Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Abstract

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

Diagnostic capacity for viral haemorrhagic septicaemia virus (VHSV) infection in rainbow trout (Oncorhynchus mykiss) is greatly increased by combining viral isolation with specific antibody detection

“…Only 17% of the fish, which were positive by 50%PNT, were also virus positive. As previously reported there is a time-lag between infection with the virus and the production of both neutralising and nonneutralising antibodies against VHSV [12,15,16]. This was evident in our data as the 50%PNT detected less antibody positive fish than the traditional diagnostic procedure of viral isolation in the initial stages of the outbreaks whereas the reverse was true for the later sampling dates producing an overall negative correlation between seropositive and virus positive samples over time (Fig.…”

Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay To Detect Antibodies to Viral Hemorrhagic Septicemia Virus

“…Additionally, we determined that methylcellulose overlay is not necessary in our VN assay because plaques are not counted to determine the titers. Complement has been shown to enhance neutralization in 50% PNTs when applied to trout serum (23,48,50). However, neutralization was not enhanced by the addition of complement in our assay, which may indicate the presence of a different immune mechanism specific to trout.…”

“…The VN assay has the advantage of recognizing antibodies that likely confer protective immunity to VHSV and can indicate recent exposure to the virus (25). The blocking ELISA is valuable for identifying nonneutralizing anti-nucleocapsid antibodies, which may persist longer and therefore extend the opportunity to detect VHSV antibodies after initial virus exposure (25,48). These assays complement viral detection methods by providing a means for determining the exposure histories of wild populations.…”

“…However, neutralization was not enhanced by the addition of complement in our assay, which may indicate the presence of a different immune mechanism specific to trout. It should be noted that the reduced sensitivity observed in our VN assay may be due to VHSV forming a complex with neutralizing antibodies in the serum, which reduces the availability of antibodies for binding to virus neutralization epitopes in the VN assay (45)(46)(47)(48). However, this concern is obviated by using the nucleocapsid protein as our target, because antibodies with nucleocapsid affinity presumably do not complex with the intact viral particle, which underscores another value of the anti-nucleocapsid ELISA.…”

“…Studies have shown that neutralizing antibodies do not persist as long as nonneutralizing antibodies in trout (25,48), and neutralizing antibody titers peak at 6 weeks postinfection in rainbow trout infected with VHSV I (24) and at 11 to 16 weeks in muskellunge infected with VHSV IVb (44). It was observed that the majority of VHSV-exposed fish with serum samples collected prior to 6 weeks postinfection had no or low neutralizing antibody titers.…”