Identification and differentiation of the nine African horse sickness virus serotypes by RT–PCR amplification of the serotype-specific genome segment 2

C. Sailleau1, C. Hamblin2, J. T. Paweska3 and S. Zientara1

Agence Française de Sécurité Sanitaire des Aliments – Alfort, Laboratoire Central de Recherches Vétérinaires, 22 rue Pierre Curie, 94703 Maisons-Alfort, France1
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK2
Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, Republic of South Africa3

Author for correspondence: Corinne Sailleau. Fax +33 1 43 68 97 62. e-mail c.sailleau{at}alfort.afssa.fr


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
This paper describes the first RT–PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT–PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT–PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT–PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
African horse sickness (AHS) is an infectious but non-contagious virus disease of equidae associated with high morbidity and mortality. The causal agent, African horse sickness virus (AHSV), belongs to the genus Orbivirus in the family Reoviridae (Borden et al., 1971 ; Verwoerd et al., 1979 ) and is transmitted by species of Culicoides midges, of which Culicoides imicola is considered the most important (Du Toit, 1944 ). There are nine AHSV serotypes, which can be distinguished in virus neutralization tests (VNT) (McIntosh, 1958 ). AHS is enzootic in southern, eastern, western and central Africa and probably in Yemen. Epizootics have occurred outside these regions on several occasions (Mellor, 1994 ); for example, Spain experienced an outbreak of AHS (1987–1989) following the importation of zebras from Namibia (Rodriguez et al., 1993 ). The disease caused by AHSV serotype 4 (AHSV 4) also spread to Portugal (1989) and Morocco (1989–1991).

The AHSV genome is composed of ten double-stranded RNA segments (Oellermann et al., 1970 ; Bremer, 1976 ), which encode at least ten viral proteins. The genome segments are numbered 1–10 in order of their migration in PAGE. Seven of the viral proteins are structural and form the double-shelled virus particle. The outer capsid is composed of two major viral proteins, VP2 and VP5, which determine the antigenic variability of the AHSVs, while the inner capsid is composed of two major (VP3 and VP7) and three minor (VP1, VP4 and VP6) viral proteins (Bremer et al., 1990 ; Grubman & Lewis, 1992 ). VP3 and VP7 are highly conserved among the nine serotypes (Oellermann et al., 1970 ; Bremer et al., 1990 ). At least three non-structural proteins, NS1, NS2 and NS3, have been identified (Huismans & Els, 1979 ; van Staden & Huismans, 1991 ; Mizukoshi et al., 1992 ).

Rapid and reliable AHSV serotype differentiation is essential at the start of an outbreak to allow for the early selection of a vaccine to help control the spread of the virus. Traditionally, laboratory confirmation of the AHSV serotype has depended on the isolation of virus by inoculation into suckling mouse brain and tissue culture (BHK21 or Vero cells) and subsequent serotyping by VNT. Confirmation of virus serotype by this route takes a minimum of 2 weeks, but often longer. Such delays can have a significant adverse effect on the extent and duration of an epizootic. This paper reports the first RT–PCR method for the rapid identification and differentiation of the nine AHSV serotypes by using nine pairs of primers, each specific for a single AHSV serotype.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Nucleic acid sample preparation.
Total RNA was extracted from 500 µl AHSV-infected tissue culture fluid by using a commercial RNA+ kit (Bioprobe system), which is a modification of the acid guanidinium thiocyanate method (Chomczynski & Sacchi, 1987 ). The RNA pellets were subsequently dried and then resuspended in 15 µl double-distilled water.

{blacksquare} Selection of primers.
Six AHSV intermediate and partial-group-specific primers were designed, based on the nucleotide sequences of segment 2 from one strain of AHSV 3 (Vreede & Huismans, 1994 ) and three strains of AHSV 4 (Sakamoto et al., 1994 ; Iwata et al., 1992 ; M. Stone-Marschat, unpublished data) (Fig. 1, step 1; Table 1). These primers were used to amplify products and identify sequences that were specific for each of the nine AHSV serotypes by using RT–PCR methods similar to those described by Zientara et al. (1995) and Moulay et al. (1995) (Fig. 1, step 2). Nine reverse primers (Table 2) were designed following analysis of the nucleotide sequences of these nine products (Fig. 1, steps 3 and 4). Primers were named as follows: P represents the forward, positive strand, N represents the reverse, negative strand and the number following P or N corresponds to the starting location on the AHSV genome.



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Fig. 1. Strategy of primer selection.

 

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Table 1. Sequences of the partial-group-specific primers

 

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Table 2. Reverse primers for use in the serotype-specific RT–PCR

 
{blacksquare} Sequencing.
Amplified products were sequenced directly by using the ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS and a 373 DNA sequencer (Applied Biosystems). Sequence analyses were carried out by using the PC/gene software (Intelligenetics).

{blacksquare} Serotype-specific RT–PCR.
Nine pairs of primers were subsequently designed, each pair corresponding to a separate AHSV serotype but including a serogroup-specific (forward) primer and a serotype-specific (reverse) primer (Table 2). The forward primer P20 used with the reverse primers 1N229, 3N228, 4N353, 6N355, 7N359 and 9N228 amplifies segment 2 of AHSV serotypes 1, 3, 4, 6, 7 and 9, respectively. The forward primer P95 used with the reverse primers 2N228 and 5N358 amplifies segment 2 of AHSV serotypes 2 and 5, respectively, and the forward primer P2100 used with the reverse primer 8N2778 amplifies segment 2 of AHSV serotype 8.

Four µl denatured RNA obtained from AHSV was added to 50 µl reaction mix containing 10 mM Tris–HCl, pH 8·8, 50 mM KCl, 200 µM of each dNTP, 1·5 mM MgCl2, 0·1 mg/ml gelatine and 5 pmol of each primer pair, 5 U avian myeloblastosis virus reverse transcriptase and 3·5 U Taq DNA polymerase. The conditions were optimized for each pair of primers (Table 3). RNA was reverse-transcribed at 42 °C for 45 min and the cDNA was amplified for either 30, 35 or 40 cycles of 95 °C for 1 min, either 48, 50 or 52 °C for 1 min and 70 °C for 2 min, with a terminal extension at 70 °C for 8 min. The amplified products from each of the reaction mixtures were electrophoresed across a 2·0% agarose slab gel and stained with ethidium bromide.


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Table 3. Optimized conditions for the AHSV serotype-specific RT–PCR

 
{blacksquare} Specificity, sensitivity and validation.
The specificity of the serotype-specific RT–PCR was determined by using nine AHS vaccine viruses. AHSV serotypes 1–8 were isolated in South Africa while serotype 9 originated from Iran. These viruses had been passaged 100 times by intracerebral inoculation of suckling mice.

The sensitivity of the RT–PCR was determined by amplifying each dilution of a tenfold dilution series prepared for each of the nine AHS vaccine viruses.

A coded panel of 46 infectious and 10 formalin-inactivated AHSV reference strains and field isolates supplied by different Office Internationale des Épizooties-designated reference laboratories and from veterinary services from widely separated countries was used to validate the RT–PCR.

{blacksquare} Virus titrations.
Fifty µl of a tenfold dilution series prepared for each AHS vaccine virus (10-1–10-7) was added to separate wells of microtitre plates. Vero cells (ATTC* CCL81) were added at a concentration of 2x105 cells/ml in RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Microplates were sealed, incubated at 37 °C for 7 days and examined microscopically each day for the appearance of a cytopathic effect (CPE) characteristic of a virus infection. Virus titres were calculated by using the methods of Reed & Muench (1938) .

{blacksquare} Virus neutralization test (VNT).
The 46 live virus samples were inoculated onto confluent monolayers of Vero cells grown in 25 cm2 flasks with RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Flasks were incubated for 5 days and examined microscopically each day for 5 days for CPE.

VNTs were carried out in microtitre plates where constant amounts of each AHSV type-specific antiserum were added to a tenfold dilution series of each virus sample. The serum–virus mixtures were incubated for 1 h at 37 °C. Vero cells were then added at a concentration of 2x105 cells/ml in RPMI 1640 medium supplemented with 5% foetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. Microplates were sealed, incubated at 37 °C for 7 days and examined microscopically each day for CPE. Virus titres were calculated by using methods described by Reed & Muench (1938) . The serotype of the type-specific antiserum that reduced the virus titre by at least two log10 compared with the virus control in the absence of any AHSV type-specific antiserum was designated the virus serotype.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Specificity
The experimental conditions for use in the serotype-specific RT–PCRs were optimized (Table 3) for each pair of primers. These primers were then used with the nine AHS vaccine viruses to confirm their specificity (Table 4 and Fig. 2). Each pair of primers amplified products to a significant extent only from the homologous AHS vaccine virus strain. However, the AHSV 4 primers (P20, 4N353) did amplify a second product of weak intensity from the AHSV 2 vaccine strain (Fig. 2, lane 10).


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Table 4. RT–PCR results for the nine selected type-specific primer pairs

 


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Fig. 2. Amplification products obtained for the nine AHS vaccine viruses with the nine serotype-specific primer sets. Lanes: M, DNA molecular mass markers VI (Roche); 1–9, AHS vaccine virus serotypes 1–9; 10, the weak-intensity amplification product obtained from AHSV 2 RNA with the AHSV 4-specific primer set.

 
Sensitivity
The sensitivity of the RT–PCR was based on the virus infectivity of the nine AHS vaccine viruses used in this study and, for each serotype, was calculated to be the last dilution of a tenfold dilution series where an amplified product could be detected. These were equivalent to 20, 15, 200, 15, 1000, 150, 50, 50 and 15 TCID50 per 500 µl for AHSV 1–9, respectively.

Validation
Table 5 shows the RT–PCR serotyping results obtained for the 46 live and 10 formalin-inactivated samples. The origin and date of isolation and the confirmation of serotype by VNT are also included for each sample. Each sample was tested by using the nine different primer sets and only one product was amplified for each sample. The size of these amplified products always corresponded to that expected for a particular AHSV serotype-specific primer set. There was a perfect correlation between the serotype designations obtained by RT–PCR and by VNT. Unlike the results obtained with the AHSV 2 vaccine strain (Table 4; Fig. 2, lanes 2 and 10), the AHSV 4-specific primer set did not amplify any visually detectable product from the three samples of AHSV 2, numbered 13, 29 and 35 (Table 5).


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Table 5. Validation of the serotype-specific RT–PCR by using a coded panel of live and inactivated AHSV reference strains and field isolates

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
AHSV causes either an acute or subacute disease, which usually results in the death of susceptible horses. The appearance of disease is dependent on the concurrent presence of the insect vector, the virus and the vertebrate host. Vector and potential vector midges are known to be present in many countries and regions that either are or have been declared free of the disease, and therefore the risk of an AHSV introduction remains. Because of the high susceptibility of non-vaccinated thoroughbred and other horses and the potential for rapid spread of the virus, it is essential to confirm the identity and serotype of any AHSV as soon as possible after the initial case of an epizootic has been clinically diagnosed. This allows for the early selection of an appropriate vaccine, which is an integral part of the recommended control strategy. Introductions of virus into AHSV-free areas are invariably caused by a single AHSV serotype. However, because the vaccine is usually only administered to animals in enzootic countries, the most widely used vaccines, with the exception of AHSV 9, are multivalent. Since these vaccines are live, attenuated viruses, it is imperative that monovalent vaccines, against each of the nine AHSV serotypes, be readily available for use during an epizootic.

Laboratory confirmation of a clinical diagnosis can be achieved rapidly and directly by the serogroup-specific ELISA (Hamblin et al., 1991 ) from necropsy tissue and by the serogroup-specific RT–PCR (Zientara et al., 1995 ) from either necropsy tissue or blood. This paper describes the development and validation of the first serotype-specific RT–PCR for AHSV, which can be used with either infectious or non-infectious laboratory-adapted viruses and necropsy tissues (Sailleau et al., 1999 ). By using these assays concurrently, AHSV can be identified and serotyped within 24 h, thereby providing the opportunity to begin monovalent vaccination with minimum delay.

Genome segment 2 was chosen as the target for the development of the serotype-specific RT–PCR as this segment encodes the serotype-specific AHSV protein VP2. The published nucleotide sequences of segment 2 from one strain of AHSV 3 (Vreede & Huismans, 1994 ) and three strains of AHSV 4 (Iwata et al., 1992 ; Sakamoto et al., 1994 ; M. Stone-Marschat, unpublished data) were compared and then used to select primers that were specific for conserved genome regions. These primers amplified nine products, each specific for one of the nine AHSV serotypes. Analysis of the nucleotide sequences of these nine products enabled the selection of nine primers. Nine pairs of primers were subsequently designed for use in the serotype-specific RT–PCR, such that each pair included one partial-group-specific and one serotype-specific primer (Tables 2 and 4). These primer pairs were shown to be specific for the nine AHSV serotypes, although the primer pair directed against AHSV 4 did amplify a product weakly from the high mouse brain passage AHSV 2 vaccine virus (Table 4). However, no cross-reaction was recorded with the other three low-passage strains of AHSV 2 isolated between 1961 and 1998. The sensitivity of the serotype-specific RT–PCR for the identification of each AHSV serotype was in accordance with previously published values (Stone-Marschat et al., 1994 ; Zientara et al., 1994 ). However, the sensitivity of the RT–PCR is undoubtedly greater than reported in this study, as these results do not take account of the non-infectious virus component of the samples.

Comparative testing by serotype-specific RT–PCR and by VNT of 56 AHSV samples showed total agreement. These included 46 infectious samples isolated between 1961 and 1998 in a number of different countries, where epidemiological links would often be unlikely. The success of the RT–PCR in identifying correctly the serotype of isolates of eight AHSV serotypes that were isolated 31–37 years apart emphasizes the genetic stability of the sequenced regions from which the primers were selected. Isolates of AHSV 5, the remaining serotype, were only available from 1993 to 1995, but were also correctly identified. It was also noted that differences in passage history did not appear to interfere with the ability of the RT–PCR to serotype the viruses.

The remaining 10 samples used to validate the RT–PCR were duplicates of some of the infectious samples that had been inactivated with formalin. Being able to identify and serotype AHSV in formalin-treated tissues is of particular importance if these are the only types of sample submitted for diagnosis. This advantage could be exploited further for the retrospective epidemiological study of previous AHSV outbreaks or suspect cases by using stored histological tissues.


   Acknowledgments
 
The authors wish to thank the following for supplying the AHSV reference and field viruses: Dr J. House, USDA Veterinary Services, Greenport, Plum Island, New York; Dr R. Madekurozwa, CVL, Dept of Veterinary Services, Causeway, Harare, Zimbabwe; Dr H. Hooghuis, Laboratorio de Sanidad y Produccion Animal de Algete, Algete, Madrid, Spain and Dr El Harrak, Laboratoire Biopharma, Casablanca, Morocco.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Borden, E. C., Shope, R. E. & Murphy, F. A. (1971). Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus – a new taxonomic group. Physicochemical and serological studies. Journal of General Virology 13, 261-271.[Medline]

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Zientara, S., Sailleau, C., Moulay, S. & Crucière, C. (1994). Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT–PCR reaction from infected organs. Journal of Virological Methods 46, 179-188.[Medline]

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Received 16 July 1999; accepted 9 November 1999.