Detecting bunyaviruses of the Bunyamwera and California serogroups by a PCR technique.
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Many bunyaviruses of the Bunyamwera and California serogroups are medically important human pathogens. The development of an effective technique to detect the viruses by using molecular biologic tools, such as PCR, improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, we evaluated eight pairs of primers for reactivity with 44 viruses of the genus Bunyavirus, using a reverse transcriptase PCR technique. With a pair of serogroup-specific primers we designed, all viruses of the serogroups tested could be detected. Further, virus-specific primer pairs were identified for California encephalitis virus, Jamestown Canyon virus, La Crosse virus, and snowshoe hare virus for use in North America. Using this technique, we could detect one La Crosse virus-infected mosquito in a pool of 100 mosquitoes with undetectable plaque titers. (+info)
The S RNA genomic sequences of Inkoo, San Angelo, Serra do Navio, South River and Tahyna bunyaviruses.
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The complete nucleotide sequences of the small (S) genomic RNA segments of five California (CAL) serogroup bunyaviruses (two Inkoo virus strains, San Angelo virus, Serra do Navio virus, South River virus and Tahyna virus) were determined. In agreement with previously published data concerning CAL serogroup viruses, the nucleocapsid (N) and non-structural (NSs) proteins were encoded in over-lapping open reading frames (ORFs). All N protein ORFs were 708 nucleotides in length and encoded a 235 amino-acid gene product. The NSs ORFs were either 279 or 294 nucleotides in length, which would encode 92 or 97 amino-acid proteins, respectively. Comparative analysis of the nucleotide sequences and amino acids corresponding to the nucleocapsid protein resulted in a predicted relationship among these viruses that generally agreed with those determined by serology. The only exception was Inkoo virus, where comparisons based on the S RNA sequence and partial M RNA sequence suggest that this virus is more similar to Jamestown Canyon virus of the Melao complex than it is to viruses such as Tahyna and La Crosse viruses of the California encephalitis complex. (+info)
Inkoo and Tahyna, the European California serogroup bunyaviruses: sequence and phylogeny of the S RNA segment.
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Inkoo (INK) and Tahyna (TAH) viruses, European representatives of the California serogroup (CAL), genus Bunyavirus, family Bunyaviridae, are transmitted by mosquitoes and frequently infect man. The S segments of INK and TAH prototype strains were amplified, cloned and sequenced. INK S consists of 986 and TAH S of 977 nucleotides (nt) coding for a nucleocapsid protein of 235 amino acids (aa) and, in an overlapping reading frame, for a nonstructural protein of 92 or 97 aa, respectively. By S segment sequences and phylogenetic analysis INK was seen to be most closely related to Jamestown Canyon virus, isolated in the USA (92.4% nt and 96.6% aa identity), which is currently classified in a different subcomplex within the CAL viruses. TAH was genetically closest to Lumbo virus, isolated in Mozambique (89.0% nt and 94.1% aa identity). The data suggest that genetic variation within the CAL viruses is less related to geographical distance than to similarity in ecological cycles. (+info)
Adsorptive endocytosis of California encephalitis virus into mosquito and mammalian cells: a role for G1.
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The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAba, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells. (+info)
Structural proteins of La Crosse virus.
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Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm). (+info)
Segmented genome and nucleocapsid of La Crosse virus.
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La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular. (+info)