Avian mortality surveillance for West Nile virus in Colorado.

We tested 1,549 avian carcasses of 104 species to identify targets for West Nile virus (WNV) surveillance in Colorado, determine species affected by WNV, compare virus isolation versus RNA detection applied to hearts and oral swabs from carcasses, and compare the VecTest WNV Antigen Assay (VecTest) to standard assays. Forty-two species tested positive. From June to September 2003, 86% of corvids, 34% of non-corvid passerines, and 37% of raptors tested positive. We developed the Target Species Index, which identified American crows as the most important avian indicator species. However, testing multiple species maximizes detection, which may be important early and late in the transmission season. This index may benefit surveillance for other zoonotic pathogens, such as highly pathogenic avian influenza H5N1 virus. VecTest using oral swabs was most sensitive for American crow, black-billed magpie, house finch, house sparrow, and American kestrel. Wildlife rehabilitation centers should be recruited to enhance WNV surveillance.

Serologic evidence of West Nile virus exposure in North American mesopredators.

Sera from 936 mammalian mesopredators (Virginia opossums, gray foxes, striped skunks, hooded skunks, raccoons, a bobcat, and a red fox) were collected during 2003 and 2004 in California, Arizona, Texas, Louisiana, Ohio, and Wyoming and screened for flavivirus-specific antibodies by an epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA). Serum samples positive for antibodies against flaviviruses were screened for West Nile virus (WNV)-specific antibodies by blocking ELISA and selectively confirmed with plaque-reduction neutralization tests. High prevalence rates were observed in raccoons (45.6%) and striped skunks (62.9%). The high WNV antibody prevalence noted in mesopredators, their peridomestic tendencies, and their overall pervasiveness make these species potentially useful sentinels for monitoring flaviviruses in defined areas.

West Nile virus quantification in feces of experimentally infected American and fish crows.

To better understand the potential environmental health risk presented by West Nile virus (WNV)-contaminated feces, we quantified the amount of WNV present in the feces of experimentally infected American crows (Corvus brachyrhynchos) and fish crows (Corvus ossifragus). Peak fecal titers ranged from 10(3.5) to 10(8.8) plaque-forming units (PFU)/g for 10 American crows and from 10(2.3) to 10(6.4) PFU/g for 10 fish crows. The presence of infectious WNV in bird feces indicates a potential for direct transmission of WNV. Thus, handlers of sick or dead birds should take appropriate precautions to avoid exposure to fecal material.

Experimental infection of fox squirrels (Sciurus niger) with West Nile virus.

Tree squirrels (Sciurus spp.) have exhibited high seroprevalence rates, suggesting that they are commonly exposed to West Nile virus (WNV). Many characteristics of WNV infections in tree squirrels, such as the durations and levels of viremia, remain unknown. To better understand WNV infections in fox squirrels (S. niger), we subcutaneously inoculated fourteen fox squirrels with WNV. Peak viremias ranged from 10(4.00) plaque-forming units (PFU)/mL of serum on day 2 post-infection (DPI) to 10(4.98) PFU/mL on 3 DPI, although viremias varied between individuals. Oral secretions of some fox squirrels were positive for WNV viral RNA, occasionally to moderate levels (10(3.2) PFU equivalent/swab). WNV PFU equivalents in organs were low or undetectable on 12 DPI; gross and histologic lesions were rare. The viremia profiles of fox squirrels indicate that they could serve as amplifying hosts in nature. In addition, viral RNA in the oral cavity and feces indicate that this species could contribute to alternative WNV transmission in suburban communities.

Susceptibility of greater sage-grouse to experimental infection with West Nile virus.

Populations of greater sage-grouse (Centrocercus urophasianus) have declined 45-80% in North America since 1950. Although much of this decline has been attributed to habitat loss, recent field studies have indicated that West Nile virus (WNV) has had a significant negative impact on local populations of grouse. We confirm the susceptibility of greater sage-grouse to WNV infection in laboratory experimental studies. Grouse were challenged by subcutaneous injection of WNV (10(3.2) plaque-forming units [PFUs]). All grouse died within 6 days of infection. The Kaplan-Meier estimate for 50% survival was 4.5 days. Mean peak viremia for nonvaccinated birds was 10(6.4) PFUs/ml (+/-10(0.2) PFUs/ml, standard error of the mean [SEM]). Virus was shed cloacally and orally. Four of the five vaccinated grouse died, but survival time was increased (50% survival=9.5 days), with 1 grouse surviving to the end-point of the experiment (14 days) with no signs of illness. Mean peak viremia for the vaccinated birds was 10(2.3) PFUs/ml (+/-10(0.6) PFUs/ml, SEM). Two birds cleared the virus from their blood before death or euthanasia. These data emphasize the high susceptibility of greater sage-grouse to infection with WNV.

Serologic evidence of west nile virus infection in free-ranging mammals, Slidell, Louisiana, 2002.

After an outbreak of West Nile virus (WNV) infections in Slidell, Louisiana, in 2002, we detected neutralizing antibodies to WNV in 13 of 120 mammals, representing five of six species sampled. Seroprevalence was measured in opossum, Didelphis virginiana (75%, n = 8), raccoons, Procyon lotor (60%, n = 5), black rats, Rattus rattus (6%, n = 36), hispid cotton rats, Sigmodon hispidus (4%, n = 24), and eastern gray squirrels, Sciurus carolinensis (2%, n = 43).

West Nile virus survey of birds and mosquitoes in the Dominican Republic.

We report West Nile virus (WNV) activity from a new area on Hispaniola, in the vicinity of Monte Cristi National Park in northwest Dominican Republic. Specific anti-WNV antibodies were detected in 12 of 58 (21%) resident birds sampled in March 2003, representing six species in the orders Cuculiformes (cuckoos), Strigiformes (owls), and Passeriformes (song birds). This seroprevalence is the highest reported from any site in the Caribbean Basin. Virus was not detected in any mosquitoes or tissues from bird specimens. Testing of 20 sick or dead birds was negative for WNV. Undetermined flavivirus antibodies were detected in four resident birds at Monte Cristi, as well as in five resident birds at Sierra de Baoruco National Park in southwest Dominican Republic. These data suggest that an unidentified flavivirus, as well as WNV, is active in the Dominican Republic.

West Nile virus infection in farmed American alligators (Alligator mississippiensis) in Florida.

In September and October 2002, an epizootic of neurologic disease occurred at an alligator farm in Florida (USA). Three affected American alligators (Alligator mississippiensis) were euthanatized and necropsied, and results confirmed infection with West Nile virus (WNV). The most significant microscopic lesions were a moderate heterophilic to lymphoplasmacytic meningoencephalomyelitis, necrotizing hepatitis and splenitis, pancreatic necrosis, myocardial degeneration with necrosis, mild interstitial pneumonia, heterophilic necrotizing stomatitis, and glossitis. Immunohistochemistry identified WNV antigen, with the most intense staining in liver, pancreas, spleen, and brain. Virus isolation and RNA detection by reverse transcription-polymerase chain reaction confirmed WNV infection in plasma and tissue samples. Of the tissues, liver had the highest viral loads (maximum 10(8.9) plaque-forming units [PFU]/0.5 cm3), whereas brain and spinal cord had the lowest viral loads (maximum 10(6.6) PFU/0.5 cm3 each). Virus titers in plasma ranged from 10(3.6) to 10(6.5) PFU/ml, exceeding the threshold needed to infect Culex quinquefasciatus mosquitoes (10(5) PFU/ml). Thus, alligators may serve as a vertebrate amplifying host for WNV.

Alligators as West Nile virus amplifiers.

Recent evidence suggests that American alligators (Alligator mississippiensis) may be capable of transmitting West Nile virus (WNV) to other alligators. We experimentally exposed 24 juvenile alligators to WNV parenterally or orally. All became infected, and all but three sustained viremia titers >5.0 log10 PFU/mL (a threshold considered infectious for Culex quinquefasciatus mosquitoes) for 1 to 8 days. Noninoculated tankmates also became infected. The viremia profiles and multiple routes of infection suggest alligators may play an important role in WNV transmission in areas with high population densities of juvenile alligators.

Poor replication of West Nile virus (New York 1999 strain) in three reptilian and one amphibian species.

Because West Nile (WN) virus primarily cycles between mosquitoes and birds, North American reptiles and amphibians have not been evaluated as reservoir hosts of this virus. We infected three species of reptiles and one species of amphibian: Iguana iguana (green iguana), Thamnophis sirtalis sirtalis (Florida garter snake), Trachymes scripta elegans (red-ear slider), and Rana catesbeiana (North American bullfrog). After inoculation with WN virus, some of the green iguanas, bullfrogs, and garter snakes showed low but detectable viral loads in the blood, oral or cloacal swabs, and/or organs.

Use of a vesicular stomatitis virus complementation system to analyze respiratory syncytial virus binding.

Respiratory syncytial virus (RSV) can be difficult to manipulate in the laboratory because it produces a fragile, filamentous virion that does not bud efficiently from the cell surface and which is sensitive to purification. These properties have complicated the studies of RSV envelope protein-host cell interactions. In this paper, we have tested the ability of the RSV attachment protein, G, to complement virus attachment of a recombinant vesicular stomatitis virus (VSVdeltaG*), which lacks any viral attachment protein. Using an enzyme-linked immunosorbent assay (ELISA) to detect bound virus, VSVdeltaG* virions were shown to incorporate the RSV G protein and to bind to Hep-2 cells. Binding of RSV G protein-complemented VSVdeltaG* virus was inhibited by incubation with RSV-specific antibodies and by the addition of exogenous sulfated glycosaminoglycans, indicating that binding by the complemented virus exhibited the characteristics of RSV binding rather than those of VSV. These results demonstrate that complementation studies with VSVdeltaG* may be useful in virus-host interaction studies of the RSV G protein.