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Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy.

Ultrastructural studies of glutaraldehyde-fixed viruses of the Bunyaviridae were performed by negative-stain electron microscopy. The surface structure of viruses of each genus was compared with that of the other genera and with Hantaan virus, the prototype of a proposed new genus of Bunyaviridae. Viruses of each genus had a surface structure distinct for that genus. In addition, Hantaan virus had a surface structure composed of a grid-like pattern of morphologic subunits not previously described for animal viruses. Careful morphologic studies of suspected Bunyaviridae may be used in considering preliminary generic assignment. This study also supports the assignment of Hantaan-related viruses to a separate generic status within the Bunyaviridae.

Bunyaviridae↗

Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae.

Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, was examined for nucleic acid characteristics which would support its previously proposed inclusion in the virus family Bunyaviridae. Nucleocapsid RNA from Hantaan virions and a control bunyavirus were examined for ribonuclease A (RNase A) sensitivity. Both viruses exhibited a similar accessibility of RNA within nucleocapsids to digestion by RNase A. Complete digestion of the RNA of both viruses was affected with high concentrations of ribonuclease. Evidence for negative strand RNA polarity was obtained by an in vitro transcriptase assay. RNA dependent RNA polymerase activity was associated with Hantaan virions. Polymerase activity required manganese and nucleoside triphosphates and was enhanced by magnesium, 2-mercaptoethanol, and sodium chloride. Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA. A common 3' terminal sequence of the three genome segments was determined to be 3' AUCAUCAUCUG. This sequence is different from those reported for viruses within the four recognized genera of the Bunyaviridae. Because all other data were consistent with nucleic acid characteristics of the Bunyaviridae, we propose a separate genus within the Bunyaviridae with Hantaan as its prototype virs.

Bunyaviridae↗

Completion of the La Crosse virus genome sequence and genetic comparisons of the L proteins of the Bunyaviridae.

La Crosse virus is a member of the Bunyavirus genus in the family Bunyaviridae, viruses with trisegmented RNA genomes of mostly negative polarity composed of large (L), medium (M), and small (S) segments. The sequences of the La Crosse/original M and S RNA segments have been previously characterized. Using reverse transcriptase in conjunction with PCR amplification, we have obtained the nucleotide sequence of the L RNA segment, which encodes the viral polymerase in a single large open reading frame. Comparison of the amino acid sequence of the LAC L protein with the sequence of other polymerases from members of the Bunyaviridae, demonstrated the presence of several conserved motifs, some of which are characteristic of many polymerase proteins. A genetic tree comparing the available polymerase proteins of the Bunyaviridae provides insights into the phylogenetic relationships within this large family. Members of the genus Bunyavirus, which are mosquito-borne and infect mammals, have a closer relationship to the plant viruses represented by tomato spotted wilt virus (Tospovirus genus) than to viruses of other genera in the family Bunyaviridae.

Amino Acid Sequence↗

Emerging infectious diseases: the Bunyaviridae.

The Bunyaviridae are a large group of viruses that infect a diversity of arthropod vectors and animal hosts. They have a worldwide distribution and can be the cause of human illness ranging from mild asymptomatic infection to hemorrhagic fever and fatal encephalitis. The growth of the human population, the expansion of agricultural and economic development, climatic changes, and the speed and frequency of global transportation all favor the emergence of bunyaviruses and other arthropod borne viruses. International monitoring of the Bunyaviridae and a greater understanding of their ecology and biology are needed to prepare for future outbreaks.

Agriculture↗

Studies on Bunyaviridae including Zaliv Terpeniya virus isolated from Ixodes uriae ticks (Acarina: Ixodidae) in Brittany, France.

Three viruses were isolated from ticks parasitizing nesting seabirds on Cape Sizun, Brittany, France, during the spring of 1979: Soldado like virus (Bunyaviridae, Nairovirus genus) from Ornithodoros (Alectorobius) maritimus (Argasidae), Zaliv Terpeniya (ZT) virus (Bunyaviridae, Uukuvirus genus) from Ixodes (Ceratixodes) uriae (Ixodidae) parasitizing chicks of the kittiwake (Rissa tridactyla), and a virus of the Sakhalin group from I. (C.) uriae. Certain virological and ultrastructural properties of ZT virus were studied and the antigenic relationships between th French isolate and other UUK serogroup viruses from I. (C.) uriae were analyzed in the complement fixation and immune diffusion tests. Cape Sizun is notable for the unusual presence of both argasied (Ornithodoros) and ixodid (Ixodes) ticks infesting seabirds in adjacent niches in a single locality, where they are infected by different viruses.

Animals↗

Analyses of the mRNA transcription processes of Punta Toro phlebovirus (Bunyaviridae).

The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293-306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NSs, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NSs mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NSs mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5' ends of the N and NSs mRNA species have shown them to be heterogeneous in sequence and some 11-18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409-6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.

Animals↗

The M RNA of impatiens necrotic spot Tospovirus (Bunyaviridae) has an ambisense genomic organization.

The nucleotide sequence of Impatiens necrotic spot virus (INSV) M RNA was determined from cDNA clones. The INSV M RNA was 4972 nucleotides in length with two open reading frames (ORFs) in an ambisense genomic organization. The larger ORF near the 3' end of the viral RNA, coding for a protein with a predicted molecular weight of 124.9 kDa, was in the viral complementary sense and produced the G2 and G1 proteins. A smaller ORF in the viral sense was capable of coding for a 34.1-kDa polypeptide, designated the NSm protein. Two subgenomic RNA species were detected in INSV-infected tissue that corresponded to the predicted sizes (3.3 and 1.0 kb) of the G2-G1 and NSm mRNAs. The ORFs were separated by a 478 nucleotide A-U-rich intergenic region similar to the regions found in other viral RNAs with ambisense ORFs. The intergenic region was predicted to form a stable stem-loop structure (-81.2 kcal/mole). The ambisense genomic organization is characteristic of the S RNA for members of the Phlebovirus, Uukuvirus, and Tospovirus genera in the Bunyaviridae family. This is the first report of an ambisense Bunyaviridae M RNA.

Amino Acid Sequence↗

Morphological identification of the agent of Korean haemorrhagic fever (Hantaan virus)as a member of the Bunyaviridae.

Korean haemorrhagic fever (KHF) (Hantaan virus), a rodent-borne viral illness, is an important cause of human disease throughout much of Asia and Eastern Europe. The agent responsible for KHF has not yet been conclusively identified. Plaque-purified KHF virus was concentrated and then banded in a potassium tartrate gradient. Material from the 1.17-1.19 g/ml band was examined by electron microscopy and particles with a morphology identical to that of the family Bunyaviridae were found. The particles were aggregated by KHF serum but not by saline solution or non-immune serum. Identification of KHF virus as a member of the family Bunyaviridae implies a potential for spread by arthropod vectors.

Bunyaviridae↗

Structural characteristics of nairoviruses (genus Nairovirus, Bunyaviridae).

Viruses from six antigenic groups of arthropod-borne viruses [Crimean--Congo haemorrhagic fever (CCHF), Nairobi sheep disease (NSD), Qalyub (QYB), Sakhalin (SAK), Dera Ghazi Khan (DGK) and Hughes (HUG) serogroups], some previously categorized as bunyavirus-like viruses and others previously ungrouped, have recently been assembled by serological analyses into a new genus of viruses (Nairovirus genus) in the Bunyaviridae. Molecular studies of the virion RNA and viral polypeptides have been undertaken with representative members of the different serogroups [Hazara (HAZ) and Congo (CON) viruses, CCHF group; Dugbe (DUG) virus, NSD group; QYB, Omo and Bandia (BDA) viruses, QYB group; Avalon (AVA) virus, SAK group; DGK and Abu Mina (AM) viruses, DGK group; and HUG virus, HUG group]. In agreement with a recent study of QYB virus and in part agreement with an earlier report on DUG virus, the results of these molecular analyses indicate that nairoviruses have: (i) three virion RNA species (large, L, medium, M, and small, S) with apparent mol. wt. of 4.1 x 10(6) to 4.9 x 10(6), 1.5 x 10(6) to 1.9 x 10(6) and 0.6 x 10(6) to 0.7 x 10(6) respectively; (ii) a 48 x 10(3) to 54 x 10(3) mol. wt. nucleocapsid (N) polypeptide; and (iii) two external glycopolypeptides, 72 x 10(3) to 84 x 10(3) mol. w. (G1) and 30 x 10(3) to 40 x 10(3) mol. wt. (G2). Cross-immune precipitation analyses have confirmed that viruses in the Nairovirus genus share antigenic determinants and are antigenically distinct from representative members of th Bunyavirus, Phlebovirus and Uukuvirus genera (Bunyaviridae).

Antigens, Viral↗

The 3' terminal RNA sequences of bunyaviruses and nairoviruses (Bunyaviridae): evidence of end sequence generic differences within the virus family.

The 3' terminal nucleotide sequences of the three virus RNA species of viruses representing eight serogroups of bunyaviruses (genus Bunyavirus, Bunyaviridae) and six serogroups of nairoviruses (genus Nairovirus, Bunyaviridae) have been characterized. Members of the Bunyavirus genus have conserved 3' end sequences (generally, 3' UCAUCACAUGA...) that differ from the conserved 3' end sequences of members of the Nairovirus genus (generally, 3' AGAGUUUCU...).

Base Sequence↗

Completion of the impatiens necrotic spot virus genome sequence and genetic comparison of the L proteins within the family Bunyaviridae.

The nucleotide sequence of the large (L) genome segment of impatiens necrotic spot virus (INSV) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. The L RNA is 8776 nucleotides long and of negative polarity, containing one large ORF on the viral complementary strand. Comparison of the deduced amino acid sequence of the INSV L RNA primary translation product (330.3 kDa) with those of the L RNAs of other members of the family Bunyaviridae reveals that this protein represents the putative viral RNA-dependent RNA polymerase. A cluster dendrogram of the (putative) RNA polymerases indicates that the genera Tospovirus and Tenuivirus, though both encompassing ambisense plant-infecting viruses, have different affinities to the animal-infecting Bunyaviridae, tospoviruses being most closely related to the genus Bunyavirus, and tenuiviruses to the genus Phlebovirus.

Amino Acid Sequence↗

Teratogenicity of Australian Simbu serogroup and some other Bunyaviridae viruses: the embryonated chicken egg as a model.

The use of embryonated chicken eggs as a model for assessing the teratogenic potential of animal viruses was investigated with 12 members of the Bunyaviridae family. Infection of 4-day-old embryonated chicken eggs via the yolk sac with 10 of the viruses resulted in deaths or congenital deformities that were similar to those observed in Akabane virus infections of fetal ruminants and included arthrogryposis, scoliosis, mandible defects, and retarded development. Statistical analysis showed that the viruses fell into three main groupings, namely, those that caused both death and deformities (Akabane, Aino, Tinaroo, and Belmont viruses), those that mainly caused death (Peaton, Thimiri, and Facey's Paddock viruses), and those that required very high doses to cause either death or deformities (Douglas and CSIR0296 viruses). In addition, two viruses (Kowanyama and Mapputta viruses) caused neither death nor deformities. A difference in the pathogenic potential between two Akabane isolates (B8935 and CSIR016) in the embryonated chicken egg model was found to correlate with differences previously observed in experimentally infected sheep; Akabane CSIR016 was the more pathogenic. It is concluded that the embryonated chicken egg model should also be of value in assessing the teratogenic potential of other Bunyaviridae and attenuated vaccine viruses, although it does not assess the ability of the virus to cross the placenta.

Animals↗

Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs.

We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5'- and 3'-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

Animals↗

The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging.

We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G(N) cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

Alanine↗

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↗

Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex.

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.

Amino Acid Sequence↗

The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice.

Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405-411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the Bunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.

Animals↗

Isolation of Bunyamwera serogroup viruses (Bunyaviridae, Orthobunyavirus) in New York state.

During routine arbovirus surveillance from 2000 to 2004 in New York state (NYS), 14,788 mosquito pools making up 36 species and nine genera were inoculated onto Vero cell cultures to test for abroad spectrum of viruses. Forty-six percent of viruses isolated in cell culture from species, excluding Culex pipiens L. and Culex restuans Theobald, were identified as Bunyamwera serogroup viruses. Here, we report the distribution and level of Bunyamwera activity in NYS detected during this period. We developed specific primers for Cache Valley virus (family Bunyaviridae, genus Orthobunyavirus, CVV) and Potosi virus (family Bunyaviridae, genus Orthobunyavirus, POTV), to facilitate rapid molecular identification of these viruses. Viral RNA was detected in 12 mosquito species by reverse transcription-polymerase chain reaction, with the majority isolated from Aedes trivittatus (Coquillet). We report the first POTV isolation in NYS and describe the development of specific primers to identify both POTV and CVV.

Animals↗