Week 12

Question 1

Rotavirus

Answer 1

• Cause more severe diarrhoea and dehydration than other agents
• Vaccination since 2006 –> reduced hospitalization by 45%

Question 2

Other disease manifestations of rotavirus

Answer 2

• Low-level viraemia
• CNS disease
• Liver involvement
• Autoimmunity modulation -T1 diabetes, celiac

Question 3

Rotavirus belongs to which family?

Answer 3

• Reovirus - dsRNA genome, multi-layered capsid, icosahedral symmetry
• Rotavirus - new genus, differences in viral proteins

Question 4

Viral proteins

Answer 4

• VP7 and VP4 = outer capsid, VP7: serotype determinants
  
  • VP4 - major receptor interaction, cleaved by trypsin into VP5 and VP8 for full infectivity
• Core: 11 segments of ds RNA + VP2 (binds RNA genome) + VP1 (RNAP)
• Inner capsid: VP6 - used to identify rotavirus species

Question 5

Rotavirus fingerprints

Answer 5

Each gene segment encodes a structural or nonstructural protein

Question 6

Lab diagnosis

Answer 6

• Stool virus detection using ELISA (VP6), RT-PCR, electron microscopy
• Seroconversion - IgG, IgM, IgA
• IgA conversion in stools

Question 7

Efects of rotavirus infection on intestinal villi

Answer 7

• Villi are blunted –> diarrhoea
• NSP4 = enterotoxin and contributes to severity of dehydration and diarrhoea
• Cells migrate from crypt to villus = new set of susceptible cells every 3 days

Question 8

Rotavirus-neutralizing antibodies

Answer 8

• Directed to outer capsid proteins - VP7 and VP4
• Serotypes - VP7 and 4 ELISA
• Genotypes: RT-PCR
• 5 VP7 (G) serotypes most common in humans
• 3 VP4 (P)
• Serotype diversity generated by virus gene segment reassortment

Question 9

Rotavirus reassortment

Answer 9

• Coninfect same cell with 2 rotavirus strains
• Different combination of virus particles
• Select for combinations using monoclonal antibodies to VP4 and VP7

Question 10

Rotavirus entry

Answer 10

• Initial attachment of VP8 to glycans
  
  • e.g. terminal (sialidase sensitive) or branched sialic acid (sialidase resistant)
  • Sialidase removes sialic acid at ends of chain
  • e.g. Histo-blood group antigens - Type A is implicated
• Binding/entry occurs via endocytosis using VP5 and VP7 recognition of integrins

Question 11

Rotaviruses and sialic acid

Answer 11

• Human rotavirus Wa and RV-3: sialidase resistant - use branched sialic acid
• RV3 also uses HBGA A type
• Monkey strain: sialidase sensitive - uses terminal sialic acid (acetyl)
• Pig rotavirus: sialidase sensitive - uses terminal and subterminal sialic acid, glycolyl

Question 12

How to study receptor interactions?

Answer 12

• Blocking interaction of virus and receptor
• Structural studies of receptor-ligand complex
• Expressing the receptor on cells that don’t normally express it –> increase infection?
• Glycan arrays
• Bioinformatics - look for presence of motifs in proteins

Question 13

Rotavirus using integrins

Answer 13

• a2B1:
  
  • bound to VP4
  • rotavirus cellular receptor
  • VP5 competes with virus for binding
• axB2, avB3:
  
  • recognised by VP7
  • cofactor

Question 14

Type 1 diabetes + rotavirus

Answer 14

• Rotavirus may accelerate progression to diabetes 1 (already have genetic predisposition)
• NOD mice used:
  
  • 5 days - diarrhoea and extraintestinal spread
  • 12 weeks - no diarrhoea, virus found in mesenteric and pancreatic lymph nodes, advanced insulitis

Question 15

Development of diabetes

Answer 15

1. Plasmacytoid DCs take up virus, activated by TLR 7 –> Type 1 interferon –>
2. Activated lymphocytes –> pancreatic lymph nodes
3. Activated CD8+ T cell
4. B cell death –> diabetes

Question 16

Rotavirus epidemiology

Answer 16

Question 17

Why vaccination within first 2 years of life?

Answer 17

• Period of most serious disease
• Natural infection protects for only 1-2 years

Question 18

Vaccination of local site

Answer 18

• Need to induce intestinal IgA
• Gut-homing T and B cells required in ANIMALS
• Most effective means of stimulation of local immunity is infection involving local site

Question 19

Live rotavirus vaccines

Answer 19

• Heterologous virus - Lamb rotavirus in China
• Partially heterologous reassortant rotavirus: Rotashield- rhesis monkey with VP7 (types 1-4) - live attenuated
• Rotateq: bovine virus with VP4 and VP7
• Rotarix: serotype P1 and G1

Question 20

Rotashield and intassusception

Answer 20

• Intasusseption also associated with natural rotavirus infection but not clear if it is true
• Most INT occurred when 1st dose given at 4-6 months

Question 21

Rotarix

Answer 21

• 85% effiacy
• human G1 isolate with most common VP4

Question 22

Rotateq

Answer 22

• Most common VP4 and VP7
• Pentavalent vaccine
• 98% efficacy
• Hospitalizations declined and notifications