Global distribution of white spot syndrome virus genotypes determined using a novel genotyping assay

White spot disease (WSD) is a serious panzootic affecting prawn aquaculture. The disease is caused by white spot syndrome virus (WSSV), a large double-stranded circular DNA virus and currently the only member of the genus Whispovirus and family Nimaviridae [1]. In intensive aquaculture systems, mortality can be rapid (3-10 days) and occurs at a rate of up to 100% [2, 3]. The economic cost of the disease on the prawn aquaculture industry worldwide has been estimated at up to US$15 billion since the emergence and initial spread of the disease, increasing at a rate of US$1 billion annually, equating to approximately 10% of global prawn production [4].

The first reports of white spot disease in penaeids were in mainland China and Taiwan in 1992 [2, 5, 6]. By the end of the decade, the disease had spread to Korea [7], Japan [8, 9], and throughout South-East Asia (Vietnam, Thailand, Malaysia, Indonesia) and India [10, 11]. This rapid proliferation of the disease was most likely through transboundary movement of infected animals. In the 1990s the disease was reported also in United States of America [12] and by 1999 WSSV was detected in Central and South America. WSSV was found in wild prawns in retrospective analysis by in situ hybridisation of histology samples from Ecuador from 1996, prior to disease reported in 1999 [13]. In 2001, WSSV was reported also in prawn farms of Khuzestan on the northern Persian Gulf coast in Iran and over several other Iranian provinces over the next decade [14]. In 2010, WSSV was observed in Saudi Arabia, greatly affecting the Penaeus indicus industry until 2013, when the industry was replaced with specific-pathogen-free (SPF) and specific-pathogen-tolerant (SPT) Penaeus (Litopenaeus) vannamei and the disease was considered eradicated [15]. By 2012, WSSV was reported to be endemic in wild penaeids from the coast of Iraq [16].

In addition, there have been incursions of the disease to other prawn-farming regions of the world where containment and biosecurity measures have resulted in reports of eradication or subsequent low levels of sporadic disease, including Spain, Mozambique and Madagascar [11]. Transmission to wild crustaceans was observed in Darwin (Northern Territory, Australia) in 1999 following inadvertent feeding of imported prawns to crustaceans in a research facility that discharged water into Darwin Harbour. The harbour and surrounding waters were declared free of WSSV in 2000, and it was considered that the infection was at a sufficiently low level as to be unsustainable [17].

In November 2016, WSSV was identified following the onset of disease in a prawn farm near Brisbane, Queensland, Australia. Previously, white spot disease had not been diagnosed in Australian prawn farms, and Australia was considered to be free of the virus (despite the aforementioned Darwin incident). The disease showed rapid spread and high mortalities, affecting seven farms by February 2017. A low number of wild-caught crustaceans in the adjacent Logan River and in Moreton Bay also tested positive for the virus. In 2018, a large surveillance program of wild crustaceans in Moreton Bay detected considerable numbers of test-positive animals in the north of Moreton Bay, but not in the south near the mouth of the Logan River (K. Beattie, personal observation).

The prawn farming industry in Queensland is valued at approximately AU$87 million annually (http://​www.​daf.​qld.​gov.​au), and the potential impact of establishment of endemic white spot disease would be severe. Hence, an important factor within the incursion investigation is the epidemiological analysis of the source, the patterns and the movement of the virus based upon strain identification and differentiation. The data are used to shape biosecurity decisions and inform risk analysis to help prevent future incursions of this and other exotic penaeid pathogens.

We recently published the whole genome sequence of WSSV-AU [18], the virus detected in a sample from the first Queensland property identified as infected with white spot disease. Analysis of the genome for genomic markers previously reported by Marks et al. (2004) [19] to show variation among WSSV strains was unable to associate the virus in South East Queensland with any previously reported genotype. The differing types of the loci hindered cumulative analysis or testing of high sample numbers, and the complexity of the markers limited their utility as a large-scale epidemiological tool. Although the scientific literature contains many reports from endemic regions with local studies using only one or a few of these markers, these were of limited epidemiological use, as many alleles were reportedly common to multiple regions. It was concluded that alternative markers were required for epidemiological tracing [18].

Examination of the WSSV-AU sequence aligned with other published WSSV genome sequences showed a number of variations in copy number of triplet-base motifs (short tandem repeats, STRs) in a similar way to microsatellite polymorphism. STRs have been used frequently to identify individuals, evolutionary processes, and kinships and for population/cluster analysis in eukaryotes [20], prokaryotes [21], and some of the larger viruses [22]. The high levels of polymorphism associated with STRs, the speed of processing, and the potential to simultaneously isolate and study large numbers of loci provide a capacity for detecting comparable differences among different levels of hierarchal clustering. Here, we describe the application of 34 STRs observed in WSSV to achieve a sensitive genotyping method. Furthermore, we demonstrate the utility of the genotyping technique to discriminate WSSV strains between, within and among the principal WSSV-affected regions of the world.

The alignment of the WSSV-AU sequence (MF768985) with Taiwanese (AF440570), Thai (AF369029), Chinese (AF332093) and Korean (JX515788) WSSV sequences was examined using Integrative Genome Viewer 2.3.98 [23, 24] to manually identify potential trimeric STR markers with variation in copy number in at least one of these reference sequences compared to WSSV-AU. Primers in the conserved sequence flanking these loci were designed using BatchPrimer3 [25], pre-selecting amplicon size less than 500 bp and with as much consistency in melting temperatures as possible among all primers. Notional size ranges for the loci were estimated up to a 30-base increase or decrease compared to the alleles observed in WSSV-AU, and hypothetical fragments were analysed in Multiplex Manager [26] to design a 4-dye multiplexed analysis protocol with as few reactions as possible while avoiding primer cross-reactivity or overlapping of fragments labelled with same dye, and using common primer annealing temperatures. Primers were redesigned as necessary to minimise the number of reactions needed. Subsequently, primers were commercially synthesised with the forward primer of each pair labelled with one of four fluorescent dyes compatible with the 3500xL Genetic Analyser (G5 dye set, Life Technologies, Thermo Fisher), leaving LIZ as the label of the commercially prepared size standard ladder. Primer sequences are listed in Table 1.

DNA was extracted, using a DNeasy Blood and Tissue Kit (QIAGEN), from the same prawn used to determine the sequence of WSSV-AU. For preliminary optimisation each STR locus, amplification was performed as a monoplex using 7.5 µL of Multiplex Master Mix (QIAGEN), 2 pmol each of forward and reverse primer, 2.5 µL of DNA, and a volume balance with sterile nuclease-free water to 15 µL. Following initial denaturation at 94 °C for 15 minutes, the reactions were cycled 40 times at 94 °C for 30 seconds, at the estimated annealing temperatures of 54, 57 or 58 °C for 30 seconds, and 72 °C for 1 minute, with a single final extension at 72 °C for 10 minutes. The reaction products were resolved using 1.5% agarose gel electrophoresis. The presence or absence of single amplicons of the expected size and the observed relative intensity were used to optimise amplification of the loci with adjustments to the annealing temperature and the inclusion of Q-solution (QIAGEN) in the mix. These empirical results were used subsequently to fine-tune and optimise multiplexed reactions.

The final optimised method targeted 34 loci in six PCRs with further multiplexing of the amplicons into three reactions prior to resolution. The loci in each PCR are shown in Table 2. PCR mixes consisted of 7.5 µL of Multiplex Master Mix (QIAGEN), 1.5 µL of Q solution (QIAGEN) where used, 2 pmol of each primer, 2.5 µL of DNA, and a volume balance of sterile nuclease-free water to 15 µL per reaction. Following initial denaturation at 94 °C for 15 minutes, the reactions were cycled 40 times at 94 °C for 30 seconds, at the respective annealing temperature (see Table 2) for 45 seconds and 72 °C for 45 seconds, with a single final extension at 72 °C for 10 minutes. Amplicons were diluted 1 in 50 using Milli-Q water and further multiplexed by combining PCRs 1, 2 and 3 (Read1), and PCRs 5 and 6 (Read3). Read2 consisted only of PCR4. Reads 1, 2 and 3 were resolved using fragment analysis by capillary electrophoresis with a 3500xL Genetic Analyser (Life Technologies, Thermo Fisher), with fragment sizes determined by comparison with the labelled size marker (GeneScan 600, Life Technologies, Thermo Fisher) using GeneMarker (Soft Genetics).

The robustness of the optimised technique was tested based on consistency in fragment lengths in repeated tests of the same DNA sample, comparison of data from re-extracted DNA from the same sample, and comparison among three operators. The sensitivity was estimated through comparison with Biosecurity Sciences Laboratory’s (BSL) standard diagnostic PCR (optimised from Sritunyalucksana et al. [27] to accommodate laboratory conditions).

Thirty-six STR markers were identified, including some with perfect tandem repeats and some with imperfect repeats but variation in copy number between reported genome sequences. Testing for robustness showed consistency in fragment lengths among repeated tests of the same DNA extract, comparison of data from re-extracted DNA from the same sample, and comparison among three operators, with 34 markers. Two markers (WSV5 and WSV9) were discarded from the locus panels because they did not work optimally at a shared annealing temperature. The sensitivity of the genotyping was determined to be equivalent to the diagnostic PCR; STR fragments were generated from samples that had diagnostic PCR Ct values as high as 38 when tested by BSL, although the larger fragments were not always observed in samples with Cts above 35. For approximately 20% of the processed retail products, more than two thirds of the loci were not amplified, and where this occurred, even when WSSV detection PCR Cts were less than 35, this was presumably because of DNA degradation as a result of the cooking, drying or other processing.

A total of seven genotypes were observed from samples taken from infected ponds in farms and in the Logan River (LG1 to LG7, Tables 3 and 5), with the majority being of genotype LG1. The seven genotypes differed in only one or two loci. Where samples were taken from the same site or pond on different occasions, and hence tested on different occasions, the results were consistent, which further demonstrates the robustness of the allele calls. A total of twelve genotypes were observed from samples taken from Moreton Bay (MB1 to MB12, Tables 3 and 5). In 2017, two genotypes were apparent. MB1 predominated and only one sample (five individuals) showed MB2. In 2018, all MB types were observed except MB2. There was no common genotype found in both the Logan area and in Moreton Bay, with one locus (WSV24) consistently showing genotypic difference between the two areas.

A large range of alleles was observed from the samples originating from outside Queensland, as indicated by the actual allele size range shown in Table 1, compared to alleles shown for Queensland samples. Most loci were highly polymorphic, while some showed only two or three alleles globally. One locus appeared monomorphic (WSV21) and was retained in the panel as a control marker. Many samples originating from regions where WSSV is endemic showed infection with multiple genotypes, seen as more than one allele at individual loci. Where this occurred, all of the possible genotype iterations were determined, as this approach would not impede subsequent analyses that rely upon allele frequencies and distances. The allelic data are summarised in Table 6 as allele frequencies for a priori given global regions. Table 7 shows Nei’s genetic identity between the same a priori regions.