Abstract
Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0297-6) contains supplementary material, which is available to authorized users.
Highlights
Cyprinid herpesvirus 3 (CyHV-3) is the taxonomical classification for koi herpesvirus (KHV) [1], the aetiological agent of an economically important and often fatal disease, koi herpesvirus disease (KHVD), in common carp and koi (Cyprinus carpio L.) worldwide [2,3,4] and is a member of the Alloherpesviridae family of the order Herpesvirales [1, 5].Analysis of herpesviruses during the infectious cycle can provide an insight into the role of the various proteins of the virus and indicate which stages of infection they are important to with regards to virulenceMonaghan et al Vet Res (2016) 47:8 characterised [11, 12]
Identification of purified CyHV‐3 proteins from SDS‐PAGE and silver staining Around 20–22 bands were detected in the purified CyHV-3 preparation with SDS-PAGE (Figure 1A), the majority of which were associated with the virus and not the uninfected cells
Serological diagnostics for other viruses have been based on immunogenic viral antigens that are abundantly expressed during infection [38]
Summary
Analysis of herpesviruses during the infectious cycle can provide an insight into the role of the various proteins of the virus and indicate which stages of infection they are important to with regards to virulence. Monaghan et al Vet Res (2016) 47:8 characterised [11, 12] Such analysis does not, take into account post-transcriptional processing such as translation initiation, elongation and termination [13], and up to 60% of the variation in protein concentration cannot be explained from mRNA analysis alone [14]. The product of ORF81 is thought to be an immunodominant protein [15], recent studies have revealed a number of envelope glycoproteins recognised by infected carp anti-sera, encoded by CyHV-3 ORFs-25, -65, -148 and -149 and the major capsid protein encoded by ORF92 [16]. Determination of which immunogenic antigens are expressed in highest abundance during the lytic infectious cycle may provide useful targets for the development of serological diagnostic tests
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