Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 11, Number 10—October 2005
Dispatch

Rapid West Nile Virus Antigen Detection

Tables
Article Metrics
11
citations of this article
EID Journal Metrics on Scopus
Author affiliations: *Centers for Disease Control and Prevention, Fort Collins, Colorado, USA

Cite This Article

Abstract

We compared the VecTest WNV antigen assay with standard methods of West Nile virus (WNV) detection in swabs from American Crows (Corvus brachyrhynchos) and House Sparrows (Passer domesticus). The VecTest detected WNV more frequently than the plaque assay and was comparable to a TaqMan reverse transcription–polymerase chain reaction.

Dead bird surveillance is an effective way to monitor the presence and spread of West Nile virus (WNV) in North America (1), and assays to detect infectious WNV virions, antigen, and RNA in tissues from infected birds are reliable techniques (24). More than 28,000 bird carcasses were tested for WNV in the United States from 1999 to 2002 (5). Processing and testing these carcasses require a substantial commitment of resources from federal, state, and local health departments. Simplifying diagnostic procedures by implementing rapid antigen-capture assays would permit increased specimen processing and, ultimately, improved surveillance.

Cloacal and oral (nasopharyngeal) swabs from dead corvids (crows and jays) are reliable sources of WNV RNA and infectious virions (6). Three field evaluations of an antigen detection assay applied to corvid carcasses collected shortly after death found that oral swabs were more sensitive than cloacal swabs for detecting WNV antigen, and that sensitivity of the VecTest WNV antigen assay (Medical Analysis Systems, Camarillo, CA, USA) applied to oral swabs was >80% for American Crows, lower for other corvids, and variable for a variety of other species (79). Several questions remain unanswered regarding the usefulness of swab specimens for WNV surveillance. How long after death of a bird can WNV be detected in swab specimens? Can swabs from noncorvid birds be used to detect WNV? Can reverse transcription–polymerase chain reaction (RT-PCR) or VecTest detect WNV in oral swab samples that have remained dry and at room temperature?

To address these questions, we compared the VecTest WNV antigen assay with standard methods of virus detection from oral and cloacal swabs taken 1–4 days postmortem from experimentally infected American Crows (Corvus brachyrhynchos) and House Sparrows (Passer domesticus). The VecTest, which was originally developed for mosquito pools as a simple, 1-step wicking assay available in a kit, requires no specialized equipment, storage conditions, or highly trained personnel and provides results in 15 minutes (10,11).

The Study

Oral and cloacal swab samples were collected daily (for 4 days) from carcasses of crows and sparrows that had been experimentally infected with either the NY99-4132 strain (30 crows and 6 sparrows) or the Kenyan KN-3829 strain (1 crow and 5 sparrows) of WNV. Carcasses were stored at ambient temperature (≈20°C) during this period. The samples were collected with standard, cotton-tipped applicators by inserting them into the cloaca or oral cavity and then placing them directly into 1 mL VecTest grinding solution A, a physiologic buffer similar to phosphate-buffered saline. Samples were subsequently frozen at –70°C until tested by a variety of methods for detecting WNV. Some oral swabs from infected crows were left at room temperature without diluent for 24 or 48 hours before testing. Negative control swab samples were collected from 25 live, healthy, uninfected crows.

All swab specimens collected from crow carcasses were positive by the TaqMan RT-PCR method, using 2 sets of WNV-specific primers (2). Several TaqMan RT-PCR–negative swabs for the sparrows were also negative by the other assays; these were disregarded in summarizing the results. Results of VecTest and Vero plaque assay (11) of the RT-PCR–positive swab specimens are shown as sensitivities (using RT-PCR as the standard for detecting WNV RNA) in Table 1. A logistic regression model accounting for anticipated correlation induced by multiple and repeated observations on each bird was used to compare sensitivities for each day postmortem, with significance determined using α = 0.05 (12). For crows evaluated 1 day postmortem, no significant difference between swab types (oral versus cloacal) (p = 0.63) and no significant difference between the 2 assays (p = 0.10) were detected. At 2 days postmortem, the effect due to swab type was not significant (p = 0.07), but a significant difference was seen in the sensitivities of the 2 assays (p = 0.004), excluding the nonsignificant effect of swab type from the logistic regression model. At 3 days postmortem, both swab type and assay differences were significant (p<0.01), with oral swabs more likely to yield a positive finding (compared with cloacal swabs) and VecTest more sensitive than plaque assay. For sparrows, no significant differences were seen between the sensitivities of the VecTest and plaque assay for either swab type on any of the 3 days (McNemar test).

VecTest detected WNV in 90% of 22 crow oral swabs that were tested after remaining dry and at room temperature for 24 hours and in 70% of 13 crow oral swabs assayed after 48 hours. By comparison, TaqMan RT-PCR detected WNV in 86% and 70% of these oral swabs, respectively, at the same time points.

Over the 4-day sampling period, geometric mean viral titers in crow oral swabs, determined by Vero cell plaque assay, decreased from 103.6 to 102.2 PFU/swab (Table 2). In contrast, the geometric mean viral titer in crow cloacal swabs decreased from 103.0 PFU/swab at 1 day postmortem to undetectable by 4 days postmortem. RNA levels, as detected by the TaqMan assay, also decreased over time.

Conclusions

VecTest has the potential to simplify dead bird surveillance for WNV by reducing required resources such as specialized equipment and costly reagent kits needed to achieve a rapid and accurate result. With appropriate biosafety measures, the assay can be conducted in the field, or in centralized regional laboratories, obviating the need for expensive shipping of bird carcasses to remote reference laboratories.

One objective of our study was to determine whether oral or cloacal swabs were preferable for WNV testing of dead birds. To answer this question, several criteria were evaluated, including the ability of 3 different assays to detect WNV, the feasibility of collecting specimens postmortem, and postmortem duration of WNV positivity. TaqMan RT-PCR detected WNV RNA and antigen in similar proportions in all cloacal and oral specimens collected from crows. However, virus isolation by Vero plaque assay was more successful when oral swabs were tested. Virus appears to be more rapidly inactivated in the cloaca compared with the oral cavity. This phenomenon was consistent for both sparrows and crows.

Fewer postmortem swab samples were available from sparrows compared with those from crows because fewer sparrow carcasses were available (sparrows are less susceptible to fatal WNV infection than crows) (13). Collecting cloacal swabs from the smaller sparrows was also more difficult after 1 day postmortem because they tended to desiccate quickly. RT-PCR detected WNV RNA in sparrows from 24/24 oral swabs, but only 11/13 cloacal swabs. Antigen was detected by VecTest from 18/24 oral and 8/13 cloacal swabs. Infectious virus was detected by plaque assay in 20/24 oral swabs but in only 6/13 cloacal swabs. Virus titers and RNA concentrations in the carcasses decayed over the 4-day period of observation and this decay was most pronounced in the cloacal swabs. Thus, oral swabs were more effective than cloacal swabs to detect WNV in both crows and sparrows.

VecTest consistently detected WNV antigen in a greater proportion of samples than Vero plaque assay detected virions. Thus, although the detection of infectious virus was inconsistent, carcasses contained sufficient quantity of viral components, both RNA and protein, to permit detection for >4 days after death. In a natural setting, carcasses most likely would decay more rapidly than in these experiments, given exposure to temperature fluctuations, microbial attack, and predation. Guidelines for WNV surveillance recommend sampling carcasses <24 hours old (14). These results suggest that older carcasses may have detectable WNV RNA and antigen that still are readily detectable with the TaqMan and VecTest assays. Thus, carcasses should be tested regardless of age, as long as they are not in a condition where sampling is impossible. In addition, swabs collected in the field can be stored at room temperature in empty cryovials for up to 48 hours and then reliably assayed for WNV antigen by VecTest.

Detecting WNV from sparrow carcasses demonstrates that swabs are useful to test species other than corvids. House sparrows, like corvids, are passerine birds that develop high levels of WNV in blood and tissues (13). Stone et al. showed that the VecTest had a sensitivity of 76% in detecting WNV in oral swabs of field-collected carcasses of house sparrows (9).

In summary, oral swabs are more useful than cloacal swabs for obtaining a reliable result with the diagnostic assays described in our study. Moreover, swabs from noncorvid birds may also be effectively assayed for WNV. Our findings suggest that large numbers of dead corvids of any age, and possibly other passerine birds, could be screened by cautiously collecting dry oral swabs in the field, storing them properly, and then testing them within 48 hours by rapid antigen detection assay or RT-PCR.

Mr Panella is a biologist in the Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado. He has participated in WNV outbreak investigations and has contributed to studies of WNV ecology in the United States.

Top

Acknowledgments

We thank Jason Velez for technical assistance, Richard Bowen for providing space for laboratory animals at Colorado State University, and Thomas Janousek and Charles Cope for providing crows for this study.

The authors have no financial interest with the manufacturers or developers of VecTest.

Top

References

  1. Eidson  M, Komar  N, Sorhage  F, Nelson  R, Talbot  T, Mostashari  F, Crow deaths a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis. 2001;7:61520. DOIPubMedGoogle Scholar
  2. Lanciotti  RS, Kerst  AJ, Nasci  RS, Godsey  MS, Mitchell  CJ, Savage  HM, 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. 2000;38:406671.PubMedGoogle Scholar
  3. Hunt  A, Hall  RA, Kerst  AJ, Nasci  RS, Savage  HM, Panella  NA, Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay. J Clin Microbiol. 2002;40:202330. DOIPubMedGoogle Scholar
  4. Steele  KE, Linn  MJ, Schoepp  RJ, Komar  N, Geisbert  TW, Manduca  RM, Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. J Vet Pathol. 2000;37:20824. DOIPubMedGoogle Scholar
  5. Komar  N. West Nile virus: epidemiology and ecology in North America. Adv Virus Res. 2003;61:185234. DOIPubMedGoogle Scholar
  6. Komar  N, Lanciotti  R, Bowen  R, Langevin  S, Bunning  M. Detection of West Nile virus in oral and cloacal swabs collected from bird carcasses. Emerg Infect Dis. 2002;8:7412.PubMedGoogle Scholar
  7. Lindsay  R, Barker  I, Nayar  G, Drebot  M, Calvin  S, Scammell  C, Rapid antigen-capture assay to detect West Nile virus in dead corvids. Emerg Infect Dis. 2003;9:140610.PubMedGoogle Scholar
  8. Yaremych  SA, Warner  RE, van de Wyngaerde  MT, Ringia  AM, Lampman  R, Novak  RJ. West Nile virus detection in American Crows. Emerg Infect Dis. 2003;9:131921.PubMedGoogle Scholar
  9. Stone  WB, Okoniewski  JC, Therrien  JE, Kramer  LD, Kauffman  EB, Eidson  M. VecTest as diagnostic and surveillance tool for West Nile virus in dead birds. Emerg Infect Dis. 2004;10:217581.PubMedGoogle Scholar
  10. Ryan  J, Davé  K, Emmerich  E, Fernández  B, Turell  M, Johnson  J, Wicking assays for the rapid detection of West Nile and St. Louis encephalitis viral antigens in mosquitoes. J Med Entomol. 2003;40:959. DOIPubMedGoogle Scholar
  11. Nasci  RS, Gottfried  KL, Burkhalter  KL, Kulasekera  VL, Lambert  AJ, Lanciotti  RS, Comparison of Vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes. J Am Mosq Control Assoc. 2002;18:294300.PubMedGoogle Scholar
  12. Diggle  P, Heagerty  P, Liang  K-Y, Zeger  S. Analysis of longitudinal data. 2nd ed. Oxford (UK): Oxford University Press; 2002.
  13. Komar  N, Langevin  S, Hinten  S, Nemeth  N, Edwards  E, Hettler  D, Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis. 2003;9:31122.PubMedGoogle Scholar
  14. Gubler  DJ, Campbell  GL, Nasci  R, Komar  N, Petersen  L, Roehrig  JT. West Nile virus in the United States: guidelines for detection, prevention, and control. Viral Immunol. 2000;13:46975. DOIPubMedGoogle Scholar

Top

Tables

Top

Cite This Article

DOI: 10.3201/eid1110.040394

Table of Contents – Volume 11, Number 10—October 2005

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.

Top

Comments

Please use the form below to submit correspondence to the authors or contact them at the following address:

Nicholas A. Panella, Arbovirus Diseases Branch, Centers for Disease Control and Prevention, PO Box 2087, Fort Collins, CO 80522, USA: fax: 970-221-6476

Send To

10000 character(s) remaining.

Top

Page created: February 21, 2012
Page updated: February 21, 2012
Page reviewed: February 21, 2012
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
file_external