Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study.

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.

Structure-function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion.

Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.

Update on viral vaccines for fish.

A substantial amount of research has been done on fish viruses affecting species in aquaculture. This review will focus on the salmonid industry, as this is the most industrialised part of fish farming where vaccines are extensively used. In spite of the amount of research performed, both in commercial companies and in academic organisations, few viral vaccines are licensed. As of today, all fish virus vaccines for sale are based upon inactivated virus or recombinant proteins. No live attenuated or DNA vaccines are currently licensed, but one DNA vaccine against IHN is being tested in controlled field trials in Canada. Vaccines against infectious pancreatic necrosis (IPN) have been sold for many years in Norway and are now also available in Chile. Most of the research on these vaccines has been performed by pharmaceutical companies, and not much information is available as scientific publications. It has also been difficult to establish reproducible IPN challenge models suitable for vaccine testing and this probably explains the lack of scientific publications. Quite the reverse is the case for the fish rhabdoviruses viral hemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). The challenge models are reproducible, and both inactivated virus and DNA vaccines offer excellent protection. Recombinant subunit vaccines have so far shown unsatisfactory effect. Little information has been published regarding vaccine development against pancreas disease (PD) and infectious salmon anaemia (ISA). PD and ISA vaccines have been tested at the laboratory scale with good results, and two commercial ISA vaccines are currently available in Canada. Regarding nodaviruses, a few publications have shown effect of recombinant subunit formulations. However, nodaviruses cause disease early in the lifecycle of marine fish, and injection of these formulations into fish of a few grams is so far difficult on a commercial scale.

Isolation and characterisation of segment 1 of the infectious salmon anaemia virus genome.

The isolation and characterisation of the largest genomic segment of infectious salmon anaemia virus (ISAV) is reported. Following identification of ISAV-specific clones from a cDNA library, a rapid amplification of cDNA ends-PCR strategy was designed to obtain the sequence of the full length mRNA transcript. The full length open reading frame (ORF) of this gene was shown to be 2169 nucleotides in length, encoding a putative protein of 722 aa. This sequence was demonstrated by RT-PCR to be specific to ISAV-infected cell cultures. The start codon of this ORF was preceded by the ISAV consensus sequence 5' GCTAAGA 3' indicating the full 5' end of the gene to have been obtained. Based on protein size and amino acid composition, this protein was shown to be similar to the PB2 protein of other orthomyxoviruses. Furthermore, a bipartite nuclear localisation signal was identified in the C-terminus of the protein as is found on all of the influenza virus P proteins. Expression of the putative PB2 as a green fluorescent marker protein-fusion protein confirmed that this protein exhibited nuclear localisation in a fish cell line. Sequences of the ISAV segment 1 gene were obtained from Scottish, Norwegian and Canadian ISAV isolates. Analyses confirmed the close genetic relationship between Norwegian and Scottish ISAV and indicated that this segment was among the most conserved of the ISAV genes identified to date. Thus, this evidence strongly suggests that the genomic segment 1 of ISAV encodes a polymerase protein which is thought to be analagous in function to the PB2 protein of influenza viruses.

An RT-PCR-based method for the diagnosis of the sleeping disease virus in experimentally and naturally infected salmonids.

The sleeping disease (SD) of rainbow trout (Oncorhynchus mykiss) is a worldwide disease for which the causative agent, the sleeping disease virus (SDV), has been recently characterized as an atypical alphavirus (Villoing et al. 2000). Up to now, no diagnostic tools were available and thus no epidemiological studies have been undertaken to evaluate the occurrence of this disease on the field. We present in this paper a sensitive and highly specific 1 working day method, which allows the detection of SDV from experimentally and naturally infected fishes. This method, based on a reverse transcriptase/polymerase chain reaction (RT-PCR) assay on total RNA extracted from SDV-infected fish organs, enables the specific DNA amplification of part of the gene encoding the SDV glycoprotein E2, as early as 2 d post-infection (d.p.i.) and as late as 70 d.p.i., at which time clinical signs of infection are no longer apparent. Moreover, we show that this RT-PCR method can be successfully used for the diagnosis of fish infected by a closely related virus, namely salmon pancreas disease virus (SPDV). This report is the first description of a very powerful diagnostic assay which could provide a more accurate replacement for the classical virological, histological and immunochemistry methods.

Rainbow trout sleeping disease virus is an atypical alphavirus.

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.