Draft of the orbivirus genus description

for the ICTV Seventh Report

Last updated: 24 February 1998

Comments to: Peter Mertens

GENUS ORBIVIRUS

Type Species bluetongue virus (BTV)

DISTINGUISHING FEATURES

Virions have an indistinct outer capsid and a genome composed of 10 segments of dsRNA. Core particles have characteristic ring shaped capsomers. Replication is accompanied by production of viral "Tubules" and viral inclusion bodies (VIB). They are transmitted between vertebrate hosts by a variety of hematophagous arthropods.

VIRION PROPERTIES

MORPHOLOGY

Virions of BTV are about 80 nm in diameter, core particles have a maximum diameter of 73 nm, sub core have a maximum diameter of 59 nm and an internal diameter of 38 nm. The virion is spherical in appearance but has icosahedral symmetry. Although no lipid envelope is present on mature virions, they can leave the host cell by budding through the cell membrane. During this process they transiently acquire an unstable membrane envelope. Unpurified virus is often associated with cellular membranes. Surface projections are only observed on virions where the particle structure is maintained (e.g., using cryoelectron microscopy). Otherwise, by conventional electron microscopy, the surface of virions is indistinct (Fig. 3). The core particle contains an inner capsid shell (the sub core layer), which surrounds the 10 ds RNA genome segments and the minor core proteins (the transcriptase complexes) that are attached to its inner surface at the 5 fold symmetry axes. Assembly of the sub core layer appears to control the overall size and symmetry of the particle. The surface layer of the core is composed of capsomeres arranged as hexameric rings (pentameric at the 5 fold axes). These rings, which are readily observed by conventional electronmicroscopy, give rise to the name of this genus.

Figure 3: (left) Image of the surface arrangement of BTV as deduced by cryoelectron microscopy (E Hewat ); (center left) electron micrograph of BTV (PPC Mertens); (center right) image of the BTV core particle (courtesy of Prasad BVV); (right) electron micrograph of BTV-derived core (courtesy of Mertens PPC). Atomic structure of the BTV1 Core , Atomic structure of the BTV1 subcore The bar represents 20 nm.

PHYSICOCHEMICAL AND PHYSICAL PROPERTIES

The virion Mr is about 10.8 x 10⁶, the core Mr is about 6.7 x 10⁶. The buoyant density in CsCl are 1.36 g/cm³ (virions) and 1.40 g/cm³ (cores). The S_20W is 550 (virions) and 470 (cores). Virus infectivity is stable at pH 8-9 but virions exhibit a marked decrease in infectivity outside the pH range 6.5 -10.2. In part, this may be related to the loss of outer coat proteins, particularly at the lower pH range. The sensitivity of the outer capsid proteins and their removal by cation treatment varies markedly with both pH and virus strain. At low pH values (less than 5.0), virions and cores are both disrupted. Unlike orthoreoviruses, at pH 3.0 virus infectivity is abolished. In blood samples, or serum, or albumin, viruses held in vitro may remain infectious for decades at less than 15 ^o C. Purified BTV virions held at 4 ^o C in 0.1M tris/HCl pH 8.0, showed no significant reduction in infectivity after 1 year. Core particles are very stable when kept at 4 ^o C. virus infectivity is rapidly inactivated on heating to 60 ^o C. In general, orbiviruses are considered to be relatively resistant to treatment with solvents, or detergents. However sodium dodecyl sulphate will disrupt the particle and destroy its infectivity. Freezing reduces virus infectivity by about 90%. When held at -70 ^o C virus infectivity remains stable.

NUCLEIC ACID

Virus particles and cores contain 12% and 19.5% RNA, respectively. The genome is composed of 10 linear dsRNA segments that are packaged in exactly equimolar ratios, one of each segment per particle. The genomic RNA is packaged as a series of ordered concentric shells within the VP3 (T2) layer of the core. Four layers of RNA, each of which has elements of icosahedral symmetry, can be detected by X-ray crystallography of the BTV core. There appears to be an association between the dsRNA molecules and protein density at the five fold axes of symmetry (vertices of the icosahedron), which is thought to represent the transcriptase complexes (TCs). From this point, the RNA appears to spiral outward around the TC for two turns until it clashes with an icosahedrally related neighbour. At this point it is thought to move inward forming the next concentric shell of RNA. For bluetongue viruses, the genome segments range in size from 3,954 to 822 bp (total size is 19.2 kbp, total M_r of 13.1 x 10⁶). There is no evidence for short ssRNA oligonucleotides in intact virions. The genomic RNAs are named "segment 1 to 10", in order of increasing electrophoretic mobility in 1% agarose gels. For bluetongue viruses, the segments migrate as three size classes 3 large (segment 1-3: 3.9-2.8 kbp), 3 medium (segments 4-6: 2.0-1.6 kbp) and 4 small segments (segments 6-10: 1.2-0.8 kbp). For other members of the genus, different sizes and size classes exist. For an individual virus species the dsRNA sizes from different isolates, or different serotypes are comparable, such that a uniform segment migration pattern is observed when genomic RNA is analysed by agarose gel electrophoresis. Variations in primary sequence can cause significant variations in rate and order of migration of genome segments during PAGE, particularly in high percentage gels (> 5% polyacrylamide). In the genome segments analysed to date, there is only a single major open reading frame (ORF), which is always on the same strand (see conserved terminal sequences below). However, the ORF may have more than one functional initiation site near to the 5' end of the RNA, resulting in production of two distinct but related proteins. For BTV-10, the 5' non-coding sequences range from 8 to 34 bp, for the 3' ends they are 31 to 116 bp in length. For other serotypes and other viruses the lengths differ; in general, however, the 5' non-coding regions are shorter than the 3' non-coding sequences. The non-coding regions of BTV and EDHV include terminal sequences of 6 bp that are identical for all 10 dsRNA segments (so far reported) and which are conserved between virus isolates. For the mRNA sense strands these sequences are 5' GUUAAA....ACUUAC 3'. Other orbiviruses have terminal sequences comparable to those of BTVs, but which are not always identical and which may not be conserved in all 10 segments, for example in AHSV these sequences are 5' GUU^A/_UA^A/_U.....AC^A/_UUAC 3'.

PROTEINS

There are 7 virus structural proteins (VP1-7). Proteins constitute 88% and 80.5% of the dry weight of virions and cores, respectively. For BTVs, the outer capsid consists of 180 copies of the 111 kDa VP2 protein arranged as triskellion structures, and 360 copies of an interdispersed and underlying VP5 protein (59kDa), which may be arranged as 120 trimers. Both VP2 and VP5 are attached to VP7. The surface of the core particle consists entirely of 780 copies of VP7 (T13), arranged with T=13 l symmetry (in a near perfect example of quasi-equivalence) as a network of hexameric and pentameric rings. Beneath the VP7 (T13) layer, the sub core capsid shell is composed of 120 copies of VP3 (T2) arranged with T=2 symmetry, displaying "quasi-quasi-equivalence". VP3 (T2) encloses the 10 dsRNA segments of the genome and the three minor proteins, which include: the 150 kDa VP 1 (Pol) which is the RNA polymerase; the 76 kDa VP4 (Cap), which is both the guanylyl transferase as well as transmethylase 1 (forming the 7 methyl guanosine of the cap structure) and transmethylase 2 (forming the 2-0 methyl guanosine of the cap structure); and the 36 kDa VP6/ VP6a (NTPase), which binds ssRNA and dsRNA, has helicase and NTPase activities.

X-ray diffraction studies indicate that the minor proteins are attached as a transcriptase complex to the inner surface of the sub core layer (VP3 (T2)) at the 5 fold symmetry axes ( at the vertices of the icosahedron). However, because there is only a single transcriptase complex at each position they do not have full icosahedral symmetry and it is not yet possible to determine their organisation at the atomic level. Other members of the genus may have proteins with significant differences in sizes.

There are three distinct non structural viral proteins, produced in cells infected with BTV or other orbiviruses. The 64 kDa NS1 (TuP) protein forms tubules that vary in length up to 4 µm and are of unknown function, but which are regarded as a characteristic feature of orbivirus replication. These tubules may have a ladder like structure, as observed in those of BTV and EHDV (68 and 52 nm in diameter) or may be finer (23 nm in diameter) with a reticular cross weave pattern, like those produced by AHSV. The 41 kDa NS2 (ViP) protein can be phosphorylated and is an important component of the matrix of VIB which are the site of virus replication and assembly. VIB also contain relative large amounts of the virus core proteins. NS2 (ViP) has ssRNA binding activity, suggesting an active role in replication and in conjunction with other virus proteins is believed to be involved in the recruitment of viral mRNA for encapsidation. The NS3/NS3a proteins are two small non structural membrane proteins (25 and 24 kDa) which are involved in the release of virus particles from cells, particularly insect vector cells which do not show CPE or high levels of cell death and become persistently infected. In this process the NS3 proteins are also released from the cell.

CARBOHYDRATES

VP5 protein may be glycosylated. NS3 and NS3a synthesised in mammalian cells can become glycosylated, forming high molecular weight products

GENOME ORGANIZATION AND REPLICATION

§: protein structure/function: RNA polymerase = "Pol"; Capping enzyme = "Cap"; Virus structural protein with T = 13 symmetry = "T13". Virus structural protein with T = 2 symmetry = "T2". Viral inclusion body matrix protein=ViP. Virus tubule protein = TuP. Protein with NTPase activity = NTPase.

The BTV genome segment coding assignments are based on the dsRNA migration in 1% agarose are: segment 1-VP1; segment 2-VP2; segment 3-VP3; segment 4-VP4; segment 5- NS1; segment 6-VP5; segment 7-VP7; segment 8 - NS2; segment 9 - VP6/VP6a; segment 10 - NS3/NS3a. Cognate genes of other strains are similar. The segment 9 and segment 10 mRNA are translated from either of 2 in-frame AUG codons. The significance of the 2 forms of the S9 and S10 gene products (NS3, NS3A; VP6, VP6A) is not known. In some cases other virus proteins form morphologically defined structures in infected cells (e.g. the flat hexagonal crystals formed of VP7(T13) of AHSVs) but of unknown functional significance.

Virus adsorption involves components of the outer capsid, although cell entry may also involve VP7 (T13). The outer capsid layer is lost during the early stages of replication. The mRNA transcription frequency of individual genes varies, with more copies produced from the smaller segments. Details of the process of virus replication are lacking. The viral inclusion bodies (VIB) are considered to be the sites of morphogenesis of transcriptionally active virus cores containing dsRNA. The smallest particles observed in VIB containing RNA are to small to be cores and may represent progeny sub core particles. The outer capsid proteins are added at the periphery of these inclusion bodies. Virus particles are transported within the cell by specific interaction with the cellular cytoskeleton and can be released prior to cell lysis through interaction with membrane-associated NS3 proteins. There is also evidence of specific association between NS1 tubules and intact virions in the cell cytoplasm. In mammalian cells, replication of orbiviruses leads to shut-off of host protein synthesis and contributes to cell lysis and the further release of virus particles. In insect vector cells there is no evidence for shut-off of host protein synthesis, or for extensive cell lysis or CPE. These cells may be persistently infected. NS3 is particularly abundant in insect cells, where it may be essential for virus release and spread. Virus particles can leave viable mammalian cells by two distinct mechanisms, extrusion (involving cell membrane damage) and budding. Only budding has been observed in cells of the BTV vector Culicoides variipennis. Continuous release from infected cells and reinfection appears to be a feature of orbivirus replication.

ANTIGENIC PROPERTIES

In common with the other genera within the family Reoviridae, the prime determinant for inclusion of virus isolates within a single orbivirus species is compatibility for reassortment of genome segments during co-infection, thereby exchanging genetic information and generating viable progeny virus strains. Nineteen species of orbiviruses are currently recognized, in addition to a number of unclassified viruses. It has either been shown or is thought likely that reassortment can occur between at least some member viruses within each species. However, dAta providing direct evidence of segment reassortment between isolates is limited and serological comparisons (primarily of the immunodominant serogroup/species specific antigen VP7 (T13)), form the usual basis of diagnostic assays for each of the virus species (serogroups). Monoclonal or polyclonal antibodies against the outer core protein VP7 (T13) can neutralise core particle infectivity, but do not appear to attach to, or neutralise intact virus particles or ISVP in aqueous suspension. Other viral proteins are also conserved between virus serotypes (in particular core and certain NS proteins). However, some of these antigens may show cross-reactions with viruses in other species/serogroups, particularly those regarded as closely related. These cross-reactions are usually at a significantly lower level than with other viruses from the same species (serogroup) and may be "one way" (e.g., between African horse sickness virus, bluetongue virus, epizootic hemorrhagic disease virus, equine encephalosis virus and Eubenangee virus species (serogroups), or between Kemerovo virus, Wad Medani virus and Great Island virus species (serogroups)).

The BTV VP2 and VP5 proteins exhibit the greatest antigenic and sequence variation. BTV VP2 has hemagglutinin activity, which is destroyed by proteases such as trypsin or chymotrypsin and is not evident on the resultant infectious sub viral particles (ISVP: containing cleaved VP2). VP2 is involved indetermination of virulence. Each species includes a number of serotypes that can be identified and distinguished by serum neutralization assays (primarily via antibodies to the outer capsid proteins). VP2 is the main neutralization antigen of BTV, while VP5 is also involved in determination of virus serotype, possibly by imposing conformational constraints on VP2. In other viruses (Kemerovo complex viruses) these roles may be reversed. There is evidence that VP2 (particularly with VP5) and VP7 can be protective antigens.

In AHSV NS3 and NS3a are variable proteins and may be divided into three groups ( , and ) based on sequence analysis. Preliminary evidence suggests that on a serological basis NS3 cross reacts poorly between these groups. NS3 can also be involved in determination of virulence (AHSV).

BIOLOGICAL PROPERTIES

BTV core particles are significantly less infectious for mammalian cell cultures than intact viruses. Core infectivity varies from 1000 fold less than intact virions, to non infectious, depending on the mammalian cell line used. In insect vector cells core particles are only slightly less infectious than intact virions

Different orbiviruses infect a wide range of mammalian hosts including ruminants (domesticated and wild), equids, rodents, bats, marsupials, birds, sloths, and primates, including humans. Orbiviruses replicate in, and are primarily transmitted by, arthropod vectors (gnats, mosquitoes, phlebotomies, or ticks, depending on the virus). Trans-stadial transmission in ticks has been demonstrated for some viruses. Infection of vertebrates in utero may also occur. Orbiviruses, particularly those transmitted by short-lived vectors (gnats, mosquitoes, phlebotomines), are only enzootic in areas where adults of the competent vector species persist and are present all, or most of the year. For example, BTV and EHDV serogroup viruses are distributed worldwide between about 50^o North and about 30^o South in the Americas and between 40^o North and 35^o South in the rest of the world. However there is evidence for persistence of these viruses over winter in the absence of overt disease. Molecular mechanisms for persistence in the host species even at low levels may be of particular importance. Virus distribution also depends on the initial introduction into areas containing susceptible vertebrate hosts and competent vector species. For this reason not all serotypes of each serogroup (e.g., BTV serogroup) are present at locations where some serotypes are endemic.

Orbivirus infection of arthropods has no evident effect. Infection in vertebrates, can be inapparent to fatal, depending on the virus and the host. Some BTV strains cause death in sheep, others cause a variety of pathologies, including hemorrhagic conditions, lameness, oedema, a transitory cyanotic appearance of the tongue, nasal and mouth lesions, etc.; still others cause no overt pathology. BTV infection of cattle may show no signs of disease but involve long-lived viraemias. AHSV, EHDV (deer) and EEV can cause severe pathology in their respective vertebrate hosts. With mortality rates in serologically naive populations (AHSV) that can be over 98% .

LIST OF SPECIES PARAMETERS IN THE GENUS

Viruses within a single species (serogroup) can be identified by:

1) Serological comparisons of antigens or antibodies (by ELISA or by more traditional assays such as complement fixation (CF), or agar gel immunodiffusion (AGID) using either polyclonal sera, or monoclonal antibodies against VP7 (T13) or other conserved antigens);

2) RNA sequence analysis (e.g. segment 3);

3) Cross hybridization assays (northern or dot blots with probes made from viral RNA or cDNA;

4) PCR, (using primers conserved segments such as segment 3 or 7, can be coupled with cross hybridisation analysis (dot blots);

5) Identification by virus serotype with a virus already in a specific orbivirus species;

6) Analysis of "electropherotype" by agarose gel electrophoresis but not by PAGE. (Similarities can exist between closely related species);

7) identification of the conserved terminal regions of the genome segments (Some closely related species can have identical terminal sequences on at least some segments).

LIST OF SPECIES IN THE GENUS

Phylogenetic trees for segment 2, 3 and for segment 10 of AHSV

CONTRIBUTORS : Peter Mertens, Allan Gould, Ian Pritchard, Miles Nunn

References