Figure 1 (Top and center) A comparison of two distinct core particle morphologies (spiked and unspiked) present amongst members of different genera of the family Reoviridae. Orbivirus: a 3D model from x-ray crystallography of the core particle of an isolate of bluetongue-1 virus. Orthoreovirus: a 3D model from x-ray crystallography studies of a core particle of an isolate of mammalian orthoreovirus 3. Cypovirus: a 3D cryoEM reconstruction of a particle of an isolate of Cypovirus 5, at 25 Å resolution. Rotavirus: a 3D cryoEM reconstruction of a double shelled particle of an isolate of rotavirus A (SiRV-A/SA11), at 25 Å resolution. Fijivirus: an electron micrograph of a core particle of an isolate of maize rough dwarf virus. Phytoreovirus: a 3D cryoEM reconstruction of the double shelled particle of an isolate of rice dwarf virus, at 25 Å resolution (highlighted in colour are a contiguous “group of 5 trimers” found in each asymmetric unit). Coltivirus: an electron micrograph of a negatively stained double shelled particle of an isolate of Colorado tick fever virus. Oryzavirus: an electron micrograph of a negatively stained core particle of an isolate of rice ragged stunt virus. Mycoreovirus: an electron micrograph of a negatively stained core particle of mycoreovirus 1 (Rosallinia necatrix mycoreovirus-1). Seadornavirus: an electron micrograph of a negatively stained core particle of an isolate of Banna virus. The reconstructions and electron micrographs are not shown to exactly the same scale. The outer capsid morphologies of members of the different genera of the family Reoviridae are more variable and may appear smooth, or with surface projects, or may even be absent. (Bottom) A diagrammatic representation of the core particles (on the left) of an orbivirus (BTV), or rotavirus (RV), which have a well defined capsomeric structure but lack large surface projections at the five-fold icosahedral axes, as compared to the turreted (spiked) core particle (on the right) of an orthoreovirus (Reo).
(Courtesy of J. Diprose.)
Figure 2 Typical virus replication cycle of a reovirus (presented for an orbivirus).
Figure 3 (Top left) Diagrammatic representation of an orthoreovirus particle in cross-section. The locations and identities of the virus structural proteins are indicated using the nomenclature scheme for both mammalian orthoreovirus (MRV) and avian orthoreovirus (ARV). The protein components of the inner and outer capsids are indicated (Duncan, R. (1999). Virology, 260, 316-328; Palmer, E.L. and Martin, M.L. (1977). Virology, 76, 109-113). (Top right) Computer-generated image of the inner capsid of mammalian orthoreovirus 1 (MRV-1), based on X-ray crystallography data. (Bottom) Electron micrograph of a negatively stained MRV-1 particle (panel A). Image reconstructions from cryoEM of MRV-1 virions (panel B), infectious subviral particles (ISVPs) (panel C) and cores (panel D). All particles are viewed from the three-fold axis of rotational symmetry.
(Courtesy of M. Nibert and T. Baker.)
Figure 4 Gene organization of the polycistronic genome segments of the five species of orthoreoviruses. The solid line indicates the dsRNA, and the numbers refer to the first and last nucleotides of the genome segment, along with the nt positions of the various ORFs (excluding the termination codons) indicated by the rectangles. The identities of the gene products encoded by the various ORFs are indicated within the rectangles. The virus species and the genome segment are indicated on the left. The code for the abbreviations can be found in the list of species.
Figure 5 Phylogenetic relationships between the five orthoreovirus species using the aa sequences of the sigma-class major outer CPs of various isolates. Sequences were aligned using ClustalX and the unrooted neighbor-joining tree constructed using MEGA4 (Dayhoff distances and 10,000 bootstrap replicates). Accession numbers used were ARV-13 (AF059271), ARV-11 (U20642), ARV-Tu (AF4645799), ARV-Md (AJ006476), ARV-Go (AY114138), BRV (AF059723), MRV-1 (X61586), MRV-2 (X60066), MRV-3 (HM159622), MRV-4 (AF368037), NBV (AF059722) and RRV (AY238886).
Figure 6 (Top panel) Structural representation of grass carp reovirus virion and core by cryoEM. Triangles represent the VP5–VP7 complex on the virion (from Cheng, L., Fang, Q., Shah, S., Atanasov, I. C. and Zhou Z. H. (2008). J. Mol. Biol., 382, 213–222). (Middle panel) Transmission electronmicroscopy (TEM) of negatively stained grass carp reovirus (GCRV) particles. From left to right: intact virion, core and top components (empty particles). The scale bar represents 100 nm (from Fang, Q., Seng, E. K., Ding, Q. Q. and Zhang, L. L. (2008). Arch. Virol., 153, 675–682). (Bottom panel) Complete atomic model of grass carp infectious subviral particle. In the right hand side of the CryoEM reconstruction the removal of the VP5 coat reveals the core proteins. Ribbon models of the atomic structures of the six conformers from four structural proteins are shown in the periphery. The black triangle encloses a VP5 trimer
(from Zhang et al. (2010). Cell, 141, 472–482).
Figure 7 Genome organization of the 11 dsRNA segments of golden shiner virus (species Aquareovirus C). Each segment has a single ORF, except Seg7 which contains two ORFs. The green arrows indicate the upstream conserved terminal sequence (+ve 5′-GUUAUUU/G….) while the red arrows indicate the downstream conserved terminal sequence (+ve ….A/UUCAUC-3′).
Figure 8 (Top left) Electron micrograph of rice ragged stunt virus (RRSV) particles (courtesy of R.G. Milne). (Bottom left) schematic of RRSV particle; (right panel) micrographs of the virus showing 2-, 3- and 5-fold symmetries (A1, B1 and C1, respectively) images of the same rotated by increments of 180° (A2), or 120° (B2), or 72° (C2) and proposed models of the 2-, 3- and 5-fold symmetries (A3, B3 and C3 respectively) (courtesy of E. Shikata). The bar represents 50 nm.
Figure 9 (Left) Negative contrast electron micrograph of maize rough dwarf virus (MRDV) virions stained with uranyl acetate showing “A” spikes; (center) smooth subcores derived from MRDV on staining with neutral phosphotungstate; (right) “B” spikes on virus-derived MRDV cores stained with uranyl acetate (courtesy of R. G. Milne). The bar represents 100 nm.
Figure 10 Neighbor-joining phylogenetic tree constructed using the amino acid sequences of outer capsid proteins of fijiviruses. (Fiji disease virus, FDV (Seg10) [AY297694]; maize rough dwarf virus, MRDV (Seg10) [L76560]; rice black streaked dwarf virus, RBSDV (Seg10) [D00606]; oat sterile dwarf virus, OSDV (Seg8) [AB011025]; Nilaparvata lugens reovirus, NLRV (Seg8) [D26127]; mal de Rio Cuarto virus, MRCV (Seg10) [AY607586]; southern rice black streaked dwarf virus, SRBSDV (Seg10) [EU784840]). Sequences were aligned using ClustalX and the tree constructed using MEGA4 (Dayhoff distances and 10,000 bootstrap replicates).
Figure 11 (Left) Electron micrograph of virus particles of Cryphonectria parasitica mycoreovirus-1 (CpMYRV-1) after purification by sucrose gradient centrifugation, stained with 1% uranyl acetate (courtesy of B. Hillman). (Right) Core particle of Rosellinia necatrix mycoreovirus-3 (RnMYRV-3) showing icosahedral arrangement surface projections (turrets or spikes), stained with 1% uranyl acetate (courtesy of C. Wei). The bar (right panel) represents 50 nm.
Figure 12 (Left) Negative contrast electron micrograph of a non-occluded virion of Orgyia pseudosugata cypovirus 5 (OpCPV-5). (Right) Negative contrast electron micrograph of empty and full “occluded” virions (purified from polyhedra) of OpCPV-5, stained with uranyl acetate (courtesy of C. L. Hill). The bars represent 20 nm.
Figure 13 CryoEM reconstructions of Orgyia pseudosugata cypovirus 5 (OpCPV-5) virions, to 26 Å resolution: (top left) non-occluded virion; (top right) occluded virion; (bottom left) cross-section of a full occluded virion; (bottom center) cross-section of a full non-occluded virion; (bottom right) cross-section of an empty virion. The cross-sections show evidence of dsRNA packaged as distinct layers and suggest localization of the transcriptase complexes at the five-fold axes of symmetry.
(Courtesy of C. L. Hill.)
Figure 14 Phylogenetic tree for polyhedrin proteins from 11 cypovirus isolates. Sequences were aligned using ClustalX and the tree constructed in MEGA4 using the neighbour-joining method and the P-distance algorithm. Branching is supported by bootstrap values >85%.
Figure 15 Electron micrographs of purified virus particles (far left) and core particles (second left) of Hyposoter exiguae idnoreovirus-2 (HelRV-2), stained with uranyl acetate (courtesy of A. Makkay and D. Stoltz). Electron micrographs of a virus particle (second right) and core particle (far right) from purified preparations of Dacus oleae idnoreovirus-4 (DoIRV-4), stained with sodium phosphotungstate (courtesy of M. Bergoin). DoIRV-4 virions have small icosahedrally arranged surface projections (estimated 12 in number). The DoIRV-4 cores have twelve large icosahedrally arranged spikes or turrets, which (like those of the cypoviruses) may lose a portion near to the tip.
Figure 16 Negative contrast electron micrograph of particles of Aedes pseudoscutellaris reovirus purified using Iodixanol (Optiprep®) gradient
(courtesy of H. Attoui).
Figure 17 Agarose gel electrophoretic profiles of genome segments of Aedes pseudoscutellaris (propagated in C6/36 cells) in 1% agarose gel. These migration patterns (electropherotype) are thought to be characteristic of each virus species.
Figure 18 Negative contrast electron micrograph of particles of Colorado tick fever virus (CTFV) (courtesy of F. A. Murphy). The bar represents 50 nm.
Figure 19 Electrophoretic profiles of the genome segments of Colorado tick fever virus isolate Florio (CTFV-Fl) and California hare virus (CTFV-Ca) in 1% agarose gel. Genome migration patterns (electropherotype) are thought to be characteristic of each virus species.
Figure 20 (Top) Diagram of the bluetongue virus (BTV) particle structure, constructed using data from biochemical analyses, electron microscopy, cryoEM and X-ray crystallography (courtesy of P.P.C. Mertens and S. Archibald). (Bottom) Electron micrographs of African horse sickness virus (AHSV) serotype 9 particles stained with 2% aqueous uranyl acetate (left) virus particles, showing the relatively featureless surface structure. (Center) Infectious subviral particles (ISVP), containing chymotrypsin cleaved outer capsid protein VP2 and showing some discontinuities in the outer capsid layer. (Right) core particles, from which the entire outer capsid has been removed, to reveal the structure of the VP7(T13) core surface layer and showing the ring shaped capsomeres
(courtesy of P. P. C. Mertens).
Figure 21 (Top left) The outer capsid layer of bluetongue virus (BTV) (from cryoEM) showing trimers of VP2 in red and trimers of VP5 in yellow, superimposed on the underlying X-ray crystallography structure for the BTV core. (Top right) The structure of the BTV core as determined by X-ray crystallography of the native core particle. The outer core surface, composed of 260 trimers of VP7 arranged with T=13 l symmetry. The chemically identical but structurally different trimers are named and colored in order of increasing distance from the five-fold axes of symmetry (P [red], Q [orange], R [green], S [yellow] and T [blue] situated at the three-fold axes). (Bottom left) The BTV 1 subcore shell (from X-ray crystallography) is composed of 120 copies of VP3, arranged with T=2 symmetry. The chemically identical but structurally different molecules are shown: “A” (green: surrounding the five-fold axis) and “B” (red: surrounding the three-fold axis). (Bottom right) Model cross-section of the BTV core showing packaging of the dsRNA as four concentric shells
(courtesy of D. I. Stuart, J. Grimes, P. Gouet, J. Diprose, R. Malby, P. Roy, B. P. V. Prasad and P. P. C. Mertens).
Figure 22 Phylogenetic tree of the T2 subcore shell proteins of members of the genus Orbivirus. The tree shows two groups: a mosquito-borne/tick-borne group where the second largest viral protein (VP2) forms the “T2” sub-core capsid layer; and a Culicoides-borne group where the third largest viral protein (VP3) forms the T2-layer. Sequences were aligned using ClustalX and the tree constructed in MEGA4 using the neighbor-joining method and the P-distance algorithm. Branching is supported by bootstrap values >85%.
Figure 23 Phylogenetic tree, constructed using complete amino acid sequences of the cell attachment protein (VP2 of the Culicoides-borne viruses, VP3 of the mosquito-borne viruses and VP4 of Great Island virus, which is tick-borne). In bluetongue virus (the Orbivirus type species) VP2 is the larger of the two outer CPs and is the most variable and primary neutralization antigen. Amino acid sequences were aligned using Clustal X and the trees were constructed using the neighbor-joining method and P-distance in MEGA4 (courtesy of H. Attoui). Names and abbreviations correspond to those used in the list of species. Sequences were aligned using ClustalX and the tree constructed in MEGA4 using the neighbor-joining method and the P-distance algorithm. Branching is supported by bootstrap values >85%.
Figure 24 Rotavirus morphology. (Left panel) Cartoon representation of a rotavirus triple-layered particle, with proteins labeled. Black corkscrews represent segments of genomic dsRNA. The precise locations of VP1 and VP3 have not been determined. (Right panel) Electron micrograph of rotavirus particles viewed by negative staining. Bar represents 100 nm.
(Provided by B. V. V. Prasad.)
Figure 25 Structure and location of protein components of the rotavirus virion. A cutaway view of a cryoEM image reconstruction of a rotavirus virion at 9.5 Å resolution (center) is a reference for enlarged, high-resolution images of specific virus components. The particle and components are colored as follows: VP4 spikes (red), VP7 layer (yellow), VP6 layer (blue) and VP2 layer (green). A portion of VP2 that extends into the interior of the core (the “hub”) and transcriptional enzymes VP1 and VP3 are colored gold in the enlarged image of a five-fold vertex (bottom, center). The VP8* and VP5* cleavage products of the VP4 spike are indicated. The trimeric foot and dimeric stalk and head of VP4 can be seen. PDB files 3KZ4 (VP6 and VP2) and 3GZT (VP7) were used to make images.
(Courtesy of B. V. V. Prasad.)
Figure 26 Phylogenetic tree comparing amino acid sequences of the rotavirus inner core shell protein VP2 (left) and intermediate capsid protein VP6 (right). Isolates and accession numbers used are: Bo/UKtc (P17462,P18610), Si/SA11 (CAA34733, AAO32085), Hu/Wa (X14942, P03530), Po/Gottfried (ADE44250, P16593), Mu/ETD_822 (ACY95261), Mu/EDIM (AAC57838), Av/PO-13 (BAA24147, BAA03836), Hu/WH-1 (AAT09117, AAT09116), Hu/Bang117 (ADF57895, ADF57898), Bo/DB176 (ADC53105, ADC53099), Mu/IDIR (AAA17401, QO1754), Hu/Bristol (CAC44890, CAA42504), Po/Cowden (P26191, AAA47097), Av/05V0049 (ADN06424, ADN06428), Hu/J19 (YP_392491, AAZ03490), and Hu/B219 (ABR32123, ABA60393). Sequences were aligned and trees calculated (neighbor-joining method) using MEGA 4 (courtesy of J. Matthijnssens). Abbreviations used to indicate host species are: Av, avian; Bo, bovine; Hu, human; Mu, murine; Po, porcine; Si, simian. Sequences were aligned using ClustalX and the tree constructed in MEGA4 using the neighbor-joining method and the P-distance algorithm. Branching is supported by bootstrap values >85%.
Figure 27 (Top) Diagram of the Banna virus (BAV) particle structure, constructed using data from biochemical analyses, electron microscopy and X-ray crystallography. (Bottom) Negative contrast electron micrograph of Banna virus particles: (left hand side) full BAV particles showing multiple of protein spikes; (right hand side) double layered cores of BAV
Figure 28 Migration patterns (electropherotypes) of genome segments from isolates of Banna virus (BAV), Kadipiro virus (KDV) and Liao ning virus (LNV) in 1% agarose gel. Electropherotype is thought to be characteristic of each virus species.
Figure 29 Phylogenetic tree for homologous genome segments from members of the species Banna virus, Kadipiro virus and Liao ning virus. The tree is based on outer-capsid and cell attachment protein sequences (encoded by Seg9 (VP9) of BAV, its homologous Seg11 (VP11) of KDV and Seg10 (VP10) of LNV). This tree also shows the two genotypes (serotypes) of BAV (A and B). The amino acid identity between VP9 of BAV and VP11 of KDV ranges between 14% and 16%. Between VP9 of BAV and VP10 of LNV amino acid identity ranges from 17% to 19%. Between VP11 of KDV and VP10 of LNV amino acid identities ranged from 17% to 18%. Between the two genotypes of BAV, it ranges from 41% to 43%. Within a given genotype of BAV it ranges between 90 and 96%. Between the two genotypes (serotypes) of LNV amino acid identity is 81%. Sequences were aligned using ClustalX and the tree constructed in MEGA4 using the neighbor-joining method and the P-distance algorithm. Branching is supported by bootstrap values >85%.
Figure 30 (Top left) Schematic diagram representing a T=13 capsid structure. (Top right) Negative contrast electron micrograph of rice gall dwarf virus particles, negatively stained with phosphotungstic acid. The bar represents 50 nm. (Bottom left) Electron cryo-microscopic image and 25 Å resolution 3D structure of the double shelled rice dwarf virus (RDV). (Bottom center) Inner shell computationally extracted with 59 nm diameter. It exhibits T=1 lattice. Dashed triangle designates one triangular face of the icosahedron. (Bottom right) Schematic diagram of fish-shaped density distribution within a triangle in a T=1 lattice.
(Courtesy of Hong Zhou and Wah Chiu, from Lu et al. (1998). J. Virol., 72, 8541–8549.)
Figure 31 Phylogenetic (neighbor-joining) tree based on phytoreovirus Seg8 sequences from the following accession numbers HoVRV-BA (GU362071), HoVRV-FI (FJ497796), HoVRV-ME (GU369689), HoVRV-NC (GU384990), HoVRV-PA (GU370369), HoVRV-RI (GU350428), HoVRV-RO (GU395195), HoVRV-SC (GU390596), HoVRV-TI (GU437834), RDV-A (D10219), RDV-B (D00536), RDV-CN (U36565), RDV-S (D13773), RGDV-BL (AY999077), RGDV-CH (AY999078), RGDV-DQ (AY999079), RGDV-GD (AY216767), RGDV-GX (DQ364683), RGDV-GZ (AY999080), RGDV-TJ (D13410), RGDV-XY (AY999081), TLEV (AY587759), WTV (J04344). Tree was produced in MEGA4 (maximum composite likelihood distances) with 10,000 booststrap replicates (values shown when >60%).
Figure 32 Negative contrast electron micrograph of CsCl purified particles of Eriocheir sinensis reovirus.
(From Zhang et al. (2004). J. Fish Dis., 27, 687–692.)
Figure 33 (A) MpRV virus particle purified on Percoll®, with a diameter of 95 nm. A damaged particle is shown at the upper left corner, showing an outer layer about 15 nm thick (indicated by a white arrow), surrounding a more compact structure with a diameter of about 75 nm (indicated by a black arrow). (B) A virus particle purified by CsCl gradient centrifugation, with a diameter of about 75 nm. (C) Particles pelleted from the clarified lyaste of infected Micromonas pusilla. Some particles (indicated by arrow) have a larger diameter. (D) Particles generated by treating whole MpRV particles (purified on Percoll®) with 1.5 M CaCl2 and subsequent purification on CsCl gradient, generating cores (or sub-cores) with smooth outline.
(Courtesy of H. Attoui and C. Brussaard.)
Figure 34 Agarose gel electrophoretic profile (electropherotype) of genome segments of MpRV in 1% agarose gel. These migration patterns (electropherotype) are thought to be characteristic of each virus species.
Figure 35 Neighbor joining tree constructed with the AA sequences of putative RdRp of representative viruses from the following genera of the family Reoviridae [accession numbers]: Seadornavirus, Banna virus: isolate BAV-Ch [AF168005], Kadipiro virus: isolate KDV-Ja7075 [AF133429], Liao ning virus: isolate LNV-NE9731 [AY317099]. Coltivirus, Colorado tick fever virus, isolate CTFV-Fl [AF134529], Eyach virus, isolate EYAV-Fr578 [AF282467]. Orthoreovirus, Mammalian orthoreovirus, serotype-1 (MRV-1) [M24734], serotype-2 (MRV-2) [M31057], serotype-3 (MRV-3) [M31058], serotype-4 (MRV-4) also known as Ndelle virus [AF368033]. Aquareovirus, Aquareovirus C, isolate golden shiner virus (GSRV) [AF403399], grass carp reovirus (GCRV) [AF260512], Aquareovirus A, isolate striped bass reovirus (SBRV) [AF450318], isolate chum salmon reovirus (CSRV) [AF418295], Aquareovirus G isolate golden ide reovirus (GIRV) [AF450323]. Orbivirus, African horse sickness virus, serotype-9 (AHSV-9) [U94887], Bluetongue virus, serotype-2 (BTV-2) [L20508], serotype-10 (BTV-10) [X12819], serotype-11 (BTV-11) [L20445], serotype-13 (BTV-13) [L20446], serotype-17 (BTV-17) [L20447], species Palyam virus, isolate CHUV [Baa76549], St Croix River virus, isolate SCRV[AF133431]. Rotavirus, Rotavirus A, strain BoRV-A/UK [X55444], strain SiRV-A/SA11 [AF015955], Rotavirus B, strain Hu/MuRV-B/IDIR [M97203], Rotavirus C, strain PoRV-C/Co [M74216], Fijivirus, species Nilaparvata lugens reovirus, strain NLRV-Iz [D49693]. Phytoreovirus, species Rice dwarf virus, isolate RDV-Ch [U73201], isolate RDV-H [D10222], isolate RDV-A [D90198]. Mycoreovirus, species Mycoreovirus-1, isolate CpMYRV-1 [AY277888], species Mycoreovirus-3, isolate RnMYRV-3 [AB102674]. Oryzavirus, isolate Rice ragged stunt virus, strain RRSV-Th [U66714]. Cypovirus, Bombyx mori cytoplasmic polyhedrosis virus-1 strain BmCPV-1 [AF323781], Dendrlymus punctatus cytoplasmic polyhedrosis virus-1 strain DsCPV-1 [AAN46860], Lymantria dispar cytoplasmic polyhedrosis virus-14 strain LdCPV-14 [AAK73087]. Dinovernavirus, species Aedes pseudoscutellaris reovirus, isolate APRV [DQ087276]. Cardoreovirus, species Eriocheir sinensis reovirus, isolate ESRV [AY542965]. Mimoreovirus, Micromonas pusilla reovirus, isolate MPRV [DQ126101]. Values at the nodes represent bootstrap confidence levels (500 replications).