A naturaly attenuated largemouth bass ranavirus strain provided protection for Micropterus salmoides by immersion immunization.

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 102.0 TCID50/mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Epidermal growth factor receptor promotes infectious spleen and kidney necrosis virus invasion via PI3K-Akt signaling pathway.

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

The composition and antiviral activity of scTRIM59 in Mandarin fish.

The tripartite motif (TRIM) proteins play critical roles in viral infection by modulating innate immunity. However, the molecular and antiviral activity of TRIM59 in mandrain fish is not fully understood. In present study, we cloned and sequenced the TRIM59 core sequence and explored its characteristics in Mandarin fish. The Siniperca chuatsi TRIM59 (scTRIM59) showed relatively high expression in immune-related organs. scTRIM59 expression was significantly down-regulated post ISKNV infection in vivo and vitro, but up-regulated at the early stages of SCRV infection in CPB cells. The overexpression of scTRIM59 inhibited ISKNV and SCRV infection, but decreased the expression of IRF3/IRF7-mediated signal genes. However, knockdown of scTRIM59 promoted the ISKNV and SCRV infection, but increased the expression of IRF3/IRF7-mediated signal genes. Those results indicated that scTRIM59 negatively regulated ISKNV, SCRV infection and IRF3/IRF7-mediated signal genes. This study provided new ideas about the function of scTRIM59.

Characterization and function of mandarin fish c-Myc during viral infection process.

c-Myc is a transcription factor and master regulator of cellular metabolism, and plays a critical role in virus replication by regulating glutamine metabolism. In this study, the open-reading frame (ORF) of c-Myc, designated as Sc-c-Myc, was cloned and sequenced. Multiple alignment of the amino acid sequence showed that the conserved domain of Sc-c-Myc, including the helix-loop-helix-zipper (bHLHzip) domain and Myc N-terminal region, shared high identities with other homologues from different species. Sc-c-Myc mRNA was widely expressed in the examined tissues of mandarin fish, and the higher mRNA levels was expressed in hind kidney. Moreover, mRNA and protein level of Sc-c-Myc was significantly increased in the Chinese perch brain (CPB) cells and spleen of mandarin fish post infection with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Sc-c-Myc overexpression promoted ISKNV and SCRV replication, on the contrary, knocking down Sc-c-Myc restrained ISKNV and SCRV replication. These results indicated that Sc-c-Myc involved in ISKNV and SCRV replication and proliferation, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

Identification of intron in ORF003 gene and its application for inactivation test of ISKNV.

The virus inactivation test is a critical skill in inactivated vaccine production. Active viruses produced viral mRNA in susceptible cells or the host can be used to infer whether a DNA virus is replicating by RT-PCR. But it is generally difficult to avoid genomic DNA contamination in the samples. However, the use of primers spanning an intron is an effective alternative for virus inactivation test. Therein, a nested RT-PCR was developed to detect active ISKNV in the inactivated vaccine. At first, the transcriptome analysis of CPB cell infected with ISKNV revealed several gaps in some viral transcripts compared to ISKNV genome. One intron in ORF003L with 80 bp (designated IN-3) was confirmed by PCR and sequencing analysis. Then, two primer sets (primer A and primer B) spanning the IN-3 intron were designed to detect ISKNV transcription. The nested RT-PCR conditions were optimized with 0.4 µM primer A and 0.2 µM primer B, and 68 °C and 55 °C for annealing temperature, respectively. The sensitivity results indicated that the nested RT-PCR could detect one copy of live ISKNV propagating in CPB cells for seven days. The nested RT-PCR method was more sensitive and accurate than the method of blind passages in cells and fish challenge experiments. Together, above results indicate that this assay is a time-saving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.

Molecular characterization and function of EGFR during viral infectionprocess in Mandarin fishSiniperca chuatsi.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase protein and plays a critical role in virus infection by modulating innate immunity. In this study, we cloned and sequenced the EGFR coding sequence of mandarin fish, designed as scEGFR, and explored its characteristics. scEGFR mRNA was widely expressed in the tested tissues of mandarin fish, and the higher mRNA levels were expressed in kidney and spleen. scEGFR expression was up-regulated in spleen and CPB cells at early stage of ISKNV and SCRV infection. Gefitinib (EGFR inhibitor) inhibited ISKNV and SCRV replication, and increased the expression of the interferon-stimulated genes (ISG). However the EGF (EGFR activator) promoted ISKNV and SCRV replication, and decreased the interferon-stimulated genes. Those results indicated that scEGFR and its signaling involved in ISKNV and SCRV infection, and EGFR activation negatively regulated the interferon response, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

Transcriptomic profiles reveal that inactivated iridovirus and rhabdovirus bivalent vaccine elicits robust adaptive immune responses against lethal challenge in marbled sleepy goby.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Siniperca chuatsi rhabdovirus (SCRV) induces autophagy via PI3K/Akt-mTOR pathway in CPB cells.

Autophagy is an important mechanism for organisms to eliminate viruses and other intracellular pathogens. Siniperca chuatsi rhabdovirus (SCRV) is an agent that has caused devastating losses in Chinese perch (Siniperca chuatsi) industry. But the role of autophagy in Siniperca chuatsi rhabdovirus (SCRV) infection is not clearly understood. In this study, we identified that SCRV infection triggered autophagy in CPB cells, which was demonstrated by the appearance of the membrane vesicles, GFP-LC3 punctuate pattern, conversion of LC3-I to LC3-II, and the co-localization of autophagosomes and lysosomes. The changes of autophagy flux in SCRV infection indicated that autophagy was inhibited at the early stage of SCRV infection, but was promoted at the late stage. UV-inactivated SCRV can induce autophagy, suggesting that SCRV replication is not essential for the induction of autophagy. Furthermore, we found inducing autophagy with Rapa inhibited SCRV proliferation, but inhibiting autophagy with 3-MA or CQ increased SCRV production in CPB cells. Then we assessed the effects of PI3K/Akt-mTOR signaling pathway on SCRV induced autophagy. We found that SCRV infection activated PI3K/AKT signaling pathway at 4 hpi, but inhibited it at 8 hpi. SCRV-N mRNA and protein level were decreased by inhibiting PI3K with LY294002, but increased by activating PI3K with 740Y-P. Those results indicated that SCRV infection induced autophagy via the PI3K/Akt-mTOR signal pathway, which will provide new insights into SCRV pathogenesis and antiviral treatment strategies.

First report of megalocytivirus (iridoviridae) in cultured bluegill sunfish, Lepomis macrochirus, in China.

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.

Development of strand-specific real-time RT-PCR for the analysis of SCRV transcription and replication dynamics.

To distinguish between three types of Siniperca chuatsi rhabdovirus (SCRV) viral RNA (vRNA, cRNA, and mRNA) and investigate SCRV transcription and replication dynamics in Chinese perch brain CPB cells, a novel, strand-specific, reverse transcriptase quantitative real-time PCR (RT-qPCR) assay was established. The method is based on strand-specific reverse transcription, using tagged primers to add a 'tag' sequence at the 5' end. We used the 'tag' sequence as the forward primer and a strand-specific reverse primer to quantify the three types of RNA. Three types of synthetic viral RNA were used as reference standards for validation and quantification. These assays were optimized to produce a standard curve from 102 to 107 copies/µL, with an efficiency of 91-101% and an R2 value of 0.9949-0.9999. The coefficients of variation for repeatability and reproducibility were less than 2.85% and 5.52%, respectively. Using this method, specific target RNA was detected at a 3500-70,000 fold higher level than other types of RNA. This method was also used to evaluate the dynamics of vRNA, cRNA and mRNA synthesis in CPB cells infected with SCRV. The results indicate that the intracellular dynamics of vRNA, cRNA and mRNA are different. In the earliest phase of SCRV infection, all three types of viral RNA increased very slowly. The copy number of vRNA and mRNA increased exponentially from 4 h post infection, while cRNA increased from 6 h post infection. The amount of cRNA was lower than vRNA and mRNA throughout the infection. The novel, strand-specific RT-qPCR method developed in this study provides critical data to aid the understanding of transcription and replication during SCRV infection.

Comparative genetic analysis and pathological characteristics of goose parvovirus isolated in Heilongjiang, China.

BACKGROUND: Goose parvovirus (GPV) causes acute enteritis, hepatitis, myocarditis and high morbidity and mortality in geese and ducks. GPV H strain was isolated from a Heilongjiang goose farm where the geese were showing signs of hemorrhage in the brain, liver, and intestinal tract. In this study, we explored the genetic diversity among waterfowl parvovirus isolates and the pathological characteristics of GPV H in Shaoxing ducklings. METHODS: The complete capsid protein (VP) and non-structural (NS) sequences of the isolated H strain were sequenced, and phylogenetic trees of VP and NS were constructed in MEGA version 5.05 using the neighbor-joining method. Three-day-old Shaoxing ducklings were inoculated with GPV and were euthanized at 1, 2, 4, 6, and 8 days post-inoculation (PI), and their organs were removed and collected. The organs of 6-day PI ducklings were fixed in formalin, embedded in paraffin, sectioned for histology, stained with HE and analyzed for pathological lesions. The distribution of the GPV H strain in the tissues of the inoculated ducklings was detected using the polymerase chain reaction (PCR) method. RESULTS: Genetic analysis of the NS and VP genes indicated that the H strain was closely related to strains circulating in China during 1999-2014, and the nucleic acid identity of those strains was 98%-99%. Classical symptoms were observed in the inoculated ducklings. GPV remained in many tissues and replicated in a majority of the tissues, leading to histopathological lesions in four tissues. CONCLUSIONS: We first reported the distribution and histopathological lesions of a Chinese strain of GPV in infected shaoxing ducklings. This H strain was moderate pathogenic for Shaoxing ducklings.

Metagenomic Analysis of the Jinding Duck Fecal Virome.

Ducks play an important role in transmitting and maintaining mammalian viruses in nature, and are a reservoir host of many animal viruses. We analyzed the fecal virome of four strains (A, B, C, and D) of ducks living in isolation by using metagenomic analysis. The feces of the ducks tested contained 18 animal virus families. The percentage values of RNA virus reads, compared to the total animal virus reads in each of the four strains were 96.96% (A), 97.30% (B), 98.01 (C), and 67.49% (D), and were mainly from Orthomyxoviridae, Mimiviridae, Bunyaviridae, Picobirnaviridae, and Reoviridae. Meanwhile, the minority of DNA virus reads were related to Herpesviridae, Adenoviridae, Iridoviridae, and other, low abundance viral families. The percentage values of Orthomyxoviridae, Mimiviridae, Bunyaviridae, Picobirnaviridae, and Herpesviridae reads were not significantly different among strains A, B, and C; however, there were marked differences in the abundance of these reads in strain D. In summary, this study provides an unbiased examination of the viral diversity in the feces of four strains of ducks in specific-pathogen-free periods, and highlights the variation in the percentage of viral families present. These results can be used as a reference for detecting duck viral pathogens and predicting zoonotic potential.

Autophagy activated by duck enteritis virus infection positively affects its replication.

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

The Stability and Efficency of CPB Cells Were Acclimated for Virus Proliferation.

BACKGROUND: Vaccinations are still the most effective means of preventing and controlling fish viral diseases, and cells are an important substrate for the production of a viral vaccine. Therefore, the rapid-stable growth and virus sensitivity of cells are urgently needed. METHODS: Chinese perch brain 100th passage (CPB p100) were acclimated in a low serum with 5% FBS L-15 for 50 passages, then transferred to 8% FBS L-15 for 150 passages. Additionally, the morphology and cell type of CPB 300th passage (CPB p300) cells were identified. We analyzed the transfection efficiency and virus sensitivity of CPB p300 cells, and then optimized the conditions of ISKNV, SCRV, and LMBV multiplication in CPB cells. RESULTS: CPB p300 cells were more homogeneous, and the spread diameter (20-30) µm in CPB p300 cells became the dominant population. The doubling time of CPB p300 was 1.5 times shorter than that of CPB p100.However, multiplication rate of CPB p300 was 1.37 times higher than CPB p100. CPB p300 cells were susceptible to ISKNV, SCRV, and LMBV, and the optimal conditions of ISKNV, SCRV, and LMBV multiplication were simultaneous incubation, 0.6 × 105 cells/cm2 and MOI = 0.1; infection at 48 h, 0.8 × 105 cells/cm2 and MOI = 0.01; simultaneous incubation, 0.7 × 105 cells/cm2 and MOI = 0.05, respectively. The time and economic costs of ISKNV, SCRV, and LMBV multiplication in CPB p300 cells were significantly reduced. CONCLUSIONS: The acquisition of CPB p300 cells laid a good material foundation for the production of ISKNV, SCRV, and LMBV vaccines.

A Novel and Effective Therapeutic Method for Treating Aeromonas schubertii Infection in Channa maculata.

Aeromonas schubertii is a pathogen that severely affects aquatic animals, including the snakehead, Channa maculata. Lytic bacteriophages have been recognized as effective alternatives to antibiotics for controlling bacterial infections. However, there have been no reports of A. schubertii phages as far as we know. In this study, a lytic bacteriophage SD04, which could effectively infect A. schubertii, was isolated from pond water cultured with diseased snakehead. The SD04 phage formed small, round plaques on Petri dishes. Electron microscopy revealed a hexagonal head and a contractile tail. Based on its morphology, it may belong to the Myoviridae family. Two major protein bands with molecular weights of 50 and 38 kilodaltons were observed after the phage was subjected to SDS-PAGE. The phage showed a large average burst size, high specificity, and a broad host range. When stored at 4 °C, phage SD04 had high stability over 12 months and showed almost no variation within the first six months. All fish were healthy after both intraperitoneal injection and immersion administration of SD04, indicating the safety of the phage. After treatment with SD04, Channa maculata in both phage therapy groups and prevention groups showed high survival rates (i.e., 83.3 ± 3.3% and 100 ± 1.3%, respectively). Phage therapy inhibits bacterial growth in the liver, the target organ of the infected Channa maculat. The experimental results indicate the potential use of phage SD04 for preventing A. schubertii infection in Channa maculata.

Role of asparagine biosynthesis pathway in Siniperca chuatsi rhabdovirus proliferation.

Viruses are non-living organisms that rely on host cellular metabolism to complete their life cycle. Siniperca chuatsi rhabdovirus (SCRV) has caused huge economic losses to the Chinese perch (Siniperca chuatsi) industry worldwide. SCRV replication is dependent on the cellular glutamine metabolism, while aspartate metabolism plays an important role in viral proliferation in glutamine deficiency. Herein, we investigated roles of asparagine metabolism in SCRV proliferation. Results showed that SCRV infection upregulated the expression of key enzymes in the aspartate metabolic pathway in CPB cells. And the key enzymes of malate-aspartic acid shuttle pathway upregulated during the virus invasion phase, and key enzymes of the asparagine biosynthesis pathway upregulated during the viral replication and release phase. When asparagine was added to the depleted medium, the SCRV copy number restored to 90% of those in replete medium, showing that asparagine and glutamine completely rescue the replication of SCRV. Moreover, inhibition of the aspartate- malate shuttle pathway and knockdown of the expression of key enzymes in the asparagine biosynthesis pathway significantly reduced SCRV production, indicating that the aspartic acid metabolic pathway was required to the replication and proliferation of SCRV. Above results provided references for elucidating pathogenic mechanism of SCRV by regulation of aspartate metabolism.

An Avirulent Largemouth Bass Birnavirus Vaccine Candidate Protects Largemouth Bass against Birnavirus Infection.

BACKGROUND: Largemouth bass birnavirus (LBBV) disease outbreaks in largemouth bass fingerlings lead to high mortality in China. Therefore, the development of immersion immunization strategies is paramount. METHODS: An avirulent LBBV strain was screened using a fish challenge assay. The proliferation dynamics of the avirulent strain were determined in vitro and in vivo. The efficacy of the avirulent vaccine was evaluated using immune gene expression, viral load, and a virus challenge, and the safety was also assessed using a reversion to virulence test. RESULTS: An avirulent virus strain, designated as largemouth bass birnavirus Guangdong Sanshui (LBBV-GDSS-20180701), was selected from five fish birnavirus isolates. The proliferation peak titer was 109.01 TCID50/mL at 24 hpi in CPB cells and the peak viral load was 2.5 × 104 copies/mg at 4 dpi in the head kidneys and spleens of largemouth bass. The largemouth bass that were immersed within an avirulent vaccine or injected with an inactivated vaccine were protected from the virulent LBBV challenge with a relative percent survival (RPS) of 75% or 42.9%, respectively. The expression levels of IL-12, MHCI, MHCII, CD8, CD4, and IgM in the avirulent group were significantly upregulated at a partial time point compared to the inactivated vaccine group. Moreover, the viral load in the avirulent vaccine group was significantly lower than those in the inactivated vaccine group and control group using real-time PCR. CONCLUSIONS: LBBV-GDSS-20180701 is a potential live vaccine candidate against LBBV disease.

Development of a New Marine Fish Continuous Cell Line Derived from Brain of Red Sea Bream (Pagrosomus major) and Its Application to Fish Virology and Heavy Metal Toxicology.

Red sea bream (Pagrosomus major) is one of the most popular farmed marine teleost fish species. Fish cell lines are becoming important research tool in the aquaculture field, and they are suitable models to study fish virology, immunology and toxicology. To obtain a Pagrosomus major cell line for biological studies, a continuous cell line from brain of red sea bream (designated as RSBB cell line) was established and has been successfully subcultured over 100 passages. The RSBB cell line predominantly consisted of fibroblast-like cells and multiplied well in M199 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome numbers of RSBB cells was 48. After transfection with pEGFP-N1, RSBB cells showed bright green fluorescence with a transfection efficiency approaching 8%. For toxicology study, it was demonstrated that metal Cd could induce cytotoxic effects of RSBB cells, accompanied with a dose-dependent MTT conversion capacity. Morphologically, cells treated with metal Cd produced rounding, shrinking and detaching and induced both cell apoptosis and necrosis. For virology study, the RSBB cells were highly susceptible to Nervous necrosis virus (NNV) and Singapore grouper iridovirus (SGIV) with steady titers (i.e., 108.0~8.3 TCID50 mL-1 and 107.0~7.2 TCID50 mL-1 respectively). Furthermore, an obvious cytopathic effect (CPE) could be observed in RSBB cells infected with Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdoviruses (SCRV). Meanwhile, all the infections were confirmed by polymerase chain reaction. The new brain cell line developed and characterized from red sea bream in this study could be used as an in vitro model for fish studies in the fields of toxicology and virology.