Getting by with a little help from their friends: how bacteria aid virus infection

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Getting by with a little help from their friends: how bacteria aid virus infection

MY NAME’S ERIC FREDE IT’S A PLEASURE AND HONOR TO INTRODUCE THE 2019 GEORGE KHOURY MEMORIAL LECTURE GEORGE RECEIVED HIS TRAINING AT PRINCETON AND HARVARD MEDICAL SCHOOLS AND CAME TO THE NIH IN 1976 AS HEAD OF THE VIRUS TUMOR BIOLOGY SECTION IN THE LABORATORY OF MOLECULAR VIROLOGY AND WAS CHIEF OF THE LABORATORY IN 1980 AND RECEIVE A FUNDING AWARD FOR OUTSTANDING GOVERNMENT SERVICE AND IN 1987 AT THE AGE OF 43, THE YEAR OF HIS DEATH, HE WAS ELECTED A MEMBER OF THE NATIONAL ACADEMY OF SCIENCES UNFORTUNATELY, I DID NOT HAVE THE PRIVILEGE OF MEETING GEORGE PERSONALLY BUT I TRAINED AS A POST-DOC WITH ONE OF HIS COLLABORATORS SHOWN ON YOUR RIGHT AND GEORGE IS ON THE FAR LEFT AND A VERY YOUNG PETER HOWLIE MANY KNOW IS IN THE MIDDLE I THINK HE’S RECEIVING AN AWARD I’M NOT CERTAIN GEORGE PUBLISHED OVER 140 PAPERS IN HIS RELATIVELY SHORT CAREER AND DECIPHERING MECHANISMS OF TRANSCRIPTION HE HAD A DISTINGUISHED GROUP OF TRAIN JEEZ AND COLLABORATORS — TRAIN JEEZ AND — TRAINEES AND COLLABORATORS AND YOU’LL RECOGNIZE MANY INDIVIDUALS THE LECTURE HAS BEEN GIVEN OWN THE YEARS BY A DISTINGUISHED GROUP OF SPEAKERS INCLUDING SEVERAL NOBEL LAUREATES AND THE INDIVIDUALS ARE LISTED THERE AND NO LESS OUTSTANDING IS TODAY’S SPEAKER, JULIE PFEIFFER SHE DID HER UNDERGRADUATE TRAINING IN OHIO AND DID HER Ph.D. AT UNIVERSITY OF MICHIGAN STUDYING RETROVIRAL RECOMBINATION AND THEN SHE WENT TO STANFORD TO DO POST-DOCTORAL WORK WITH OUR NEXT YEAR’S KHOURY LECTURER SHE WENT TO U.T. SEARCH WARRANT WARRANT — U.T SOUND SOUTHWESTERN AND HAS BEEN ELECTED TO THE AMERICAN ACADEMY OF MICRO BIOLOGY AND A JOURNAL BOARD MEMBER OF SCIENCE JOURNAL OF VIROLOGY AND SO ON AND SHE’S REALLY QUITE WELL KNOWN FOR A NUMBER OF CONTRIBUTIONS TO VIROLOGY BUT I THINK PROBABLY FOREMOST AMONG THEM IS HER PIONEERING WORK IN HER PLAY BETWEEN PATHOGENS AND THE HOST MICROBIOME AND IT’S HOW BACTERIA AIDS VIRUS INFECTION WE’RE LOOKING FORWARD TO YOUR SEMINAR >> THANK YOU SO MUCH TO ERIC FOR THE REALLY NICE INTRODUCTION IT’S A HUGE HONOR FOR ME TO BE HERE TODAY THIS IS IN HONOR OF DR. KHOURY SO I THINK IT’S KIND OF FIT IG START WITH JUST A LITTLE BIT OF VIROLO VIROLOGY AND THERE WAS A SAYING SAID IT’S BAD NEWS WRAPPED IN PROTEIN WHICH IS ASTUTE DR. KHOURY WORKED ON A SMALL VIRUS SHOWN ON THE TOP I’VE BEEN INTERESTED IN VIRUSES WITH RNA GENETIC MATERIAL SHOWN ALONG THE BOTTOM AND I’VE BEEN REALLY INTERESTED IN ENTERIC RNA VIRUSES THAT INFECT THROUGH THE FECAL-ORAL ROUTE THESE ARE VIRUSES THE KINDS I’LL TELL YOU ABOUT TODAY HAVE A PROTEIN SHELL SURROUNDING THEIR NUCLEIC ACID AND THE CLASSES I’LL MOSTLY TALK ABOUT TODAY HAVE VERY SMALL GENOMES 7500 NUCLEOTIDES IS ENOUGH TO ACCOMPLISH WHAT THEY NEED THEY’RE COMPLETELY RELIANT ON THE HOST CELL THIS IS MY HAND DRAWN SCHEMATIC OF THE REPLICATION CYCLE IT’S INCREDIBLY SIMPLE FOR THE VIRUSES THEY ATTACH AND DUMP THEIR RNA GENOME IN, IT’S TRANSLATED TO MAKE VIRAL PROTEINS AND YOU THEN MAKE LOTS OF VIRAL RNAs AND NEW VIRIONS THE VIRUSES I’LL TALK ABOUT

TODAY ARE TOXIC TO THE HOST CELLS SO THEY WILL KILL THE HOST CELL THEY INFECT WE CAN USE SO-CALLED PLAQUE ASSAYS WHERE EACH OF THE WHITE CIRCLES IS A WHOLE IN A CELL MONOLAYER WHERE A VIRUS LANDED A VERY SIMPLE NICE SYSTEM TO WORK WITH WE HAVE A VARIOUS OF RESEARCH INTERESTS IN THE LAB SOME RELATED TO LONG-STANDING INTEREST IN VIRAL EVOLUTION SO THE IDEA THESE ARE RNA VIRUSES AND HAVE POLYMERASES AND HAVE DIVERSE POPULATIONS WE’RE ALSO INTERESTED IN HOW THIS POPULATIONS MOVE WITHIN AND BETWEEN ANIMALS AND WE MOSTLY USE MOUSE MODELS FOR THAT WORK WE’VE FOCUSSED A LOT IN THE LAST FEW YEARS ON VIRUS MICROBIOTA INTERACTIONS AND THAT WILL BE THE MAIN FOCUS OF TODAY’S TALK I WILL TOUCH ON AT THE END OF THE TALK A VERY NEW PROJECT IN OUR LAB THAT IS SO NOT READY FOR PRESENTATION THAT IT’S A LITTLE BIT TERRIFYING BUT I LIKE PRESENTING UNPUBLISHED WORK IN AN EARLY PHASE BECAUSE SOMETIMES YOU CAN GET KEEN INPUT FROM THE FIELD SO THAT PROJECT HAS TO DO WITH INNATE IMMUNE MATH — PATHWAYS IN THE SINGLE CELLS SO I’LL TOUCH ON THAT IN THE FEW SLIDES AT THE END MOST WILL BE ABOUT HOW VIRUSES INTERACT WITH BACTERIA IN THE GUT SO ENTERIC VIRUSES SPREAD BY THE FECAL-ORAL ROUTE MANY HERE ARE FAMILIAR WITH NOROVIRUSES AND OTHER VIRUSES AND THERE’S SEVERAL GREAT INVESTIGATORS WORKING ON THOSE VIRUSES AND THOSE ARE VERY IMPORTANT HUMAN PATHOGENS IN MY LAB WE TEND TO FOCUS ON VIRUSES NOBODY CARES ABOUT BECAUSE THEY’RE GREAT MODEL SYSTEMS AND HIGHLY TRACTABLE WE HAVE POLLOVIRUS AND THE MAIN MODEL I’LL USE THROUGH THE BULK IS POLLOVIRUS MOST HAVE PROBABLY HEARD OF POLIO VIRUS BECAUSE OF PICTURES THAT SHOW AN IRON LUNG WARD SO IT CAN INDUCE PARALYSIS AND IT WAS INCREDIBLY SCARY BUT IT ENDS UP PARALYSIS HAPPENED IN LESS THAN 1% OF INFECTED INDIVIDUALS EVEN BEFORE THE VACCINES WAS AVAILABLE FOR THE MOST PART THE VIRUS SPREAD THROUGH THE FECAL-ORAL ROUTE LOTS OF PEOPLE DIDN’T KNOW THEY WERE INFECTED IT HAS AN FORTUNATE SIDE SEQUENCES AND HERE’S A ROAD MAP SHOWING IN RED COUNTRIES WHERE POLIOVIRUS WAS ENDEMIC AND YOU SEE HOW EFFECTIVE THE CAIN HAS BEEN ARGUABLY, ONE OF THE MOST EFFECTIVE THINGS BIOMEDICAL SCIENCE HAS EVER DONE SO POLIOVIRUS SAY MODEL SYSTEM I GET THIS QUESTION SHOULDN’T YOU STUDY SOMETHING CLINICALLY IMPORTANT I WANT FRAME THIS AND TELL YOU WHY WE STUDY IT AND THROUGHOUT THE TALK I’LL ILLUSTRATE THINGS WE WERE ABLE TO DO WITH POLIO WE WOULDN’T HAVE BEEN ABLE TO DO WITH ANYTHING ELSE FIRST OFF, IT’S BEEN STUDIED OVER 100 YEARS THAT MEANS WE HAVE A HUGE KNOWLEDGE BASE TO WORK WITH SECOND, IT’S ACTUALLY INCREDIBLY SAFE SO WE CAN VACCINATE LAB MEMBERS FREQUENTLY I WANT THEM TO START ON POLIOVIRUS BECAUSE IT’S THE SAFEST WE HAVE IN THE LAB KNOWLEDGE OF THE VIRUS HAS RENDERED A HUGE DEAL OF TRACTABILITY AND IF YOU ARE GOING TO TACKLE A TOUGH MESSY QUESTION IT’S NICE TO HAVE TOOLS IN YOUR TOOLBOX TO ILLUSTRATE THAT IN THE HISTORY OF POLIO I’M PUTTING UP A PUB MED TIME LINE AND NUMBER OF PUBLICATIONS PER YEAR SHOWN HERE AND YOU CAN SEE THE FIRST PAPERS APPEARED IN 1879 THERE WERE A NUMBER OF PAPERS CONTINUING ON THROUGH THE 1940s AND THIS IS WHEN THE MAJOR PARALYTIC EVENT STARTED HAPPENING TO THE TWO VACCINES WERE DEVELOPED THE INACTIVATED VACCINE GIVEN IN THE UNITED STATES AND THE ATTENUATED VACCINE, THE LIVE ORAL VACCINE FREQUENTLY GIVEN BACK IN THE DAY THIS WAS THE QUENCE OF TWO MAJOR THINGS THE FIRST IS THE MARCH OF DIMES ARGUABLY ONE OF THE MOST SUCCESSFUL FUND-RAISING CAMPAIGNS IN HISTORY AND LOTS OF

SMALL CONTRIBUTIONS FROM A LOT OF PEOPLE CAN MAKE A HUGE DIFFERENCE THE SECOND INCREDIBLY IMPORTANT ADVANCE ARE HELLE CELLS AND WITHOUT THOSE CELLS MON OF THIS WOULD HAVE BEEN POSSIBLE SO INCREDIBLY IMPORTANT CAN SEE THE DOWNSWING IN PUBLICATIONS BECAUSE WE HAD THE VACCINES HOWEVER, PEOPLE CONTINUED TO USE POLIO VIRUS BECAUSE IT WAS EASY TO WORK WITH AND MADE A GREAT MODEL SYSTEM AND WE HAD A GOLDEN ERA WHERE PEOPLE WORKED OUT MOLECULAR VIROLOGY I JOINED THE FIELD RIGHT HERE IN 2001 MY TIMING ISN’T ALWAYS THE BEST BUT WE’RE STILL CONTINUING TO LEARN NEW THINGS ABOUT THE VIRUS I WANT TO COMPARE AND CONTRAST THIS WITH ZIKA VIRUS AND WHEN IT CAME ON THE SCENE RECENTLY THERE WERE HARDLY ANY PREVIOUS PUBLICATIONS WE KNEW VERY LITTLE ABOUT THAT VIRUS IN COMPARISON SO WHEN I STARTED MY INDEPENDENT LAB IN 2006, WE ASKED A COUPLE QUESTIONS BUT ONE OF THE MAIN ONES WAS WHY DID SO FEW PEOPLE DEVELOP PARALYSIS FROM POLIOVIRUS IT LIKELY CIRCULATED IN THE COMMUNITIES AND LOTS OF PEOPLE WERE EXPOSED BUT ONLY RARELY DID IT CAUSE PARALYSIS AND THERE ARE A LOT OF NEUROTROPIC VIRUSES THAT CAN SPREAD TO THE CENTRAL NERVOUS SYSTEM THROUGH THE ROUTES POLIO USES AND IT COULD BE APPLICABLE TO OTHER SYSTEMS AS WELL AND ENROVIRUS D68 IS OF INTEREST TOO AND IN THINKING ABOUT THE QUESTION WHY DID SO FEW PEOPLE INFECTED WITH POLIO DEVELOP PARALYSIS AND A FEW PEOPLE TACKLED THIS QUESTION IN THE LAB AND USED WHAT I’M CALLING BAR-CODED VIRUS TO TACKLE THIS THEY’RE GENETICCALLY TABBED AND ONCE IT MAKES IT TO THE SYSTEM IT REPLICATES LIKE CRAZY EVEN IF IT’S HARD TO GET THERE IT HAS TIDERS IF YOU HAD A MIXED POPULATION TO START WITH AND HAD A LIMITED POPULATION IN THE CENTRAL NERVOUS SYSTEM YOU COULD IDENTIFY THE BARRIERS FOR THE FIRST TIME SO THEY CREATED THIS SYSTEM IS AND THEY DON’T ALTER VIRAL PREPLICATION CAPACITY AND WE CAN TELL THEM APART WE DID THIS ON THE OLD SCHOOL TECHNIQUE ON A HYBRIDIZATION ASSAY AND WHAT YOU CAN SEE IS ALL 10 VIRUSES WERE SHED IN FECES BUT ONLY ONE TOOK OLD IN THE GUT AND MADE IT TO THE BRAIN WHAT THAT LOOKS LIKE IN PICTURE FORM WAS THEY PUT ALL 10 IN ORALLY AND WHAT CAME OUT WAS ALL 10 BUT IN TERMS OF WHAT TOOK HOLD IN THE TISSUES IT WAS JUST ONE OF THOSE VIRUSES NOWADAYS WE USE NEXT GENERATION SEQUENCING TECHNOLOGY TO TACKLE THESE QUESTIONS SO A POST-DOC IN THE LAB CREATED A PANEL OF 150 VIRUSES AND HE CAN ANALYZE THEIR MOVEMENT THROUGH NEXT-GENERATION SEQUENCING BUT A LOT OF THE OUTCOMES FROM THE EXPERIMENTS ARE SIMILAR SO IN THE BEND WHAT SHARON AND KAREN FOUND WERE THERE WERE MULTIPLE BARRIERS THAT LIMITED IT FROM THE GUT TO THE CENTRAL NERVOUS SYSTEM IMPORTANTLY IS INNATE IMMUNITY A REALLY INTERESTING ONE WE FOUND WAS THERE’S INEFFICIENT VIRAL MOVEMENT UP NEURONS SO THE VIRUS CAN GET INTO THE NEURONS FINE BUT HAS DIFFICULT TRAVELS LONG DISTANCES IN A RETRO GRADE MANNER AND WE USED THE SCIATIC NERVE MODEL FOR THAT WORK AND THE INTESTINAL BARRIER ITSELF WAS IMPORTANT SO THIS LED TO A QUESTION AND A MY BOTH SIS WE WONDERED WHETHER GUT BACTERIA INHERITED MOVEMENT FROM OTHER VIRUSES IT SHOWS A MAP INTESTINE AND THE VIRUS IS BLUE AND THE BACTERIA ARE STAINED IN GREEN MEMBERS OF THE MICROBIOTA AND IT’S THE SUBJECT OF A HUGE NUMBER OF STUDIES THESE DAYS ARE IMPORTANT FOR HOST PHYSIOLOGY, IMMUNE DEVELOPMENT, NUTRITION AND SO FORTH

AND BACK BACK TEARIOLOGISTS WERE AHEAD OF US THERE WAS A KNOWN PHENOMENON OF COLONIZATION RESISTANCE WHERE THE GOOD BACTERIA WOULD INHIBIT PATHOGENS LIKE SALMONELLA AND THIS WAS PREVIOUSLY KNOWN IN THE FIELD WE THOUGHT IT WON’T BE A STRETCH IF IT’S ALSO TRUE FOR VIRUSES THE SECOND REASON WE KIND OF MADE THIS HYPOTHESIS WAS A MORE SOCIAL REASON AND THAT WAS BACTERIOLOGISTS AND VIROLOGISTS WILL CLUSTER INTO THEIR GROUPS AND NOT COMMUNICATE VERY MUCH AND IT’S AN IMMUNOLOGY IN MY DEPARTMENT AND HOWEVER IN 2007 I FOUND MYSELF AS THE ONLY VIROLOGIST IN OUR DEPARTMENT KIND OF IN MY SECOND YEAR OF THE FACULTY POSITION SO I WAS LONELY BUT THANKFULLY THE BACTERIOLOGISTS GOT INTERESTED IN ME AND I LEARNED A LOT FROM THEM IN 2019 WE HIRED A LOT OF FACULTY AND THERE’S A HUGE AMOUNT OF COLLABORATION BETWEEN VIROLOGISTS AND BACTERIOLOGISTS AND WHO WOULDN’T WANT TO COLLABORATE WITH THESE PEOPLE I’M LUCKY TO BE IN THAT ENVIRONMENT IT’S STIMULATED 24 JOINT PUBLICATIONS IN THE LAST 10 YEARS SOME OF WHICH ARE SHOWN HERE OKAY SO THAT WAS OUR HYPOTHESIS THAT IT INHIBITS VIRAL APPLICATION AND THEY ORALLY INFECTED UNTREATED MICE WITH INTESTINAL MICROBES INTACT OR TREATED THEM WITH ANTIBIOTICS TO DEPLETE THEIR INTESTINAL FLORA AND SHE FOUND IN THE CONVENTIONAL MICE THE VIRUS REPLICATED AND CAUSED DISEASE BUT CONTRARY TO OUR EXPECTATION IN THE ANTIBIOTIC-TREATED MICE, POLIO VIRUS DISEASE WAS DIMINISHED AND ASTUTE LISTENERS WILL REALIZE IT’S THE OPPOSITE OF OUR HYPOTHESIS IT SEEMS LIKE GUT BACTERIA PROMOTE PATHOGENESIS SO THERE’S SEVERAL QUESTIONS THAT FALL OUT OF THIS THE FIRST IS IS THIS APPLICABLE TO OTHER VIRUSES OR IS THIS A POLIO SPECIFIC ANYONE — PHENOMENON I DON’T KNOW HOW MANY WOULD HAVE CAED IF IT WAS JUST POLIO SPECIFIC THE QUESTION IS YES IN COLLABORATION WITH ANOTHER LAB WE SHOW THIS IS ALSO TRUE FOR REOVIRUS WHICH SO DOUBLE-STRANDED — WHICH IS A DOUBLE-STRANDED VIRUS AND WE LOOKED AT THE MOUSE RETROVIRUS SPREAD FROM MOUSE MOTHER TO THEIR OFFSPRING THROUGH MILK SO IT TOO HAS AN ENTERIC PHASE AND IT RELIES ON THE MICROBIOTA AND THERE’S ACTIVITY WITH NOROVIRUSES THEY TOO RELY ON INTESTINAL MICROBES WE HAVE FOUR FAMILIES OF ENTERIC FAMILIES THAT ALL HAVE RELIANCE ON MICROBIOTA THE OTHER QUESTIONS ARE WHY AND HOW HOW IS THIS ALL WORKING? THAT’S THE HARDER QUESTION IT COULD BE THE BACTERIA ARE DOING SOMETHING TO THE HOST ENVIRONMENT TO PERHAPS MAKE IT MORE PERMISSIVE OR COULD BE THE VIRAL PARTICLES ARE DIRECTLY INTERACTING WITH THE BACTERIA OR BOTH SO I’M JUST GOING TO SUMMARIZE THE MECHANISMS THAT PEOPLE HAVE EVALUATED OVER THE LAST SEVERAL YEARS FIRST EFFECTS OF BACTERIA ON VIRAL PARTICLES AND THEIR INEFFECTIVITY WE HAVE SHOWN VIRUSES CAN DIRECTLY INTERACT WITH BACTERIA I’LL SAY MORE IN A MINUTE THIS STABILIZE THE VIRAL PARTICLES AND MAKES THEM RESISTANT TO HEAT AND BLEACH AND SURVIVE MORE READILY TO SURVIVE THAT SECOND IS IT ENHANCES THEIR ABILITY TO ATTACH TO THEIR HOST CELL SO THEIR INTERACTIONS WITH BACTERIA STIMULATES THE ATTACHMENT PROCESS AND THAT’S TRUE FOR POLIO AND HUMAN NOROVIRUS SEVERAL GROUPS HAVE SHOWN AN AFFECT ON THE HOST RESPONSE THE BACTERIA GLYCAN ITS STIMULATES CERTAIN IMMUNE CASCADES TO SO THE VIRUS CAN REP KATE UNDER THE RADAR —

REPLICATE UNDER THE RADAR AND BACTERIA SEEM TO DAMPEN INTERFERON RESPONSES AND THAT SEEMS TO PROMOTE THEM AND WE’VE LOOKED AT THE DIRECT INTERACTIONS MECHANISMS SO THAT WILL BE THE FOCUS THE NEXT LITTLE BIT THIS IS MY DETAILED MOLECULAR MECHANISM SLIDE FOUR TODAY — FOR YOU TODAY AND A DRAWING SHARON MADE TO DEPICT HER THESIS WORKS AND SHOWS A POLIO VIRUS PARTICLE DRESSED AS A COWBOY SHARON IS A NATIVE TEXAN IT’S RIDING THE BACTERIA WHILE HOLDING ON TO BACTERIA SURFACE GLYCAN RING TO INITIATE INFECTION SO WE THINK THIS IS A PRETTY ACCURATE DEPICTION OF WHAT SHE FOUND SO POLIOVIRUS AND THE POLYSACCHARIDES ON THE SURFACE OF THE NEGATIVE BACTERIA AND THIS HAS TWO CONSEQUENCES THAT IT ENHANCES RECEPTOR BINDING AND AIDS VIRAL STABILITY I WANT TO SAY A COUPLE THINGS ABOUT THE STABILIZATION OF VIRAL PARTICLES OUR WORK HAS SHOWN BACTERIA BLOCKS PREMATURE RNA RELEASE AND THIS SHOWS VIRION UNDER GOING RNA RELEASE AND THE VIRUS IS NO LONGER INFECTIOUS AND WE IDENTIFIED WHAT SPECIFIC LIGANDS ARE INVOLVED AND THAT’S WORK FROM CHRIS ROBINSON WHO NOW HAS HIS OWN LAB AND A CURRENT POST-DOC AND FIRST I’LL TALK ABOUT HOW CHRIS ROBINSON AND OTHERS WERE ABLE TO MAP VIRAL BINDI BINDING DETERMINATE WHAT’S IMPORTANT FOR THE INTERACTIONS? COULD THEY IDENTIFY MUTANT VIRUSES WITH BINDING AND WOULD WE PROBE CONSEQUENCES OF THE INTERACTIONS CHRIS SET OUT TO DO THIS AND SEARCHING FOR VIRAL MUTANTS WITH REDUCED BINDING TO THE GLYCANS AND YOU CAN DO THIS IN A COUPLE WAYS YOU CAN ENRICH FOR NON-BINDING MUTANTS ITERATIVELY TAKING THE VIRUS DON’T BIND AND EXPANDING KNOWN MUTANTS AND THIS IS WHERE WORKING ON POLIO IS IMPORTANT WE HAVE 100 YEARS OF CHARACTERIZED MUTANTS AVAILABLE SO THE STORY I’LL TELL YOU NEXT IS ABOUT THE ATTENUATED SABIN STRAIN LIVE ORAL VACCINE STRAIN AND THIS LIVE ORAL VACCINE STRAIN HAS A LOT OF AMINO ACID CHANGES COMPARED TO THE VIRULENT COUNTERPART AND SEVERAL HERE SHOWN IN RED ARE SURFACE EXPOSED AND WE SHOOK OUT OUR FREEZER AND SCREENED A BUNCH OF MUTANTS SO THE STORY IS REALLY ABOUT THESE PARTICULAR MUTANTS THE ASSAY WE DO IS INCREDIBLY SIMPLE JUST TAKE VIRUS AND INCUBATE IT WITH BUFFER OR THE BACTERIAL LIGAND AND INCUBATE THEM FOR SIX HOURS AND THE ACTIVITY WILL STABILIZE AND WE HAVE A PLAQUE ASSAY TO ENUMERATE THE AMOUNT LEFT AND CHRIS LUCKED OUT OR I WOULDN’T BE TELLING YOU THE STORY OBVIOUSLY THE WOUND THE WILD TYPE VIRUS IN THE ABSENCE OF LPS YOU LOSE AFTER THE INEFFECTIVITY BUT FOR THE ATTENUATED SABIN STRAIN THERE WAS NO STABILIZATION YOU WANTED TO MAP WHICH SPECIFIC MUTATION AND IT ENDED UP ONE WAS RESPONSIBLE FOR THIS THE T99K AND IT WAS NOT STABILIZED AT TEMPERATURES WE HAVE A MUTANT VIRUS WITH REDUCED BINDING AND IMPORTANTLY THEY FOUND THERE IS A

CONSEQUENCE FOR THIS VIRUS AND IT SUFFERS A DEFECT FROM VIRALIN STABILITY IN FECES THIS IS ONE OF THE GROSSER EXPERIMENTS WE’VE EVER DEVISED AND THAT’S SOMETHING FOR US INOCULATING MICE WITH THIS AND THE WILD TYPE VIRUS AND LET THEM POOP IN A CAGE 24 HOURS AND AGEING THE FECES SEVERAL DAYS LIKE A STEAK AND WE SPECIFICALLY FOUND THAT AFTER THE AGING THIS MUTANT IN PARTICULAR HAD A DEFECT THEN YOU CAN FEED THESE TO NEW MICE AND IT COMPOUNDS OVER TIME THIS SEEMS TO BE IMPORTANT SOW WHAT ARE THE DETERMINATES IN THE INTERACTION? WE WANTED TO SCREEN TO SEE WHAT MOLECULES IDENTIFY AND STABILIZE POLIO VIRUS AND WE CHOSE STABILITY BECAUSE IT’S AN EASY READ OUT HERE SIMILAR ASSAY AS BEFORE, WE INCUBATE VIRUS WITH AND HONESTLY WE STOLE CHEMICALS FROM PEOPLE WALKING UP AND DOWN THE HALL AND WE UP THE INCUBATION TO 42 AND DO THE PLAQUE ASSAY TO DETERMINE HOW MUCH VIRAL ASSAY IS LEFT WE’RE LOOKING AT THE PERCENT OF INPUT VIRUS YOU CAN SEE THE BUFFER TREATED VIRUSES, YOU LOSE ALMOST THE INEFFECTI INEFFECTIST — INEFFECTIVITY AS I ALLUDED TO BEFORE, LPS IS ONE AND WE FOUND A FEW OTHER MOLECULES SUFFICIENT FOR THIS AFTER THINKING ABOUT WHAT IS IT THE THINGS THAT WORK HAVE IN COMMON, WE REALIZED THERE ARE ALL GLYCANS AND THEY HAPPEN TO BE CONTAIN ONE SPECIFIC KIND OF GLYCAN GLUCOSAMINE SO I’M DEPICTING THAT HERE WITH A PURPLE HEXAGON INTERESTINGLY, HERE THE PURE OLIGOMER WAS SUFFICIENT BUT MONOMERIC WAS NOT BUT THAT’S ALL WE KNEW AT THAT POINT SO WE KNEW THE SHORT GLYCANS DO NOT STABILIZE POLIOVIRUS AND THERE’S A COUPLE HYPOTHESES FOR WHY THEY MIGHT BE MAYBE THEY JUST DON’T BIND THE VIRUS OR MAYBE THEY BIND BUT IN ORDER TO HAVE ACTIVITY YOU HAVE TO BRIDGE MULTIPLE BINDING SITES SO THERE ARE 60 POTENTIAL BINDING SITES BECAUSE THERE’S 60 INDIVIDUAL PROTEINS PER VIRAL PARTICLE AND MAYBE YOU NEED TO BRIDGE MULTIPLE SITES TO LOCK THE VIRUS INTO A CONFIRMATION THAT IS STABLE WE SAT ON THAT FOR A WHILE UNTIL A NEW POST-DOC CAME IN AND HE’S LIKE A LEGIT GLYCAN GUY AND HE CAN DO THINGS WE HAVEN’T BEEN ABLE TO DO BEFORE HE’S BEEN PURIFYING GLYCANS AND EXAMINING THEIR BINDING AND ACTIVITY AND USES A TECHNIQUE WHERE YOU CAN VISUALIZE GLYCANS ON GELS HE WOULD DO THINGS LIKE GET DIFFERENT LENGTHS AND RUN FRACTIONS AND THEN QUERY EACH FRACTIONS FOR BINDING AND ACTIVITY AND SO FORTH AND I’M JUST GOING TO SUMMARIZE HIS DATA HERE BECAUSE IT’S STILL VERY MUCH ONGOING IT’S LOOKING LIKE HYPOTHESIS 2 SEEMS TO BE AT PLAY WHERE WE DON’T SEE BINDING WITH THE GLYCANS BUT SIX ARE ABLE TO BIND HOWEVER THEY DO NOT HAVE BINDING IN THE STABILITY ASSAY AND THE SHORT ONES MAYBE DIDN’T BRIDGE BECAUSE YOU HAVE TO BRIDGE MULTIPLE BINDING SITES NEXT WE WANTED TO VISUALIZE THE INTERACTIONS AND HE DID EM AND A THOUGHT HE WAS CRAZY BECAUSE POLIOVIRUS IS ABOUT THE SIZE OF A RIBOSOME AND HE WOULD SEE AND HE LIKED TO GO TO THE CORE WITH REOVIRUS BECAUSE IT’S LARGER THAN POLIOVIRUS AND YOU CAN SEE THE PARTICLES STUCK TO DIFFERENT

KINDS OF BACTERIA WE CALL THIS THE E. COLI REOVIRUS NECKLACE THE IMAGES KIND OF CHANGED OUR THINKING PEP — WHAT IS THE INFECTIOUS UNIT? JURE INGEST — YOU’RE INGEST ING BACTERIA COATED WITH VIRUS AND ARE BACTERIA BOUND VIRUS THE TRANSMITTED FORM AND MIGHT THERE BE INTERESTING CONSEQUENCES OF THE INTERACTIONS THE IDEA THAT MAYBE YOU COULD SHOEHORN IT INTO A VIRUS AND THAT’S AN AREA WE’VE BEEN WORKING ON I WANT TO TELL YOU WHY I THINK THIS IS IMPORTANT THE IDEA OF DELIVERING MORE THAN ONE VIRUS PER CELL RNA VIRUSES HAVE NO PROF PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME.ROF PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME.F PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME.PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME.PROOF-READING ON AVERAGE THEY HAVE ONE NUCLEOTIDE MISTAKE PER GENOME MOST OF THESE REDUCE MUTATION AND SOMETIMES INACTIVATE EVERY ONCE IN A WHILE YOU’LL GET A BENEFICIAL MUTATION TO ALLOW RESISTANCE TO NEUTRALIZING ANTIBODY BUT FOR THE MOST PART THE MOU — MUTATIONS ARE BAD NEWS BUT YOU CAN USE SEVERAL GENETIC PROCESSES THE IDEA IS IF YOU CAN SHOVE TWO VIRUSES INTO THE SAME CELL AT THE SAME TIME, THEY CAN GET BY WITH A LITTLE HELP WITH THEIR FRIENDS FOR EXAMPLE, THERE’S COMPLIMENTATION IF THEY HAVE A PINK AND BLUE VIRUS AND DIFFERENT GENOME EFFECTS MAYBE IF THEY’RE BOTH IN THE SAME CELL AT THE SAME TIME YOU CAN FACILITATE ENOUGH REPLICATION TO GET REVERSION THERE’S GENETIC RECOMBINATION WHERE IF THEY HAVE TWO DIFFERENT ATTENUATING MUTATIONS YOU CAN RESTORE THE WILD TYPE AND FOR VIRUSES LIKE INFLUENZA AND REOVIRUS, THEY CAN UNDER GO REASSORTMENT TO GET A NOVEL VARIANT HOWEVER THE PROBLEM IS EACH PROCESSES KNOWN TO BE BENEFICIAL TO VIRUSES REQUIRE SYNCHRONIZATION THINK OF THE EXCESS OF YOUR CELLS IN COMPARISON TO THE NUMBER OF VIRUSES PRESENT WHAT ARE THE CHANCE THE PINK AND BLUE WOULD EVER BE AT THE SAME CELL IN THE SAME TIME HOWEVER IF THEY’RE RIDING IN TOGETHER, MAYBE THIS COULD HAPPEN LET ME WALK THROUGH LOGIC WE THINK THE FIRST INFECTION EVENT IN THE GUT IS SPECIAL AND UNIQUE OBVIOUSLY DIFFERENT THAN REPLICATION IN CULTURED CELLS I WOULD ALSO ARGUE THAT IT’S VERY DIFFERENT FROM OTHER CYCLES OF REPLICATION WITHIN THE HOST SO YOU’RE TALKING ABOUT VIRUSES GOING FROM THIS BACTERIA FINDING THE FIRST CELL AND THEN REPLICATE AND GET LOCAL RELEASE OF THOUSANDS OF PROGENY VIRUSES IT’S POSSIBLE EVERY SUBSEQUENT CYCLE MAY BE EASIER BUT THE FIRST INFECTION EVENT IS SPECIAL AND UNIQUE AND PROBABLY DIFFICULT SO WE MADE THIS HYPOTHESIS MAYBE BACTERIA FACILITATES THE WHOLE CO-INFECTION IDEA WHERE MULTIPLE VIRUSES ARE DELIVERED EVEN WHEN HAVE YOU VERY FEW VIRUSES PLENTY LIKE DURING INTERHOST TRANSMISSION SO TO START EXAMINING THIS WE AMASSED A COLLECTION OF CULTURABLE AL BACTERIA USING AN ANAEROBIC CHAMBER AND STARTED MEASURES IN THE ABSENCE OF BACTERIA WITH FEW VIRAL PARTICLES TO SEE IF BACTERIA COULD FACILITATE THIS HERE AGAIN WE USED RE-AGENT THE POLIO FIELD THESE ARE VIRUSES DEVELOPED HERE AT NIH AND SHE MADE A POLIOVIRUS EXPRESSING FLUORESCENCE PROTEINS AND WE HAVE A GREN AND RED ONE — GREEN AND RED ONE AND CAN MIX IT AND INFECT HELA CELLS SO ONLY 1% OF THE CELLS ON THE PLATE ARE INFECTED AND CAN DO FLEU FLEU FLEURO FU FLUOROCYTEOMTRY AND

WE HAD NEGATIVE STRAINS AND GRAM POSITIVE STRAINS WAND WE SEE A LOW LEVEL OF CO-INFECTION BUT WHAT WAS PREDICTED SO YOU CAN SEE WHILE SOME BACTERIAL STRAINS DON’T DO MUCH IN THE FREQUENCY SOME INCREASE CO-INFECTION QUITE A BIT CERTAIN BACTERIAAL STRAINS EFFECTIST SO IT WOULD BE OUCH IT BINDS MAYBE THE ONES THAT INDUCE CO-INFECTION BIND MORE OR MAYBE EXPECT LIKE BACTERIAL ADHERENCE TO CELLS SOME ADHERE BETTER THAN OTHERS TO HOST CELLS OBVIOUSLY, YOU HAVE TO BIND TWO VIRUSES TO EVEN SCORE IN THE ASSAY BUT MAYBE YOU DON’T NEED A HUGE NUMBER OF VIRUSES BOUND IN FACT, THAT’S WHAT WE FOUND WE FOUND THERE WASN’T MUCH OF AN EFFECT OF BINDING EFFICIENCY AND IT WAS MORE HAVING TO DO WITH THE ABILITY OF THE BACTERIAL STRAIN TO SLAP DOWN AND THE HELA CELL SO THEN WE THOUGHT IT WOULD BE IMPORTANT TO EXAMINE THE GENETIC CONSEQUENCES OF THIS SO COULD IT INFLUENCE SOMETHING LIKE GENETIC RECOMBINATION AND AGAIN THIS IS WHERE WE HAD A HUGE TOOLBOX USING OUR MODEL SYSTEM VIRUSES AND WE HAD DRUG RESISTANT MUTANTS SO WE HAD RE-AGENT DEVELOPED BY MY POST-DOC MENTOR WHEN SHE WAS A POST-DOC AND THEY DEVELOPED THE TOOLS TO STUDY GENETIC RECOMBINATION IT’S A SIMPLE SYSTEM HERE’S THE WILD TYPE VIRUS SENSITIVE TO A DRUG AND CAN GROW AT HIGH TEMPERATURE AM HERE WE HAVE A MUTANT VIRUS AND IT CAN’T FORM FLAK PLAQUES AT THE DUAL-RESTRICTIVE CONDITION BUT IF YOU GET A RE RECUMBENT EVENT IT READILY FORMS PLAQUES SO WHAT AN EASY READOUT IF I’M GOING ASK ANDREA TO SCREEN 40 BACTERIAL SCREENS IT’S A SIMPLE PLAQUE ASSAY WE CONFIRM GENETICALLY THE EXPERIMENT’S SUPER SIMPLE WE TAKE THE RE-AGENT AND INCUBATE WITHOUT BACTERIAL STRAINS AND INFECT THEM UNDER DUAL PERMISSIVE CONDITIONS SO RECOMBINATION CAN HAPPEN IF IT’S GOING TO HAPPEN AND TIDER THE PROGENY VIRUSES TO DETERMINE THE NUMBER OF RECOMBINANTS VERSUS THE NUMBER OF VIRUSES I WON’T WALK YOU THROUGH THE DATA BUT SAY BACTERIA THAT PROMOTED CO-INFECTION THE BARS BIGGEST ON THE YELLOW BAR CHART ALSO PROMOTED GENETIC RECOMBINATION THIS IS IMPORTANT BECAUSE THE VIRUSES ARE DEAD WITHOUT THAT RECOMBINATION EVENT SO IT WAS ABLE TO RESURRECT THEM AND THIS WAS DEPICTED HERE WHERE WE HAVE MULTIPLE VIRAL GENOMES BEING DELIVERED TO THE MAMMALIAN CELL AND THAT CAN RESCUE FITNESS OF A VIRUS THIS PROMPTED ONE TWITTER USER TO SAY I ALWAYS EXPECTED THE LAST BASTARDS WERE IN COULD COHOOTS WHICH I APPRECIATE AND WE THOUGHT IT WOULD BE IMPORTANT TO EXAMINE THE PROCESS IN MICE AND OTHER VIRAL SYSTEMS ANDREA WAS DEVELOP REOVIRUS SYSTEM IT ALSO HAS A SEGMENTED GENOME AND READILY UNDER GOES REASSORTMENT AND WORK IN THE ’80s SHOWS THEY WERE DETECTABLE AND THOUGHT IT WOULD BE A GREAT SYSTEM TO LOOK AT THE HYPOTHESIS IN MICE THE WAY THIS WORKS FAIRLY SIMPLE, WE HAVE TO TWO DIFFERENT REOVIRUS STRAINS, T3D AND 26 — T10 AND WE CAN TELL THEM APART BY ANTIBODY AND THE GENETIC SEGMENTS IN A GEL I’LL SHOW YOU IN A MINUTE WE MIXED THEM TOGETHER AND INOCULATED MICE AND HARVEST TISSUE AND FECES AND QUERY THE

RESULTING VIRUSES WITH A GEL-BASED SYSTEM IF YOU JUST RUN GELS YOU CAN SEE THE 10 GENOME SEGMENTS AND SEE THE T3D AND T1L RUN DIFFERENTLY AND THERE’S A CONSTELLATION OF DIFFERENT ASSORTMENTS SO IT’S AN EASY READOUT THE MOUSE CONDITIONS TO USE, WE CAN USE CONVENTIONAL MICE WAY NORMAL MICROBIOTA OR USE STERILE GERM-FREE MICE WHERE THEY HAVE BUBBLE ISOLATORS AND YOU HAVE GREAT ISOLATOR FOR THAT HERE AND THE IDEA IS IF WE CAN FIND BACTERIAL STRAINS THAT PROMOTE OR DO NOT THE CO-INFECTION PROCESSES RECAN — WE CAN RECOLONIZE THE MICE AND FIRST DO THE ORAL INOCULATIONS OF GERM-FREE MICE AND FIRST DO PLAQUE ASSAYS TO FIGURE HOW MUCH VIRUS IS THERE AND EXAMINE THE REASSORTMENT FREQUENCY THIS IS THE AMOUNT OF VIRUS PRESENT SO FECAL VIRUSES THE CONVENTIONALS AS EXPECTED INCREASING VIRUS OVER TIME AND THEN WE WERE IN FOR A HUGE SHOCK THAT WAS THAT FOR A FEW OF THESE TIME POINTS THE TIDERS FROM THE GERM-FREE MICE WERE HIGHER AND THIS WAS CONTRARY TO A LOT OF OUR PREVIOUS WORK AND THE SAME WAS TRUE IN TISSUES YOU SEE HERE IN TURQUOISE, THE TIDERS IN THE GERM-FREE MICE WERE HIGHER SO OUR PREVIOUS WORK WITH ANTIBODY TREATED MICE SUGGEST THE OPPOSITE RESULT SO WE’RE STILL TRYING TO WRAP OUR HEAD AROUND THE RESULTS WE THINK IT HAS SOMETHING TO DO WITH IMMUNE DEFICIENCY IN THE GERM-FREE MICE SO WE’RE FOLLOWING UP AND STILL WE THOUGHT WHAT WOULD GET THE FREQUENCY USING THE GEL-BASED ASSAY AND WE’RE USING AT THE RELATIVE REASSORTMENT FREQUENCY AND YOU CAN SEE THE FREQUENCY IN THE MICRO IOTA HOVERING MICE IS DIFFERENT THAN THE GERM-FREE MICE THIS IS INTERESTING BECAUSE REMEMBER THE TIDERS IN THE GERM-FREE MICE IS ACTUALLY HIGHER SO IT SUGGESTED IN SPICE OF THE HIGHER VIRAL TIDERS THEY HAVE LOWER RESORTMENT FREQUENCIES SUGGESTING BACTERIA MAY BE PROMOTING THIS PROCESS IN VIVO WE WONDER IF VIRAL CO-INFECTION IS MORE COMMON THAN WE THOUGHT THE IDEA OF INFECTION IS SINGLE PARTICLES IN AND OUT BUT WE THINK BACTERIA MAY BE THAT SCAFFOLD FOR INFECTION AND THAT’S WHAT’S TRANSMITTED IN BETWEEN HOSTS WHICH ACTUALLY CHANGES THE WAY YOU THINK ABOUT THE IMMUNE RESPONSE AS WELL WE KNOW THERE ARE OTHER MECHANISMS OF CO-INFECTION SPEARHEADED HERE WITH A LAB THAT ID THE IDEA THEY COULD BE SHED AND THEY CAN INDUCE CO-INFECTION AND MY LAB HAS SHOWN VIRUSES CAN CLUMP UNDER AGGREGATES UNDER PHYSIOLOGICAL CONDITIONS AND THEY TOO CAN INDUCE CO-INFECTION TO SUMMARIZE THIS PART, I’VE SHOWN YOU BACTERIA SEEM TO PROMOTE ENTERIC REPLICATION AND TRANSMISSION THERE’S A COMMON THEME, WE HAVE FOUR VIRUSES OF ENTERIC VIRUSES THAT ALL BIND BACTERIAL SURFACE GLYCANS POLIO BINDS PEPTOGLYCAN AND REOVIRUS WE KNOW BINDS LPS AND THE MOUSE RETROVIRUS BINDS LPS AND WORK HAS SHOWN NOROVIRUS CAN COMBINED HISTOANTIGENS ON SELECT BACTERIAL STRAINS THERE’S DISTINCT MECHANISMS AND THEY’RE NOT MUTUALLY EXCLUSIVE BUT IT’S AN ACTIVE AREA OF INVESTIGATION FOR A LOT OF LABS I WANT TO GIVE A SHUTOUT TO THE FACULTY DRIVING THE FIELD FORWARD WE’VE BEEN DUBBED THE POOP SISTERS BY A SENIOR COLLEAGUE IN THE FIELD AND THE NAME STUCK SO THESE ARE THE PEOPLE THERE’S A FEW NOT DEPICTED HERE SO IT’S BEEN A WONDERFUL COMMUNITY I WANT TO TELL ONE SHORT STORY ABOUT STEPHANIE ON THE END SHE WORKS IN POLIO VIRUS AND THE STORY IS ABOUT HUMAN NOROVIRUS IT’S A TERRIBLE VIRUS THOSE WHO HAVE HAD IT KNOW THIS

INCREDIBLY UNCOMMON, MISERABLE AND UNCULTURABLE FOR FOUR DECADES OR MORE WHICH MAKES YOU DEPENDENT ON HUMAN VOLUNTEERS AND CULTURE ATTEMPTS HAVE BEEN ONGOING AND PEOPLE WOULD FILTER STOOL BECAUSE YOU DON’T WANT TO CONTAMINATE OUR CELL CULTURE AND STEPHANIE SAW OUR WORK AND FOUND INDEED IF SHE ADD THE BACTERIA BACK, FINALLY FOR THE FIRST TIME IN 40 YEARS, SHE COULD GET HUMAN NOROVIRUS TO REPLICATE IN CULTURED B CELLS IT’S TRICKY AND DOESN’T WORK IN EVERYBODY’S HANDS BUT ROBUST IN HER HANDS AND THIS IS THE FIRST EVIDENCE FOR THIS AS THE BEEN MORE EFFORTS MADE AND THIS IS AN EXAMPLE OF A FIND FROM A BASIC SCIENCE NOBODY CARES ABOUT CAN DO SOMETHING MORE CLINICALLY IMPORTANT SO WE CONTINUE TO DO A LOT OF WORK ON THE VIRUS MICROBIOTA PROJECTS BUT I WANT TO END WITH A COUPLE SLIDES ON THE NEW PROJECT DEALING WITH THIS INNATE MONTGOMERY SHOW OF HANDS, HOW MANY PEOPLE HEARD OF THIS? MORE THAN I WOULD HAVE THOUGHT THAT’S AMAZING THESE ARE SINGLE-CELL EUKARYOTIC PROTIZOA AND THE REASON THEY BECAME OF INTEREST IN TERMS OF THEIR INNA INNATE IMMUNITY MAMMALIAN AND IMMUNE RESPONSES ARE ECONOMY KATED YOU HAVE LIGANDS THAT ARE RECOGNIZED PRODUCING CYTOKINES AND INTERFERON AND INNATE AND ADOPTIVE CELLS AND WHAT A NIGHTMARE AND WHY I’VE AVOIDED IMMUNITY MY ENTIRE LIFE SO HOW DID WE GET HERE AND BUILD UP THIS COMPLEXITY IN EVOLUTIONARY HISTORY AND A PATHWAYS ARE MISSING IT’S AN IMPORTANT PATHWAY BUT ALL THE REDUNDANCY MAY BE LIMITING OUR DISCOVERY OF SOME OF THESE THINGS SO WE SPENT A LOT OF TIME LOOKING AT THE MODIFIED VIEW OF THE TREE OF LIFE WE HAVE BACTERIA AND PLANT, FUNGI, AND THEY’RE RIGHT THERE I’LL GET BACK IT AND THEN WE START WITH ANIMALS, SPONGES MOVING UP TO MICE AND HUMANS THIS PART OF THE TREE OF LIFE IS REALLY IGNORED BY A LOT OF BIOLOGY BUT THERE’S BEEN MAJOR INNA INNATE IMMUNITY AND SO WE THINK THERE’S AN UNTAPPED POTENTIAL IN THESE KINDS OF ORGANISMS SO THIS PROJECT REALLY IS COMPLETELY DRIVEN BY A TALENTED POST-DOC WHO RECENTLY GOT A FANCY FELLOWSHIP AND SHE DID HER Ph.D. WITH NICOLE KING KIND OF THE KING OF THIS FIELD SO WHAT ARE THESE THINGS? THEY’RE AQUATIC EUKARYOTIC PROTOZOA ANIMALS ARE MULTICELLULAR ORGANISMS AND THESE ARE UNICELLULAR BUT CAN BECOME MULTICELLULAR AND I’LL GET BACK TO THAT IN A MINUTE SOME CAN BECOME MULTICELLULAR THEY’RE UBIQUITOUS AND YOU CAN GO TO ALMOST ANY WATER AND THIS IS THE DISGUSTING CREEK THAT FLOWS THROUGH OUR BUILDING AND THIS IS COLLECTION FROM THE CREEK AND WE CAN PURIFY THEM AND GROW THEM IN THE LAB SO SHE CAME IN MY OFFICE ONE DAY AND SAID THERE MUST BE A VIRUS THAT INFECTS THESE THINGS AND THEY HAVE UNKNOWN IMMUNITY WE THOUGHT WHAT AN INTERESTING UNTAPPED SYSTEM AND THE DOWNSTREAM PATHWAYS THEY’RE NOT HERE BECAUSE THEY’RE NOT MULTICELLULAR BUT WE WONDERED IF THEY HAVE SENSING PATHWAYS AND A UNIQUE WAY OF RESPONDING TALKING BRIEFLY HOW MAMMALS UNDERGO INNATE IMMUNE SENSING

THEIR BACTERIAL VIRAL SIGNALS AND GLYCANS OR DOUBLE-STRANDED RNA CAN INITIATE CYTOKINES AND WE HAVE SYTOSTOLIC DNA CAN COME THROUGH THIS PATHWAY SO THAT’S ALL GOOD SO WE PUBLISHED THE TRANSCRIPTOMES OF THESE SPECIESES AND COMPARED TO THE MAMMALIAN COMPLEXITY THIS IS WHAT THE CHON ANOS HAD A COUPLE HAVE GAS AND STING AND DON’T HAVE A LOT OF THE FUNCTIONAL RNAI OR TYPICAL RNA OR INTERFERON OR CYTOKINES HOWEVER, IF WE TREAT IT WITH THE KNOWN MICROBIAL LIGANDS KILLS THEN AND IT CAN KILL SOME BUT NOT ALL THE CHOANO SPECIES IN PROGRESS SHE OBVIOUSLY YOU DO TRANSCRIPTOMICS AND HAS A BUNCH OF HITS SOME CONFIRMED ONES ARE SHOWN THE IFI44L THE IF STANDS FOR INTERFERON WHAT IS IT DOING AN ORGANISM THAT DOESN’T HAVE INTERFERON? COULD IT BE AN EARLY BUILDING BLOCK THERE’S INTERESTING HITS TO FOLLOW-UP ON AND I’M EXCITED ABOUT HER FORWARD GENETIC SCREENS YOU CAN SELECT FOR THE SYSTEMS AND MAN — MAP FOR LESIONS THIS ARTICLE WAS PUBLISHED ON IT TINY SWAMP CREATURES TO CAN LEAD TO IMMUNOLOGY WATCH FOR AERIAL SHE IS SPECIAL I TRIED TO THINK THE LAB MEMBERS AS WE WENT ALONG AND FUNDING FROM NIAID AND SEVERAL FOUNDATIONS, THE FOUND FUND — THE FUNDING HAS BEEN PIVOTAL SO THIS HAS BEEN HELPFUL I’D LIKE TO THANK THE U.T SOUTHWESTERN ENVIRONMENT IT’S BEEN AN INCREDIBLE ENVIRONMENT TO DO THIS WORK IN PARTICULAR WITH LAURA HOOPER AND SEBASTIAN WINTER’S HELP I’LL STOP THERE AND I’M HAPPY TO TAKE QUESTIONS IF THERE ARE TRAINEES IT WOULD BE AWESOME IF YOU WENT FIRST >> I HAD A QUESTION ABOUT THE RISK FOR THE VIRUS TO USE THE BACTERIA FOR THE CO-INFECTION MODEL DOES THAT RISK ACTIVATION OF TLRs AND I’M SURE YOU’VE LOOKED AT THAT >> SO FOR OUR POLIO WORK, ALL OF THAT WORK WAS DONE IN VITRO FIRST OF ALL AND ALL DONE IN HELA CELLS THAT LACK THE TLRs SO IN SOME SWENS — SENSE WE MAY HAVE CLEANED THE SYSTEM TOO MUCH IN VIVO ALL BETS ARE OFF AND WE LIKE THE EXPERIMENTS IN VIVO AND WE CAN COLONIZE GERM-FREE MICE WITH MUTANT DO OR DON’T HAVE THE PROPERTIES SO THAT’S GOING TO BE A BETTER ANSWER TO THAT >> THAT WAS A BEAUTIFUL TALK I’M INTERESTED IN VIRUSES MOVING AROUND AND YOUR VIRUS HAS THE PROBLEM IT HAS TO BE STABLE IN A SENSE TO SURVIVE ITS TRIP UNSTABLE ENOUGH TO UNCOAT I’M LOOKING TO SEE IF YOU SEE CAN SEE VIRUS AND EVEN JUST LPS HAVE YOU LOOKED TO SEE IF IT

STILL MOVES AND B, DO THESE BACTERIAL ASSOCIATIONS REMOVE A RETIREMENT FOR POCKET FACTOR IN BIOLOGY >> POCKET FACTOR LINGO IS LIPID IN THE PARTICLES AND STABILIZES THEM AND IS INVOLVED IN THE UNCODING PROCESS THAT WAS INITIALLY OUR FIRST THOUGHT WHEN WE SAW LPS WAS ONE OF THE LIGANDS WE HAD EVIDENCE IT’S AT PLAY BUT IT IS THE POCKET FACTOR MUTANTS IF WE WANTED TO IT’S THAT PART IN DETAIL AND WE HAVEN’T GOTTEN THERE YET BUT WE LIKE THE IDEA A BACTERIAL LIPID COULD PLAY A ROLE ON THE OTHER QUESTION YOU HAVE THE INTERFACE WITH BACTERIA AND HANDING OFF TO THE RECEPTOR AND HOW’S THAT WORK WITH THE DYNAMIC WE HAVEN’T LOOKED AT ALL EXPOSURE OF BP4 THERE WAS A COLLECTION OF ANTIBODIES THAT CAN RECOGNIZE SOME OF THE LIGANDS NORMALLY INTERM AND WE THOUGHT ABOUT THOSE EXPERIMENTS AND HAVEN’T GOTTEN TO THEM WE HAVE 60 BINDING SITES FOR THE SECTOR AND FOR THE BACTERIAL LIGANDS ON EACH PARTICLE ONE PHASE WOULD BE OCCUPIED BY THE BACTERIA AND THE OTHER PHASE WOULD BE FREE TO BIND TO THE CELLS BUT IT’S HAND WAVING AT THIS POINT >> THANK YOU >> I’M STILL TRYING TO WRAP MY HEAD AROUND THE DISCORDANTS BETWEEN THE ANTIBIOTIC TREATED AND GERM-FREE MICE WONDERING IF THERE’S AN OPPORTUNITY TO LOOK AT THE BACTERIA YOU SHOWED WOULD ENHANCE THE RECOMBINATION AND THINK WHICH ANTIBIOTICS YOU’RE USING BECAUSE THERE MAY BE SOME GREATER SPECIFICITY THERE AND IT ALSO STRIKES ME THAT WHEN WE GIVE THE ANTIBIOTICS THERE’S AFFECTS ON THE HOST IN TERMS OF THE MUCOSAL LINE THAT MAY PLAY INTO THIS I’M NOT SURE I HAVE A QUESTION EXCEPT TO SAY ABOUT USING DIFFERENT CLASSES OF ANTIBIOTICS AND IF I HAD A QUESTION, WHICH MAYBE YOU COULD ANSWER, IT DID GET ME THINKING A LOT ABOUT WHEN SOMEONE PRESENTS AND YOU’RE NOT SURE IF IT’S A BACTERIAL OR VIRAL INFECTION AND YOU GIVE THEM ANTIBIOTICS AND WHETHER THIS KIND OF IDEA MIGHT THEN BE EXACERBATING THAT IN THE ANTIBIOTICS MAY BE A GOOD PLACE TO TEST >> THE IMPLICATION OF SHARON’S WORK IS MAYBE ANTIBIOTICS CAN HAVE ANTIVIRAL EFFECTS BOUGHT WE WE — BECAUSE WE HAVE FIVE ANTIBIOTICS THAT WOULD NEVER BE CLINICALLY ACHIEVABLE FOR POLIO WE THINK IT’S A NUMBERS GAME AND YOU HAVE TO KNOCK DOWN A THE FLORA A LOT BUT FOR THE CLOSE COUSIN VIRUS YOU CAN GIVE THE MICE A SINGLE DOSE OF STREPTOMYOCIN AND IT DROPS IT TWO LOGS AND WE’RE CHASING THAT IN THE LAB IN TERMS OF YOUR EARLIER QUESTION WE THINK A LOT ABOUT ARE THERE OFF-TARGET THINGS REVEALING THE FEENPHENOTYPES AND WE WERE CAREFUL TO CONTROL FOR THAT AND WE TOOK AN ANTIBODY RESISTA RESISTANT ANTIBIOTIC AND THEY WERE ON THE ANTIBIOTIC REGIMENT AND THERE WAS SUFFICIENT TO RESTORE VIRAL APPLICATION WE THINK IT’S NOT STRICTLY SOME OFF-TARGET THING BUT MESSY >> SO YOU MENTIONED ONLY ONE PERCENT EACH LESS THAN ONE PERCENT OF NUMBER OF PATIENTS REALLY AFFECTED SO WHAT HAPPENS? BASICALLY OUR PRESIDENT ROOSEVELT HAD THESE LIFE LONG PARALYZATION >> WE THINK IT HAS TO DO WITH BREAKDOWN OF BARRIER FUNCTION WE THINK THERE’S MULTIPLE LINES OF DEFENSE IN MOST INDIVIDUALS LIMIT VIRAL HOME TO THE CENTRAL NERVOUS SYSTEM THERE’S INNATE IMMUNITY AND THE GUT BARRIER ITSELF THERE IS INEFFICIENT VIRAL MOVEMENT AND NEURON AND WE THINK THERE HAS TO BE SOME BREAKDOWN AT LEAST IN A

COUPLE OF THOSE THINGS IF NOT ALL THREE FOR THE VIRUS THEN TO REACH THE CNS THAT MIGHT EXPLAIN WHY IT WAS PRETTY RARE BUT NOT SO RARE IT’S A RARE HUMAN MUTATION THIS ONE PERCENT FREQUENCY DOESN’T REALLY SPEAK OF THINGS YOU’D EXPLAIN BUT PERHAPS A MULTIMANY OF MULTI-LAYERED BARRIER BREAKDOWN >> WHEN YOU SUPPRESS THE REPCATION DO YOU SEE VARIANTS LESS DEPEND EN FOR THEIR PROPAGATION? >> THAT’S A GREAT QUESTION AND SOMETHING TO SEE IF YOU CAN EVENTUALLY GET A RESISTANT VARIANT TO EMERGE WE TRIED A COUPLE PASSAGES DIDN’T GET ANYTHING BUT WE DIDN’T REALLY TRY >> IF THERE’S NO MORE QUESTIONS LET’S THANK JULIE FOR A FANTASTIC TALK THERE’LL BE A RECEPTION IN THE LIBRARY WE HOPE YOU CAN JOIN US