In field use of water samples for genomic surveillance of infectious spleen and kidney necrosis virus (ISKNV) infecting tilapia fish in Lake Volta, Ghana.

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

Expression and purification of S5196-272 and S6200-317 proteins from Tilapia Lake Virus (TiLV) and their potential use as vaccines.

Tilapia Lake Virus Disease (TiLVD) is caused by Tilapia Lake Virus (TiLV), and it has a cumulative mortality rate of up to 90% in Nile tilapia (Oreochromis niloticus). TiLV is a negative enveloped single-stranded RNA virus with 10 genomic segments. Segment 5 (S5) and segment 6 (S6) were predicted to include a signaling peptide, suggesting that the encoded proteins of these two segments may exist as part of the virus envelope. Based on bioinformatic predictions, the S5 and S6 proteins in this study were produced, including S527-343, S527-172, S5196-272, S630-317, S630-190, and S6200-317. All proteins were tested for their expression in Escherichia coli. Only S5196-272 and S6200-317 were expressed as soluble and insoluble proteins, respectively. The soluble protein was purified using affinity chromatography, whereas the insoluble protein was solubilized using 6 M urea lysis buffer before purification. Both proteins were further purified using gel filtration chromatography, and the results showed a symmetric peak of both proteins suggested a high degree of uniformity in the conformation of these proteins. Antigenicity results indicated that these proteins were recognized by serum from TiLV-infected fish. The immunization tests revealed that serum antibodies levels in Nile tilapia produced by S5196-272 and S6200-317 were significantly increased (p-value < 0.05) at 7 days post-immunization (dpi) compared to antibody levels on Day 0 (D0). All the results combined suggested a potential vaccine candidate of S5 and S6 for TiLV protection in Nile tilapia.

Refolded recombinant major capsid protein (MCP) from Infectious Spleen and Kidney Necrosis Virus (ISKNV) effectively stimulates serum specific antibody and immune related genes response in Nile tilapia (Oreochromis niloticus).

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1ß and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.

Tilapia lake virus downplays innate immune responses during early stage of infection in Nile tilapia (Oreochromis niloticus).

Tilapia lake virus (TiLV) causes high mortality and high economic losses in tilapines. We describe an experimental challenge study focusing on early post challenge innate immune responses. Nile tilapia (Oreochromis niloticus) were infected with 105 TCID50/mL TiLV intraperitoneally, followed by virus quantification, histopathology and gene expression analysis in target (brain/liver) and lymphoid (spleen/headkidney) organs at 3, 7, 12, 17, and 34 days post challenge (dpc). Onset of mortality was from 21 dpc, and cumulative mortality was 38.5% by 34 dpc. Liver and kidney histopathology developed over the period 3-17 dpc, characterized by anisocytosis, anisokaryocytosis, and formation of multinucleated hepatocytes. Viral loads were highest at early time (3 dpc) in liver, spleen and kidney, declining towards 34 dpc. In brain, viral titer peaked 17 dpc. Innate sensors, TLRs 3/7 were inversely correlated with virus titer in brain and headkidney, and IFN-ß and Mx showed a similar pattern. All organs showed increased mRNA IgM expression over the course of infection. Overall, high virus titers downplay innate responses, and an increase is seen when viral titers decline. In silico modeling found that TiLV segments 4, 5 and 10 carry nucleolar localization signals. Anti-viral effects of TiLV facilitate production of virus at early stage of infection.

Resistance of pearlspot larvae, Etroplus suratensis, to redspotted grouper nervous necrosis virus by immersion challenge.

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty-day-old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post-infection, and the infected larvae were subjected to nested RT-PCR and histology. The virus was isolated from infected larvae using SSN-1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT-PCR and the virus was isolated using SSN-1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT-PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.

Production-level risk factors for syncytial hepatitis in farmed tilapia (Oreochromis niloticus L).

Syncytial hepatitis (SHT) is an emerging viral disease of tilapia characterized by significant morbidity and mortality. This study aimed to establish the production-level risk factors associated with presence and severity of SHT. Production factors were analysed during multiple outbreaks of SHT that occurred between 2011 and 2013 on a single tilapia farm in Ecuador and compared with the year 2010 before the SHT outbreaks. Relative risks, t tests, modified Poisson and forward stepwise linear regression analyses were performed using EPIINFO™. Compared to other strains, Chitralada had an elevated risk of SHT [RR = 2.1 (95%CI 1.8-2.4)]. Excessive mortality associated with the presence (and severity) of SHT increased by 611 (365), 6,814 (5,768) and 388 (340) deaths per 100,000 fry when stocking density, dissolved oxygen and pond production cycles were raised by 1 fish/m2 , 1 mg/L and 1 cycle, respectively. Excessive mortality associated with the presence (and severity) of SHT decreased by 337 (258) and 1,354 (1,025) deaths per 100,000 when stocking weight and water temperature increased by 1 g and 1°C, respectively. Time (season and stocking year) was not significantly associated with SHT. This study shows that some production factors increase the risk incidence and severity of SHTon a farm.

Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm-raised tilapia Oreochromis niloticus (L.) in Thailand.

Betanodavirus infection was diagnosed in larvae of farm-raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous necrosis virus (RGNNV).