Genetic analysis of infectious salmon anaemia virus (ISAV) from Scotland

The nucleotide sequences of segments 2 and 8 of infectious salmon anaemia virus (ISAV) isolates from Scotland were determined. These were used to predict amino acid sequences of the products of these segments. The sequences were compared with those previously published for ISAV from Norway and North America. Nucleotide and amino acid variations were found in the Scottish isolate, indicating that it is not identical to those Norwegian or North American strains. Although phylogenetic analysis of the sequences was not possible, it was clear that the Scottish isolate is more closely related to the Norwegian strain than the North American. Sequencing further isolates that are geographically or temporally separated will be important in advancing understanding of the epidemiology of this important disease.     

Identification of infectious salmon anaemia virus in Atlantic salmon from Nova Scotia (Canada): evidence for functional strain differences

Infectious salmon anaemia (ISA) is a serious disease responsible for high morbidity in farmed Atlantic salmon Salmo salar in Norway, Scotland and New Brunswick, Canada. Recent attempts to identify different strains of ISA virus (ISAV) based on nucleotide sequence variation have shown that the Norwegian and Scottish samples are similar to one another but markedly different from New Brunswick samples. These data may suggest the presence of different strains on each side of the Atlantic but no functional difference has been found with either strain. We describe the first identification and characterisation of ISAV in Atlantic salmon from Nova Scotia, Canada. Further, salmon infected with the Nova Scotia ISAV do not show typical ISAV pathology or mortality. Sequencing of this new strain showed it to possess greater similarity to ISAV from Norway and Scotland than to ISAV from New Brunswick. These findings are discussed in terms of a possible origin of the Nova Scotia ISAV strain and the existence of an avirulent ISAV strain. The impact of current strain variation studies on our knowledge of ISAV is also discussed.     

Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis

The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.     

Experimental herpes-like viral infections in marine bivalves: demonstration of interspecies transmission

The sequences of gene segments 2 and 8 from 10 different isolates of infectious salmon anaemia virus (ISAV) sampled in Norway, Canada and Scotland between 1987 and 1999 were determined and compared. Pairwise comparisons revealed a high degree of homology between the European isolates, with identities of 98 to 100% for both genes examined. The Canadian isolate showed identities of 84 and 87 to 88% with the European isolates for the nucleotide sequence of segments 2 and 8, respectively. Phylogenetic analyses were performed to establish the interrelationship between the European virus isolates. The evolutionary rate based on 4 Norwegian isolates clustered together in the analysis of segment 2 was calculated to be 0.96 x 10(-3) nucleotides site(-1) yr(-1). On the basis of this mutation rate it was estimated that the Norwegian Glesvaer 90 and Canadian Bay of Fundy 97 isolates diverged around 1900, which coincides with transportation of salmonids between Europe and North America starting in the late nineteenth century.     

Infectious salmon anaemia virus in wild fish from Scotland.

Following the outbreak of infectious salmon anaemia (ISA) at salmon farms in Scotland, UK, a survey was established to determine the extent of infection in wild fish. All fish tested were free from the clinical symptoms of ISA. Isolations of ISAV were made from 5 sea trout within areas where ISA affected salmon farms were located. Evidence for ISAV in other sea trout was provided by ISA RT-PCR diagnostic tests. Results from ISA RT-PCR tests reveal evidence for ISAV being present in salmon parr, adult salmon and juvenile brown trout in rivers distant from salmon farms and indicate that, at the time of the survey (1998-1999), ISAV may have been widely distributed. Nucleotide sequence analysis of segments 2 and 8 showed that for most sequences from wild fish there was 100% homology with ISAV isolated from clinically affected farmed fish although evidence is presented which indicates variability in ISAV sequences from wild fish. Modelling the RT-PCR findings indicates that ISAV among salmonid fish was spatially non-random. Brown trout, sea trout and salmon (adult and parr) show a pattern of occasionally large numbers of positive samples against a background of very low numbers.     

Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway

Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.     

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

马传染性贫血病毒白细胞弱毒疫苗株及其亲本毒驴强毒株前病毒基因组比较分析

为阐明马传染性贫血白细胞弱毒疫苗株(EIAVDLV)的致弱和免疫保护机理,对EIAVDLV121及其亲本驴强毒株(EIAVDV117)前病毒全基因组序列进行了测定,并结合准种理论,分析了EIAV疫苗致弱过程中基因组进化特点。利用LA-PCR技术对EIAVDV117和EIAVDLV121的前病毒基因组分两段进行扩增,分别获得4个和10个前病毒全基因组序列。EIAVDV117前病毒基因组平均为8236bp,G C含量38.0。EIAVDLV121前病毒基因组平均8249bp,G C含量37.3。两者的前病毒基因组平均差异率为2.8。其中S2、LTR和env基因差异较大,分别为4.1、3.9和3.1。此外,S2、S3和env推导的氨基酸的差异明显,分别为10.4、5.6和4.8(gp90为6.8)。EIAVDLV121各基因的异质性均显著高于EIAVDV117。研究发现体外培养的EIAVDLV121至少有5种类型的LTR混合存在。在gp90推导的氨基酸序列上,EIAVDV117比EIAVDLV121平均多2个N-糖基化位点,总数为19,其中3个为EIAVDV117特有。EIAVDLV121有1个疫苗株特有N-糖基化位点。研究结果为进一步探讨马传染性贫血弱毒疫苗生物学特性提供信息。     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。     

Infectious salmon anemia virus specifically binds to and hydrolyzes 4-O-acetylated sialic acids

Infectious salmon anemia virus (ISAV) is the causative agent of infections in farmed Atlantic salmon. ISAV presumably represents a new genus within the Orthomyxoviridae. ISAV has been shown earlier to exhibit a receptor-destroying activity, which was defined as an acetylesterase with unknown specificity. We have analyzed the substrate specificity of the ISAV esterase in detail. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid. In addition, the purified 9-O-acetylated sialic acid derivative was also hydrolyzed, but at lower rates. When we used a glycosidically bound substrate, ISAV was unable to hydrolyze 9-O-acetylated sialic acid, which represents the major substrate for the influenza C virus esterase. ISAV completely de-O-acetylated glycoprotein-bound 5-N-acetyl-4-O-acetyl neuraminic acid. Thus, the enzymatic activity of the hemagglutinin-esterase of ISAV is comparable to that of the sialate-4-O-esterases of murine coronaviruses and related group 2 coronaviruses. In addition, we found that ISAV specifically binds to glycoproteins containing 4-O-acetylated sialic acids. Both the ISAV esterase and recombinant rat coronavirus esterase specific for 4-O-acetylated sialic acids hydrolyzed ISAV receptors on horse and rabbit erythrocytes, indicating that this sialic acid represents a receptor determinant for ISAV.     

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus

Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.     

Comparative aspects of infectious salmon anemia virus,an orthomyxovirus of fish,to influenza viruses

Infectious salmon anaemia (ISA) is a viral disease that was first recorded in 1984 in farmed Atlantic salmon. The infectious salmon anaemia virus (ISAV) is classified as the type species of the genus Isavirus in the Orthomyxoviridae family and is evolutionary remote to the influenza viruses. The genome consists of eight negative single-stranded RNA segments, and it utilises the same mechanisms as influenza viruses to enter and exit cells. Although a common ancestor of ISAV and other genera of Orthomyxoviruses could be dated back several millions of years, there are still many similarities between ISAV and the influenza viruses regarding morphology, replication cycles and interactions with their respective hosts.     

Nucleotide sequence variation in salmonid alphaviruses from outbreaks of salmon pancreas disease and sleeping disease

We compared 18 salmonid alphaviruses (SAV) including the reference F93-125 salmon pancreas disease virus (SPDV) and S49p sleeping disease virus (SDV) isolates by nucleotide sequence analyses of regions within the E1, nsP4 and nsP3 genes, and found these to comprise 3 distinct groups, which we have designated Subtypes 1, 2 and 3: Subtype 1, which comprised SAVs with sequences closely similar to the reference SPDV isolate, included SAVs from pancreas disease (PD) outbreaks in farmed salmon in Ireland and Scotland over a 10 yr period; viruses from recent outbreaks of sleeping disease (SD) in freshwater-reared trout farmed in England, Scotland and France were closely similar to and were grouped with the reference SDV isolate in Subtype 2; 3 viruses isolated from PD-affected salmon in Norway were genetically different from viruses belonging to Subtypes 1 and 2 and have been assigned to Subtype 3; 1 virus isolated from PD-affected salmon in the Western Isles, Scotland, in 2003 showed consistent nucleotide sequence differences from SAV Subtypes 1, 2 and 3, but was more closely related to the Subtype 1 SAVs. The occurrence of the different subtype SAVs appeared to have a geographical basis, which may prove useful in future molecular epidemiology studies of SAV-induced disease outbreaks.