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A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes

Protein synthesis in Bunyamwera virus-infected cells.

In Vero cells infected with Bunyamwera virus there is a rapid inhibition of cell RNA and protein synthesis to levels of 30 and 3% respectively of the control rate, both the rate of inhibition and the time lag before its initiation being multiplicity dependent. Using u.v.-irradiated virus, investigation of the mechanism of inhibition of host cell protein synthesis indicates that synthesis of new virus components is required for inhibition to occur. Quantitative comparison of the proteins synthesized in infected cells shows that at higher m.o.i. synthesis of virus, as well as cellular proteins, is inhibited. Bunyamwera virus-infected Vero cells synthesized three virus-specific proteins identified as the structural virion proteins. Nucleoprotein is synthesized predominantly early in infection while the major envelope glycoprotein and the minor glycoprotein are synthesized predominantly late in the infection cycle.

Animals

Relationships of bunyamwera group viruses by neutralization.

A standardized serum dilution plaque reduction neutralization test was used for cross-neutralization studies of 23 strains of Bunyamwera serogroup viruses. Antigenic relationships were determined by inspection of the neutralization tests results as well as by numerical taxonomic analysis. Based on these analyses five complexes, containing 1-11 viruses, were distinguished. Little or no cross-reactivity was observed between viruses of different complexes. Three of the viruses tested were indistinguishable from prototypes and probably represent strains or varieties of those prototypes. A tentative classification scheme for the Bunyamwera group is presented.

Antigens, Viral

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

Aedes

Probing orthobunyavirus reassortment using Bunyamwera and Batai viruses as models.

Reassortment is a critical evolutionary mechanism for segmented viruses, enabling the exchange of intact genome segments during co-infection and driving orthobunyavirus evolution; however, the molecular mechanisms underpinning this process remain unclear. With over 100 orthobunyavirus species, many of which are significant human and veterinary pathogens, understanding how reassortment influences transmissibility and virulence is essential for preempting the emergence of novel pathogens. Here, we use Bunyamwera virus (BUNV) and Batai virus (BATV) as models to explore orthobunyavirus reassortment through reverse genetics. We established the first reverse genetics system for BATV, generated reassortants, and employed minigenome assays to assess replication machinery compatibility. Additionally, we developed a novel hybridization chain reaction assay for high-resolution visualization of viral RNA segments. Our findings revealed that all six reassortants between BUNV and BATV are viable, exhibiting notable phenotypic differences in interferon-deficient (IFNAR-/-) mice. This work introduces essential tools and new insights into orthobunyavirus reassortment and pathogenesis, laying the groundwork for understanding this critical evolutionary process.

Animals

Serologic examination of birds from the area of Southern Moravia for the presence of antibodies against arboviruses of the groups alfa, flavo, Bunyamwera supergroup, and the virus Yaba 1-Lednice 110. I. Domestic fowls.

196 geese, 141 ducks, and 100 hens from 9 localities of the South Moravian region (District of Breclav) were examined by means of the haemagglutination-inhibition test (HIT) for the presence of antibodies against 12 arbovirus antigens of the groups Alfavirus (Western Equine Encephalitis (WEE), Eastern Equine Encephalitis (EEE), Semliki, Sindbis, Chikungunya, O'nyong-nyong), Flavovirus (Tick-borne Encephalitis (TBE), Dengue and West Nile Virus (WN), Bunyamwera Supergroup (Tahna and Beta Calovo), and the Yaba 1-Lednice 110 Virus. In tested fowls antibodies were mostly found against Yaba 1-Lednice 110 Virus, namely 3.6% in geese and 17.7% in ducks. Antibodies against Calovo Virus were found in only 1 of the tested ducks. All sera of hens were negative. Antibodies against arboviruses of the Alfa-and Flavovirus groups were not detected.

Animals

Sunday Canyon virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas.

A new arbovirus was isolated from Texas, U.S.A., populations of the Cliff Swallow parasits Argas (Argas) cooleyi Kohls and Hoogstraal, 1960. The virus, named Sunday Canyon, is serological urelated to any of 185 arbovirus strains or 20 other viral agents with which it was compared. Morphologically it resembles Bunyamwera viruses and, in common with them, is sensitive to lipid solvents and acid pH, and apparently possesses RNA. Although considerably resistant to a temperature of 41.5 degrees C, it rapidly loses infectivity when incubated at 56 degrees C. It is lethal for newborn white mice and infective for the Vero and Antheraea eucalypti cell lines. Sundays Canyon virus is the second tick-associated, Bunyamwera virus-like agent known from North America and the third virus to be reported from A. cooleyi in Texas.

Animals

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Animals

[A serological survey of arboviruses in the human population of Senegal].

2,457 serum samples collected during an epidemiological survey on treponematosis in Senegal by the W. H. O. IR051 team in five areas of Senegal between 1972 and 1975 were studied for arbovirus antibodies using hemagglutination inhibition test. In addition 998 were studied using CF test and 1012 in neutralization test with yellow fever virus. The prevalence of arbovirus antibodies is important in all the country and does not vary basically between the different areas. In group A, chikungunya antibody is the most frequent and Sindbis antibody was found only in areas where migrant birds are resting. There is evidence of recent yellow fever infection in Upper Casamance and in Eastern Senegal. Most of the children are not immune and have to be immunized. Zika and Wesselsbron viruses show activity in all the areas and West-Nile, as Sindbis, is related to areas where birds are numerous. Bunyamwera virus activity is low.

Adult

Discontinuous counter-immunoelectrophoresis in the study of viruses.

A method for the use of the discontinuous counter-immunoelectropnoresis (DCIE) technique in the study of viruses of the major taxonomic groups is reported. The method was successfully employed in detection of antigen of viruses in the bunyavirus, bunyavirus-like, flavivirus, orbivirus, and rhabdovirus groups, and/or of antibody to these agents. Although less sensitive than the complement fixation test, the DCIE test was found to be rapid and in some cases more specific in differentiating related viruses or detecting antigenic similarity between strains of the same virus.

Animals

Mosquito-borne arboviruses in arctic america.

Mosquito-borne arboviruses are prevalent throughout subarctic regions of Canada and Alaska, principally in the boreal forest extending between latitudes 53 and 66 degrees N, but they have been identified in tundra regions as far north as 70 degrees N. All mosquito-borne agents have been bunyaviruses, comprising principally the snowshoe hare subtype of California encephalitis (CE) virus, but also Northway virus. Mosquito vectors comprise several Aedes species and Culiseta inornata, all of which have supported replication of CE virus following incubation at 13 degrees C or lower temperatures. Isolation of virus from wild-caught larvae points towards transovarial transfer. Principal vertebrate reservoirs of infection are mammals, especially snowshoe hares (Lepus americanus) and ground squirrels (Citellus undulatus). Where the boreal forest merges into prairie grassland around 53 degrees N, Culex tarsalis mosquitoes become prevalent, and an alphavirus, western equine encephalomyelitis, is detected more frequently than CE virus.

Aedes

International arbovirus research.

International cooperation in arbovirus research became increasingly prominent shortly after it was found in the early 1940's, that antigenic relationships existed among certain viruses isolated in different areas of thw world. The interest of a number of scientists and of a private organization led to the establishment of an international information exchange and to the preparation of an international catalogue of arboviruses including certain other viruses of vertebrates. International cooperation was fostered by the interest taken in these agents by the World Health Organization and the creation by this agency of an international net of collaborating laboratories with assigned areas of the world. A number of recent problems have been revealed and solved, or partly solved by this international effort, among which are questions relating to: the Marburg agent disease, the existence of an antigenic relationship between rabies virus and some arboviruses, aspects of the ecology or viruses in the California, Uukuniemi and Kemerovo groups, as well as several unanticipated antigenic relationships among viruses. Numerous questions still remain the solution of which might be expedited by continued and increased cooperative international effort such as overwintering or survival of viruses under unfavorable circumstances, pathogenic potential for man and domestic animals of certain viruses, role on chronic illnesses, establishment of the etiology of certain diseases and investigations of the basic properties of viruses, particularly of the Bunyaviridae.

Animals

Biochemical studies on the Phlebotomus fever group viruses (Bunyaviridae family).

Analyses of the virion polypeptides and genomes of several Phlebotomus fever group viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have established that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 X 10(6), 2.2 X 10(6), and 0.8 X 10(6), respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 X 10(6), 1.8 X 10(6), and 0.75 X 10(6), respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 X 10(3). A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 X 10(3) and 50 X 10(3) in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% beta-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 X 10(3). Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses.

Arboviruses

Virus neutralizing antibodies to arboviruses in birds of the order Anseriformes in Czechoslovakia.

Sera from birds of the order Anseriformes in Czechoslovakia were examined for virus neutralizing (VN) antibodies to arboviruses. VN antibodies to Sindbis, Calovo and Tahyna viruses were found in 15, 5 and 6 out of 106 greylag goose (Anser anser) sera. Out of 38 ducks, 6 mallards (Anas platyrhynchos) and 1 garganey (Anas querquedula) contained VN antibodies to Sindbis virus, 6 mallards to Calovo virus, 4 mallards and 1 garganey to Tahyna virus, 2 mallards and 1 garganey to tick-borne encephalitis (TE) virus and 1 mallard to West Nile (WN) virus.

Animals

Bunyavirus development in arctic and Aedes aegypti mosquitoes as revealed by glucose oxidase staining and immunofluorescence.

Northway virus replication has been detected in salivary glands of wild-caught Culiseta inornata and Aedes communis mosquitoes from the western Canadian Arctic after incubation at 4 degrees C for 9 to 11 months, and after incubation at 13 degrees C for 3 to 4 months after they received virus by oral ingestion or intrathoracic injection. Aedes hexodontus supported Northway virus replication after incubation at 13 degrees C for one month after intrathoracic injection. Aedes aegypti supported Northway virus replication after incubation at 13 degrees C or 23 degrees C for 6 to 28 days following intrathoracic injection. A larval isolate of California encephalitis virus (snowshoe hare subtype) multiplied in all 3 species of arctic mosquito after incubation at 13 degrees C for 1 to 3 months after virus was administered by oral ingestion or intrathoracic injection. Virus was detected in salivary glands of Cs. inornata after 329 days incubation at 4 degrees C after intrathoracic injection. Bunyavirus antigens in salivary glands of arctic and domestic mosquitoes were detected by the glucose oxidase immunoenzyme technique somewhat less frequently than by assay for virus infectivity.

Aedes

Cache Valley virus: isolations from mosquitoes in Saskatchewan, 1972-1974.

Eighteen isolations of Cache Valley virus (Bunyaviridae) were obtained from a total of 113,694 mosquitoes collected in Saskatchewan during the summers of 1972 to 1974. Most of the isolations were from mosquitoes collected during August. Culiseta inornata, the most abundant mosquito (38% of total collected), had the highest minimum vector-infection rate (0.83 isolations per 1000 mosquitoes). The virus was also isolated from Culex tarsalis and Aedes vexans. It is indicated in the isolations that the prairie grasslands of the province are enzootic for Cache Valley virus.

Aedes