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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.

SUBMITTER: Hunt AR

PROVIDER: S-EPMC130729 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

Hunt Ann R AR Hall Roy A RA Kerst Amy J AJ Nasci Roger S RS Savage Harry M HM Panella Nicholas A NA Gottfried Kristy L KL Burkhalter Kristen L KL Roehrig John T JT

Journal of clinical microbiology 20020601 6

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%) ...[more]

PMID: 12037058

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Project description:The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.

Project description:BackgroundWest Nile virus (WNV), primarily vectored by mosquitoes of the genus Culex, is the most important mosquito-borne pathogen in North America, having infected thousands of humans and countless wildlife since its arrival in the USA in 1999. In locations with dedicated mosquito control programs, surveillance methods often rely on frequent testing of mosquitoes collected in a network of gravid traps (GTs) and CO2-baited light traps (LTs). Traps specifically targeting oviposition-seeking (e.g. GTs) and host-seeking (e.g. LTs) mosquitoes are vulnerable to trap bias, and captured specimens are often damaged, making morphological identification difficult.MethodsThis study leverages an alternative mosquito collection method, the human landing catch (HLC), as a means to compare sampling of potential WNV vectors to traditional trapping methods. Human collectors exposed one limb for 15 min at crepuscular periods (5:00-8:30 am and 6:00-9:30 pm daily, the time when Culex species are most actively host-seeking) at each of 55 study sites in suburban Chicago, Illinois, for two summers (2018 and 2019).ResultsA total of 223 human-seeking mosquitoes were caught by HLC, of which 46 (20.6%) were mosquitoes of genus Culex. Of these 46 collected Culex specimens, 34 (73.9%) were Cx. salinarius, a potential WNV vector species not thought to be highly abundant in upper Midwest USA. Per trapping effort, GTs and LTs collected > 7.5-fold the number of individual Culex specimens than HLC efforts.ConclusionsThe less commonly used HLC method provides important insight into the complement of human-biting mosquitoes in a region with consistent WNV epidemics. This study underscores the value of the HLC collection method as a complementary tool for surveillance to aid in WNV vector species characterization. However, given the added risk to the collector, novel mitigation methods or alternative approaches must be explored to incorporate HLC collections safely and strategically into control programs.

Dataset Information

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

Publications

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

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