Persistent detection of Tilapia lake virus in wild tilapia and tinfoil barbs.

Background and Aim: One of the emerging viral diseases in freshwater fish is Tilapia lake virus (TiLV), which infects all stages of fish and results in mass mortalities. Previously, a TiLV case was detected in the wild environment in Malaysia that involved tilapia and tinfoil barb. Hence, this study aimed to determine the presence of TiLV in wild tilapia (Oreochromis niloticus) as well as tinfoil barbs (Barbonymus schwanenfeldii) at the similar lake after the initial outbreak in year 2017. Materials and Methods: Both fish species were sampled from this lake at a month interval for two years and subjected to TiLV detection using reverse transcriptase-polymerase chain reaction and cell culture isolation. Concurrently, bacterial isolation and water quality measurements were performed to deduce their correlation with TiLV occurrence. Other wild fish species and mollusk were also occasionally sampled during the fish inventory activity at this lake. The fish's weight, length, and associated clinical signs were noted throughout the entire study period. Results: Mortality was not observed throughout the whole study period, and results indicated a moderate to high prevalence of TiLV infection in both tilapia and tinfoil barbs. There was no correlation between TiLV infection with the isolation rate of opportunistic bacteria such as Aeromonas spp., Plesiomonas spp., and Edwardsiella spp. in the study site. At the same time, the Pearson correlation test revealed a moderate negative correlation between the water pH with the presence of TiLV (R=-0.4472; p<0.05) and a moderate positive correlation between the water iron content with the monthly detection of Aeromonas spp. in wild tilapia. This is contrary to tinfoil barbs, where there was a moderate negative correlation between the water iron content with the monthly isolation of Aeromonas spp. (R=-0.5190; p<0.05). Furthermore, isolation of TiLV on cell culture-induced viral invasion was resulted in the cytopathic effects. Conclusions: Our results suggest that the wild fish may harbor TiLV for an extended period following a massive die-off event in 2017 without any obvious clinical signs and mortality. The persistency of viruses in the wild may need continuous and effective control as well as prevention strategies.

Current status and advances of fish vaccines in Malaysia.

Fish diseases have a significant negative influence on the Malaysian aquaculture industry. Since the 1980s, the sector has grown in size, which has resulted in a rise in the prevalence of infectious outbreaks affecting both freshwater and marine cultured fish species. Demand for commercially available fish vaccinations is predicted to increase as infectious disease outbreaks continue to occur. In Malaysia, aquaculture vaccine research and development (R&D) are still in its infancy, with most efforts concentrating on producing vaccines against bacterial infections, most notably streptococcosis, vibriosis, and motile Aeromonas septicemia. Despite several attempts, no homegrown vaccine has been effectively introduced into the manufacturing pipeline to date. At the moment, only three imported aquatic vaccines have received full permission, a far cry from the 314 and 60 vaccines licensed in the poultry and porcine industries, respectively. This review will describe recent findings regarding the development of aquaculture vaccines for certain fish species and diseases in Malaysia. In our opinion, R&D on fish vaccines is critical to the aquaculture industry's viability.

Risk factors associated with viral nervous necrosis in hybrid groupers in Malaysia and the high similarity of its causative agent nervous necrosis virus to reassortant red-spotted grouper nervous necrosis virus/striped jack nervous necrosis virus strains.

BACKGROUND AND AIM: Viral nervous necrosis (VNN) is a serious disease of several marine fish species. VNN causes 100% mortality in the larval stages, while lower losses have been reported in juvenile and adult fish. This study aimed to detect the occurrence of VNN while identifying its associated risk factors and the genotypes of its causative agent in a hybrid grouper hatchery in Malaysia. MATERIALS AND METHODS: A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors. RESULTS: A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNN-associated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage. CONCLUSION: This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain.

A polyprotein-expressing salmonid alphavirus replicon induces modest protection in atlantic salmon (Salmo salar) against infectious pancreatic necrosis.

Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation.

A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses.