Orbivirus NS4 Proteins Play Multiple Roles to Dampen Cellular Responses

Non-structural protein 4 (NS4) of insect-borne and tick-borne orbiviruses is encoded by genome segment 9, from a secondary open reading frame. Though a protein dispensable for bluetongue virus (BTV) replication, it has been shown to counter the interferon response in cells infected with BTV or African horse sickness virus. We further explored the functional role(s) of NS4 proteins of BTV and the tick-borne Great Island virus (GIV). We show that NS4 of BTV or GIV helps an E3L deletion mutant of vaccinia virus to replicate efficiently in interferon-treated cells, further confirming the role of NS4 as an interferon antagonist. Our results indicate that ectopically expressed NS4 of BTV localised with caspase 3 within the nucleus and was found in a protein complex with active caspase 3 in a pull-down assay. Previous studies have shown that pro-apoptotic caspases (including caspase 3) suppress type I interferon response by cleaving mediators involved in interferon signalling. Our data suggest that orbivirus NS4 plays a role in modulating the apoptotic process and/or regulating the interferon response in mammalian cells, thus acting as a virulence factor in pathogenesis

Detection of a Fourth Orbivirus Non-Structural Protein

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4

Isolates of Liao Ning Virus from Wild-Caught Mosquitoes in the Xinjiang Province of China in 2005

Liao ning virus (LNV) is related to Banna virus, a known human-pathogen present in south-east Asia. Both viruses belong to the genus Seadornavirus, family Reoviridae. LNV causes lethal haemorrhage in experimentally infected mice. Twenty seven isolates of LNV were made from mosquitoes collected in different locations within the Xinjiang province of north-western China during 2005. These mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns where sheep represent the main livestock species. The regions where LNV was isolated are affected by seasonal encephalitis, but are free of Japanese encephalitis (JE). Genome segment 10 (Seg-10) (encoding cell-attachment and serotype-determining protein VP10) and Seg-12 (encoding non-structural protein VP12) were sequenced for multiple LNV isolates. Phylogenetic analyses showed a less homogenous Seg-10 gene pool, as compared to segment 12. However, all of these isolates appear to belong to LNV type-1. These data suggest a relatively recent introduction of LNV into Xinjiang province, with substitution rates for LNV Seg-10 and Seg-12, respectively, of 2.29×10−4 and 1.57×10−4 substitutions/nt/year. These substitution rates are similar to those estimated for other dsRNA viruses. Our data indicate that the history of LNV is characterized by a lack of demographic fluctuations. However, a decline in the LNV population in the late 1980s - early 1990s, was indicated by data for both Seg-10 and Seg-12. Data also suggest a beginning of an expansion in the late 1990s as inferred from Seg-12 skyline plot

Inhibition of Orbivirus Replication by Fluvastatin and Identification of the Key Elements of the Mevalonate Pathway Involved

International audienceThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC B

Micromonas pusilla reovirus: a new member of the family Reoviridae assigned to a novel proposed genus (Mimoreovirus)

Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90–95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV

Circular genomes related to anelloviruses identified in human and animal samples by using a combined rolling-circle amplification/sequence-independent single primer amplification approach

A combined rolling-circle amplification (RCA) and sequence-independent single primer amplification (SISPA) approach was applied to four samples of human plasma and one sample of saliva from a cat. This approach permitted the characterization of nine anelloviruses. Most of them were identified as highly divergent strains that were classified into species of the genus Anellovirus. The smallest anellovirus described so far in humans was characterized (2PoSMA, 2002 nt; 'small anellovirus' species). Two highly divergent sequences belonging to the species Torque Teno Mini Virus (LIL-y1, 2887 nt; LIL-y2, 2871 nt), which clustered into a new phylogenetic branch, were also identified in human plasma samples. Finally, two genomes that are separated by a genetic divergence of 46 % were characterized in the cat's saliva, one of these creating a distinct phylogenetic branch (PRA1, 2019 nt). These results highlight the potential of RCA-SISPA for detecting circular (or circularized) genome

Full-genome characterisation of Orungo, Lebombo and Changuinola viruses provides evidence for co-evolution of orbiviruses with their arthropod vectors

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein 'outer-capsid protein 1' (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 'subcore-shell protein' as the third largest protein 'VP3(T2)' in the first of these groups, but the second largest protein 'VP3(T2)' in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors

Liao ning virus, a new Chinese seadornavirus that replicates in transformed and embryonic mammalian cells

Seadornaviruses are emerging arboviral pathogens from the south-east of Asia. The genus Seadornavirus contains two distinct species, Banna virus (BAV) isolated from humans with encephalitis and Kadipiro virus. BAV replicates within insect cells and mice but not in cultured mammalian cells. Here, the discovery of Liao ning virus (LNV), a new seadornavirus from the Aedes dorsalis mosquito, which was completely sequenced and was found to be related to BAV and Kadipiro virus, is reported. Two serotypes of LNV could be distinguished by a serum neutralization assay. According to amino acid identity with other seadornaviruses, and to criteria set by the ICTV for species delineation, LNV was identified as a member of a new species of virus. Its morphology was characterized by electron microscopy and found to be similar to that of BAV. LNV is the first reported seadornavirus that replicates in mammalian cells, leading to massive cytopathic effect in all transformed or embryonic cell lines tested. LNV- and BAV-infected mice producing a viraemia lasting for 5 days was followed by viral clearance. Mice infection generated virus quasi-species for LNV (the first reported observation for quasi-species in the family Reoviridae) but not for BAV. Challenge with BAV in mice immunized against BAV did not lead to productive infection. However, challenge with LNV in mice immunized against LNV was lethal with a new phase of viraemia and massive haemorrhage

Potential duplications in YUOV VP3(OC1).

<p>OC1 sequence of YUOV VP3 with repeats identified by REPRO. The sequences in blue font represent the NH2 terminal domain while the sequences in the red font represent the COOH terminal domain. Superimposed hydrophobicity profiles of the two domains are shown below each alignment. The amino acid identity between the two identified repeats is 28%. Hydrophobicity profiles of repeats are shown below the alignment. In YUOV VP3, aa 11 to 448 were identified as a repeat of aa 45 to 851. Finer sequence analysis identified that aa 60–448 have highly similar hydrophobicity plots to aa 462–851.</p