Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.     

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.     

Seven infection-specific polypeptides in BHK cells infected with Bunyamwera virus.

Virus-specific polypeptide synthesis was examined in BHK cells and Vero cells infected with Bunyamwera virus. In BHK cells, in addition to the four previously reported virus-coded proteins (L, G1, G2, and N), three other infection-specific proteins were detected. These proteins, of nominal molecular weight 50,000 (p50), 16,000 (p16), and 13,000 (p13), were not labeled in mock-infected cells, were first synthesized between 4 and 8 h after infection, and were relatively prominent among the limited number of proteins generated late in infection. In preparations of purified Bunyamwera virus from BHK cell supernatants, p16 was detected but not p50 or p13. In Vero cells infected with Bunyamwera virus, both p50 and p13 were labeled strongly. Maprik virus, a member of the Mapputta group of arboviruses, is a member of the Bunyavirus genus (S.E. Newton, unpublished data). Maprik virus did not induce the synthesis of p50, p16, or p13; however, two smaller proteins (p17 and p15) which may correspond to p16 and p13 were labeled late in Maprik infection. Our data argue that p16 is a virus-coded component of the Bunyamwera virus particle and that p50 and p13 are virus-coded, nonstructural proteins.     

Mutant identifying a third recombination group in a bunyavirus.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.     

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.     

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

Isolation of a bunyamwera group arbovirus from a naturally infected caribou

A Bunyamwera group arbovirus was isolated from the blood and from the brain of a female caribou parasitized with meningeal worms. The virus passed through a 0.45 micron filter; was ether sensitive; possessed no hemagglutination properties; could be propagated in suckling mice, 6-day old chick embryos, and BHK-21 tissue culture; and produced plaques in chick embryo fibroblast tissue culture. Neither complement-fixation or neutralization tests were sensitive enough to determine the serotype of the virus.     

Serologic evidence of arboviral infections in white-tailed deer from central Wisconsin

A survey conducted during 1979-1980 on white-tailed deer (Odocoileus virginianus) in central Wisconsin revealed serological evidence of infection by selected arboviruses. Among sera from 41 deer, antibody was detected for Jamestown Canyon virus (56%) and Bunyamwera group virus (80%), demonstrating their continuing endemic activity. Antibody for La Crosse virus, not found previously in sera from deer in central Wisconsin, also was detected (5%) in this study.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.     

Detection of Orthobunyavirus in mosquitoes collected in Argentina

Bunyamwera virus (BUNV) (Bunyaviridae, genus Orthobunyavirus, serogroup Bunyamwera) is considered an emerging pathogen for humans and animals in American countries. The CbaAr‐426 strain of BUNV was recovered from mosquitoes Ochlerotatus albifasciatus (Diptera: Culicidae) collected in Córdoba province (Argentina), where serological studies detected high seroprevalences in humans and animals. Molecular detection of Orthobunyavirus was performed in mosquitoes collected in Córdoba province. Seventeen mosquito pools of Oc. albifasciatus, Ochlerotatus scapularis and Culex quinquefasciatus (Diptera: Culicidae) showed positive results; four of these positive pools, all of Oc. scapularis, were sequenced. All amplicons grouped with BUNV in the Bunyamwera serogroup. The findings highlight the circulation of BUNV in Córdoba province and represent the first report of BUNV‐infected Oc. scapularis mosquitoes in Argentina.     

Virus isolations from mosquitoes in southern Ontario, 1976 and 1977

Following the 1975 epidemic of St. Louis encephalitis (SLE) in Ontario, programs were instituted to monitor virus activity in mosquito populations during 1976 and 1977. Mosquitoes were trapped with CDC light traps and CO2 cone traps, pooled by species, and tested for virus by intracerebral inoculation of suckling mice. In 1976, 51 175 mosquitoes were tested. SLE virus was isolated from two mixed pools of Culex pipiens--C. restuans mosquitoes. Five isolations of California serogroup viruses were made. Three of these were trivittatus virus, which has not been demonstrated previously in Canada, and the other two were snowshoe have virus. Other viruses isolated in 1976 were a virus antigenically identical to the virus of infectious bursal disease of chickens and 34 Flanders viruses. In 1977, 34 428 mosquitoes were tested. Flanders virus was isolated most frequently, from pools of mixed C. pipiens--C. restuans mosquitoes. The only other isolate was a Bunyamwera group virus, Cache Valley virus. This virus has not been reported previously in Ontario.     

Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus

Baby hamster kidney (BHK) cells were infected with Semliki Forest virus (SFV) and, 2 h later, were treated for 4 h with 10 microM monensin. Each of the four to six flattened cisternae in the Golgi stack became swollen and separated from the others. Intracellular transport of the viral membrane proteins was almost completely inhibited, but their synthesis continued and they accumulated in the swollen Golgi cisternae before the monensin block. In consequence, these cisternae bound large numbers of viral nucleocapsids and were easily distinguished from other swollen cisternae such as those after the block. These intracellular capsid-binding membranes (ICBMs) were not stained by cytochemical markers for endoplasmic reticulum (ER) (glucose-6-phosphatase) or trans Golgi cisternae (thiamine pyrophosphatase, acid phosphatase) but were labeled by Ricinus communis agglutinin I (RCA) in thin, frozen sections. Since this lectin labels only Golgi cisternae in the middle and on the trans side of the stack (Griffiths, G., R. Brands, B. Burke, D. Louvard, and G. Warren, 1982, J. Cell Biol., 95:781-792), we conclude that ICBMs are derived from Golgi cisternae in the middle of the stack, which we term medial cisternae. The overall movement of viral membrane proteins appears to be from cis to trans Golgi cisternae (see reference above), so monensin would block movement from medial to the trans cisternae. It also blocked the trimming of the high-mannose oligosaccharides bound to the viral membrane proteins and their conversion to complex oligosaccharides. These functions presumably reside in trans Golgi cisternae. This is supported by data in the accompanying paper, in which we also show that fatty acids are covalently attached to the viral membrane proteins in the cis or medial cisternae. We suggest that the Golgi stack can be divided into three functionally distinct compartments, each comprising one or two cisternae. The viral membrane proteins, after leaving the ER, would all pass in sequence from the cis to the medial to the trans compartment.     

Antiviral activity released from Aedes albopictus cells persistently infected with Semliki forest virus.

Aedes albopictus (mosquito) cells persistently infected with Semliki Forest virus released an agent which inhibited virus production by A. albopictus cells infected with homologous virus. Inhibition of virus production was accompanied by a marked reduction in the synthesis of viral RNA and viral proteins. Expression of the antiviral effect was prevented by pretreatment of cells with actinomycin. No analogous antiviral activity was detected in culture fluids of A. albopictus cells persistently infected with a flavivirus (Kunjin virus) or a bunyavirus (Bunyamwera virus).     

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.     

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.     

TUBULES IN DILATED CISTERNAE OF ENDOPLASMIC RETICULUM OF THLASPI ARVENSE (CRUCIFERAE)

Tubular inclusions in dilated cisternae of endoplasmic reticulum (ER) were found in healthy and virus diseased pennycress (Thlaspi arvense L., Cruciferae). The dilated cisternae were encountered more often in virus-infected cells than in comparable cells from healthy control plants. Dilated ER cisternae were found in vascular parenchyma cells and were shown to be interconnected. The tubules measured 30 nm in diam and cisternae that contain them may be more than 10 μm in length.     

Biochemical requirements of virus wrapping by the endoplasmic reticulum: involvement of ATP and endoplasmic reticulum calcium store during envelopment of African swine fever virus

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae.     

Self-protection and survival of arbovirus-infected mosquito cells

Arboviruses are serious pathogens for men but cause little damage to their arthropod vectors. We have studied how a mosquito cell line derived from one of the relevant vectors for arboviruses responds to Bunyamwera virus, a well-characterized arbovirus. Confocal, live cell microscopy and electron microscopy showed that Bunyamwera virus induces deep changes in mosquito cells. Early in infection these cells develop long projections and create new intercellular connections where cell organelles and viral proteins are detected. Live cell microscopy shows that these connections are developed before viral protein can be detected by immunofluorescence. Interestingly, their proliferation is accompanied by a progressive trapping of the nucleocapsid and RNA polymerase viral proteins into large cytoplasmic aggregates. A significant drop in the release of infectious virions then follows. Before that, numerous viruses assemble in peripheral Golgi stacks and they apparently exit the cells immediately since they do not accumulate intracellularly. This mechanism of assembly seems to cause little damage to the integrity of cell endomembranes. The characterization of the antiviral mechanisms operating in mosquito cells can be of great help in the fight against pathogenic arboviruses.     

Filamentous structures in dengue type 3 virus infected mouse neurones

Dengue type 3 (H-87) virus was inoculated into suckling mouse brain and animals were sacrificed at 24 hr intervals. Parallel filamentous structures were found in infected neurones in close association with virus particles in the distended endoplasmic cisternae. They were usually arranged in a crystalloid pattern, oriented in different directions within the cisternae. Faint helical features were sometimes observed. These filamentous structures measured 15-25 nm in width and varied in length. Their possible involvement with viral material or a viral core is postulated.