleman, Johnston, & Work, 1969; Hayes, 1981; Howard & Wallis, 1974).

After peak EEE infectivity in Cs. melanura during the summer of 2001, the drought in the fall of 2001 and the summer of 2002 dramatically reduced the number of Cs. melanura pools and their EEE infectivity. The variation in rainfall and temperature affected the availability of mosquito populations that could maintain the enzootic transmission cycle (Hayes & Hess, 1964). Drought has been reported to disrupt any zoonotic outbreak of EEE by interfering with Cs. melanura development (Komar & Spielman, 1994). In one study, a drought in the spring followed by a wet summer was an indicator for high WNV transmission (Shaman, Day, & Stieglitz, 2005). In Florida, when little water was available, the bird and mosquito populations were brought together seeking water. Thus the risk of WNV transmission was increased.

Although Ae. vexans had been implicated as a bridge vector for EEE (Hayes, 1981), the authors did not identify this mosquito species as a vector for EEE in southeastern Virginia. With abundant rainfall in the fall of 2002 and in the summers of 2003 and 2004, including a hurricane in September of 2003, Cs. melanura flourished, maintaining the enzootic transmission cycle of EEE.

The highest WNV infection rates in Cs. melanura occurred in 2003, with a marked reduction in 2004. In 2002, Cx. pipiens was the primary enzootic vector of WNV in southeastern Virginia (no Cs. melanura pools tested positive for WNV). These findings were consistent with those of previous reports, from the northeastern United States (Andreadis et al., 2000; Bernard et al., 2001). Aedes vexans and Cx. salinarius were also implicated as bridge vectors for WNV (Hayes, 1981; Moncayo & Edman, 1999; Turell et al., 2001; Vaidyanathan, Edman, Cooper, & Scott, 1997), and WNV-infected pools of these species were detected in southeastern Virginia. Although the potential for a major epizootic transmission cycle existed in southeastern Virginia, the data reported here did not support the occurrence of one during the five-year study period. Continued mosquito control practices in southeastern Virginia contributed to these results.

The appearance of WNV infection in Cx. pipiens in 2004 at the level of infection in Cs. melanura in 2003 might indicate a shift in vector preference or availability in 2004 in southeastern Virginia. It may also be the result of all districts more consistently using gravid traps for Cx. pipiens. Since Cx. pipiens had been shown to be a potential bridge vector (Turell et al., 2005), this shift might increase the risk of epizootic transmission in future seasons.

In 2002, the number of Cx. pipiens trapped and screened was similar to the number in 2001, but only one-sixth of the number in 2003. This result might be attributed to less-than-average rainfall during the summer months of 2002, as compared with rainfall in 2003. Another factor was that not all the health districts used gravid traps consistently during the 2000-2002 seasons. Whether the dramatic increase in the virus activity was merely a result of increased precipitation in 2003 and 2004 (Hayes, 1981), or an indication of a permanent increase in the distribution of these viruses in southeastern Virginia, will be determined in the next few years. The mounting evidence that arboviruses persist in their vertebrate hosts during the winter (Kuno, 2001) would suggest the latter. When the MIRs from 2000 to 2003 were compared, a fivefold (0.1 to 0.56) increase in EEE activity was observed, with a comparable number of mosquitoes tested. This result might be in part due to improved molecular methods of virus detection.


Peak EEE virus infections in southeastern Virginia were generally observed before peak infections of WNV. WNV was detected later in the summer, peaking in August, at a time when Cx. pipiens (the primary enzootic vector of WNV) were most abundant (Turell et al., 2001).

The immune status of the wild bird population against arbovirus infection is the other variable to consider. The IgM seroconversion percentages of sentinel chickens indicate that the peak arboviral activity occurred in 2003. This result did not coincide with the peak WNV MIR among Cx. pipiens in 2002 or the peak EEE MIR in Cs. melanura, which was recorded in 2001. The number of Cs. melanura pools tested was markedly reduced in 2002, indicating that fewer mosquitoes were available to maintain the transmission cycle. Hence, fewer birds, including the sentinel chickens, would have been exposed to EEE or WNV. The number of Cs. melanura pools increased dramatically in 2003, however, and the study findings for the sentinel chickens indicate that more birds were exposed to arboviruses. In 2004, the wild bird population had greater herd immunity because of the 2003 exposures (Chamberlain, 1958), and therefore bird infectivity was reduced.

In conclusion, the EEE and WNV surveillance program demonstrated an increase in the incidence of arbovirus infections in mosquitoes and sentinel chickens in southeastern Virginia from 2000. The primary enzootic vector of EEE was Cs. melanura, and that of WNV was Cx. pipiens. Both rainfall and temperature variations affected the vector populations and the arbovirus infection rates in southeastern Virginia.

Acknowledgements: The authors would like to acknowledge technical contributions related to mosquito trapping and sera collection by the Tidewater Regional Arbovirus Surveillance Team, as well as the processing and sample testing done by laboratory personnel: Sharon Rittman, Bridget Dalton, Woodnard Givens, Barbara A. McCormick, Josephine Eballar, Virginia Chan, Evelyn Engada. This work would not have been possible without their dedicated support. Thanks also are due to Dr. Lanciotti for RT-PCR confirmation of infection in mosquito pools in 2000 and 2001. In addition, the authors thank Dr. Michael Brown for his comments about the manuscript. The study was partially supported by a West Nile surveillance grant from CDC through the Virginia Department of Health.

Corresponding Author: Dongxiang Xia, Director of the Norfolk Public Health Laboratory, Commonwealth of Virginia, 830 Southampton Ave., Norfolk, VA 23510. E-mail:


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Karin C. Loftin, Ph.D.

Alpha A. Diallo, Ph.D.

Marcia W. Herbert

Priyarshadan G. Phaltankar, M.Sc.

Christine Yuan, M.P.H.

Norman Grefe

Agnes Flemming

Kirby Foley

Jason Williams, M.S.

Sandra L. Fisher, M.P.H.

Michael Elberfeld

Juan Constantine

Mitchell Burcham

Valerie Stallings, M.D., M.P.H.

Dongxiang Xia, M.D., Ph.D.
TABLE 1 Number of Pools Submitted Each Year for WNV and EEE Vector

 Number of Pools Percentage of WNV-Positive
Year Genus and Species Submitted Pools Submitted Pools

2000 Culiseta melanura 1,293 59.5% 0
 Anopheles crucians 144 6.6% 0
2001 Culiseta melanura 714 36.3% 1
2002 Culex pipiens 190 9.9% 6
 Culiseta melanura 125 6.5% 0
2003 Culiseta melanura 1,634 35.7% 18
 Culex pipiens 394 8.6% 7
 Culex salinarius 730 15.9% 1
 Aedes vexans 350 7.6% 1
2004 Culiseta melanura 3,015 66.1% 5
 Culex pipiens 262 5.7% 15
 Culex salinarius 677 14.8% 1

 WNV- and EEE-Positive
Year Genus and Species EEE-Positive Pools Pools

2000 Culiseta melanura 6 0
 Anopheles crucians 1 0
2001 Culiseta melanura 27 0
2002 Culex pipiens 0 0
 Culiseta melanura 2 0
2003 Culiseta melanura 40 1
 Culex pipiens 0 0
 Culex salinarius 1 0
 Aedes vexans 0 0
2004 Culiseta melanura 50 2
 Culex pipiens 0 0
 Culex salinarius 0 0

TABLE 2 Yearly Occurrence of WNV and EEE Activity in Mosquito Pools and
Sentinel Chickens, 2000-2004

Virus Activity in Mosquito Pools
 Number of Number of
 WNV-Positive EEE-Positive
Year Pools Cx. pipiens (MIR) Pools Cs. melanura (MIR)

2000 0 0 7 0.1
2001 1 0.03 27 0.9
2002 6 2.7 2 0.5
2003 28* 0.64 42* 0.56
2004 23** 1.8 52** 0.37

Virus Activity in Sentinel Chickens
 Number of Chicken Number of Chicken
 WNV-Positive Seroconversion EEE-Positive Seroconversion
Year Chickens Percentage Chickens Percentage

2000 0 0% 5 6.8%
2001 0 0% 5 5.1%
2002 5 5.6% 0 0%
2003 49** 51% 21** 22%
2004 28** 24% 15** 13%

*One mosquito pool reacted with both WNV and EEE.
**Two mosquito pools and two chickens reacted with both WNV and EEE.

TABLE 3 Average Temperature and Precipitation

Temperature ([degrees]F)
Year May June July August September October

2001 66.5 75.8 76.3 78.8 70.4 61.3
2002 66.7 76.8 80.8 79.5 74.1 64.4
2003 65.7 75.0 80.1 80.3 74.1 62.0
2004 73.1* 75.1 79.2 76.0 72.6 60.9

Precipitation (Inches)
Year May June July August September October

2001 2.89 6.91 4.40 4.21 2.46 0.75
2002 3.50 4.28 3.59 4.27 6.69 6.55
2003 4.66 3.43 8.56 6.08 9.54 3.91
2004* 4.67 4.86 10.89 11.11 3.3 1.88

*The 2nd warmest May and 3rd wettest summer ever recorded in Norfolk.
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Author:Xia, Dongxiang
Publication:Journal of Environmental Health
Geographic Code:1U5VA
Date:May 1, 2006
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