Experimental infection of North American birds with the New York 1999 strain of West Nile virus - Research

Emerging Infectious Diseases, March, 2003 by Nicholas Komar, Stanley Langevin, Steven Hinten, Nicole Nemeth, Eric Edwards, Danielle Hettler, Brent Davis, Richard Bowen, Michel Bunning

To evaluate transmission dynamics, we exposed 25 bird species to West Nile virus (WNV) by infectious mosquito bite. We monitored viremia titers, clinical outcome, WNV shedding (cloacal and oral), seroconversion, virus persistence in organs, and susceptibility to oral and contact transmission. Passeriform and charadriiform birds were more reservoir competent (a derivation of viremia data) than other species tested. The five most competent species were passerines: Blue Jay (Cyanocitta cristata), Common Grackle (Quiscalus quiscula), House Finch (Carpodacus mexicanus), American Crow (Corvus brachyrhynchos), and House Sparrow (Passer domesticus). Death occurred in eight species. Cloacal shedding of WNV was observed in 17 of 24 species, and oral shedding in 12 of 14 species. We observed contact transmission among four species and oral in five species. Persistent WNV infections were found in tissues of 16 surviving birds. Our observations shed light on transmission ecology of WNV and will benefit surveillance and control programs.

**********

West Nile virus (WNV) is a mosquito-borne flavivirus (family: Flaviviridae) that uses birds as primary vertebrate reservoir hosts (1). WNV emerged in North America in New York City in 1999 (2,3) and has since spread throughout much of the North American continent (4). The virus affects the health of the public as well as domestic animals and wildlife. In 1999-2001, WNV was associated with 149 cases of clinical neurologic disease in humans (e.g., encephalitis and meningitis) (2,4,5), 814 cases of equine encephalitis (4-6), and 11,932 deaths in birds in the United States (4,5,7). Most reported fatal infections in birds occurred in crows. The American Crow (see Table 1 for scientific names of birds) has been proposed as the basis for a national surveillance system for avian deaths attributed to WNV (7,8). Since 1999, >150 species of dead birds have been reported as WNV positive to the Centers for Disease Control and Prevention (CDC) ArboNET surveillance program (unpub. data). Although the precise cause of death in these birds may not be proven, WNV has been isolated from the carcasses or WNV-specific RNA sequences have been detected. However, not all birds die from infection with the New York 1999 strain of WNV. Many birds sampled in 1999 and 2000 in New York City survived natural WNV infection and developed humoral immunity (9,10).

Although crows are commonly reported as infected with WNV (11), the identity of the avian reservoirs for WNV remains unknown. Surveillance data on avian deaths and seroprevalence studies suggest hypotheses about reservoir host species but do not indicate the competence of a particular species to infect a culicine vector. Furthermore, birds may be involved in transmission by means other than mosquito bites, yet little is known about contact or oral transmission among birds.

To better understand the role of birds in WNV transmission, we exposed 25 species of birds, representing a wide range of avian orders and families, to infectious mosquito bites. We then monitored viremia titers, clinical outcomes, viral shedding in cloacal and oral cavities, persistence of viral infections in organs, and development of neutralizing antibodies. The viremia data generated were used to quantitate reservoir competence. We also evaluated susceptibility to oral and direct contact transmission when possible.

Methods

Source of Birds

Birds were obtained commercially when possible or as nonreleasable injured birds (raptors only), or from the wild (Table 1). Only seronegative birds were used. Information on the plaque-reduction neutralization assay used is available in Appendix A (online only; available from: URL: http:// www.cdc.gov/ncidod/EID/vol9no3/02-0628-appA.htm).

Source and Infection of Mosquitoes

We used colonized mosquitoes (Culex tritaeniorhynchus) originally obtained from Taiwan in 1997. Adult female mosquitoes (<10 days old), used for infecting birds, were inactivated by chilling at approximately 4[degrees]C and inoculated intrathoracically with 1 [micro]L of an aqueous solution containing [10.sup.7] PFU WNV (NY99-6480) per 1 mL. Mosquitoes were then incubated at 16:8 h light:dark, 28[degrees]C, 80% relative humidity for 6-10 days before they were exposed to birds. Successful infection of mosquitoes was confirmed by plaque assay of whole mosquito homogenates (after incubation).

Source of Virus

Two isolates of WNV (New York 1999) were used. The NY99-6480 strain was isolated from mosquitoes (C. pipiens) and passed once in Vero cell culture. The NY99-4132 strain was isolated from brain of an American Crow and passed one to three times in Vero cell culture. The TBH-28 strain of St. Louis encephalitis virus (SLEV; family: Flaviviridae) was obtained from the CDC reference collection.

Experimental Infection

We exposed birds to WNV-infectious mosquito bites by holding their exposed skin (usually of the breast) against a screened carton containing 5-15 mosquitoes. Birds were considered sufficiently exposed when one mosquito had engorged to repletion. In the few cases when birds were probed extensively by mosquitoes but no visible blood was imbibed, we considered them infected if viremia developed. When possible, at least one uninfected conspecific bird (contact-exposed group) was placed in a cage with a mosquito-exposed bird as a control for direct transmission (in the absence of mosquito-borne transmission). Some birds (orally exposed group) were exposed to per os infections by using a variety of techniques; our objective was to show that per os transmission is possible. Techniques used included placing 200 uL water (containing a suspension of WNV [NY99-4132]) in the back of the oral cavity to stimulate the swallow reflex; placing a dead infected mosquito (containing approximately [10.sup.7] PFU) in the bird's oral cavity and stimulating the swallow reflex with 200 uL of water; and placing a dead infected adult House Mouse (Mus musculus) or House Sparrow (euthanized 3-5 days after subcutaneous injection of 2,000-8,000 PFU) in the cage. Viral loads in the mice and House Sparrows were inferred from infected cohorts and estimated at >[10.sup.5] PFU per animal. Information about methods for venipuncture is available in Appendix B (online only; available from: URL: http://www.cdc.gov/ncidod/EID/vol9no3/02-0628-appB.htm).

Collection of Oral and Cloacal Samples

For some birds, daily cloacal or nasopharyngeal (oral) swabs were collected concurrently with blood samples during the first 7 days postinoculation (dpi). Cotton- or Dacron-tipped applicators were used, and contaminated swabs were dipped in cryovials containing 0.5-mL BA1 to transfer any virus to the cryovial. These cryovials were placed immediately on wet ice (temporarily) and stored at -70[degrees]C for subsequent titration by Vero plaque assay (described in Appendix C, online only; available from: URL: http://www.cdc.gov/ncidod/EID/vol9no3/020628-appC.htm).

Illnesses, Deaths, and Euthanasia

Exposed birds were observed twice a day for signs of severe illness, such as neurologic irregularities and recumbency. Birds unable to ambulate or consume food and water were euthanized by C[O.sub.2] asphyxiation or intravenous inoculation of sodium pentobarbitol at a dose of approximately 80 mg/kg. We recorded fatal cases to determine estimates of mortality rates for each species.