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Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes . Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR (-/-) ) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR (-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

Vaccinia virus ( Orthopoxvirus ) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17 - particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17 - particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17 - particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17 - particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17 - particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17 - particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17 - particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17 - particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

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.

At least 12 serotypes of 'atypical' bluetongue virus (BTV-25 to BTV-36) have been identified to date. These atypical serotypes fail to infect/replicate in Culicoides -derived cell lines and/or adult Culicoides vectors and hence can no longer be transmitted by these vectors. They appear to be horizontally transmitted from infected to in-contact ruminants, although the route(s) of infection remain to be identified. Viral genome segments 1, 2 and 3 (Seg-1, Seg2 and Seg-3) of BTV-26 were identified as involved in blocking virus replication in KC cells. We have developed Culicoides -specific expression plasmids, which we used in transfected insect cells to assess the stability of viral mRNAs and protein expression from full-length open reading frames of Seg-1, -2 and -3 of BTV-1 (a Culicoides -vectored BTV) or BTV-26. Our results indicate that the blocked replication of BTV-26 in KC cells is not due to an RNAi response, which would lead to rapid degradation of viral mRNAs. A combination of degradation/poor expression and/or modification of the proteins encoded by these segments appears to drive the failure of BTV-26 core/whole virus-particles to assemble and replicate effectively in Culicoides cells.

Bluetongue is an economically important disease of domesticated and wild ruminants caused by bluetongue virus (BTV). There are at least 36 different serotypes of BTV (the identity of which is determined by its outer-capsid protein VP2), most of which are transmitted by Culicoides biting midges. IFNAR (-/-) mice immunised with plant-expressed outer-capsid protein VP2 (rVP2) of BTV serotypes -1, -4 or -8, or the smaller outer-capsid protein rVP5 of BTV-10, or mock-immunised with PBS, were subsequently challenged with virulent strains of BTV-4 or BTV-8, or with an attenuated clone of BTV-1 (BTV-1RG C7 ). The mice that had received rVP2 generated a protective immune response against the homologous BTV serotype, reducing viraemia (as detected by qRT-PCR), the severity of clinical signs and mortality levels. No cross-serotype protection was observed after challenge with the heterologous BTV serotypes. However, the severity of clinical signs, viraemia and fatality levels after challenge with the attenuated strain of BTV-1 were all increased in mice immunised with rVP2 of BTV-4 and BTV-8, or with rVP5 of BTV10. The possibility is discussed that non-neutralising antibodies, reflecting serological relationships between the outer-capsid proteins of these different BTV serotypes, could lead to 'antibody-dependent enhancement of infection' (ADE). Such interactions could affect the epidemiology and emergence of different BTV strains in the field and would therefore be relevant to the design and implementation of vaccination campaigns.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae , which is available at ictv.global/report/spinareoviridae.

Tick-borne viruses are responsible for various symptoms in humans and animals, ranging from simple fever to neurological disorders or haemorrhagic fevers. The Kemerovo virus (KEMV) is a tick-borne orbivirus, and it has been suspected to be responsible for human encephalitis cases in Russia and central Europe. It has been isolated from Ixodes persulcatus and Ixodes ricinus ticks. In a previous study, we assessed the vector competence of I. ricinus larvae from Slovakia for KEMV, using an artificial feeding system. In the current study, we used the same system to infect different tick population/species, including I. ricinus larvae from France and nymphs from Slovakia, and I. persulcatus larvae from Russia. We successfully confirmed the first two criteria of vector competence, namely, virus acquisition and trans-stadial transmission, for both tick species that we tested. The estimated infection rates of engorged and moulted ticks suggest specificities between viral strains and tick species/developmental stages.

Bluetongue virus serotypes 1 to 24 are transmitted primarily by infected Culicoides midges, in which they also replicate. However, "atypical" BTV serotypes (BTV-25, -26, -27 and -28) have recently been identified that do not infect and replicate in adult Culicoides , or a Culicoides derived cell line (KC cells). These atypical viruses are transmitted horizontally by direct contact between infected and susceptible hosts (primarily small ruminants) causing only mild clinical signs, although the exact transmission mechanisms involved have yet to be determined. We used reverse genetics to generate a strain of BTV-1 (BTV-1 RG C7 ) which is less virulent, infecting IFNAR (-/-) mice without killing them. Reassortant viruses were also engineered, using the BTV-1 RG C7 genetic backbone, containing individual genome segments derived from BTV-26. These reassortant viruses were used to explore the genetic control of horizontal transmission (HT) in the IFNAR (-/-) mouse model. Previous studies showed that genome segments 1, 2 and 3 restrict infection of Culicoides cells, along with a minor role for segment 7. The current study demonstrates that genome segments 2, 5 and 10 of BTV-26 (coding for proteins VP2, NS1 and NS3/NS3a/NS5, respectively) are individually sufficient to promote HT.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana , based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus ), dengue virus ( Flavivirus ), African swine fever virus ( Asfarviridae ) and poliovirus ( Picornaviridae ). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR (-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus , a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2 n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides -borne pathogens., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Bluetongue is a severe, economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world. It is associated with major production losses, restrictions of animal movements and trade, as well as costs associated with developing and implementing effective surveillance and control measures. Mammalian hosts infected with bluetongue virus (BTV) generate a protective neutralising antibody response targeting the major BTV outer-capsid protein and serotype-specific antigen, VP2. BTV VP2 proteins that have been expressed in plants are soluble, with a native conformation displaying neutralising epitopes and can assemble with other BTV structural proteins to form virus-like particles (VLPs). His-tagged VP2 proteins of BTV serotypes 4 and 8 were transiently expressed in Nicotiana benthamiana then purified by immobilised metal affinity chromatography (IMAC). Antisera from IFNAR -/- mice prime/boost vaccinated with the purified proteins, were shown to contain VP2-specific antibodies by Indirect ELISA (I-ELISA), western blotting and serum neutralisation tests (SNT). Vaccinated mice, subsequently challenged with either the homologous or heterologous BTV serotype, developed viraemia by day 3 post-infection. However, no clinical signs were observed in mice challenged with the homologous serotype (either prime-boost or single-shot vaccinated), all of which survived for the duration of the study. In contrast, all of the vaccinated mice challenged with a heterologous serotype, died, showing no evidence of cross-protection or suppression of viraemia, as detected by real-time RT-qPCR or virus isolation. The induction of protective, serotype-specific neutralising antibodies in IFNAR -/- mice, indicates potential for the use of plant-expressed BTV VP2s as subunit vaccine components, or as a basis for serotype-specific serological assays.

BTV-4 structural proteins VP2 (as two domains: VP2D1 and VP2D2), VP5 (lacking the first 100 amino acids: VP5Δ1-100) and full-length VP7, expressed in bacteria as soluble glutathione S-transferase (GST) fusion-proteins, were used to immunise Balb/c and α/β interferon receptor knock-out (IFNAR(-/-)) mice. Neutralising antibody (NAbs) titres (expressed as log10 of the reciprocal of the last dilution of mouse serum which reduced plaque number by ≥50%) induced by the VP2 domains ranged from 1.806 to 2.408 in Balb/c and IFNAR(-/-) mice. The immunised IFNAR(-/-) mice challenged with a homologous live BTV-4 survived and failed to develop signs of infection (ocular discharge and apathy). Although subsequent attempts to isolate virus were unsuccessful (possibly reflecting presence of neutralising antibodies), a transient/low level viraemia was detected by real time RT-PCR. In contrast, mice immunised with the two VP2 domains with or without VP5Δ1-100 and VP7, then challenged with the heterologous serotype, BTV-8, all died by day 7 post-infection. We conclude that immunisation with bacterially-expressed VP2 domains can induce strong serotype-specific NAb responses. Bacterial expression could represent a cost effective and risk-free alternative to the use of live or inactivated vaccines, particularly if viruses prove to be difficult to propagate in cell culture (like BTV-25). A vaccine based on bacterially expressed VP2 and VP5 of BTV is also DIVA-compatible., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 (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.

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.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a -1 frameshift. Elements favouring -1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.

The complete nucleotide sequence of Middelburg virus (MIDV) was determined for strain MIDV-857 from Zimbabwe. The isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that MIDV can cause severe disease in equids. Full-length cDNA copies of the viral genome were successfully synthesized by an innovative RT-PCR amplification approach using an 'anchor primer' combined with the SMART methodology described previously for the synthesis of full-length cDNA copies from genome segments of dsRNA viruses. The MIDV-857 genome is 11,674 nt, excluding the 5'-terminal cap structure and poly(A) tail (which varies in length from approximately 180 to approximately 220 residues). The organization of the genome is like that of other alphaviruses, including a read-through stop codon between the nsP3 and nsP4 genes. However, phylogenetic analyses of the structural protein amino acid sequences suggested that the MIDV E1 gene was generated by recombination with a Semliki Forest virus-like virus. This hypothesis was supported by bootscanning analysis using a recombination-detection program. The 3' untranslated region of MIDV-857 also contains a 112 nt duplication. This study reports the first full-length sequence of MIDV, which was obtained from a single RT-PCR product.

Family Reoviridae is known, by definition, to contain dsRNA viruses with 10-12 genome segments. We report here the characterization of the first member of this family with a nine-segmented genome. This virus was isolated from Aedes pseudoscutellaris mosquito cells and designated aedes pseudoscutellaris reovirus (APRV). Virions are single-shelled with turrets but are non-occluded by contrast to cypoviruses. APRV replicates in various mosquito cell lines, but not in mice or mammalian cells. Complete sequence analysis showed that APRV is phylogenetically related to cypoviruses, fijiviruses and oryzaviruses. The maximum amino acid identities with cypoviruses, oryzaviruses or fijiviruses in the polymerase, are compatible with values observed between these genera and lower than values within a given genus. This suggests that APRV should be classified within a new genus that we designated Dinovernavirus (sigla from D: Double-stranded, i: insect, nove: nine from the latin "novem", rna: RNA, virus) in family Reoviridae.

Coltiviruses are tickborne viruses of the genus Coltivirus. The type species, Colorado tick fever virus (from North America), has been isolated from patients with flulike syndromes, meningitis, encephalitis, and other severe complications. Another coltivirus, Eyach virus, has been isolated from ticks in France and Germany and incriminated in febrile illnesses and neurologic syndromes. Seadornaviruses are endemic in Southeast Asia, particularly Indonesia and China. The prototype virus of the genus, Banna virus (BAV), has been isolated from many mosquito species, humans with encephalitis, pigs, and cattle. Two other seadornaviruses, Kadipiro and Liao Ning, were isolated only from mosquitoes. The epidemiology of seadornaviruses remains poorly documented. Evidence suggests that BAV is responsible for encephalitis in humans. Infection with BAV may be underreported because it circulates in regions with a high incidence of Japanese encephalitis and could be misdiagnosed as this disease.

Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 A resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked beta sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.

Background: Eyach virus (EYAV) is a tick-borne virus belonging to genus Coltivirus, family Reoviridae. It was isolated in Germany and France and has been suspected to be responsible for neurological diseases in humans. To date, there has been no relatively rapid and relatively specific serological assay for EYAV., Objectives: To develop an ELISA for EYAV, suitable for epidemiological and/or diagnostic purposes. This ELISA should allow to distinguish between infections with EYAV and the related Colorado tick fever virus (CTFV)., Study Design: VP6, VP7 and VP12 of Eyach virus (the three proteins most divergent between EYAV and CTFV) were expressed in bacteria using the pGEX-4T-2 vector. A partial sequence of VP6 (designated pVP6) was chosen to develop an ELISA for detecting anti-EYAV IgG antibodies in serum. This choice was based on two observations: (i) the homologous VP7 protein of CTFV was successfully used as a target for detecting antibodies to CTFV (the VP7 showed the highest reactivity to an anti-CTFV antibody among all CTFV expressed proteins); (ii) to distinguish infection with EYAV from a CTFV infection: the expressed sequence was chosen within a region which is highly divergent (49% of amino acid identity) from the homologous VP7 sequence of CTFV., Results and Conclusions: pVP6 was shown to be the most reactive among the three expressed proteins. The elaborated pVP6 ELISA was evaluated with 340 sera of French blood donors, and found to exhibit a specificity of 100% (no false positives). Furthermore, no cross reaction was detected with antibody to CTFV, thus permitting us to distinguish between infections by either virus. The use of this recombinant protein for serological assays is a good alternative to the use of native EYAV antigen due to the extremely low productivity of the virus in cell culture, and the requirement for suckling mice. This ELISA will be useful to clarify the epidemiological status and the suspected pathogenicity of the virus.

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9. The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid. The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past. The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells. The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.

VP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genus Coltivirus, family Reoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol. 22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral genera Orbivirus, Orthoreovirus, and Rotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus.