Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) have been identified as the causative agents for white tail disease (WTD) of M. rosenbergii. In this study, the gene sequences encoding MrNV and XSV capsid proteins were separately ligated into the pGEX-4T-3 expression vector and transformed into Escherichia coli. After induction, glutathione-S-transferase (GST)-tagged MrNV and XSV fusion proteins were obtained with molecular masses of 68 and 43 kDa, respectively. Specific polyclonal antibodies for MrNV and XSV against viral recombinant proteins and infected prawn tissues were verified using Western blotting. According to immunodot blot assay results, the detection sensitivities of antibodies were approximately 5 ng μL(-1) for both recombinant proteins GST-MrNV and GST-XSV. In additional, MrNV and XSV were detected at dilution levels of 1:2560 and 1:640 in the infected prawn tissues, respectively. No cross-reactions with white spot syndrome virus or grouper nervous necrosis virus were observed using immunodot blot assays. MrNV and XSV in infected muscle tissues were detected using immunohistochemistry. Although the detection limit of the immunodot blot assay was lower than that of nested reverse transcription polymerase chain reaction, these polyclonal antibodies can be useful for confirming MrNV and XSV infections in field tests.

The icp11 gene encoding the highly abundant DNA mimic protein of white spot syndrome virus (WSSV) was cloned into the pTYB1 and pGEX-6P-1 expression vectors and introduced into E. coli by transformation. After induction, C-terminally intein-tagged ICP11 (ICP11-intein) and N-terminally glutathione-S-transferase (GST)-tagged ICP11 (GST-ICP11) proteins with molecular masses of 64 and 35 kDa were obtained. These proteins were purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific for ICP11 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei by dot blotting, western blotting or immunohistochemistry without cross-reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was approximately 0.7 fmole/spot of GST-ICP11 as determined by dot blotting. These MAbs showed stronger immunoreactivity than other MAbs from previous studies that are specific for VP28 and VP19. A combination of MAbs specific for ICP11, VP28 and VP19 increased the detection sensitivity of WSSV during early infection to a sensitivity 250 times lower than that of one-step PCR. Therefore, the MAbs specific for ICP11 could be used to confirm and enhance the detection sensitivity for WSSV infection in shrimp using various types of antibody-based assays.

vp1, a gene encoding one of the capsid proteins of Taura syndrome virus, was cloned into the pGEX-6P-1 expression vector, and the resulting construct was then used to transform E. coli strain BL21. After induction, an N-terminally glutathione-S-transferase-tagged VP1 (GST-VP1) protein with a molecular mass of 80 kDa was obtained. This protein was purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three MAbs specific for the VP1 protein were selected that were suitable for detecting natural TSV infection in Penaeus vannamei by dot blotting, western blotting and immunohistochemistry. This detection occurs without cross-reaction to other shrimp tissues or other common shrimp viruses. As determined by dot blotting, the detection sensitivity of the MAbs was approximately 2 fmole/spot of the GST-VP1. These MAbs showed detection sensitivity comparable to that of MAbs specific for VP2, but they exhibited stronger immunoreactivity than previously studied MAbs specific for VP3. Although the sensitivity of the MAbs to VP1 was 1,000 times lower than one-step RT-PCR, they could be used in various types of antibody-based assays to confirm and enhance the detection sensitivity of TSV infection in shrimp.

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.

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.

The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248-3045), CP-I (nucleotides 3046-3954) and CP-C (nucleotides 3955-4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes.

Taura syndrome virus (TSV) is one of the major pathogens causing mortality in the whiteleg shrimp, Litopenaeus vannamei. In this study, the gene sequence encoding the VP2 capsid protein (40 kDa) of TSV was cloned into pMAL-C2 expression vector. Five monoclonal antibodies (MAbs) were produced against the VP2 capsid protein, which was expressed heterologously in the form of a fusion protein with maltose binding protein and called MBP-VP2. All MAbs belonged to the IgG1 subclass and could bind MBP-VP2 at 400-800 pg/spot in immuno-dot blot assays. The MAbs could detect VP2 both in extracts from shrimp infected naturally in western blotting and dot blotting and in shrimp tissues in immunohistochemistry. Additionally, these MAbs did not exhibit cross-reactivity to extracts from uninfected shrimp or shrimp infected with several other common viruses. However, the dot blot assay sensitivity for TSV was approximately 10,000 times lower than that of one step RT-PCR. The MAb TSV2-88 specific to VP2 obtained in this study demonstrated an approximately twofold higher sensitivity than that of the MAb specific to VP3 from a previous study. In immunohistochemistry, the MAb TSV2-88 specific to VP2 demonstrated stronger immunoreactivity than the MAb TSV3-601 specific to VP3. A combination of the VP2 and VP3 MAbs could be used to more easily detect TSV infections in field samples of L. vannamei with better sensitivity and fidelity than using a single MAb.

Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei.