Evaluation of the vector competence for Batai virus of native Culex and exotic Aedes species in Central Europe.

BACKGROUND: Batai virus (BATV) is a zoonotic arbovirus of veterinary importance. A high seroprevalence in cows, sheep and goats and infection in different mosquito species has been observed in Central Europe. Therefore, we studied indigenous as well as exotic species of the genera Culex and Aedes for BATV vector competence at different fluctuating temperature profiles. METHODS: Field caught Culex pipiens biotype pipiens, Culex torrentium, Aedes albopictus and Aedes japonicus japonicus from Germany and Aedes aegypti laboratory colony were infected with BATV strain 53.3 using artificial blood meals. Engorged mosquitoes were kept under four (Culex species) or three (Aedes species) fluctuating temperature profiles (18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C) at a humidity of 70% and a dark/light rhythm of 12:12 for 14 days. Transmission was measured by testing the saliva obtained by forced salivation assay for viable BATV particles. Infection rates were analysed by testing whole mosquitoes for BATV RNA by quantitative reverse transcription PCR. RESULTS: No transmission was detected for Ae. aegypti, Ae. albopictus or Ae. japonicus japonicus. Infection was observed for Cx. p. pipiens, but only in the three conditions with the highest temperatures (21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C). In Cx. torrentium infection was measured at all tested temperatures with higher infection rates compared with Cx. p. pipiens. Transmission was only detected for Cx. torrentium exclusively at the highest temperature of 27 ± 5 °C. CONCLUSIONS: Within the tested mosquito species, only Cx. torrentium seems to be able to transmit BATV if the climatic conditions are feasible.

Comparison of Immunogenicity Between a Candidate Live Attenuated Vaccine and an Inactivated Vaccine for Cache Valley Virus.

Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses.

Oral susceptibility of aedine and culicine mosquitoes (Diptera: Culicidae) to Batai Orthobunyavirus.

BACKGROUND: A number of zoonotic mosquito-borne viruses have emerged in Europe in recent decades. Batai virus (BATV), a member of the genus Orthobunyavirus, is one example of a relatively newly emerged mosquito-borne virus, having been detected in mosquitoes and livestock. We conducted vector competency studies on three mosquito species at a low temperature to assess whether Aedes and Culex mosquito species are susceptible to infection with BATV. METHODS: Colonised lines of Aedes aegypti and Culex pipiens and a wild-caught species, Aedes detritus, were orally inoculated with BATV strain 53.2, originally isolated from mosquitoes trapped in Germany in 2009. Groups of blood-fed female mosquitoes were maintained at 20 °C for 7 or 14 days. Individual mosquitoes were screened for the presence of BATV in body, leg and saliva samples for evidence of infection, dissemination and transmission, respectively. BATV RNA was detected by reverse transcription-PCR, and positive results confirmed by virus isolation in Vero cells. RESULTS: Aedes detritus was highly susceptible to BATV, with an infection prevalence of ≥ 80% at both measurement time points. Disseminated infections were recorded in 30.7-41.6% of Ae. detritus, and evidence of virus transmission with BATV in saliva samples (n = 1, days post-infection: 14) was observed. Relatively lower rates of infection for Ae. aegypti and Cx. pipiens were observed, with no evidence of virus dissemination or transmission at either time point. CONCLUSIONS: This study shows that Ae. detritus may be a competent vector for BATV at 20 °C, whereas Ae. aegypti and Cx. pipiens were not competent. Critically, the extrinsic incubation period appears to be ≤ 7 days for Ae. detritus, which may increase the onward transmissibility potential of BATV in these populations.

Infection and transmission of Cache Valley virus by Aedes albopictus and Aedes aegypti mosquitoes.

BACKGROUND: Cache Valley virus (CVV; Bunyavirales, Peribunyaviridae) is a mosquito-borne arbovirus endemic in North America. Although severe diseases are mainly observed in pregnant ruminants, CVV has also been recognized as a zoonotic pathogen that can cause fatal encephalitis in humans. Human exposures to CVV and its related subtypes occur frequently under different ecological conditions in the New World; however, neurotropic disease is rarely reported. High prevalence rates of neutralizing antibodies have been detected among residents in several Latin American cities. However, zoophilic mosquito species involved in the enzootic transmission are unlikely to be responsible for the transmission leading to human exposures to CVV. Mechanisms that lead to frequent human exposures to CVV remain largely unknown. In this study, competence of two anthropophilic mosquitoes, Aedes albopictus and Ae. aegypti, for CVV was determined using per os infection to determine if these species could play a role in the transmission of CVV in the domestic and peridomestic settings of urban and suburban areas. RESULTS: Aedes albopictus were highly susceptible to CVV whereas infection of Ae. aegypti occurred at a significantly lower frequency. Whilst the dissemination rates of CVV were comparable in the two species, the relatively long period to attain maximal infectious titer in Ae. aegypti demonstrated a significant difference in the replication kinetics of CVV in these species. Detection of viral RNA in saliva suggests that both Ae. albopictus and Ae. aegypti are competent vectors for CVV under laboratory conditions. CONCLUSIONS: Differential susceptibility to CVV was observed in Ae. albopictus and Ae. aegypti, reflecting their relatively different capacities for vectoring CVV in nature. The high susceptibility of Ae. albopictus to CVV observed in this study suggests its potential role as an efficient vector for CVV. Complemented by the reports of multiple CVV isolates derived from Ae. albopictus, our finding provides the basis for how the dispersal of Ae. albopictus across the New World may have a significant impact on the transmission and ecology of CVV.

Vertical transmission of naturally occurring Bunyamwera and insect-specific flavivirus infections in mosquitoes from islands and mainland shores of Lakes Victoria and Baringo in Kenya.

BACKGROUND: Many arboviruses transmitted by mosquitoes have been implicated as causative agents of both human and animal illnesses in East Africa. Although epidemics of arboviral emerging infectious diseases have risen in frequency in recent years, the extent to which mosquitoes maintain pathogens in circulation during inter-epidemic periods is still poorly understood. This study aimed to investigate whether arboviruses may be maintained by vertical transmission via immature life stages of different mosquito vector species. METHODOLOGY: We collected immature mosquitoes (egg, larva, pupa) on the shores and islands of Lake Baringo and Lake Victoria in western Kenya and reared them to adults. Mosquito pools (≤25 specimens/pool) of each species were screened for mosquito-borne viruses by high-resolution melting analysis and sequencing of multiplex PCR products of genus-specific primers (alphaviruses, flaviviruses, phleboviruses and Bunyamwera-group orthobunyaviruses). We further confirmed positive samples by culturing in baby hamster kidney and Aedes mosquito cell lines and re-sequencing. PRINCIPAL FINDINGS: Culex univittatus (2/31pools) and Anopheles gambiae (1/77 pools) from the Lake Victoria region were positive for Bunyamwera virus, a pathogenic virus that is of public health concern. In addition, Aedes aegypti (3/50), Aedes luteocephalus (3/13), Aedes spp. (2/15), and Culex pipiens (1/140) pools were positive for Aedes flaviviruses at Lake Victoria, whereas at Lake Baringo, three pools of An. gambiae mosquitoes were positive for Anopheles flavivirus. These insect-specific flaviviruses (ISFVs), which are presumably non-pathogenic to vertebrates, were found in known medically important arbovirus and malaria vectors. CONCLUSIONS: Our results suggest that not only ISFVs, but also a pathogenic arbovirus, are naturally maintained within mosquito populations by vertical transmission, even in the absence of vertebrate hosts. Therefore, virus and vector surveillance, even during inter-epidemics, and the study of vector-arbovirus-ISFV interactions, may aid in identifying arbovirus transmission risks, with the potential to inform control strategies that lead to disease prevention.

Culex tarsalis is a competent vector species for Cache Valley virus.

BACKGROUND: Cache Valley virus (CVV) is a mosquito-borne orthobunyavirus endemic in North America. The virus is an important agricultural pathogen leading to abortion and embryonic lethality in ruminant species, especially sheep. The importance of CVV in human public health has recently increased because of the report of severe neurotropic diseases. However, mosquito species responsible for transmission of the virus to humans remain to be determined. In this study, vector competence of three Culex species mosquitoes of public health importance, Culex pipiens, Cx. tarsalis and Cx. quinquefasciatus, was determined in order to identify potential bridge vector species responsible for the transmission of CVV from viremic vertebrate hosts to humans. RESULTS: Variation of susceptibility to CVV was observed among selected Culex species mosquitoes tested in this study. Per os infection resulted in the establishment of infection and dissemination in Culex tarsalis, whereas Cx. pipiens and Cx. quinquefasciatus were highly refractory to CVV. Detection of viral RNA in saliva collected from infected Cx. tarsalis provided evidence supporting its role as a competent vector. CONCLUSIONS: Our study provided further understanding of the transmission cycles of CVV and identifies Cx. tarsalis as a competent vector.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase.

The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.

Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection.

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Culicoides sonorensis (Diptera: Ceratopogonidae) is not a competent vector of Cache Valley virus (family Bunyaviridae, genus Orthobunyavirus).

We investigated the susceptibility of Culicoides sonorensis to Cache Valley virus (CVV) (family Bunyaviridae, genus Orthobunyavirus) infection and the potential that it could be a vector or site of virus reassortment. CVV is native to the New World and causes disease in livestock. Infected blood meals were fed to both a competent vector, Anopheles quadrimaculatus, and Culicoides sonorensis. All Anopheles mosquitoes were infected as expected, but only 21 % of the C. sonorensis insects were susceptible to infection. These appeared to present a midgut barrier, because virus persisted but did not disseminate. This means Culicoides sonorensis is not likely to be a vector of CVV but could be involved in viral reassortment. Schmallenberg virus (SBV) (family Bunyaviridae, genus Orthobunyavirus) was recently discovered in Europe and probably is a novel virus resulting from a reassortment of two orthobunyaviruses, and an ongoing epizootic in cattle and small ruminants has caused significant economic damage.

Attenuation of bunyamwera orthobunyavirus replication by targeted mutagenesis of genomic untranslated regions and creation of viable viruses with minimal genome segments.

Bunyamwera virus (BUNV) is the prototype virus for both the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite, negative-sense RNA genome. The coding region of each segment is flanked by untranslated regions (UTRs) that are partially complementary. The UTRs play an important role in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Using reverse genetics, we generated recombinant viruses that contained deletions within the 3' and/or 5' UTRs of the L or M segments to determine the minimal UTRs competent for virus viability. We then generated viruses carrying deleted UTRs in all three segments. These viruses were grossly attenuated in tissue culture, being significantly impaired in their ability to produce plaques in BHK cells, and had a reduced capacity to cause host cell protein shutoff. After serial passage in tissue culture, some viruses partially recovered fitness, generating higher titers and producing larger plaques. We determined the complete nucleotide sequence for each virus. The deleted UTR sequences were maintained, and no amino acid changes were observed in the nonstructural proteins (NSs and NSm), the nucleocapsid protein (N), or the Gn glycoprotein. One virus had a single amino acid substitution in Gc. Three viruses contained amino acid changes in the viral polymerase that mostly occurred in the C-terminal domain of the L protein. Although the role of this domain remains unknown, we suggest that those changes might be involved in the evolution of the polymerase to recognize the deleted UTRs more efficiently.

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.

Amino acid changes within the Bunyamwera virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates.

The genome of Bunyamwera virus (BUNV) comprises three RNA segments that are encapsidated by the virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation.

Visualizing the replication cycle of bunyamwera orthobunyavirus expressing fluorescent protein-tagged Gc glycoprotein.

The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also of the Orthobunyavirus genus, are encoded by the medium (M) RNA genome segment and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the endoplasmic reticulum (ER) and transit to the Golgi compartment for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (X. Shi, J. Goli, G. Clark, K. Brauburger, and R. M. Elliott, J. Gen. Virol. 90:2483-2492, 2009.). In this study, the coding sequence for a fluorescent protein, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, was fused to the N terminus of truncated Gc, and two recombinant BUNVs (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages, whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP, resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi compartment at later stages of infection. The fluorescent protein-tagged viruses will be valuable reagents for live-cell imaging studies to investigate virus entry, budding, and morphogenesis in real time.

Bunyamwera orthobunyavirus S-segment untranslated regions mediate poly(A) tail-independent translation.

The mRNAs of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, possess a 5' cap structure but lack a 3' poly(A) tail, a common feature of eukaryotic mRNAs that greatly enhances translation efficiency. Viral mRNAs also contain untranslated regions (UTRs) that flank the coding sequence. Using model virus-like mRNAs that harbor the Renilla luciferase reporter gene, we found that the 3' UTR of the BUNV small-segment mRNA mediated efficient translation in the absence of a poly(A) tail. Viral UTRs did not increase RNA stability, and polyadenylation did not significantly enhance reporter activity. Translation of virus-like mRNAs in transfected cells was unaffected by knockdown of poly(A)-binding protein (PABP) but was markedly reduced by depletion of eukaryotic initiation factor 4G, suggesting a PABP-independent process for translation initiation. In BUNV-infected cells, translation of polyadenylated but not virus-like mRNAs was inhibited. Furthermore, we demonstrate that the viral nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm early in infection, followed by nuclear retention of PABP. Our results suggest that BUNV corrupts PABP function in order to inhibit translation of polyadenylated cellular mRNAs while its own mRNAs are translated in a PABP-independent process.

The unique architecture of Bunyamwera virus factories around the Golgi complex.

Viral factories are novel structures built by viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyavirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. Numerous ribonucleoproteins were released from purified tubes disrupted in vitro. Actin and myosin I were identified by peptide mass fingerprinting in isolated tubes while actin and the viral NSm non-structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. Studies with NSm deletion mutants and drugs affecting actin showed that both NSm and actin are key factors for tube and virus assembly in Golgi. Three-dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to Golgi stacks and make contacts with intracellular viruses. We propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of Golgi stacks, the actin-containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories.

Genetic elements regulating packaging of the Bunyamwera orthobunyavirus genome.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) comprises three segments of negative-sense, single-stranded RNA. The RNA segments are encapsidated by the viral nucleocapsid (N) protein and form panhandle-like structures through interaction of complementary sequences at their 5' and 3' termini. Transcription and replication of a BUN genome analogue (minireplicon), comprising the viral non-coding sequences flanking a reporter gene, requires just the viral RNA polymerase (L protein) and N protein. Here, sequences of Bunyamwera serogroup M segment RNAs were compared and conserved elements within nt 20-33 of the 3' and 5' non-coding regions that can affect packaging of minireplicons into virions were identified. RNA-folding models suggest that a conserved sequence within nt 20-33 of the 5' end of the genome segments maintains conserved structural features necessary for efficient transcription. Competitive packaging experiments using M, L and S segment-derived minireplicons that encode different reporter genes showed variable packaging efficiencies of the three segments. Packaging of a particular segment appeared to be independent of the presence of other segments and, for the S segment, packaging efficiency was unaffected by the inclusion of viral coding sequences in the minireplicon.

Identification of the Bunyamwera bunyavirus transcription termination signal.

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3' ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5' terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3'-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5' non-translated region (NTR) of the S segment. A 6 nt region (3'-GUCGAC-5') within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3'-UGUCG-5', that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5' NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.

The Bunyamwera virus mRNA transcription signal resides within both the 3' and the 5' terminal regions and allows ambisense transcription from a model RNA segment.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of RNA viruses. BUNV genomic strands are templates for both replication and transcription, whereas the antigenomic RNAs serve only as templates for replication. By mutagenesis of model templates, we showed that the BUNV transcription promoter comprises elements within both the 3' and the 5' nontranslated regions. Using this information, we designed a model ambisense BUNV segment that transcribed BUNV S mRNA from the genomic strand and green fluorescent protein (GFP) mRNA from the antigenome. Demonstration of GFP expression showed that this ambisense strategy represents a viable approach for generating BUNV segments able to express additional proteins.