Scottish Marine and Freshwater Science Volume 4 Number 3: Epidemiology and Control of an Outbreak of Viral Haemorrhagic Septicaemia in Wrasse Around Shetland Commencing 2012

5 Laboratory Methods

5.1 Background

Marine Scotland Science ( MSS) is a designated national reference laboratory for fish, shellfish and crustacean diseases in accordance with EC Council Directive 2006/88/ EC. This includes accreditation ( ISO 17020 and ISO 17025) and participation in ring-tests organised by the EU Reference Laboratory for Fish Diseases.

5.2 Virus Isolation with ELISA Confirmation

Virus isolation with ELISA confirmation as recommended by the OIE ^[10] was used to test individual wrasse for VHS on sites operating under IDN. The same procedure was used to test individual Dover sole, European seabass, turbot and whiting from the temporary holding facility operating under an IDN, individual Atlantic salmon on farms operating under CDN, individual wild gadoids in the pens of one farm operating under a CDN, and pooled free-ranging wild fish from south-west Shetland mainland.

Tissue comprising kidney, spleen, heart and brain from each sampled individual was placed in viral transport medium ( VTM) and shipped to the laboratory in cool boxes containing frozen freezer blocks. Tissues were homogenised on receipt, centrifuged, the supernatant incubated with an equal volume of a laboratory-specific antiserum to Infectious pancreatic necrosis virus and inoculated onto a fathead minnow ( Pimephales promelas) cell line ^[16] within 48 hours of sampling. The cells were incubated and inspected for a cytopathic effect ( CPE) by phase-contrast microscopy three times a week and, in its absence after seven days, subcultured with continued inspection for a further seven days. A site for which no cultures showed CPE was declared VHS negative. Cultures showing CPE were further tested by ELISA using antibody MAb IP5B11 ^[17] . A site for which the ELISA absorbance of one or more cultures exceeded a defined threshold value was declared VHS positive; otherwise the site was declared VHS negative.

The use of virus isolation with ELISA confirmation to test for VHS has not previously been subjected to a formal evaluation of its diagnostic specificity (Sp) and sensitivity (Se). Some information on the Sp of virus isolation with ELISA confirmation is available from the results of tests using the same procedure carried out at this laboratory in the five years prior to this outbreak. There was no outbreak of VHS in Scotland over this time and the methods, equipment and staff at the laboratory were relatively stable. None of the 293 VHS tests generated a positive result indicating a Sp for the laboratory procedure of greater than (>) 99% with a 95% confidence interval ( CI) equal to or greater than (≥) 99%. There is insufficient laboratory data to estimate the Se of virus isolation with ELISA confirmation laboratory per se, although it is possible to estimate the probability of identifying a hypothetically infected farm out-with Shetland as being VHS positive ^[18] . Such an analysis involves assumptions including, for example, that the distribution of within farm infection prevalences on farms hypothetically infected with VHSV out-with Shetland would be the same as that observed on VHS positive farms around Shetland. It is concluded that there was a 99% (95% CI of 95% to >99%) chance of detecting a single farm out-with Shetland if it was hypothetically VHS positive.

5.3 Quantitative RT-PCR

Quantitative RT-PCR was used to screen individual wrasse and lumpsucker ( Cyclopterus lumpus) at the first-reported site. Tissue comprising heart and kidney from each sampled individual was stored in the proprietary solution RNALater ^®. The test ^[19] , developed by MSS, is a two-step procedure which detects a part of the nucleocapsid (N-) gene of VHSV genotypes I-IV.

Quantitative RT-PCR was also used to determine the genotype of the VHSV infecting VHS positive wrasse at the first-reported site. The test ^[20] , developed by MSS, categorises VHSV on the nucleic acid sequence variation of the N-gene enabling a rapid initial evaluation of the genotype and the risks to farmed and wild fish based on their relative susceptibility.

5.4 Nucleic Acid Sequencing

Partial nucleic acid sequences for the VHSV N- and glycoprotein (G-) genes were obtained for isolates propagated in cell culture. The isolates originated from four of the five species of wrasse and from all three localities around Shetland. Sequencing involved extracting RNA from VHSV infected cell culture material, conversion of RNA into complementary- DNA ( cDNA), amplification of VHSV cDNA using the polymerase chain-reaction ( PCR) with primers 5'ATGGAAGGA

GGAATTCGTGAAGCG-3' and 5'GCGGTGAAGTGCTGCAGTTCCC-3' for the N-gene ^[21] and primers 5'-CATTTGTGCACACAACAAGCTAG-3' and 5'-GGTCATT CGGACAGGTGTGCTCAG-3' for the G-gene, agarose-gel purification of PCR products, and quantification of DNA prior to sequencing. The nucleic acid sequences were obtained using the amplification primers on a Beckman Coulter CEQTM 8800 Genetic Analysis System utilising the specialist software Sequencher ^® v4.9 ^[22] .

Nucleic acid sequence data for the N- and G-genes for a representative isolate of each of the four VHSV genotypes were obtained from the European Molecular Biology Laboratory nucleic acid sequence database ^[23] . These isolates ^[24] had previously been used to demonstrate the utility of N- and G-gene nucleic acid sequence data for genotyping ^[25] .

All sequences were aligned using the specialist software Clustal Omega ^[26] prior to statistical analysis, resulting in comparative nucleic acid sequences, containing no deletions or insertions, of 413 nucleotides from position 33 of the VHSV genome for the N-gene and 513 nucleotides from position 9134 of the VHSV genome for the G-gene.