SUMMARYFour structural polypeptides of Hazara virus, an agent closely related to the Crimean-Congo haemorrhagic fever (C-CHF) viruses, were resolved by SDSpolyacrylamide gel electrophoresis. Three glycoproteins were identified (mol. wt. 84 000, 45 000 and 30 000) and were found to be associated with the virion envelope. A fourth polypeptide (mol. wt. 52000) was non-glycosylated and associated with the nucleocapsid. The structural proteins of Hazara virus differ markedly from those reported for other bunyaviruses.Hazara virus was isolated in 1964 from ticks collected in the Hazara district of West Pakistan (Begum et al., 1970a, b) and has aroused interest in recent years due to its close serological relationship with the Crimean-Congo haemorrhagic fever (C-CHF) viruses (Casals & Tignor, 1974). Both Hazara and C-CHF viruses are classified as members of the family Bunyaviridae, primarily on the basis of their morphological appearance by electron microscopy (Murphy et aL, 1973; Jelifikov~t et al., 1975;Korolev et al., 1976;Smirnova et al., 1977) and, together with others, comprise one of the many unique serogroups within the family (Porterfield et al., 1975/76), Studies of the molecular structure of these viruses have been stimulated by the desire for an effective vaccine against C-CHF. Efforts have focused on Hazara virus since this agent elicits cross-protection in mice against C-CHF virus challenge, may be safely handled in the laboratory and replicates to 10-fold higher titres in cell culture than C-CHF strains (R. S. Foulke et al., unpublished observations).Hazara virus, strain JC280, in the 8th suckling mouse brain (smb) passage was obtained from J. Casals (YARU, New Haven, Conn., U.S.A.), passaged in suckling mice and cloned from the l lth smb passage by three terminal dilution passes in BHK-21 cells. Virus was propagated by inoculation of BHK-21 cell monolayers (5 × 108 cells) with virus (0.1 p.f.u./cell) and incubation under medium 199 (Earle's) containing 1/40 normal amino acids (Gibco), 5% dialysed foetal calf serum (FCS), 0.01 M-Hepes buffer and antibiotics. Radiolabelled metabolites (New England Nuclear) were added 4 h post-infection (~H-labelled amino acids, glucosamine or uridine, 10 ~tCi/ml; ~4C-labelled amino acids, 4 ¢tCi/ml) and infected supernatants were harvested 24 h post-infection, briefly centrifuged (380 g, 10 rain) and then clarified (8000 g, 30 rain). Virus samples were concentrated by direct pelleting (SW27, 116000 g, 60 min) or by (NH4)2SO 4 precipitation (Rosato et al., 1974); each procedure yielded similar amounts of virus. Concentrates were resuspended in TNE (0.01 M-tris-HCl, 0.1 M-NaC1, 0.001 M-EDTA) and purified by equilibrium centrifugation [SW50.1, 250000 g, 60 rain for pellets; SW27, 116000 g, 4 h for (NH4)2SO4 precipitation] on two successive continuous gradients of 20 to 50% (w/v) sucrose in TNE. Gradient fractions were assayed for radioactivity by addition of Scintilute containing 10% (v/v) Scintisol (Isolab, Akron, Ohio, U.S.A.) before using a Beckman LS8000 beta counter. I...
