Lecture 26 Flashcards
(39 cards)
1
Q
When did we start caring about flaviviruses?
A
It all started with the construction of the Panama cana which was just a a pathway between Caribbean Sea and Pacific Ocean in 1881 by France and this took 25 years to build. Then in 1904 over 85% of canal workers git sick and many hospitalized and this was due to yellow fever and malaria. The diagnosis of yellow fever was clear and symptoms were known but the infectious agent was not known.
2
Q
What are the clinical features of yellow fever?
A
There is a3-6 day incubation period characterized by fever and flu-like symptoms (headache, myalgia, lumbosacral pain, nausea, malaise, prostration, dizziness, conjunctival infection, furred tongue, red at tip, bradycardia). Symptoms get better for 2 to 24 hours (period of remission) and then about 20% of cases move to a period of intoxication (3-8 days) characterized by high fever, hematotonesis (vomiting blood), low urine output and general weakness (headache, epigastric pain, vomiting, prostration, malaise, jaundice, oliguria -> anuria, tender liver hypotension -> shock, stupor -> coma, hypothermia, haemorrhage, convulsions. and then some will recover but 20% of those in intoxication will die.
3
Q
How is yellow fever transmitted?
A
In 1881, right before Panama Canal construction, Carlos Juan Finlay (cuban physician) proposed that yellow fever could be mosquito-borne illness (novel idea at the time)
- Walter Reed (American army surgeon) led a team to prove this theory and this was the “yellow fever commission)
- Human subjects were recruited and given 100$ to be part of study and an extra hundred if you became ill (at the time this was a good deal)
4
Q
What was the outcome of the yellow fever commission?
A
- Clara Maass (nurse) volunteered to be bitten by mosquitos (aides aegypti) that had fed on yellow fever patients (contracted mild disease and quickly recovered)
- Researchers were certain that mosquitoes were the route of transmission, but lacked sufficient evidence to prove to the world
- In an effort to demonstrate that Maass was immunized, she allowed herself to be bitten again and died 10 days later, public sentiment over her death put an end to yellow fever experiments on humans
5
Q
What were the mosquito brigades?
A
- Colonel Gorgas arrived in Panama was the Chief Medical Office (1904) after the insect-borne novel idea
There had been discoveries linking mosquitos with malaria so he decided to act on possibility that insects could be transmitting yellow fever - In 1905 starts the “mosquito brigades” a campaign to fumigate and eradicate the disease by eliminating the mosquito vector
- By august 1906, there were just 7 cases of yellow fever along the Panama Canal versus 10s of 1000s the year prior
- Yellow fever had actually been described 300 years prior but it took them a while to figure out it was an insect and deal with that, partial reason that Washington DC is the American capital because George Washington lived in Philadelphia but then there was an outbreak (killed 10% of the population) and moved to Washington DC
6
Q
How did they attempt to control yellow fever and were they successful?
A
- In 1915, Rockefeller foundation funded a project to eliminate breeding places for aides aegypti in areas where yellow fever was prevalent
However yellow fever remained in some settings because the transmission cycle more complicated than initially though (before only insect to human, no human to human) and there was a third reservoir, monkeys by other mosquitos passing it around and occasionally it would spill back over t humans
7
Q
What is the global distribution of yellow fever virus?
A
These viruses tend to be in tropical and subtropical areas due to rainfall and presence of mosquitoes that transmit (still prevalent today). Reported outbreaks in most of northern South America and central Africa in recent years, and subsaharan Africa is at risk (risk is based on the presence of the transmitting vector mosquito)
8
Q
About the flaviviridae family?
A
- Flavus (latin = yellow due to yellow eyes and skin jaundice) and type virus is YFV
- Enveloped virus
- Icosahedral capsid (40-60 nm in diameter)
- Genome (+)ssRNA, monopartite and linear, 9.6 to 12.3 kb in length or around 10 kb on average
9
Q
Different genera in Flaviviridae family?
A
- Mosquito-borne flavivirus (YFV, zika, dengue, JEV, West Nile virus)
- Tick borne flavivirus
- NKV (no known vector)
- Insect specific flavivirus (just insect to insect transmission)
- Hepacivirus (HCV)
- Pestivirus
10
Q
What is the global distribution of Japanese Encephalitis Serogroup?
A
South of 49th parallel will probably find a mosquito borne borne encephalitis virus
11
Q
About Japanese Encephalitis Virus?
A
- Arbovirus (insect transmission), transmitted by Culex mosquitoes vs YFV aides
- Primary host is birds, pigs / humans are incidental or dead-end hosts because can’t human to human as not enough virus in blood to be picked up by mosquito again to go infect other humans so really from birds, pig to mosquitos to us
- 30 to 50k cases each year (25-30% mortality so about 10k deaths a year)
- Symptoms: fever, headache, vomiting, neurologic symptoms (mental status, movement disorders, etc.) common to many of the insect-borne viruses
- No treatment (supportive care) like for all flaviviruses
- Vaccine (formalin inactivated or live attenuated) recommended for travel to south east Asia specifically over 1 month before travel (only flavivirus with a vaccine)
12
Q
Increasing distribution of JEV?
A
- 1924 => Japan
- 1954 => Japan and some of china
- 1990 => pretty much all of south east Asia, India and Indonesia
- many flaviviruses start localized and then increase distribution
13
Q
Clinical feature of JEV?
A
- Majority (80%) are asymptomatic or cause nonspecific febrile illness
- JEV primarily affects children or young adults (0-15 years)
- More severe cases (20% of symptomatic cases):
- meningoencephalitis, aseptic meningitis in blood, polio like acute flaccid paralysis, parkinsonian syndromes (tremor, cogwheel rigidity, ask like facies)
14
Q
Age difference JEV vs WNV?
A
- WNV less virulent than JEV
- JEV lots of young humans infected and then goes down but seroprevalence is high where endemic since most people affected young
- WNV particularly affects elderly and immunocompromised so only later on in life and seroprevalence is very low
14
Q
Age difference JEV vs WNV?
A
- WNV less virulent than JEV
- JEV lots of young humans infected and then goes down but seroprevalence is high where endemic since most people affected young
- WNV particularly affects elderly and immunocompromised so only later on in life and seroprevalence is very low
15
Q
About WNV?
A
- Arbovirus, transmitted by Culex as well, primary host is Birds and humans are incidental dead end hosts as well as other mammals
- Only about 20-6k cases each year, less prevalent than JEV (3-15% mortality) and primarily elderly, immunocompromised
- Symptoms: fever, headache, vomiting, muscle weakness, lower back pain, neurologic symptoms (loss of motor function, encephalitis, meningitis)
- 80% are asymptomatic, 20% of symptomatic cases develop neuroinvasive disease
- No treatment (supportive care); no vaccine
16
Q
Spread of WNV in the Americas?
A
- 1st found in Eastern Africa then 90s spread to New England and over following decade been found in every state, province in North America
- one flavivirus we could be worried about cuz mosquitos that transmit are found here
17
Q
About dengue virus?
A
- arbovirus, transmitted by aides mosquitos (aides aegypti same as yellow fever and aides albopictus)
- primary hosts humans
- Symptoms: fever, headache, vomiting, abdominal pain, low blood pressure, tachycardia, seizures and encephalitis
- No treatment (only supportive care), no vaccine
18
Q
Clinical features of DENV?
A
viremia 4 days after mosquito bite, fever starts a day after detectable in blood then shock hemorrhage at day 4 of illness
- vaccine would be useful because about 1 million cases a year and about 2.4 billion at risk of infection about 1/3 of population
- 50k to 100k cases of dengue hemorrhagic fever and dengue septic shock (about 10% of all cases) and this lead to 50% lethality
- No age distinction, much more virulent than WNV and JEV (encephalitis viruses)
19
Q
Spread of dengue?
A
- Near the Florida and a bit northern South America, but then removed fumigation measures and BAM spread also climate supports aides ageypty expansion in practically all of central and South America
- Correlates with incidence of dengue hemorrhagic fever
20
Q
Geographic distribution of DENV subtypes?
A
4 Dif subtypes all Dif serotypes, one of the reasons why it’s difficult to make a vaccine
- Before one den1 and den2 in americas and Africa, all 4 only in south east Asia but now all 4 types everywhere
- novel outbreaks on islands, climate change could increase range, right now particularly at risk are south US and South Europe (dengue would go to naive populations)
21
Q
What about the emergence of ZIKV?
A
- We’ve known about the virus for a long time them rapid concern about it, it spread so fast because the mosquitos were all already present but the populations were completely naive, increase in transmissibility and spread like wildfire but kind of to its own detriment because rapid immunity of the herd
- 1947: first detected in monkeys in Uganda, rare infection of humans => mild illness, even asymptomatic so nobody really cared and very little monitoring or basic research
- 1960: first human cases in nigeria
- 1970s: cases in Pakistan, India, Malaysia and Indonesia
- 2007: epidemic n island of Yap, Micronesia, 1st time really transmissible, nearly 50% of the population got it
- 2013: epidemic on French Polynesia here very transmissible and very virulent, quite scary due to the fatal neurological microcephaly and guillon Barré syndrome
- 2014-2016: zika appears in northern Brazil and spreads through the americas and that’s when it became a pathogen of global concern
22
Q
About ZIKV?
A
- Arbovirus, aedes aegypti (like dengue and YFV)
- Symptoms (20%): fever, maculopapular rash (bumps on bumps), headaches, joint and muscle pain, edema (fluid filling into Dif cavities in the body), vomiting
- Recent outbreaks (2013-present): guillon Barré syndrome (autoimmune disorder in CNS) and fetal microcephaly
- No treatment (supportive care)
- No vaccine
23
Q
What is needed to tackle an emerging virus?
A
- Model systems to study: reverse genetics models, in vitro and ex vivo models, in vivo pathogenesis and immunocompetent models
- Diagnosis to limit spread: rapid, point of care diagnostics, ZIKV specific immunoassays
- Vector control to limit spread: transmission modelling, entomology surveillance, integrated vector control strategies
- Therapeutic strategies to help: ZIKV specific antivirals, therapeutic antibodies, vaccine development
24
What is the genome organization of flaviviruses?
- Highly structured 5' and 3' UTRs that perform a number of functions and flank poly protein coding sequence, about 10 kb on average
- cap-dependent translation, Structural proteins (C capsid, prM/M membrane protein also antagonizes immune respones, E envelope glycoprotein) and non structural proteins necessary to replicate viral genome and antagonize innate antiviral response (NS1, NS2A and 2B, NS4A and B RNA replicase proteins, NS3 protease (cleavage of poly protein along cytoplasmic membrane) /helicase (unwind dsRNA intermediate from genome replication), NS5 RdRp and methyltransferase for capping of the genome)
25
Flavivirus structure?
In nature gradient between immature and mature virion
Immature has prM before pre membrane protein cleaved so prM takes on an arrow head type topology so virus is spiky
Mature has M which flattens out post cleavage and gives a smoother viral particle
26
How does the virus enter the cell?
E (envelope) protein mediates attachment to receptor (endocytosis)
receptors are unknown all that is known is that probably Dif receptors and co receptors for Dif affected cell types, don't localize to one cell type, immune cells, epithelial cells, and neural cells, however best receptor candidates are highly sulphated form of heparin sulphate protein and dC Sine lectin protein
- In late endosome, low pH -> E protein conformational change so it sticks to the endosomal membrane and results in fusion and the release into the cytoplasm (class II fusion protein)
27
How do flaviviruses maximize coding capacity?
Polyprotein
28
About poly protein processing?
The cap recruits ribosome and translation machinery, ribosome scans for start codon and then translates whole poly protein which is pretty much entirely intermembrane proteins which helps keep them all localized together, on the side of the ER lumen the poly protein is processed by the signalase protease and on the cytoplasm side, cleavage by NS3 protease and 2B is its Cofactor
29
In what two forms does the genome exist?
- Enters linear and competent for translation, when enough proteins are made, replication happens and the genome circularizes which facilitates replication and forming the replication organelle where the genome must enter to replicate
- It facilitates because form long range RNA interactions from 3' and 5' UTR and the termini are brought together forming long range duplex, disrupting some of those highly structured UTRs and facilitating replication
30
How does replication occur?
There is a stem loop A near the 5' UTR which recruits NS5 (RdRp) which thanks to genome circularization can transfer to the 3' end although how this happens is unknown
The entire negative strand intermediate is then transcribed leaving dsRNA behind so NS3 (helicase) needed to unwind allowing NS5 to make more new copies of (+) ssRNA genome
Multiple polymerases can go to this (-) strand and as it moves along, it displaces other (+) ssRNA which can then be assembled or translated, this results in asymmetric replication where 1 (-) strand can make up to 50 (+) ssRNA
It's known how NS5 gets to +strand but unknown how it get recruited to the (-) intermediate to make more genome
31
What is the difference between (-) strand and (+) strand?
1 (+) ssRNA makes 1 ambisense dsRNA but 1 ambisense dsRNA makes many (+) ssRNA (about 50:1 ratio), so differences in the process of synthesis even though it's the same polymerase and helicase
32
What are sfRNAs?
Subgenomic flaviviral RNAs
There are 4 stem loop structures in the 3' UTR that form knots by winding around the RNA and there is a hist exonuclease that degrades uncapped RNA since the RNA becomes uncapped during replication and XRN1 can do its thing (not the best way to clear viral infection but It is one of the ways
But because of the knots XRN1 stalls and causes different lengths of 3' UTR which can then inhibit antiviral responses in both hosts (type I IFN in mammals and RNAi pathway in mosquitos which is the primary antiviral pathway in mosquitos)
Also modulates cytopathic effects and cell death in mammals like in mice, eliminating sfRNAs was enough to remove mortality of ZIKV
33
How do flaviviruses adapt to two hosts?
- conserved deletions in DENV 3'UTR upon mosquito-adaptation from multiple passes in mosquitoes, disrupts one of the XRN1-resistant structures, leads to different primary sfRNA species (1 more abundant in humans)
- increased fitness in mosquitos, reduced in mammals so one of the proposed ways for naturally live-attenuated in humans
- Reverse of deletions when re-adapted to humans so eventually end up with full-length sequence (even though initial deletion)
34
How can the deletion be reversed?
- Infection involves a diverse population of viruses, flavivirus RdRp's introduce 1 mutation per new strand, highly error prone, all virions are slightly different even in just one infection and there is no repair mechanism, mutations sometimes don't affect, sometimes bad cuz affects some steps of the viral life cycle => selected agains, sometimes beneficial => participates in emergence
- each new host/tissue/cell type will create different selective pressures like for example virus must first escape gut to be disseminated so some may be selected for, then must enter salivary glands to infect humans and at each step different section, find cocktail quite different (mix) => then when gets reinfected => reselection of human adapted virus
35
Flaviviruses induce some odd membranes which ones?
- Rough ER derived vesicle packets (VPs) that have room for a sing dsRNA intermediate and have pores which are the presumed exit site for newly synthesized viral RNAs for translation or assembly)
ALos convoluted membranes (CMs) not sure why or how impacts replication
- They are sites of replication, dsINTERMEDIATE and nsps localize there
36
About Flavivirus assembly sites?
- genome extruded through pore then immediate capsid and structural protein interaction on opposing membranes => starts to bud into ER membrane
- budding viruses can be observed next to pores of VPs, visions stack within the lumen of the VP-containing ER network
- Virions egress through the Golgi
37
How does the virions assembly, maturation and release happen?
The immature virion is necessary so it doesn't reinfect the same cell, if mature would just leave the ER and rerelease the genome and reinfect the cell, the change in pH and cleavage of prM by furring help the conformational change and mature virion can now fuse with the membrane and will be secreted through the general secretory pathway
38
Why is it hard to make a dengue vaccine?
- Dif subtypes BUT ALSO
- DENV antibody dependent enhancement which is yet another way a flavivirus may use host immune response for down benefit
- Increased severity of secondary infection
- Abs from primary infection where they worked super well and neutralized and stuff, now recognize different serotypes weakly and facilitate DENV binding and internalization => free entry into cells since macrophages recognize fc region of Ab without neutralizing
- Leukocytes and BBB cells lead to encephalitis
- Inflammation and cytokine secretions (cytokine storm)
- Compromises CNS function
- Even evidence that zika and dengue so similar that the infection of one may make the infection by the other worse through the same mechanism so antivirals may be the best option for pan flavi scale and no ADE, if we want a vaccine it must really be pan-flavivirus
