Development of a real time polymerase chain reaction assay for equine encephalosis virus

Highlights

• The S7 genes of 38 EEV isolates were sequenced.

• A real-time RT-PCR assay for equine encephalosis virus was developed.

• The 95% limit of detection of the EEV assay was 10^2.9 TCID₅₀.

• The assay was specific for EEV and did not detect other orbiviruses (BTV, AHSV).

Abstract

Equine encephalosis virus (EEV) is the cause of equine encephalosis. The disease is similar to mild forms of African horse sickness (AHS) and the two diseases are easily confused. Laboratory identification and serotyping of EEV is based on viral isolation in BHK-21 cells and a viral plaque inhibition neutralisation test. These procedures are time-consuming and therefore a more rapid diagnostic assay for EEV that can distinguish EEV from African horse sickness virus (AHSV) infections was developed.

The S7 (VP7) gene from 38 EEV isolates representing all seven serotypes was amplified and sequenced. A conserved region at the 5′ end of the gene was identified and used to design group-specific EEV primers and a TaqMan^® MGB™ hydrolysis probe.

The efficiency of the EEV real-time RT-PCR assay was 81%. The assay was specific, as it did not detect any of the nine serotypes of AHSV, nor 24 serotypes of bluetongue virus (BTV) and sensitive, with a 95% limit of detection of 10^2.9 TCID₅₀/ml blood (95% confidence interval: 10^2.7 to 10^3.3).

The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly.

Introduction

Equine encephalosis was first described by Sir Arnold Theiler, who described a fever in horses that simulated African horse sickness (AHS), which he called “ephemeral fever” (Theiler, 1910). Theiler differentiated the disease from AHS on clinical signs (incubation period and temperature characteristics) and transmission experiments. Equine encephalosis virus (EEV) was isolated in 1967 from a Thoroughbred mare named Cascara from the Kimberley district of South Africa. Clinical signs of the affected horse included listlessness, tightening of the muscles of the face, a high temperature and an elevated pulse rate about 24 h before death. The virus was also recovered from blood samples taken from other horses on the same farm, which had exhibited no clinical signs except a febrile reaction (Erasmus et al., 1970).

Equine encephalosis is endemic to southern Africa (Barnard, 1997, Venter et al., 1999) and the seroprevalence is more than 75% in horses and 85% in donkeys (Venter et al., 1999). In Thoroughbred horses the seroprevalence of neutralising antibodies against one or more serotypes of the EEV was 56.9% (Howell et al., 2002). Antibodies against EEV have been demonstrated in zebra and African elephant (Williams et al., 1993, Barnard, 1997).

EEV infection in horses was been reported recently in Israel, and involved approximately 150 cases with no reported mortalities (Mildenberg et al., 2009, Aharonson-Raz et al., 2011). Circulation of EEV in Ethiopia, Ghana and The Gambia has also been reported recently (Oura et al., 2012).

EEV is transmitted between equid hosts by the bites of Culicoides spp. midges (Diptera: Ceratopogonidae), specifically C. imicola, which is regarded as the main vector of EEV (Paweska et al., 1999, Venter et al., 1999). C. imicola is the most abundant vector of the Culicoides species associated with livestock in the summer rainfall region of southern Africa. The first isolation of an EEV strain from Culicoides species in South Africa was done by Theodoridis et al. (1979). Since then, C. bolitinos has also been confirmed as a vector for EEV (Paweska and Venter, 2004).

EEV is a member of genus Orbivirus in the family Reoviridae, subfamily Sedoreovirinae consisting of species such as AHSV, bluetongue virus (BTV), and epizootic haemorrhagic disease virus (EHDV) with similar structural morphology and functional properties. The genome of EEV is similar to those of other orbiviruses and consists of ten double-stranded (ds) RNA segments encapsulated by a double-layered icosahedral shell. Each of the segments codes for a viral protein, namely seven structural proteins (VP1 to VP7) and non-structural proteins (NS1, NS2, NS3/NS3a, NS4) (Mertens et al., 1984, Firth, 2008, Belhouchet et al., 2011, Ratinier et al., 2011).

There are seven serotypes of EEV. These are EEV-1 (Bryanston), EEV-2 (Cascara), EEV-3 (Gamil), EEV-4 (Kaalplaas), EEV-5 (Kyalami), EEV-6 (Potchefstroom), and EEV-7 (E21/20) (Gorman et al., 1983, Howell et al., 2002).

Most EEV infections are subclinical in nature and mild forms are confused easily with mild forms of African horse sickness virus (AHSV) infections, as both infections exhibit similar clinical signs (Howell et al., 2004). This makes diagnosis difficult and laboratory tests are needed to differentiate the diseases. There are various laboratory methods used in the diagnosis of EEV infection. Isolation of the virus is performed in baby hamster kidney (BHK) cells, suckling mice brains, or embryonated hen's eggs (Erasmus et al., 1970). The virus is serotyped by the plaque inhibition neutralisation assay (Quan et al., 2008). A serological group-specific, indirect sandwich enzyme-linked immunosorbent assay (ELISA) is available for the detection of EEV antigen (Crafford et al., 2003). Tests for antibody detection include complement fixation (CF), agar gel immunodiffusion (AGID) or indirect immunofluorescent antibody (IFA) tests (Howell et al., 2004). The disadvantages of these methods are that they are time-consuming and only provide a retrospective diagnosis.

To date, no reverse transcription polymerase chain reaction (RT-PCR)-based assay for the detection of EEV nucleic acid has been described. Real-time RT-PCR provides several advantages over the use of conventional PCR and ELISA, including rapid turn-around with high analytical specificity, sensitivity and a reduced risk for contamination. As the clinical signs of AHSV and EEV infections in equines may be difficult to distinguish a rapid and reliable diagnostic real-time RT-PCR assay for EEV is needed to for rapid diagnosis of this infection.

This paper describes the development and optimisation of a real-time RT-PCR assay for the sensitive and specific detection of EEV in samples from horses infected naturally with EEV. This was accomplished by sequencing the S7 (VP7) gene of 38 EEV isolates representing all seven serotypes and identifying a conserved region for the design of an EEV real-time RT-PCR assay using a TaqMan^® MGB™ hydrolysis probe. Critical control parameters of the assay, as well as the repeatability, analytical sensitivity and specificity of the assay were estimated.