Coding strategy of the S RNA segment of Dugbe virus (Nairovirus; Bunyaviridae).

The S RNA segment of Dugbe (DUG) virus (Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141-148). The 49.4-kDa product was expressed as a fusion protein with beta-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3' to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Physical and biological properties of Nairobi sheep disease virus.

Thermal and pH stability of Nairobi sheep disease (NSD) virus were studied. The 180th mouse brain passage lost infectivity at a higher rate than "wild" virus at 4 degrees C. At 37 degrees C and neutral pH, "wild" virus again was more stable than cell culture and mouse brain attenuated strains with half-life periods of 104, 87 and 51 min, respectively. At 0 degrees C the cell culture attenuated virus was most stable at pH 7.4 with an estimated half-life of 164 h. The density of the virus in sucrose gradients came to 1.195 g cm -3. Metabolic growth inhibition studies using a halogenated nucleoside, and staining of RNase and DNase-treated infected cell cultures with acridine orange, indicated that NSD virus has a single stranded RNA genome. The growth of the cell culture adapted virus was assayed in monolayers of BHK21/13 cells at low multiplicity of infection. Cell-associated virus (CAV) was first detected at 6 h post-inoculation (PI). The titre increased rapidly until CPE appeared at 48 h and declined after 72 h PI. Cell-free virus (CFV) was first detected at 10 h PI. The titre of CFV increased up to 72 h, but on average was two log units less than the CAV titre.

Nairobi sheep disease in Kenya. The isolation of virus from sheep and goats, ticks and possible maintenance hosts.

Nairobi sheep disease was seen principally upon movement of susceptible animals into the enzootic areas. This occurred most frequently for marketing purposes near the main centres of population. Other outbreaks followed local breakdowns in tick control measures. The disease did not occur in epizootic form during the period under consideration. Nairobi sheep disease was isolated from pools of Rhipicephalus appendiculatus but not from many pools of other tick species. No virus was isolated from the blood or tissues of a range of wild ruminants and rodents.

The laboratory diagnosis of Nairobi sheep disease.

The laboratory methods available for the isolation and identification of Nairobi sheep disease virus have been compared. The results show that inoculation of tissue culture (BHK 21 C 13) with suspensions of infected organs or plasma followed by fluorescent antibody tests on coverslip preparations gave the quickest means of identification. This test did not depend on the production of a cytopathic effect. Primary isolation of the virus in infant mouse brain and identification either by fluorescent antibody methods or by complement fixation with antigen prepared from the mouse brain offers a slightly more sensitive isolation system and would be recommended where no tissue culture facility exists.