Detection of West Nile Virus Antigen in Mosquitoes and Avian Tissues by a Monoclonal Antibody-Based Capture Enzyme Immunoassay

Abstract

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10 2.1 to 10 3.7 PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n ‫؍‬ 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10 3 PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

West Nile virus infection of horses

“…Antigen capture ELISA tests have been used to confirm the presence of WNV in avian tissues and mosquito pools [40] but are unsuitable for use in horses due to the low level of viraemia.…”

Evaluation of Antigen-Capture ELISA and Immunohistochemical Methods for Avian Surveillance of West Nile Virus

“…Immunohistochemical analysis (IHC) and antigen-capture ELISA (ACE) have been used to detect West Nile virus in birds. 9,18,20 Both assays are amenable to high sample load, and the cost is approximately 30% of that of real-time RT-PCR analysis in this laboratory.…”

“…As originally reported, the threshold level for detection of WNV by ACE was 10 3 viral particles or a cycle-threshold value (Ct) Յ30 by real-time RT-PCR analysis. 9 The real-time RT-PCR results for these false-negative samples had Ct values between 30 and 40, implicating low viral load. There was 1 false-positive result for cloacal swab specimens, presumably owing to fecal matter; no false-positive results for skin samples; and 2 false-positive results for feathers, possibly due to contaminants on the feather calamus or in the pulp.…”

“…The cloacal swab specimen and skin and feather samples were tested using a previously published ACE protocol. 9 The only modification was decreasing the concentration of the stop solution to 0.18 M sulfuric acid. b The positive control was a lysate of WNV recombinant protein expressed in transfected monkey kidney (COS) cells, and the negative control was a lysate of COS cells without recombinant protein.…”

“…4,5,[9][10][11] Diagnostic tests such as VI, RT-PCR, IHC on ear notches, AC-ELISA on serum, or ear-notches supernatant are some of the most commonly used tests to detect the presence of a PI animal. 15 Virus isolation and RT-PCR are costly when used on individuals within a population, whereas IHC and AC-ELISA are cost friendly, with charges…”