Microalgal extracts induce larval programming and modify growth and the immune response to bioactive treatments and LCDV in Senegalese sole post-larvae.

Immunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days post-hatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast ß-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL-1) enriched for 30 min with MAe or Y (at 2 mg mL-1 SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The post-larvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total anti-protease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN post-larvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast ß-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses.

Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration.

Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5µg) at 2days before infection (1.1×107) held at 26°C died even though mortality was delayed from 8days (1µg) and 4days (5µg). Similarly, CpG ODN 1668 administered (5µg) at 2days before infection (1.2×106) held at 23/20°C had 100% mortality; the mortality was delayed from 9days (23°C) and 11days (20°C). Moreover, when CpG ODN 1668 administered (1/5/10µg) at 2/4/7days before infection or virus concentration was decreased to 1.1×104 and held at 20°C had mortality rates of 20/60/30% (2days), 30/40/60% (4days) and 60/60/20% (7days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1×107) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20°C and infection dose of 1.1×104).

Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus.

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

Cynoglossus semilaevis ISG15: a secreted cytokine-like protein that stimulates antiviral immune response in a LRGG motif-dependent manner.

ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation.

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses.

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C(60) fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1 h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID(50)/mL. The influence of the C(60) concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001 mg/mL. It has been found that C(60) is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C(60) fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.

Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish Cells by AmiRNA.

BACKGROUND: Envelope protein 53R was identified from frog Rana grylio virus (RGV), a member of the family Iridoviridae, and it plays an important role in the virus assembly. Although inhibition of iridovirus major capsid protein (MCP) by small hairpin RNAs (shRNAs) has been shown to cause resistance to viral infection in vitro, RNA interference (RNAi) to inhibit aquatic animal virus envelope protein gene product has not been reported. METHODOLOGY: We devised artificial microRNAs (amiRNAs) that target a viral envelope protein gene RGV 53R. By incorporating sequences encoding amiRNAs specific to 53R of RGV into pre-miRNA155 (pSM155) vectors, which use the backbone of natural miR-155 sequence and could intracellularly express 53R-targeted pre-amiRNAs. The pre-amiRNAs could be processed by the RNase III-like enzyme Dicer into 21-25 nt amiRNAs (amiR-53Rs) in fish cell lines. The levels of 53R expression were analyzed through real-time PCR and RGV virions assembly were observed by electronic microscopy in fish cells transfected with or without amiR-53Rs at 72 h of RGV infection. CONCLUSION/SIGNIFICANCE: The results argue that viral envelope protein RGV 53R can be silenced and the virions assembly was deficient by amiR-53R-1, and further identified the first amiRNA of envelope protein gene from iridovirus that was able to cause resistance to virus infection in fish cells. The data demonstrate that the viral infection is efficiently suppressed (58%) by amiR-53R-1 targeting positon 36-57 of RGV 53R. Moreover, electron microscopic observations revealed virion assembly defect or reduced virions assembly capacity was closely correlated to expression of amiR-53R-1. Based on real time PCR of the Mx gene, we found no evidence of activation of IFN by amiR-53R-1.

Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment.

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.

A new iridovirus isolated from soft-shelled turtle.

A virus was isolated from soft-shelled turtle (Trionyx sinensis) with 'red neck disease' on a farm in Shenzhen, China, the virus multiplied and caused a cytopathogenic effect (CPE) at 15-30 degrees C in CO, FHM, CK and BF-2 cells. The optimum conditions for replication was in CO cells at 25-30 degrees C. The virus was sensitive to chloroform treatment, acid (pH 3) or alkaline (pH 10) conditions and heating at 56 degrees C for 30 min. Treatment with 5-iodo-2-deoxyuridine (IUDR) inhibited viral replication, indicating the presence of a DNA genome. Electron microscopy of infected CO culture fluid revealed spherical particles measuring 120-160 nm in diameter. Observation of ultra-thin sections showed numerous hexagonal viral particles in the cytoplasm and nucleus of cells typical of an iridovirus. This virus was moderately virulent for turtles in infection tests. We suggest that this virus is named soft-shelled turtle iridovirus (STIV).

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations.

Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV3) and chilo iridescent virus (CIV), two other well known viruses of the family Iridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A2. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.

Effect of inhibitors of the host cell RNA polymerase II on African swine fever virus multiplication.

The role of the host cell RNA polymerase II in African swine fever (ASF) virus growth has been examined using inhibitors of this enzyme. The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of mRNA precursor synthesis in mammalian cells, strongly inhibits the production of infectious progeny virus in Vero cells, but does not significantly affect the synthesis of virus-specific macromolecules. On the other hand, virion assembly seems to proceed normally in the presence of DRB, as virus particles can be seen in electron micrographs with a morphology indistinguishable from that observed in the absence of the inhibitor. However, taking into account the inhibition of the infectivity caused by the drug, most of these particles must be defective. In contrast with this effect of DRB on ASF virus replication, the toxin alpha-amanitin does not inhibit the production of infectious ASF virus in Vero cells or porcine alveolar macrophages. This result indicates that the host RNA polymerase II does not transcribe viral genes and that active transcription of the cell genome is not needed for ASF virus replication.

Phosphonylmethoxyalkylpurines and -pyrimidines as inhibitors of African swine fever virus replication in vitro.

Several phosphonylmethoxyalkylpurine and -pyrimidine derivatives related to (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated as inhibitors of African swine fever virus (ASFV) replication in Vero cells. (S)-HPMPA has previously been shown to inhibit ASFV replication at a minimum inhibitory concentration (MIC) of 0.01 microgram/ml with a selectivity index of 15000. Of the new compounds tested, the following emerged as the most potent and selective inhibitors of ASFV replication: the cyclic phosphonate of (S)-HPMPA [(S)-cHPMPA] with an MIC of 0.2 microgram/ml and a selectivity index of 2500, the 2,6-diaminopurine analogue of (S)-HPMPA [(S)-HPMPDAP] with an MIC of 0.5 microgram/ml and a selectivity index of 1400, and the cytosine [(S)-HPMPC] and guanine [(RS)-HPMPG] analogues with an MIC of 1 microgram/ml and a selectivity index of 600-700.

Comparative efficacy of broad-spectrum antiviral agents as inhibitors of African swine fever virus replication in vitro.

Various nucleoside analogues, selected on the basis of their previously established broad-spectrum antiviral properties, were evaluated for their potency and selectivity as inhibitors of the in vitro replication of the iridovirus, African swine fever virus (ASFV). The test compounds included (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)adenine, (RS)-3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, (S)-9-(2,3-dihydroxypropyl)adenine, carbocyclic 3-deazaadenosine (C-c3Ado), 3'-azido-2',3'-dideoxythymidine, pyrazofurin and ribavirin. As the most efficacious inhibitors of ASFV replication emerged (S)-HPMPA followed by C-c3Ado. The minimum inhibitory concentration of (S)-HPMPA for ASFV replication was 0.01 microgram/ml, and its selectivity index was 15,000. The corresponding values for C-c3Ado were 0.025 micrograms/ml and 8000, respectively. It would seem justified to further pursue these compounds for their anti ASFV activity in vivo.

New agents active against African swine fever virus.

Actinobolin, atropine, carrageenan, megalomycin C, suramin, and tetracenomycin C were tested for their activity against African swine fever virus replication. Both viral inhibitory potency and cytotoxicity were investigated. Megalomycin C, suramin, atropine, and carrageenan exhibited significant activity. Megalomycin C was the most active of the four agents with respect to the concentration of compound that blocked the formation of infectious virus by 50%. Suramin was the next most active agent in this respect, but because of its lower cytotoxicity, it had the most favorable therapeutic index.

Insect iridescent virus type 6 induced toxic degenerative hepatitis in mice.

The toxic effect of insect iridescent virus type 6 - chilo iridescent virus - (CIV) was investigated using Balb/c mice (strain ByJ Ico and Kisslegg). The animals were inoculated with CIV intraperitoneally (1 X 10(9) to 9.2 X 10(11) TCID50/animal). The animals which were administered with 1 X 10(11) to 9 X 10(11) TCID50 of CIV per animal, developed acute clinical illness and died during 18 to 80 h post infection. Histopathological and electronmicroscopic examinations of the liver tissues of those animals which died and/or were sacrificed when moribund showed acute degenerative hepatitis leading to death. No evidence for viral replication was found in the liver cells affected. A mortality rate between 21.1% and 100% was recorded for CIV, depending on the strain and number of mice used and the dose of virus administered. The toxic effect of CIV was eliminated or reduced extensively using heat denaturation or treatment of CIV with sodium dodecylsulphate or proteinase K. This indicates that the nature of the factor causing toxic degenerative cell damage is a protein.

Interaction of frog virus 3 with the cytomatrix. III. Role of microfilaments in virus release.

The role of microfilaments in the release of frog virus 3 (FV3) from the plasma membrane was studied. Scanning electron microscopic study of FV3-infected baby hamster kidney (BHK) cells showed that late in infection (15 hr), numerous microvillus-like projections containing virions and microfilaments occur on the cell surface. Two microfilament-disrupting drugs, cytochalasin B and cytochalasin D, inhibited both the formation of microvillus-like projections and virus release. In the drug-treated cells, the virions accumulated in large numbers beneath the plasma membrane (transmission electron microscopy), suggesting that both drugs affected the release of the virus at the level of plasma membrane rather than the traverse of the virus to the plasma membrane. Two-dimensional gel analysis of actin from FV3-infected and uninfected cells revealed the following. There was no difference in the synthesis of actin in infected versus uninfected cells. However, the actin of infected cells is more acidic than its counterpart in uninfected cells. Temporally, the change in actin preceded the formation of microvilli-like projections involved in virus release. The change in actin is virus induced and is linked to virus maturation since a ts mutant of FV3 (ts9467), which is deficient in virus production at the restrictive temperature (30 degrees), did not modify actin. The mutant, at the permissive temperature (25 degrees), produced virions and altered the actin. Together, the above results attribute an active role for microfilaments in virus release.

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine.

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Isolation and characterization of a frog virus 3 variant resistant to phosphonoacetate: genetic evidence for a virus-specific DNA polymerase.

A variant of frog virus 3 (FV3) resistant to 200 micrograms/ml phosphonoacetate was isolated, and used to establish that the DNA polymerase induced in FV3-infected cells was virus coded. In addition, inhibitor studies showed that the FV3 polymerase is similar to eukaryotic polymerase alpha in its sensitivity to aphidicolin, and that resistance to phosphonoacetate does not confer cross-resistance to thymidine arabinoside or acycloguanosine.

Antiviral action of 5-amino-2-(2-dimethyl-aminoethyl)benzo-[de]-isoquinolin-1,3-dion e.

A newly synthesized imide derivative of 3-nitro-1,8-naphthalic acid, 5-amino-2-(2-dimethylaminoethyl)benzo-[de]-isoquinolin-1,3-dione (designated M-FA-142), was tested on chick embryo cells against herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV), and on Vero cells against African swine fever virus (ASFV). At a concentration of 4 micrograms/ml the drug inhibited VV replication by about one order of magnitude, and that of HSV-1 by about three orders of magnitude. A minor effect was shown against ASFV. Virus inhibition was found to depend on the amount of drug and multiplicity of infection. No virucidal effect was observed on the viruses tested, except for a slight effect on HSV-1. Inhibition of virus growth could be reversed when the drug was removed from the cell culture medium. Serial passages of HSV-1 and VV in the presence of the drug caused the appearance of drug-resistant viruses.

Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene.

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.