Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus.
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Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-coding sequences into a bacterial expression plasmid utilizing a beta-galactosidase fusion protein system was employed for epitope localization. A nuclease BAL 31 plasmid expression library, in which processive regions of the 3' end of the G2 glycoprotein coding sequences were deleted, allowed for approximation of the carboxy-terminal limit of the antigenic determinants. Further subcloning of limited G2 polypeptide sequences into the bacterial expression vector permitted more refined localization of the epitopes. The characteristics of the immunoreactivity of these small peptide regions (between 11 and 34 amino acids) produced in bacteria as G2-beta-galactosidase fusion proteins were similar to those of the authentic Rift Valley fever virus G2 glycoprotein. These defined antigenic determinants and their importance in virus infectivity are discussed. (+info)
Global survey of antibody to Hantaan-related viruses among peridomestic rodents.
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A global serological survey of rodents was conducted to determine the distribution and prevalence of antibody to Hantaan-related viruses, which are the causative agents of haemorrhagic fever with renal syndrome (HFRS) in man. Over 1700 rodent sera from more than 20 sites worldwide were examined by immunofluorescent antibody assay. High-titred positive sera were further tested by plaque reduction neutralization tests with prototype Hantaan virus and urban rat-associated Hantaan-like virus. Antibody-positive rodents were found in most, but not all, sites sampled. The highest antibody prevalence rates were found in Baltimore, MD, USA and Belem, Brazil, and Rattus norvegicus was the species most often found positive. Bandicota indica and B. bengalensis, species previously not recognized as hosts of hantaviruses, were also positive. Neutralization tests detected antibody in Rattus sera specific for urban rat-associated Hantaan-like virus, but failed to establish the specificity of antibody in Bandicota sera. These results indicate that Hantaan-related viruses exist beyond the currently recognized boundaries of HFRS in man and suggest that human HFRS-like disease might be occurring in other areas of the world where rodent-human contact is common. (+info)
Host-adaptive antigenic variation in Bunyaviruses.
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Bunyamwera virus and snowshoe have virus (family Bunyaviridae) were passaged up to six times through mosquito cells in culture and the resultant viruses were compared to the input, mammalian cell-passed virus using monoclonal antibodies raised against the input virus. The mosquito cell-adapted virus population consisted of mutants which were better adapted to replication in the new host than was the input mammalian cell-passed virus and were differentially susceptible to neutralization by antibody. (+info)
Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens.
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The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent.
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The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells. (+info)
La Crosse virus nucleocapsid protein controls its own synthesis in mosquito cells by encapsidating its mRNA.
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Within 24 to 48 h of La Crosse virus infection of mosquito cells, greater than 75% of the S mRNA was found to band in CsCl density gradients at the position of genome or antigenome nucleocapsids. The encapsidation of the S mRNA correlates with the repression of N protein synthesis in vivo, and the encapsidated S mRNA cannot be translated in vitro. Unlike genome and antigenome assembly, S mRNA assembly is a relatively slow process, which is not coupled to its synthesis. Within the encapsidated S mRNA population, three forms could be distinguished, those with intact primers which were or were not also assembled with N protein and those in which the primer and up to 3 template bases had been lost. We suggest that genome replication, but not transcription, is down regulated with time in mosquito cells for reasons that are unclear. The pool of unassembled N protein then increased to the point at which it began to interact with its own mRNA, as this mRNA also contains what is considered to be the assembly site, i.e., the conserved sequences at the 5' ends of all genome and antigenome chains. This lead to the assembly of the entire mRNA, except for the nontemplate primer. Some of the primers were then also assembled with N protein, whereas others were digested to produce truncated mRNAs. (+info)
Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture.
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A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells. (+info)