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.

Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum).

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.

Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2.

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.

Rapid detection of porcine reproductive and respiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus recognised world wide as an important cause of reproductive failure and pneumonia in pigs. American and European strains of PRRSV, differentiated antigenically and genomically, have been reported. PRRSV infections are currently diagnosed using serology, virus isolation and/or immunocytochemistry. In order to overcome various drawbacks associated with these techniques, conventional, block-based RT-PCR methods for the detection of PRRSV nucleic acid in clinical samples have been described. These methods require gel electrophoresis for analysis of PCR products and present high risk of DNA carry-over contamination between the samples tested. We describe the detection of PRRSV RNA in serum samples and in blood impregnated filter disks (FDs), obtained from experimentally inoculated pigs, using a closed-tube, fluorimeter-based PCR assay. The assay eliminates the use of gel electrophoresis, and is as sensitive and specific as the conventional block-based PCR assay, detecting positive samples as early as 1 day post-inoculation. We also report a rapid fluorimeter based PCR method for differentiating American and European strains of PRRSV.

The reverse transcription polymerase chain reaction for the diagnosis of porcine reproductive and respiratory syndrome: comparison with virus isolation and serology.

A single-tube reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of porcine reproductive and respiratory syndrome (PRRS) virus in blood samples from infected pigs was developed. This test was assessed for sensitivity and application as a rapid diagnostic tool by comparison with virus isolation and detection of PRRS virus antibody in blood. The RT-PCR test was slightly more sensitive than virus isolation for detection of virus in serum and markedly more sensitive than virus isolation from plasma from experimentally infected pigs. The RT-PCR test was also applicable when using whole blood-impregnated filter paper discs, with 94% of the specimens taken by this procedure being positive when compared to RT-PCR performed on serum. PRRS viral nucleic acid was detected in blood samples as early as 24 h after infection and persisted for some time, whereas circulating antibody to PRRS virus was not detected in the same animals until 9 days after infection. These results indicate that the RT-PCR may be an useful technique for the early identification of PRRS viral nucleic acid in blood samples of infected pigs.

Serological evidence for pneumovirus infections in pigs.

A serological survey was carried out on pig sera from herds in Northern Ireland to investigate the incidence of reactivity to bovine respiratory syncytial virus (BRSV) antigens. A total of 529 pig sera from 61 herds were tested and 219 (41 per cent) were found to be reactive with BRSV-infected cell cultures in an indirect immunofluorescence test. None of the BRSV-reactive sera immunostained turkey rhinotracheitis virus-infected cell cultures, indicating specificity for BRSV epitopes. The specificity of this reactivity for BRSV antigen was confirmed by double immunolabelling, using monoclonal antibodies to BRSV and two pig sera with different reactivities to BRSV antigens. A longitudinal serological investigation of two litters of pigs indicated that BRSV-serum reactivity developed between six and 11 weeks after birth. The immunofluorescent staining pattern observed with the majority (73 per cent) of the BRSV-reactive pig sera was typical of that observed with known BRSV-reactive bovine sera. The other immunoreactive pig sera stained BRSV-infected cell cultures in an atypical staining pattern. These different reactivity patterns, combined with the results of the serum neutralisation tests, suggest that more than one serotype of a porcine pneumovirus may exist.