Molecular epidemiology of African horse sickness virus based on analyses and comparisons of genome segments 7 and 10.

This paper describes a method to rapidly identify African horse sickness virus (AHSV), using a single tube reverse transcription polymerase chain reaction (PCR). This method was used to amplify cDNA copies of genome segments 7 and 10 from several different AHSV strains, of different serotypes, which were then analysed by sequencing and/or endonuclease digestion. AHSV VP7 (encoded by genome segment 7) is one of the two major capsid proteins of the inner capsid layer, forming the outer surface of the core particle. VP7 is highly conserved and is the major serogroup specific antigen common to all nine AHSV serotypes. Digestion of the 1179 bp cDNA with restriction enzymes, allowed differentiation of several strains of different serotypes and identified six distinct groups containing AHSV-1, 3, 6 and 8; AHSV-2; AHSV-4; AHSV-5; AHSV-7; and AHSV-9. Differences were detected between wild type viruses and vaccine strains that had been attenuated by multiple passage in suckling mouse brain or in tissue cultures. RFLP analysis was also used to study variation the 758 bp cDNA copies of AHSV genome segment 10, which encodes the two small non-structural membrane proteins NS3 and NS3a. In this way it was possible to distinguish each of the strains tested, except AHSV 4 (USDA) and AHSV 9 (USDA). However, these isolates could be distinguished by RFLP analysis of genome segment 7 cDNA. Using sequence analysis of genome segment 10 we were able to classify the virus isolates into three groups: AHSV-1, 2 and 8; AHSV-3 and 7; AHSV 4, 5, 6 and 9. These studies confirmed that the virus which first appeared in central Spain in July 1987, subsequently spread into northern Morocco in October 1989.

Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.

Detection of African horse sickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay.

A reverse transcription-polymerase chain reaction (RT-PCR) assay followed by dot-blot hybridisation was used to detect African horse sickness virus (AHSV); the primers employed amplified the S7 gene that encodes the VP7 protein. The RT-PCR assay was compared with virus isolation for detecting AHSV in blood samples form horses experimentally infected with AHSV-4 and AHSV-9. The influence of sample storage and transportation and the effects of two anticoagulants (EDTA and heparin) were also studied. RT-PCR results were obtained within 48 hours as opposed to a minimum of 15 days for virus isolation. RT-PCR and virus isolation were equally sensitive for detection of AHSV-4. Viraemia was detected more consistently by RT-PCR than by virus isolation from horses infected with the less virulent AHSV-9 isolate except from one animal in which virus was detected only by virus isolation. The sensitivity of virus isolation was increased by passaging samples five times. This study indicates that RT-PCR is a sensitive and rapid method for use in the face of an outbreak of this serious disease, although it has also some limitations as a diagnostic technique.

Nucleotide sequence comparison of the segments S10 of the nine African horsesickness virus serotypes.

Segments 10 (S10) of the double-stranded RNA (ds RNA) genomes from African horsesickness virus (AHSV) serotypes 2, 4, 5, 6 and 7 were cloned and sequenced. Direct sequencing of previously reverse transcribed amplified (RT)-PCR segments S10 was also performed. Nucleotide sequences of two strains (the virulent Moroccan strain and a vaccine strain) of the same serotype (4) were determined. Sequences of the viral serotypes were analysed and compared to each other. Two in-phase ATG codons were conserved in the S10 of AHSV-2, AHSV-4, AHSV-5, AHSV-6 and AHSV-7 and were considered candidate translation initiation codons of NS3 and NS3A respectively. A close relationship between serotypes 4, 5 and 9 and between serotypes 3 and 7 were described. Closer relationships of serotype 2 to the 1 and 8 group on one hand and of serotype 6 to the 4, 5 and 9 group on the other hand were observed.

Differentiation of African horse sickness viruses by polymerase chain reaction and segments 10 restriction patterns.

This paper describes a single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV). The genomic segments 10 of viruses of the 9 AHSV serotypes were amplified. The 758bp products were digested to completion by restriction enzymes. The restriction fragment length polymorphisms of segments 7 and 10 cDNA allowed the differentiation of the nine serotypes.

Detection of African horse sickness viruses by dot-blot hybridization using a digoxigenin-labelled probe.

In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other equine virus or orbivirus isolates. The sensitivity and specificity of this probe, together with the simplicity and rapidity of this technique, suggest that a non-radioactive dot blot assay may be useful as a method for the routine and rapid diagnosis of viral infections.

Application of the polymerase chain reaction to the detection of African horse sickness viruses.

The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.

Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs.

A single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method is discussed. Application of the RT-PCR method should improve detection and shorten the time required to confirm a clinical diagnosis of AHSV infection.

Diagnosis and molecular epidemiology of the African horse sickness virus by the polymerase chain reaction and restriction patterns.

African horse sickness is a viral disease caused by an orbivirus belonging to the Reoviridae family. This paper describes a polymerase chain reaction (PCR) for amplifying segments 7, which encode for VP 7, a protein common to the 9 known serotypes of this virus. A reverse transcription step is necessary before amplification. No amplified product could be observed in cell cultures infected with other equine viruses. The amplified DNAs were digested to completion by 8 different restriction enzymes. The restriction fragment length polymorphisms allowed the differentiation of the group of serotypes AHSV-1, 3, 6, 8 and the viruses AHSV-2, AHSV-4, AHSV-5, AHSV-7 and AHSV-9. Differences could also be described between vaccinal strains of the same serotype produced in cell cultures or in brains of suckling mice.