Pradit Wangman

Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test

An immunochromatographic strip test is described for detection of the polyhedrin protein of Penaeus monodon nucleopolyhedrovirus (PemoNPV). The test employs one monoclonal antibody (MAb MBV5) conjugated to colloidal gold to bind to polyhedrin protein and a 1:1:1 mixture of 3 other MAbs (MBV8, 14 and 21) to capture colloidal-gold MAb-protein complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (GAM) antibody is used to capture excess free colloidal-gold conjugated MBV5 to validate test performance. Heating of homogenates of PemoNPV-infected P. monodon postlarvae prepared in PBS for 30min was necessary to maximize T line color intensity, and homogenates of infected postlarvae could still be scored as PemoNPV-positive when diluted 1:64. A strip test result was obtained within 15min of sample application, and although about 200-fold lower than a one-step PCR test for PemoNPV, its detection sensitivity was comparable to a dot blot. Due to its simplicity not reliant on sophisticated equipment or specialized skills, the strip test could be adopted to screen easily for PemoNPV infections at shrimp hatcheries and farms.

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus.

The capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia coli as a 42 kDa glutathione S-transferase (GST)-fusion protein (GST-XCP) or a 20 kDa His6-fusion protein (His6-XCP) were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), combined, and used to immunize Swiss mice to produce monoclonal antibodies (MAbs). Using dot blot, Western blot, and immunohistochemistry (IHC) methods, 4 MAbs specific to the XSV CP detected XSV in the freshwater prawn Macrobrachium rosenbergii without cross-reaction to host proteins or to proteins of Macrobrachium rosenbergii nodavirus (MrNV) or 5 of the most pathogenic viruses of penaeid shrimp. In dot blots, the combined MAbs could detect down to ~10 to 20 fmol µl-1 of purified GST-XCP protein, which was somewhat more sensitive compared to any single MAb. Used in conjunction with an MrNV-specific MAb, white tail disease (WTD) was diagnosed more effectively. However, the sensitivity at which the combined 4 MAbs detected XSV CP was 1000-fold lower than XSV RNA detected by RT-PCR. IHC analysis of M. rosenbergii tissue sections using the MAbs showed XSV infection to co-localize at variable loads with MrNV infection in heart and muscle cells as well as cells of connective tissues in the hepatopancreas. Since XSV histopathology remained prominent in tissues of some prawns in which MAb reactivity for MrNV was low compared to MAb reactivity for XSV, XSV might play some role in WTD severity.