Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination.

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Nanoencapsulation of inactivated-viral vaccine using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in olive flounder (Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection.

Viral haemorrhagic septicaemia virus (VHSV), a (-) ssRNA virus belonging to the genus Novirhabdovirus of rhabdoviridae family, is the aetiological agent of viral haemorrhagic septicaemia (VHS) disease which causes huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. Previously, we developed an inactivated vaccine viz., formalin-inactivated VHSV mixed with squalene as adjuvant which was effective in conferring protective immunity (58-76% relative percentage survival) against VHSV but the mode of administration was intraperitoneal injection which is not feasible for small sized fingerling fish. To overcome this limitation, we presently focused on replacing the injection route of vaccine delivery by oral and immersion routes. In this context, we encapsulated the inactivated VHSV vaccine with chitosan nanoparticles (CNPs-IV) by water-in-oil (W/O) emulsification method. After encapsulation, two sets of in vivo vaccination trials were conducted viz., preliminary trial-I and final trial-II. In preliminary trial-I, olive flounder fingerlings (10.5 ±â€¯1.7 g) were vaccinated with CNPs-IV by different delivery strategies involving oral and immersion routes (single/booster dose) followed by challenge with VHSV (1 × 106 TCID50 virus/fish) to evaluate an effective method amongst different applied delivery strategies. Subsequently, a final trial-II was conducted to better understand the immune mechanism behind the efficacy of the employed delivery strategy and also to further improvise the delivery mechanism with prime-boost (primary immersion and oral boosting) combination in order to improve the transient anti-VHSV response in the host. Evaluation of RPS analysis in trial-I revealed higher RPS of 46.7% and 53.3% in the CNPs-IV (immersion) and CNPs-IV (immersion/immersion) groups, respectively compared to 0% RPS in the CNPs-IV (oral) group and 20% RPS in the CNPs-IV (oral/oral) group when calculated against 100% cumulative mortality percentage in the NVC (non-vaccinated challenged) control group, whereas, in the trial-II, RPS of 60% and 66.6% were obtained for CNPs-IV (immersion/immersion) and CNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, CNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to control, in both the systemic (kidney) and mucosal (skin and intestine) immune compartments of the host post immunization as well as post challenge. To conclude, mucosal immunization with CNPs-IV vaccine can orchestrate an effective immunization strategy in organizing a coordinative immune response against VHSV in olive flounder thereby exhibiting higher protective efficacy to the host with minimum stress.

Differences of Viral Hemorrhagic Septicemia Virus Loads among Organs of Dead and Surviving Olive Flounder Infected by Intramuscular Injection and Immersion Challenge.

Viral hemorrhagic septicemia virus (VHSV) is an important viral pathogen in the culture of Olive Flounder Paralichthys olivaceus. Based on cumulative mortality, the virulence of VHSV was found to be highly different depending on challenge routes and exposure doses (using tissue culture infectious dose with 50% endpoint [TCID50]). Olive Flounder were injected with VHSV at 102.5 , 104.5 , 106.5 , and 108.5 TCID50/100 µL/fish. A second group of fish was immersed at 103.5 , 105.5 , and 107.5 TCID50/mL at 10°C for 1 h in this study. The cumulative mortality was observed at 15 d postinfection. Immersion challenge at 103.5 TCID50/mL caused no mortality, while intramuscular injection challenge resulted in high levels of mortality with all VHSV exposure doses. Overall, Olive Flounder was susceptible to VHSV, with cumulative mortality of 90% or 100% in fish intramuscularly injected with high or low doses of VHSV. The cumulative mortality was 40% and 70% at 105.5 and 107.5 TCID50/mL, respectively, in the immersion challenge group. The VHSV titration and copy numbers were estimated by TCID50 and quantitative reverse transcription PCR methods. From dead Olive Flounder, VHSV titration was consistently detected in all tested organs, ranging from 105 to 109 TCID50/mL. The VHSV titration was under the detection limit from surviving Olive Flounder, but the VHSV N gene was detected.

Development of double labeling in situ hybridization using RNA probes for genome detection of nervous necrosis virus (NNV).

In situ hybridization (ISH) of genomic segments using RNA-RNA hybrid for nervous necrosis virus (NNV) detection has not been reported yet. The objective of this study was to develop RNA-ISH using RNA probes for the detection of NNV in infects SSN-1 cells or sevenband grouper Hyporthodus septemfasciatus. Two viral RNA segments viz., RNA1 and RNA2 were synthesized by in vitro transcription and labeled with fluorescein UTP and dignoxigenin dUTP, respectively. These labeled RNA probes specifically detected NNV in infected SSN-1 cells. We also applied double labeling RNA-ISH with two-color staining of RNA probes. The results showed that these two viral genomic segments were localized in same regions although RNA1 was also expressed separately. These findings suggest that RNA1 overexpression may be important for sufficient assembly of infectious particles. The RNA-ISH showed that both RNA segments were localized in the tectum opticum, torus semicircualris, cerebellum, thalamus, hypothalamus, and medulla of experimentally infected brain tissues. Especially, RNA segments were highly localized around the ventricle, suggesting that ventricle might play a vital role in the spread of NNV. This technique can be useful for understanding the localization of NNV and the relationship between clinical sign and viral expression.

Development of PCR method for detecting Kudoa iwatai (Myxozoa: Multivalvulida) from rock bream Oplegnathus fasciatus.

We developed a PCR assay targeting the 28S rDNA of Kudoa iwatai (Multivalvulida: Myxozoa) and investigated the prevalence of infection in rock bream Oplegnathus fasciatus, which is commercially an important aquaculture species in Korea, with this assay. Detection limit of the PCR assay was 2.5 fg/µl with plasmid DNA and 8.6 × 103 spores/ml with purified spores, respectively. This PCR assay did not amplify DNA of other Kudoa species (Kudoa septempunctata, Kudoa lateolabracis, Kudoa thyrsites) tested. Sliced muscles of whole body from 318 rock bream (wild and cultured) were examined by this PCR assay and also with the naked eyes. All of the wild fish did not produce amplicons nor did harbor visible Kudoa cysts (0/70). Three of the cultured fish were PCR-positive and also harbored visible Kudoa cysts (3/248, 1.2%). The sequences of amplicons (574 bp) were 100% identical with those of the K. iwatai already registered in Genbank. When the visceral organs of these three fish were examined, visible cysts were not found, but one stomach sample was found to be PCR-positive. There was no difference in the prevalence of infection estimated by PCR assay and the presence of visible Kudoa cysts in our samples. This is thought to be because the development of K. iwatai is already completed and only mature Kudoa cysts existed in our samples.

Amoebic gill infection in coho salmon Oncorhynchus kisutch farmed in Korea.

About 70% mortality occurred in cultured coho salmon Oncorhynchus kisutch at a marine farm in the South Sea of Korea in 2014. Diseased fish showed greyish or pale patches on the gills, with no internal signs of disease. No bacteria or viruses were isolated from diseased fish, but numerous amoebae were found on the gills. Histopathological examinations revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae had a 630 bp partial 18S rRNA gene fragment specific to Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that this Korean amoeba belonged to the N. perurans group. This is the first report of N. perurans infection in Korea.

Hirame rhabdovirus (HIRRV) as the cause of a natural disease outbreak in cultured black seabream (Acanthopagrus schlegeli) in Korea.

In 2015, a high mortality rate of about 40% was observed in black seabream (Acanthopagrus schlegeli) on a farm on the southern coast of Korea. Most of the diseased fish showed a hemorrhage of the mouth, pale liver, petechial hemorrhaging in the internal fat, and an enlarged spleen. Other than Alella sp., no parasites or bacteria were isolated from the diseased fish, and all of the tissue filtrates produced cytopathic effects (CPEs) in FHM and CHSE-214 cells. A polymerase chain reaction analysis revealed that the cell culture supernatants with CPE expressed specific 730-bp fragments for the hirame rhabdovirus (HIRRV) phosphoprotein gene. The nucleotide sequences showed a minimum of 95.8% identity to five other known isolates of HIRRV, including CA-9703 and 8401-H from olive flounder (Paralichthys olivaceus) in Korea and Japan. An experimental challenge was conducted in which the virus was delivered by injection, and the cumulative mortalities of black seabream challenged with this new HIRRV isolate at 10(4.8) TCID50/fish and 10(3.8) TCID50/fish were 100% and 20%, respectively. This fulfilled Koch's postulates and confirmed that HIRRV was the cause of disease and mortality for both the natural and experimental infection of black seabream.

Genetic positioning of aquabirnavirus isolates from cultured Japanese eel Anguilla japonica in Korea.

Aquabirnavirus is an epizootic virus in Japanese eel Anguilla japonica farms in Korea, although its origin is unclear. In the present study, nucleotide sequences of the VP2/NS junction region of 9 Korean aquabirnaviruses from cultured eel in various areas of Korea during 2000-2009 were analyzed to evaluate their genetic relatedness to worldwide isolates. The nucleotide sequences showed more than 94.2% identity among the 9 Korean eel isolates, 71.2% identity among 16 Korean isolates from freshwater and marine fish, and 71.1% identity among 25 worldwide isolates. All 9 isolates in this study were phylogenetically classified into genogroup II, including isolates from Denmark, Spain, Taiwan and Japan, and were discrete from salmonid and marine fish isolates (genogroup I and VII) in Korea. These results suggest that the Korean eel isolates have most likely been introduced from outside the country and not from coastal areas of Korea.

Infection of wild mullet (Mugil cephalus) with Myxobolus episquamalis in Korea.

Wild mullet (Mugil cephalus) with white cysts on their scales were obtained from Yeosu on the south coast of Korea in 2009. Cyst-like plasmodia consisted of a large number of mature myxosporean spores and numerous sporogonic stages. Spores were oval-shaped in their front view, tapering anteriorly to a blunt apex, and lenticular in their lateral view. They measured 7.0 µm (6.2-7.6) in length, 5.2 µm (4.0-6.2) in width, and 4.9 µm (3.8-6.0) in thickness. Polar capsules contained a polar filament with five to six turns and measured 3.5 µm (2.5-4.5) in length and 2.0 µm (1.6-2.3) in width. Nucleotide sequences of the 18S rRNA gene of the myxosporean parasites in our study showed 99.8 % identity with Myxobolus episquamalis Egusa, Maeno and Sorimachi, 1990 from mullet in Tunisia. These results suggest that the Myxobolus sp. found on the scales of wild mullet is M. episquamalis. In the histopathological examination, spores were observed not only in the plasmodia on the scales, but also in the intestine, pancreas, heart, kidney, stomach, gill, skin, spleen, and liver, suggesting the possibility of the coinfection by different Myxobolus species.

Morphologic and genetic evidence for mixed infection with two Myxobolus species (Myxozoa: Myxobolidae) in gray mullets, Mugil cephalus, from Korean waters.

The present study was performed to trace the decisive evidence for mixed infection of 2 Myxobolus species, M. episquamalis and Myxobolus sp., in the gray mullet, Mugil cephalus, from Korean waters. Mullets with whitish cyst-like plasmodia on their scales were collected near a sewage plant in Yeosu, southern part of Korea, in 2009. The cysts were mainly located on scales and also found in the intestine. The spores from scales were oval in a frontal view, tapering anteriorly to a blunt apex, and measured 7.2 µm (5.8-8.0) in length and 5.3 µm (4.7-6.1) in width. Two polar capsules were pyriform and extended over the anterior half of the spore, measuring 3.5 µm (2.3-4.8) in length and 2.0 µm (1.5-2.2) in width. In contrast, the spores from the intestine were ellipsoidal, 10.4 µm (9.0-11.9) in length and 8.4 µm (7.3-10.1) in width. The polar capsules were pyriform but did not extend over the anterior half of the spore, 3.7 µm (2.5-4.5) in length and 2.2 µm (1.8-2.9) in width. The nucleotide sequences of the 18S rDNA gene of the 2 myxosporean spores from scales and intestine showed 88.1% identity to each other and 100% identity with M. episquamalis and 94.5% identity with M. spinacurvatura from mullet, respectively. By the above findings, it is first confirmed that mullets from the Korean water are infected with 2 myxosporean species, M. episquamalis and Myxobolus sp.

Production and Characterization of Monoclonal Antibodies Against Structural Proteins of Hirame Novirhabdovirus.

Hirame novirhabdovirus (HIRRV) is a significant viral pathogen of Japanese flounder (Paralichthys olivaceus). In this study, seven monoclonal antibodies (mAbs) against HIRRV (isolate CA-9703) were produced and characterized. Three mAbs (1B3, 5G6, and 36D3) were able to recognize nucleoprotein (N) (42 kDa) and four mAbs (11-2D9, 15-1G9, 17F11, and 24-1C6) recognized matrix (M) protein (24 kDa) of HIRRV. Western blot, Enzyme-linked immunosorbent assay, and indirect fluorescent antibody technique (IFAT) results indicated that the developed mAbs were specific to HIRRV without any cross-reactivity against other different fish viruses and epithelioma papulosum cyprini cells. All the mAbs comprised IgG1 heavy chain and κ light chain except 5G6, which has a heavy chain of IgG2a class. These mAbs can be very useful in development of immunodiagnosis of HIRRV infection.

Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome.

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.

Temperature-dependent viral replication and antiviral apoptotic response in viral haemorrhagic septicaemia virus (VHSV)-infected olive flounder (Paralichthys olivaceus).

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia virus (VHSV) in the winter and spring but has zero mortality over 20 °C. In this experiment, we studied the effect of rearing temperature on viral replication, viral transcription and antiviral apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive flounder were given intra-peritoneal injections of VHSV (10(7.8) TCID(50)/ml) and were reared at 15 °C or 20 °C. Five fish were randomly sampled for head kidney at 3, 6 and 12 h post-infection (hpi) and 1, 2, 4 and 7 days post-infection (dpi). Total RNA extracted from the tissue was reverse transcribed and used as template for real-time PCR. In the 15 °C group, the number of viral gRNA copies peaked after 2 dpi and remained high through 7 dpi, while in the 20 °C group, the copy number was at the highest at 1 dpi but drastically declined at later stages. Viral mRNA levels in the 15 °C group gradually increased starting at 3 hpi to reach their maximum value at 12 hpi and remained high until 2 dpi, whereas the other group showed much lower copy numbers that were undetectably low at 4 and 7 dpi. Type II IFN expression increased as the viral copies increased and the 20 °C group showed quicker and stronger expression than the 15 °C group. The MHC class I and CD8 expression was high in both the groups at early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15 °C group expression reduced below control levels, while they expressed higher to control in 20 °C group. The expression of granzyme in 15 °C fish showed a single peak at 2 dpi, but was consistently expressing in 20 °C fish. Individuals expressed very high levels of perforin expressed very high levels of caspase 3. In 15 °C fish, TNFα, FasL and p53 expressed significantly higher than 20 °C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15 °C fish whereas it was significantly elevated in 20 °C group. Interestingly individual fish with high caspase 3 expression contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus).

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1ß, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Detection of megalocytivirus from imported tropical ornamental fish, paradise fish Macropodus opercularis.

Megalocytivirus was detected from paradise fish Macropodus opercularis imported from Indonesia. Four of 11 fish (36%) in 2006 and 40 of 117 fish (34%) in 2008 were found to be PCR-positive for megalocytivirus. Phylogenetic analysis based on partial major capsid protein (MCP) gene nucleotide sequences revealed that the sequences detected in paradise fish were classified as Genotype II, which includes freshwater fish isolates from Southeast Asian countries, closely related to infectious spleen and kidney necrosis virus (ISKNV), Murray cod iridovirus (MCIV), and dwarf gourami iridovirus (DGIV-2004). Paradise fish was added as a new host for megalocytivirus based on this study.

Investigation of nervous necrosis virus (NNV) replication in vitro using RNA in situ hybridization.

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus of family Nodaviridae. Its genome consists of two RNA segments, RNA1 and RNA2. Several studies have investigated NNV detection by in situ hybridization (ISH), but these have typically focused on the detection of the RNA2 gene. In this study, we localized both RNA1 and RNA2 NNV segments in viral-infected cells by ISH, using labeled RNA probes (RNA-ISH). Also, immunocytochemistry (ICC) assay was carried out for localization of viral particle by targeting the coat protein. Further, viral quantification assays were performed by quantitative RT-PCR and viral infectivity (TCID50) in SSN-1 cells. Viral segments were observed by RNA-ISH at 6 h post infection (hpi), while NNV particles were detected at 24 hpi by ICC. Use of double labeling RNA-ISH revealed the co-expression of the two viral segments in the same area of the cells, while RNA1 was also detected separately. Comparison of the level of viral genomic segments and viral infectivity revealed significantly more copies of RNA1 at each time points than copies of RNA2 and greater NNV titers. The results suggest that RNA1 might be expressed in the early stages of replication, with RNA2 expressed later. The virions then assemble through initially expressed viral genomic segments. Even though infectious particles displayed very efficient packaging, the RNA1 segment was still over-produced.

PLGA encapsulated inactivated-viral vaccine: Formulation and evaluation of its protective efficacy against viral haemorrhagic septicaemia virus (VHSV) infection in olive flounder (Paralichthys olivaceus) vaccinated by mucosal delivery routes.

Viral haemorrhagic septicaemia virus (VHSV), an OIE listed viral pathogen, is the etiological agent of a contagious disease, causing huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. In continuation with our previous work, where injection vaccination with inactivated VHSV mixed with squalene (as adjuvant) conferred higher protective immunity to olive flounder, the present study focused on replacing the injection route of vaccine delivery by immersion/oral route to overcome the limitations of the parenteral immunization method. Here, we encapsulated the inactivated VHSV vaccine with PLGA (poly lactic-co-glycolic acid) nanoparticles (PNPs-IV) and evaluated its ability to induce protective immunity in olive flounder (12.5 ±â€¯1.5 g) by initially immunizing the fishes by immersion route followed by a booster with the same dose two weeks later with half of the fish through immersion route and other half through oral route (incorporated into fish feed). Cumulative mortalities post-challenge (1 × 106 TCID50 virus/fish) with virulent VHSV-isolate, were lower in vaccinated fish and RPS of 60% and 73.3% were obtained for PNPs-IV (immersion/immersion) and PNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, PNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to controls, in both the systemic (kidney) and mucosal (skin and intestine) immune compartment of the host post immunization as well as post challenge. Thus it can be inferred that the adopted immunization strategy efficiently protected and transported the inactivated viral antigen to target immune organs and positively stimulated the protective immune response against VHSV in olive flounder.

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

Characterization of the Transcriptome and Gene Expression of Brain Tissue in Sevenband Grouper (Hyporthodus septemfasciatus) in Response to NNV Infection.

Grouper is one of the favorite sea food resources in Southeast Asia. However, the outbreaks of the viral nervous necrosis (VNN) disease due to nervous necrosis virus (NNV) infection have caused mass mortality of grouper larvae. Many aqua-farms have suffered substantial financial loss due to the occurrence of VNN. To better understand the infection mechanism of NNV, we performed the transcriptome analysis of sevenband grouper brain tissue, the main target of NNV infection. After artificial NNV challenge, transcriptome of brain tissues of sevenband grouper was subjected to next generation sequencing (NGS) using an Illumina Hi-seq 2500 system. Both mRNAs from pooled samples of mock and NNV-infected sevenband grouper brains were sequenced. Clean reads of mock and NNV-infected samples were de novo assembled and obtained 104,348 unigenes. In addition, 628 differentially expressed genes (DEGs) in response to NNV infection were identified. This result could provide critical information not only for the identification of genes involved in NNV infection, but for the understanding of the response of sevenband groupers to NNV infection.

Amoebic gill disease outbreak in marine fish cultured in Korea.

In 2015, 6.7-60% mortality was observed in black seabream ( Acanthopagrus schlegelii), rock bream ( Oplegnathus fasciatus), and gray mullet ( Mugil cephalus) farmed in the southern coast of Korea. On examination, numerous amoebae were found on the gills of these 3 fish species with detection rate of 100%. Some rock bream and gray mullet were coinfected with bacteria ( Pseudomonas anguilliseptica, Vibrio tapetis, or Vibrio anguillarum). Histologic examination revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae collected from the 3 fish species had specific 630 bp of a partial 18S rRNA gene fragment for Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that these Korean amoeba isolates belonged to the N. perurans group. Based on our results, black seabream, rock bream, and gray mullet were added as new hosts for N. perurans.