NEXTED SLIDE, PLEASE. SO THIS
BRINGS US TO TODAY'S SEMINAR
SPEAKER, DR. SUSAN WEISS. SUSAN
RECEIVED HER BACHELOR'S DEGREE
FROM ARE BRANDEIS, HER PHD WORK
WITH MICHAEL BRIAN AT HARVARD
AND BETWEEN 1976-1980 WAS A POST
DOC WITH CAROL FARM AND MICHAEL
BISHOP. IN 1980 RECEIPTED TO
RECRUITED TO UNIVERSITY OF
PENNSYLVANIA, WHICH SHE BECAME A
FULLY TENURED PROFESSOR IN 1982
IN THE SCHOOL OF MEDICINE. 2010
AND 2019 WAS POST DOCTORATE IN
TRAINING AND PROGRAMS IN THE
SCHOOL OF MEDICINE AT UPENN.
SINCE 2018, SHE SERVED AS VICE
CHAIR OF THE DEPARTMENT OF
MICROBIOLOGY AND SINCE 2020
CO -DIRECTOR OF PEP
CO -DIRECTOR OF PE -- PENN
CENTER. IN 1994 ELECTED FELLOW
OF AMERICAN ACADEMY OF
MICROBIOLOGY, AND FELLOW OF AAAS
AND LAST YEAR ELECTED MEMBER OF
THE NATIONAL ACADEMY OF
SCIENCES. NEXT, PLEASE. HER WORK
FOCUSED ON PATHOGENGENESIS OF
CORONAVIRUS AND ZIKA AND RNA
INDUCED PATHWAYS AND ENDOGENOUS
PATHOGENISIS. HER TALK IS
ENTITLED CORONAVIRUS ACTIVATION,
ANTAGONIST FROM MHV TO SARS-COV
2. IF YOU CLICK ONE MORE WE WILL
SEE THE CME CODE, 50127. SO WITH
THAT LET'S PLEASE WELCOME SUSAN
WEISS FOR THE 2024 KHOURY
LECTURE.
[ APPLAUSE ]
>> SUSAN WEISS: THANK YOU, ERIC.
I'M REALLY HONORED TO BE INVITED
FOR THIS LECTURE. I DIDN'T
PERSONALLY KNOW GEORGE R. BROWN
BUT REMEMBER HIM. I HA-- NOT KNN
GEORGE KHOURY BUT REMEMBER HIS
WORK OVER THE YEARS. OKAY. THERE
IS MY TITLE AGAIN. I BRIEF
OVERVIEW OF MY TALK -- IS THIS
OKAY? THE SOUND OKAY? I WILL
GIVE A BRIEF INTRODUCTION TO
CORONAVIRUS AND INTERFERON
SIGNALLING PATHWAYS AND TALK
ABOUT ROLL OF 2 PRIME
PHOSPHODIESTERASE, OR PDE IN RNA
CELL ANTAGONISTISM AND ENDURING
CORONAVIRUS PATHOGENISIS, THE
MODEL CORONAVIRUS MHV THEN TALK
ABOUT VRS AND SARS ENCODED
MULTI
MULTIPLE ENABLED AND PROBABLY
THE SECOND HALF IS INFECTION OF
PRIMARY NASAL EPITHELIAL
CULTURALS WE USE AS MODEL FOR
THE EPITHELIAL WITH LETHAL AND
INFECTIOUS CORONAVIRUS AND FOCUS
ON ROLE OF SIGNALLING RESPONSES
IN THIS MODEL SYSTEM. THEN THIS
SORT OF BOTTOM LINE IS INTER FER
RON DIFFERENTIATES AND IS
CRITICAL FOR CONTROL OF HUMAN
CORONAVIRUS VIRAL IN NASAL
EPITHELIAL AREA. EVERYONE HAS
SEEN THIS PICTURE. CORONAVIRUSES
ARE A FAMILIAR WITHIN THE NIDAL
VIRUS NAMED FOR MESSENGER RNAS
GENERATING DURING INFECTION
MEANING NESTED. THE PARTICLE HAS
GOTTEN FAMOUS AS WELL. THE
GENOME IS A VERY LONG 31-KILO
B ASE, PROBABLY THE LARGEST
RNA-VIRUS GENOME AND COMPLEX
WITHIN PLATFORM TO FORM A
HELICAL CAPSID, SURROUNDED BY A
MEMBRANE DERIVED FROM HOST CELL,
IN THE BLUE. IN THAT MEMBRANE
THERE ARE THREE PROTEINS , THE
SPIKE PROTEIN THAT HAS GOTTEN A
LOT OF PUBLICITY. THIS IS THE
PREE TEEN THAT MEDIATES --
PROTEIN THAT IMMEDIATES
ATTACHMENT AND CELL-TO-CELL
FUSION AND WITH SPREAD AND
CONTAINS MAJOR EPITOPES FOR THE
IMMUNE RESPONSE.
THERE ARE TWO OTHER, THE M
PROTEIN, MEMBRANE AND THEY ARE
IMPORTANT FOR STRUCTURE OF THE
VIRION AND OTHER STEPS DURING
VIRAL REPLICATION. SOME
CORONAVIRUSES ALSO IN HEMOGLUTEN
AND ESTERASE. NOT FOR THIS BUT
THIS IS NONESSENTIAL HOMOLOGUS
TO THE ESSENTIAL PROTEIN. I WILL
DO A BRIEF HISTORY OF
CORONAVIRUSES. OKAY. CAN SEE.
YEAH, CAN SEE THIS. OKAY. SO
CORONAVIRUSES FIRST APPEAR IN
LITERATURE IN THE 60S. AT THAT
POINT THE ONLY HUMAN VIRUSES WE
KNOW ABOUT WERE THE 43 AND 2290.
THEY BOTH CAUSE THE COMMON COLD
BUT VERY MANY ANIMAL
CORONAVIRUSES KNOWN AT THAT
TIME. INFECTIOUS BRONCHI SIS,
BOVINE, GASTRO INTER IE TIS AND
THESE WERE IMPORTANT FOR
ANIMALS. FROM THE LATE 60s UNTIL
THE FIRST SARS EPIDEMIC IN 2 #
02 THERE IS A LOT OF BASIC
BIOLOGY. NOBODY KNOWS A LOT BUT
THERE WAS A SMALL GROUP OF CELLS
AND HOW THEY COME OUT AND THE
IMMUNE RESPONSE. THE CYCLES
IDENTIFIED PROBABLY IN EARLY 80s
IN THE SPIKE PROTEIN. SO WHEN
SARS -- THE FIRST SARS -- THIS
IS A LIST OF VIRUSES WE ARE
WORKING ON. BASIC BIOLOGY WAS
DONE ON ANIMAL VIRUS, LITTLE ON
HUMAN. SO WHEN SARS, THE FIRST,
EMERGED IN CHINA IN 2002
EVERYBODY WAS SHOCKED. OUR FIELD
WAS TOTALLY SURPRISED THIS COULD
HAPPEN THAT, A HUMAN CORONA
COULD CAUSE DISEASE LIKE THIS.
IT SOON BECAME APPARENT -- I
SHOULD MENTION THE FIRST
INTERNATIONAL MEETING WAS HELD
IN GERMANY IN 1980, ABOUT 60 AT
THE MEETING, THE ENTIRE FIELD AT
THE TIME. AFTER THE FIRST SARS
EPIDEMIC HUNDREDS OF PEOPLE
STARTED WORKING ON THESE
VIRUSES. WE LEARNED SOON AFTER
THAT THAT THEY HAD THEIR ORIGIN
IN BATS AND HAD TRANSMITTED TO
HUMANS THROUGH AN INTERMEDIATE
SPECIES. THE OTHER THING THAT
HAPPENED RIGHT AFTER THAT
EPIDEMIC WAS THAT PEOPLE REALLY
LOOK FOR MORE HUMAN CORONA
VIRUSES. THESE TWO IDENTIFIED
HKU1A, AND 163 AND TO CATEGORIZE
HUMAN CORONA THOUGH THE 1 WAS
ISOLATED WITH OLDER PERSON WITH
PNEUMONIA AND 63 CAN CAUSE KROOP
AND BRONCHI IE TIS. THINGS WERE
QUIET UNTIL 2012 WHEN MERS
RESPIRATORY SYNDROME VIRUS
EMERGED IN SAUDI, CAUSING A
SMALLER EPIDEMIC, WHICH STILL
GOES ON. THERE ARE STILL PEOPLE
INFECTED BY MERS CORONAVIRUS. IT
WAS LESS TRANCE MMISSIBLE IN
HUMANS BUT IS IN CAMELS AND
TRANSMITTED TO HUMANS. THOUGH
DIFFERENT FROM SARS. CAMELS ARE
A LARGE RESERVOIR FOR MERS SO
THEY ARE IN MIDDLE EAST AND
AFRICA THAT HAVE HIGH LEVELS OF
THE POSITIVITY TO MERS
CORONAVIRUS. THINGS AGAIN WERE
QUIET UNTIL 2019 WHEN AS WE ALL
KNOW SARS-COV 2 EMERGED ON A
DIFFERENT PART OF CHINA. MOSTLY
LIKELY HAS BAD ORIGIN. THERE
HAVE BEEN VIRUSES IDENTIFIED
CLOSE TO THE-COV 2 BUT WE DON'T
KNOW THE PRECURSOR YET. WE DON'T
KNOW IF THERE WAS AN
INTERMEDIATE SPECIES. THERE MAY
BE, BUT THAT HASN'T REALLY BEEN
PROVEN YET. OF COURSE WE KNOW
THIS IS THE AGENT OF COVID-19.
SO COMMON CORONAVIRUSES ARE Z
ONOTIC AND SPREAD TO HUMANS.
OC43 IS BELIEVED TO HAVE
ORIGINATED IN CATTLE OR PIGS AND
TRANSMITTED TO HUMANS IN LATE
1890s. IT IS QUITE HOMOLOGOUS TO
BOVINE AND MOUSE HEPATITIS VIRUS
AND THOUGHT TO BE MUCH MORE
SEVERE INFECTION AND SORT OF
MORPHED INTO A COMMON COLD, VOL,
SHOULD SAY. 2-2-90 IS BELIEVED
TO HAVE ORIGIN IN TRANSMITTED TO
CAMELS TO HUMANS IN LATE 18TH
CENTURY. FINALLY NL63 ALSO IS
BELIEVED TO HAVE ORIGIN IN BATS
SO NOT SURPRISING THESE WERE
TRANSMITTED TO HUMANS FROM
ANIMALS. SO HUMAN CORONAVIRUSES
BELONG TO DIFFERENT GENERA SO
EITHER ALPHA OR BETA VIRUSES.
2290 AND 63 ARE ALPHA. BETAS,
HUMAN VIRUSES FALL INTO THREE
SUBCATEGORIES. I WILL TALK ABOUT
THESE. THE SARBEKA INCLUDE TWO
SARS AND RUBEKO INCLUDE MRSA AND
BAT VIRUSES. ALL THESE HAVE
SIMILAR GENOME STRUCTURE AND
ORGANIZATION WITH THIS VERY LONG
OPEN FRAME 1A AND B AT FIVE
PRIME END OF GENOME COMPRISING
TWO-THIRDS OF GENOME. THESE
PROTEINS ARE TRANSLATED
EVENTUALLY INTO 16 ON STRUCTURE
OF PROTEINS, WHICH I'M NOT GOING
TO TALK ABOUT. DESPITE THIS THEY
ARE ARRAYED IN SAME ORDER. WHAT
DIFFERENTIATES AMONG THESE
DIFFERENT GIVE NERA AND SURES
ARE ACCESSORY GENES THAT ENCODE
SMALL PROTEINS WITH NUMBER BY --
HERE LIKE 4A, 4B, ET CETERA.
THESE ARE PROTEINS THAT INTERACT
WITH SORT OF ANTAGONIZE THE
HOSTS. SOME ARE WELL
CHARACTERIZED. I WILL TALK ABOUT
SOME. THAT IS WHAT -- SO YOU CAN
SEE THE ALPHA CORONAVIRUS S HAVE
MORE SIMPLE GENOME, JUST THIS
WAS EVENTUALLY CAN BE ONE OR TWO
OPEN READING FRAMES. WE SEE THAT
THE BETA CORONAVIRUSES HAVE A
LOT MORE PARTICULARLY SARS AND
MERS HAVE QUITE A FEW ACCESSORY
PROTEINS.LY
PROTEINS.LY. AND I WILL TALK
ABOUT THEM IN A BIT. I WILL TALK
ABOUT OLD FINDING WHAT WE
LEARNED FROM THE EMBEKO AND
MOUSE. MHV COMES IN MANY
STRAINS. SOME ARE SHOIN HERE.
INFECT VERY DIFFERENT ORGANS,
LIVER, BRAIN, LUNG, GF GI. THE
ANIMALS ARE GITROPHIC. SOME CAN
BE DUAL LIKE A-59 HERE. BECAUSE
IT WAS FOUND EARLY ON AND
IDENTIFIED THE RECEPTOR FOR MHV
IN EARLY 90s, IF I REMEMBER
CORRECTLY. IT WAS FOUND ALL THE
STRAINS WAS SAME RECEPTOR,
CCAM1. THAT SUGGESTS RECEPTOR IS
NOT ONLY KEY TO PATHOGENISIS OR
TROEKISM, SO WAY BACK IN 80s AND
90s WE CONSTRUCTED CHIMERIC
VIRUSES EXCHANGING BETWEEN 59
AND JHM AND SOMETIMES J52 AND WE
FOUND POST ENTRY EVENTS WERE
LARGELY DETERMINE T V ROPHISM.
SO IT WASN'T ALL ABOUT
RECEPTORS. WE WANTED TO KNOW THE
ROLE OF INTERFERON SIGNALLING IN
DETERMINING PATHOGENISIS SO
INFECTED INTERFERON KNOCK-OUT
MICE WITH SOME OF THE STRAINS.
BASICALLY WHAT WE FOUND IS THAT
THE MICE DIED VERY QUICKLY,
WITHIN TWO DAYS. THAT VIRUS
INFECTED ORGANS THAT WERE NEVER
INFECTED IN OTHER TYPE MICE. SO
THIS JHM THAT IS HIGHLY
NEUROTROPHIC COULD ONLY INFECT
KIDNEY, LIVER, HEART OR ANY
ORGAN. SO THAT INTERFERON WAS
IMPORTANT IN SIGNALLING ORGAN
TROEP PISTOL M. THEY HAVE
GENERATED CON MREKSS AND THE
DOUBLE-STRANDED RNA IS PART OF
THE NATURAL REPLICATION CYCLE.
THIS IS TRUE FOR ALL RNA
VIRUSES. SOME DNA ALSO PRODUCE
DOUBLE-STRANDED RNA. THIS IS
WHEN SENSED BY HOST A SIGNAL TO
INITIATE ANTI -VIRAL SIGNALLING
OR INNATE RESPONSES. SO
CORONAVIRUSES PRODUCE THESE
DOUBLE-STRANDED RNAS AND THE
ENDOPLASTIC. THIS IS HUMAN CALU
CELLS. DESPITE THE FACT -- IT IS
THOUGHT THE RS REPLICATES TO
PROTECT DOUBLE-STRANDED RNA FROM
BEING SENSED BY HOST AS DANGER
SIGNAL TO INITIATE ANTI VIRAL
PATHWAYS. DESPITE THAT THESE
VIRUSES DO INDUCE ANTI -VIRAL
SIGNALLING. SO I WANT TO TALK
ABOUT THE DOUBLE-STRAND RNA AND
ANTI -VIRAL PATHWAYS I'M TALKING
ABOUT. CORONAVIRUSES PRODUCE
DOUBLE-STRANDED RNA SHOWN HERE
DURING THE REPLICATION AND
TRANSCRIPTION. IN THAT SENSE BY
THESE DIFFERENT SENSORS SO MDA5
SENSES DOUBLE AS CORONAVIRUS
RNA, RIDI DOES NOT. THE MDA
SENSING SIGNALS TO PRODUCE
INTERFERON WHICH THEN SIGNALS
THROUGH STAT TRANSCRIPTION
FACTORS TO INDUCE EXPRESSION OF
ENDOCRINE GENES AS WELL AS SITE
PSYTOCHINES. OASs AND SYNTHESIS,
TWO IN HUMANS AND PRODUCE 2RA
THAT ACTIVATES RYE BOW NUKE USE
A AND LEAD TO APOPTOSIS AND
INFLAMMATION. THE THIRD DOUBLE
STRANDED RNA IS SENSED BY PKR,
THAT AUTO PHOSPHOROLATES AND
ALPHA, TRANSLATION INITIATION
FACTOR AND ONCE DONE THE
INITIATION IS SHUT DOWN LEAPING
TO A POP TOE SIS AND
INFLAMMATION. THESE ARE THREE
INDEPENDENT PATHWAY, OAS AND PKR
ARE STIMULATED SO SUPER-INDUCED
AND ALSO INDUCED ONCE INTER
INTERFERONS ARE PRODUCED. THIS
IS SHOWN IMPORTANT IN TERMS OF
COVID. THE GWA STUDIES EARLY
DURING THE PANDEMIC SUGGESTED --
IDENTIFIED SUSCEPTIBILITY GENES.
AMONG WERE TYPE 1 INTERCEPTOR
AND THE OLOGOIC SYNTHETASIS.
VIRUSES IN RESPONSE HAVE EVOLVED
PROTEINS OR ACCESSORY PROTEINS
TO COMBAT THESE PATHWAYS,
RESPONSES. SO I WANT TO TALK
ABOUT MHV ACCESSORY PROTEIN NS2,
WHICH ANTAGONIZES THE RNA CELL
ACTIVATION PATHWAY. THIS WORK
WAS ALL DONE WITH BOB
SILVERMAN , A CLOSE COLLABORATOR
FOR MORE THAN TEN YEARS. HE
RECENTLY RETIRED. SO TOGETHER
WITH BOB WE STUDIED THIS
PROTEIN. WE FOUND A VERY
INTERESTING ACTIVITY FOR IT, I
THINK. IT HAS TWO PRIME
PHOSPHODIRESTERATES. ONS IS 2A5
WHICH ACTIVATE RNA WHICH CLEAVE
SINGLE STRANDED RNA, VIRUS AND
HOST, LEADING TO REDUCTION OF
REPLICATION AND ALSO BUT
INCREASE IN INFLAMMATION AND
CELL DATASET. IT IS AN ENZYME
AND CLEAVES THIS CELL AND SHUTS
DOWN THIS PATHWAY COMPLETELY. IN
THE CASE OF MHV THIS IS A REALLY
IMPORTANT ANTI VIRAL --
ANTI -HOST ANTAGONIST ACTIVITY.
I WILL SEW Y SHOW YOU WHY. USIN
THE A59 STRAIN AND MUTANT THAT
WAS UNABLE -- HAS INACTIVE NS2
EN SOO ENZYME WE COMPARED
MACROPHAGES AND FOUND IT
REPLICATES 10 TO THE 7 PER MIL,
A LOG SCALE ON THE LEFT. WHEREAS
THE MUTANT BARELY REP CANDIDATES
AT ALL. THIS ACTIVITY WAS
ESSENTIAL TO REPLICATE IN MACRO
PHAGES FOR THIS RESPONSE, FIE
FIBROBLASTS, NO NEED. AND WHEN
WE DO THIS COMPARISON IN
KNOCK-OUT MICE THE MUTANT
RECOVERS REPLICATION. SO THIS
ACTIVITY IS REALLY ESSENTIAL FOR
REPLICATION FOR MHV. WE CAN SEE
HERE THIS IS A BIOANALYZER ASSAY
OF THE RNA FROM INFECTED
WILD-TYPE BONE MARROW DERIVED M
MA CEO PHAGES. WE SEE A TYPE OF
RNA WHEREAS IN MUTANT INFECTED
THERE IS ACTIVATION OF RNA CELL,
EVIDENCED BY THIS DEGRADED OR
CLEAVED MICROCELL RNA. BECAUSE
THIS IS A HOUSE VIRUS WE CAN
TRANSLATE TO NATURAL HOSTS AND
LOOK AT WHAT HAPPENS IN THE
MOUSE. TAKING THE SAME WILD TYPE
AND MUTANT VIRUSES WE INFECT A
MOUSE. CAN INFECT IT AS
HEPATITIS OR INTERCRANIALLY, IN
WHICH CASE WE GET ENCEPHALITIS
OR HEPATITIS. WE INFECT THE
MOUSE. WE SEE DRAMATICALLY THAT
-- WE MEASURE VIRUS IN THE
LIVERS FIVE DAYS AFTER
INFECTION, THE PEAK OF
REPLICATION. WE SEE THE WILD
TYPE VIRUS PRODUCES AGAIN 10 TO
THE AND THE MUTANT BARELY REP
CANDIDATES. WE DO THIS IN
KNOCK-OUT MICE THE MUTANT
RECOVERS. EVEN INVIVO S2 IS
VITAL. SAME THING, WE HAVE THE
SUSTAINING OF H & E OF LIVER
SECTIONS FROM THESE MICE. WE CAN
SEE THE WILD SIDE VIRUS CAUSES
INFLAMMATION IN WILD TYPE OR
MUTANT. LIVERS WHEREAS MUTANT
VIRUS SHOWS NO CITOPATH IK IC IN
WILD BUT IS IN MUTANT. ONE OF
THE INTERESTING THINGS IS WHEN
WE LOOK AT SAME INFECTION BUT
LOOK AT THE BRAIN WE SEE THAT
THERE'S EQUAL REPLICATION IN
MUTANT OR WILD TYPE MICE WITH
EITHER VIRUS SO NS2 AND RNA
ACTIVATION IS IRRELEVANT IN
BRAIN. I WILL TELL YOU IN A
MINUTE WHY THAT IS TRUE. THIS IS
AN ORGAN-SPECIFIC ANTAGONIST IN
MHV. JUST TO SUMMARIZE WE WORKED
ON THIS PATHWAY A LONG TIME.
LOTS OF ASPECTS. I'M GOING TO
SUMMARIZE A FEW OF THE THINGS WE
FOUND OUT. SO MHV INFECTION OF
MICE, ANTAGONIST OF RNA IN TURNS
OUT IN MYELOID IS REQUIRED FOR
PATHOGENISIS IN LIVER, NOT IN
THE BRAIN. THE REASON WE THINK
THAT IS THE BRAIN HAS VERY LOW
LEVELS OF OLIGOSYNTETASIS AND
OTHER GENES AND TURNS OUT THIS
IS TOTALLY DEPENDENT ON OAS
GENES. RNA CELLS ARE
CONSTITUENTIVEL EXPRESS
SIMILARLY AND CON STITCH TIFLY
IN ALL TYPES BUT VARIABLE AMONG
CELL TYPES AND LOW IN BRAIN AND
ALSO THAT RNA CELL COULD BE
ACTIVATED INDEPENDENTLY OF MDA5
AND MAD SIGNALLING IN HUMAN
MOUSE AND BAT CELLS SO IS
INDEPENDENT PATHWAY THAT CAN BE
ACTIVATED EVEN WHEN INTERFERON
CAN BE SHUT DOWN BY VIRUS
VIRUSES. THE P THE
PHOSPHODIESTERASES ARE ENCODED
WITH MERS AND UNRELATED ROTO
VIRUSES AND BELIEVED IT WAS
PICKED UP FROM THE CELL AT SOME
POINT. THE CELLULAR ACAP7 HAS A
PHOSPHODIESTERASE STRAIN OF
UNKNOWN FUNCTION IN HOST.
FINALLY RNA CELL ACTIVATION FOR
MANY VIRUSES IS DEPEN DONATE
SYNTHESIS 3 NOT IN 1 OR 2. FOR A
WHILE THERE WAS PUBLICATION
SUGGESTING THAT OAS1 WAS
IMPORTANT RESTRICTION FOR
SARS-COV 2 AND I THINK IT IS NOT
CLEAR WHAT IS THE OAS THAT IS
IMPORTANT. SOMETHING WE ARE
INTERESTED IN WORKING ON.
CLEARLY FOR OTHER VIRUSES OAS3
WAS THE ONLY ISOFORM THAT REALLY
MATTERED. SO FROM HERE I'M GOING
TO MOVE ON TO STUDYING MERS. WE
INITIALLY GOT INTO THE INNATE
IMMUNITY BECAUSE THERE IS HOM
MOLL GOUS PE. BUT LOOKED AT THE
BROAD ABILITY TO ANTAGONIZE HOST
RESPONSES AND MOVED TO SARS
DURING THE PANDEMIC. I WANT TO
TALK A LITTLE ABOUT MERS AND
SARS ENCODED ANTAGONIST OF
DOUBLE-STRANDED INDUCED
PATHWAYS. HERE IS THE MERS
GENOME. AS I MENTIONED BEFORE
THE MERS GENOME IS MORE COMPLEX
THAN THE -- SOME OF THE OTHER
VIRUSES AND HAS THESE SMALL OPEN
READING FRAMES. MERS HAS AT
LEAST THREE ANTAGONISTS AND THIS
IS CONSERVATIVE FOR ALL
CORONAVIRUS VIRUSES. THIS IS
INTERESTING. ENDONUCLEASI THAT
CLEAVES TO PREVENT ACCUMULATION
OF DOUBLE-STRANDND RNA SO ALMOST
SACRIFICES ITSELF BUT IMPORTANT
TO FRE VENT ACCUMULATING
DOUBLE-STRANDED RNA BECAUSE IT
WILL INDUCE HOST RESPONSES. BUT
THERE IS THE RNA BINDING PROTEIN
THAT FURTHER SHIELDS FROM HOST
DETECTION. NS4B IS HOM MOLL GOUS
TO NS2 AND THE V SO A PRIME
PHOSPHODIESTERASE WHICH
ANTAGONIZING RNA CELL AND
LOCALIZATION SIGNAL THAT
PREVENTS NUCLEAR TRANSFER OF
CAPAB AND INFLAMMATION DOWN
STREAM. USING RECOMBINANT
VIRUSES IN THESE DIFFERENT
PROTEINS WE WERE ABLE TO SHOW
THAT MERS PRETTY MUCH COMPLETELY
SHUTS DOWN INNATE RESPONSES VIA
THESE THREE PROTEINS. SARS IS A
LITTLE DIFFERENT. HAS ENDOU AND
15 LIKE MERS BUT DOESN'T HAVE A
PHOSPHO OR DOUBLE-STRANDED
BINDING PROTEIN AND LESS ABLE TO
SHUT DOWN THE IMMUNITY. HAS OPEN
READING FRAMES WHICH THE R6
ENCODED PROTEIN IS BELIEVED TO
PREVENT NUCLEAR TRANCE
TRANSLOCATION OF STATS AND SHUT
DOWN THE SIMULATED GENE
EXPRESSION, ALTHOUGH COUPLE
PUBLISHED STUDIES, AS WELL AS
OUR OWN WORK DOESN'T SHOW THAT
REALLY HAPPENS. SO BUT THERE'S
LOTS TO LOOK AT IN THESE OTHER
PROTEINS, AND IS STILL TO BE
EXPLORED. SO JUST TO QUICKLY
LOOK HOW MERS AND SARS INCREASED
INTERFERON SIGNALLING WE USED
LUNG DERIVED CALU AND HER IS
LAMDA AND BETA AND STIMULATED
GENE. THESE ARE QCPR LOOKING AT
INDUCTION OVER MOCK-INFECTED
CELLS AND CAN SEE THE SARS IN
THE BLUE, WILD TYPE SARS INDUCES
VERY LITTLE INTERFERON TYPE 3 OR
TYPE 1. A SMALL AMOUNT OF THIS
INTER FE IN/* F -- INTERFERON
STRAIN WITHDRAWS THIS PRODUCES
MORE IN THESE GENES. WHEN WE
LOOK AT MERS, MERS INDUCES
ALMOST UNDETECTABLE AMOUNTS OF
INTERFERON BUT IF WE MUTATE 15
OR 15-PLUS, NS4A WE CAN SEE A
LOT MORE INDUCTION, UP TO
9,000-FOLD OF INTERFERON DURING
INFECTION OF THE CELL TYPE.
SIMILARLY MERS DOESN'T INDUCE
ANY OF THESE PKR OR RNA CELL BUT
DOUBLE-MUTANTS DO. SO THESE
ANTAGONIST PROTEINS ARE
IMPORTANT TO MAINTAIN -- TO KEEP
THE HOST RESPONSES DOWN. IF WE
LOOK AT REPLICATION, WE CAN SEE
IN THE CASE OF SARS A SMALL
DEFECT IN THE NSP15 MUTANT AND
LARGER IN MERS MUTANTS. I WILL
GET BACK TO THIS LATER ON. SO
THE POINT IS THAT BOTH THESE
ADVISORS INDOUGS -- EXPRESS
INTERFERON PROTAGONIST PROTEINS
BUT MERS MUCH MORE THAN SARS.
PERHAPS CORRELATING WITH IT
BEING MORE PATHOGENIC. JUST TO
SUMMARIZE THIS PART HERE, SO
HERE IS SAME PATHWAYS. WE KNOW
THE 15 OR ENDO %CONFLPREVENTS
DOUBLE-CLEAVING AND THE NS4A
DOUBLE-BINDING PROTEIN FROM MERS
HERE. THEN THE MERS ENCODES THE
PROTEIN TO SPECIFICALLY SHUT
DOWN THIS PATHWAY, AS DOES MHV
AND OC43 SO MERS TAKES CARE OF
EVERYTHING. SARS IS DIFFERENT IN
THAT IT DOES INTRODUCE
SIGNALLING, NOT A LOT COMPARED
TO OTHERS. I WILL SHOW YOU BUT
DOES INDUCE THESE PATHWAYS. IT
IS KIND OF AMAZING. KEEPS THE
PHOS ALPHA THROUGHOUT
REPLICATION BUT CONTINUES
PROTEINS AND REPLICATES
EFFICIENTLY WHEREAS OTHER
VIRUSES -- I'M NOT GOING TO SHOW
YOU, THEY NEED TO KEEP THIS
ALPHA UNPHOSPHORELATED OR BAD
FOR THEIR REPLICATION. WE DON'T
UNDERSTAND HOW SARS WORKS
DESPITE INDUCING THE PATHWAYS.
IT SUGGESTS THAT IT SIMULATES
THIS GENE EXPRESSION THOUGH WE
HAVEN'T SEEN THAT
EXPERIMENTALLY. THERE ARE OTHER
PROTEINS WE ARE INTER TESINTERED
AND WORKING ON THE RF6 ON THAT
PROTEIN. AT THIS POINT I WANT TO
SWITCH TO THE SORT OF -- TO TALK
ABOUT OUR MOST RECENT STUDIES.
ALL THEY STARTED DURING THE
PANDE
PANDEMIC. THE QUESTION IS HOW
DOES IT INCREASE RESPONSE IN
NASAL EPITHELIALIUM. WE USED
DIFFERENT VIRUSES THAT AFFECTED
THE RESPIRATORY TRACT AND CAUSES
DIFFERENT DISEASES. MERZ CAUSES
A LETHAL PNEUMONIA AND INFECTS
MOSTLY LOWER RESPIRATORY, THE
LUNGS. SARS-COV 2 CAUSES A REAL
RANGE OF DISEASES FROM
ASYSTEMATIC TO MILD UPPER
RESPIRATORY DISEASE TO LOWER
RESPIRATORY TO LETHAL DISEASE
THIS THE LUNG AS WELL. THEN NL63
AND 2290 THAT BOTH CAUSE
PRIMARILY UPPER RESPIRATORY
DISEASE. KIND OF CALL THEM THE
COMMON COLD CORONA VIRUSES. WE
ALSO USE FOR THESE EXPERIMENTS
HUMAN RHINO VIRUS 16, A
COMPLETELY DIFFERENT TYPE VIRUS.
WE USE IT KIND OF AS CONTROL FOR
HOW COMMON COLD VIRUS WILL
BEHAVE. SO WHY DO WE THINK THE
NASAL EPITHELIAL YUM IS
IMPORTANT? IT IS THE FIRST LINE
OF DEFENSE AGAINST RESPIRATORY
PATHOGENS. INITIAL SIDE OF VIRAL
REPLICATION. PRIMARILY COMPOSED
OF SILLIATED AND GOBLET CELLS
THAT PRODUCE MUSIC CUSS AND HAS
THESE TIGHT JUNCTIONS AT THE
APICAL SIDE OF THE CELLS SHOWN
HERE THAT CONTRIBUTE TO
EPITHELIAL BARRIER. THE MUCUS --
IS AN EARLY RESPONSE SO THE
PSYLLIA PUSH MUCUS AROUND. WE
BELIEVE THAT IS TO KEEP VIRUS
FROM BEING ASPIRATED INTO THE
LUNG. THERE IS A HIGH BASAL
LEVEL OF EXPRESSION OF
INTERFERON AND OTHER GENES IN
THE NASAL EPITHELIAL YUM SO MAY
BE PRIMED TO PREVENT AGAINST
INVADING VIRUSES. THERE ARE
CLINICALS PUBLISHED WHERE THEY
LOOK BY RNA SIK BASAL LEVELS IN
RESPONSE LEVELS IN NOSE AND
FOUND HIGHER, BETTER LEVELS OF
THESE GENES WAS ASSOCIATED WITH
LESS SEVERE COVID 19. SO I THINK
THE INTERFERON RESPONSE IN THE
BRAIN IS QUITE IMPORTANT. WITH
THE EPITHELIAL CELL MODEL
CULTURES ON AIR LIQUID INTERFACE
TO RECAPITULATE THE AIRWAY. ALL
THIS WORK IS DONE IN
COLLABORATION WITH NOEM CULLEN,
AN EMT SURGEON AT PENN. HE HAS
COLLECTION FROM HUNDREDS OF
PATIENTS OF PRIMARY EPITHELIAL
CELL IN A KIND OF BIOBANK SO WE
HAVE AN UNENDING SUPPLY OF CELLS
TO WORK WITH. WHEN WE USE THESE,
WE PUT THEM ON TRANSWELLS,
EXPAND IN LIQUID CULTURAL AND
REMOVE THE LIQUID AND
DIFFERENTIATE UNDER AIR LIQUID
INTERFACE TO RECAPITULATE EN
VIVO AIRWAY. HERE IS A PICTURE.
THEY LOOK A LOT LIKE NASAL
EPITHELIAL YUM. THIS IS THE
CILIA HERE . YOU SHOULD BE ABLE
TO SEE THE CILIA BEATING. THIS
IS A CULTURE WHERE MUCUS HAS
BEEN REMOVED SO YOU CAN SEE WHAT
IS GOING ON WITH CILIA. THIS
THIRD HAS NOT BEEN WASHED OFF.
YOU CAN SEE THE CILIA KIND OF
PUSHING AROUND THE MUCUS. SO
AFTER SEVERAL WEEKS OF
DIFFERENTIATION THEY ARE READY
TO BE INFECTED. SO THIS IS THE
KINETICS OF REPLICATION IN
PRIMARY NASAL CULTURES. WE CAN
SEE DIFFERENT PATTERNS. ALL
THESE ARE DONE WITH -- WE NOW
POOL YELLS FROM FOUR TO SIX
DONORS AND REPEAT THAT ABOUT
THREE TIMES. BY THE TIME WE ARE
DONE WE HAVE LOOKED ABOUT
SOMEWHERE BETWEEN 12 AND 18
DONORS. THIS IS THE AVERAGE
REPLICATION KINETICS. IF WE TAKE
THAT AN PART WE CAN SEE THAT
SARS AND MERS HAVE SIMILAR
KINETICS. THESE ARE LONG
CULTURE, GO UP FOR EIGHT DAYS,
192 HOURS. IF WE LOOK AT THE
COMMON COLD CORONAVIRUSES THEY
REPLICATE EARLY AND CLEARED FROM
CULTURES. THEN FINALLY IF WE ADD
HUMAN RHINO VIRUS 16 IT BEHAVES
PRETTY MUCH LIKE THE COMMON COLD
VIRUSES SO HAVE SIMILAR
REPLICATION PEAK THEN ARE CLE
CLEARED. SO THERE ARE DIFFERENT
TEMPERATURES IN THE UPPER AND
LOWER TRACT. WITH NOSE HAVING
TEMPERATURES IN RANGE OF THE --
OR THE NASAL CAVITY, LIKE 32, 33
DEGREES. THE LUNGS WITH MORE
LIKE A 37 DEGREES BODY
TEMPERATURE. SO WE WANTED TO SEE
HOW THESE VIRUSES REPLICATED AT
THE DIFFERENT TEMPERATURES GIVEN
THAT THEY REPLICATE IN DIFFERENT
PARTS OF THE RESPIRATORY TRACT.
WHAT WE FOUND IS COLD VIRUSES,
98, 63 AND HRV16 ALL SORT HAVE
SIMILAR PATTERN, THEY REPLICATED
MUCH BETTER AT LOWER TEMPERATURE
SHOWN HERE, 33 DEGREES THAN AT
37 DEGREES. IT WAS PRETTY
DRAMATIC FOR NL63, FOR EXAMPLE.
SO THIS IS MORE THE NASAL
AIRWAY, NOT THE LUNG. WHEN WE
LOOKED AT THE SAME DIFFERENCE IN
TEMPERATURE FOR SARS AND MERS WE
SAW FOR SARS UP UNTIL 144 HOUR,
NO PREFERENCE FOR EITHER
TEMPERATURE BUT CRASHES AT 37.
REALLY NEEDS 33 DEGREES TO
CONTINUE TO REPLICATE. I WILL
GET BACK TO THIS LATER. MERS
REALLY HAS NO PREFERENCE FOR
TEMPERATURE. IT CAN REPLICATE
EQUALLY AT 33 OR 37. SO THE 16
PEAKS AT EARLY TIMES, FOLLOWED
BY CLEARANCE. THE REPLICATION OF
SARS AND MERS PLATEAUS WITHOUT
EVIDENCE OF CLEARANCE. SIMPLY
COMMON COLD VIRUSES REPLICATE
MORE EFFICIENTLY AT 33, COMPARED
TO 37. SARS ALSO REPLICATES
OPTIMALLY AT 33. ONLY AT LATE
TIME POINTS AND MERS IS NOT
IMPACTED BY TEMPERATURE AT ALL.
SO WE WANTED TO KNOW WAS HOW DO
INTERFERENCE SIGNALLING
RESPONSES VARY AMONG INFECTION
WITH VIRUS VIRUSES, RESPIRATORY
VIRUSES. SO WE DID AN RNA SIK
EXPERIMENT TO INFECTED CULTURALS
WITH ALL THESE AND EXTRACTED RNA
AND SUBJECTED IT TO RNA SEEK.
THESE ARE VOLCANO PLOTS -- I
WILL PUT THEM ALL UP FIRST.
WHERE REGULATION OF GENES IS TO
THE RIGHT AND DOWN REGULATION TO
THE LEFT. THE INTERFERON SIGNALS
ARE HIGHLIGHTED IN GREEN. WE CAN
SEE IF WE LOOK AT MERS, THERE'S
VERY LITTLE INDUCTION OF
INTERFERON SIGNALLING GENES. A
FEW THAT ARE DOWN REGULATED.
SARS A BIT MORE, AS WE SAW IN
THE CALU CELLS. IF YOU LOOK AT
COLD VIRUSES, JUST WAY, WAY MORE
GENES INDUCED IN THE SIGNALLING
GENES INDUCED AND HIGHER LEVELS
AS WELL. WHEN WE QUANTIFIED THE
NUMBER OF INTERFERON RESPONSE
GENES INDUCED REGULATED NICHE IK
FEBRUAR
INFECTION, MERS LOW AND SARS
MINIMAL AND OTHER THREE HIGHER.
ONE OF THE TRADEMARKS OF COLD IS
INTRODUCE A LOT OF INTER FOR RON
SIGNALLING. THE IS A KEY PIECE
OF VALIDATION. I TRUST PCR
BETTER, IT IS QUANTITATIVE SO WE
DO THIS TO MAKE SURE THE RNA
SEEK ACTUALLY MAKES SENSE OR
CORRESPONDS WITH IT. WE CAN SEE
AGAIN VERY LITTLE SARS --
INDUCED BY SARS, ALMOST NOTHING
DETECTABLE BY MERS AND OTHER
VIRUSES TO DIFFERENT EXTENTS ARE
INDUCING MORE INTER INTERFERON
AND STIMULATED GENES. THE OTHER
WAY WE LOOK, WE LIKE THIS, IS
LOOKING AT THE INTERFERON
PROTEIN EXPRESSION BY WESTERN
BLOT. WE CAN SEE IF WE LOOK AT
-- THIS IS THE FIRST SUSTAINING
FOR PHOS STAT TYPES FOR GENES.
MERS REALLY INDUCES NO RESPONSE
AT ALL. EVEN IF WE LOOK AT ANY
TIME POINT, WE DON'T SEE IT.
SARS WE SEE LATE TIME POINTS,
SOME 92 HOURS. EIGHT DAY
POST-INFECTION, NOTHING UNTIL
THEN. WHEREAS OTHER VIRUSES BY
48 TO 96 HOURS ARE INDUCING STAT
PHORELATION. WHEN WE LOOK AT A
BUNCH OF STIMULATED GENES WE SEE
SAME. NOTHING FOR MERS. SARS
LATE. OTHER VIRUSES MUCH
EARLIER. THEN WE ALSO LOOK AT
PKR ACTIVATION JUST TO REMIND
YOU, WE MEASURE THIS BY PHOSPR
AND ALPHA. WE SEE THE SAME
PATTERN. NOTHING FOR MERS, SARS
LATE, OTHER VIRUSES AT EARLIER
TIME POINTS ARE INDUCING
PHOSPHORELATION OF THE PATHWAY.
THIS IS BLOTTING BOARD THAT CAPS
AND PROTEINS OF EACH VIRUS TO
SHOW THEY ARE REPLICATING TO
REASONABLE EXTENT. SO FROM THIS
WE CONCLUDE THERE ARE THREE
DIFFERENT KINDS OF PATTERNS THAT
FOR MERS REALLY NO SPONRESPONSER
SARS 2 DELAYED AND 6290 AND
RHINO, EARLY AND ROBUST
RESPONSES. THE NEXT THING WE
WANT TO KNOW WHAT HAPPENS IF WE
TAKE AWAY INTERFERON SIGNALLING
RESPONSE, WHAT WILL HAPPEN TO
REPLICATION OF THESE VIRUSES? SO
WE USED A DRUG CALLED
RUXALITANEM, IMPAIRING
INTERFERON SIGNALLING AND REACTS
WITH INTERCEPTOR CAUSING
ACTIVATION OF CHIN
CHINASEPHOSPHOROLATES. AND RUX
INHIBITS JACK ACTIVITY,
PREVENTING PHOS OF STAT AND
GREATLY REDUCING
INTERFERON-SIMULATED GENE
TRANSCRIPTION. WHEN WE TREAT
CELLS WITH RUX AND THEN INFECT
THEM WE SEE THIS PATTERN FOR THE
THREE COLD VIRUSES THAT WERE
CLEARED FROM INFECTION, NO
LONGER CLEARED. THEY PLATEAU,
SORT OF RESEMBLE MORE KINETICS
OF VIRULENT VIRUSES OF MERS AND
SARS. WHEN WE DO SAME TREATMENT
TO THESE WE SEE LITTLE EFFECT ON
SARS-LATENT INFEBRUARYING SHN
AND NO EFFECT ON MERS. SO THIS
SUBSTANTIATES THAT THE RESPONSE
IS WHAT IS LIMITING COLD VIRUSES
IN NOSE. WHEREAS TWO VIRTUAL
LENT DON'T INDUCE MUCH RESPONSE.
TO SUMMARIZE HERO HUMAN
CORONAVIRUSES INDUCE A WIDE
RANGE OF DIFFERENT SIGNALLING
RESPONSES AND SARS SIGNIFICANTLY
DELAYS INTERFERON RESPONSES. THE
INHIBITION OF INTERFERON WITH
RUX IMPAIRS CLEARANCE OF COMMON
COLD BUT MINIMAL EFFECT ON
REPLICATION OF LETHAL VIRUSES.
SO WE SUGGEST FROM ALL THIS THAT
THE EXTENT OF INTERFERON
SIGNALLING IN NOSE MAY IN PART
DETERMINE IF INFECTION CAN
PROGRESS. SO THE VIRUS ISN'T
CLEARED FROM THE NOSE AND CAN BE
MORE EASILY ASPIRATED FROM LUNG.
SO WE WANTED TO KNOW THE ROLE OF
VIRUS ENCODED ANNING THE KNISS I
TALKED ABOUT IN REGULATING
REPLICATION AND CLEARANCE IN THE
NASAL CELL MODEL. SO THESE ARE
THE SAME VIRUSES I SHOWED YOU
BEFORE, THE MERS MUTANTS. THEY
ARE EVEN MORE AT TEN YOU WAITED
IN THE MODEL. SAME FOR SARS
WHICH DIDN'T SHOW MUCH DEFECT
AND CELL LINE IS LOOKING AT
ATTENUATED. WE CAN LOOK AT THIS
AGAIN BY WESTERN BLOT AND WE SEE
THAT SARS MUTANT INDUCES MORE
PSTAT THAN WILD BUT BOTH DO
INDUCE WHEREAS MERS, THERE IS
NOTHING, NO INDIRECTION BY MERS.
THEY INDUCE EARLY AND ROBUST
COMPARED TO WILD TYPE. SAME
THING AGAIN FOR INTERFERON
STIMULATED. THE MUTANT INDUCES
QUITE WELL. THE MUTANT SARS MAY
INDUCE EARLIER BUT STILL THIS
DELAYED INDUCTION, WHEREAS MERS
IS NOT DELAYED AT ALL. IF WE
LOOK AT PKR WE SEE SAME PATTERN
THAT MUTANT VIRUSES INDUCE MORE
PKR, NOT SURPRISINGLY, THAN WILD
TYPE. AGAIN, JUST THIS IS JUST
TO SHOW THAT ALL THE VIRUSES ARE
REPLICATING. OKAY. SO WE THEN
TREATED -- WE TREATED CELLS WITH
RUX AND INFECTED WITH WILD TYPE
AND MUTANT VIRUSES AND SEE
INTERESTINGLY HERE IS THE SOLID
LINES ARE UNTREATED AND DOTTED
LINES ARE TREATED WITH RUX AND
WE CAN SEE THAT THE MUTANT VIRUS
THAT WAS -- DIDN'T REPLICATION
VERY WELL IN PRESENCE AND
WITHOUT THE DRUG NOW IS RESCUED
FOR REPLICATION. IN OTHER WORDS,
THE SAME THING IS TRUE WITH THE
MERS MUTANTS. SO TREATING OR
INHIBITING THE INTERFERON SIGNAL
ALLOWS MUTANT VIRUSS TO BEHAVE
AS WILD TYPE SO JUST SHOWS AGAIN
THAT THESE ANTAGONISTS PREVENT
THE EFFECTS OF INTERFERON
SIGNALLING AND ONCE GONE, THERE
ARE WILD TYPE VIRUSES. SO OKAY.
SO IN ADDITION TO INDUCING
DIFFERENT PATTERNS OF
SIGNALLING, AS I SHOWED YOU,
THERE'S DIFFERENT PATTERNS OF
TEMPERATURE DEPENDENCE FOR
REPLICATION. WE WONDERED WHETHER
DOES INTERFERON SIGNALLING
RESPONSE CONTRIBUTE TO
TEMPERATURE DEPENDENCE. WE SAW
THESE THREE PATTERNS BOTH FOR
INTERFERON SIGNALLING AND
TEMPERATURE DEPENDENCE THAT SORT
OF ALONG THE SAME LINES. SO
STARTING WITH SARS WE ASKED
WHETHER INTERFERON SIGNALLING TO
SARS WAS MORE ROBUST AT 37
VERSUS 33 AND WE FOUND IT WAS.
IF WE LOOK HERE WE CAN SEE AT
EACH TEMPERATURE THERE IS MORE
PSTAT AND STIMULATED GENES AND
MORE PPKR WHEN THE EXPERIMENT IS
DONE AT 37 DEGREES COMPARED TO
33. HOWEVER WHEN WE TREAT THE --
THESE CELLS WITH RUX, WE CAN SEE
THAT THE 37-DEGREE REPLICATION
IS NOW RESCUED, SO THE VIRUS NO
LONGER IS TEMPERATURE-SENSITIVE
WHEN WE GET RID OF INTERFERON
SIGNALLING. THESE TWO ARE VERY
ENTER TWINED. AND THIS IS TRUE
WITH NL63. SO NL63 IS HIGHLY
TEMPERATURE DEPENDENT, LOWER
TEMPERATURE DEPENDENT FOR
REPLICATION. IF WE LOOK AT --
AGAIN, AT THE INTERFERON
SIGNALLING RESPONSE WE SEE EARLY
IN INFECTION IT IS MUCH MORE
ROBUST AT 37. AT 24 AND 48
HOURS. WHEN WE GO TO THE LATER
TIMES IT BECOMES MORE ROBUST AT
33 BECAUSE THE VIRUS CLEARLY
DOESN'T REPLICATE OUT HERE AT
37. REPLICATES SO POORLY. AGAIN,
IF WE TREAT THE CELLS WITH RUX,
IT CAN NOW REPLICATE AT 37
DEGREES, SHOWN HERE. JUST LIKE
THE WILD TYPE VIRUS. JUST LIKE
THE 33-DEGREE REPLICATION. SO
INHIBITION OF THE SIGNALLING
RESCUES REPLICATION AT 37. SO
JUST TO SUMMARIZE, SO THE LETHAL
CORONAVIRUS ENCODE ANTAGONISTS
THAT PREVENT INDUCTION OF
INTERFERON SIGNALLING AND
CLEARANCE AND CELLS AND
ACTIVATION OF THE ANTAGONIST
RESULTS IN FINE TYPES THAT
MIRROR COMMON COLD VIRUSES SO WE
CAN FLIP FROM VIRAL LENT TO
COMMON COLD WITH GETTING RID OF
THESE OR ACTIVATING THE
ANTAGONIST. THE OPTIMAL
REPLICATION OF COMMON COLD
CORONAVIRUSES AT 33 IS AT LEAST
PART DUE TO
TEMPERATURE-DEPENDENT INTERFERON
RESPONSES. THIS WAS ALSO SHOWN
EARLIER BY FAIL AND FOXMAN FOR
RHINO VIRUS, SIMILAR KIND OF
FINDING. SO THIS IS ONE MORE --
THIS IS THE LAST VIGNETTE I'M
GOING TO TALK ABOUT. THAT IS THE
QUESTION OF HAVE SARS
CORONAVIRUS MUTANTS EVOLVED TO
BE MORE SIMILAR TO A COMMON COLD
VIRUS. SO IT SEEMS, FROM THE
LITERATURE, FROM OBSERVATIONS,
THAT THE LATER VARIANTS ARE MORE
UPPER RESPIRATORY AND MAYBE LESS
VIRAL LENT SO WE WANTED TO KNOW
WHETHER THEY ACTUALLY HAD THE
CHARACTERISTICS WE HAVE
IDENTIFIED OF THE COMMON COLD
VIRUSES. SO REPLICATING MORE
EFFICIENTLY AT LOWER
TEMPERATURE, INDUCING A ROBUST
INTERFERON RESPONSE AND
CLEARANCE FROM THE NASAL
EPITHELIAL CELLS. SO WE
INITIALLY INFECTED THESE
CULTURES WITH WILD-TYPE
WASHINGTON STRAIN OF SARS COVID
2, ALPHA, BETA, DELTA AND EARLY
OMNICHRON VARIANT. ALL VARIANTS
REPLICATED BETTER THAN WILD TYPE
TO SOME EXTENT, THOUGH ALL
REPLICATE QUITE WELL. WE DIDN'T
GO FAR OUT, BUT OMNICHRON WINS
THE REPLICATION FOR KINETICS. WE
WANTED TO KNOW WHAT KIND OF
INTERFERON RESPONSE DID IT
INDUCE. WE USE THE RNA SEEK AND
VOLCANO PLOTS. HERE IS WILD TYPE
SARS, USED DELTA AS COMPARISON
AND HERE IS OMNICHRON. I BOXED
IN THE AREAS. SARS INDUCES A
SMALL RESPONSE. WILD TYPE SARS,
DELTA MAYBE SIMILAR BUT OMNICRON
INDUCES MORE. SO DID SEEM LIKE
A COMMON COLD VIRUS, THOUGH NOT
QUITE TO THE EXTENT. WE WANTED
TO LOOK AT REPLICATION. WE
THOUGHT IT WOULD PROBABLY HAVE
REQUIREMENT FOR LOW TEMPERATURE
REPLICATION BUT WE WERE
COMPLETELY WRONG. HERE IS
KINETICS OF WILD TYPE SARS AND
KINETICS OF BA1. SEEMS TO BE
INSENSITIVE TO HIGH TEMPERATURES
SO WE DON'T UNDERSTAND HOW IT
DOES THAT. THAT SEEMED SORT OF
QUIZZICAL BECAUSE HOW COULD IT
SURVIVE AND SPREAD SO WELL. BUT
THEN WE FOUND IT IS LESS
SENSITIVE TO TYPE 1 AND 3
INTERFERON TREATMENT. THAT IS
SHOWN HERE. HERE IS TYPE 1
INTERFERON TREATMENT THAT WILD
TYPE VIRUS IS QUITE SENSITIVE
WHEREAS OMNICRON IS NOT. FOUND
BE PRETREATING CELLS. SAME FOR
LAMBDA AND TYPE 3 INTERFERON,
MUCH LESS SENSITIVE SO SOMETHING
WE CAN'T UNDERSTAND. SO IN
SUMMARY OF THE WHOLE THING,
CORONAVIRUS ENCODE NONSTRUCTURAL
PROTEINS AND SUB-GENEROUS
ACCESSORY PROTEINS THAT
ANTAGONIZE NATIVE IMMUNE
RESPONSES. IN FACT, THERE ARE
LOT MORE ACTIVITIES WITHIN THE
REPLICASE. LOCIS ON THIS. HAVE
BUNCH OF CAPPING AND
METHYL IZATION ENZYMES. COMMON
COLD LIKE RHINO 16 PREFER LOWER
AND INDUCE SIGNALLING WHICH
LEADS TO CLEARANCE. LETHAL VIRUS
AND MARIE INDUCE AND PERSIST IN
AIR LIQUID INTERFACE AND ARES AR
RATED INTO THE LUNG WE BELIEVE
AND THIS CONTRIBUTES TO 33
DEGREE FOR COMMON COLD VIRUSES
AND FOR SARS LATENT INFECTION.
OMNICRON HAS EVOLVED UNIQUE FINE
PHENOTYPES AND WE ARE TRYING TO
LOOK AT THOSE TO SEE HOW THEY
BEHAVE WITH RESPECT TO
INTERFERON INDUCTION AND
TEMPERATURE REQUIREMENTS. THEN
SO FINALLY INDUCTION OF
INTERFERON SIGNALLING IN
EPITHELIAL YUM CAN CONVERT
LETHAL VIRUS INTO COMMON COLD
VIRUS. WE ARE WONDERING IF
INTERFERON IS A POSSIBLE
THERAPEUTIC APPROACH FOR THESE
VIRUSES TO SORT OF CONVERT --
CONVERT THEM BACK AND FORTH WITH
INTERFERON TREATMENT. SO JUST
ACKNOWLEDGE THE PEOPLE IN MY
LAB. THIS IS MY CURRENT GROUP OF
PEOPLE. SOME OF THESE -- CLAYTON
OTTER DID ALMOST ALL THE WORK I
DESCRIBED IN THE SECOND HALF OF
THE SEMINAR. HE IS AMAZING. MD
PHD STUDENT WHO JUST WENT BACK
TO MEDICAL SCHOOL. THESE ARE MY
-- NOEM'S BEEN A CLOSE
COLLABORATOR. HE IS RIGHT HERE.
BOB SILVERMAN FOR MANY, MANY
YEARS AND THESE ARE MORE RECENT
COLLABORATORS, ALL OF THEM GREAT
COLLABORATORS AND FUNDING IS
FROM NIH. I WILL STOP THERE.
[ APPLAUSE ]
>> IF ANYONE HAS QUESTIONS, USE
THE MICS. YEAH, QUESTIONS. MAYBE
I WILL GET STARTED. SO YOU
MENTIONED IN THE INTRODUCTION
THAT ONE OF THE EARLY SEASONAL
CORONAVIRUSES THAT EMERGED IN
THE 1890s, OC43 WAS HIGHLY
PATHOGENIC, OR THOUGHT TO BE. IS
THERE ANYTHING KNOWN ABOUT
WHETHER THE VIRUS CHANGED
SUBSEQUENT TO THAT INITIAL -- OR
WHETHER IT WITH WAS THE
POST-POPULATION ISSUE OR DRIFT
OR IS THERE ANY REMNANTS OF ANY
ISOLATES AVAILABLE FROM THOSE --
>> I DON'T KNOW. IT IS SORT OF
LIKE WONDERING WHETHER OMNICRON
BECOMING LESS UPPER RESPIRATORY
HAS TO DO WITH VACCINE OR
CHANGES IN THE VIRUS. I DO THINK
FOR EXAMPLE 229A, THEY HAVE TWO
SMALL OPEN READING FRAMES THAT
USED TO -- IN THE MORE CLINICAL
ISOLATES WERE ONE AND ANTAGONIST
SO IT IS POSSIBLE THEY HAVE LOST
SOME OF THOSE ANTAGONIST --
>> INOCULATING THE HOST?
>> THAT IS WHY OMNICROM
SURVIVES, IT DOESN'T KILL THE
HOST. I MEAN, SIMPLISTICALLY.
>> YES.
>> THANK YOU. VERY CLEAN AND
CLEAR LECTURE. I WAS WONDERING A
COUPLE THINGS. SO ONE, THE
INTERFERON ISSUE CANNOT BE THE
SOLE DRIVER FOR TEMPERATURE
DIFFERENTIAL, RIGHT, BECAUSE
ISN'T WORKING IN CASE OF THE
LAST VARIANT --
>> SAY THAT AGAIN?
>> THE INTERFERON RESPONSES
CANNOT BE THE ONLY DRIVER OF
TEMPERATURE DIFFERENTIAL. I WAS
WONDERING IF THERE ARE OTHER
HYPOTHESIS YOU ARE WORKING ON.
>> OKAY, SO -- WELL, COULD BE A
MAJOR DRIVER OF IT, I THINK. WE
DID -- WE HAVE LOOKED AT LIKE
THE THERMAL STABILITY OF THE
ACTUALLY VIRUS PARTICLES, FOR
ALL THESE. THEY ARE REALLY ALL
THE SAME. SO IT IS NOT INHERENT
IN THE VIRION. IF THAT IS
UNTABLE -- ORIGINALLY I THOUGHT
IT WOULD BE EN ENZYMES BUT THE
FACT WE TREAT WITH RUX AND GET
RID OF INTERFERON SIGNALLING,
LOSES THAT SENSITIVITY, TELLS ME
IT IS PROBABLY A MAJOR
EXPLANATION.
>> THAT DOESN'T WORK FOR THE
OMNI CRON THOUGH?
>> I DON'T UNDERSTAND OMNICRONS
SO --
>> MUST BE OTHER ISSUE.
>> THEY ARE NOT TEMPERATURE
SENSITIVE. I DON'T KNOW. I HAVE
NO IDEA WHAT GENES OR WHAT
PROTEINS IN OMNICRON ARE GIVING
THAT. I COULDN'T BELIEVE THAT
RESULT. SEEMED VERY STRANGE.
>> THE OTHER QUESTION I HAD, THE
CULTURE DATA CLEARLY DEMONSTRATE
THAT WHEN THE INFECTION IS UPPER
RESPIRATORY CONFINED, IS
SELF-LIMITING, WITH CONTRIBUTION
WITH INTERFERON RESPONSE. IN
TERMS OF THE ADAPTIVE IMMUNE
RESPONSE, WHAT IS THE DEAL?
>> SO WE ARE -- THAT IS ONE OF
THE THINGS WE HAVE BEEN TRYING
TO DO. PUT MACROPHAGES OR OTHERS
INTO THE CELL CULTURES. NOT
EASY. THIS CAN ONLY TELL US
ABOUT EARLY STEPS BEFORE THERE
IS ADAPTIVE IMMUNITY. A
LIMITATION, FOR SURE.
>> YOU MENTIONED YOU USE NUMBER
OF DIFFERENT DONORS IN THESE
PRIMARY NASAL EPITHELIAL
CULTURES. IF YOU LOOK AT
INDIVIDUAL DONOR, DO YOU SEE
EVIDENCE OF STRONG DONOR
DEPENDENCE? IS THERE ANY WAY
POTENTIALLY TO LINK THAT BACK TO
PATHOGENISIS AND THE
INDIVIDUAL'S HOME CELLS DRIVE.
>> I'M REALLY GLAD YOU ASKED
THAT QUESTION. THE MOST STRIKING
THING WE FOUND -- IN BEGINNING
WE DID INDIVIDUAL DONORS. TURNS
OUT WHEN YOU AVERAGE IT IS THE
SAME. THE MOST STRIKING THING IS
THERE ARE A LOT OF DONORS THAT
SEEM ALMOST RESISTANT TO MERS
INFECTION COMPLETELY. THAT IS
SOMETHING WE ARE REALLY TRYING
TO UNDERSTAND NOW. WE ARE ALSO
LOOKING AT BRONCHIAL CULTURES
FROM SAME PERSON, NASAL AND
BRONCHIAL AND HOW THEY MAY
DIFFER. BUT THE MERS -- IN THE
PATIENTS OR DONORS THAT ARE SO
INSENSITIVE TO MERS SEEM MORE
SUSCEPTIBLE TO SARS. THE OTHER
THING WE DID FIND IS IF WE TREAT
THESE WITH IL13, A SORT OF
ASTHMA MIMIC -- MIMICS ASTHMA,
WE GET A TILTING TOWARD GOBLET
CELLS. I DIDN'T MENTION THIS BUT
MERS INFECTS GOBLET, SARS
AFFECTS CILIA CELLS. SO MAY BE
PEOPLE WITH MORE GOBLET CELLS,
THAT MAY BE A DIFFERENCE -- ALSO
LOOKING AT PEOPLE WITH SORT OF
CHRONIC RHINO SINUSITIS SO THERE
IS DIFFERENCES AMONG THE
INDIVIDUALS.
>> I'M SURE YOU KNOW TOGETHER
TRIALS SHOWED THAT LAMBDA 1 WAS
PRETTY EFFECTIVE OF GIVEN EARLY.
WONDERING YOUR THOUGHTS ON THAT.
WHETHER YOU THINK INTRA NASAL,
LAMBDAL OR OTHER INTERFERENCE
WOULD BE SUPERIOR.
>> I DON'T REALLY -- FROM MY OWN
EXPERIENCE I DON'T KNOW. SOUNDS
LIKE SOMETHING THAT COULD BE
TRIED. I MEAN, IT DOES SEEM TO
WORK.
>> WE HAVE A COUPLE QUESTIONS
ONLINE. HOWARD YOUNG IN
FREDERICK. LOW DOSE ORAL
INTERFERON IS REPORTED TO HAVE
ANTI VIRAL ACTIVITY.
>> WHAT KIND?
>> LOW DOSE ORAL INTERFERON.
>> WE HAVEN'T DONE THAT. I THINK
IT SOUNDS LIKE IT MAKES SENSE.
>> ADAM K, PERHAPS I MISSED BUT
IN NASAL EPITHELIAL YUM BEHAVING
IN UNIQUE WAYS WITH CORONAVIRUS
COMPARED TO OTHERS OR IS IT JUST
DOING ITS REGULAR DUTY?
>> I THINK IT IS DOING ITS
REGULAR DUTY? THE REASON WE USED
THE RHINO WAS TO KIND OF HAVE A
CONTROL THAT WOULD BE AN
ORDINARY COMMON COLD VIRUS,
COMPLETELY DIFFERENT TYPE OF
VIRUS. ONE'S MEMBRANE, ONE IS
NON-MEMBRANE, REALLY DIFFERENT.
TO THAT EXTENT I WOULD THINK IT
IS DOING ITS NORMAL JOB.
>> ANYBODY ELSE? ALL RIGHT.
LET'S THANK SUSAN FOR A
BEAUTIFUL SEMINAR.
[ APPLAUSE ]
>> THERE WILL BE A RECEPTION
OUTSIDE. THANK YOU.
HOPEFULLY IT IS THERE.