No abstract

Cell culture isolates of salmon pancreas disease virus (SPDV) of farmed Atlantic salmon and sleeping disease virus (SDV) of rainbow trout were compared. Excluding the poly(A) tracts, the genomic nucleotide sequences of SPDV and SDV RNAs include 11,919 and 11,900 nucleotides, respectively. Phylogenetic analysis places SPDV and SDV between the New World viruses of Venezuelan equine encephalitis virus and Eastern equine encephalitis virus and the Old World viruses of Aura virus and Sindbis virus. When compared to each other, SPDV and SDV show 91.1% nucleotide sequence identity over their complete genomes, with 95 and 93.6% amino acid identities over their nonstructural and structural proteins, respectively. Notable differences between the two viruses include a 24-nucleotide insertion in the C terminus of nsP3 protein of SPDV and amino acid sequence variation at the C termini of the capsid and E1 proteins. Experimental infections of Atlantic salmon and rainbow trout with SPDV and SDV confirmed that the disease lesions induced by SPDV and SDV were similar in nature. Although infections with SPDV and SDV produced similar levels of histopathology in rainbow trout, SDV induced significantly less severe lesions in salmon than did SPDV. Virus neutralization tests performed with sera from experimentally infected salmon indicated that SPDV and SDV belonged to the same serotype; however, antigenic variation was detected among SDV and geographically different SPDV isolates by using monoclonal antibodies. Although SPDV and SDV exhibit minor biological differences, we conclude on the basis of the close genetic similarity that SPDV and SDV are closely related isolates of the same virus species for which the name Salmonid alphavirus is proposed.

Abstract. We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.Porcine circovirus (PCV) was first identified in 1974 as a noncytopathic contaminant of a continuous pig kidney (PK-15) cell line. 34 The virus was later shown to possess a single-stranded circular DNA genome of 1.76 kb 8,25,31 and is now classified in a new virus family, the Circoviridae. 23 Serum antibodies to the PCV contaminant of PK-15 cell cultures have been demonstrated in pig populations worldwide, 1,11,12,18,20,32 However, experimental infection of pigs with this virus has failed to produce clinical disease. 4,32 Postweaning multisystemic wasting syndrome (PMWS) in pigs, first identified in western Canada in 1991, 9 is characterized by progressive weight loss, respiratory signs, and jaundice. 9,17 Similar syndromes have also been described in pigs in the USA, 10 France, 22 Northern Ireland, 21 Spain, 27 the Republic of Ireland, 29 Denmark, 6 and Germany. 19 The demonstration of PCV antigen and nucleic acid in lesions sug- Received for publication September 3, 1999. gested that a new, more virulent PCV may have emerged in pig populations in these countries. 5,13,22 All PCV isolates characterized from cases of PMWS form a closely related group at the nucleotide sequence level, exhibiting Ͼ96% intragroup nucleotide sequence identity, 26 but are quite distinct from the PCV isolated from the PK-15 cell line, exhibiting less than 80% nucleotide sequence identity. 16,24,26 It has been proposed that the PCV associated with PMWS should be referred to as PCV type 2 (PCV2) and the PCV conta...

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.KEY WORDS: Salmon pancreas disease · Sleeping disease · Salmonid alphavirus · SAV · Subtypes 1, 2 and 3 Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [105][106][107][108][109][110][111] 2005 are relatively low (Weston et al. 2002). In contrast, the reference SPDV isolate F93-125 (Nelson et al. 1995) and reference SDV isolate S49p (Castric et al. 1997) showed 91.1% nucleotide sequence identity along their 11919 nucleotide (nt) and 11900 nt genomes respectively (Weston et al. 2002). In a comparative study of alphavirus sequences, Powers et al. (2001) suggested that, on the basis of this limited nucleotide sequence divergence, the 2 reference SPDV and SDV isolates constitute 2 subtypes of a new alphavirus species, for which the name Salmonid alphavirus (SAV) was proposed (Weston et al. 2002).To date, sequence data have been reported for the 2 reference isolates only. The objective of this study was to gain further insight into the molecular diversity exhibited by SAVs with the view to applying our findings epidemiologically. In this paper we compare partial nucleotide sequences of 16 additional SAVs originating in France, Ireland, Norway and the United Kingdom. Our results support the occurrence of 3 genetically different SAV groups, which we have called Subtypes 1, 2 and 3. MATERIALS AND METHODS Viruses.The SAVs compared by nucleotide sequencing in this study mainly comprised viruses present in viraemic serum samples that were submitted to the Veterinary Sciences Division for serological diagnosis, and cell culture isolates (Table 1). In the case of 2 SAVs (F02-67, F02-85), viruses were present in tissue homogenates obtained from clinical outbreaks of SD. The reference F93...

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.