Assays to Detect West Nile Virus in Dead Birds

Using oral swab samples to detect West Nile virus in dead birds, we compared the Rapid Analyte Measurement Platform (RAMP) assay with VecTest and real-time reverse-transcriptase–polymerase chain reaction. The sensitivities of RAMP and VecTest for testing corvid species were 91.0% and 82.1%, respectively.

antigen in samples. VecTest uses antibodies bound to gold sol particle labels, while the RAMP test uses antibodies bound to fluorescently labeled latex particles. Development of a visible reddish-purple line in both the test and control zones on the VecTest strip indicates a positive result. The RAMP test strip, enclosed within a cartridge, is inserted into a reader that calculates the ratio between the fluorescence emitted at the test and control zones and displays the results as RAMP units. Values above a background threshold are recorded as positive.
This study compared WNV results from the RAMP and VecTest on oral swab samples from dead birds, with RT-PCR on brain tissue as the standard. Brain swab samples were also tested as an alternate antigen source in the RAMP and VecTest.

The Study
Birds included in this study were received from mid-May to late November 2004 and from mid-February through May 2005 from counties in New York State. Oral swab samples for the RAMP and VecTest were collected with 2 sterile, polyester fiber-tipped plastic applicators held together and moved around the oral cavity and proximal esophagus. One swab sample was twirled in 1.0 mL of VecTest buffer solution in a 5-mL plastic tube. The second swab sample was either twirled in 1.0 mL of RAMP buffer solution in a separate 5-mL plastic tube or placed in an empty 5-mL plastic tube, capped, and frozen at -20ºC for later testing. RAMP tests were run the same day on fresh material or later on frozen samples. Before being tested, all frozen samples were thawed at room temperature; swabs not previously mixed in solution and swabs from thawed carcasses were then mixed in RAMP buffer solution. Samples were taken from the brains of a subset of corvid species by swabbing cerebral parenchyma and processing as for oral samples. The RAMP and VecTest were run according to manufacturers' directions in a class II biosafety cabinet at the New York State Department of Environmental Conservation's Wildlife Pathology Unit. RAMP test values >50, calculated by the RAMP reader, were recorded as positive. Differences in test performance were assessed by chi-square analysis. Data are expressed as a percentage in text and tables only when n is >10.
Brain samples for RT-PCR were taken at necropsy and frozen at -20ºC. Brain tissue was analyzed at the Arbovirus Laboratory, Wadsworth Center, New York State Department of Health, as described previously (2,3). RT-PCR was repeated on 54 birds for which results from RAMP, VecTest, or both, contrasted with RT-PCR results. Retests of 6 birds yielded different results from the original tests. Three of these were initially positive and retested negative; the original values were low, which indicated infectivity was focal and undetected on a different sample, and the level was below RAMP and VecTest limits of detection. Three originally negative samples retested positive; 2 were highly positive, which indicated a technical error, and 1 kidney tissue sample was positive, although results of a retest with brain tissue were negative.
RAMP confirmed more Common Grackles (Quiscalus quiscula) (3/3) and House Sparrows (5/6) as positive than did VecTest (2/3 and 3/6, respectively). With the exception of a few species, both tests performed poorly overall on small sample sizes of other noncorvid species.
To determine if RAMP results were affected by freezing the sample, samples from 13 corvids (10 positive, 3 negative) were retested by using swabs taken from frozen carcasses. Six initially were tested with fresh swabs and 7 with frozen swabs; all retests yielded results similar to initial results. The same results for fresh versus frozen samples were obtained with VecTest (4).

Conclusions
Although RAMP was more sensitive than VecTest, both appear adequate for WNV surveillance in dead corvids. RAMP also performed well with oral swabs from Common Grackles and House Sparrows, although sample sizes were small. These findings are similar to previous results for VecTest, which also tested well with House Finches (Carpodacus mexicanus), Northern Cardinals (Cardinalis cardinalis), and American Kestrels (Falco sparverius) (4). As in the previous study, both tests did poorly in RT-PCR-positive raptors.
In the previous New York study, VecTests successfully tested brain, kidney, blood, feather pulp, and cloacal samples from corvids and House Sparrows (4). In the current study, RAMP and VecTest worked well with brain as the antigen source. Brain swab samples may be the preferred antigen source when the oral cavity is compromised. Further testing of alternative swab samples is warranted and may identify a superior antigen source; however, testing internal organs may pose greater risks and may not be applicable in field work and nonlaboratory-based surveillance. In addition, further testing, including immunohistochemical tests, on noncorvids should be conducted to accurately assess these tests and identify the distribution of WNV in the oral cavity and internal tissues.
In this study, VecTest produced no false-positive results. Although its specificity was high, RAMP produced 8 false-positive results (range 50.9-147.6). Four of these were near the >50 positive indicator level and may have been due to other sources of fluorescence. The remaining 4 false-positives (3 American Crows and 1 Blue Jay), with scores from 74.4 to 147.6, came from birds with oral cavities compromised by blood or fly eggs, which may have biased results.
VecTest results are easily distinguished when a true WNV-positive reaction occurs, but the reddish-purple line may appear faint or thin in other cases and may be subject to interpretation (4). RAMP quantitative results eliminate subjective interpretation, which helps assure replication but limits confidence in lower RAMP-positive scores.
The RAMP system requires an initial purchase of an electronic reader (≈US $3,500), and materials cost $13-$15 per test; VecTest costs $8 per test. If large numbers of specimens are tested, the cost of the RAMP reader per test is minimal. The RAMP test requires a minimum of 1.5 h to run because of the required cartridge drying time; VecTest takes 15-30 min to run after the test strip is placed in the sample solution.
In conclusion, both RAMP and VecTest are useful alternatives to RT-PCR for WNV surveillance in dead corvids and some passerine species when immediate turn-around of large numbers of specimens is valuable. Testing with RAMP is advantageous because of its increased sensitivity; however, follow-up testing with RT-PCR is recommended for low RAMP-positive results near the positive indicator level. Using both tests in a system in which initial testing is conducted with VecTest may also be useful; RAMP could be reserved for high-priority cases in which VecTest results are negative. RT-PCR should still be used to confirm initial viral activity in a new period and area and for research requiring more definitive results.