Ribavirin stimulates the immune response of Atlantic salmon.

Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral infections in mammals and also in fish. It has been seen by others that ribavirin by itself is able to stimulate the immune system of mammals, causing a differentiation of T-cells to T helper 1 cells (Th)-1. In this work, we evaluated the immune effect of ribavirin in vitro on kidney cells from Atlantic salmon and in vivo by oral administration of ribavirin to Atlantic salmon. For this purpose, the transcripts of immune molecules Tbet, GATA3, CD8, CD4, IFNα, IFNγ, IL-4/13, IL-10, IL-12, IL-15 and TGF-B were quantified. The results show that ribavirin administered orally in food to Atlantic salmon increased IFNγ and CD4 transcripts in the in vivo assays and, in addition, increased IL-12, IL-15 and CD8 in the in vitro analyses, indicating that the treatment stimulates a Th1 type response in salmon.

Evaluation of the immune response against immature viral particles of infectious pancreatic necrosis virus (IPNV): a new model to develop an attenuated vaccine.

Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish. Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response. Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.

Bioinformatic analysis of the genome of infectious salmon anemia virus associated with outbreaks with high mortality in Chile.

The infectious salmon anemia virus (ISAV), an orthomyxovirus, is the major cause of outbreaks of high mortality rates in salmon in Chile. It has been proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase and fusion glycoproteins. However, based on current information, the contribution of other viral genes cannot be ruled out. To study this, we isolated and determined the complete coding sequence of two high-prevalence Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and ISAV901_09. These isolates were compared to 15 Norwegian isolates that exhibit differences in their virulence. For this purpose, we performed bioinformatic analyses of (i) functional domains, (ii) specific mutations, (iii) Bayesian phylogenetics, and (iv) structural comparisons between ISAV and influenza virus glycoproteins by using molecular modeling. Phylogenetic analysis shows two genogroups for each protein, one of them containing the Chilean isolates. The gene sequence of the polymerase complex and nucleoprotein indicated that they are closely related to homologues from highly pathogenic Norwegian viruses. Notably, seven of the eight mutations that are present only in the Chilean isolates are on the polymerase complex and nucleoprotein. Structural modeling of hemagglutinin-esterase shows patches of variable residues on its surface. Fusion protein modeling shows that insertions are flexible regions that could affect proteolytic processing, increasing either the accessibility or the number of recognition sites for specific proteases. We found antigenic drift processes related to insertion into the isolated segment 5 of the ISAV752_09. Our results confirm the European origin of Chilean isolates to be the result of reassortments from Norwegian ancestors.

In vivo effect of EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide) on experimental infected rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) fry with infectious pancreatic necrosis virus.

The in vivo antiviral effect of 5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide (EICAR) was evaluated in coho salmon and rainbow trout fry, experimentally infected with infectious pancreatic necrosis virus (IPNV). Treatment consisted of a daily bath of 2 h in 0.4 microg ml(-1) or 0.8 microg ml(-1) of EICAR, for approximately 20 days. The behavior of the fish was studied for 45 days post-infection. The survival of the infected treated groups was compared with the survival of non-infected and infected untreated control groups. The results showed that the survival of coho salmon and rainbow trout fry in the infected group treated with both doses of EICAR was similar to the survival observed in the healthy control group (approximately 94%). While, the survival of the infected and untreated control fish was 56% for salmon and 28% for trout, there were no significant difference in the weight of coho salmon fry between those treated with EICAR and non-infected and infected untreated control groups. However, in rainbow trout there was a statistically significant weight decrease in infected untreated group. Finally, the analysis of tissue samples of the fish by reverse transcription associated with the polymerase chain reaction (RT-PCR) suggest that EICAR have decreased the viral load in infected treated fry. Consequently, the results indicate that EICAR is an effective inhibitor of IPNV replication in vivo and could be a promissory antiviral compound for the treatment of IPNV disease.

Inhibitory effects of EICAR on infectious pancreatic necrosis virus replication.

Recently, the antiviral 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) was shown to inhibit the replication of the infectious pancreatic necrosis virus (IPNV). In order to obtain more information about the mechanism of the antiviral action of EICAR we studied its effect on viral macromolecules synthesis. EICAR was found to inhibit IPNV messenger and genomic RNA synthesis. To inhibit viral RNA synthesis, EICAR must be added at least 3 h before the start of RNA synthesis. This suggests that EICAR does not directly affect the viral RNA polymerization process. Moreover, the antiviral action of EICAR was reversed by the exogenous addition of guanosine (5-50 microg/ml), but not adenosine or cytidine (10-100 microg/ml). Our findings suggest that the antiviral action of EICAR is mediated by a reduction of the intracellular guanosine 5'-triphosphate (GTP) pool level, as has been observed with ribavirin and EICAR in other biological systems.

Detection of the infectious hematopoietic necrosis virus directly from infected fish tissues by dot blot hybridization with a non-radioactive probe.

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.

Polyacrylamide gel electrophoresis of viral genomic RNA as a diagnostic method for infectious pancreatic necrosis virus detection.

A rapid and simple technique for the diagnosis of IPNV from cell culture and infected fish tissues has been developed. It is based on the observation of IPNV bisegmented double-stranded RNA genome in silver stained polyacrylamide gels after electrophoresis. The method is highly specific and can detect as little as 1 ng of viral RNA, which corresponds to 1 x 10(5) pfu, making it possible to visualize the viral genome as soon as the initial cythopatic effect appears. Furthermore, the RNA viral genome could be detected directly from fish tissues when fish showed clear clinical signs of the disease. The method has the advantage of detecting any strain of IPNV. An acrylamide concentration of 6% and bisacrylamide concentration of 0.13% give a rapid and definitive result in less than 6 h.

Inhibition of in vitro reconstitution of rotavirus transcriptionally active particles by anti-VP6 monoclonal antibodies.

Six monoclonal antibodies specific for the major capsid protein of rotavirus, VP6, previously characterized, were tested in a biological assay for their capacity to block the transcriptase activity associated with the single-shelled particles. The results showed that two MAbs (RV-50 and RV-133), specific for distinct antigenic sites, were able to block the transcription when they were incubated with a purified baculovirus-expressed group A VP6, prior to the reconstitution of the single-shelled particles from the cores, suggesting that at least two domains are involved in active single-shelled particle reconstitution. The results obtained previously from immunochemistry of synthetic peptides did not allow us to attribute this biological activity to a particular linear sequence of the protein, the domain involved being probably complex and dependent on the folding of the protein. However, the C-terminal end, which is necessary for binding into single-shelled particles could be necessary but not sufficient to restore the transcription, since neither of these two MAbs reacted significantly with peptides of this region. These two MAbs will be useful reagents to study the interactions between VP6 and the cores.

Structure of rotavirus particle: interaction of the inner capsid protein VP6 with the core polypeptide VP3.

The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able to reconstitute the particle with both the mutant cores and VP6. These results suggest the existence of various steps for the assembly of single shelled particles, where the VP6-VP3 interaction seems to be important for recovery of RNA polymerase activity.

Function of rotavirus VP3 polypeptide in viral morphogenesis.

The phenotype of the rotavirus SA-11 mutant tsB carrying a thermosensitive mutation in gene 3, which encodes VP3, was characterized further from both infected cells and purified viral particles. The mutant phenotype was initially identified as negative for in vivo double- and single-stranded RNA synthesis. Our results show that the in vitro transcriptional properties of the tsB mutant at the restrictive temperature were identical to those of the wild-type strain. Similar results were obtained with respect to the VP3-associated guanylyl-transferase activity. Analysis of viral particles made by mutant-infected cells at the restrictive temperature showed that only empty single-shelled particles were assembled. This indicates that viral morphogenesis is halted after the initial viral transcription and before RNA replication, suggesting that VP3 may be required as part of the replicase system but not for subviral particle assembly. These data suggest that such a phenotype is not due to alteration of a VP3 function related to transcription.

In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP6.

Purified baculovirus-expressed group A rotavirus VP6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to bind as a monomer to viral cores, indicating that trimerization of VP 6 may be not required for reconstitution of the polymerase activity.

Respiratory syncytial virus detection by dot blot hybridization with a nonradioactive synthetic oligo deoxynucleotide probe.

A synthetic oligodeoxynucleotide corresponding to a region of the nucleocapside gene (N) of respiratory syncytial virus (RSV), was used as a DNA probe to develop a nonradioactive hybridization assay for the detection of RSV. The probe was labeled by incorporation of biotin-7-dATP to the 3' end by a reaction catalyzed by terminal deoxynucleotydil transferase. The dot blot hybridization assay was found to be specific for RSV when tested against RSV isolates (subgroups A and B) obtained from cell cultures and isolates of adenovirus, reovirus, rotavirus, and pararotavirus. The assay detected both RSV subgroups (A and B) without significant differences. The dot blot hybridization assay using the nonradioactive probe led to similar results to indirect immunofluorescence (IFI) when tested against a panel of 64 clinical samples from nasopharyngeal secretions of infants with clinical symptoms of respiratory disease. This assay may provide the basis for a rapid, simple, and inexpensive method for routine RSV diagnosis.

Effect of nucleotide analogues on rotavirus transcription and replication.

The effects of several nucleoside and nucleoside triphosphate analogues were studied on rotavirus replication and transcription. Nucleoside triphosphate analogues modified at sugar residues were capable of inhibiting in vitro transcription, including adenosine-9-beta-D-arabinofuranoside 5'-triphosphate, 3'-deoxyadenosine 5'-triphosphate, adenosine 5'-triphosphate 2',3'-dialdehyde, guanosine 5'-triphosphate 2',3'-dialdehyde, and cytosine-9-beta-D-arabinofuranoside 5'-triphosphate. Two dialdehyde derivatives, adenosine 5'-triphosphate 2',3' dialdehyde and guanosine 5'-triphosphate 2',3'-dialdehyde, were irreversible inhibitors, forming a stable complex with the viral polypeptide VP3. The effect of the corresponding nucleosides of the inhibitory analogues was studied in SA-11 rotavirus-infected MA-104 cells. Adenosine-9-beta-D-arabinofuranoside and 3'-deoxyadenosine were effective inhibitors of RNA synthesis, an effect that could be due to their inhibition of viral transcription.

Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase.

Rotavirus single-shelled particles have several enzymatic activities that are involved with the synthesis of capped mRNAs both in vivo and in vitro. Because single-shelled particles must be structurally intact to carry out transcription, it has proven to be difficult to identify the protein within such particles that possesses associated RNA polymerase activity. One approach for characterizing the function of the individual proteins within single-shelled particles is to use nucleotide analogs to specifically label those proteins, such as the viral RNA polymerase, that have affinity for nucleotides. In this study, 8-azido-ATP (azido-ATP), a photoreactable nucleotide analog, was used to identify the viral RNA polymerase on the basis of the ability of the analog to inhibit transcription activity associated with rotavirus particles on exposure to UV light. When single-shelled particles were treated with UV light in the presence of [alpha-32P]azido-ATP, the structural protein VP1 became radiolabeled because of cross-linking of the nucleotide analog, and there was a corresponding decrease in the ability of the particles to synthesize mRNA. In parallel experiments in which single-shelled particles were not exposed to UV light, VP1 was not radiolabeled and the particles successfully used azido-ATP as a substrate for the synthesis of viral mRNAs. Taken together, these results are consistent only with the conclusion that VP1 is the rotavirus RNA-dependent RNA polymerase.

Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3.

Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5' end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.

Characterization of rotavirus electropherotypes excreted by symptomatic and asymptomatic infants.

Human rotavirus isolates from 1100 stool samples were analyzed by polyacrylamide gel electrophoresis, and 48 different migration patterns were detected. Heterogeneity in the migration of segment 10 was observed in both long and short electropherotypes in which three long and two short patterns were identified. In spite of these variations all short and long electropherotypes were subgrouped by enzyme immunoassay as subgroups I and II respectively. Mixed infections were detected in 17% of cases and the subgrouping correlated with the corresponding electropherotypes. The same electropherotypes were present in severe, mild and asymptomatic cases and no electropherotype was particularly associated with greater virulence. Furthermore, the electropherotypes isolated from nosocomial asymptomatic cases were the same as those detected from those admitted with severe diarrhea. It seems unlikely that electropherotyping can be used to identify more virulent strains of rotavirus.

Involvement of structural and nonstructural polypeptides on rotavirus RNA synthesis.

Rotavirus are segmented double stranded RNA viruses with a double protein capsid around a central core. The virus replicates in the cell cytoplasm. After infection, eleven mRNAs are transcribed from the viral genome. To characterize further the infection cycle, viral polypeptide synthesis and RNA replication were studied using labelled precursors. The involvement of nonstructural polypeptides NS34 and NS35 was determined by the kinetics of the appearance of viral polypeptides in infected cells. Experiments in which cycloheximide was used showed that the synthesis of both polypeptides was required to begin RNA replication. The isolation of subviral particles at 8 hours postinfection indicates that there is a particle containing the nonstructural polypeptides and the structural polypeptides VP1, VP2, and VP6 that seem to be able to transcribe the viral genome to produce other RNA species. The results suggest that there is a core-like particle similar to one obtained in vitro which upon the addition of VP6 is able to transcribe the virus genome. This seems to indicate that core-like particles may alter their specificity for plus or minus RNA synthesis depending upon the polypeptides that interact with it. The interaction between VP6 and the viral core was analyzed by means of antibodies raised against the viral core and VP6. The results suggest that VP6 contains a specific binding site to the core complex and this interaction allows the synthesis of mRNA.

Faecal excretion of rotavirus and other enteropathogens in newborns of the high and low socio-economic stratum in Santiago, Chile.

Faecal excretion of enteropathogens was studied in newborns in their first week of life. Rotavirus was investigated in 225 neonates, of whom 107 belonged to the low socio-economic stratum (SES) and 118 to the high SES. Half of each group were delivered by caesarean section. Rotavirus was detected in 10 infants (4.4%). Eight of them had been in the same ward and excreted the same viral electrophoretype. Enteropathogenic bacteria were isolated from 8 out of 57 (14.0%) newborns. Positive cultures were equally distributed by SES and route of delivery. Giardia lamblia was the only parasite detected, in one infant (2.6%) of the high SES. None of the children developed symptoms. Faecal excretion of enteropathogens ended spontaneously within a week in all cases. It is suggested that the lack of symptomatology and the spontaneous termination of the faecal excretion are related to immaturity of the small intestinal mucosa, that does not allow the completion of the steps that must take place during a successful infectious event.

Role of the inner protein capsid on in vitro human rotavirus transcription.

The inner protein shell of human rotavirus consists of a single polypeptide called VP6 which was removed from the single-shelled virus by treatment with CaCl2, leaving the viral core. The core thus obtained was unable to transcribe. However, the addition of a supernatant containing VP6 in the absence of Ca2+ restored the transcriptional activity. VP6 obtained from different electropherotypes and serotypes was able to restore transcriptional activity to homologous and heterologous cores. Viral cores obtained after incubation with purified VP6 had electron microscopic characteristics, polypeptide compositions, and transcription products similar to those of the single-shelled virus. The results suggested the successful in vitro reconstitution of the single-shelled virus.