Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity

Abstract

Introduction: Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells.

Methods: CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated.

Results: The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA.

Conclusion: The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

Keywords: Avian nephritis virus; Microplate assay; Neutral red uptake; Neutralizing antibody; Plaque assay; Sigmoidal four-parameter logistic regression.