Alan P. Dupuis

High-Throughput Detection of West Nile Virus RNA

ABSTRACT The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

West Nile Virus and Wildlife

Abstract West Nile virus (WNV) has spread rapidly across North America, resulting in human deaths and in the deaths of untold numbers of birds, mammals, and reptiles. The virus has reached Central America and the Caribbean and may spread to Hawaii and South America. Although tens of thousands of birds have died, and studies of some bird species show local declines, few regionwide declines can be attributed to WNV. Predicting future impacts of WNV on wildlife, and pinpointing what drives epidemics, will require substantial additional research into host susceptibility, reservoir competency, and linkages between climate, mosquitoes, and disease. Such work will entail a collaborative effort between scientists in governmental research groups, in surveillance and control programs, and in nongovernmental organizations. West Nile virus was not the first, and it will not be the last, exotic disease to be introduced to the New World. Its spread in North America highlights the need to strengthen animal monitoring programs and to integrate them with research on disease ecology.

Serologic Evidence of West Nile Virus Transmission, Jamaica, West Indies

In spring 2002, an intensive avian serosurvey was initiated in Jamaica, Puerto Rico, and Mexico. We collected >1,600 specimens from resident and nonresident neotropical migratory birds before their northerly migrations. Plaque reduction neutralization test results indicated specific neutralizing antibodies to West Nile virus in 11 resident species from Jamaica.

Detection by Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds

We adapted an indirect immunoglobulin G enzyme-linked immunosorbent assay to facilitate studies of West Nile virus (WNV) and evaluated its application to taxonomically diverse avian species. Anti-WNV antibodies were detected in 23 bird species, including many exotic species, demonstrating its value in studies of WNV epizootiology.

Virus Detection Protocols for West Nile Virus in Vertebrate and Mosquito Specimens

ABSTRACT The recent outbreaks of West Nile virus (WNV) infection in the northeastern United States and other regions of the world have made it essential to develop efficient, sensitive, and rapid protocols for virus surveillance. Laboratory testing is the backbone of any surveillance program. Protocols to detect the presence of WNV have been refined since 1999 for sensitivity, speed, efficiency, and specificity. This paper presents the protocols currently used by the New York State Department of Health to handle vertebrate and mosquito specimens that have been submitted for WNV testing to the Arbovirus Laboratories of the Wadsworth Center.

West Nile Virus, Venezuela

To the Editor: West Nile virus (WNV; genus Flavivirus; family Flaviviridae) has been perpetuating in North America since 1999 (1). However, its status as a self-perpetuating pathogen in South America remains uncertain. Infected horses and birds have been reported in various Caribbean Islands, Mexico, and northern Central America (2,3). In South America, isolated reports of infected dead-end hosts (horses) have come from northern Colombia and Argentina but they lack evidence for infection in avian amplifying hosts (4,5). We report serologic evidence of establishment of WNV in South America. Serum samples from birds and horses from 33 locations in Venezuela (Appendix Table) were screened for immunoglobulin G (IgG) antibodies against WNV antigen by ELISA (6) and confirmed by plaque reduction neutralization test (PRNT) as previously described (7). The flavivirus generating the IgG response was identified by using the following criteria: 90% inhibition of virus in serum diluted at least 1:40 and 4-fold greater neutralizing antibody titer compared with closely related flaviviruses. IgG antibody against flavivirus was detected by ELISA in 14 of 576 resident birds, including 5 Turdus leucomelas, 3 Gallus gallus (captive), 2 Campylorhamphus trochilirostris, and 1 each of Elaenia flavogaster, Coereba flaveola, Thraupis palmarum, and Anisognathus flavinucha. WNV was confirmed as the etiologic agent of infection in 5 adult birds (3 T. leucomelas [pale-breasted thrush], 1 C. flaveola [bananaquit], and 1 G. gallus [domestic chicken] with the earliest collection date in February 2006); virus neutralization titers ranged from 80 to 320. One serum sample cross-reacted with other flaviviruses tested, with equivalent titers to WNV, Saint Louis encephalitis virus (SLEV), and Ilheus virus (ILHV) and was thus considered infected with an undetermined flavivirus. Seven serum samples were negative (antibody titers <20), and 1 sample was not tested because of insufficient sample volume. Antibody against flavivirus was detected by ELISA in 141 of 791 horses, and 34 (4.3%) were confirmed positive for WNV infection by PRNT; viral titers ≥640 occurred in half of these horses. The earliest collection date for a WNV-positive horse was February 2004 and the most recent was May 2006. Specific WNV-reactive equine serum samples were distributed in valley regions (prevalence 1.3%), savannah grasslands (2.4%), the western region of Zulia (0.4%) and the Central Lake Basin (0.3%). A total of 46 (5.8%) equine serum samples were positive for neutralizing antibody to SLEV, and 8 (1.0%) samples were positive for neutralizing antibodies to ILHV. Forty-nine samples neutralized at least 2 of the 3 viruses and were classified as undetermined flaviviruses. Serum samples from 2 horses were negative in neutralization assays; 2 others were not tested because of insufficient sample volume. WNV-infected resident birds, rather than an importation event, are the basis of establishment of WNV in South America. We hypothesize that ornithophilic mosquitoes (such as some Culex spp.), which are present in the area in consistently high numbers, acquired the virus through hematophagous feeding on recently infected, migrating birds. Once introduced to local mosquitoes, virus is amplified among susceptible resident birds fed upon by ornithophilic mosquitoes. This pattern allows perpetuation and subsequent establishment of virus in a continuous transmission cycle, as opposed to infection of dead-end hosts, e.g., horses. This is the first report of WNV infection in South American birds and definitive establishment of the virus in South America. We observed varying WNV seroprevalence rates in birds and horses across regions in Venezuela (Figure). These differences reflect the focal and stochastic nature of arbovirus transmission, which depends upon many ecologic factors. One possible explanation for the greater seroprevalence in the central and eastern llanos (savannahs) and valley regions, compared with the coastal western region of Zulia State (p<0.0001, by Pearson’s χ2 test) would be virus introduction by migrating birds by an eastern migration route. Figure Collection sites for West Nile virus (WNV) in Venezuela. Symbols represent results of tests for specific antibodies to WNV in serum samples of birds and horses (viral titers in a 90% plaque reduction neutralization test >40 and a 4-fold differential ... Existence of several closely related flaviviruses in the American tropics (8–10) may convey cross-protection in animals (e.g., ILHV and SLEV) or humans (dengue viruses, yellow fever virus), thereby potentially diminishing disease caused by a newly introduced flavivirus such as WNV. Although ILHV infection has not been detected in Venezuela, this flavivirus is prevalent in Brazil, Peru, French Guyana, Trinidad, and Colombia. Our study demonstrated widespread distribution of ILHV in Venezuela. Other South American flaviviruses, such as Bussuquara, Cacipacore, and Iguape, and as yet undiscovered viruses may also circulate in Venezuela. We encourage those involved in the public and animal health systems in Venezuela to consider zoonotic flaviviruses in the differential diagnoses of human and equine cases of encephalitis and to consider ecologic surveillance for zoonotic flaviviruses in mosquito and vertebrate host populations. We recommend monitoring blood and organ donations for flavivirus infections. Our study sheds light on flavivirus distribution in Venezuela. However, nothing else is known about the ecology of zoonotic flaviviruses in this country. Such knowledge will be essential for designing effective surveillance and control should these viruses be shown to cause human illnesses.

Land Use and West Nile Virus Seroprevalence in Wild Mammals

We examined West Nile virus (WNV) seroprevalence in wild mammals along a forest-to-urban gradient in the US mid-Atlantic region. WNV antibody prevalence increased with age, urbanization, and date of capture for juveniles and varied significantly between species. These findings suggest several requirements for using mammals as indicators of transmission.

Prevalence of West Nile Virus in Migratory Birds during Spring and Fall Migration

To investigate the role of migratory birds in the dissemination of West Nile virus (WNV), we measured the prevalence of infectious WNV and specific WNV neutralizing antibodies in birds, principally Passeriformes, during spring and fall migrations in the Atlantic and Mississippi flyways from 2001-2003. Blood samples were obtained from 13,403 birds, representing 133 species. Specific WNV neutralizing antibody was detected in 254 resident and migratory birds, representing 39 species, and was most commonly detected in northern cardinals (Cardinalis cardinalis) (9.8%, N = 762) and gray catbirds (Dumetella carolinensis) (3.2%, N = 3188). West Nile virus viremias were detected in 19 birds, including 8 gray catbirds, and only during the fall migratory period. These results provide additional evidence that migratory birds may have been a principal agent for the spread of WNV in North America and provide data on the occurrence of WNV in a variety of bird species.

DNA Vaccination of American Robins (Turdus migratorius) Against West Nile Virus

West Nile virus (WNV) has caused at least 1150 cases of encephalitis, 100 deaths, and an estimated 30,000-80,000 illnesses in 6 of the last 7 years. Recent evidence from several regions has implicated American robins (Turdus migratorius) as an important host for feeding by Culex mosquitoes, and, when integrated with their host competence for WNV, demonstrates that they are a key WNV amplification host. We evaluated the efficacy of a DNA plasmid vaccine at reducing the viremia and infectiousness of hatch-year American robins. We found that a single dose of vaccine injected intramuscularly resulted in more than a 400-fold (10(2.6)) decrease in average viremia. Although sample sizes were small, these results suggest that vaccinated robins exhibit viremias that are likely to be mostly noninfectious to biting Culex mosquitoes. More broadly, if an orally effective formulation of this vaccine could be developed, new control strategies based on wildlife vaccination may be possible.

West Nile Virus Infection of Birds, Mexico

Birds of 2 of 3 passerine species died after experimental infection with 2 strains from Mexico.