Origin of 1997-98 Rift Valley fever outbreak in East Africa.
Explore the source record for details and available documents.
Africa, Eastern↗
Biomedical subjects
M Bouloy
Publications and source records attributed to M Bouloy.
Recombinant Ebola virus nucleoprotein and glycoprotein (Gabon 94 strain) provide new tools for the detection of human infections.
After cloning and sequencing the glycoprotein (GP) gene of one of the Gabonese strains of Ebola virus isolated during the 1994-1996 outbreak, it was shown that the circulating virus was of the Zaire subtype. This was confirmed in this study by cloning and sequencing the nucleoprotein (NP) gene of this strain. These two structural proteins were also expressed as recombinant proteins and used in ELISA tests. NP was expressed as a His-tagged fusion protein in Escherichia coli and was purified on resins charged with nickel ions. GP was expressed by means of recombinant baculoviruses in Spodoptera frugiperda cells. Both recombinant proteins reacted positively in ELISAs for the detection of IgG antibodies in convalescent human sera from Gabon and Zaire. The difference in the relative titres of anti-NP and -GP antibodies was variable, depending on the sera. In addition, the recombinant NP reacted with heterologous sera from Côte d'Ivoire and was used successfully to detect IgM antibodies by mu-capture ELISA in sera from Gabonese patients.
Amino Acid Sequence↗
Emergence of subtype Zaire Ebola virus in Gabon.
Gabon has recently been struck three times by Ebola hemorrhagic fever. The first isolate originating from the 1994 outbreak has been subjected to molecular characterization of its GP and VP24 genes. Sequence analysis demonstrates that the agent, Gabon-94 virus, belongs to subtype Zaire of Ebola virus. The isolate is closely related to the Kikwit-95 isolate, and both viruses seem to have evolved from a progenitor virus different from that of the Zaire-76 isolates. The relatively close relationship of all subtype Zaire viruses isolated at different geographical locations and up to 20 years apart suggests an extreme conservation in the yet unknown natural reservoir of Ebola viruses. The level of genetic variability in the human host might be different as indicated by the comparison of isolates from a single outbreak (Mayinga-76 and Eckron-76), but needs further investigation on clinical material of patients by PCR since both isolates have different levels of passages in tissue culture.
Animals↗
Identification of the Ebola virus in Gabon in 1994.
Explore the source record for details and available documents.
Adult↗
Analysis of the 3' terminal sequence recognized by the Rift Valley fever virus transcription complex in its ambisense S segment.
A reconstituted transcription system composed of the Rift Valley fever phlebovirus (Bunyaviridae family) proteins L and N expressed via recombinant vaccinia viruses and an S-like model RNA containing the CAT gene in the antisense orientation, has been described previously by Lopez et al. (J. Virol., 1995, 69, 3972-3979). We extended the use of this in vivo system to determine the sequence at the 3' end of the ambisense S segment recognized by the transcription complex. A mutational analysis of the sequences at the 3' end of the S-like genomic or antigenomic RNA was undertaken. The data indicated that the minimal sequence required for transcription resides in the 13 first 3' nucleotides of the genomic or antigenomic RNA. In these sequences, two regions appeared crucial: the bases at positions 3 to 8 and the purine at position 13. In addition, the terminal repeat ...GU could be deleted without affecting significantly the template activity of the RNA. These data support the prime and realign mechanism proposed recently for Bunya- and Arenaviruses
Chloramphenicol O-Acetyltransferase↗
Mapping of the mutations present in the genome of the Rift Valley fever virus attenuated MP12 strain and their putative role in attenuation.
The MP12 attenuated strain of Rift Valley fever virus was obtained by 12 serial passages of a virulent isolate ZH548 in the presence of 5-fluorouracil (Caplen et al., 1985. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J. Gen. Virol., 66, 2271-2277). The comparison of the M segment of the two strains has already been reported by Takehara et al. (Takehara et al., 1989. Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus. Virology 169, 452-457). We have completed the comparison and found that altogether a total of nine, 12 and four nucleotides were changed in the L, M and S segments of the two strains, respectively. Three mutations induced amino acid changes in the L protein but none of them was located in the recognized motifs conserved among RNA dependent polymerases. In the S segment, a single change modified an amino acid in the NSs protein and in the M segment, seven of the mutations resulted in amino acid changes in each of the four encoded G1, G2, 14 kDa and 78 kDa proteins. Characterization of the MP12 virus indicated that determinants for attenuation were present in each segment and that they were introduced progressively during the 12 passages in the presence of the mutagen (Saluzzo and Smith, 1990. Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine. Vaccine 8, 369-375). Passages 4 and 7-9 were found to be essential for introduction of temperature-sensitive lesions and attenuation. In an attempt to correlate some of the mutations with the attenuated or temperature-sensitive phenotypes, we determined by sequencing the passage level at which the different mutations appeared. This work should help to address the question of the role of the viral gene products in Rift Valley fever pathogenesis.
Animals↗
Variability of the NS(S) protein among Rift Valley fever virus isolates.
Eighteen strains of Rift Valley fever (RVF) virus collected over a period of 38 years and isolated from diverse localities in Africa and from various hosts (human, animal and arthropod) were investigated by RT-PCR followed by sequencing of the NS(S) protein coding region. This region was chosen to analyse variability because, in contrast to the N protein, the NS(S) protein differs in various phleboviruses and there exists an RVF virus (clone 13) in which 70% of the NS(S) ORF is deleted, suggesting that this sequence is under a weak selective pressure. Sequence data indicated that percentage divergence among isolates ranged from 0 to 9.6% at the nucleotide level and from 0 to 9.5% at the amino acid level. Phylogenetic analysis based on the NS(S) gene revealed two major lineages: Egyptian and sub-Saharan. This led to the establishment of the relatedness between strains and insights into the NS(S) protein, the function of which is still undetermined. Alignment of the deduced amino acid sequences indicated that the cysteine residues are conserved, as are several motifs representing potential phosphorylation sites.
Animals↗
Persistent infection of mammalian cells by Rift Valley fever virus.
Infection of mammalian cells with Rift Valley fever virus (RVFV) leads generally to the production of virus and cell death. In this paper we examined the fate of Vero cells infected with three strains of RVFV and observed that, while a large proportion of cells exhibited a clear cytopathic effect (CPE), a small but significant fraction did not undergo a lytic infection but was able to proliferate and establish a persistent infection. Several independent RVFV persistently infected cell lines have been established and passaged for more than 1 year after infection with a virulent strain (ZH548) and two attenuated strains (C13 and MP12). Although the viruses used for the primary infection were plaque-purified, we do not know whether defective-interfering particles were responsible for the establishment of the persistent infection. The persistently infected cells became resistant to superinfection with RVFV but not with other viruses and shed low amounts of infectious, lytic and non-lytic virus during a limited number of passages. In all the passages tested, the three genomic segments or related products were synthesized as well as the structural nucleoprotein N and glycoproteins G1 and G2. Abnormal defective RNAs were detected, migrating faster or slower than their respective counterparts. The faster-migrating RNAs were internally deleted, some of them possessing only the very terminal part of the 5' genomic end.
Animals↗
The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules.
Overlapping cDNAs representing the complete L segment of Rift Valley fever virus were assembled, and the L protein was expressed via a recombinant vaccinia virus. The transcriptase activity of the L protein was assayed with two types of templates: natural ribonucleoproteins (RNPs) and artificial genome-like RNAs. RNPs purified in a CsCl gradient did not retain the RNA polymerase function, but the activity was restored when the L cDNA was expressed in mammalian cells via a recombinant vaccinia virus. Indeed, after transfection of transcriptase-depleted RNPs in cells infected with the recombinant vaccinia virus expressing the L protein, the mRNAs coding for the N and NSs proteins and to a lesser extent, those coding for the glycoproteins were synthesized as well as the corresponding proteins. The transcriptase activity of the recombinant L protein was then investigated by using synthetic templates containing the reporter chloramphenicol acetyltransferase gene in the antisense orientation flanked by the 3' and 5' noncoding region of the S genomic segment. Our results indicate that after transfection of the RNA templates, transcription was achieved in cells coexpressing both the L and N proteins. Together, the experiments demonstrate that the two proteins N and L are absolutely required and sufficient to reconstitute the transcriptase activity.
Base Sequence↗
Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment.
The 74HB59 strain of Rift Valley fever (RVF) virus, isolated from a human case in the Central African Republic, was shown to be composed of a heterogeneous population of viruses when plaque-purified clones were analyzed for their reactivity with monoclonal antibodies (MAbs) directed against the nucleocapsid (N) protein or the nonstructural (NSs) protein. One of these clones, C13, was of particular interest in that it proved to be avirulent in mice and hamsters, and highly immunogenic. Although C13 showed normal reactivity with a large panel of MAbs directed at the glycoproteins, it failed to react with specific MAbs or polyclonal antibodies directed at the NSs protein and with a specific MAb recognizing the N protein of the Egyptian strains. Consequently, the small RNA segment, which encodes the N and NSs proteins in an ambisense strategy, was sequenced and compared with the existing sequence of the attenuated MP-12 RVF virus strain. We found that the NSs gene contained, in addition to two conservative coding changes, a large internal deletion of 549 nucleotides that removes 69% of the open reading frame but conserves in-frame the N and C termini of the predicted translation product. In addition, the sequence revealed that the N protein of C13 contained a single amino acid change. Clone C13 replicated normally in certain cell types in vitro and in Culex pipiens mosquitoes after intrathoracic inoculation, but established abortive infections in MRC-5 human fibroblasts.
Animals↗
Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.
The sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called 'polymerase module', we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the 'polymerase module', we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.
Amino Acid Sequence↗
Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins.
We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.
Amino Acid Sequence↗
Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs.
The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha- or beta-globin mRNA.
Animals↗
Completion of the genome sequence of Rift Valley fever phlebovirus indicates that the L RNA is negative sense and codes for a putative transcriptase-replicase [corrected].
Explore the source record for details and available documents.
Base Sequence↗
Bunyaviridae: genome organization and replication strategies.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Recombinant baculoviruses expressing yellow fever virus E and NS1 proteins elicit protective immunity in mice.
Recently, we showed that yellow fever virus (YFV) E and NS1 proteins in Spodoptera frugiperda cells infected with a recombinant baculovirus are similar, if not identical to those produced during YFV infection. To study the role of E and NS1 in the induction of protective immunity against fatal YFV challenge, these viral antigens were expressed either alone or in tandem via recombinant baculoviruses Ac-E. NS1, Ac-E1 and Ac-NS1. Swiss mice were immunized with lysates of insect cells infected with the recombinant baculoviruses. Solid protection against lethal YFV encephalitis was achieved after immunization with cell lysates containing the E protein with or without the NS1 protein. Mice inoculated with recombinant protein NS1 alone were not significantly protected but showed an increased survival time. Recombinant E protein expressed alone or in tandem with NS1 elicited a low but significant level of neutralizing antibodies. Although protein NS1 synthesized by recombinant baculovirus expressing E plus NS1 was more immunogenic than that expressed alone, neither strategy induced NS1-specific antibodies with complement-mediated cytolytic activity.
Animals↗
Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells.
The cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat-labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent Mr of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV.
Animals↗
Characterization of the 5' and 3' ends of viral messenger RNAs isolated from BHK21 cells infected with Germiston virus (Bunyavirus).
The 3' ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3' end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3' ends, but were not polyadenylated. They have acquired at their 5' end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5' terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.
Animals↗