Orbiviruses
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The document discusses the genus Orbivirus, which contains 22 virus species. Orbiviruses have segmented double-stranded RNA genomes and non-enveloped capsids with distinctive doughnut-shaped capsomeres. They primarily infect animals but can occasionally cause illness in humans through tick bites. Orbiviruses replicate cytoplasmically in host cells and are able to cause a range of outcomes from subclinical infection to severe disease depending on virus and host factors.
Orbiviruses
- 1. Orbiviruses By- Sanju Sah St. Xavier’s college, Maitighar, Kathmandu Department of Microbiology
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- 4. Introduction • The genus Orbivirus is a member of the Reoviridae family, in the subfamily Sedoreovirinae. • Orbiviruses are named for their doughnut-shaped capsomeres (orbi means “ring” in Latin). • This genus currently contains 22 species (including the type species Bluetongue virus). • The name of ‘bluetongue’ was later used to describe the distinctive cyanotic tongue of some severely affected sheep. • The only orbiviruses believed to infect humans are the tick-borne Kemerovo viruses of Siberia and the related Tribec virus found in Slovakia. 4
- 5. • The primary reservoirs are rodents and birds, and infection of humans following a tick bite can lead to a febrile meningoencephalitis but without paralysis, similar to the better-known tick-borne encephalitis (TBEV) a flavivirus. • The BTV group, which consists of at least 24 different serotypes (BTV-1, -2, etc.), infects Culicoides species and a number of wild and domestic ruminants (e.g., sheep, cattle), occasionally causing high morbidity and mortality, but often with no apparent clinical symptoms. • Other Orbiviruses that can cause disease in animals include African horse sickness virus (AHSV; nine serotypes, AHSV-1, -2, etc.) and epizootic hemorrhagic disease virus of deer (EHDV; nine serotypes, EHDV-1, 2, etc.). 5
- 6. There are currently 22 recognized virus species as well as 13 unassigned viruses in this genus • Species (with constituent viruses): • African horse sickness virus • Bluetongue virus • Chuzan virus • Corriparta virus • Epizootic haemorrhagic disease virus • Equine encephalosis virus • Eubenengee virus • Eubenengee virus • Ngoupe virus • Tilligerry virus • Great Island virus • Broadhaven virus • Great Island virus • Kemerovo virus • Lipovnik virus and Tribec virus 6
- 7. •Lake Clarendon virus •Palyam virus CSIRO Village virus •Pata virus •Peruvian horse sickness virus Elsey virus •St Croix river virus •Umatilla virus Stretch Lagoon virus Umatilla virus •Wallal virus •Warrego virus •Wongorr virus •Yunnan virus Middle Point virus 7
- 8. Structure • Orbiviruses are non enveloped viruses with two protein shells and a genome consisting of double-stranded RNA (dsRNA) segments. • The viron size is 70–80 nm in diameter. • The outer capsid has an icosahedral configuration, and the two proteins of the capsid have distinctive shapes, one globular and almost spherical, the other sail-shaped. • The globular structures, 120 in number, sit neatly in the spaces formed by each of the six-membered rings of VP7 trimers. 8
- 9. • The sail-shaped spikes, which project 4 nm beyond the globular proteins, are located above 180 of the VP7 trimers and form 60 triskelion-type motifs that cover nearly all the VP7 molecules. • These spikes are believed to be the hemagglutinating protein VP2 that also contains the neutralizing epitopes. • The underlying globular proteins are probably composed of VP5. • The outer layer of the core is made up of clusters of VP7 trimers, which exist as prominent triangular protrusions, and are located at all the local and strict threefold axes. 9
- 10. • There are a total of 780 VP7 molecules per particle, forming 260 trimers, or triangular capsomers, that are arranged around distinctive 132 channels, which are present at all 5- and 6-coordinated positions of the T=13 lattice • VP1, VP4 and VP6 are minor enzymatic proteins that are packaged along with the ten genome segments within the central space of the virus core. • The virion is constructed of two concentric protein shells, the sub- core layer which contain 120 copies/particle of the VP3. • The inner layer, which is made up of the second major protein, VP3 is relatively featureless and exhibits a smooth bed of density, with almost spherical shape but it has an angular appearance owing to variations in thickness of the protein shell. 10
- 11. • The orbivirus outer-capsid layer is composed of two additional structural proteins (VP2 and VP5) which mediate cell- attachment and penetration during initiation of infection. • The outer-capsid proteins are more variable than the core proteins and most of the non-structural proteins and the specificity of their reactions with neutralizing antibodies determines the virus serotype. • No lipid envelope is present. 11
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- 13. Genome • Segmented linear dsRNA genome and therefore are classified as Class III viruses. • It contains 10 segments coding for 12 proteins. • One copy of each gene segment is packaged per virion. • Segments size ranges from 822 to 3,954 bp (BTV). • Genome total size is 19,200 bp (BTV). • In most cases each gene segment encodes a single open reading frame (ORF). 13
- 14. • The 10 dsRNA segments of BTV have conserved terminal sequences. • Both strands of each of the 10 dsRNA segments are entirely complementary, forming complete duplexes. • However, the 5' terminus of the coding strand of each duplex is capped and methylated, as are the BTV mRNA transcripts . • The opposing, noncoding strands in the virion dsRNA species are neither capped nor methylated. • For the mRNA sense strand of all segments, the conserved terminal sequences involve eight nucleotides at the 5' ends and six nucleotides at the 3' ends. 14
- 15. • The 3'-noncoding sequences are generally longer, varying from 31 (M5) to 116 (S10) nucleotides. • The genome encodes 7 major structural proteins (VP1–VP7) and 3 major non structural proteins (NS1-NS3). • Each genome segment of BTV codes for the synthesis of one major protein, except segment S10, which codes for two related proteins: NS3 and NS3A. • In addition to the seven structural proteins, four nonstructural proteins (NS1, NS2, NS3, and NS3A) are also synthesized in infected cells. 15
- 16. • The structural proteins are numbered VP1 to VP7 in order of their decreasing sizes. • The outercapsid proteins VP2 and VP5 are encoded by RNA segments L2 and M5, respectively. • The VP2 protein of BTV is a hemagglutinin and is directly involved in the attachment of virus to cells. • VP5 is capable of inducing membrane destabilization and permeabilization. • In virus-infected cells, parental BTV virions (550S) are converted to core particles (470S) containing VP1, VP3, VP4, VP6, and VP7 and exhibiting RNA-dependent RNA polymerase activity. 16
- 17. • This enzymatic activity allows the virus to synthesize mRNA from virion RNA templates. • The three minor proteins VP1, VP4 and VP6, together with 10 dsRNAs, form the innermost part of the core. • All three proteins are highly conserved across the different BTV serotypes that have been analyzed to date. • The primary sequence of VP1 contains a GDD motif characteristic of other RNA polymerases. • The second minor protein of the core, VP4 is encoded by M4 RNA. 17
- 18. • The protein has a high content of charged amino acids and exists as a dimer in solution and within the core. • A leucine zipper in the center of the molecule is responsible for the dimerization of VP4. • The dimeric form of VP4 is essential for encapsidation of the protein within the core. • The third minor protein, VP6, is encoded by S9 RNA and is very rich in basic amino acids. • VP6 may bind to nucleic acids. • Like other helicase proteins,VP6 appears to form hexameric oligomers in the presence of RNA. 18
- 19. • VP6 binds dsRNA (as well as ssRNA) may be relevant to the encapsidation process and sequestering of dsRNA species into precursor cores and virions. • The VP3 and VP7 proteins are the two major proteins of the core; VP7 is the more abundant . • The larger protein, VP3, forms an inner scaffold for the deposition of the surface layer of VP7 in the core. • Four different nonstructural proteins have been identified in BTV-infected cells. • The two major nonstructural proteins, NS1 and NS2, are synthesized abundantly, whereas the two minor, closely related proteins, NS3 and NS3A, are barely detectable. 19
- 20. • The sequences of each NS protein are highly conserved (96%) among BTV serotypes. • The synthesis of NS1 and NS2 in BTV-infected cells coincides with that of two virus-specific structures, tubules, and the granular inclusion bodies (IBs), that are characteristic for orbivirus-infected cells. • NS1 is the most abundant protein in BTV infected cells. • It forms tubules that may be involved in translocation of progeny virus particles to the cell membrane • The second nonstructural protein, NS2, is the only virus-specific phosphoprotein that is present in BTV-infected cells . 20
- 21. • NS2 is encoded by RNA segment S8 and is rich in charged amino acids. • Unlike the NS1 and NS2 proteins, NS3 and NS3A are the two smallest nonstructural proteins. • Exceptions to the one gene one protein rule are segment 9 (Seg-9) and segment 10 (Seg-10) both of which encode two nearly identical proteins initiated from in-phase AUG codons close together near the upstream termini (VP6 and VP6a encoded by Seg-9: NS3 and NS3a encoded by Seg-10). • VP6(a), NS3a and NS4 are presumably expressed by leaky scanning. 21
- 22. Epidemiology • Internationally, only infrequent cases of clinical illness in humans have been reported in Russia, Eastern Europe, Africa, and South and Central America. • To date, fewer than 50 cases have been described. • All age groups may be infected with orbiviruses. • However, sero-prevalence studies suggest that most infections occur in childhood. 22
- 24. Replication • Replication is cytoplasmic that means it occurs in cytoplasm. • Virus adsorbs rapidly to susceptible cells at both 4°C and 37°C, with the maximal adsorption occurring within 20 minutes. • The cell receptors involved in orbivirus infection have not been identified, although some information is available suggesting their nature. • In early studies, analysis of BTV–erythrocyte interactions indicated that the virus binds to a sialoglycoprotein, the glycophorin component of human. 24
- 25. • Virus is subjected to endocytosis after binding to a receptor on a cell plasma membrane through VP2 of the outer capsid . • After invagination and detachment from the cell surface, a coated vesicle containing the virus is formed. • The clathrin coat of this vesicle is rapidly lost, leading to the formation of a large translucent, endocytic vesicle . • BTV is converted to core particles that appear to lack all the VP2 and most of the VP5 of the outer capsid layer. • Removal of both outer capsid proteins appears to be required to activate the virion transcriptase. • In the cytoplasm, the partially or completely uncoated particles bind to the cell cytoskeleton (specifically the intermediate filaments) and initiate transcription of viral mRNA species. 25
- 26. • mRNA synthesis can be activated by proteolytic digestion of the outer capsid of the virus . • This facilitates access of NTPs to the genome within the core and the extrusion of the newly synthesized mRNA to the cytoplasm through the channels along the fivefold symmetry axes. • VP6 helicase unwinds the genomic duplex RNA molecules, whereas VP1 polymerase initiates the synthesis of positive- stranded RNA species from the 3' end of each negative- stranded RNA segment of the dsRNA molecule. • Negative-stranded RNA species are synthesized from the 3' end of the positive-stranded RNA molecule, and VP1 alone is able to initiate the replicase activity. 26
- 27. • Once the positive-stranded RNA molecules are synthesized, the 5'-termini of these molecules are modified by the enzymatic activities of VP4. • The class I methylated cap structure is formed solely by VP4, and thus both genomic positive-stranded RNAs and the 10 mRNAs possess the 5'-methylated cap structure . • The mRNA species are capped and methylated during the transcription process. • They are extruded from subviral particles into the cytoplasm and program the synthesis of virus proteins. 27
- 28. • The secondary structures of the mRNAs may facilitate mRNA egress from particles and may also contribute to the overall stability of the mRNA species within the cytoplasm of infected cells. • The smaller genome segments are generally the most frequently transcribed. • The packaged positive strand serves as a template for synthesis of a negative strand, and once the negative strand is synthesized, it remains within the nascent progeny particle. • Infection of cells leads to a rapid inhibition of cellular macromolecular synthesis. • Most proteins are synthesized throughout the infection cycle and accumulate during infection until cell lysis. 28
- 29. • Recent data have shown that the two proteins are derived from initiation of protein synthesis at two distinct initiation codons in the S9 mRNA and not by post-translation modification. • During infection of cells, a reduction in the number of microtubules occurs with some fragmentation of the microtubule network near the cell membrane. • Microfilaments also change during infection. • The involvement of the cytoskeleton in multiplication has been indicated by its close association with BTV antigens and virus-specific structures, including IBs, tubules, and 55-nm diameter, virus-derived particles. 29
- 30. • During infection, a fibrillar network develops around the infecting core particle in the cytoplasm. • Viral capsid proteins, such as core proteins VP3 and VP7, are also present in the IB matrix, indicating that this may be the assembly site of core particles either within or at the periphery of the IBs. • The presence of electron-dense virus-derived immature particles inside IBs has led to the belief that core particles may be synthesized within IBs and that the further stages of mature virion development occur either in the cytosol or after binding of the particles to the cytoskeleton. • A hallmark of orbivirus infections is the presence of tubules throughout the cytoplasm. 30
- 31. • Expression of VP3 and VP7 in the presence of VP1, VP4, or VP6, or any combination of these, leads to the encapsidation of these minor proteins. • Once the stable core is assembled, the two outer capsid proteins VP2 and VP5 are acquired by interaction with the core surface, both proteins being attached to the VP7 layer of the core. • The mechanism whereby virion is released from infected cells has been the subject of some speculation and appears to depend on the cell type. 31
- 32. • Egress of virus particles from infected cells is accomplished by a process of extrusion in which individual or groups of virus particles move through a locally disrupted plasma membrane. • Assembled particles were released from infected cells in the presence of the recombinant NS3 proteins. 32
- 33. Pathogenesis • Orbiviruses produce a spectrum of consequences from infection, reaching from severe and fatal disease to subclinical infection, depending on virus or host strain. • Virus is transmitted to vertebrate hosts through blood- feeding insect vectors, and infectious particles migrate to lymph nodes where they initially replicate and subsequently spread to spleen, thymus, and other lymph nodes, becoming associated with circulating blood cells, where virus may persist for weeks or months. • A distinct feature of orbivirus infections is their capacity to induce prolonged cell-associated viremias in vertebrate hosts, including both the species involved in virus transmission cycles and clinical hosts. 33
- 34. • Infectious virus can be detected for up to 8 weeks, and viral nucleic acid can be detected in erythrocytes for up to 20 weeks. • Kemerovo complex virus of genus Orbivrus may cause maningoencephalitis in humans. • Many orbiviruses have a predilection for the vascular endothelial cells, which can lead to disruption in blood flow and subsequent ischemia. • Orbiviruses also have a tropism for the nervous system, leading to encephalitis and, possibly, neuropathies. 34
- 35. Clinical manifestations • In cases of Oklahoma tick fever or Kemerovo or Lipovnik virus infections, a tick transmits the virus. • Oklahoma tick fever is now considered a new arboviral disease in the western United States that is related to the Kemerovo serogroup of orbiviruses. • Although incubation time from exposure to clinical illness is unknown in humans, it has been estimated at 6-9 days in animals. • Based on serologic studies, orbiviral infections often are asymptomatic or present as a mild nonspecific febrile illness for which medical care is not sought. 35
- 36. • Patients who present for medical evaluation often report fever, myalgia, nausea, vomiting, diarrhea, and confusion. • Seizures and, rarely, focal neurologic deficits may also develop. • Several patients had concomitant cytopenia (transient thrombocytopenia, anemia, and/or leukopenia). • Each patient recalled tick exposure; however, the specific causative tick is currently unknown. 36
- 37. Laboratory diagnosis • Specimen: Blood, serum and CSF • The serum may be used for viral isolation and for serology. • In orbiviral infections, the protein and white blood cell count may be slightly elevated. • In patients presenting with neurologic infection, collect cerebrospinal fluid (CSF) for cell count and differential, protein, glucose and viral culture. • The CSF may be examined for evidence of an orbiviral infection with serology and/or viral isolation. 37
- 38. • Testing specifically for orbiviral infection may be done via serological studies or viral isolation from the serum or CSF. • The presence of viral-specific immunoglobulin M (IgM) in the CSF suggests acute infection. • Serologic studies may include complement fixation, enzyme immunoassay, and neutralizing antibodies. • The serologic diagnosis requires a 4-fold rise in acute and convalescent antibody titers. • Viral isolation may be accomplished by the inoculation of suckling mice or by cell cultures (ie, Vero or BHK-21 cells). BHK means Baby Hamster kidney cells 38
- 39. Vaccines • Mixtures of recombinant proteins and multicomponent empty particles, such as CLPs and VLPs, have potential for development of efficacious and safe vaccines against orbivirus infections and disease. 39
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