THE GOOD AFTERNOON, EVERYBODY. I'M DIRECTOR OF THE H.I.V. DYNAMICS, REPLICATION PROGRAM AT THE NCI FREDERICK AND IT'S A PLEASURE TO INTRODUCE THE 2016 MEMORIAL LECTURE I'M NOT SURE HOW I ADVANCE THE SLIDES. SO I DIDN'T HAVE THE GOOD FORTUNATE OF KNOWING GEORGE PERMANENTLY, BUT I KNOW MANY OF HIS ACQUAINTANCES AND COLLABORATORS AND HERE HE IS AROUND 1986 WITH HIS POST DOCTORAL MENTOR, MALCOM ARTOIN, AND HIS COLLEAGUE, PETER HOWLEY. GEORGE. A GRADUATE OF PRINCETON AND HARVARD MEDICAL SCHOOLS. CAME TO THE NIH IN 1976 TO HEAD UP THE VIRUS TUMOR BIOLOGY SECTION AND IN 1980, BECAME CHIEF OF THAT LABORATORY. IN 1981, HE RECEIVED THE ARTHUR S. FLEMING AWARD FOR OUTSTANDING GOVERNMENT SERVICE. AT AGE 43, IN 1987, THE YEAR THAT HE TRAGICALLY DIED OF CANCER, HE WAS AN ELECTED MEMBER OF THE NATIONAL ACADEMY OF SCIENCES GEORGE PUBLISHED OVER 140 PAPERS, DECIPHERING MECHANISMS OF EUKARYOTIC TRANSCRIPTION. HE HAD A VERY DISTINGUISHED GROUP OF COLLABORATORS AND TRAINEES, SOME OF WHICH ARE LISTED BE L. HIGHLY DISTINGUISHED, THESE GROUPS OF SPEAKERS, INCLUDE SEVERAL NOBLE LAUREATES AND BE MISS OF THE NATIONAL ACADEMY OF SCIENCES. CONSISTENT, WE ARE VERY PLEASED AND FORTUNATE, THIS YEAR, TO HAVE CHARLIE RICE, THE 2016 GEORGE CORY LECTURE. CHARLIE IS PROFESSIONAL AND HEAD OF THE LABORATORY OF INFECTIOUS DISEASE. Ph.D. AND POST DOCTORATE RESEARCH AT CAL TECH AND THEN THE LATE 1980'S WAS THE FACULTY AT WASHINGTON UNIVERSITY AT ST. LOUIS, AND IN 2,000, HE CONTINUED HIS EASTWARD MIGRATION TO JOIN THE FACILIT FACULTY IS ROCKER FELLER UNIVERSITY. CHARLIE HAS MANY HONORS, TOO NUMEROUS TO LIST HERE. HE IS A NATIONAL MEMBER OF THE ACADEMY SCIENCES, LAST YEAR, HE WON THE ROBERT COKE,A WARD, HE'S PAST PRESIDENT OF AMERICAN SOCIETY OF VIROLOGY AND A FELLOW OF AAAS. CHARLIE HAS PUBLISHED OVER 400 PAPERS IN THE FIELD OF VIRALLOLOGY, AND HE HAS BEST KNOWN FOR CLONING AND SEQUENCES OF RNA VIRUSES, DEVELOPING IN-VITRO -- RECEPTORS AND KORE SEPTUORS FOR INNECK AND ILLUMINATION-BASED LOSEDATING MECHANISMS OF THE ANTIVIRAL INMATE IMMUNITY. IT'S A GREAT PLEASURE TO INTRODUCE CHARLIE RICE FOR THE 2016 GEORGE CURRY LECTURE. HEPATITIS C VIRUS AND HIS FELLOW, I THINK IS SOMETHING LIKE, NEVER A DULL MOMENT, RIGHT? >> SO THANKS VERY MUCH, ERIC AND FOR ALL OF YOU FOR COMING. I'VE HAD A WONDERFUL DAY HERE AND IN FACT, IN SORT OF PREPARATION FOR THIS LECTURE, I SORT OF TOOK A LOOK AT GEORGE'S ACCOMPLISHMENTS BY THE TIME HE WAS IN HIS EARLY 40'S, AND REALLY, THE NUMBER OF DISCOVERIES THAT HE MADE, AND THE PEOPLE THAT HE INFLUENCED IS REALLY QUITE AMAZING AND MADE ME FEEL LIKE KIND OF A SLACKER. SO I WAS DECIDING TODAY, WHETHER OR NOT TO TALK TO YOU MORE ABOUT THE HISTORY OF HEPATITIS C OR COVER SOME RELATIVELY NEW WORK IN THE LAB AND I DECIDED ON THE LATTER BECAUSE I WOULD GUESS THAT NIH. PROBABLY THE HISTORY OF HEPATITIS C, AND THE CONTRIBUTIONS OF PLANE OF THE PEOPLE WHO ARE HERE AT THE NIH OR HAD BEEN AT THE NIH ARE WELL KNOWN. SO THIS REALLY IS QUITE A REMARKABLE TIME, IN THE STUDY OF THIS VIRUS. WHEN YOU THINK ABOUT WHERE WE COME, IT WAS IF THE 1980'S, THERE WAS THIS MYSTERIOUS AGENT OF POST TRANSFUSION HEPATITIS, NOT DUE TO HEPATITIS A OR B, AND IT WAS REALLY THE SORT OF KEEN EYE OF HARVEY ALTER, WHO'S HERE IN THE AUDIENCE THAT SORT OF NOTICED THIS. IN 1989, AGAIN, HARVEY, WITH MICHAEL HOW THEON AND HIS GROUP IDENTIFIED THE MAJOR CAUSATIVE AGENT OF THIS, CAUSED HEPATITIS C. THIS ALLOWED DIAGNOSTICS TO BE PUT IN PLACE QUICKLY, BLOOD SUPPLY, ATV-FREE, SHORTLY THEREAFTER AND TODAY IN 2011, WE WENT FROM A SUBFRACTION OF THOSE TREATED, TOO THE FIRST AGENTS THAT WERE DIRECTED AGAINST THE VIRAL PROTEASE, CURING 75%, IN COMBINATION WITH INTERFEREON AND [INDISCERNIBLE] AND TODAY, IN 2015 AND BEYOND, WE HAVE SEVERAL APPROVED THERAPIES THAT ARE REALLY QUITE REMARKABLE IN THEIR ABILITY TO ACHIEVE ELIMINATION OF THE VIRUS, IN MORE THAN 95% OF THOSE THAT ARE INFECTED. SO IN TERMS OF OF A STORY OF MEDICAL RESEARCH, THAT HAS SORT OF IMPACT IN THE CLINIC, THIS IS REALLY QUITE A TRAJECTORY. AND THING JUST SHOWS YOU, SOME OF THE EARLY DATA FROM SOME OF THESE COMPOUND, AND THIS SHOWS YOU -- THIS IS ONE OF THE NONSTRUCTURAL PROTEINS FOR VIRUS REPLICATION, AND PARTICLE ASSEMBLY AND NS5B, THE RNA-DEPENDENT ON POLYMERASE. A NUCLEOTIDE PRO DRUG. THISY SHOWS YOU SOME OF THE EARLY DATA, THIS SORT OF REMARKABLE, SUSTAINED BIOLOGIC RESPONSE RATE IN PATIENTS THAT ARE TREATED WITH THIS COMBINATION THERAPY. AND THIS IS ARVONI IS ONE OF THE APPROVED DRUGS FOR HEPATITIS C. BASICALLY, ONE PILL A DAY, SORT OF WITH REMARKABLE RESPONSE RATES IN THIS MOST OF THE JEEPO TYPES THAT ARE TREATED. SO YOU KNOW, I THINK THIS IS FOR THE FIELD, AND THAT INCLUDES THE CLINICIANS, AND THE BASIC SCIENTISTS, AND PEOPLE IN BIOTECH AND PHARMA, AND THE PATIENTS, THIS IS REALLY FANTASTIC NEWS, BUT I JUST WANT TO POINT OUT LAWYER REALLY, STILL SOME SIGNIFICANT CHALLENGES THAT ARE ASSOCIATED WITH REALLY IMPLEMENTING THIS. FIRST OF ALL, YOU ASKED AND WE DON'T KNOW MOST OF THE PEOPLE THAT ARE INFECTED SO OBVIOUSLY, IF YOU CAN'T IDENTIFY THE PEOPLE THAT ARE INFECTED, YOU CAN'T THE REALLY SORT OF ACHIEVE CURE AND IN THE MAJORITY OF THE POPULATION THE OTHER THING IS IMPLEMENTATION. ADVANCED LIVER DISEASE AND CANCER, ACTUALLY, SORT OF IMPLEMENTING THOSE ADVANCES IS REALLY, SORT OF PRETTY SLOW. AND THIS JUST SHOWMAN YOU, A COMPILATION OF THE HISTORY OF ATV TREATMENT IN THE U.S. UP TO 2014, SO REALLY, BEFORE THESE DIRECT ACTING ANTIVIRAL THERAPIES WERE EN VOGUE. AND YOU CAN SEE, APPROACHING 4 MILLION PEOPLE, WE ACTUALLY SUCCEEDED IN TREATING AND CURING ONLY ABOUT 10% OF THOSE. SO NOT A VERY GOOD TRACK RECORD. THE OTHER THING, WHICH I THINK MANY OF YOU PROBABLY NOTICED IN "THE WALL STREET JOURNAL" AND "THE TIMES" AND OTHER PAPERS IS THE COST OF THESE TREATMENTS. THIS NUCLEOTIDE PRO DRUG, WHEN IT WAS ORIGINALLY RELEASED WAS $84,000, BASICALLY, A THOUSAND DOLLARS A PILL FOR A FULL COURSE OF TREATMENT. FIRST PROTEINS JAYS INHIBITORSES, IF YOU LOOK AT THE GAP BETWEEN THE ACTUAL PRICE THESE WERE RELEASED AT IN THE U.S., AND THE ACTUAL COST OF MANUFACTURING THEM, YOU CAN SEE THERE'S A BIG GAP THERE. WE SHOULD REMEMBER, THAT A MAJORITY OF THE HEPATITIS C CASES ARE NOT IN COUNTRIES THAT CAN AFFORD THESE TREATMENT PRICES. THE GOOD NEWS IS THE PRICES ARE COMING DOWN, BUT PERHAPS NOT AS FAST AS WE WOULD LIKE THEM TO BE. THE CHALLENGING PATIENT GROUPS USED TO BE THE TREATMENT OF PEOPLE THAT WERE CO-INFECTED WITH H.I.V., A LARGE FRACTION OF THOSE IN THE U.S. WERE MORE DIFFICULT TO TREAT. THOSE WITH ADVANCED LIVER DISEASE. PEOPLE WITH MORE ADVANCED SIRROSIS AND SO ON, WERE DIFFICULT TO TREAT. FORTUNATELY, THOSE TREATMENTS, THESE NEW TREATMENTS ARE REALLY NORMALIZING THE TREATMENT RESPONSE. SO MANY OF THE DIFFICULT-TO-TREAT PATIENTS CAN BE TREATED. BUT ONE OF THE OTHER CHALLENGES WE HAVE IS WE REALLY DON'T KNOW IF IN THE THEY'RE CASE OF TREATING SOMEONE, AND GETTING RID OF THE VIRUS, IF THAT'S GOING TO ELIMINATE THEIR RISK OF LIVER DISEASE. AND THE PEOPLE WITH ADVANCED LIVER DISEASE. IT DOESN'T COMPLETELY ELIMINATE THE POSSIBILITY THAT THEY WILL DEVELOP LIVER CANCER. AND THE OTHER THING WE DON'T UNDERSTAND, IS REALLY SORT OF HOW THE DISEASE PROGRESSES. WE BELIEVE THAT THIS IS AN IMMUNE MEDIATED, SORT OF CHRONIC INFLAMMATION, THAT IS ASSOCIATED WITH THE DEVELOPMENT OF CANCER. WE DON'T UNDERSTAND HOW THIS MIGHT BE SORT OF REVERSED OR REVERSIBLE. AND, I GUESS THE OTHER THING IS VIRUS RESISTANCE. MANY HAVE IOLOGISTS IN THE AUDIENCE, -- VIROLOGIST, AND HEPATITIS C, WITH ABOUT 1 70 MILL ONE PEOPLE INFECTED. CHRONICALLY INFECTED INDIVIDUAL, AND THESE SEVEN GENOTYPES THAT WE ACTUALLY HAVE SOME INHIBITORS LIKE THE NUCLEO SIGHT INHIBITOR THAT HAVE VERY GOOD PANGENEO COVERAGE AND HIGH BARRIER TO CLINICAL RESISTANCE AND SOME OF THE OTHER COMPONENTS OF THE COMBINATION THERAPIES AREN'T THE QUITE AS GOOD, BUT THEY'RE GETTING BETTER. THERE ARE GOING TO BE TREATMENT FAILURES SO I THINK EVEN IF IT'S A SMALL FRACTION, 2% OR 5%. THAT'S STILL A LOT OF PEOPLE I HAVE TO WORRY ABOUT IN TERMS OF COMING UP WITH SALVAGE THERAPIES. I THINK MOST PEOPLE BELIEVE THAT A VACCINE MAY BE NECESSARY TO COMPLETELY ERADICATE THIS VIRUS FROM THE WORLD AND REALLY WORK ON A VACCINE, THE LAG BEHIND THE DEVELOPMENT, AND I THINK THAT'S ONE IRREVERSIBLE YEAH, THE ECONOMIC DRIVING FORCE, THE FARM SUIT IAL COMPANIES HAVE FELT FOR NEW THERAPIES, BUT STILL, AN IMPORTANT GOAL. BEING A MOLECULAR BIOLOGIST, WHAT SHOULD I DO NOW. AND IF YOU'RE ACTUALLY SORT OF SUCCESSFUL IN A FIELD, YOU KNOW, WHAT DO YOU DO NEXT? CONTINUE BAKE WORK ON ATV, SWITCH TO OTHER VIRUSES. MAYBE ZIKA SEEMS TO BE QUITE POPULAR THESE DAYS. BUILD ON A HEPATITIS C WORK. BUT TWIERSIFY, MAYBE CONSIDER AN ALTERNATIVE CAREER. THIS MIGHT BE A LITTLE LATE IN MY CASE OR ALL OF THE ABOVE. AND I WOULD JUST SORT OF MAKE THE ARGUMENT THAT WE TEND TO OVER REACT WHEN THERE ARE THESE KINDS OF ADVANCES MADE AND GO BACK TO HEPATITIS B, WHERE THE PROVINCE LAPPIC VACCINE WAS MADE, AND THE RECOMBINANT FORM IN 1986. EFFECTIVE CHEMOTHERAPY WAS DEVELOPED AND CONTINUED TO BE DEVELOPED CAN REALLY SORT OF CONTROL VIREMIA. AND RESEARCH HAS REALLY DEPRIORITIZED IN THE 90'S. I THINK THE HPV LABS WERE REALLY HAVING A HARD TIME. EVEN THOUGH IT WAS HARD TO UNDERSTAND THE LIFE CYCLE TO GET THE -- WE'RE LEFT WITH 240 MILLION PEOPLE, WHO ARE CHRONICALLY INFECTED BECAUSE HEPATITIS B CHRONIC INFECTION ARE TYPICALLY NONE CURATIVE. SO IF YOU WITHDRAW TREATMENT, THE VIRUS COMES BACK. AND NOW, WE'RE SEEING SORT OF A RESURGENCE OF HPV, RESEARCH IN 2013, WITH SOME OF THESE ADVANCES. SO SOME PEOPLE, MOVING BACK IN THE ATV WORD INTO HPV AND OTHERS, REALLY JUST SORT OF HANGING IN THERE AND CONTINUING TO WORK ON THIS VIRUS. SO I THINK A LOT OF REASONS WHY WE SHOULD CONTINUE TO STUDY HEPATITIS C, AND THERE ARE UNANSWERED QUESTIONS, THAT WE HAVE, FANTASTIC SYSTEMS, INCLUDING THIS ARRAY OF CHEMICAL TOOLS, GIVEN TO US BY THE PHARMACEUTICAL INDUSTRY AND OTHERS, AND I THINK MY SORT OF PUNCH LINE WOULD BE, REALLY, MAINTAINING, SORT OF BASIC RESEARCH DIVERSITY IS REALLY IMPORTANT AND WE NEED TO BE TRANSLATIONAL AND FOCUSED WHEN WE DO THAT. WE ALSO REALLY NEED TO ENCOURAGE BASIC SCIENCE RESEARCH IN THE PIPELINE, WHICH IS REALLY SORT OF THE GENERATOR OF REAL TRANSFORMATIVE DISCOVERIES. AND SO YOU NEVER REALLY KNOW WHAT'S COMING NEXT. SO I WANT TO TELL YOU, SORT OF THREE, WHAT NEXT STORIES THAT SORT OF STEM FROM OUR WORK, AND THE FIRST ONE REALLY HAS TO DO WITH WHY WE CAN'T CULTURE PRIMARY ISOLATES MUCH ATV. THIS IS SORT OF A DIRTY LITTLE SECRET IN THE SENSE THE H.I.V. FOLKS CAN ACTUALLY TAKE THE CLINICAL ISOLATE AND PUT IT IN CULTURE AND STUDY IT. WWE HAVEN'T DONE THAT DRIVEN BY HEPATITIS C'S AT LEAST AT THE TIME, FAIRLY UNIQUE EXPLOITAGES OF ACELLULAR MICRONASE A POSITIVE HOST FACTOR FOR REPLICATION. AT THE END, IF I HAVE TIME, I'LL TALK A LITTLE BIT MORE ABOUT PATHOGEN SIS, VIRUS ILMENITE AND PROMISING NEW MODELS THAT ARE POPPING UP. SO I HAVE TO BACKTRACK A LITTLE BIT ON THIS FIRST STORY, TOO TALK ABOUT WHY WE CAN'T CULTURE PRIMARY ISOLATES AND SORT OF GO BACK IN HISTORY A LITTLE BIT ABOUT THE DISCOVERY, WHICH I MENTIONED BEFORE. THIS WAS A COLLABORATION WITH DAN BRADLEY, AT THE CDC, WHO CREATED THIS POOL OF HIGHLY INFECTIOUS CHIMP PLASMA, AND MIKE [INDISCERNIBLE] USED THIS TO MAKE AN EXPRESSION LIBRARY, AND THAT LED TO THE SORT OF DISCOVERY OF HEPATITIS C, WHICH I MENTIONED EARLIER. THAT WAS IN 1989. IN 1997, USING AS A SORT OF FAMOUS PATIENT H, THE MATERIAL THAT HARVEY ALTER HAD PRODUCED THIS H-77 STRAIN, FROM A TRANSFUSION CASE AND THIS WAS DONE BY BOB PURCELL AT NIH, SORT OF MADE THE FIRST INFECTIOUS CLONE FOR HEPATITIS C. THE PROBLEM WITH THAT WAS THE ONLY WAY WE COULD ACTUALLY TEST THE INFECTIVITY OF TRANSCRIPTS FROM THE CLONE IS A POSITIVE-STRAND RNA VIRUS WAS TO INJECT IT HEPATICALLY INTO THE LIVER OF A CHIMPANZEE, AT THAT TIME, WE THOUGHT WE WERE ON A HOME STRETCH, WE CAN MAKE MILLIGRAM QUALITIES OF AN RNA THAT MIMIC THE STRUCTURE. WE SHOULD BE ABLE TO FIND A CELL CULTURE SYSTEM THAT WOULD ALLOW US TO GET DOWN TO BASIC STUDIES ON THIS VIRUS. AND IT TURNED OUT. ALL THE ATTEMPTS THAT OTHERS MADE, SORT OF FAILED TO SHOW, TOO DEMONSTRATE REPLICATION FROM THESE TRANSCRIPTS, WHICH WERE INFECTIOUS IN VIVO, BUT NOT ABLE TO REPLICATE IN VITRO. IN 1999, RALPH BARTONSLOGER, K--V THIS IS A HACK TUB VERGED OF THE GENOME WHERE THEY REPLACED THE PROTEIN CODING PORTION OF THE OPEN READING FRAME WITH NEOMI SIN RESISTANCE, AN INTERNAL SITE THAT DROVE PRODUCTION OF THE PROTEINS THAT WERE BELIEVED TO BE THE RNA AMPLIFICATION MACHINE -- MACHINERY. IF YOU CAN TRANSFECT THAT, SELECT G418, AND AT A VERY LOW FREQUENCY, ABOUT 1 IN A MILLION TRANSINFECTED CELLS, CAN YOU GET A COLONY GROUPS UP, THAT HAD CONSISTENTLY GROWING RTNA. WHEN THESE WERE RE-ENGINEERED BACK INTO THE ORIGINAL CLONES, YOU COULD INCREASE THE EFFICIENCY OF THE SYSTEM BY AS MUCH AS 50,000 FOLD. SO THIS GAVE US ONE OF THE FIRST SYSTEMS WHERE WE COULD BEGIN TO STUDY PHENOTYPIC ANALYSIS, THE SORT OF REPLICATION PROTEINS, IN THE RNA ELEMENTS AND THE GENOME. AND WE WERE ALSO ABLE TO MAKE HIGHLY PERMISSIVE CELL LINES. THE BAD NEWS IS WHEN THESE ADAPTED MUTATION MUTATIONS WERE ENGINEERED INTO FULL GENOMES, THAT SHOULD BE ABLE TO MAKE VIRUS. WE WERE UNABLE TO DEMONSTRATE THE PRODUCTION OF INFECTIOUS VIRUS. THAT CAME ALONG IN 2005. MORE THAN 15 YEARS AFTER THE DISCOVERY OF HEPATITIS C AND IT CAME FROM A RARE CASE OF [INDISCERNIBLE] HEPATITIS. JAPANESE COLEMAN PEP TIGHTS, A STUDY BY TASHI TAKITA IN JAKNOW PA, AND THIS HAD THE UNIQUE ABILITY OF BEING ABLE TO REPLICATE IN TEASE HEPATITIS BA TOMIA CELLS, WITHOUT ADAPTIVE MUTATIONS. WE WERE BEGINNING TO THINK, THEY WERE DELETERIOUS FOR THE REPLICATION IN THE VIRUS CYCLE. IN LINE WITH THAT, IT URN TODAY OUT WHEN THESE TRANSCRIPTS WERE USED ELECTED TO [INDISCERNIBLE] INFECTIOUS VIRUS WAS PRODUCED. THIS VIRUS COULD BE USED TO INFECT NAIVE CELLS AND HUMANIZED MICE. MICE THAT HAD BASICALLY, A HUMANNITESSED LIVER, WITH HEPATOCYTES OR THE WHICH I AMIA PANSY MODEL. THIS WAS THE FIRST TIME WE HAD A COMPLETE SYSTEM. WHERE WE COULD STUDY THE LIFE CYCLE IN THE LABORATORY. SO A LINGERING MYSTERY, SORT OF DURING THIS TIME IS WHY DON'T MOST PRIMARY ATBI'S SO IT'S REPLICATED IN CELL CULTURE AND WHY ARE ADAPTIVE MUTATIONS NEEDED. YOU COULD THINK OF A COUPLE OF DIFFERENT POINTS, THESE HEPATITIS BA TOMIA CELLS COULD HARBOR AN ENVIRONMENT, THAT SORT OF BLOCKED REPLICATION, AS THESE NONE ADAPTIVE ISOLATES, THE CELLS MIGHT BE MISSING SOMETHING HATHESE NATURAL ISOLATES REQUIRED. AND SAEED WHO JOINED THE LAB AS A POST DOC COMING FROM THE WAKITO LAB IN JAPAN, WAS, I GUESS, FAIRLY NAIVE AT THE TIME, AND HE DECIDED HE WOULD TACKLE THIS PROJECT I HAD BEEN TRYING TO GET SOMEBODY TO WORK ON FOR A NUMBER OF YEARS, WITHOUT SUCCESS. SO WE USUALLY START THINGS OFF IN THE LAB WITH BET. MY BET WAS, SINCE THESE ADAPTIVE MUTATIONS ARE ALL OVER THE PLACE, AND THE GENOME REFLECTS MULTIPLE WAYS THAT THE VIRUSES ARE OVER COMING SOME SORT OF INHIBITTORY FACTOR IN THESE CELLS. AND THIS JUST SHOWS YOU A MAP IN THE REPLICATES REGION OF THE GENOME, OF HCB, OF THE SPECTRUM OF ADAPTIVE MUTATIONS THAT HAVE BEEN IDENTIFIED THAT I MENTIONED THAT ARE ABLE TO INCREASE THE ABILITY THIS VIRUS TO REPLICATE, AND YOU CAN SEE, THEY ARE IN THE NS THERE PROTEINS JAYS, THIS NS5A PROTEIN THAT I MENTIONED, THAT IS A TARGET FOR SOME OF THE POTENT ANTIVIRAL DRUGS THAT OF COURSE LICENSED AND THE R RNA-DEPENDENT POLYMERASE. AND SCIENTISTS TOOK THE OPPOSITE VIEW ON THIS AND SAID NO WAY, THERE MUST BE SOMETHING MISSING. I WAS CONVINCED THAT I WAS RIGHT, SO HE SCREENS LIBRARY, AND ACTUALLY, SEVERAL TIMES, AND CAME UP WITH ABSOLUTELY NOTHING. HE GOT TO DO HIS OVER EXPRESSION SCREEN, SO WE HAD HEPATITIS B TOMEIS CELLS THAT WERE TRANCE DUCED WITH A VECTOR OR A LIBRARY OF HUMAN C DNA'S, THESE WERE SHORT OF SELECTED WITH TERAMYCIN AND ELECTROPARADED WITH A DRUG-SELECTABLE REP CON, WHICH REQUIRED AT LEAST TWO ADAPTIVE MUTATIONS TO REPLICATE. THE SORT OF BACKGROUND OF THIS WOULD BE ESSENTIALLY, ZERO AND DIDN'T POTENTIALLY EXPRESSION SOME FACTOR THAT WOULD ALLOW THIS TO REPLICATE. AND THEN YOU COULD SELECT FOR ME MEO M MICE INRESISTANCE. SO THESE ARE TWO GENOTYPES. FROM 3A AND 4A. 3 SORT OF DIFFERENT REPRESENTATIONS OF THIS LIBRARY. YOU CAN SEAT G4NA RESISTANT COLLIE 92S GROWING UP. THIS IS POLYMERASE AND NOTHING ON THE VECTOR CONTROL PLAIN. SO THEN THE QUESTION WAS, WELL, WHAT IS IT OR WHAT ARE THEY THAT THESE CELLS ARE EXPRESSING THAT SORT OF MIGHT ALLOW THESE GUYS TO REPLICATE. THEY PICKED INDIVIDUAL CLONE FROM THESE GUYS AND EVERY SINGLE ONE OF THESE CLONES HAD A LENGTHY VIRUS, THAT EXPRESSED THIS PROTEIN SEC14, THAT I'LL TELL YOU ABOUT IN A MOMENT. NOW, THE OTHER QUESTION, WHAT ABOUT THESE CLONES. ARE THEY REPLICATING IN THEM, THAT DONE THE HAVE ADAPTIVE MUTATIONS. WHEN HE SEQUENCED THREE QUARTERS HAD THE WILD TYPE REPLICON SEQUENCE, WHICH WE WERE HOPING FOR. THERE WERE SOME MUTANT AND MIXED SEQUENCE. NONE OF THESE MUTANTS CORRESPONDED TO THE ADAPTIVE MUTATION. THEY THINK THESE WERE MUTATIONS, PROBABLY PICKED UP IN THE TRANSCRIPT POPULATION OR IN THE SELECT. SO IT REALLY SEEMS LIKE THESE GENOMES COULD REPLICATE SORT OF WITHOUT ADAPTIVE MUTATIONS IN THIS BACKGROUND. IF YOU EXTEND THIS TO OTHER A CV TYPES IF YOU TAKE CELLS, AND EXPRESS, IT BASICALLY ALLOWS PAN GENOTYPE REPLICATIONS FOR ALL OF THESE DIFFERENT GENOTYPE ICELETS AS SHOWN HERE. WITHOUT ANY ADAPTIVE MUTATIONS. A NUMBER OF PATIENT ICELETS, PROVIDED BY ANDREA BRANCH AT MOUNT SINAI, AND IN CELLS THAT EXPRESSED SEC 14 INTERIOR 2, WE SEE EVIDENT OF REPLICATION, OVER THE LEVEL YOU SEE, WHEN REPLICATION IS BLOCKED, BASICALLY, WITH THIS VERY POTENCY END THE ANTIVIRAL WHICH TARGETS. HOW DOES THIS WORK AND THIS IS STILL A WORK IN PROGRESS. WE DON'T KNOW THE ANSWER TO THIS YET. WHAT IS SEC1402. IT'S ALSO CALMED C22 PROTEIN FACTOR. OR TAP 1. IT BELONGS TO A FAMILY OF LIPID BINDING PROTEINS, UBIQUITOUS, BUT EXPRESSED IN THE LIVER, BRAIN OR PROFIT ATE. THERE'S REDUCE-- OR PROSTATE. NOTHING THAT WOULD SAY, THIS MUST BE THE MAGIC OF GETTING NONE ADAPTED ICELETS TO REPLICATE. IT URNS IT OUT, THIS TAKAFERA-ASSOCIATED PROTEIN, SOME OF YOU PLAY KNOW THAT TECOFEROL IS VITAMIN E. AND THERE WAS PREVIOUS DATA, ENHANCED THE UPTAKE OF VITAMINE. AS WE WERE WRITING UP THIS, WONDERING. THERE WAS A NICE PAPER THAT'S ACTUALLY SHOWN HERE, AND WHAT THEY HAD FOUND WAS THAT SOME OF THESE NON-GFH ISOLATES COULD BE STIMULATED BY THE ADDITIONAL OF VITAMIN E TO THE CULTURE MEDIUM. AND THAT'S SHOWN ON THE LEFT. THEIR HYPOTHESIS WAS VITAMIN E WAS PREVENTING LIPID PROXIMATE DIAGNOSED, THIS WAS A NEGATIVE REELING LATER OF ATV REPLICATION. IF YOU COMPARE THE RESULTS WITH VITAMIN E SUPPLEMENTATION, TOO OVER EXPRESSIONS, YOU REQUEST SEE THEY BOTH DO THIS FOR THIS H77 GENOME THAT'S BEING AS AD HERE. IF YOU COMBINE T YOU SEE A SLIGHT INCREASE IN EFFICIENCY, ABOUT YOU NOTHING REALLY SORT OF AILED AD AND SYNERGISTIC. WE CAN SHOW BY USING DILIP DATED SERUM THAT, SUPPLEMENTATION OF THAT, WITH VITAMIN E WOULD ALLOW REPLICATION OF THIS GENOME, AS WOULD SEG14L TWO BUT ONLY IF YOU HAD VITAMIN EPRESENT. EXPRESSIONS, SEC14 ALLOWS THAT TO HAPPEN, WHERE'S VITAMIN E DOES NOT. SO SOMETHING ABOUT SUPPLEMENTING WITH VITAMIN E, VERSUS EXPRESSION OF THIS CELLULAR FACTOR IN HEPATOMA CELLS. SO THE SORT OF SIMPLE MODEL THAT WE HAVE, AND I THINK WE STILL NEED TO BE CONFIRMED IS THAT HEPATITIS B TAUPE IS CELLS INTEXTED WITH HEPATITIS C. AND THESE NONE ADOPTIVE ISOLATES, INDUCE -- THIS IS BAD SOMEHOW, FOR THE FORMATION AND MAINTENANCE OF A FUNCTIONAL HEPATITIS C VIRUS REP CATION COMPLEX AND EITHER BY SUPPLEMENT WITHING VITAMIN E, THIS CREATES AN ANTIOXIDANT ENVIRONMENT, WHICH CAN ALSO BE MIMICKED BY ADDING KINASE INHIBITORS, OTHER THINGS THAT ARE GOING TO ACT AS ANTIOCH AT THAT POINTS AND THAT SORT OF RELIEVES THE STRESS AND. [CHEERING] RNA VIRUSES AND MICRO R IN, A. VICTIMS, ADDICTS OR EXPLOITERS. AND THIS IS REALLY A STORY THAT WAS DRIVEN BY AN AMAZINGLY TALENTED GRADUATE STUDENT SHOWN HERE, AND JOE WAS ONE OF THE STUDENTS THAT REALLY SORT OF COMES TO THE LAB WITH HIS OWN IDEAS AND HE WAS VERY INTERESTED IN LOOKING AT THE INTERACTION OF MICRONASE AND -- HOW THAT INTERPLAY, MIGHT INFLUENCE HOST BIOLOGY, HE THOUGHT THAT HEPATITIS C MIGHT BE, YOU KNOW, SORT OF A GOOD PLACE TO START. AND THE REASON FOR THAT IS BASED ON A DISCOVERY BY PETER SARFO AT STANFORD, WHO NOTICED. ATV SHOWN HERE, ACTUALLY HAS TWO BEHINDING SITES FOR MERE 22. THE MOST ABUNDANT THE RNA AND HEPATO SIGHTS IN THE LIVER. WHAT PETER'S GROUP SHOWED AND THE OTHER GROUP SHOWED, THESE INTERACTIONS OF HEPATITIS C. DON'T EXERT THE NORMAL SORT OF SUSUPPRESSIVE EFFECT. BUT THERE WERE REQUIRED FOR ATV REPLICATION. THE MICRO RNA INTERACTION WITH THE RNA SILENCING COMPLEX, COULD PROTECT THE [INDISCERNIBLE] OF THE ATV GENOME, NOT UPON CAD FROM EXONUKE LYSIS THAT WOULD BE THERE TO DEGRADE IT. TH AND OTHERS, SOME RECENT WORK. WOULD SUGGEST THIS MIGHT BE INVOLVED IN ORCHESTRATING THE TRANSITION OF THIS POSITIVE STRAND GENOME, WHICH ACTS AS THE MAG FOR TRANSLATION, BEING A SUBSTRATE FOR MINER TRANCEITHESIS. ANDY JO WAS INTERESTED IN THIS I WON'T GO YOU THAT'S DETAILS BUT UV CROSS INK WELL, THESE COMPLEXES AND EITHER CONTROL CELLS OR INFECTED CELLS, BREAK OPEN THE CELLS, IMMUNOPRECIPITATE WITH AN ANTIBODY AGAINST ORGANAUT. CUT OUT THESE CROSS LINK RNA PROTEIN SPECIES, SEQUENCE THESE BY ILLUME NUMB SEQUENCING AND ALIGN THE READS, EITHER ON A VIRAL OR HOST GENOMES. AND THIS GIVES YOU SORT OF A MAP OF LEADS ACROSS THE VIRAL GENOME, AND ALSO, ON CELLULAR GENOMES, AND IT GIVES YOU KIND OF AN INCORRECT PICTURE OF WHAT MICRO RNA'S MIGHT BE BEHINDING. YOU GET ORGANIZE NAUGHT, SORT OF MICRO RNA READS AND ARGONAUT CROSS, AND YOU TRY AND LINK THESE TO SEE IF THERE'S A SEED SEQUENCE IN THIS MICRO RNA THAT WOULD BE CONSISTENT WITH RECOGNIZING THIS PARTICULAR MESSENGER, VIRAL TARGET. YOU CAN FIND EVIDENT OF THESE CHIMERAS, WHERE YOU SEE A LINKAGE BETWEEN THE MICRO RNA AND THE TARGET RNA AND THAT ALLOWS YOU TO UNAMBIGUOUSLY ASSIGN WHERE A PARTICULAR MICRO RNA IS BEHINDING. THIS SHOWS YOU SOME OF THE DATA JOE PRODUCED ACROSS THE GENOME, SHOWING THIS HUGE PEAK AT THE 122 BINDING SITES, A NUMBER OF OTHER ARGUE NAUGHT THAT INVOLVE OTHER MICRO RNA'S, MOST OF WHICH WE HAVEN'T TESTED YET. THIS ALLOWING YOU TO UNAMBIGUOUSLY, ASSIGN THESE TO PARTICULAR TARGET SITES ON VIRAL AND MESSENGER RNA'S, THE INTERESTING THING THAT JOE FOUND WITH THE STUDIES ON HEPATITIS C WAS THAT A CV INFECT, ACTUALLY BOUND ENOUGH OF THIS MICRON RNA122, TOO EXERT A SPONGE EFFECT SO THAT WE ACTUALLY FOUND A LESS BINDING TO 122 TARGET IN-INFECTED CELLS, COMPARED TO CONTROL CELLS. AND THAT RESULTED, ACTUALLY IN AN INCREASED LEVEL OF THESE 122 TARGET RNA'S, IN INFECTED CELLS, SO THAT THIS VIRUS, ACTUALLY BY BINDING TO THESE MICRO RNA'S, IT APPEARED, SOAK UP ENOUGH. THAT IT CAN DEREGULATE, SORT OF NORMAL TRUNNELSLATIONAL AND DEGRADATIVE EFFECTS ON HIS HOST ACTS. NOW, THIS WAS REALLY CORRELATIVE, AND THE WAY YOU PROVE THIS IS TO ACTUALLY SEE IF CAN YOU TEST IT EXPERIMENTALLY. YOU COULD ACTUALLY, POTENTIALLY, STEIN A MUTAN THE ATV GENOME, THAT CAN NEUTRALIZE, BECAUSE IT HAD DIFFERENT THE SEEDS, BUT WOULD FOE LONGER BE ABLE TO UTILIZE 122. HE NOT LUCKY IN THE SENSE THAT THIS VIRUS WAS VIABLE AND THAT'S SHOP HERE ON THE LOWER LEFT AND THAT SHOWS YOU THE ABILITY OF THE SORT OF WILD TYPE VIRUS TO REPLICATE IN THE PRESENCE OF 122, AND THE MIR15 DERIVATIVE TO, EPIICATE IN HOH 7 CELLS. THAT VIRUS IS ABLE TO REPLICATE N CELLS THAT WE HAVE KNOCKED OUT MIR122. AND BASICALLY, IF YOU USE NUCLEIC ACIDS TO SOAK UP THE RNA'S, IT WOULD SHOW THAT WOULD BLOCK THE ABILITY OF 122 TO BE UTILIZED BY HEPATITIS C AND IN THIS, YOU REQUESTULE TOOY SEE, THE NERVE 15 VIRUS IS INHINTED BY A MIV15, ANTISENSE, NUCLEIC ACID, BUT NOT ONE WHICH INTERACTS WITH 122, AND VICE VERSA FOR THE WILD TYPE VIRUS. BUT THEN QUESTION WAS, WHAT ABOUT THE SPONGE EFFECT, AND I SHOWED YOU THIS BEFORE, WHERE MIR122, WITH WILD TYPE VIRUS, ACTUALLY GAVE YOU THE SHIFT OF THE RIGHT, THE INCREASE AND THE FREQUENCY OF 122 TARGET RNA'S, AND IN THE CASE OF MIR15, WE NO LONGER SAW THAT EFFECT FOR MIR. BUT INSTEAD, SAW SUBTLE SHIFT, BUT STATISTICALLY SIGNIFICANT SHIFT OF MIR15 TARGETED MESSAGE MESSAGES. SO IT REALLY SEEMS THIS SORT OF CLOSES THE LOOP, SHOWING THAT ATV IS REALLY SORT OF RESPONSIBLE FOR SEQUESTERING THESE RNA'S, AND SORT OF DEREGULATING THEIR ABILITY TO SORT OF MODULATE THE HOST GENE TARGETS. WE THINK THIS IS AN INTERESTING OBSERVATION IN THE SENSE THAT FOR THESE VIRUSES THAT HAVE RELATIVELY, LITTLE INFORMATION IN THEIR GENOMES T ALLOWS THEM TO REGULATE CONSULAGESES OF HOST CHAINS, WITH RELATIVELY LITTLE GENOMIC INFORMATION. AND, I GUESS THE QUESTION, AT THAT POINT IS, IS THIS SORT OF SOMETHING THAT'S, SORT OF SPECIFIC ATV OR IS THIS A GENERAL VIRAL STRATEGY. SO SHOWN HERE, FROM DENMARK, JOINED THE LAB AS A POST DOC, AND WAS BASICALLY APPRENTICEDS WITH JOE, AND HAS BASICALLY EXTENDED THIS ANALYSIS TO A NUMBER OF DIFFERENT VIRUSES AND I WON'T GO THROUGH ALL THE DATA THAT HE HAS SHOWN. HE FOUND ANOTHER GROUP OF VIRUSES, THAT HAD A PHENO TYPE ARE -- A VERY PREDOMINANT LEAK, AND THE LOCUS IN THE VIRAL GENOME, OTHER -- THIS IS A VIRUS, WE HAVE SEEN SOMETHING SIMILAR FOR CLASSICAL SWINE FEVER VIRUS. UNLIKE HEPATITIS C. THE HOTSPOT FOR BINDING FOR THE VIRUS IS IN THE THREE-PRIME NONE CODING, NOT IN THE 5 PRIME. BOTH OF THESE INTERACTIONS, AS I'LL SHOW YOU IN THE NEXT SLIDE, ARE ABSOLUTELY REQUIRED WE HAVE THE 3 PRIME UTR, AND THE MIR17, RNA THAT'S RECOGNIZING IT. IF YOU INTRODUCE MUTATIONS IN THE GENOME, LIKE THOSE THAT WE PUT INTO THE M IRK R-15 VIRUS FOR HEPATITIS C. THAT SHOULD DISRUPT THIS INTERACTION, AND IF IT'S REQUIRED. YOU CAN RESTORE THAT BY BASICALLY SUPPLYING A MIMIC, WHICH ALLOWS THE BASE PAYING AT NUCLEOTIDE'S 3 AND 4 TO OCCUR. WHERE'S AGAIN, THE MUTAN THE CAM REPLICATE, IF YOU SLY THIS, BY TRANSFICTION. ACTUALLY, WE DO SEE REPLICATION OF THE MUTANT VIRUS HERE BUT IT TURNS OUT, THAT IS ACTUALLY REVERSION, BACK TO THE WILD TYPE SEQUENCE. SIMILAR RESULTS WE'RE SEEING FOR LED 7. AND THE REPORTER SHOWN HERE, AND THIS IS WHAT THE MICRO RNA BINDING SITE IN THE 3-PRIME UTR. THIS IS SORT OF A WAY YOU LOOK FOR SORT OF FUNCTIONAL IMPACT OF DOWN REGULATINGLY, AND YOU CAN SEE IN THE CASE OF SORT OF CELLS THAT HAVE ENDOGENOUS MIR-17 EXPRESSION, , I GUESS IN A CASE OF HEPATITIS C, YOU MIGHT ASK, WHAT MIGHT BE THE IMPACT OF SEQUESTERING MIROLIN 22 IN VIVO. WE DON'T KNOW THIS FOR SURE. THIS IS STILL SPECULATION, BUT THE 15O TYPE OF LIVER-PACIFIC MIR122 KNOCKOUT, BIOSIS, THE PROGRESSIVE FIBROSE LIVER DISEASE AND LIVER CANCER. SO IT'S POSSIBLE THAT THIS SORT OF DEREGULATION OF MIR-122, BIOLOGY, BY CHRONIC ATV INFECTION IS PROVIDING AT LEAST ONE OF THE SORT OF FACTORS THAT'S INVOLVED IN THE ASSOCIATE OF THIS VIRUS, WITH LIVER CANCER. IMMUNITY AND PATHOGENESIS. I THINK THESE ARE INTERESTING TOPICS IN HEPATITIS C WE HAVEN'T THE GOTTEN AS MANY ANSWERS AS I'D LIKE. WE DON'T HAVE A VACCINE FOR THE VIRUS AND WE DON'T KNOW ALL THE FACTORS TRYING DISEASE PROGRESSION. SO THIS IS A DOG. THIS IS A PICTURE OF HATE, AND SHE'S ACTUALLY RUNNING ON THE SORT OF BEACH OF LONG ISLAND. THIS IS OUR LAB MASCOT. MY AUSTRALIAN SHEPHERD. THE REASON I SHOW THIS IS THAT SOME YEARS AGO, I GOT A CALL FROM IAN LIPKIN AND IAN HAD IDENTIFIED A NONE PRIMATE CAPACITY VIRUS IN SAMPLES FROM A RESPIRATORY OUT BREAK IF DOG, AND THIS VIRUS, ACTUALLY, TOO DATE IS THE SORT OF CLOSEST RELATIVE OF HEPATITIS C. AND IT TURNED OUT WHEN PEOPLE WORKED OUT, ASSAYS AND SOME OF THIS WAS DONE BY THE ROBELLOLAB HERE AT NIH, THAT THIS WHAT WAS ORIGINALLY CALLED CANINE CAPACITY VIRUS WAS NOT COMMON IN DOGS, IT WAS COMMON NON-HUMAN HORSES SO IT WAS RENAMED NONE PRIMATE CAPACITY VIRUS. ABOUT A THIRD OF THE HORSES EXAMINED HAD ANTIBODIES TO THIS. A SUBSTANTIAL FRACTION, THOSE WERE ON A POSITIVE. ONE OF THESE ECMOMACHINES 89 ISOLATES WERE IDENTITY CAL TO THE CANINE SAMPLES THAT WERE ORIGINALLY REPORTED, AND THE RELATIONSHIP BETWEEN THE DOG SAMPLES, AND THE HORSE VIRUS WERE STILL SORT OF SPECULATING ON BUT IT URNS IT OUT. YOU KNOW, HAVE IOLOGISTS ARE BEING SEQUENCING. MORE CLOSELY, RETROSPECTIVELIED, NON-PRIMATE CAPACITY VIRUS AND WITH ADDITIONAL SEQUENCES, THERE WERE PEGGY VIRUSES FOUND IN HORSES, EQUINE PEGGY VIRUSES, AND A NUMBER OF RODENT CAPACITY VIRUSES AND PEGGY VIRUSES, WHICH GOT US QUITE EXCITED. BATS SEEMED TO ALSO BE SORT OF A HOT SOURCE OF THESE VIRUSES AND THERE HAVE ALSO BEEN, I GUESS, WHAT WE WOULD HAVE EXPECTED, AN ISOLATE IN GIBBONS, REPORTED BY LOKUN IN 2013 AND VIRUSES FOUND IN DOMESTIC CATTLE. SO REALLY, FROM, YOU KNOW, SORT OF THIS VERY ISOLATED MICROCOSM, THING VISION THAT WE HAD OF HEPATITIS C, BASED ON THE DISEASE CAUGHTED BY HEPATITIS C. THERE ARE A LOT OF RELATED VIRUSES, OUT THERE IN NATURE. EVEN THOUGH CHIMPANZEES OF COURSE THE CORNERSTONE OF HUMAN HEPATITIS B HEPATITIS RESEARCH, WE REALLY CAN'T THE WORK WITH THEM FOR THE KINDS OF STUDIES WE WANT TO DO ANYMORE. IT'S GETTING HARDER AND "HARDBALL"ER AND ORDER HAER TO GET LIVER SAMPLES BECAUSE PEOPLE ARE MOVING TO NONINVASIVE METHODS AND EXTREMELY DIFFICULT TO GET SAMPLES FROM,A CUTE INFECTS AND THE HUMAN CHIMERIC LIVER MOUSE MODELS, WHICH WE HAVE WORKED ON A LOT, AS HAVE OTHERS, THE ONES WHICH ARE HIGHLY SUSCEPTIBLE TO HEPATITIS C OR B, THOSE GUYS REALLY DON'T HAVE A FUNCTIONAL IMMUNE SYSTEM. SO IF YOU BELIEVE THAT THE PATHOGEN SIS IS DRIVEN BY SORT OF IMMUNE MECHANISMS, YOU'VE STILL GOT A WAYS TO GO. SO A LOT OF REASONS WHY WE WOULD LIKE TO HAVE RODENT CAPACITY VIRUS MODEL. WE LOOKED AT THE POTENTIAL TO ESTABLISH A SMALL ANIMAL MODEL. THIS IS A PICTURE OF THE DEER MASK, AND THE LAB MASK. THAT I LOOK TOTALLY DIFFERENT AND TURNS OUT IN A MOUSE PHYLOGENY, THEY ARE VERY DIFFERENT. AND EVA BILLERBRICK TOOK SOME OF THE DEER MOUSE VIRUS AND INFECTED A COUPLE OF DIFFERENT SPECIES. SHE COULD EASILY ESTABLISH CHRONIC INFECTIONS IN THESE SORT OF WILD MOUSE HOSTS. BUT WHEN WE TRIED TO OKAY PUT THESE IN HIGHLY IMMUNOEFFICIENT STRAINS OF LEGIBILITY MICE. KNOCKOUTS THAT ARE BASICALLY DEFECTIVE AND ADAPTIVE IMMUNITY, IF OTHER AG129 MICE,HAR KNOCKOUTS OF THE TYPE 1 AND TYPE 2 INTERFERON RECEPTORS. NO LUCK IN TERMS OF GETTING AN INFECTIOUS ANIMAL. SO WE WERE FACED WITH REALLY TRYING TO WORK OUT A SET OF RE-AGENTS THAT WOULD ALLOW US TO WORK WITH TEASE TEAR MICE OR, YOU KNOW, SOR--WITH THESE DEER MICE OR WOR K ON HORSES WHICH WE'RE ACTUALLY DOING A LITTLE WORK ON OR WAIT FOR THE NEXT VIRUS TO EMERGE. TRAP A BUNCH OF WILD RATS THERE WERE RUNNING AROUND THERE. SO HE AND HIS TEAM, SUITED UP, IN SUITABLE BIOCONTAINMENT APPAREL, AND WENT OUT AND TRAPPED ABOUT 120 OF THESE NEW YORK RATS AND SWOBBED THEM AND SLICED THEM AND DICED THEM. AND SEQUENCED THEM AND THEY BASICALLY FOUND TWO LINEAGES OF FOREWAY RAT HEALTHY TISSUE PASSITY VIRUSES WHICH ARE SHOWN HERE AND THEY SORT OF LOOKED IN THE TISSUES OF THESE ANIMALS AND FOUND VERY HIGH LEVELS OF THIS NORWAY RAT PASIVIRUS RNA IN THE SEAROOM, BUT NO NEGATIVE NEGATIVE STRAND RNA WHICH IS SORT OF INDICATIVE OF REPLICATION IN ANY OF THESE TISSUES, EXCEPT THE LIVER, AND THIS WAS TRUE FOR BOTH OF THESE LINEAGES. WE BEGAN TO LOOK AT THIS IN LABORATORY MICE AND THIS IS REALLY SORT OF NEW DATA, NOT PUBLISHED AND AND FAIRLY EARLY ON. EVA BILLBECK HAS BEEN IN THE POST LAB DRIVING THIS. AND BASICALLY, WHAT EVA HAS BEEN ABLE TO SHOW, IF YOU TAKE THESE IMMUNODEFICIENT MICE AND THESE ARE IN, RG KNOCKOUTS, THE AG129 TYPE 1 AND TYPE 2 RECEPTOR KNOCKOUTS OR THE TYPE 1 RECEPTOR KNOCKOUTS ON THE 129 BACKGROUND. ALL THESE OF THESE IMMUNE OPEN DEFICIENT STRAINS CAN BE INFECTED WITH THIS VIRUS AND IN FACT, ESTABLISH A CHRONIC INFECT. IF YOU LOOK IN CONTRAST IF IMMUNOSTRAINS, USING THE ORIGINAL RAT SERUM OR SERUM THAT HAS ACTUALLY BEEN PASSAGE IN THESE IMMUNODEEFFICIENT MICE. YOU CAN VIENNESE ANIMALS, ALSO GET INFECTED BUT THEY'RE ABLE TO RESOFT INFECTION, MORE QUICKLY IN THE CASE OF ANIMALS INFECT INFECTED WITH THE RAT VIRUS, AND SLOWLY WITH POTENTIALLY, SORT OF A MAS ADAPTED SORT OF VARIANT SHOWN HERE. THIS IS REALLY AN INFECTIOUS VIRUS AND ONE OF THE THINGS THAT WAS DONE IN THE OLD DAYS WITH HEPATITIS C IS TO ACTUALLY DO REVERSE TITRATION IN CHIMPANZEE, TOO MEASURE THE DOSE OF THESE NON-A, NON-B HEPATITIS SAMPLES AND HEPATITIS C, SAMPLES. MICE CAN BECOME INFECTED AND THESE ARE 10 GENOME COPIES SORT OF INOCULATED I.V. INTO THESE MICE AND THESE ARE WILD TYPE MICE. YOU CAN SEE THAT EARLY ON, AFTER INFECTION, WITH LOW DOSE, YOU SEE LOWER LEVELS AND SORT OF UNDETECTABLE. BUT 5 DAYS AFTER INFECTION, ALL OF THESE ANIMALS, REGARDLESS OF 10,000 OR 10 GENOMES, BEING USED FOR INOCULATION HAVE HIGH TIGHTERS OF VINY EMIA. OLDER MICE ARE ABLE TO CLEAR MORE QUICKLY, AND YOUNGER MICE SHOW A LITTLE BIT MORE SCATTER. THAT'S SOMETHING WE HAVEN'T THE EXAMINED TOO QUICKLY, BUT ALSO, SOMEWHAT TYPICAL OF VIRAL INFECTIONS IN NEARING MODELS. WE BELIEVE THAT THIS IS HEPATO HEPATOTROPIC VIRUS. THE IF YOU TAKE KNOCKOUT MICE THAT HAVE MIR122, SELECTIVELY KNOCKED OUT IN THE LIVER AND ASK IF THESE CAN BE INFECTED BY THIS VIRUS, WE SEE NO EVIDENT OF PERIPHERAL VIREMIA AND WE CAN FIND VERY HIGH LEVELS OF VIRUS IN THE LIVER, BUT EITHER UNDETECTABLE LEVELS OR VERY LOW LEVELS IN OTHER TISSUES THAT MAY BE DUE TO CONTAMINATION WITH SORT OF LOW LEVELS OF BLOOD IN THESE ANIMALS. AND WE CAN READILY DETECT MINUS STRAINED RNA IN THE LIVER. ONE OF THE THINGS EVA STARTED TO LOOK AT WAS THE IMPACT OF T-CELLS ONE EXPERIMENT, SHE'S USED 18 THE BODIES TO DEPLETE AND ASKED WHAT HAPPENS IN TERMS OF OF [INDISCERNIBLE] OVER TIME. WHAT YOU CAN SEE IN WILD TYPE MICE, AND LET'S JUST LOOK AT THE BLACK SIX MICE, IN THE SORT OF CONTROL ANIMALS, THE VIRUS IS CLEARED RELATIVELY QUICKLY, AS SHOWN HERE. IF YOU DECLEAT CDH CELLS, SLOWLY, AND IF YOU DEPLETE CD4 CELLS, BEFORE INFECTION, THESE ANIMALS BECOME CHRONICALLY INFECTED. AND THIS CHRONIC INFECTION PERSISTS, AND YOU SEE THE DATA BEFORE IS THE CD4 CELLS COME BACK. SO THAT WE SEE NO -- IN TERMS OF THE FREQUENCIES, NO DISTINGUISHED DIFFERENCE BETWEEN THESE AND THE CONTROL ANIMALS AND YET, THEY ARE THE RECONSTITUTION OF THE CD4 COMPARTMENT IN THESE CELLS. WE DON'T HAVE EPITAUPE SUFFICIENT DATA YET. IT'S NOT SUFFICIENT TO SORT OF ALLOW THIS VIRUS TO BE CLEARED. WE BELIEVE THAT CD8'S ARE PROBABLY THE MOST IMPORTANT BECAUSE IF YOU DO, RATHER THAN THE SORT OF TRANSIENT CDA DEPLETION, WHERE THE ANIMALS CAN RECOVERY IN THE THREE TO FOUR-WEEK PERIOD, AND CLEAR THE VIRUS, IF YOU DO, SORT OF TRANSIENT, YOU SEE THIS DELAY. IF YOU CONTINUE TO DEPLETE CD8'S, THE ANIMALS REMAIN INFECTED, ALL BE IT, THERE'S SOME KIND OF IMMUNE CONTROL GOING ON HERE, IN TERMS OF OF THE INFECTION, WHICH COULD BE MEDIATED BY ANTIBODIES. WE THINK THE ULTIMATE INFECTOR CELLS ARE LIKELY TO BE AT LEAST IN PART, CD8'S. IF YOU ARE TAKE THE ANIMALS AND RE-INFECT THEM. WE DON'T REALLY KNOW YET IF THIS IS STERILIZING. SO THING ACTUALLY MAY GIVE US A MODEL OF WHERE WE CAN BEGIN TO STUDY SOME OF TEASE ASPECTS OF ATV ASSOCIATED DISEASE, AND JUST TO SUMMARIZE THIS LAST PART, WE HAVE A MOUSE MODEL THAT SHOWS IMPORTANT IMMUNOLOGICAL CHARACTERISTICS. THE CLEARANCE, SEEMS TO BE CD4 AND CDAP CELLS. I DIDN'T SHOW YOU ALL THE DATA FOR THIS. SEEM TO BE THE ESSENTIAL FACT INFECTORS. THEY CONTAIN A A SECONDARY FICTION. I DIDN'T SHOW YOU THAT. AND ONE THING I THINK IS INTERESTING, AND THIS IS SORT OF SIMILAR TO WHAT IS SEEN IN CHRONIC -- IS VIRAL RESISTANCE IS CHARACTER USED BY ACCUMULATION OF T-CELLS THAT ARE HIGH AND SORT OF CHECKPOINT MARKER PD1. AND REGULATORY SEQUENCE IS SUGGESTING THESE T-CELL ARE THERE, BUT THEY MAY HAVE AN EXHAUSTIVE PHENO TYPE, AND AND EVA HAS GONE ON TO SHOW, IF YOU BLOCK THIS CHECKPOINT YOU ACTUALLY CAN GET AN EFFECT. ALTHOUGH WE DON'T KNOW YET, IF IT ULTIMATELY LEADS TO RESURRECTION OF T-CELL RESPONSES THAT ARE ABLE TO CLEAR THE VIRUS. IF YOU LOOK AT THE ACUTE INFECTION, AND I DIDN'T SHOW YOU THE LIVER DATA OR THE PATHOLOGY FOR THIS. WE DO SEE QUICK INFILTRATION NK CELLS WHICH DON'T SEEM TO BE IMPORTANT FOR CONTROL AND ACUTE ANIMALS IN THE IMMUNOCOMPETENCE MOLDEL BUT WE SEE INFILTRATION OF T-CELLS THAT, SORT OF CORRELATES WITH CLEARANCE OF THE VIRUS, WHICH IS VERY MUCH OF WHAT WE SEE IN AN ACUTE CASE OF ATV. IN TERMS OF OF THESE LONG-TERM CHRONIC INFICTIONS THAT ARE ESTABLISHED, SO FAR, WE ONLY SEE EVIDENT OF MILD TO MODERATE INFLAMMATION AND NO SIGNS OF FIBROSE SO FAR. SO WE THINK THIS IS GOING TO BE A VERY INTERESTING ANIMAL MODEL TO STUDY SOME OF THE BASIC IMMUNOLOGY, OF THE TISSUE SPECIFIC VIRUS INFECT. COULD BE A PLATFORM FOR TESTING APPROACHES. THE EFFECTS OF ANTIVIRAL MEDIATED CLEARANCE ON FUNCTION, ARE SOMETHING THAT EVA IS VERY INTERESTED IN. RIGHT NOW, WE DON'T KNOW IF THIS VIRUS IN MICE, IS SUSCEPTIBLE TO SOME OF THESE POTENT ANTIVIRALS THAT ARE BEING USED TO TREAT A, V. AND WE'RE HOPING THAT THIS CAN EVENTUALLY BE ESTABLISHED FOR A MODEL FOR CHRONIC INFECTION AND LIVER DISEASE. SO WE'RE BEGINNING TO EXAMINE OTHER COMORBIDITIES ASSOCIATED WITH LIVER DISEASE PROGRESSION, IN THE CASE OF CHRONIC ATV. AGE, MALE SEX. ALCOHOL AND OTHER FACTORS. ALONG WITH OUR COLLABORATORS. CASH APUR IS NOW AT OHIO STATE. EVA HAS REALLY PICKED UP THE RODENT VIRUS STUDIES AND IS DOING ALL THE IMMUNE CHARACTERIZATION OF THAT. I DIDN'T TALK ABOUT THE HORSE VIRUS. BUT WE HAVE BEEN WORKING ON THAT, IN COLLABORATION WITH A FANTASTIC GROUP AT CORNELL, BUT TOM DEAFERS, THIS IS MAX OUR SORT OF SUPERSTAR HORSE. HE ACTUALLY VOLUNTEERED TO TEST THE FIRST INFECTIOUS TRANSCRIPTINGS FOR THIS NON-PRIMATE CAPACITY VIRUS. HE RESOLVED THE INFECTION, AND IS PROTECTED AGAINST RE-INFECTION. GREAT SOURCE OF ACUTE -- HE WAS DESTINED TO BE I THINK, SORT OF PUT OUT TO PASTURE, PERMANENTLY, BUT NOW, HE GETS OATS AND CARROTS. IT HAS BEEN A COLLABORATION WITH BOB DARNELL, AND HELLS LAB. I DIDN'T TALK ABOUT THE WORK ON THE OTHER PESTY VIRUSES, CLASSICAL [INDISCERNIBLE] VIRUS, WHICH WE CAN'T WORK WITH IN THIS COUNTRY. SO NICK WAS AT THE IBIIN SWITSER LAND. I DID THOSE STUDIES AND CAME UP WITH VERY PARALLELED DATA TO WHAT I SHOWED YOU BEFORE. VIRAL DIARRHEA VIRUS AND REALLY A FANTASTIC POST DOC WHO WORKED ON THIS PROJECT, AS WELL AS A BE NUMBER OF OTHER ONCE, I HAVEN'T HAD TIME TO TALK ABOUT. THE LIBRARY WHY JOSE SILVA, WHO'S NOW AT COLUMBIA UNIVERSITY, AND SOME AGENTS FROM OTHERS AND YOU KNOW, ALSO, JUST THANK THE HEPATITIS C COMMUNITY. THE PEOPLE IN THIS FIELD, I THINK HAVE BEEN JUST FANTASTIC TO WORK WITH. IT'S BEEN A VERY COLIEGE IAL SORT OF ENTERPRISE. A LOT OF SORT OF GREAT EXCHANGE OF IDEAS AND IRREVERSIBLE SHAKING OF RE-AGENTS. WE BENEFITED FROM A GREAT DEAL OF FUNDING FROM THE NIH FROM THIS WORK OVER THE YEARS. ACTUALLY, THE NATIONAL CANCER INSTITUTE WAS FACT THE FIRST ONE THAT WOULD TAKE A RISK ON US, SORT OF WORKING ON THIS MORE THAN 20 YEARS AGO. AND A LOT OF PHILANTHROPIC ORGANIZATIONS, IN PARTICULAR, THE GREENBERG MEDICAL RESEARCH INSTITUTE, AND THE STAR FOUNDATION FOUNDATION THAT, REALLY ALLOWED US TO DO SOME OF THESE THINGS THAT ARE A LITTLE BIT MORE OUT OF THE BOX. AND WITH THAT, I WILL STOP, AND IF THERE ARE TIME FOR QUESTIONS, I WOULD BE HAPPY TO ANSWER THEM, AND IF NOT, I WOULD BE HAPPY TO ANSWER THEM AT THE RECEPTION. THANK YOU. [Applause]. >> [SPEAKING AWAY FROM MICROPHONE] >> YEAH, I THINK -- QUESTION WAS, DO YOU SEE LIVER PATHOLOGY IN THE RATS. WITHOUT ANY KIND OF IMMUNOCOMPROMISING, YOU KNOW, THEY DO BECOME CHRONICALLY INFECTED AND I DON'T REALLY HAVE THE DATA FOR THAT. THAT'S A MIDST THING, AND AT LEAST I DON'T THINK THERE ARE ANY OVERT DISEASE, BUT I THINK HE'S DOING THAT IN A CONTROLLED FASHION NOW, AND HOPEFULLY, HE'LL SEE SOMETHING AND BE ABLE TO SORT OF DO THE RAT STUDIES TO SORT OF PARALLEL THE MOUSE STUDY THAT IS WE'RE PLANNING ON. >> YOU JUST SPOKE OF THE HISTO PATHOLOGY OF THE RIVER AND THE RAT. BUT YOU DIDN'T REALLY SHOW A PICTURE OF IT. SO I'M NOT REALLY SURE WHAT YOU'RE TALKING ABOUT. IN THE HUMAN, THE HEPATITIS C IS CLASSICAL. INFILTRATE AND IT'S LIKE A BALL, RIGHT? AROUND THERE AND -- IT'S ALMOST PATHO MNEMONIC. >> YOU MEAN IN THE ACUTE RESOLVING INFECT OR THE CHRONIC? >> GEE, YOU GOT ME THERE. WHEN YOU SEE A BIOPSY -- >> WE'LL HAVE HARVEY ANSWER THAT QUESTION HERE SHORTLY. >> NO. NO. BUT USUALLY, LIKE FOR ANATOMIC PATHOLOGY ET CETERA, WHEN YOU THINK OF HEPATITIS B C, YOU THINK OF THE LYMPHOCYTES JUST BUMMING BULGING OUT OF THERE. IS THAT WHAT YOU SEE IN THE RAT? >> WELL, WE REALLY VOLUNTARY LOOKED AT THE RAT DATA. IN THE MOUSE -- WE RELEVANT LOOKED AT THE RAT DATA INFRASTRUCTURA. IN THE MOUSE DATA, WE SEE THE INPARAMETERY, IN THE ACUTE RESOLVES, YOU KNOW, SORT OF PART OF THE INFECTION, I THINK IT'S A MUCH DIFFERENT SITUATION THAN WHAT YOU SEE IN CHRONIC C. BUT ALSO, IN THE MOUSE MODEL, WE HAVEN'T THE ACTUALLY SORT OF DEVELOPED THE REAGENTS OR THE TECHNIQUES TO SORT OF BEGIN TO LOOK AT THE LOCALIZATION OF A LYMPHOCYTE AND WHERE THE ACTUAL CYTES AND VIRUSES ARE. THIS IS STILL PRETTY EARLY AT A SO I CAN'T REALLY GIVE YOU A GREAT ANSWER TO YOUR QUESTION. >> OKAY. THANK YOU. >> CHARLIE, GOING BACK TO THE BEGINNING OF YOUR TALK. WERE THE PRICE OF DRUGS TO DROP TO WHERE GOVERNMENTS COULD AFFORD TO TREAT THEIR POPULATIONS, DO YOU THINK H CV COULD BE ERADICATED OVER THE NEXT THREE, FOUR TECH AIDS, -- OVER THE NEXT THREE, FOUR DECADES, WITHOUT A VACCINE >> WE'RE NOT GOOD AT ERADICATING VIRUSES, IN MANY CASES, EVEN WHEN WE DO HAVE A VACCINE. I THINK THE COMPOUNDS WE HAVE, HAVE THAT POTENTIAL. BUT WHETHER OR NOT THEY CAN REALLY ACHIEVE IS DISTRIBUTION AND PENETRANS, THAT WOULD BE REQUIRED TO DO THAT. I THINK IT'S GOING TO TAKE SORT OF A LONG TIME. SO I THINK WE SHOULDN'T ASSUME THAT THAT'S GOING TO BE THE CASE, AND I THINK YOU KNOW, DEVELOP HAPPENING A VACCINE WILL BE A VERY GOOD SORT OF ADJUNCT. BUT AS I SAID BEFORE, EVEN A VACCINE, IF YOU LOOK AT THE KINETICS WITH WHICH HPV IS BEING ELIMINATED, WE HAVE A VACCINE, THERE ARE STILL GLOBAL CHALLENGES TO THAT VIRUS AS WELL. AND I GO BACK TO MY ROOTS WITH YELLOW FEVER AND, YOU KNOW, MAX TYLER, IN DEVELOPING THE YELLOW FEVER VACCINE, IN 1937, AND IT'S BEEN IN A HALF A BILLION PEOPLE. NOW WE'RE SEEING URBAN OUT BREAKS OF YELLOW FEVER IN AFRICA. SO SORT OF MAKING THESE THINGS, AND IT'S IMPLEMENTING THEM, AND STICK WITHING THEM ARE OTHER FACTORS THAT WE CAN DO BETTER AT. IT'S A WISHY WASHY ANSWER TO YOUR QUESTION, BUT WE SHOULDN'T ASSUME THAT THE GAME IS OVER UNTIL THE GAME IS OVER. >> THANK YOU. THANK YOU. [Applause]