MAb‐based competitive ELISA for the detection of antibodies against influenza D virus

Abstract

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb-based competitive ELISA for the detection of antibodies against IDV. Thirty-one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724-3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid-phase competitive ELISA (IDV-cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase-conjugated MAb as competitor, which had shown wide intratype cross-reactivity and positivity in HI. To evaluate the diagnostic performances of IDV-cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut-off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV-cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI -0.899 to 0.961) between HI test and IDV-cELISA. ROC analysis showed that IDV-cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993-1.000). A cut-off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1-99.9) and 98.75 (95% CI 97.1-99.6). These results proved excellent diagnostic performances of IDV-cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti-IDV antibodies in different animal species.

Risk Mapping of Influenza D Virus Occurrence in Ruminants and Swine in Togo Using a Spatial Multicriteria Decision Analysis Approach

“…Clearly, increasing sampling intensity and prioritizing young cattle with respiratory clinical signs is necessary; both these factors were strongly associated with IDV according to expert's opinion. Seropositivity rates we calculated may also have been underestimated because HI assays are less sensitive than ELISAs, which should be preferred for further serological analyses [31], even if previous IDV seroprevalence in Africa were calculated from HI assays data as well [2].…”

Emerging Influenza D Virus Threat: What We Know so Far!

“…In a most recent study, Henritzi et al developed a tetraplex real-time RT-PCR assay for screening influenza virus types A, B, C and D simultaneously [77]. Moreno et al focused on the development and validation of MAb-based competitive ELISA for the detection of antibodies against IDV which may be advantageous when compared to the traditional hemagglutination inhibition (HI) test [78].…”

Formal estimation of the seropositivity cut‐off of the hemagglutination inhibition assay in field diagnosis of influenza D virus in cattle and estimation of the associated true prevalence in Morocco

“…The choice of any strategy is based on the estimation of the cost-benefit ratio (e.g. Moreno et al, 2019) followed by inference of VNT using the methodology developed in this study or by the direct use of VNT as reference test is recommended.…”