Preparation of Polyclonal Antiserum to the Recombinant TiLV-S8 Protein and Its Application in the Detection of Naturally Tilapia Lake Virus (TiLV) Infected Tilapia

Tilapia lake virus (TiLV) is classified as a negative-sense, single-stranded RNA virus in the family Amnoonviridae. It is an enveloped virus with 10 genomic RNA segments, each coding for a protein. TiLV causes disease in tilapia, and outbreaks can lead to significant economic losses for the tilapia aquaculture industry. In this study, the gene encoding the segment 8 protein of TiLV was cloned into the expression vector pET15-b and then transformed into Escherichia coli strain BL21. After induction, the recombinant TiLV-S8 protein (rTiLV-S8), with a molecular mass of 20 kDa, was expressed, purified, and used to immunize mice. The mouse antiserum against rTiLV-S8 protein demonstrated specific immunoreactivity for the viral protein, approximately 19 kDa in TiLV-infected fish tissues, as determined by Western blotting. According to the results of the dot blotting assay, the antiserum was about 80 times less sensitive than one-step RT-PCR in detecting TiLV in homogenates of infected fish samples and showed no cross-reaction with uninfected fish tissues, other common fish viruses, or prevalent bacterial species found in aquatic animals. Furthermore, this polyclonal antiserum could be employed to identify TiLV-infected fish in the field using dot blotting assay, and the results can be confirmed by immunohistochemistry

Generation of Monoclonal Antibodies against Major Capsid Protein (MCP) of Nervous Necrosis Virus (NNV)

Viral encephalopathy and retinopathy (VER), a serious disease that affects several species of fish all over the world, is caused by nervous necrosis virus (NNV). In this study, two monoclonal antibodies (MAbs), namely 5C1 and 5C4, were generated from a mouse immunized with recombinant major capsid protein (MCP) of red-spotted grouper nervous necrosis virus (RGNNV). These MAbs displayed immunoreactivity against MCP and culture fluid of NNV-infected E-11 cell culture tested by dot blotting. Western blot analysis against recombinant MCP and the culture fluid of E-11 cells infected with NNV revealed immunoreactivity at approximately 63 and 37 kDa, respectively. Isotyping test revealed that all the MAbs were IgG2a. According to immunohistochemistry analysis, the MAbs immunoreactivities staining were found in viral assembly sites in the cytoplasm of targeted tissues such as gills and eye of NNV-infected Asian sea bass. The MAbs did not display any cross-reactivity with the recombinant capsid proteins of other fish viruses, including the infectious spleen and kidney necrosis virus (ISKNV), scale drop disease virus (SDDV), tilapia lake virus (TiLV), or other bacterial species commonly found in diseased fish. The immunoreactivity was observed when the MAbs were used for NNV detection by dot blotting in NNV-infected fish as verified by RT-PCR. These results indicated that the MAbs were useful in the development of more specific rapid and simple diagnostic technique for NNV infection in the future