• View in gallery

    Mean daily viremia titers plotted for three groups of eight house sparrows, with each group infected with a different strain of West Nile virus. Strains are coded as follows: New York (NY99) = solid black line; Kenya (KEN) = solid gray line; Australia (Kunjin [KUN]) = dashed line. Error bars represent standard deviations of the mean. pfu = plaque-forming units.

  • View in gallery

    Survival of house sparrows as a function of infection with West Nile virus (WNV) strain type. The number of house sparrows that survived WNV infection is plotted by day post-inoculation (dpi) for each of three strains. No mortality occurred after 8 dpi. All birds were euthanized at 14 dpi. All eight sparrows that were mock inoculated with diluent, and blood sampled on the same schedule as the other groups, survived the 14-day period of observation. The survival profiles for each strain are coded as follows: New York (NY99) = solid black line; Kenya (KEN) = solid gray line; Australia (Kunjin [KUN]) = dashed line.

  • 1

    Komar N, 2003. West Nile virus: Epidemiology and ecology in North America. Adv Vir Res 61 :185–234.

  • 2

    Malkinson M, Banet C, Weisman Y, Pokamunski S, King R, Drouet MT, Deubel V, 2002. Introduction of West Nile virus in the Middle East by migrating white storks. Emerg Infect Dis 8 :392–397.

    • Search Google Scholar
    • Export Citation
  • 3

    Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, McLean R, West Nile Virus Avian Mortality Surveillance Group, 2001. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis 7 :615–620.

    • Search Google Scholar
    • Export Citation
  • 4

    Taylor RM, Work TH, Hurlbut HS, Rizk F, 1956. A study of the ecology of West Nile virus in Egypt. Am J Trop Med Hyg 5 :579–620.

  • 5

    Work TH, Hurlbut HS, Taylor RM, 1955. Indigenous wild birds of the Nile Delta as potential West Nile circulating reservoirs. Am J Trop Med Hyg 4 :872–878.

    • Search Google Scholar
    • Export Citation
  • 6

    Komar N, Panella NA, Burns JE, Dusza SW, Mascarenhas TM, Talbot TO, 2001. Serologic evidence for West Nile virus infection in birds in the New York City vicinity during an outbreak in 1999. Emerg Infect Dis 7 :621–625.

    • Search Google Scholar
    • Export Citation
  • 7

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 8

    Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ, 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286 :2333–2337.

    • Search Google Scholar
    • Export Citation
  • 9

    Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, Lanciotti RS, Peters CJ, 2000. First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley Province, Kenya. Am J Trop Med Hyg 62 :240–246.

    • Search Google Scholar
    • Export Citation
  • 10

    Beaty BJ, Calisher CH, Shope RE, 1995. Arboviruses. Lennette EH, Lennette DA, Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. Seventh edition. Washington, DC: American Public Health Association, 189–212.

  • 11

    Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, Bowen M, McKinney N, Morrill WE, Crabtree MB, Kramer LD, Roehrig JT, 2002. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 298 :96–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Charrel RN, Brault AC, Gallian P, Lemasson JJ, Murgue B, Murri S, Pastorino B, Zeller H, de Chesse R, de Micco P, de Lamballerie X, 2003. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe. Virology 315 :381–388.

    • Search Google Scholar
    • Export Citation
  • 13

    Scherret JH, Poidinger M, Mackenzie JS, Broom AK, Deubel V, Lipkin WI, Briese T, Gould EA, Hall RA, 2001. The relationship between West Nile and Kunjin viruses. Emerg Infect Dis 7 :697–705.

    • Search Google Scholar
    • Export Citation
  • 14

    Beasley DW, Li L, Suderman MT, Barrett AD, 2001. West Nile virus strains differ in mouse neurovirulence and binding to mouse or human brain membrane receptor preparations. Ann N Y Acad Sci 951 :332–335.

    • Search Google Scholar
    • Export Citation
  • 15

    Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff B, Miller BR, Komar N, 2004. West Nile viral strains exhibit differential virulence for American crows (Corvus brachyrhynchos). Emerg Infect Dis 10: (in press).

  • 16

    Hayes CG, 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci 951 :25–37.

  • 17

    Eidson M, Kramer L, Stone W, Hagiwara Y, Schmit K, New York State West Nile Virus Avian Surveillance Team, 2001. Dead bird surveillance as an early warning system for West Nile virus. Emerg Infect Dis 7 :631–635.

    • Search Google Scholar
    • Export Citation
  • 18

    Eidson M, Miller J, Kramer L, Cherry B, Hagiwara Y, West Nile Virus Bird Mortality Analysis Group, 2001. Dead crow densities and human cases of West Nile virus, New York State, 2000. Emerg Infect Dis 7 :662–664.

    • Search Google Scholar
    • Export Citation
  • 19

    Julian KG, Eidson M, Kipp AM, Weiss E, Petersen LR, Miller JR, Hinten SR, Marfin AA, 2002. Early season crow mortality as a sentinel for West Nile virus disease in humans, northeastern United States. Vector Borne Zoonotic Dis 2 :145–155.

    • Search Google Scholar
    • Export Citation
  • 20

    Mostashari F, Kulldorff M, Hartman JJ, Miller JR, Kulasekera V, 2003. Dead bird clusters as an early warning system for West Nile virus activity. Emerg Infect Dis 9 :641–646.

    • Search Google Scholar
    • Export Citation
  • 21

    Guptill SC, Julian KG, Campbell GL, Price SD, Marfin AA, 2003. Early-season avian deaths from West Nile virus as warnings of human infection. Emerg Infect Dis 9 :483–484.

    • Search Google Scholar
    • Export Citation
  • 22

    Theophilides CN, Ahearn SC, Grady S, Merlino M, 2003. Identifying West Nile virus risk areas: the Dynamic Continuous-Area Space-Time system. Am J Epidemiol 157 :843–854.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

VARIATION IN VIRULENCE OF WEST NILE VIRUS STRAINS FOR HOUSE SPARROWS (PASSER DOMESTICUS)

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  • 1 Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado; Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado

The observation of avian mortality associated with West Nile virus (WNV) infection has become a hallmark epidemiologic feature in the recent emergence of this pathogen in Israel and North America. To determine if phenotypic differences exist among different WNV isolates, we exposed house sparrows (Passer domesticus) to low passage, lineage 1 WNV strains from North America (NY99), Kenya (KEN), and Australia (KUN; also known as Kunjin virus). House sparrows inoculated with the NY99 and KEN strains experienced similar mortality rates and viremia profiles. The KUN strain elicited significantly lower-titered viremia when compared with the other strains and induced no mortality. This study suggests that natural mortality in house sparrows due to Old World strains of WNV may be occurring where the KEN strain occurs.

INTRODUCTION

West Nile virus (WNV) is a mosquito-borne flavivirus (Flaviviridae) that has emerged as an important human, veterinary, and wildlife health threat. Avian hosts are the primary vertebrate reservoir in the maintenance of WNV, with ornithophilic mosquitoes serving as the principal vectors.1 Avian morbidity and mortality is a new hallmark of WNV outbreaks in the Middle East and North America.2,3 Whether WNV historically has caused disease in birds is unknown. However, no reports of natural WNV-associated mortality in adult birds occurred during the first six decades after the discovery of the virus in 1937. Interestingly, an Egyptian strain, Ar-248, was isolated from a sick pigeon (Columba livia) squab in a field study conducted from 1952 to 1954.4 This strain caused severe morbidity and mortality in hooded crows (Corvus corone) and house sparrows (Passer domesticus) in the laboratory.5

House sparrows, which are found world-wide, were implicated as an important amplifying vertebrate reservoir host during the 1999 outbreak of WNV in New York City, where they were frequently exposed to WNV and were relatively more abundant than other bird species.6 Laboratory studies confirmed that they are highly competent for transmission of the NY99 strain of WNV to mosquitoes.7 The recognition of their apparent involvement in WNV transmission cycles, and their ubiquitous presence throughout the geographic range of WNV, make the house sparrow a good animal model for virulence studies using different WNV strains.

We wished to determine whether WNV-derived avian mortality observed in recent years in North America and the Middle East is a new phenomenon related to the NY99 strain of WNV (which has a ≥ 99.7% nucleotide identity to the Israel 1998 strain), or whether other WNV strains not previously associated with avian mortality also can be fatal to house sparrows.8 Thus, to determine the phenotypic differences between the bird-virulent NY99 strain of WNV and other WNV genotypes, we measured magnitude and duration of viremia, and survival, for the NY99 strain, a Kenyan strain, and an Australian strain, in an important avian reservoir host, the house sparrow.

MATERIALS AND METHODS

Birds and animal care.

House sparrows were captured using mist nets (Avinet, Inc., Dryden, NY) in Larimer County, Colorado and housed in commercial cages (Safeguard, Inc., New Holland, PA) with perches and sufficient space for limited flight, in accordance with institutional guidelines. Mixed bird seed and water were provided ad libitum. All birds were confirmed seronegative for neutralizing antibodies to both WNV and St. Louis encephalitis (SLE) virus (Flaviviridae) prior to use in experimental infection studies. The maintenance and care of experimental animals in this study complied with the National Institutes of Health guidelines for the humane use of laboratory animals.

Viruses.

The WNV strains and anti-sera were obtained from the reference collection at the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention (Fort Collins, CO), except for the Australian strain, which was acquired from the University of Texas Medical Branch (Galveston, TX). The NY99-4132 strain (NY99) was isolated from the brain of an American crow (Corvus brachyrhynchos) and passaged twice in Vero cell culture. The Kenyan strain KN-3829 (KEN) was obtained from a pool of male Culex univittatus mosquitoes and passaged twice in Vero cell culture.9 The Australian strain Kunjin-6453 (KUN) was isolated from a pool of Cx. annulirostris mosquitoes and passaged once in Vero cells. Virus stocks were titrated by Vero plaque assay.

Inoculation and sampling of house sparrows.

Birds were inoculated subcutaneously on the breast with 0.1 mL of minimal Eagle’s medium containing 1,700 plaque-forming units (pfu) of the assigned viral genotype for the treatment group or 0 pfu for the control group. All birds were monitored every 12 hours during the study for clinical signs of illness. Daily 0.1-mL blood samples were collected for seven days post-inoculation (dpi) and diluted in 0.45 mL of BA-1 diluent (Hanks M-199 salts, 0.05 M Tris, pH 7.6, 1% bovine serum albumin, 0.35 g/L of sodium bicarbonate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, 1 μg/mL of Fungizone) in 2-mL cryovials. The samples were allowed to coagulate at ambient temperature for 30 minutes, centrifuged for separation of serum, and stored at −80°C until titrated for infectious viral particles by plaque assay. The detection and titration of viruses in blood samples were performed using a double-overlay Vero plaque assay.10 The second overlay, containing neutral red, was added on the second dpi and the plaques were counted at three and four dpi. Each sample was titrated in duplicate using serial 10-fold dilutions in BA-1 diluent.

Serology.

Serum samples were assayed using the plaque-reduction neutralization test with the NY99 strain and the TBH-28 strain of SLE virus.10 At 14 dpi, all surviving house sparrows were bled from the jugular vein using a 1-mL syringe with a 27-gauge needle. Whole blood was taken (0.6 mL) and placed in microtainer serum separators (Becton Dickinson, Franklin Lakes, NJ). The specimens were allowed to coagulate at room temperature for 30 minutes, centrifuged for separation of serum, stored at −20°C, and heat inactivated at 56°C for 30 minutes prior to testing. Serum samples were diluted 1:10 in BA-1 for testing in duplicate and endpoint antibody titers were determined using serial two-fold dilutions.

RESULTS

All sparrows (except controls) developed detectable viremias within 1 dpi, except for three birds inoculated with the KUN strain, which developed viremias within 2 dpi (Figure 1). The NY99 strain elicited peak serum viremia titers ranging from 5.6 to 10.8 log10 pfu/mL (log-transformed mean = 9.9). The duration of viremia ranged from four to six days with a mean duration of 5.0 days. House sparrows infected with the KEN strain developed similar viremias with peak titers ranging from 6.4 to 10.8 log10 pfu/mL (log-transformed mean = 9.9). Viremias were detected for 4–6 days (mean duration = 5.4 days). The KUN strain elicited lower viremia titers with peaks ranging from 3.6 to 5.7 log10 pfu/mL (log-transformed mean = 5.1), with a duration of 3–5 days (mean = 3.8 days). All surviving birds (except controls) developed 90% neutralizing antibody titers (≥ 1:20) at 14 dpi.

Fatal infections occurred in 3 (38%) of 8 sparrows inoculated with the NY99 strain, 4 (50%) of 8 inoculated with the KEN strain, 0 of 8 inoculated with the KUN strain, and 0 of 8 controls (Figure 2). Fatalities were observed between 5 and 8 dpi. Severe clinical manifestations related to WNV infection were not observed during the study. All mortality occurred between routine checkups.

No significant differences in virus titer, duration of viremia, and survival between the NY99 and KEN strains were observed. The KUN strain generated lower amounts of circulating virus (P < 0.05, by Student’s t-test). There was no morbidity/mortality observed in the KUN strain-inoculated group, indicating that there is a difference in virulence among Lineage 1 WNV genotypes (Pearson’s χ2 statistic = 4.941, degrees of freedom = 1, P = 0.0262; combining the mortality data from KEN and NY99 into one group for the comparison with the KUN group).

DISCUSSION

House sparrows are abundant passerine birds in North America and regions of Europe, Africa, and Asia where WNV strains circulate. They are probably important amplifying hosts for WNV in many of these regions, and therefore serve as useful animal models for studying WNV infections in birds. We evaluated the viremia profiles and mortality rates in sparrows exposed to three WNV strains from Lineage 1. Phylogenetic trees group WNV strains into two major lineages.8 Lineage 1 constitutes strains from North America, Africa, the Middle East, Asia, and Australia (Kunjin virus). Lineage 2 WNV strains are restricted to sub-Saharan Africa. This genetic distinction has played an important role in that all WNV strains isolated from outbreaks causing human encephalitis have been classified as Lineage 1. Complete genomic sequencing of the bird-virulent Israel 1998 and NY99 strains of WNV group them in the same clade (≥ 99.7% nucleotide identity).11,12 Kunjin viruses are considered by some as a subtype of WNV in its own clade (1b), distinct from other Lineage 1 WNV strains.13 We chose the three strains in our study because they were available in low passage and because the KEN strain aligns in the same clade (1a) as the NY99 strain (but phylogenetically closer to recent epidemic strains isolated from Romania and Russia), whereas the KUN strain is genetically distant (but still within Lineage 1). The KEN strain differs in nucleotide identity from the NY99 strain by 3.4%, whereas the KUN strain differs from both the KEN and NY99 strains by 12%.12 We hoped to determine whether bird virulence was an attribute of Lineage 1 or restricted to the United States/ Israel genotype.

No significant differences in viremia titer, duration of viremia, and survival were observed among the North American and Afro-European strains in the house sparrows. The Australian strain generated lower amounts of circulating virus. Whereas the Afro-European and North American strains caused significant mortality in the sparrows, the Australian strain appeared non-virulent. Thus, we conclude that phenotypic differences with respect to virulence do occur among Lineage 1 WNV genotypes. These data coincide with an infection study in mice using different KUN and WNV strains in which KUN viruses were non-neuroinvasive and caused no mortality.14 In a parallel WNV strain evaluation using American crows, we also observed greater virulence of the NY99 and KEN strains compared with the KUN strain.15 However, the crows were more susceptible to fatal infection after inoculation with the NY99 strain compared with the KEN strain. Infection of crows with the KEN strain resulted in lower-titered viremia and significantly reduced mortality relative to infection with the NY99 strain, which caused 100% mortality. The different response of crows to infection with the NY99 strain may be partially attributed to the fact that the American crow is a naive species with no historical exposure to WNV transmission at the population level. The house sparrow of North America, on the other hand, is a population recently introduced from Europe where WNV has likely been circulating for hundreds of years.16 Genomic selection that increases host resistance to pathogens may contribute to the difference observed in WNV (NY99) virulence between the two species of birds. Thus, it may be a combination of host susceptibility and expressed phenotypic differences in WNV genotypes that contribute to virulence in birds.

Further investigation is necessary to truly understand the mechanism involved in the expression of phenotypic differences in avian virulence observed in Lineage 1 WNV strains. However, our finding that the KEN strain is, for house sparrows, similarly virulent to the NY99 strain suggests that surveillance of bird mortality may be useful in Eurasia and Africa using house sparrows as a target species. Surveillance of bird mortality has proven to be a useful tool in monitoring virus activity and dissemination in the United States3,17–19 and predicting risk of human infection.18–22

Figure 1.
Figure 1.

Mean daily viremia titers plotted for three groups of eight house sparrows, with each group infected with a different strain of West Nile virus. Strains are coded as follows: New York (NY99) = solid black line; Kenya (KEN) = solid gray line; Australia (Kunjin [KUN]) = dashed line. Error bars represent standard deviations of the mean. pfu = plaque-forming units.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.99

Figure 2.
Figure 2.

Survival of house sparrows as a function of infection with West Nile virus (WNV) strain type. The number of house sparrows that survived WNV infection is plotted by day post-inoculation (dpi) for each of three strains. No mortality occurred after 8 dpi. All birds were euthanized at 14 dpi. All eight sparrows that were mock inoculated with diluent, and blood sampled on the same schedule as the other groups, survived the 14-day period of observation. The survival profiles for each strain are coded as follows: New York (NY99) = solid black line; Kenya (KEN) = solid gray line; Australia (Kunjin [KUN]) = dashed line.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.99

Authors’ addresses: Stanley A. Langevin and Aaron C. Brault, Center for Vector-Borne Diseases, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, Telephone: 530-754-8359, Fax: 530-752-3349, E-mails: acbrault@ucdavis.edu and salangevin@ucdavis.edu. Nicholas A. Panella and Nicholas Komar, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, PO Box 2087, Fort Collins, CO 80522, Telephone: 970-221-6400, Fax: 970-221-6476, E-mails: nap4@cdc.gov and nck6@cdc.gov Richard A. Bowen, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80521, E-mail: rbowen@colostate.edu.

Acknowledgements: We thank Jason Velez and staff for preparation of Vero cell monolayers, and private property owners in Larimer County, Colorado for permission to capture sparrows. Bob Tesh and David Beasley graciously provided the Kunjin virus strain. Susan Beckett and Paula Schneider assisted with manuscript preparation. Roger Nasci, Michel Bunning, John Roehrig, and Duane Gubler reviewed the manuscript.

REFERENCES

  • 1

    Komar N, 2003. West Nile virus: Epidemiology and ecology in North America. Adv Vir Res 61 :185–234.

  • 2

    Malkinson M, Banet C, Weisman Y, Pokamunski S, King R, Drouet MT, Deubel V, 2002. Introduction of West Nile virus in the Middle East by migrating white storks. Emerg Infect Dis 8 :392–397.

    • Search Google Scholar
    • Export Citation
  • 3

    Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, McLean R, West Nile Virus Avian Mortality Surveillance Group, 2001. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis 7 :615–620.

    • Search Google Scholar
    • Export Citation
  • 4

    Taylor RM, Work TH, Hurlbut HS, Rizk F, 1956. A study of the ecology of West Nile virus in Egypt. Am J Trop Med Hyg 5 :579–620.

  • 5

    Work TH, Hurlbut HS, Taylor RM, 1955. Indigenous wild birds of the Nile Delta as potential West Nile circulating reservoirs. Am J Trop Med Hyg 4 :872–878.

    • Search Google Scholar
    • Export Citation
  • 6

    Komar N, Panella NA, Burns JE, Dusza SW, Mascarenhas TM, Talbot TO, 2001. Serologic evidence for West Nile virus infection in birds in the New York City vicinity during an outbreak in 1999. Emerg Infect Dis 7 :621–625.

    • Search Google Scholar
    • Export Citation
  • 7

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 8

    Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ, 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286 :2333–2337.

    • Search Google Scholar
    • Export Citation
  • 9

    Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, Lanciotti RS, Peters CJ, 2000. First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley Province, Kenya. Am J Trop Med Hyg 62 :240–246.

    • Search Google Scholar
    • Export Citation
  • 10

    Beaty BJ, Calisher CH, Shope RE, 1995. Arboviruses. Lennette EH, Lennette DA, Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. Seventh edition. Washington, DC: American Public Health Association, 189–212.

  • 11

    Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, Bowen M, McKinney N, Morrill WE, Crabtree MB, Kramer LD, Roehrig JT, 2002. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 298 :96–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Charrel RN, Brault AC, Gallian P, Lemasson JJ, Murgue B, Murri S, Pastorino B, Zeller H, de Chesse R, de Micco P, de Lamballerie X, 2003. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe. Virology 315 :381–388.

    • Search Google Scholar
    • Export Citation
  • 13

    Scherret JH, Poidinger M, Mackenzie JS, Broom AK, Deubel V, Lipkin WI, Briese T, Gould EA, Hall RA, 2001. The relationship between West Nile and Kunjin viruses. Emerg Infect Dis 7 :697–705.

    • Search Google Scholar
    • Export Citation
  • 14

    Beasley DW, Li L, Suderman MT, Barrett AD, 2001. West Nile virus strains differ in mouse neurovirulence and binding to mouse or human brain membrane receptor preparations. Ann N Y Acad Sci 951 :332–335.

    • Search Google Scholar
    • Export Citation
  • 15

    Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff B, Miller BR, Komar N, 2004. West Nile viral strains exhibit differential virulence for American crows (Corvus brachyrhynchos). Emerg Infect Dis 10: (in press).

  • 16

    Hayes CG, 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci 951 :25–37.

  • 17

    Eidson M, Kramer L, Stone W, Hagiwara Y, Schmit K, New York State West Nile Virus Avian Surveillance Team, 2001. Dead bird surveillance as an early warning system for West Nile virus. Emerg Infect Dis 7 :631–635.

    • Search Google Scholar
    • Export Citation
  • 18

    Eidson M, Miller J, Kramer L, Cherry B, Hagiwara Y, West Nile Virus Bird Mortality Analysis Group, 2001. Dead crow densities and human cases of West Nile virus, New York State, 2000. Emerg Infect Dis 7 :662–664.

    • Search Google Scholar
    • Export Citation
  • 19

    Julian KG, Eidson M, Kipp AM, Weiss E, Petersen LR, Miller JR, Hinten SR, Marfin AA, 2002. Early season crow mortality as a sentinel for West Nile virus disease in humans, northeastern United States. Vector Borne Zoonotic Dis 2 :145–155.

    • Search Google Scholar
    • Export Citation
  • 20

    Mostashari F, Kulldorff M, Hartman JJ, Miller JR, Kulasekera V, 2003. Dead bird clusters as an early warning system for West Nile virus activity. Emerg Infect Dis 9 :641–646.

    • Search Google Scholar
    • Export Citation
  • 21

    Guptill SC, Julian KG, Campbell GL, Price SD, Marfin AA, 2003. Early-season avian deaths from West Nile virus as warnings of human infection. Emerg Infect Dis 9 :483–484.

    • Search Google Scholar
    • Export Citation
  • 22

    Theophilides CN, Ahearn SC, Grady S, Merlino M, 2003. Identifying West Nile virus risk areas: the Dynamic Continuous-Area Space-Time system. Am J Epidemiol 157 :843–854.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Nicholas Komar, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, PO Box 2087, Fort Collins, CO 80522.
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