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

10 Genetic Analysis

10.1 Background

Nucleic acid sequence variation between the VHSV isolates from this outbreak is less than or equal to (≤) one percent. This variation was used to assign isolates to haplotypes and to carry out a phylogenetic analysis using the specialist software PAUP* (version 4.0b10) ^[34] .

10.2 Confirmation of VHSV Genotype

Two viral isolates were recovered from corkwing and goldsinny wrasse at the first-reported site. The partial nucleic acid sequences for both isolates are the same and constitute a single haplotype designated, for the purposes of this report, hap _D.

The nucleic acid sequence of hap _D was used to confirm the initial assignment (section 2.2) of the outbreak to VHSV genotype III. Percentage dissimilarities between representative isolates (footnote 24) of each VHSV genotype and hap _D for the N- and G- genes are presented in Table 4 on the following page. The smallest difference is between hap _D and VHSV genotype III (1%). This similarity was further confirmed by neighbor-joining ^[35] ; the clustering of hap _D with VHSV genotype III is supported by a bootstrap value ^[36] approaching 100%. Analyses using other haplotypes from the other VHS positive sites involved in this outbreak generated the same result. This confirms that the virus responsible for the outbreak is VHSV genotype III.

Table 4. Nucleic acid sequence percentage dissimilarity between the haplotype from the first-reported site and VHSV genotypes

^α 413 nucleotides compared;
^β 513 nucleotides compared;
^γ haplotype of isolates from the first-reported site;
^δ a representative (footnote 24) of each VHSV genotype.

10.3 VHSV from Stocked Wrasse

Isolates with the same partial nucleic acid sequences as hap _D were recovered from rockcook wrasse from two VHS positive farms in north-west and east Shetland Mainland. A further three VHSV haplotypes (no codes assigned) were also identified from VHS positive wrasse.

Nucleic acid sequences for the four haplotypes were compared to the reference VHSV genotype III isolate UK-H17/2/95 which had been isolated from a haddock caught during a research cruise off eastern Scotland in 1995 ^[37] as used to confirm the genotype of hap _D (section 10.2). The four outbreak haplotypes are more similar to each other (one to 10 nucleotide differences) than to the reference isolate (11 to 14 nucleotide differences).

The haplotypes were also subjected to a phylogenetic analysis using maximum parsimony ^{[38] , [39]} . The phylogenetic relationship between the reference isolate and the four outbreak haplotypes is undetermined with a bootstrap value of ≤ 50%. The four outbreak haplotypes cluster into two groups both supported by bootstrap values of ≥ 95% (Figure 4 on the following page). One group consists of hap _D and an isolate differing by two nucleotides recovered from rockcook wrasse on one of the two VHS positive farms in North-West Shetland mainland. The second group consists of two haplotypes differing from each other by a single nucleotide recovered from four species of wrasse (ballan, corkwing, goldsinny and rockcook) on two farms located close to each other in south-west Shetland; these two farms are closely linked because some stock on one farm was moved onto the other. The two groups of haplotypes differ from each other by seven to 10 nucleotides. These differences may be a consequence of either molecular evolution following a single infection event or of multiple infection events followed by more modest molecular evolution; there is insufficient data to resolve this.

Figure 4. Maximum parsimony 50% majority rule consensus tree of phylogenetic relationships between haplotypes

10.4 VHSV from Gadoids Within Farm Pens

Isolates from two VHS positive poor cod on one of the farms (section 9.2) are assigned to haplotype hap _D, which is the same as isolates from two VHS positive rockcook wrasse stocked on the same farm. There is insufficient information to determine whether the poor cod contracted the infection from VHS positive wrasse on the farm or were representatives of the VHS positive free-ranging wild fish which infected the farm.

10.5 VHSV from Free-ranging Wild Fish

Partial VHSV nucleic acid sequences were obtained from four pooled groups of herring, two pools of Norway pout, and one pool each of grey gurnard, plaice, sprat and whiting (section 9.4). Two haplotypes both of which differed from the VHSV isolates recovered from stocked wrasse (and poor cod within farm pens) were observed. The most frequent haplotype was common to all the wild fish species except whiting from which it differed by three nucleotides (<1%). The nucleic acid sequences are the consensus of VHSV from cell cultures of an unknown number of VHSV infected fish in each VHS positive pools and this constrains further inference regarding the amount of VHSV variation between VHS positive wild fish.

10.6 Comparison of VHSV Isolates Recovered from Sites and the Wild

The number of nucleotide sequence differences between VHSV haplotypes from stocked wrasse and wild species (three to nine nucleotides) is within the range of variation between haplotypes from stocked wrasse (one to 10 nucleotides). Nucleic acid sequences for the six haplotypes were subjected to a phylogenetic analysis using maximum parsimony as previously described ^[39] . The association between the wild fish and stocked wrasse haplotypes is uncertain (Figure 4) and more sequence data would be required to resolve this.