Replication of ds RNA Viruses Flashcards
Family Reoviridae
Genera
- Orbivirus (Bluetongue virus); epizooty
- Rotavirus (human RV); diarrhea associated diseases in children
- Orthoreovirus
- a.o.
Genome
- segmented dsRNA, 10 - 12 segments
- 18 - 27 kB
- e.g. Bluetongue: 19.2 kb in 10 segments
Virion structure
- Complex, 3-layered protein shell
- Insensitive to detergents (non-enveloped)
- Glycoproteins
Reovirus architecture
- Infectious sub viral particle (2 layers)
- Core (1 layer)
Replicative cycle
- Receptor binding and endocytosis
- Acid-induced proteolysis of the virion within the endosome (see 6) = conversion of the virion into the infectious subviral particle (ISVP)
- Proteolytic conversion can take place already in the intestine
- Can penetrate through endosomal membrane into cytoplasm
- Cytoplasm: Conversion form IVSP to core particle
- Synthesis of capped viral mRNAs takes place in the core particle
- Association of mRNAs with newly translated proteins to RNase-sensitive sub viral particles SVP
- “Transcription” of mRNAs into (-)-strand RNA and formation of RNase resistent SVP with 10 dsRNA segments (reassortment?)
- Formation of virions by assembly of preformed outer capsid proteins
Cell entry of reoviruses
- Hemagglutinin binds cellular receptor
- Receptor mediates endocytosis into the endosome
- Proteolysis leads to ISVP and allows penetration through endosomal membrane
- Conversion into core particle; place of mRNA synthesis
- RNase insensitive
- Allows entry of nucleotides and start of mRNA transcription
3-layered reovirus-particle
insensitive to detergents
Stepwise dissociation:
Virion: VP1, 2, 3, 4, 6, 7
ISVP: VP1, 2, 3, 6
Core: VP1, 2, 3
Function of rotaviral structural proteins
VP1: RdRp, forms protein bridge between 5 ́and 3 ́ends of the genome segments
VP2: RNA binding; essential for activity of VP1
VP3: Guanylyltranserfase, methyltransferase, ssRNA binding, in complex with VP1
VP4: Hemagglutinin, fusion protein (?), cleavage required before fusion
VP6: Trimer, essential for transcription, transcriptional pore
VP7: Ca2+ dep. trimeric membrane glycoprotein in the outer capsid
Functions of reoviral proteins
VP1: RdRp, forms protein bridge between 5 ́and 3 ́ends of the genome segments, in complex with VP3
VP2: RNA binding; inner layer in the core, essential for activity of VP1/VP3 complex
VP3: Guanylyltranserfase, methyltransferase, ssRNA binding, in complex with VP1 (capping!)
VP4: Hemagglutinin, fusion protein, cleavage into VP5,VP8 is required for fusion
VP6: Trimer, essential for transcription
VP7: Ca2+ dep. trimeric membrane glycoprotein in the outer capsid
NSP1: Zn-finger, RNA binding, IRF3 (interferon system) antagonist
NSP2: RNA binding, oligomer, helicase/NTPase, forms viroplasm with NSP5
NSP3: Dimer, binds 3 ́end of viral mRNAs, competes with PABP for eIF4G-1 binding, so prevents cyclization and translation of cell. mRNAs
NSP4: Membrane glycoprotein at ER, involved in assembly, interacts with viroplasm
NSP5: Phosphoprotein, RNA binding, protein kinase, forms viroplasm with NSP2
NSP6: Interacts with NSP5, is inside the viroplasm
Naked RNA ist not infectious! dsRNA is never naked, always together with viral proteins
RNA Transcription in double layered core particle of Rotaviruses
- newly synthesised mRNAs emerge from particles
- mRNAs leaves core particles through channels
- at the bottom of each channel resides a complex of VP1 (polymerase) and VP3 (guanylyltransferase, capping enzyme)
- at each five fold axis one genome segment may be replicated (max. 12, due to 12-fold symmetry)
-> Orthoreoviruses in contrast have ISVPs with one protein layer!
Genome replication
- Conserved UGUG motif recognized in all segments by RdRp
- Panhandle structure: interaction of 5 ́and 3 ́end of each segment (base pairing)
- Non-coding sequences contain packaging- and assembly-signals
- dsRNA is synthesized in simultaniously forming subviral particles
- Viroplasm: area of RNA replication in the cytoplasm
- Subviral particles mature at the viroplasm, bud into the ER and receive thereby the outer capsid proteins (glycoproteins)
-> VP4 and VP7 are essential for infection process, but not for RNA replication
Fusion of reovirus ISVP with endosome membrane
- μ1N peptide and theta peptide, both cleavage products of μ1 protein contribute to pore formation
- μ1N peptide alone is sufficient to form a pore in the target membrane
- ISVP are then recruited to the pores, docking
Blue tongue disease
A mainly acute developing, seasonal disease of sheep, cattle, goat and wild ruminants
transmission via gnats
Pathogen is very variable (24 serotypes with different virulence)
Distribution: Africa, North- and Middle-America, Asia, Australia, southern Europa (Spain, Portugal, Greek, Italy, Bulgary, Turkey, …)
Bluetongue Virus
Taxonomy
Family: Reoviridae
Genera: Orthoreovirus
Orbivirus
Rotavirus
Coltivirus
Aquareovirus
Virions
Size: 60-80 nm; non enveloped
Capsid (Icosahedron)
several protein shells; ISVP
Genome: dsRNA, 18-27 kb 10-12 segments; reassortment
Replication: in cytoplasm
Blue tongue disease -> Clinics
Incubation periode: 3-7 days
Acute progression (sheep)
Fever, hyperemia of oral and nasal mucosa, Lips- and tongue-edema, blue colouring of the tongue (rare), purulent rhinitis, dyspnea, erosion and ulceration at the mucosa, claw inflammation, degenerative changes in the skeletal-muscles, lameness, redness of skin (wool becomes brittle or sheds); diarrhea in young animals, death often within a week
Subacute progression
Symptoms similar, but less grave, also clinical inapparent infections
Cattle
mostly mild progression
Guiding symptoms: ulceration, necrosis, salivation, redness, edema, inappetence, lameness (muscle- / claw inflammation), erythema, edema at the udder and vulva, decrease of milk yield
Morbidity
sheep: high (depending on the race)
cattle: ca. 5% (can be higher)
Letality
very variable (sheep: 2-40%)
Blue tongue disease -> Pathogenesis
Commuication via gnat bites -> primary proliferation in the lymphatic tissue -> Viremia (erythrocytes), affinity to the endothelium small bloodvessel -> destruction of endothelial cells
-> vasoconstriction, stasis, exudation
-> circulation failure, edema, diapedesis-
bleeding, inflammational hyperemia
Claw changes due to blood stasis epithel- and muscle damages due to capillary coaggluation necrosis
After diaplacentary infection (5./6. week of pregnancy): misscarriages, fetopathy
Blue tongue disease -> Epidemiology
Transmission of the pathogen via gnats (Culicoides)
Proliferation in salivary gland of arthropods
(arbovirus = artropode borne virus)
Spreading of the virus mainly during rainy season in summer (dep. on the time of flight of the gnats). Contact infections are unknown.
Viral reservoir:
Inapparently infected cattle, goats and wild ruminants
Hibernation: larvea of gnats? Yes!
Blue tongue disease -> Entry of BTV-serotypes into Europe since 1998
- 6 BTV-strains (1, 2, 4, 8, 9, 16)
- 16 countries
Reasons: - climatic changes
- spreading of C. imicola
- local punkies as vectors
-> Illegal vaccine triggers a second blue tongue outbreak
