Rapid detection of human pathogenic orthobunyaviruses.

Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10(4) to 10(1) molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 x 10(7) OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies.

Infection of Israeli culicoides with African horse sickness, blue tongue and akabane viruses.

Type 9 African horse sickness virus and type 4 bluetongue virus multiplied to a high titre in an Israeli strain of Culicoides puncticollis after intrathoracic inoculation. Akabane virus persisted for at least 10 days in this midge after intrathoracic inoculation but with little evidence of virus multiplication. All 3 viruses failed to multiply in C. puncticollis after ingestion by the oral route and all were inactivated by 4 days post infection. Five other species of Israeli Culicoides supported multiplication of bluetongue virus after intrathoracic inoculation.

An attenuated strain of Akabane virus: a candidate for live virus vaccine.

An attempt was made to attenuate the high virulent OBE-1 strain of Akabane virus by adaptation to low temperature. In it the virus was subjected to passage through HmLu-1 cell cultures at 30 degrees C. Cloning was carried out on the virus which had undergone 20 passages through these cultures to select a strain adapted to low temperature. Finally, ten clones were obtained. As a result, nine strains of clone in which virus replication was poor in HmLu-1 cell cultures at 40 degrees C were obtained. Of them, five strains of clone produced uniform plaques. Of these strains, one, or the TS-C2 strain, was selected. It was considerably lower both in peripheral infectivity to suckling mice and in intracerebral infectivity to 3-week-old mice than the OBE-1 strain. Calves and pregnant cows inoculated with the TS-C2 strain by the intracerebral, intravenous, or subcutaneous route were free from pyrexia, leukopenia, and viremia. Virus recovery was negative from various organs and fetuses. All the animals inoculated, however, were found to have neutralizing antibody produced. The results mentioned above suggested that the TS-C2 strain might have been so attenuated as to be available as a candidate strain for a live virus vaccine.

The growth of akabane virus in chicken embryos.

Akabane virus produced either death or developmental abnormalities including arthrogryposis in chicken embryos inoculated by the yolk sac route when four days old. The effects depended on the dose of virus. Yolk sac inoculation of seven-day-old embryos did not result in abnormal embryos, but some chickens were viraemic when they hatched. Inoculation of older embryos intravenously, into the allantoic cavity, or onto the chorioallantoic membrane had no effect on the embryo and akabane virus was not recovered.