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Lecture 6

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Microbiology and Immun (Sci)
MIMM 465
Edith Zorychta

Lecture 6 Contents - Gastroenteritis  Viral gastroenteritis (4 viruses causes gastroenteritis - Rotavirus – major cause of viral diarrhea in young children (also high mortality) - Norovirus – major cause of viral diarrhea in ALL ages Gastroenteritis: an acute infectious syndrome of the stomach lining and the intestine - Syndromes: diarrhea, vomiting, abdominal pain, nausea, fever and chills - Causes can be infection by:  Viruses – rotavirus, adenovirus, calicivirus, astrovirus  Bacteria – E. coli, salmonella, shigella and cholera  Parasites – Giardia lamblia, Entamoeba histolytica and Cryptosporidium - Gastroenteritis is triggered EITHER by 1. Toxins that are secreted by microbes and lead to excessive loss of water and electrolytes OR 2. By direct invasion of the wall of the gut by microbes, followed by inflammation - Inflammation gives the symptoms of diarrhea - Transmission: highly contagious, oral-fecal route Impacts: - >2 million deaths annually, second only to pneumonia for children under 5 yrs of age - 1 in 200 children who contract diarrhea die from it = high mortality in children - 4,500 children die of diarrhea every day in low-income countries Mortality caused by acute diarrheal disease - Pie chart from WHO - Rotavirus (860,000) > Typhoid (600,000) > Shigella (600,000) > ETEC (380,000) > Cholera (120,000)  Except for rotavirus, rest are bacteria - Total deaths: 2.5 million (mostly in young children) Treatment: - Mostly resolve w/o specific treatment - Reduce symptoms and prevent dehydration - Tips for treatment  Allow gastrointestinal tract to settle by not eating for a few hours.  Sip small amounts of clear liquids or suck on ice chips if vomiting is still a problem.  Give infants and children oral rehydration solutions to replace fluids and lost electrolytes.  Gradually reintroduce food, starting with bland, easy-to-digest food, like toast, broth, apples, bananas, and rice.  Avoid dairy products, caffeine, and alcohol until recovery is complete.  Get plenty of rest. Prevention: washing hands!!! - Wet hands  soap (20seconds)  scrub backs of hands, writs, b/w fingers and under fingernails  rinse  towel dry  turn off taps with towel Acute gastroenteritis viruses: - Rotavirus: leading cause among children 3-15 months old, also infect adults - Adenovirus: mainly infect children under 2 yrs of age, one of the 49 types of adenovirus affects gastrointestinal tract - Caliciviruses: infect people of all ages, the most common cause, and Norovirus infection leads to epidemic  The most common cause of viral diarrhea - Astrovirus: primarily infects infants, young children and elderly  In people with weak immune system Rotavirus (morphology of ‘wheel’) - Belongs to Reoviridae  There are quite a few number of other family under Reoviridae - Infect gastrointestinal and respiratory tracts (ALL the tract we have to digest the food and breathe) - Non-enveloped particles - Icosahedral capsid (T = 13) with an outer and inner protein shell - 11 segments dsRNA genome  ALL Reoviridae have dsRNA genome - Species A to G (7 species), rotavirus A causes >90% of the infections - Transmission: fecal-oral route - Tissue tropism: small intestinal enterocytes - Mainly infect infants and young children.  >500,000 children under 5 yrs of age die of rotavirus infection in low income countries, per year - Prevention and treatment: vaccines and oral rehydration (no special treatment) Rotavirus vaccines - The first rotavirus vaccine Rotashield was licensed in 1998, was unfortunately withdrawn from market due to the increased risk for intussusception (1 in 12,000 vaccinated infants) = sharp pain in the intestine - In 2006, two new vaccines against rotavirus A were tested to be safe and effective.  These are rotarix by GlaxoSmithKline and RotaTeq by Merck.  In 2009, WHO recommended inclusion of these two rotavirus vaccines in national immunization program. 11 RNA segments of rotavirus - Each segment encode one viral protein except for segment 9 and 11 - Alternative initiation of translation in segments 9 and 11 = translate two different proteins - VP: structural protein - NS: non-structural protein (not involved in virus particle assembly) Proteins and basic functions - NSP4, translated from segment 10  It is an enterotoxin, that cause diarrhea symptom  If you can neutralize this toxin, you can relieve such symptom Structure of rotavirus particles - Two shells – inner and outer shells - Outer layer – VP4 and VP7 - Middle layer of the outer shell VP6 - Inner layer – VP2 - VP1 and VP3 form a channel  VP1 and VP3 are responsible for transcribing viral RNA and add 5’ cap to the RNA  RNA could leave into the cytosol to make viral protein and viral genome - dsRNA NEVER leave the core Rotavirus life cycle - Many details are quite unknown - Viral particle has 3 layers – Outer, middle and inner layer – with spikes on the surface - Following the entry, transcription (+) strand RNA occurs  these can be used to make viral proteins  viral proteins can be assembled to form the core structure - RNA’ll be converted into dsRNA inside the core - The particle will mature  virion assembly at ER membrane  leave the cell Entry: - VP4 and VP7 = spike - VP4 is cleaved into VP8* (aa 1-247) and VP5* (aa 248-776)  VP8 and VP5 ARE NOT directly made from viral RNA  VP8: binds to sialic acid  VP5: binds to integrins  There are two receptors, sialic acid and integrin that are needed for the rotavirus to enter the cell - Entry leads to the loss of outer layer, release of DLPs (double layered particles) into the cytoplasm;  This uncoating process can be recapitulated by treating virus particles with calcium chelators, such as EDTA - Can destabilize the outer layer RNA transcription (I) - VP6 form the intermediate layer to coordinate the outer layer (VP7) and the inner layer (VP2); - 260 trimers of VP6 form a T=13 icosahedral capsid with 132 aqueous channels spanning the VP7 and VP6 layers  This channel is used so the viral RNA could leave to the host cytoplasm - DLPs are transcriptionally competent - RNA transcripts exit from the channels RNA transcription (II) - The inner core contains VP1, VP2 and VP3 - 60 dimers of VP2 form the shell structure with 12 five-fold axis, is the only viral protein with self- assembly property - VP1 is the RdRP, VP3 is guanylyl and methyl transferase  VP3 responsible for adding 5’ cap to the mRNA RNA transcription (III) - Packing of viral RNA segments w/in the VP2 capsid RNA transcription (IV) - Structure of VP1 (RdRP) DIAGRAM ON RIGHT  It has four channels  dsRNA’s 5’ cap is anchored on the surface of the polymerase  dsRNA separates into two single strands  (-) strand will go into one channel and (+) strand to another channel  (-) strand is used to make more (+) strand RNAs - When the (+) mRNA is made it is used to translate VPs - Specifically selected 11 segments of RNA will form the core in the ER lumen  Conversion of (+) strand RNA into dsRNA  particle will mature (addition of outer layer)  true particle How does this viral particle leave the cell? - This virus is non-enveloped membrane  It has to BREAK the host cell (lyse) in order to leave the cell - Acquire outer layer in the ER NOVOVIRUS - The most common cause of viral gastroenteritis in ALL AGES - Responsible for 90% of non-bacterial outbreaks of gastroenteritis - Belongs to Caliciviridae  Includes, Norovirus, sapovirus, vesivirus, lagovirus – 4 members - Non-enveloped, icosahedral virus - 38nm in diameter (VERY SMALL) - (+) sense, ssRNA genome – can be readily translated into VPs as the virus enters the host cell - Lack of reliable cell culture system and small animal models – can’t study this virus well - Norovirus is divided into five genogroups (GI-GV), share 51-56% genomic nucleotide similarity; = quite diverse - Each genogroup is further divided into different genotypes, share higher homology of 69-97% genomic nucleotide similarity; - GI has 8 genotypes, GII has 17 genotypes; = GI and GII are responsible for most of the infections - GII.4 viruses account for 70-80% of norovirus outbreaks; - major genogroup’s genotype - Norwalk virus is the prototypical norovirus (GI.1-NV) and it is the first isolated viral agent causing diarrhea. - Norovirus has caused at least four global epidemics of gastroenteritis over the past 15 years (1995–1996, 2002–2003, 2004–2005, 2006–2007). Phylogenetic analysis of norovirus - GI and GII has majority of the genotypes Outbreaks of Noroviruses in the US - Proportion of Non-GII : GII other than GII.4 : GII.4 are different every year  Meaning that the norovirus come and go each year and there is a genotype which dominates all the infections  Ex. 2006, GII.4 dominated most of the infections Features of norovirus infection: - Highly contagious - Viral shedding precedes the onset of illness/symptom and continues long after illness - Withstand a wide range of temperatures - Great diversity of the virus, lack of cross- protection and long-term immunity – reason why it has been staying with us for a LONG TIME - Easily undergo mutations - Important symptom of norovirus: sudden onset of vomiting and diarrhea Structure of Norovirus and genome - Non-enveloped and very small - VP1 is cleaved to form 3 proteins, P2, P1 and S, on the surface  P2 (protrusion 2) binds to the receptor - Two different particles - Empty and filled particles - Genome has 3 ORFs - (-) strand will be transcribed into (+) strand - Important technique to study the gene function = you mutate them and see how the function is altered HBGAs - Histo-blood group antigens are receptors of norovirus; - Histo-blood group antigens are a family of complex glycans that are expressed on the surfaces of red blood cells, gut, respiratory epithelia and biological secretions in humans. - Three major groups of histo-blood group antigens: ABO, Lewis, and secretor families, all involved in binding of norovirus for ENTRY of the virus;  P2 of norovirus binds to the histo-blood group Ags Association of VP1 with HBGAs - AAs of the P2 protein binds to the glycan complexes (very small) of the HBGAs  trigger the entry of the virus High diversity in the binding site of the receptor - Different Noroviruses use different HBGAs as a receptor - Different norovirus strains exhibit variable HBGA-binding patterns Host susceptibility - A subgroup of people are resistant to norovirus infection; - This natural protection mechanism involves the polymorphism of histo-blood group antigens (HBGAs) in human population; - 20% of Europeans who are non-secretors are naturally resistant to infection with Norwalk virus; - Nonsecretors do not have a functional fucosyltransferase 2 gene (for the norovirus to bind – virus can’t recognize) Contents - The impact of respiratory diseases on human health - Viral causes of respiratory diseases - Respiratory syncytial virus Impact on human heal
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