Isolation of Kaeng Khoi virus (KKV) from Eucampsipoda sundaica bat flies in China.

A virus strain (WDBC1210) was isolated from specimens of bat flies (Eucampsipoda sundaica) associated with Leschenault's Rousette (Rousettus leschenaultii) in the China-Myanmar border area of Yunnan Province, China. Both BHK-21 and VeroE6 cells infected with WDBC1210 showed evident cytopathic effects (CPE), and the highest propagation titer was 1×105.6. The virus particles were spherical, 70nm in diameter. Virus plaques could be observed in BHK-21 cells. The whole genome of WDBC1210 contained three RNA segments: the small gene (S), 975 nucleotides long; the medium gene (M), 4568 nucleotides long; and the large gene (L), 6866 nucleotides long. The nucleotide homologies of the S, M, and L genes between WDBC1210 and the original isolate of Kaeng Khoi virus (KKV; PSC-19 strain) were 96.9%, 94.1%, and 95.2%, respectively. Phylogenetic analyses based on the S, M, and L segments indicated that WDBC1210 belongs within the same clade as the KKV strain PSC-19, a member of the Bunyaviridae family. This is the first report on the isolation of KKV from bat flies (Eucampsipoda sundaica) and from an inland area, nearly 2000km north from the original isolation site of KKV in Thailand, suggesting that KKV virus not only has a diverse set of vectors, but also a wide geographic distribution.

Immunohistochemical study of age-dependent brain lesions in mice infected intracerebrally with Kasba (Chuzan) virus.

When Kasba (Chuzan) virus (an orbivirus) was injected intracerebrally into 1-, 2- or 4-week-old mice, non-purulent necrotizing encephalitis developed and the mice showed nervous symptoms and became moribund. The necrotic lesions were more severe in younger animals. In 1-week-old mice, viral titres rose until 7 days post-infection, while in 2- and 4-week-old animals the titres reached a peak on day 3 and then declined gradually. Glial fibrillary acidic protein-positive astrocytes increased in the white matter, hippocampus and subpial area of the cerebral cortex of infected animals, and lectin-RCA-1-positive cells, thought to be microglial cells, increased in the necrotic lesions. The number of these glial cells increased even after viral titres had declined. In this study there were no survivors in any age group, but survival time increased with age.

Encephalopathy in suckling mice infected with Kasba (Chuzan) virus.

Kasba (Chuzan) virus (an orbivirus), strain K-47, produced encephalopathy with severe necrosis in suckling mice inoculated intracerebrally. On day 3 after inoculation with 10(3)TCID50, the mice showed severe focal encephalomalacia and meningitis. On day 4, necrosis had spread to the midbrain, cerebellum and spinal cord. From one day after inoculation, virus was recovered from the brain and the titre rose over the next 3 days. Immunohistochemical examination demonstrated viral antigens in the cytoplasm of both degenerate and intact neurons, and ependymal cells in or around necrotic lesions. The study indicated that the virus has an affinity for immature nerve cells in the brains of suckling mice and causes primary encephalomalacia. Since the lesions resembled those of the hydranencephaly-cerebellar hypoplasia syndrome in calves (Chuzan disease), the system described should prove useful in studies on pathogenesis.

Characterization of JKT-7400, an orbivirus which grows in Aedes albopictus mosquito cells: evidence pointing to a minor virion protein, VP6, as the RNA guanylyltransferase.

JKT-7400 virus, an orbivirus originally isolated from Culex mosquitos, was plaque purified and adapted to Aedes albopictus mosquito cells. Conditions which enhance viral cytopathic effect and optimize plaque formation are described. In contrast to bluetongue virus, the prototype orbivirus, no replication of JKT-7400 virus in vertebrate cells was observed. The core particle of JKT-7400 virus contains 10 segments of dsRNA and three minor proteins, VP1, VP4, and VP6. The inner shell contains two major proteins, VP2 and VP7, and the outer shell consists of the other two major proteins, VP3 and VP5. Evidence is presented suggesting that the viral protein associated with the capping of virus mRNA, i.e., the guanylyltransferase, is VP6, one of the core proteins.

Sensitive plaque assay and propagation of Chuzan (Kasba) virus, a Palyam serogroup orbivirus, in BHK-21 cells.

Various factors influencing plaque formation of Chuzan virus in BHK-21 cell monolayers were studied and a practical method for plaque assay was developed. On addition of trypsin (5 micrograms/ml) and/or diethylaminoethyl (DEAE)-dextran (50 micrograms/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 micrograms/ ml DEAE-dextran was added to the diluent and overlay medium, plaques were produced in about 10-fold higher numbers than without trypsin and DEAE-dextran. Based on these results, a practical plaque assay method for Chuzan virus was established. Using this method, one-step growth of Chuzan virus was performed at an input multiplicity of 25 plaque-forming units (PFU) per cell. Cytopathic effects were first observed at 7.5 h post-inoculation (p.i.), and were complete at 12 h p.i. The titre of cell-associated virus, after gradual decline during the first 3 h of incubation, showed a rise within 4.5 h p.i. and a rise to a plateau of 10(6.3)PFU/0.2 ml at 12 h p.i. By indirect immunofluorescence, virus-specific antigen was detected in the cytoplasm of the cells at 4.5 h p.i., and all the cells fluoresced at 6 h p.i. Haemagglutination activity was first detected in infected whole cultures at 7.5 h p.i. reaching a plateau of 1:64 at 15 h p.i. Plaque formation and haemagglutination by the virus were specifically inhibited by antisera against the original and the plaque-cloned virus.

Polypeptides induced in chick embryo cells by Kemerovo virus.

Fifteen polypeptides induced by Kemerovo virus were detected in chick embryo cells (Mr 140, 98, 89, 72, 65, 62, 57, 54, 50, 47, 43, 41, 39, 31 kD, and 30 kD). Nine of them, namely the 140, 98, 65, 62, 57, 54, 50, 47 kD, and 41 kD polypeptides were also found in the partially purified virus. However, the latter contained also considerable amount of host cell proteins, predominantly the 205 kD, 45 kD, and 37 kD polypeptides. In the electron microscope the spherical viral particles exhibited a poorly defined surface structure of a diameter of 70-75 nm.

Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae).

Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41 degrees C as the nonpermissive temperature and 36 degrees C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus.

Viral superinfection in cells carrying an arenavirus and/or a togavirus.

Four lines of the same L-cell clone were transferred 60 times in parallel: uninfected cells, a line carrying lymphocytic choriomeningitis virus (LCMV), another one carrying tick-borne encephalitis virus (TEV) and one carrying both viruses. In double persistency, LCM and TEV were suppressed and stimulated, respectively. Cell multiplication rates were comparable in all four lines. Single LCMV persistence caused marked resistance of L cells to superinfecting viruses from various taxonomic groups, but this phenomenon was abolished or even reversed to increased sensitivity in the cell line with co-persisting LCMV plus TEV.