Date Published: January 17, 2017
Publisher: Public Library of Science
Author(s): Amy M. Hurwitz, Wanzhi Huang, Baijun Kou, Mary K. Estes, Robert L. Atmar, Timothy Palzkill, Ellen R. Goldman.
Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.
Norovirus is the leading cause of foodborne illnesses in the United States and is commonly known as the cruise ship or winter bug. Infections spread rapidly and result in roughly 267 million unique cases and 200,000 deaths annually around the globe in addition to significant health and economic repercussions [1–3]. Although evaluation of a candidate vaccine is underway [4,5], there are currently no treatments available for infection beyond rehydration and symptom-directed therapy. Further, the standard diagnostic assays present significant limitations that hinder their ability to identify infection in order to inform effective prevention strategies, especially at the point-of-care. These shortcomings in detection can lead to otherwise preventable outbreaks, while immunocompromised and other especially vulnerable patients who become infected by norovirus can suffer from chronic symptoms and inappropriate treatments . To ameliorate the effects of norovirus in people, developing improved diagnostics can help to prevent outbreaks and inform appropriate clinical responses that will protect sensitive patient populations.
Improved point-of-care tests (POCT) for detecting norovirus are needed to alleviate the health and economic burdens of infections. While many tests can specifically identify GII.4 with strong sensitivity, the primary limitation with existing diagnostics revolves around the lack of ability to reliably detect and distinguish among the full range of human-infecting strains, including those classified amongst the genotypes appearing in genogroups GI or GII for epidemiological studies . Although the updated RIDA®QUICK immunochromatographic assay (Version N1402) has expanded its specificity to detect some GI genotypes, there is still no evidence that it can detect GI.7 or GI.8 to follow-up on results from the previous version that indicated it could not detect either genotype [15,29]. In addition to having sufficient genotype specificity and sensitivity, the ideal POCT should be easy and rapid to perform and cheap to produce. These qualities would enable individual diagnosis that would inform appropriate treatment of gastrointestinal symptoms, prevention of the rapid spread of outbreaks, and more accurate recording of infections for epidemiological studies. Other diagnostic methods, such as electron microscopy, RT-PCR, and ELISAs are limited by the equipment, expertise and time they require, and thus do not facilitate POC detection. Therefore, improved diagnostic tools are still needed to fulfill the call for an accurate POCT.