Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of european and north american genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcripts

<p>Abstract</p> <p>Background</p> <p>Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (<it>Salmo salar</it>); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family <it>Orthomyxoviridae</it>, genus, <it>Isavirus</it>. The <it>Isavirus </it>genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family <it>Orthomyxoviridae </it>such as <it>Influenzavirus A </it>have been extensively analyzed, those of <it>Isavirus </it>remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts.</p> <p>Results</p> <p>Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%.</p> <p>Conclusions</p> <p>We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CA^T/_ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.</p

Three Rs Approaches in the Production and Quality Control of Fish Vaccines

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants -- experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry -- agreed that efforts should be undertaken to replace the vaccination challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum

Effects of early rearing history on selected endocrine and immune functions in juvenile Pacific salmonids

The effects of different early rearing conditions on plasma Cortisol concentration, immune function, hematological profile, and disease resistance were examined in populations of hatchery-reared and wild Chinook salmon (Oncorhynchus tshawytscha) and hatchery-reared, wild, and colonized coho salmon (O. kisutch). These features were examined during smoltification and after an acute stress. The effect of initiating feeding with a wild-type diet as compared to a commercially prepared diet was also examined in chinook salmon fry as one aspect of rearing history that is different between fish reared in the hatchery and those in the wild. During smoltification and following periods of acute stress, wild chinook salmon and wild and colonized coho salmon had significantly higher concentrations of plasma Cortisol. Hatchery-reared juveniles showed less sensitivity to stress and lower concentrations of plasma Cortisol during the smoltification period and after an acute stress. Antibody producing cell (APC) number and disease resistance to Vibrio anguillarum were not significantly different between the hatchery and wild chinook salmon. These features were also similar among the hatchery, wild and colonized coho salmon smolts, despite significantly higher levels of circulating Cortisol in the wild and colonized smolts. White blood cell to red blood cell (wbc/rbc) ratios were slightly higher in wild fish than in their hatchery-reared counterparts in chinook and coho salmon juveniles. Significant elevations in plasma Cortisol concentration after an acute stress was still evident retained in the wild and colonized coho salmon juveniles even after holding them for 6 months in an artificial rearing environment. Disease resistance in the wild fish significantly decreased over that time. Following the 6-month rearing period, the initial numbers of APC in the wild fish were significantly higher than those in their hatchery counterparts. This difference precludes the ability to conclude that a cause-effect relationship exists between a high Cortisol response and decreased specific immune function. The use of a live diet to initiate feeding in chinook salmon fry compares favorably to feeding a commercial diet in that the activity of lysozyme and wbc/rbc ratios were higher in this group. Specific immune function was correlated with body weight while non-specific immune defense was not. There appear to be physiological differences between hatchery-reared, wild and colonized coho and chinook salmon. Rearing history may be a determinant in the survival of hatchery-reared salmonids released into the natural environment.Land and Food Systems, Faculty ofGraduat

Reduced Infestation Levels of <i>Lepeophtheirus salmonis</i> in Atlantic Salmon (<i>Salmo salar</i>) following Immersion Exposure to Probiotic <i>Aliivibrio</i> spp.

Salmon lice (Lepeophtheirus salmonis) constitute a major challenge during the production of farmed Atlantic salmon in Norway. Preventive measures are considered to have a higher impact on sustainable control than lice treatment. Therefore, the studies presented here aimed to document the preventive effects of probiotic Aliivibrio spp. on lice infestation in experimental challenges. A reduction in salmon lice attachment success (58–65%) was observed in two separate aquarium trials, where Atlantic salmon were exposed to different compositions of Aliivibrio species 91 and 155 days prior to lice challenge. In a third trial, no difference in attachment was observed in groups exposed to probiotics 58 days prior to lice challenge compared to controls. However, a relative reduction in lice counts was seen on movable stages later in the trial. High levels of probiotic bacteria had no impact on lice viability in an in vitro bioassay on the preadult life stage; thus, the mechanism behind the preventative effect remains unknown. In conclusion, probiotic Aliivibrio bacteria can likely be used as a preventive tool to reduce salmon louse infestations in the salmon industry. The mechanism is still unknown, and this novel approach to lice control warrants further investigation to understand its optimal use and potential

Three Rs approaches in the production and quality control of fish vaccines

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.JRC.DG.I.3-In-vitro method