Molecular Typing of Epizootic Hemorrhagic Disease Virus Serotypes by One-Step Multiplex RT-PCR

Epizootic hemorrhagic disease virus (EHDV) is one of the most important viral diseases of white-tailed deer (Odocoileus virginianus) worldwide (Allison et al. 2010). Infection often results in a fatal hemorrhagic disease with high mortality that can have a significant impact on deer breeding and hunting (Sleeman et al. 2009). Epizootic hemorrhagic disease virus is closely related to bluetongue virus (BTV), which can affect deer (Cervidae), sheep (Ovis aries), and cattle (Bos taurus), with the clinical disease produced by EHDV and BTV being indistinguishable. Bluetongue virus and EHDV are in the genus Orbivirus, family Reoviridae. Both are transmitted by Culicoides midges (Stallknecht et al. 1995). Their genomes consist of 10-segments of double-stranded RNA. The translated product of the segment 2 gene (VP2) is considered as the major serotype-specific antigen (Anthony et al. 2009). A TaqMan real-time reverse transcriptase (RT)-PCR capable of detecting all eight serotypes of EHDV has been described (Clavijo et al. 2010); however, the assay was not designed to differentiate the most common North American serotypes of EHDV. Identification of the specific serotypes of EHDV in clinical samples is important to better understand the epidemiology of the disease and to facilitate the development and use of vaccines. An emerging serotype in North America, EHDV-6, is detected regularly during EHD outbreaks and in surveillance samples from white-tailed deer (Wildlife Management Institute 2012). There is increased concern that the geographic distribution of EHDV-6 in North America may be changing and that there is a potential for other EHDV serotype to emerge (Ruder et al. 2012). Therefore, it is critical to continue monitoring the distribution of endemic EHDV serotypes.

The most common method for EHDV serotype identification is virus isolation followed by virus neutralization assay (Tokuhisa et al. 1981). The method requires the use of monospecific reference sera and is time consuming and costly. A few molecular techniques were reported for the characterization of EHDV serotypes (Wilson et al. 1990; Harding et al. 1996); however, they were for EHDV serotypes 1 and 2 only and required additional steps of various endonuclease treatments, which made the test more expensive and time consuming.

We developed a rapid, cost-effective, sensitive, and specific assay for direct typing EHDV serotypes 1, 2, and 6 using a one-step multiplex RT-PCR accompanied with a high-throughput viral RNA extraction method. This method had been used to identify EHDV serotypes from 427 EHDV-positive samples during 2009–12.

We collected tissue (spleen, lung, or lymphatic tissue) or ethylenediaminetetraacetic acid (EDTA) blood samples from white-tailed deer, submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL, College Station, Texas, USA), and found EHDV positive by TaqMan real-time RT-PCR (Clavijo et al. 2010). Viral RNA was extracted by a high-throughput Kingfisher 96 magnetic particle processor (Thermo Scientific, Waltham, Massachusetts, USA) using the Ambion MagMax RNA viral isolation kit (AM1836; Life Technologies, Carlsbad, California, USA). Briefly, tissue preparation (100 µL) or EDTA blood (50 µL) were transferred to a 96-deep well plate and mixed with 400 µL of lysis/binding buffer contained RNA carrier and binding beads mixture according to manufacturer's instruction. The samples were washed once with wash buffer 1 and once with wash buffer 2. Final viral RNA was eluted with 90 µL of elution buffer.

Primers used for typing EHDV serotypes were designed using available sequences of EHDV segment 2, encoding VP2 protein of serotypes from GenBank (Benson et al. 2013). All primers, based on the high conserved region of the EHDV segment 2 of serotype 1, 2, and 6, were aligned and compared through multiple sequence alignment using ClustalW (Larkin et al. 2007) to ensure the specificity of primers to the targets. The primers were synthesized commercially. Primer sequences for EHDV-1, 2, and 6 are listed in Table 1.

A one-step multiplex RT-PCR assay was performed using a Qiagen OneStep RT-PCR kit (no. 21012; Qiagen, Valencia, California, USA); 10 pmol of each primer and 5 µL of template RNA in a 25 µL final reaction volume were used for RT-PCR reaction. A no-template control and three PCR-positive amplification controls containing RNA from EHDV-1, 2, and 6, respectively, were included. The reaction mixture consisted of 1× RT-PCR buffer, 2 mM of each dNTPs, 2.5 mM MgCl₂, 6.5 U Rnasin inhibitor, and 1 µL of enzyme mix. The RT-PCR amplification was carried out using a 9700 GenAmp PCR thermo cycler (Perkin-Elmer, Waltham, Massachusetts, USA) and consisted of initiation at 50 C for 50 min and 95 C for 10 min, followed by 45 cycles at 95 C for 20 sec, 57 C for 30 sec, and 72 C for 45 sec. The final extension was 72 C for 5 min. The amplification products were visualized by electrophoresis on 1.5% agarose gels using a fluorescent nucleic acid gel staining method (GelRed™, Phenix Research Products, Candler, North Carolina, USA).

Sanger's method of DNA sequencing was used to verify the identity of the PCR products. Briefly, PCR products were purified by QIAquick PCR purification kits (Qiagen). Cycle Sequencing kit (Life Technologies) was used for sequencing cycle reaction. The profile of sequencing cycle reaction consisted of 96 C for 1 min followed by 25 cycles of 96 C for 10 sec, 50 C for 5 sec, and 60 C for 4 min. The sequence data were collected by a 3130XL Genetic Analyzer (Applied Biosystems, Foster City, California, USA) and analyzed using sequence analysis software (Geneious 6R, Biomatters, Auckland, New Zealand).

The relative analytic sensitivity of this EHDV subtyping assay was determined by testing 10-fold serial dilutions from each known single EHDV serotype (EHDV-1, 2, and 6) RNA with initial RNA concentration of 2.4–3.2 ng/µL. The results were compared with TaqMan real-time RT-PCR assay (detection limit of 0.3 to 29fg RNA; Clavijo et al. 2010). Our one-step multiplex RT-PCR subtyping assay gave positive results for each known RNA to the seventh 10-fold dilutions in which the TaqMan real-time PCR assay had positive results with threshold cycle values of 36.42–37.28. A similar result was demonstrated by testing clinical samples. Individual sets of primers (EHDV-1, 2, and 6) can be used to confirm the EHDV serotype when a mixed infection is suspected in the initial testing by a one-step multiplex RT-PCR.

The PCR products from 107 EHDV-positive samples and three known reference RNA of EHDV serotype 1, 2, and 6 were directly sequenced to evaluate the assay's analytical specificity. All sequences were subjected to BLAST search. Based on sequence data, some genetic variations among strains within the same serotype were observed (sequence identity ≥95%); however, all serotypes identified by our one-step multiplex RT-PCR subtyping assay matched with corresponding EHDV reference serotypes after alignment with the GenBank database. In addition, 32 clinical samples confirmed as BTV positive by real-time PCR (Hofmann et al. 2008) and five North American strains of BTV isolates (BTV-2, 10, 11, 13, and 17; TVMDL) were also tested against EHDV serotype-specific primers. No cross-reaction was observed from any of these samples. The specificity of the assay was further examined by testing reference RNA of eight EHDV serotypes obtained from the Arthropod Borne Animal Disease Research Laboratory (US Department of Agriculture, Agricultural Research Service, Manhattan, Kansas, USA). No cross-reaction was found among currently existing serotypes of EHDV (Fig. 1).

Among samples tested during the 4-yr period, 76 were identified as EHDV-1 (18%), 250 were EHDV-2 (59%), 90 were EHDV-6 (21%), and 11 were mixed serotypes (2%). The data, based on sample submission histories, also displayed the seasonal variations of EHDV serotypes distribution in white-tailed deer (Fig. 2). Epizootic hemorrhagic disease virus-6 was first detected as exotic reassortant Orbivirus in 2006 (Allison et al. 2010). Recently EHDV-6 has been increasingly and regularly detected from clinical samples from white-tailed deer in Texas. Mixed infections detected in this study were mostly mixed serotypes of EHDV-1/2 or 2/6 (Fig. 3), with only one case as EHDV-1/6. Mixed-serotype infections have been reported in BTV (BTV-11/17 or 10/17) in sheep and cattle (Samal et al. 1987; Stott et al. 1987). This is the first report of mixed-serotype infections with EHDV in white-tailed deer. Our data show that mixed-serotype EHDV infection in white-tailed deer is not uncommon and provide evidence of a mechanism by which reassortment of genome segments could occur within a natural host. We also found coinfections of BTV and EHDV in EHDV-infected white-tailed deer populations (data not shown). Similar evidence was also reported in cattle (Sailleau et al. 2012).

Epizootic hemorrhagic disease virus is widespread in white-tailed deer. The EHDV infection is attributed to serious epidemics in wild deer populations with economic impact on deer breeding and captive hunting. Diagnostic testing can provide important information for mapping distribution of EHDV outbreaks, estimating infection and mortality rates, and monitoring changes in deer populations. The one-step multiplex RT-PCR assay described here is a simple, sensitive, specific method that can simultaneously identify each EHDV endemic serotype. The assay is an efficient tool for molecular typing of EHDV serotypes and is useful in studying serotype distribution, emergence, and evolution of EHDV.

We thank William C. Wilson in USDA for providing 8 serotypes EHDV RNA for validating this study. We thank Jennifer Meier and the Molecular Diagnostic section at Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for technical assistance, the Information Technology section in TVMDL for their technical support, and the Virology section for providing BTV references strains. We thank Roger Parker and Pam Ferro at TVMDL for reviewing the manuscript and the veterinarians and wildlife professionals for providing samples for this development. This work was supported by funds from TVMDL.