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PubMed1981

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↗
PubMed2004

The high genetic variation of viruses of the genus Nairovirus reflects the diversity of their predominant tick hosts.

The genus Nairovirus (family Bunyaviridae) contains seven serogroups consisting of 34 predominantly tick-borne viruses, including several associated with severe human and livestock diseases [e.g., Crimean Congo hemorrhagic fever (CCHF) and Nairobi sheep disease (NSD), respectively]. Before this report, no comparative genetic studies or molecular detection assays had been developed for this virus genus. To characterize at least one representative from each of the seven serogroups, reverse transcriptase-polymerase chain reaction (RT-PCR) primers targeting the L polymerase-encoding region of the RNA genome of these viruses were successfully designed based on conserved amino acid motifs present in the predicted catalytic core region. Sequence analysis showed the nairoviruses to be a highly diverse group, exhibiting up to 39.4% and 46.0% nucleotide and amino acid identity differences, respectively. Virus genetic relationships correlated well with serologic groupings and with tick host associations. Hosts of these viruses include both the hard (family Ixodidae) and soft (family Argasidae) ticks. Virus phylogenetic analysis reveals two major monophyletic groups: hard tick and soft tick-vectored viruses. In addition, viruses vectored by Ornithodoros, Carios, and Argas genera ticks also form three separate monophyletic lineages. The striking similarities between tick and nairovirus phylogenies are consistent with possible coevolution of the viruses and their tick hosts. Fossil and phylogenetic data placing the hard tick-soft tick divergence between 120 and 92 million years ago suggest an ancient origin for viruses of the genus Nairovirus.

Amino Acid Sequence↗
PubMed1990

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Animals↗
PubMed2002

Dugbe nairovirus S segment: correction of published sequence and comparison of five isolates.

The sequence of the S (small) RNA segment of the ArD 44313 isolate of Dugbe nairovirus (DUG) has been redetermined, and a number of apparent errors in the previously reported sequence (V. K. Ward, A. C. Marriott, A. A. El-Ghorr, and P. A. Nuttall, 1990, Virology 175, 518-524) were revealed. Our results indicate that the S RNA is 1716 nucleotides (nt) in length and contains one large open reading frame spanning 1449 nt. This can encode a 483 amino acid polypeptide, M(r) 53.9 kDa, corresponding to the viral nucleocapsid protein N. The DUG N protein is thus similar in length to the N proteins of Hazara (HAZ) and Crimean-Congo haemorrhagic fever (CCHF) nairoviruses, which are 485 and 482 amino acids in length, respectively. S segment RNA sequences were also determined for DUG isolates IbAr 1792, IbH 11480, ArD 16095, and KT 281/75; only the KT 281/75 sequence differed markedly from that of ArD 44313. Phylogenetic trees were constructed for these nairovirus S segment sequences.

Amino Acid Sequence↗
PubMed1992

Expression of the nucleocapsid protein of Dugbe virus and antigenic cross-reactions with other nairoviruses.

The small (S) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) encodes a single protein, the nucleocapsid (N) protein, of M(r) 49.4 kDa. cDNA derived from the complete coding region for the N protein was cloned into Autographa californica nuclear polyhedrosis virus (AcNPV) under control of the polyhedrin promoter and used to infect Spodoptera frugiperda insect cells. Western blotting analysis using monoclonal antibodies demonstrated the production of DUG N protein in the infected cells. Monoclonal and polyclonal antibodies to the N protein of Crimean-Congo haemorrhagic fever (CCHF) virus were found to cross-react weakly with the baculovirus expressed DUG N protein by Western blotting. When used in an enzyme linked immunoassay (ELISA), the DUG N protein reacted with polyclonal mouse immune ascitic fluids raised against either CCHF or Hazara viruses (both members of the CCHF serogroup of nairoviruses). Cross-reactions between DUG virus (Nairobi sheep disease serogroup) and members of other nairovirus serogroups were not detected.

Antigens, Viral↗
PubMed1996

Large RNA segment of Dugbe nairovirus encodes the putative RNA polymerase.

The nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.

Amino Acid Sequence↗
PubMedDate not supplied

Infection and coding strategies of arenaviruses, phleboviruses, and nairoviruses.

The infection and coding strategies of three groups of negative-stranded RNA viruses (arena viruses, phleboviruses, and nairoviruses) that include the etiologic agents of hemorrhagic disease in humans have been studied. Arenaviruses have two viral RNA species. The smaller RNA species (S) codes for the viral nucleoprotein (N protein) and for the viral glycoprotein species (G1 and G2, which are derived from a precursor glycoprotein, GPC). The S RNA has an ambisense arrangement. The proteins are translated from subgenomic mRNA species (viz., N protein from a viral-complementary mRNA and glycoprotein from a viral-sense mRNA). The larger arenavirus RNA species (L) is presumed to code for the viral transcriptase/replicase. Phleboviruses and nairoviruses are members of the Bunyaviridae. They both have three species of viral RNA. Other than the sizes of the viral proteins and the viral RNA species, virtually nothing is known about the coding strategy of nairoviruses. Phleboviruses have an ambisense coding arrangement to their smallest (S) RNA species. This S RNA codes for the viral N protein (translated from a viral-complementary mRNA) and a nonstructural protein (translated from a viral-sense mRNA). The middle-size (M) RNA of phleboviruses codes for a precursor to the viral glycoproteins (translated from a viral-complementary mRNA). The largest viral RNA (L) is presumed to code for the viral transcriptase/replicase.

Animals↗
PubMed1982

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↗
PubMed1990

Studies on the pathogenicity of a nairovirus, Dugbe virus, in normal and immunosuppressed mice.

Susceptibility to lethal infection with the KT281/75 strain of the tick-borne nairovirus, Dugbe virus, was similar in an outbred strain and several inbred strains of mice. For the outbred strain, both neural and extraneural routes of virus inoculation resulted in lethal infection, but susceptibility decreased with age and only intracerebral inoculation produced a lethal infection in adults. In newborn mice, subcutaneous (s.c.) inoculation of virus (analogous to a tick-bite) produced a disseminated infection, titres being highest in the upper respiratory tract (URT), spleen and liver at 5 days post-inoculation (p.i.), the heart at 7 days p.i. and brain by 8 days p.i. In neonates inoculated intranasally (i.n.), by contrast, virus spread rapidly from the URT to the brain by 2 days p.i., in the absence of a detectable viraemia. Virus was undetectable in the blood of s.c. and i.n. inoculated adults; in the former, virus replication was limited to the site of inoculation, and in the latter virus grew in the respiratory tract and again spread to the brain. Immunosuppression of i.n. inoculated adult mice with cyclophosphamide produced some mortality indicating that host defences are important in protecting the adult, especially as newborn and adult lung tissue were equally able to support the growth of Dugbe virus in culture. The similarity between the pattern of Dugbe virus infection in the mouse and that of other, more pathogenic nairoviruses suggests that, although haemorrhagic disease was not observed, this may be a useful model for studying the genetic basis of nairovirus virulence and for testing vaccines and anti-viral drugs.

Animals↗
PubMed1991

Structure and morphogenesis of Dugbe virus (Bunyaviridae, Nairovirus) studied by immunogold electron microscopy of ultrathin cryosections.

We have studied the structure and morphogenesis of Dugbe (DUG) virus (Bunyaviridae, Nairovirus) in cultured porcine kidney (PS) cells and a tick cell line (Ra 243) using immunogold electron microscopy. DUG virus is a tickborne arbovirus, considered to be a low health hazard, that is antigenically and genetically related to Crimean Congo haemorrhagic fever (CCHF) virus (Marriott et al., 1990). We have investigated the maturation and intracellular transport of DUG virus particles as a model for other more pathogenic nairoviruses using monoclonal antibodies for immunogold labelling of ultrathin cryosections and immunofluorescence techniques. The spherical DUG virus particle measures about 90 nm in diameter, with a 5 nm thick membrane covered by 5-7 nm long projections or "spikes". These projections form hollow cylindrical morphological units, about 5 nm in diameter. DUG virus infection caused only a slight cytopathogenic effect in mammalian cells and none in tick cells. DUG virus particles assembled by budding from the Golgi complex, where the DUG virus glycoprotein G1 accumulated in vesicles originating from Golgi cisternae. The nucleocapsid protein N accumulated in scattered foci throughout the cytoplasm, and this appears to be related to the limited maturation of DUG virus particles that occurred. The reduced number of budding virus particles observed in tick cells was correlated with the reduced cytopathology observed.

Animals↗
PubMed2004

Inhibition of Dugbe nairovirus replication by human MxA protein.

Sensitivity to the interferon-induced protein, MxA, has previously been demonstrated for viruses belonging to the Orthobunyavirus, Hantavirus and Phlebovirus genera of the Bunyaviridae family. We have extended these findings to a member of the fourth and remaining genus containing viruses that infect man and other animals, the nairovirus Dugbe virus (DUGV). Indirect immunofluorescence experiments using VA9 cells (Vero cells permanently transfected with MxA cDNA) revealed strongly reduced DUGV antigen expression, suggesting that MxA inhibited DUGV replication. Western and Northern blot analyses showed significantly lower DUGV nucleocapsid (N) protein expression and DUGV genomic RNA, respectively, in the presence of MxA. Viral titres were also reduced by more than two orders of magnitude in VA9 cells compared with control VN36 cells. This finding may have application to nairovirus therapeutics.

Animals↗
PubMed1993

Non-viral sequences at the 5' ends of Dugbe nairovirus S mRNAs.

Analyses of the mRNA transcription processes of viruses in four genera (Bunyavirus, Hantavirus, Phlebovirus and Tospovirus) of the family Bunyaviridae have revealed a common mechanism of initiation using host-derived primers, known as cap-snatching. To provide similar information on the fifth genus in the family, the 5' ends of Dugbe nairovirus S mRNA species were specifically cloned and sequenced. This revealed the presence of non-viral heterogeneous sequences, five to 16 nucleotides in length (average of 10 nucleotides) at the 5' ends, confirming that cap-snatching to prime mRNA synthesis is a familial characteristic of the Bunyaviridae. Inspection of the sequences in the primers on nairovirus, bunyavirus and phlebovirus mRNAs suggests that in some cases polymerase slippage occurs shortly after initiation, resulting in a partial reiteration of the 5'-terminal nucleotides of the viral RNA.

Base Sequence↗
PubMed2005

Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells.

We report the successful infection of the cell line ISE6 derived from Ixodes scapularis tick embryos by the tick-borne Hazara virus (HAZV), a nairovirus in the family Bunyaviridae. Using a recombinant Semliki Forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of HAZV, and we showed that this blockage is mediated by the replication of the Semliki Forest alphavirus replicon; the vector containing the HAZV nucleoprotein gene in sense or antisense orientation efficiently inhibited HAZV replication. Moreover, expression of a distantly related nucleoprotein gene from Crimean-Congo hemorrhagic fever nairovirus failed to induce HAZV silencing, indicating that the inhibition is sequence specific. The resistance of these cells to replicate HAZV correlated with the detection of specific RNase activity and 21- to 24-nucleotide-long small interfering RNAs. Altogether, these results strongly suggest that pathogen-derived resistance can be established in the tick cells via a mechanism of RNA interference.

Animals↗
PubMed1996

Ultrastructural studies on the replication and morphogenesis of Nairobi sheep disease virus, a Nairovirus.

The Nairovirus Nairobi sheep disease virus (NSDV) affects sheep and goats causing severe hemorrhagic gastroenteritis and high mortality. Replication and morphogenesis of NSDV was determined by electron microscopic examination of ultra-thin sections of 143B and BHK-21 cells at varying times after infection. By 4 h post-infection (p.i.) of 143B cells, virions budding from the luminal side of the bilayer membrane of smooth membrane vesicles were observed. Morphologically mature virus particles were electron-dense, spherical and of uniform size (100 nm diameter) and accumulated in smooth membrane vesicles associated with the Golgi complex. In BHK-21 clone 13 cells, mature virus particles in smooth membrane vesicles were present by 8 h p.i. The morphogenesis of NSDV was restricted to the smooth membrane vesicles of Golgi complex, and budding of virus from other sites was not detected. Extracellular virus particles were observed by 10 h p.i., before expression of cytopathic effects. The cytopathic effects were observed at 24 h p.i. in 143B cells and at 36 h p.i. in BHK-21 cells. The morphology and morphogenesis of NSDV in BHK-21 cells and in 143B cells resembles that of other members of the family Bunyaviridae.

Animals↗
PubMed1992

Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy.

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

Amino Acid Sequence↗
PubMed1992

Mechanisms of neutralization of a nairovirus (Dugbe virus) by polyclonal IgG and IgM.

Dugbe virus is a member of the nairovirus genus of the Bunyaviridae. Purified polyclonal anti-Dugbe virus IgG, which neutralized greater than 99.5% of virus, reduced attachment of virus to BSC-1 cell monolayers by only 36%. A 100-fold lower concentration neutralized virus by 88%, and had no effect upon attachment. Neutralizing IgG did not affect the ability of Dugbe virus to be internalized by or to fuse with BSC-1 cells. This suggests that IgG neutralization occurs largely at a stage subsequent to primary uncoating. Purified polyclonal anti-Dugbe virus IgM neutralized infectivity and had no effect on the attachment of virus to cells, but inhibited internalization of virus by about 50%. Thus IgM neutralizes partly by interfering with entry of virus and partly by a post-entry event. Neutralization by intermediate concentrations of IgM was enhanced 20-fold in the presence of complement. At high concentrations of IgM, complement-dependent neutralization declined. This is probably due to IgM binding in a planar rather than crab conformation, which does not expose the complement binding sites. Aggregation occurred only at relatively low concentrations of immunoglobulin. Electron microscopy and reactivation of infectivity by vortexing suggested that aggregation makes only a minor contribution to neutralization by IgG or IgM.

Animals↗
PubMed1990

Coding strategy of the S RNA segment of Dugbe virus (Nairovirus; Bunyaviridae).

The S RNA segment of Dugbe (DUG) virus (Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141-148). The 49.4-kDa product was expressed as a fusion protein with beta-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3' to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

Amino Acid Sequence↗
PubMed1985

Polypeptide synthesis of Dugbe virus, a member of the Nairovirus genus of the Bunyaviridae.

The replication of Dugbe (DUG) virus, a member of the Nairovirus genus of the Bunyaviridae, has been investigated. During the infection of BS-C-1 cells a virus-specific c.p.e. was initially observed followed by recovery of the cell monolayer but with continued production of infectious virus. Six DUG virus-induced polypeptides were identified with apparent molecular weights, determined by gel electrophoresis, of 92000 (p92), 82000 (p82), 77000 (p77), 52000 (p52), 48000 (p48) and 34000 (p34). The polypeptides p77 and p34 were detected in purified DUG virions but not in extracts of virus-infected cells pulse-labelled with [3H]leucine. Polypeptides p48 and p52 were found in both purified virus preparations and in extracts of infected cells. p82 and p92 were found only in lysates of infected cells. When two-dimensional gel electrophoresis was used to analyse infected cells, p48 was found to have a net positive charge.

Animals↗