Development of a Focus-Reduction Neutralizing Test for Detecting Equine Herpesvirus Type-1-Neutralizing Antibodies

Abstract

ABSTRACT. Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n=3) and in those infected naturally (n=16). The FRNT for EHV-1 was high-throughput and time saving. The FRNT can be used in large-scale seroepidemiological studies of EHV-1 and in evaluating vaccine efficacy. KEY WORDS: EHV-1, focus-reduction, virus-neutralizing test.doi: 10.1292/jvms.13-0043; J. Vet. Med. Sci. 75(9): 1209-1212, 2013 Virus-neutralization (VN) is a classical test used for the serological diagnosis of equine herpesvirus type-1 (EHV-1) infection. In addition to having diagnostic applications, the VN test is used to evaluate humoral immune responses to EHV-1 infection or EHV-1 vaccines [3][4][5]. Although the importance of cell-mediated immunity in protection against EHV-1 infection has been confirmed, induction of systemic VN antibodies is also essential for this purpose [1].There are two conventional methods of VN testing for EHV-1. One is the 50% tissue culture infective dose (TCID 50 ) method, which has been used by many researchers investigating 13]. This method has been employed as an OIE (World Organisation for Animal Health) standard protocol for the diagnosis of EHV-1 infection [8]. The method can be applied to large numbers of samples, because it uses 96-well plates (two columns of wells for testing one sample). However, it takes 4 or 5 days of culture to see clear cytopathic effects. Another method is the plaquereduction neutralizing test (PRNT) using 24-well plates [10]. The PRNT has shown greater sensitivity than the TCID 50 method in VN tests for measles virus and rubella virus [6,11], and we have confirmed the same tendency with EHV-1 (data not shown). However, the PRNT is laborious and has limitations for screening the large numbers of serum samples needed for epidemiological investigations, because it uses 24-well plates (one and a half plates for testing one sample). Furthermore, it takes 4 or 5 days of culture to see plaques, as is the case with the TCID 50 method.A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps. Because of such differences, the FRNT has several advantages over the PRNT. The use of 96-well plates makes it possible to...

Epizootiological investigation of equine herpesvirus type 1 infection among Japanese racehorses before and after the replacement of an inactivated vaccine with a modified live vaccine

“…The horses that were confirmed not to show seroconversion by natural infection with EHV-1 or EHV-4 in either of these tests ( n = 50, each period) were selected for further analysis. The virus-neutralizing (VN) titer for EHV-1 was measured for the sera by using a focus-reduction method [12]. An antibody response to the vaccination was considered significant if a ≥ 4-fold increase occurred in VN titers between the first sera and any one of the post-vaccination sera.…”

Successful Control of Winter Pyrexias Caused by Equine Herpesvirus Type 1 in Japanese Training Centers by Achieving High Vaccination Coverage

“…The sera were subjected to EHV-1 and EHV-4 gG ELISAs, and horses confirmed not to show seroconversion in both of these tests (n ϭ 106) were selected for further analysis. They were subjected to virus neutralization (VN) testing for EHV-1 by using a focus reduction method, as described previously (16). An antibody response to the vaccination was considered to have occurred if a Ն4-fold titer rise between the presera and any one of the postvaccination sera occurred.…”

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“…Serological-based assays, such as the virus neutralization test (VNT), plaque reduction, and peptide-based ELISA assays, are also available. Acute and convalescent serum samples with significant increase in antibody titer (four-fold) can indicate recent infection [ 19 , 20 , 21 ]. In addition, virus isolation on primary cell culture system is considered the gold standard assay, which facilitates further comparative and molecular analyses.…”