Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout ( Oncorhynchus mykiss )

Abstract

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1-505 bp) gene was amplified by RT-PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS-PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.

Imunobiológicos para o diagnóstico laboratorial de ranavirose: clonagem e expressão do gene MCP de isolado brasileiro de >i<Ranavirus>/i< e produção de anticorpos policlonais anti-proteína MCP recombinante

“…Como demonstrado por estes trabalhos, os métodos sorológicos apresentam grandes vantagens principalmente quando a doença é endêmica ou está presente em baixa prevalência na população sem sinais clínicos graves, visto que, muitas vezes, não é possível a identificação direta do vírus, pois as partículas virais não estão mais presentes, diferentemente dos anticorpos que podem ser detectados por um longo período de tempo (JARAMILLO et al, 2017;RAHNAMA et al, 2019). Em psiculturas e raniculturas, onde os animais são submetidos a condições de manejo adequadas, por exemplo com água e ração de qualidade, podem ocorrer casos assintomáticos de ranaviroses, que possivelmente seriam detectados por análises sorológicas.…”