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West Nile Virus Infection in Birds and Mammals LAURA D. KRAMER AND KRISTEN A. BERNARD Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, Slingerlands, New York 12159, USA ABSTRACT: West Nile virus (WNV) was found throughout New York State in year 2000. The epicenter was located in New York City with a high level of activity in the immediately surrounding counties, including Rockland, Westchester, Nassau, and Suffolk. During 2000, WNV testing was performed by the Wadsworth Center on 3,687 dead birds, representing 153 species, 46 families, and 18 orders. There were 1,203 WNV-positive birds, representing 63 species, 30 families and 14 orders. The percentage of WNV-positive birds was 33% for all birds tested throughout the state, with no significant difference in infection rates in migratory versus resident birds, although significantly more resident birds were submitted for testing. The highest apparent mortality for the entire season was observed in American crows in Staten Island, a location that also showed the highest minimal infection rate in Culex pipiens complex mosquitoes. Studies examining tissue tropism of WNV in corvids and noncorvids from the epicenter and from remote locations indicated that the kidney was the most consistently infected tissue in birds, regardless of level of infection. The brain was the next most consistently positive tissue. The differences in infection among the tissues were most apparent when low levels of virus were present. Experimental mouse inoculation demonstrated a classical flavivirus infection pattern. KEYWORDS: West Nile virus; tissue tropism; birds; mammals INTRODUCTION West Nile virus (WNV) is a newly emerging mosquito-borne virus now found on four continents, Africa, Asia, Europe, and North America. The infection in humans has been characterized by a broad range of clinical symptoms, from asymptomatic infection to mild nonspecific symptoms, including fever and headache, to more serious cases with myocarditis and encephalitis.1 In 1999, 62 cases of WNV were confirmed in New York City and in 2000, 14 cases. Serosurveys conducted in 1999 demonstrated that 2.6% of the population in an area of high viral activity had antibody to WNV,2 and in 2000, approximately 1% of the population demonstrated seroconversion.2 The introduction of WNV to the northeastern United States in 1999 was accompanied by unprecedented mortality in indigenous and captive birds. This was unexAddress for correspondence: Laura D. Kramer, Ph.D., Director, Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159. Voice: 518-869-4524; fax: 518-869-4530. [email protected] 84 KRAMER & BERNARD: WEST NILE VIRUS IN BIRDS AND MAMMALS 85 pected since, historically, in countries where WNV is enzootic, the virus has led to minimal disease in avian species. WNV has been demonstrated to infect a wide variety of vertebrate species besides man and birds, as indicated by evidence of antibody to the virus and virus isolation.3 Studies during the 1950s in Egypt indicated that the hooded crow and the house sparrow had antibody rates of 65 and 42%, respectively, and virus was isolated from the blood of an adult crow.4 The work presented here is aimed at elucidating WNV infection in vertebrates other than man. Data from 2000 in New York State were analyzed to determine which bird species were found infected most frequently with WNV in 2000, and which avian tissues were most often infected. Five out of 149 wild mammals were found to be infected with WNV in year 2000 in New York State. These included one little brown bat (Myotis lucifugus), one big brown bat (Eptesicus fuscus), one squirrel (Sciurus carolinensis), one domestic rabbit (Oryctolagus cuniculus), and one eastern chipmunk (Tamias striatus). A preliminary study delineating infection in white mice as a model for infection in mammals was undertaken to begin to define the pathogenesis of WNV. MATERIAL AND METHODS Tissue Submission and Processing Dead birds and mammals were submitted for necropsy to the Department of Environmental Conservation, Wildlife Pathology Unit, New York State. Multiple tissues from suspect animals were forwarded to the Arbovirus Laboratories of the Wadsworth Center, New York State Department of Health, for viral assay. Protocols for handling tissues and for viral assays have been described previously.5 Briefly, 50 mm3 sections of tissue were excised and triturated directly in lysis buffer containing guanidium isothiocyanate (Qiagen, CA). RNA was extracted as per the manufacturer’s instructions, and reverse transcription–polymerase chain reaction (RT-PCR) was conducted using an ABI Prism 7700 Sequence Detector using TaqMan One-Step RT-PCR master mix (Applied Biosystems, Foster City, CA). Two sets of primers/probes (1160; 3111) were used as described.5,6 Ct values—the PCR cycle at which an increase in the fluorescence rises above the threshold value determined by the standard curve—were recorded; from this the TaqMan score was calculated, that is, + to ++++. Determination of Minimum Infection Rate (MIR) in Mosquitoes MIRs were calculated assuming one infected mosquito per pool per 1000 mosquitoes tested. Mosquito pools were processed as described.5 Briefly, pools were triturated using SpexCertiPrep 8000-D mixer mill (Metuchen, NJ). A 350 µL aliquot was removed immediately for RNA extraction, which was conducted as described above for vertebrate tissue, as was RT-PCR. The remainder of the triturated pool was held for inoculation of cell culture to detect live infectious virus. Cell Culture Assay African green monkey kidney (Vero) cell cultures in 25-mm flasks were inoculated with 0.1 mL homogenized and clarified mosquito suspension. Monolayers were 86 ANNALS NEW YORK ACADEMY OF SCIENCES checked daily for seven days for evidence of cytopathology (cpe). After seven days or when cpe was observed, cells were spotted on slides, fixed and stained with primary antibody, H5.46 WNV monoclonal antibody, and secondarily with goat antimouse IgG fluorescein-conjugated antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Fluorescence was evaluated using an Olympus BH-2 microscope equipped with FITC filter set. Plaque Assay Cell culture plaque assays to quantitate infectious virus were conducted as previously described.7 Briefly, six-well Costar plates with Vero cell monolayers were inoculated with homogenized and clarified tissue suspensions, 0.1 mL per well. A single overlay of 2.0% oxoid agar with 0.8 mL of 1.0% neutral red was added; plaques were read from day 3 to day 7. Pathogenesis of West Nile Virus in Adult Mice Female Balb/C mice, five weeks old, were inoculated intraperitoneally with 103 PFU WNV isolated from a pool of 50 Cx. pipiens mosquitoes from New York in 2000. Virus was diluted in low endotoxin phosphate-buffered saline containing 1% fetal bovine serum (diluent). Three control mice were inoculated with diluent alone, and three additional mice were not inoculated, but were housed with inoculated mice as contact controls. The mice were weighed and observed for clinical signs daily. Three mice each were sacrificed at 19 and 28 hours, days 2, 3, 4, 5, 8, and 9. Control mice were held for 24 days. Heart blood was collected, and the brain, liver, spleen, kidney, and heart were harvested for assay of infectious virus. All tissues were weighed, and diluent was added to make a 20% homogenate for brain or 10% homogenate for all other organs. The limit of detection was 250 PFU/g for brain and 500 PFU/g or mL for other organs and serum. RESULTS Avian Infection A comparison of infection rates in year-round residents, migrants, mixed populations (in which only a portion of the population migrates), and captive birds indicated that the predominant number of birds that died and were tested for WNV were permanent residents (TABLE 1). However, there was no difference in the proportion of birds from all categories that were infected, 30 to 43%. The proportion of American crows found infected with WNV was compared with the number of human8 and equine9 cases in the five boroughs of NYC, Long Island (Nassau and Suffolk counties), and two other locations within the epicenter, Westchester and Rockland counties (FIG . 1). Ten of 14 cases in humans in New York State were detected on Staten Island (SI), where more than 90% of the crows tested were infected, and the minimal infection rate in Cx. pipiens complex mosquitoes (Cx. pipiens and Cx. pipiens-restuans combined) was 10.9. Three other boroughs in NYC—Brooklyn, Manhattan, and Queens—had one to two human cases, and all other locations had none. The greatest numbers of dead crows were submitted by KRAMER & BERNARD: WEST NILE VIRUS IN BIRDS AND MAMMALS 87 FIGURE 1. Relative infection rates of crows, humans, and equines by location in epicenter of WNV outbreak, 2000. MIR, minimal infection rate in Culex pipiens and Culex pipiens-restuans complex mosquitoes combined. Number of human and equine cases and percentage positive crows are reported. Numbers above bars represent number of crows tested. TABLE 1. Infection in birds with different migration patterns Behavior pattern Number tested Species Positive Individuals Percent Year-round residents 2924 28 1116 38 True migrants 51 14 22 43 Mixed population 175 15 53 30 Captive birds 25 6 10 40 Rockland county, of which 75% were infected with WNV; the MIR in Cx. pipiens complex mosquitoes in Rockland was 2.0, but no human or horse cases were observed. Suffolk had the second highest number of dead crows submitted for testing and had the greatest number of equine cases in NYS (8), but infected horses also were detected in Staten Island, the Bronx, and Nassau. More detailed information on WNV infection in birds and mosquitoes can be found in Bernard et al.10 The MIRs of Cx. pipiens and Cx. pipiens-restuans complex mosquitoes were combined. Other Culex species were not included in this summary because of problems encountered with identification of other species of Culex mosquitoes (unpublished data, Kramer). Vero cell cultures inoculated with positive mosquito pools of all species indicated that infectious virus could be detected in 111/266 (41.7%) 88 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 2. WNV cell culture results for mosquitoes collected in New York State during 2000 Mosquito species Ochlerotatus cantator Oc. japonicus Oc. triseriatus Ae. vexans Ae. species An. punctipennis Culex pipiens Cx. pipiens-restuans Cx. salinarius Cx. species Psorophora ferox No. positive pools by No. positive pools by cell culture RT-PCR (n tested) 1 5 3 10 1 1 79 212 31 19 1 0 (1) 0 (5) 1 (3) 1 (10) 0 (0) 0 (1) 25 (60) 61 (146) 13 (26) 9 (13) 1 (1) FIGURE 2. Relative TaqMan score of brain, heart, and liver compared to kidney. The numbers above the bars indicate the number of tissues compared in parallel with the kidney. 1+ represents Ct value 32–36; 2+ to 3+, 24–32; 4+, 14–25. (TABLE 2). Passage through cell culture did not allow for amplification of initially undetectable levels of infectious virus. Tissue Tropism in Birds TaqMan RT-PCR results from kidney, heart and brain tissues from 49 crows were compared in order to determine tissue tropisms of WNV (FIG . 2). Birds found infected with low, medium, and high concentrations of viral WNV RNA during 2000 were selected. Multiple tissues from the crows were tested in parallel, but not all tissues were available from all birds. Each tissue was scored as its TaqMan score relative to the TaqMan score of the kidney of the same bird, and the mean was taken. Kidney was the most consistently positive tissue, regardless of the level of infection. The heart, brain and liver of birds with TaqMan scores of 3–4+ in the kidney had levels KRAMER & BERNARD: WEST NILE VIRUS IN BIRDS AND MAMMALS 89 of viral RNA equal to that of the kidney, and these tissues therefore were equally good indicators of infection. Similarly, heart and brain of birds with TaqMan scores of 2+ in the kidneys were infected to an equal extent; liver, however, had 0.7 (70%) of the level of viral RNA. The heart, brain, and liver of birds with kidneys that were low positives (1+) had approximately 0.2 (20%) of the kidney’s level of viral RNA. Focal Infection of Tissues A study was conducted to determine the uniformity of viral infection in various organs of crows. The level of viral RNA was determined in 8 to 15 separate sections excised from each of 12 individual paired crow brains and kidneys (TABLE 3). There was insufficient tissue available to excise 15 sections in every case. All but four positive tissues demonstrated variable levels of viral RNA (i.e., from Ct value 36 to 14, TaqMan score 1+ to 4+) in excised tissue sections. Four tissues demonstrated Ct values < 25 (i.e., 4+ levels of viral RNA in all sections: 3 brains, crows 10, 11, 12 and one kidney, crow 12). In two of the 12 crows (crows 1 and 3), brains were negative when the corresponding kidneys had several sections that were low positives (Ct 36 to 32); in one additional crow (crow 2), the brain was negative (− or ±) when the kidney was highly positive (Ct 29 to14). In one crow (crow 4), 13 of 15 brain sections were highly positive with Ct values < 29, but only one of 11 kidney sections was a low positive with a Ct value of 34. The variability observed among sections of both brain and kidney was more than 10-fold in some instances. Tissue Tropism in Mammals The WNV-inoculated mice appeared healthy until day 6 when mild clinical signs were evident in one mouse. Neurologic signs, ranging from ataxia and weakness to bilateral hindleg paralysis, were observed on days 7 through 9 in two of the six remaining mice. The maximum weight loss was 9%. No clinical signs were observed in the mock-inoculated or contact control mice. The following gross pathologic changes were observed in some, but not all mice: myocarditis (days 4, 5, 8, and 9); splenomegaly and lymphadenopathy (day 5); hemorrhagic meninges, hepatic lipidosis, lymphonecrosis of spleen and lymph nodes, reduced thymic size, and intestinal distention (days 8 and 9). Virus in the serum reached a peak of 105.4 PFU/mL after three days and was cleared rapidly with only one mouse having a detectable viremia day 4 (FIG . 3). Infectious virus was also present early in the spleen of two mice at 19 h; one spleen contained 103 PFU/mL in the absence of detectable viremia. The titer in the spleen then increased to a peak at day 3 to 4. The heart was first positive at 28 h, and then increased in titer until day 4. On each of days 5, 8, 9, one heart was positive with low levels of virus, 103.2–103.5 PFU/g. Virus was first detected in the kidney after 2 days. The level of infectious virus in this tissue increased slowly, reaching a peak on day 5, with 105.2 PFU/g virus. The brain of one mouse was infected at 28 hours. The concentration of virus in the brain remained low until day 5, when one mouse had a significantly increased titer, 104.8 PFU/g. By day 8, one mouse was very sick, with 106.6 PFU/g virus in its brain, and 103.2 PFU/g virus in its heart. Another mouse, which was mildly ill on day 8 with ruffled fur but no neurologic signs, was found dead on day 9. The kidney and brain of this mouse had detectable virus, but with titers below the peak, 102.7 and 104.9 PFU/g, respectively. TABLE 3. Focus of West Nile virus in the brain and kidney of infected American crows 90 ANNALS NEW YORK ACADEMY OF SCIENCES KRAMER & BERNARD: WEST NILE VIRUS IN BIRDS AND MAMMALS 91 FIGURE 3. Experimental infection of Balb/C mice with WNV. Mice were inoculated intraperitoneally with 103 PFU WNV isolated from a pool of Culex pipiens in NY, 2000. Each bar represents results for a tissue from an individual mouse. White bar = serum; diagonally hatched ba r= spleen; black ba r = brain; stippled ba r= heart; horizontally hatched ba r= kidney. No bar indicates that the sample was below the limit of detection (250 to 500 PFU/g or mL). DISCUSSION WNV has a very broad host range. In 2000 in the northeastern US, more than 60 species of birds became infected and died from this virus. This is in sharp contrast to the biology of WNV in Europe, Asia and Africa, where there are few reports of bird mortality. Laboratory experiments with Hooded Crows demonstrated a high mortality in young birds and development of immunity in adult birds.11 Fourteen orders of birds were found infected with WNV in New York State in 2000.10 The predominant number of dead birds tested were members of the Order Passeriformes, followed by Galliformes, Columbiformes, and Charadriiformes. When species that had a minimum of 10 individuals tested were compared, the percent of positive birds ranged from 15% (Ciconiformes) to 44% (Psittaciformes).10 In New York State, within the epicenter of viral activity—the five boroughs of NYC, Long Island (Nassau and Suffolk counties), Rockland and Westchester counties—American crows demonstrated significantly greater mortality from WNV than did other species of birds.10 In other locations in the state, no difference in infectivity was observed among bird species found dead. A sharp increase in sightings of dead crows preceded the first human case detected in NYC,12 possibly providing an early 92 ANNALS NEW YORK ACADEMY OF SCIENCES warning of increased risk to man. The highest MIR in mosquitoes on Staten Island correlated with the greatest proportion of infected crows and the greatest number of human cases. Other locations had lower MIRs, but five of eight had MIRs greater than 1. An MIR of 1 with St. Louis encephalitis virus, a related flavivirus, in Florida has been understood to signify increased risk to humans.13 It is important to recognize, however, that the WNV MIRs reported here are a measure of the number of mosquitoes with detectable viral RNA and not in many cases, infectious virus. This is not surprising since the limit of detection of the TaqMan RT-PCR assay was determined to be 0.08 PFU or 10−1.1 log10 PFU.5 Thus the significance of the MIRs must be interpreted with caution. Mosquitoes that have evidence of viral RNA only and no infectious virus represent incompetent vectors, but theoretically they might have become competent with an increased extrinsic incubation period had they lived out their lifespan. Calculation of MIRs for each species also is dependent on correct identification of mosquitoes, which in some cases was problematic in 2000 (unpublished data, Kramer). Although the greatest number of birds submitted for testing were year-round residents of New York State, there was no difference in infection rates among birds with different breeding behavior. Viremic migratory birds are suspected to contribute to the movement of WNV in southern Europe.14 Similarly, migrating species on the East Coast of the US may provide a mechanism of viral transport over distance and thereby a means of viral spread to new areas throughout both temperate and tropical regions of the Western Hemisphere. However, the true likelihood that this would happen is unclear because the length of time that birds have viremic titers sufficient to infect vector mosquitoes may be short in duration. Birds that will be most important to the transmission cycle probably gather in large groups in locations with high numbers of ornithophilic mosquitoes. The analysis of RNA levels in brain, heart, kidney and liver indicated that the kidney, and secondarily the brain, was the avian tissue most consistently infected with WNV. These results confirmed those of Steele et al.15 They examined birds infected with WNV in 1999, following introduction of the virus into New York, and found that the kidney was the tissue most frequently infected, but that the brains of most birds were also infected with WNV. Furthermore, the examination of the focal infection within the kidney and brain, as determined by TaqMan assay of multiple sections, suggests that the kidney displays consistently higher levels of viral antigen than does the brain. This observation is further supported by immunofluorescent staining of frozen tissue sections of the brain and kidney (unpublished data). WNV antigen appears much more focal in the brain than in the kidney, which showed evidence of staining throughout. The experimental infection in mice demonstrated a classical flavivirus pattern of infection. After peripheral inoculation, virus was found in lymphoid tissues, followed by the heart and kidney. Peak viremia occurred on day 2 to 3. Virus was found in the brain as early as 28 h and consistently on days 2 and 3. It is important to note that the viremia was high for all three of these time points; therefore, the virus measured in the brain may represent virus in the blood. By day 4, virus unambiguously had invaded the brain. Thus, the NY strain of WNV was neuroinvasive in adult mice. Using three-week-old outbred mice, Halevy et al. found similar results after peripheral inoculation using an Israeli strain of WNV.16 KRAMER & BERNARD: WEST NILE VIRUS IN BIRDS AND MAMMALS 93 ACKNOWLEDGMENTS We would like to acknowledge the considerable contributions of the Arbovirus Laboratories’ staff, including Elizabeth B. Kauffman, Susan A. Jones, Alan P. Dupuis II, Kiet A. Ngo, Greg D. Ebel, Donna M. Young, and David Nicholas. We thank Ward Stone and the Wildlife Pathology Unit of the Department of Environmental Conservation, the Division of Epidemiology of the New York State Department of Health, the New York City Department of Health, and the Centers for Disease Control and Prevention. REFERENCES 1. SAMPSON, B.A., C. AMBROSI, A. CHARLOT, et al. 2000. The pathology of human West Nile Virus infection. Hum. Pathol. 31: 527–531. 2. CDC. 2001. Serosurveys for West Nile virus infection—New York and Connecticut counties, 2000. Morb. Mortal. Wkly. Rep. 50: 37-39. 3. HAYES, C.G. 2000. West Nile Fever. In The Arboviruses: Epidemiology and Ecology, Vol. V. T.P. Monath, Ed.: 59–88. CRC Press, Inc. Boca Raton, FL. 4. TAYLOR, R.M., T.H. WORK, H.S. HURLBUT & F. RIZK. 1956. A study of the ecology of West Nile virus in Egypt. Am. J. Trop. Med. Hyg. 5: 579. 5. SHI, P-Y., E.B. KAUFFMAN, P. REN, et al. 2001. High throughput detection of West Nile virus RNA. J. Clin. Microbiol. 39: 1264–1271. 6. LANCIOTTI, R.S., A.J. KERST, R.S. NASCI, et al. 2000. Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J. Clin. Microbiol. 38: 4066–4071. 7. REISEN, W.K., R.P. MEYER, S.B. PRESSER & J.L. HARDY. 1993. Effect of temperature on the transmission of western equine encephalomyelitis and St. Louis encephalitis viruses by Culex tarsalis (Diptera:Culicadae). J. Med. Entomol. 30: 151–160. 8. CDC. 2000. Update: West Nile Virus activity—Eastern United States, 2000. Morb. Mortal. Wkly. Rep. 49: 1044–1047. 9. TROCK, S.C., B. MEADE, A.L. GLASER, et al. 2001. West Nile outbreak among horses in New York State, 1999 and 2000. Emerg. Infect. Dis. In press. 10. BERNARD, K.A., J.G. MAFFEI, S.A. JONES, et al. 2001. Comparison of West Nile virus infection in birds and mosquitoes in New York State in 2000. Emerg. Infect. Dis. 7: 679–685. 11. WORK, T.H., H.S. HURLBUT & R.M. TAYLOR. 1953. Isolation of West Nile virus from hooded crow and rock pigeon in the Nile Delta. Proc. Soc. Exp. Biol. Med. 84: 719–722. 12. EIDSON, M., L.D. KRAMER, W. STONE & I. GOTHAM. 2001. Dead bird surveillance as an early warning system for West Nile virus. Emerg. Infect. Dis. 7: 631–635. 13. DAY, J.F. & L.M. STARK. 1996. Transmission patterns of St. Louis encephalitis and eastern equine encephalitis viruses in Florida: 1978–1993. J. Med. Entomol. 33: 132–139. 14. RAPPOLE, J.H., S.R. DERRICKSON & Z. HUBALEK. 2000. Migratory birds and spread of West Nile virus in the Western Hemisphere. Emerg. Infect. Dis. 6: 319–328. 15. STEELE, K.E., M.J. LINN, R.J. SCHOEPP, et al. 2000. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet. Pathol. 37: 208–224. 16. HALEVY, M., Y. AKOV, D. BEN-NATHAN, et al. 1994. Loss of active neuroinvasion of West Nile virus: pathogenicity in immunocompetent and SCID mice. Arch. Virol. 137: 355–370.