Rift Valley fever virus

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.     

Batai and Ngari viruses: M segment reassortment and association with severe febrile disease outbreaks in East Africa

Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.     

Iquitos virus: a novel reassortant Orthobunyavirus associated with human illness in Peru

Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of "Oropouche fever" during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.     

Crimean-Congo hemorrhagic fever virus genomics and global diversity

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high case fatality that occurs in Africa, Europe, the Middle East, and Asia. The complete genomes of 13 geographically and temporally diverse virus strains were determined, and CCHF viruses were found to be highly variable with 20 and 8%, 31 and 27%, and 22 and 10% nucleotide and deduced amino acid differences detected among virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments, respectively. Distinct geographic lineages exist, but with multiple exceptions indicative of long-distance virus movement. Discrepancies among the virus S, M, and L phylogenetic tree topologies document multiple RNA segment reassortment events. An analysis of individual segment datasets suggests genetic recombination also occurs. For an arthropod-borne virus, the genomic plasticity of CCHF virus is surprisingly high.     

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality.Cases are reported in several countries in Africa,Europe,the Middle East,and Asia.Phylogenetic analyses based on the virus S (nucleocapsid),M (glycoprotein),and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation.In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of the...     

Genome Sequence Analysis of In Vitro and In Vivo Phenotypes of Bunyamwera and Ngari Virus Isolates from Northern Kenya

Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP) and large plaque (LP) and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I) that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies.     

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.     

Epidemiology and mutational analysis of global strains of crimean-congo haemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality. Cases are reported in several countries in Africa, Europe, the Middle East, and Asia. Phylogenetic analyses based on the virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation. In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples. We then analyzed the alignment using entries from AAIndex, the Amino Acid Index database, to identify amino acid mutations that performed significant changes in charge, pka, hydropathy and side chain volume. Finally, we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage. Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.     

Complete Genome Sequencing of Mosquito and Human Isolates of Ngari Virus

Ngari virus (NRIV) is a recently described, naturally occurring reassortant between two other orthobunyaviruses, Bunyamwera virus (BUNV) and Batai virus (BATV). Intriguingly, this reassortment was associated with the acquisition of heightened virulence, although the molecular basis for this is not understood. Here we report the first complete genome sequences of Ngari virus. We include five isolates from various geographical locations, as well as samples isolated from both mosquitos and human cases. Based on an analysis of these sequence data, NRIVs are clearly genetically distinct from all known BUNV and BATV strains but are very closely related to one another regardless of their source.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Virulence of La Crosse virus is under polygenic control.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.     

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality.Cases are reported in several countries in Africa,Europe,the Middle East,and Asia.Phylogenetic analyses based on the virus S (nucleocapsid),M (glycoprotein),and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation.In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples.We then analyzed the alignment using entries from AAIndex,the Amino Acid Index database,to identify amino acid mutations that performed significant changes in charge,pka,hydropathy and side chain volume.Finally,we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage.Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.     

Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'- and 5'-terminal sequences

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3'- and 5'-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3' and 5' NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3'- and 5'-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3' and 5' NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3' and 5' NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3' and 5' NTRs were selected.     

Creation of a recombinant Rift Valley fever virus with a two-segmented genome

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed.     

Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.

A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.     

Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein

The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreGN and PreGC precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of GN and GC, respectively. However, the resulting polypeptide defined by the N termini of GN and GC is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreGN that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreGN C-terminal cleavage events, a panel of epitope-tagged PreGN truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the GN ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreGN C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the GN glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreGN allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.     

Creation of a nonspreading Rift Valley fever virus

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.