Replication of ds RNA Viruses

Question 1

Name the characteristics and genera of Reoviridae

Answer 1

Genera: Orbivirus (bluetounge virus), Rotavirus, Orthovirus
genome: segmented ds RNA, 10-12 segments, 18-27kb, eg Bluetongue 19.2 kB in 10 segments
Virion structure: komplex, 3-layered protein shell, insensitive to detergents, glycoproteins

Question 2

Describe the replicative cycle of reoviridae

Answer 2

• 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 (see 3)
• Can penetrate through endosomal membrane into cytoplasm
• Cytoplasm: Conversion from ISVP to core particle
• Synthesis of capped viral mRNAs takes place in the core particle
• Association of mRNAs with newly translated proteins to RNase-sensitive subviral 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

Question 3

Where does the mRNA synthesis take place in reoviridae?

Answer 3

The RNA synthesis occurs right at the tunnel structures (Turret) of the core particle. Each turret has a polymerase bound and the synthesised mRNA leaves the particle stretchy through the tunnel.

Question 4

What are the steps of the cell entry of reovirus?

Answer 4

• 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
  →turrets have to be selective due to dsRNA being a danger signal for the host cell, functional homolog to membrane system of +ssRNA viruses

Question 5

What are the functions of the rotavital structural proteins?

Answer 5

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

Virion: VP1, 2, 3, 4, 6, 7
ISVP: VP1, 2, 3, 6
Core: VP1, 2, 3

Question 6

What are the functions of the non-structural proteins of rotavirus?

Answer 6

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

Question 7

explain the genome replication process of reovirus.

Answer 7

• 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→hiding dsRNA from innate immune system
• 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)

Question 8

What are the characteristics of the Blue tongue virus (orbivirus)?

Answer 8

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

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) need of 24 different vaccines
Distribution: Africa, North- and Middle-America, Asia, Australia, southern Europa (Spain, Portugal, Greek, Italy, Bulgary, Turkey, …)

Question 9

How can blue tongue disease be detected?

Answer 9

• RT-PCR: detection of viral genome - in currently infected animals detectable between 2 days to 8 months past infection (hidden genome)
• ELISA: detection of viral proteins and antibodies- can be detected during infection and after recovery from disease (detectable from 14 days past infection onwards)

Question 10

What actions are required after detection of blue tongue virus in cattle?

Answer 10

-disclosure
-introduction of hazard zone and monitoring zone
-prohibition of animal transport
-monitoring of suspected animals:
1. Sentinel animals
Serological testing of BTV-negative cattle since March 2007 (monthly)
2. Wild animal monitoring
3. Entomological monitoring
- Federal territory wide
- Morphological sorting and characterisation of gnats - Monitoring of the gnat pools via real-time RT-PCR

-insect killing
→immunisation of farm animals with killed vaccine