Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.     

Experimental infection of turbot, Psetta maxima (L.), with strains of viral haemorrhagic septicaemia virus isolated from wild and farmed marine fish

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.     

Betanodavirus infection in bath‐challenged Solea senegalensis juveniles: A comparative analysis of RGNNV,SJNNV and reassortant strains

Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3–4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole.     

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass,Dicentrarchus labrax (L.)

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red‐spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV‐type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.     

鱼类神经坏死病毒研究进展与发展趋势

神经坏死病毒(nervous necrosis virus,NNV)是一种世界范围内流行、严重危害多种海水和淡水鱼类的传染性病原。NNV为单一正链、2节段RNA病毒,基因组由RNA1(3.1 kb)和RNA2(1.4 kb)组成。在病毒复制过程中,会合成亚基因组RNA3。RNA1编码RNA聚合酶。RNA2编码衣壳蛋白,为病毒的唯一结构蛋白。RNA3编码B1和B2两种非结构蛋白。根据病毒衣壳蛋白的基因序列,神经坏死病毒可以分成4种基因型,分别为拟鲹、红鳍东方鲀、条斑星鲽和赤点石斑神经坏死病毒基因型。但是,目前只发现A、B、C三种病毒血清型,A对应拟鲹神经坏死病毒基因型,B对应红鳍东方鲀神经坏死病毒基因型、C对应条斑星鲽神经坏死病毒和赤点石斑神经坏死病毒基因型。病毒存在垂直和水平两种传播途径,而且广泛分布于养殖和野生鱼类中。阻断病毒在野生与养殖鱼类之间的传播和开展新型鱼类疫苗研发是将来研究趋势。     

Evaluation of the therapeutic effect of potassium permanganate at early stages of an experimental acute infection of Flavobacterium columnare in channel catfish,Ictalurus punctatus (Rafinesque)

The efficacy of potassium permanganate (KMnO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus, was evaluated. Fish were experimentally challenged by waterborne exposure for 2 h to F. columnare after cutaneous abrasion, and treated with KMnO₄ at 2.0 mg L^?1 above the KMnO₄ demand at 0, 1, 2 or 4 h postchallenge for 24 h. Challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged and treated with KMnO₄ at 0, 1, 2 or 4 h postchallenge had mortalities of 26%, 63%, 64% and 83% respectively. The mortality of challenged fish treated with KMnO₄ at 0 h postchallenge (26%) was significantly less than the positive control (77%). The mortalities of challenged fish treated at 1, 2 or 4 h postchallenge were not significantly different from the positive control fish. The results suggest that KMnO₄ has a clear therapeutic value in early stages of columnaris infection but limited therapeutic value once the infection has progressed.     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.     

Dietary L-Carnitine Supplementation Protected Turbot (Scophthalmusmaximus) Against Streptococcus parauberis Infection

The protective effect of dietary L-carnitine supplementation was accessed in turbot (Scophthalmusmaximus) challenged with Streptococcus parauberis. Infected fish fed 240mg L-carnitine showed significantly (P < 0.01) higher survival (78%) than infected fish fed 40mg L-carnitine (11%). When dietary L-carnitine supplementation was suspended, a significant increase in mortality was observed in infected fish. The present report documents that dietary L-carnitine fed on a continuous basis may act as an immunostimulant in turbot.     

Investigation on protective effect of recombinant protein (OmpTS) of Aeromonas hydrophila in Common carp (Cyprinus carpio)

The outer membrane protein of Aeromonas hydrophila is a potential candidate for vaccine development. In this study, after cloning and expression of ompTS, 270 common carp, weighing 44 ± 5.7 g divided into five groups, were injected intraperitoneally twice with 3‐week intervals. Groups included the following: PBS, PBS plus Freund's adjuvant, recombinant protein, recombinant protein plus Freund's adjuvant and 20 fish as negative control. Two weeks after the second injection, 30 fish of each group were challenged with a dose of 2 × LD₅₀ of Aeromonas hydrophila and RPS was measured. The antibody level was measured using ELISA test. The protection of recombinant protein in the immunized fish with and without adjuvant, respectively, was about 82.61% and 78.26% (the protection of recombinant protein electroeluted from an SDS–PAGE with and without adjuvant, respectively, was about 78.62% and 69.57%). The average of antibody level in recombinant protein with and without adjuvant was significantly higher than the PBS group (p < .05). The ability of recombinant ompTS to increase the antibody level and to protect the fish from challenge by A. hydrophila demonstrated that recombinant ompTS protein injection can be used to immunize common carp against A. hydrophila infection.     

Aeromonas salmonicida infection in wrasse (Labridae), used as cleaner fish, on an Atlantic salmon, Salmo salar L., farm

Abstract. Typical Aeromonas salmonicida with similar biochemical characteristics to A. salmonicida from Atlantic salmon, was isolated from wrasse, Ctenolabrus rupestris (L.) and Centrolabrus exoletus (L.), stocked as cleaner fish with these salmon. Although no external clinical signs were apparent, localized bacterial microcolonies were observed in muscle, gills, intestine, kidney and myocardial tissue. Mortalities attributed to A. salmonicida comprised 55% (n = 32) of total mortalities. No carriers of A. salmonicida were found in wild wrasse following stress testing. Although salmon post-smolts died when challenged with 1 × 10⁵ ml^-1 of a virulent strain, there were no mortalities in challenged wrasse. An oral route of infection is suggested rather than water-borne transfer as wrasse browsed on salmon mortalities. Wrasse were treated for A. salmonicida infection by injection with antibiotic and were also vaccinated, and in the latter case, elevation of antibody levels was noted.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.     

Disease resistance and immune‐relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus‐like agent

Infectious spleen and kidney necrosis virus (ISKNV), family Iridoviridae, genus Megalocytivirus, may cause high mortality rates such as those seen in mandarin fish, Siniperca chuatsi. ISKNV has attracted much attention due to the possible environmental threat and economic losses it poses on both cultured and wild populations. We have investigated the pathogenicity of ISKNV‐like agent Megalocytivirus, isolated from infected pearl gourami, in golden mandarin fish, Siniperca scherzeri – a member of the Percichthyidae family – and in another Percichthyidae species, S. chuatsi. Fish were challenged with four different doses of ISKNV‐like agent Megalocytivirus (1, 10, 100 or 1000 μg per fish) over a 30‐day period, and cumulative fish mortalities were calculated for each group. No significant mortality was observed for fish challenged with the lowest dose (1 μg per fish) relative to a control group. However, all other challenged groups showed 100% mortality over a 30‐day period in proportion to the challenge dose. Quantitative real‐time PCR was performed to measure mRNA expression levels for six immune‐related genes in golden mandarin fish following ISKNV‐like agent challenge. mRNA expression levels for IRF1, Mx, viperin and interleukin 8 significantly increased, while mRNA levels for IRF2 and IRF7 remained constant or declined during the challenge period.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.     

Development of a reproducible infectious pancreatic necrosis virus challenge model for Atlantic salmon, Salmo salar L.

Atlantic salmon smolts, previously unexposed to infectious pancreatic necrosis virus (IPNV), were placed into tanks of sea water at 10 °C. After 4 weeks, 40 fish were injected intraperitoneally (i.p.) with homogenized and filter‐sterilized kidney material obtained from salmon with clinical IPN in a marine farm in Shetland. The injected fish were cohabited with 40 untreated fish. Mortalities began in the injected fish on day 7 and reached a peak of 48% on day 14. In the cohabitation group, mortalities began on day 14 and reached a peak of 70% on day 27. The IPNV in the Shetland kidney homogenate was cultured in Chinook salmon embryo (CHSE) cells and passed twice. This cultured virus was injected i.p. into fish at various doses ranging from 10 to 10⁷ TCID₅₀ fish^?1 4 weeks after seawater transfer. Challenge tanks contained 30 injected fish and 30 cohabitees. Mortality rates and levels were dose‐dependent. The highest dose used resulted in a similar mortality pattern as obtained with a similar dose of the Shetland kidney homogenate, indicating that virulence was retained after two passes in tissue culture. Even with the lowest dose, mortality reached 12% in the injected group and 23% in the cohabitees. The IPNV titres were high (10⁶?10⁹ i.u. g^?1 kidney) in fish which died during the experiment and low (<10⁵ i.u. g^?1 kidney) or undetectable in surviving fish. The cultured virus (pass 3) was used in a challenge model where the population density of fish in the tanks was high (50 injected and 50 cohabitees) or low (15 injected and 15 cohabitees). In the high stocking density tank, mortalities peaked at about 35% in the injected group and at 52% in the cohabitees. In the low stocking density tank, mortalities peaked at about 40% in the injected fish but no mortality occurred in the cohabitees. However, IPNV was detected (up to 10⁴ i.u. g^?1 kidney) in 82% of cohabitees sampled on day 30. These data suggest that lethal lateral transmission of the virus is dependent on the infectious pressure from the injected group. A further trial was conducted to investigate the effect of time post‐seawater transfer on the susceptibility of post‐smolts to IPN. Groups of fish were challenged every 2 weeks from week 0–10. Few mortalities occurred at week 0 and virus titres were high in these fish. Most survivors became carriers, some with titres >10⁶ i.u. IPNV g^?1 kidney. From 2 to 10 weeks after seawater transfer, mortalities in both injected and cohabitees were substantial with viral titres >10⁷ i.u. g^?1 kidney. Survivors had lower titres and in many virus was undetectable. Throughout the experiments, moribund fish were sampled for histology and all showed typical IPN histopathology.