Emergency response to emerging diseases: TiLV in tilapia

Tilapia lake virus (TiLV) is a novel RNA virus resembling Orthomyxovirus. It has been recently re-classified to Tilapia tilapinevirus species, under Tilapinevirus genus, Amnoonviridae family (ICTV, 2018). Since the first discovery in Israel in 2014, so far TiLV has been reported from 14 countries in three continents (Asia, Africa, and South America). Thailand is one of the affected countries that reported emergence of this virus in 2017. Initially, we employed nested RT-PCR primer sequences previously published for TiLV diagnosis. However, the resulting amplification of nonspecific fish genes led us to modify the nested RT-PCR protocols into a semi-nested RT-PCR by omitting a non-specific primer to avoid false positive results. Subsequently, our molecular work together with histopathology and sequence analysis confirmed the presence of TiLV infection in Thailand. Prior to the publication of our manuscript, we informed the Thai Department of Fisheries of our discovery of TiLV in Thailand. Our publication was preceded by a brief article at the website of the Network of Aquaculture Centers in Asia-Pacific in which we warned of the spread of TiLV and offered free use of a newly improved, semi-nested RT-PCR method and positive control plasmid for detection of TiLV. To date, we have provided positive controls in response to 44 requests from 24 countries who have expressed their appreciation for our attempt to help in emergent controlling the spread of this fish pathogen. Our current study focuses on genetic diversity of TiLV and development of detection method that covers all genetic variants

False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia

<p>Abstract</p> <p>Background</p> <p>Infectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp <it>Penaeus (Litopenaeus) vannamei </it>are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours.</p> <p>Findings</p> <p>Our continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome.</p> <p>Conclusions</p> <p>Results from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.</p

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp

<p>Abstract</p> <p>Background</p> <p>From 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.</p> <p>Results</p> <p>Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number <ext-link ext-link-id="FJ498866" ext-link-type="gen">FJ498866</ext-link>). <it>In situ </it>hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and <it>in situ </it>hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM.</p> <p>Conclusions</p> <p>ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp.</p

RRP20, a componenet of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20

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

Co-Interactive DNA-Binding between a Novel, Immunophilin-Like Shrimp Protein and VP15 Nucleocapsid Protein of White Spot Syndrome Virus

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly

Scale Drop Disease Virus (SDDV) and Lates calcarifer Herpes Virus (LCHV) coinfection downregulate immune-relevant pathways and cause splenic and kidney necrosis in barramundi under commercial farming conditions

Marine farming of barramundi (Lates calcarifer) in Southeast Asia is currently severely affected by viral diseases. To better understand the biological implications and gene expression response of barramundi in commercial farming conditions during a disease outbreak, the presence of pathogens, comparative RNAseq, and histopathology targeting multiple organs of clinically “sick” and “healthy” juveniles were investigated. Coinfection of scale drop disease virus (SDDV) and L. calcarifer herpes virus (LCHV) were detected in all sampled fish, with higher SDDV viral loads in sick than in healthy fish. Histopathology showed that livers in sick fish often had moderate to severe abnormal fat accumulation (hepatic lipidosis), whereas the predominant pathology in the kidneys shows moderate to severe inflammation and glomerular necrosis. The spleen was the most severely affected organ, with sick fish presenting severe multifocal and coalescing necrosis. Principal component analysis (PC1 and PC2) explained 70.3% of the observed variance and strongly associated the above histopathological findings with SDDV loads and with the sick phenotypes, supporting a primary diagnosis of the fish being impacted by scale drop disease (SDD). Extracted RNA from kidney and spleen of the sick fish were also severely degraded likely due to severe inflammation and tissue necrosis, indicating failure of these organs in advanced stages of SDD. RNAseq of sick vs. healthy barramundi identified 2,810 and 556 differentially expressed genes (DEGs) in the liver and muscle, respectively. Eleven significantly enriched pathways (e.g., phagosome, cytokine-cytokine-receptor interaction, ECM-receptor interaction, neuroactive ligand-receptor interaction, calcium signaling, MAPK, CAMs, etc.) and gene families (e.g., tool-like receptor, TNF, lectin, complement, interleukin, chemokine, MHC, B and T cells, CD molecules, etc.) relevant to homeostasis and innate and adaptive immunity were mostly downregulated in sick fish. These DEGs and pathways, also previously identified in L. calcarifer as general immune responses to other pathogens and environmental stressors, suggest a failure of the clinically sick fish to cope and overcome the systemic inflammatory responses and tissue degeneration caused by SDD

Efficacy of heat-killed and formalin-killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia ( Oreochromis niloticus )

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture‐based heat‐killed (HKV) and formalin‐killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 µl of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 10(6) TCID(50‐)inactivated virus. A booster vaccination was carried out at 21‐day post‐vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV‐specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival of 71.3% and 79.6% for HKV and FKV, respectively. Significant up‐regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia

Discovery of wild populations of Betta smaragdina Ladiges, 1972 (Teleostei, Osphronemidae) in a western province of Thailand

The bubble-nesting Betta smaragdina Ladiges, 1972 can be distinguished from the 4 other species of bubble-nesting Betta Bleeker, 1850 by being reportedly confined to Northeastern Thailand. We found large populations of fish in Western Thailand that closely resemble those from the Northeastern Region. The new populations inhabit a variety of places encompassing lakes, marshes, and streams. Morphological studies of Betta fish from the 2 well-separated regions, together with DNA analyses, show that the western and the northeastern populations are the same species

Immunization of Nile Tilapia (Oreochromis niloticus) Broodstock with Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing four males and eight females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 × 10(6) TCID(50) per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3 weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6–14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1–3 day old larvae but were undetectable in 7–14 day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination