Studies were conducted to define the natural host range of the Korean henorrhagic fever (KHF) agent in South Korea, and to identify colonized rodents susceptibl64o this infection. Eight species of field rodents were captured in areas of Korea endemic for yKHF and their tissues were examined by immunofluorescence for the presence of KHF antigen. One hundred and fourteen of 817 Apodemus agrarius coreae captured between 1974 and 1978 had one or more positive organs. No positive organ was found in 239 rodents of the other seven species examined. Two hundred and thirty-eight specimens of Apodemus agrarius Ck jejuensis captured on Jeju Island, an area thought to be free of disease, were also negative. Attempted laboratory infection of nine species of rodents captured in the field but maintained in the laboratory was successful only in the two subspecies of Apodemus. The 46 specimens of A. a. jejuensis tested in this manner were all uniformly susceptible to infection as determined by immunofluorescence. Serial sacrifice of experimentally infected A. a. jejuensis revealed viremia of short duration terminating on day 10 postinfection. In contrast, other tissues of this animal, including lung, kidney, liver and parotid gland were positive on day 10 and remained so through the 100-day observation period. When 12 species of colonized laboratory rodents were inoculated with KHF agent five were found to develop KHF antibody by indirect immunofluorescence and two, Calomys callosus and Apodemus agrarius ningpoensis, developed detectable KHF antigen in their tissues. The first successful isolation and propagation demic areas of disease on the Korean peninsula, LA.) of the etiologic agent of Korean hemorrhagic fever was demonstrated utilizing the indirect fluores-... i (KHF) was described in a previous communicacent antibody (IFA) technique and human KHF 1 tion. The presence of KHF agent-specific antigen convalescent sera. Numerous isolations of the in frozen sections of various tissues of Apodemus agent have been made from rodents of this agrarius coreae, a vesper mouse indigenous to en-species, and successive passage of the agent in a boow-on_ subspecies, Apodemus agrarius jejuensis captured in areas free of natural disease, has been report

The etiologic agent of Korean hemorrhagic fever has been propagated in a human cultured cell line derived from a carcinoma of the lung. The cells, described as type II, alveolar epithelial, support replication of the agent and successive passages. Antigen of the Korean hemorrhagic fever agent is readily detected in infected cells by means of direct or indirect fluorescent antibody techniques. Previous attempts to propagate this agent in vitro had been unsuccessful.

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...