Gastroenteritis Viruses Prof. Mary K. Estes

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1 Gastroenteritis Viruses Mary K. Estes, Ph.D. Professor of Molecular Virology and Microbiology and Medicine-GI, BCM Director, Texas Medical Center DDC 1 Outline Gastrointestinal viruses Rotaviruses Noroviruses 2 Diarrhea is a health problem worldwide The amount of diarrheal water equals the amount of water over Victoria Falls in 1 min Diarrhea is caused by many infectious agents, including viruses It has been estimated that in any given 24 hr period, 200 million people on Earth have gastroenteritis 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Viruses causing diarrhea major pathogens in humans Rotaviruses Norwalk virus/norovirus (Calicivirus) Sapovirus (Calicivirus) Astroviruses Adenoviruses (serotypes 40 and 41) Aichi virus 4 Rotavirus Viral gastroenteritis Dehydrating diarrhea in young children Almost every child (< 5 years) gets infected 5 Epidemiology World wide infection and disease 111 million (M) episodes of gastroenteritis 25 M clinic visits, 2 M hospitalizations ~ 440,000 deaths in children < 5 years (Parashar et al., 2003, Emerg Infect Dis 9: ) 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Rotavirus pathogenesis Disease is age-restricted: Virus replication in all ages Generally asymptomatic infection in adults Symptomatic infection occurs in the young children 3 months to 2 years of age; < 2 week old mice and rats Main tissue tropism enterocytes Diarrhea caused by multiple factors: Malabsorption NSP4 enterotoxin Enteric nervous system 7 Rotaviruses infect mature enterocytes of villus epithelium of the small intestine 8 RV-gnotiobiotic piglets 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Slide 7 mke1 mestes, 5/22/2007

5 Rotavirus infection in calves Villus blunting & loss of absorptive capacity Uninfected 4 dpi 10 Rotavirus pathogenesis Rotavirus infection Time: 0 12 hpi hpi 7 dpi Virus replication: Diarrhea: 0 0 Histopathology: None Patchy v iral antigen duo<j ej <ileum 4+ None Enterotoxin High titers duo<j ej = ileum 4+ Villus atrophy and blunting, inflammation, mononuclear infiltration of lamina propria, vacuolation of epithelial cells ENS 0 0 None 11 Rotavirus genes and proteins dsrna segment Protein VP1 VP2 VP3 VP4 VP2 VP4 Neutralization antigen NSP1 VP6 NSP3 NSP2 VP7 NSP4 - enterotoxin NSP5, NSP6 VP6 Subgroup antigen VP7 Neutralization antigen Subcore VP1, VP3 12 The screen versions of these slides have full details of copyright and acknowledgements 4

6 Rotavirus replication Occurs in the cell cytoplasm Viral RNA always is inside a protein shell Many aspects not well understood A unique process occurs during the assembly of newly made particles 13 α 2 β 1 α v β 3 α x β 2 14 Unique particle assembly by budding into the ER Is Ca 2+ -dependent ( requires [Ca 2+ ] i ) Is NSP4-dependent Participating proteins: VP4, VP7, and NSP4 Transient lipid bilayer formation Following dissolution, mature triple layered particles are formed 15 The screen versions of these slides have full details of copyright and acknowledgements 5

7 NSP4: an intracellular receptor and enterotoxin NH 2 H2 H1 47 CHO CHO L25 Lumen ER membrane H3 93 Cytoplasm AAH Cleavage COOH ? Toxin 16 NSP4-inoculated mouse The screen versions of these slides have full details of copyright and acknowledgements 6

8 Properties of Rotavirus enterotoxin First described viral enterotoxin (NSP4) A novel toxin; No sequence similarity to other know n toxins Induces age-dependent diarrhea Mobilizes intracellular Ca 2+ not age-dependent Does not induce histologic changes in the intestine Induces age-dependent chloride secretion in intestinal mucosa of mice May be a target for treatments or vaccines 19 How does the Rotavirus enterotoxin NSP4 act? NSP4 is released from virus-infected cells Cells have a receptor for NSP4 Binding to this receptor causes cell signaling that leads to chloride secretion and diarrhea Antibody to NSP4 will partially protect against disease Some avirulent rotaviruses have mutated NSP4 genes 20 Effects of Rotavirus infection of polarized epithelial cells Disrupts Tight junctions Microvillar, microfilament network Perturbs protein targeting Sucrase-isomaltase Induces chemokine responses Evokes intestinal secretion Enterotoxin Enteric nervous system 21 The screen versions of these slides have full details of copyright and acknowledgements 7

9 NSP4 Cl - Cl - Cl -? Ca 2+ Enterocytes Nucleus Courtesy of A. Einerhand 22 New unexpected information about Rotavirus replication Rotavirus infections are not limited to intestinal enterocytes Rotavirus can be found in the serum of infected animals and children (viremia) Extraintestinal rotavirus can be seen in the absence of diarrhea Does extraintestinal Rotavirus infect other tissues and cause non-diarrheal disease? 23 Treatment and vaccines 24 The screen versions of these slides have full details of copyright and acknowledgements 8

10 Treatment? 25 Oral rehydration therapy Per liter of clean water 90 meq sodium 20 meq potassium 80 meq of chloride 30 meq of citrate 20 g glucose OR ¾ T. table salt 1 T. baking soda 1 c. orange juice or 2 bananas 4 T. sugar The screen versions of these slides have full details of copyright and acknowledgements 9

11 Vaccines? Live-attenuated reassortant vaccines Three currently approved and others being tested Not yet clear if these vaccines will protect in developing countries where needed most Inactivated virus Virus-like particles Need to be tested as backup plan 28 Conclusions Rotaviruses cause diarrhea by several mechanisms - Enterotoxin, Malabsorption, ENS Why is disease age-restricted? Age-dependent chloride channel, Immunity Rotaviruses cause extraintestinal infectionsdo they cause other diseases? Live, attenuated vaccines are safe and their effectiveness in developing countries needs to be established New, effective vaccines and therapies may target the rotavirus enterotoxin 29 Norwalk virus (NV) First identified human Calicivirus Prototype strain of genetically related Noroviruses Noroviruses 23 million cases of gastroenteritis per year in USA Cause illness in babies, children, adults and elderly Cause outbreaks in daycare centers, schools, nursing homes, hospitals, military Cause >96% of all outbreaks of nonbacterial gastroenteritis 30 The screen versions of these slides have full details of copyright and acknowledgements 10

12 Norovirus gastroenteritis Highly stable Highly infectious (1-10 virions) 31 Norovirus background Many asymptomatic infections and prolonged post-symptomatic shedding Some people resistant to infection (20%) Possibly due to genetic factor? Persistent infections Transplant patients, immunosuppressive therapy Unusual clinical presentation Disseminated intravascular coagulation Spread by contaminated food, water and environmental surfaces 32 Norovirus pathogenesis Incubation period 1 2 days Produces shortening of microvilli in small intestine with infiltration of mononuclear cells Transient malabsorption and delayed gastric emptying may occur Cause of vomiting unknown Immune responses poorly characterized 33 The screen versions of these slides have full details of copyright and acknowledgements 11

13 Clinical responses of 2 adult volunteers given NV Dolin et al., JID 123: (1971) s NV studies 12 volunteers NV 6 ill 6 not ill > 2 yrs NV 6 ill 6 not ill Immunity due to previous exposure? Repeated susceptibility Repeated resistance Genetic factor may determine susceptibility or resistance to NV 35 Challenges studying Norwalk virus (NV) Infection and disease only in humansno small animal model No cell culture no in vitro system for infection and replication Low concentration of virions in stool 36 The screen versions of these slides have full details of copyright and acknowledgements 12

14 Virus-like particles (VLPs) Expressed capsid proteins VLPs 50 nm (Prasad et al., Science 1999) - Virus surrogate - Structurally and antigenically similar to Norw alk virion capsids - Model for Norw alk virus-cell binding 37 Norwalk VLPs hemagglutinate human red blood cells Model for Norwalk virus-host cell binding: Discovered the Norwalk VLP hemagglutination receptor is the H antigen Hutson et al., J Virol (2003) 38 What is the H antigen? Carbohydrate histo-blood group antigen on the cell surface and on secreted mucins Presence controlled by genetically determined expression of a carbohydrate modifying enzyme, a fucosyltransferase, FUT2 FUT2 -/- = 20% of population FUT2 +/+ = secretor Henry et al., Vox Sang (1995) 39 The screen versions of these slides have full details of copyright and acknowledgements 13

15 FUT2 genotype versus NV challenge outcome Non-infected Infected FUT2 -/- 8 0 FUT2 +/-, FUT2 +/ n = 51 FUT2 -/- individuals are resistant to NV infection (0/8 infected, p < ) The H antigen is a host susceptibility factor for Norwalk virus infection Hutson et al. (J. Med. Virol. 77: 116; 2005) 40 Is host susceptibility similar for all Noroviruses? NO Hemagglutination patterns and CHO binding patterns are strain specific Secretor status does not influence susceptibility to Snow Mountain virus infection All people will be susceptible to some norov irus! 41 Summary: Noroviruses Clinical significance is increasing numbers and settings H 329 H histo-blood group CHO antigen is host susceptibility factor W 375 C N 331 Knowledge of NV-CHO binding site (P domain) or the structure of other viral proteins may lead to antivirals to help prevent transmission N 42 The screen versions of these slides have full details of copyright and acknowledgements 14

16 43 44 The screen versions of these slides have full details of copyright and acknowledgements 15