Expression and characterization of the two outer capsid proteins of African horsesickness virus: the role of VP2 in virus neutralization.

African horsesickness virus (AHSV) is a gnat-transmitted member of the Orbivirus genus of the Reoviridae family. The virus has a genome of 10 double-stranded RNA species (L1-L3, M4-M6, S7-S10). The L2 and M6 genes of AHSV serotype 4 (AHSV-4) which encode the outer capsid proteins VP2 and VP5, respectively, were inserted into recombinant baculoviruses downstream of the baculovirus polyhedrin, or p10 promoters. Recombinant baculoviruses expressing VP2, VP5, or VP2 and VP5 proteins of AHSV-4 were isolated. The expressed AHSV proteins were similar in size and antigenic properties to those of viral AHSV-4. Expressed VP2 and VP5 proteins were purified to homogeneity and utilized to differentiate sera from vaccinated and infected horses. Antigens were also used to determine whether any other AHSV serotypes are related to AHSV-4. The results indicated that AHSV-4 is distantly related to some serotypes (e.g., AHSV-2, -6, and -9) but not to others (e.g., AHSV-5 and -7). Hyperimmune monospecific antisera raised in rabbits with purified VP2 neutralized the infectivity of a virulent strain of AHSV-4 isolated from an infected horse during a recent outbreak of the disease in Spain.

Further studies on the efficacy of an inactivated African horse sickness serotype 4 vaccine.

The immunity induced by two inoculations of a commercial inactivated African horse sickness (AHS) serotype 4 (AHSV-4) vaccine was studied. No adverse reaction was observed in five horses following vaccination. Following challenge-inoculation, no clinical signs attributable to AHS, no viraemia indicating infection, and no anamnestic response was observed in the vaccinated ponies. Two control ponies developed clinical signs typical of AHS, high levels of viraemia, and died 7 and 8 days postchallenge-inoculation. The quality of immunity induced by the two-dose regimen was compared with a one-dose regimen from a previous study; in the one-dose study following challenge-inoculation, six of nine ponies were protected from clinical signs of AHS, seven of the nine vaccinated ponies developed an anamnestic response, and one pony had a viraemia about 10(3) 50% mouse lethal dose of AHSV-4 per ml of blood for 3 days following challenge-inoculation. The utility of an efficacious inactivated AHS vaccine in the control and eradication of AHS from a non-endemic area is discussed. The lack of viraemia following vaccination with an inactivated vaccine and the prevention of vector infection by animals exposed to field virus are important in the eradication of AHS.

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.

Isolation and identification of African horse sickness virus during an outbreak in Lagos, Nigeria.

An outbreak of African horse sickness involving two horse stables in Lagos, Nigeria, was investigated. Inoculation of blood from infected horses into suckling albino mice resulted in isolation of a virus which was identified as African horse sickness virus by the complement fixation test. The clinical, pathological and epizootiological findings (reported elsewhere) were consistent with African horse sickness. Potential threats of the epidemic to international horse trade are briefly highlighted.

Characterization of virulence variants of African horsesickness virus.

There are three clinicopathologic syndromes associated with African horsesickness (AHS) virus infection in horses. These different forms of AHS (pulmonary, cardiac, and fever forms) vary in the organs affected, the severity of lesions, time of onset of clinical signs and mortality rates. We have studied the effects of infection with three cell culture passaged variants of AHS virus in naive North American horses. One of these viruses, AHS/4SP, consistently caused the pulmonary form of AHS with rapid onset of severe pulmonary edema and 100% mortality. A second variant, AHS/9PI, resulted in signs and lesions typical of the cardiac form of AHS: pericardial effusion, subendocardial hemorrhage and widespread subcutaneous edema. Mortality was approximately 70%. The third variant, AHS/4PI, produced mild to subclinical disease in horses, usually expressed only as transient mild fever. No mortality occurred in horses due to infection with AHS/4PI. All surviving infected animals did, however, seroconvert with both neutralizing and ELISA-reactive antibodies. The results of these studies indicate clearly that in naive horses the form of disease expressed is a property of the AHS virus inoculum.

African horse sickness in Spain.

The aetiology, pathogenesis and epizootiology of African horse sickness (AHS) are reviewed with special reference to recent outbreaks in the Iberian peninsula. AHS is a highly fatal insect-borne viral disease of Equidae. It is caused by an Orbivirus (family Reoviridae) and nine serotypes are recognised. Outbreaks occurred in central Spain in 1987 and in southern regions of the Iberian peninsula in 1988, 1989 and 1990. All were associated with serotype 4 of the virus, whereas other occurrences of AHS outside Africa have all been caused by serotype 9. The clinical picture in the outbreaks was mainly of the acute (pulmonary) form except in 1988 when the subacute (cardiac) form of disease predominated. Several hundred horses died or were destroyed as a result of the outbreaks. Further spread was contained by a combination of slaughter of sick animals, movement controls, and vaccination which was extended over an increasingly wide area in successive years. The 1987 outbreak is believed to be associated with infected zebras imported from Africa. Possible explanations for the recurrence of disease in Spain in successive years are considered to include (a) the climatic conditions in Southern Spain, which could permit continuous vector activity, (b) the relative clinical resistance of mules and donkeys, which may permit subclinical circulation of the virus, (c) incomplete population immunity among horses due to possible gaps in the vaccination strategy.

The characterization of equine encephalosis virus and the development of genomic probes.

Equine encephalosis virus (EEV) is an orbivirus associated with a peracute illness of horses in southern Africa. The virus has now been partially purified for the first time and characterized on a molecular level. The virion is composed of 10 dsRNA segments and a protein capsid consisting of at least seven structural proteins that vary in Mr from 36,000 to 120,000. Partial clones of six of the dsRNA segments of EEV serotype Cascara were obtained and analysed for possible use as serotype-specific or group-specific probes in the detection of EEV dsRNA. Cloned fragments of genome segments 3, 8 and 10 were found to show high conservation of these segments, hybridizing to dsRNA from the six EEV serotypes under conditions that indicated more than 90% sequence homology. The genome segment 2-specific probe did not hybridize with dsRNA from any of the other EEV serotypes, suggesting that this segment encodes the serotype-specific antigen of EEV. Cross-hybridization of probes from genome segments 3 and 5 with dsRNA from bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) indicated that EEV is more closely related to BTV and EHDV than to AHSV. Both probes can be used to distinguish between EEV and AHSV dsRNA.

[Various aspects of horse sickness]. / Enkele aspecten van paardepest.

The aetiology, symptoms, diagnosis and control of African horse sickness are described. Special attention is paid to the introduction and epizootiology of the disease in Spain and its consequences in respect to the international trade of horses.

Isolation and identification of African horsesickness virus from naturally infected dogs in Upper Egypt.

African horsesickness virus was isolated from blood samples of street dogs in Aswan Province in Arab Republic of Egypt. Of six isolated "dog strain" African horsesickness viruses, three viruses designated D2, D6 and D10 have been identified as type 9 African horsesickness virus. Methods of isolation, tissue culture adaptation, serological indentification and typing are described. Horses experimentally infected with dog viruses showed febrile reaction and characteristic clinical and pathological signs of African horsesickness. Reisolation of African horsesickness virus type 9 was achieved from the horses during serial passages.