Exam 1 Flashcards
1
Q
virus definition
A
obligate intracellular parasites
all viruses have a NUCLEIC ACID GENOME (DNA or RNA, ss or ds) and a CAPSID
(protein coat encoded by virus)
about 1/2 have envelope
2
Q
what key features do viruses lack?
A
- enzymes that produce basic chemical building blocks
- enzyme systems that generate usable energy
- enyzmes / tRNAs / ribosomes that direct protein synthesis
- membranes that concentrate and localize key molecules
3
Q
virion
A
a single, complete, infectious virus particle
4
Q
nucleocapsid definition
A
genome (DNA or RNA) + capsid protein
5
Q
naked capsid virus
A
nucleocapsid only, no membrane
6
Q
enveloped virus
A
nucleocapsid + lipid membrane and glycoproteins
7
Q
sensitivity of naked vs enveloped viruses
A
Enveloped are more sensitive to most insults (ie drying, heat, detergents, acid), can be disinfected with ethanol
Naked capsids needs bleach
8
Q
pros and cons of naked capsids
A
Pros: 1. Retain infectivity on drying, 2. Survive well on environmental surfaces, 3. Spread easily via fomites, 4. Can survive the acidic environment of the gastrointestinal tract
Cons: 1. Must kill host cells for release of mature virus particles (can cause self limiting infection) 2. Humoral (Ab) imm resp may be sufficient to neutralize infection
9
Q
fomite
A
inanimate object that can cause spread of pathogens (door handles, shared cup, elevator buttons, utensils, etc)
10
Q
enveloped virus pros and cons
A
Pros: 1. Do not need to kill cells in order to spread 2. May require both humoral (Ab) and cellular (T cell) imm responses to kill off the virus ‘factories’
Cons: 1. Can not survive in the gastrointestinal tract, 2. Must stay wet during transmission, 3. Transmission in large droplets and secretions
11
Q
general capsid properties
A
symmetrical
simple (1-3 proteins)
icosahedral or helical
less proteins = less genes = smaller genome size = capsid can be smaller to fit genome
Self assembly, no E needed to form capsid
12
Q
simplest icosahedral capsids composition
A
3 protein complex to form 1 triangle, then 20 triangles self assemble in into icosahedral (60 identical proteins)
remember that enveloped viruses have BOTH capsids and membranes
13
Q
5:3:2 rule
A
5 is vertex, 3 is center of triangle, 2 is the fold between base of 2 triangles
14
Q
helical capsid composition
A
Made of single proteins
Symmetrical helix around single axis
OPEN no closed like icosahedron
remember that enveloped viruses have BOTH capsids and membranes
15
Q
bacteriophage capsids
A
Hybrid – icosahedral head and helical tails
Tail attaches to bacterium
16
Q
purpose of virus genome
A
caries virus genes
genes code for all viral proteins
17
Q
required viral protein functions
A
virus genome replication
formation of capsid
virion assembly
18
Q
optional viral protein functions
A
evasion of intracellular defense systems
evasion of extracellular imm resp
19
Q
DNA vs RNA genome transcription / translation
A
DNA > mRNA > protein
vs
RNA > protein
20
Q
what does the Baltimore classification system use to group viruses
A
genome structure, plus reverse transcriptase
21
Q
Baltimore classification in order I to VII
A
dsDNA
ssDNA
dsRNA
pos sense RNA
neg sense RNA
retroviruses (RT)
ss/dsDNA (RT)
22
Q
general stages of virus lifecycle
A
- binding, 2. entry and uncoating, 3. early gene expression, 4. replication of genome, 5. late gene expression, 6. assembly of virions, 7. exit
23
Q
key factors of binding
A
Specific protein on virus particle (capsid or membrane)
Specific protein on cell surface
“lock and key” = virus receptor
virus can also bind things other than its receptor (ie co-receptors or proteins on surface that allow them to aggregate)
24
Q
dsDNA genome replication
A
regular dsDNA replication
use host machinery
25
ssDNA genome replication
ssDNA > dsDNA > ssDNA gets packaged
Use host cell DNA Pol to make dsDNA
Virions can have sense OR antisense strand bc first step is making other strand of the virus
26
pos sense RNA genome replication
pos RNA > neg RNA > pos RNA
facilitated by Rdrp
pos strand can immediately make proteins (act as mRNA) to make Rdrp (early gene)
Rdrp makes neg RNA, which acts as template for pos
27
neg sense RNA genome replication
neg RNA > EITHER mRNA or pos RNA
mRNA > proteins
pos RNA > template for neg RNA genome
RDRP IS PACKAGED INSIDE OF CAPSID, RELEASED ON ENTRY TO HOST CELL
28
dsRNA virus genome replication
dsRNA > pos mRNA > dsRNA
uses RDRP
dsRNA packaged into virion
29
Retrovirus genome replication
pos RNA > ssDNA (in capsid) > dsDNA (in capsid) > integration > EITHER mRNA or pos RNA
mRNA makes proteins
posRNA gets packaged
RETROVIRUSES ARE POS STRAND RNA
RT enters with pos RNA genome, RT activated, makes ssDNA WITHIN CAPSID
ssDNA in capsid, RT also makes antisense DNA IN CAPSID
INTEGRATION into host genome, then follows normal dsDNA genome after
30
genome strucutre of retroviruses
pos sense RNA
31
naked vs enveloped virus exit
naked = rupture and kill host cell
enveloped = budding, usually do not directly kill host cell
32
methods for quantifying virus
biological assays: 1. plaque assay, 2. endpoint calculations, 3. focus formation assay (transformation)
physical assays: 1. hemagglutination assay, 2. direct particle count, 3. nucleic acid (genome) testing
33
virus growth in embryonated egges
eggs 11-12 days post fertilization
Inject into chorioallantoic membrane (amniotic fluid), seal, incubate, harvest fluid
still used to make flu vaccines
34
list the CPEs (cytopathic effects)
cell lysis
rounding (or other morphology changes)
syncytia
inclusion bodies
35
ways to measure virus growth
CPE
hemadsorption
transformation
36
what is a syncytia
a single cell or cytoplasmic mass containing several nuclei, formed by fusion of cells or by division of nuclei
37
what is an inclusion body
dense areas in cell where virus components accumulated; aggregate of virus particles or virus-induced proteins characteristic of virus infection; Crystalline array of viral components
are often sites of assembly
38
hemadsorption
Virus makes proteins that bind carbs on surface of RBCs, when cell is infected virus makes it express that protein
Virus added to cells > cell expresses protein > add RBCs > wash > if RBCs stuck, those cells are infected
used for viruses with no obvious CPE
Usually enveloped viruses
39
transformation
property of some oncogenic viruses
exhibit one or more of the following: 1. Immortalization, 2. Colony formation, 3. Tumor formation in immunocompromised mice, 4. Loss of anchorage dependence, 5. Loss of contact inhibition, 6. loss of polarization
40
what is a transformation focus
area where cells have been transformed and display oncogenic properties; colony formation, loss of contact inhibition, etc.
41
plaque assay
"gold standard"
Virus added to monolayer > overlay with agar on top (only infect nearby cells) > after replication it spreads to neighboring cells, count the number of plaques
42
plaque assay method
Serial 10x dillutions, Allow to infect for hr or so, Add agar, At termination point count plaques.
Concentrated virus kills all cells, dilute virus kills few cells
Number of plaques * dilution = PFU
43
plaque assay titer
number of plaques * dilution factor = PFU
44
endpoint titration TCID50 assay
Can be used to quantify virus by measuring CPE, transformation, or viral fluorescence (eg, GPF) per well. ea well gets yes/no with many replicates per dilution
45
focus forming assay (transformation)
foci formed from loss of contact inhibition
count number of foci relative to titer
46
hemagglutination assay
Viruses that bind to sialic acid on surface of RBCs
Bc virus has multiple binding sites, can bind more than one RBC ea, get ‘bridges’
Round bottom well --> RBCs form dot in middle
In presence of virus NO DOT bc RBCs are stuck to virus and not all in one spot, form pink halo
47
hemagglutination assay method
2 fold dilutions of virus with equivalent number of RBCs per well
Highest dilution that hemagglutinates = 1 HA unit
ie if the 8, 16, and 32x dilution has NO DOT, but the 64 has a dot, the titration is 32 HA units
48
direct particle count
Count particles using EM
Add exact conc of beads, count beads and virus particles, calc number particles per ml
49
nucleic acid testing
qPCR for virus genes
can quantify using standard dilutions
50
what is MOI
MOI (multiplicity of infection) = the number of INFECTIOUS PARTICLES per CELL
they must be infectious!!
determined by plaque assay
51
notes on different quantitative assays
remember what the assay is measuring;
there are more genomes than particles, and there are more particles than virions
52
direct vs indirect virus diagnosis
direct:
1. virus particles, 2. virus proteins (Ags), 3. virus nucleic acids (genomes)
indirect:
serology (Abs)
53
high confidence vs easy access diagnostic methods
high confidence: virus isolation, genome detection, Ag detection
easy access: serology
54
methods for direct detection of virus
EM, IF, ELISA, PCR
55
EM
visualizations of virus particles
used to detect viral particles in lesions or type is unknown
don't need spec Abs
56
IF
immunofluorescence visualization of virus proteins (particles or Ags)
Uses virus-specific antibodies to detect a specific viral protein, usually in TISSUE SECTION OR CELLS
1' Ab binds Ag, 2' Ab (conjugated to fluorophore) binds 1' Ab,
57
ELISA
Ab detection of virus proteins (particles or Ags)
virus-specific antibodies to detect virus particles or secreted viral proteins in FLUID
capture ELISA = sandwich, indirect Ag to 1' Ab to 2' Ab
58
rapid ELISA: lateral flow assay
covid tests, pregnancy tests
analyte added to one end, moves through and binds labeled Ab, moves to 1' Ab (test line) and then 2' Ab (control line)
sandwich on test line if Ag present
control line recognizes the labeled Ab and will always show color
59
PCR
molecular detection of virus na (genomes or transcripts)
realtime qPCR (DNA) or RT-qPCR (RNA) can be used to quantify virus genomes
60
Serology
indirect detection
Ab to virus proteins are generated by BCs in response to infection, Serology is detection of Ab in serum
if Ab exists for virus, the imm sys has in contact w that virus
most commonly use ELISA
61
RNA vs protein vs serology
Early during infection cannot detect Abs (takes about 7 days for full Ab response)
Pos serology test does NOT mean ongoing infection
Serology shows they have been exposed, not necessarily actively sick (tho they can be depending on how long the virus causes disease for)
62
Serology ELISA
Instead of capturing virus, you are capturing the Ab THE HOST PRODUCED
sandwich with viral protein on well, then serum, then 2' Ab
1' Ab is in the serum if the patient has come into contact w virus
63
Serology neutralization assay and hemagglutination inhibition assay
add test serum to virus, see if it can prevent CPE (virus infecting monolayer) or hemagglutination
64
syndromic testing
multiple pathogens within a single panel, based symptoms
65
pathogenesis
the process by which one organism causes disease in another
66
2 components of viral disease
1. Effects of virus replication on the host
2. Effects of host response on virus and the host
67
outcome of most viral infections
subclinical, resolve on their own w/o symptoms
no disease DOES NOT mean unsuccessful replication
there is no inherent value to the virus to make a person sick
68
tamiflu
neuraminidase inhibitor
After virus buds, virus is stuck to host cell, needs to cleave interaction between sialic acid and the viral receptor to be released
Does so through neurominidase
Block neurominidase > block viral budding > virus cannot propagate
69
why study pathogenesis
identify targets for
1. antiviral drugs
2. immunomodulation
3. vaccines
70
most common point of viral entry
mucosal surfaces: GI, respiratory tract, urogenital
71
key features of the skin
outer layer is dead keratinized cells > no virus replication
entry via breaks or punctures
Epidermis has no blood/lymphatics – local infection
Dermis and sub-dermal tissues are highly vascularized – infections may disseminate
72
common skin viral infections
Herpes, pox, papilloma, arbo
(arbo requires an insect vector)
73
key features of respiratory tract
most common route of viral entry
high absorptive area and high turnover
barriers: mech - mucous, cilia, sneezing/coughing
cellular: MO
humoral: IgA
Entry via aerosolized droplets or contact w saliva
74
common respiratory viral infections
upper: rhino, corona, influenza, parainfluenza, RSV, herpes, adeno, boca, coxsacki
lower: influenza, parainfluenza, RSV, adeno, boca, metapneumo
75
key features of GI tract
entry via eating/drinking
contents always in motion - allow for more virus-host interactions
barriers: environmental - acidic stomach, alkaline intestine, digestive enzymes, mucus
homoral - IgA
76
common GI tract viral infections (and how they survive the GI)
Reoviruses – require proteases for entry; use microbiota to enhance infection
Noroviruses – stable at large pH range; may use IgA to facilitate uptake by intestinal epithelial cells; use microbiota to enhance infection
Coronaviruses – enveloped; resistance mechanism(s) not clear
77
M cells in the GI
Host needs to sample material in gut lumen
M cells - Able to transfer gut lumen materials into the imm cells in peyers patch, important for imm resp, virus can use it tho :(
M cells do NOT have microvilli
78
key features of urogenital tract
Barriers: mech - mucus, environment - low pH
Entry via minute abrasions from sex
Some viruses produce local lesions (HPV)
Some viruses spread from urogenital tract (HIV, HSV)
79
common urogential tract viral infections
HPV, HIV, HSV
80
key features of eyes
barrier: blinking
entry: minute abrasions, environment (swimming pools)
localized infection - conjunctivitis
disseminated infection - spread to CNS (HSV-1)
81
common eye viral infections
conjunctivitis (just means eye inflamm, caused by many viruses)
HSV-1 (can spread to CNS)
82
possible outcomes of infection
cell death
abortive infection
persistant infection
transformation
83
abortive infections
virus can enter but cannot produce infectious particles
lack of right conditions or host antiviral activity
cells may still be damaged, killed, or transformed, but no new virions
84
cell death after viral infection
most common outcome
apoptosis and necrosis collectively called CPE
diversion of cell E
shutoff host synthesis
competition of mRNAs for ribosomes
competition for cellular transcription factors
cell lysis
85
pyrogens
trigger fever
IL1 IL6 TNF
86
what causes pyrogen release
cell lysis
pyrogens induce fever, trigger stronger imm resp, impairs normal cell funct
disease is coming from imm resp to virus, not directly from virus
87
persistent infection
no cell death and cells are not altered significantly in their growth
virus enters but no cell death or new virion formation
3 types: chronic, latent, and recurrent
88
chronic persistent infections
constant production of virus
long incubation period before disease
HBV (hep B), HCV (hep C)
89
latent persistent infections
no progeny virus but virus genome is maintained
small number of virus proteins produced (genome maintenance)
may have constant low-level reactivation that is pathogenically silent
herpesviruses
90
recurrent persistent infections
reactivation of latent virus infection
e.g., stress can reactivate latent HSV to produce virus (cold sore)
91
mechanisms for transformation
encode viral oncogene derived from cellular gene (papilloma blocks tumor suppressor)
integration into host chromosome causes disruption in normal genes (retroviruses)
chronic infections causing constant injury/repair cycle (HCV (hep C) liver damage) - virus is not directly causing cancer
92
tropism
tissues infected by a particular virus
limited vs pantropic (many tissues susceptible)
93
determinants of tropism
1. Accessibility of the permissive cell, 2. Presence of appropriate cell surface receptors, 3. Presence of intracellular host factors required for virus rep, 4. Absence of suppressive antiviral mediators (most important is interferon)
94
disseminated infection
virus spreads beyond 1' site of infection, usually to a 2' target tissue
polio enters through GI, disseminates through lymph to blood to CNS
95
systemic infection
many organs infected
flavi enter bloodstream via insect bite, spread to many tissues
96
localized infection
1' infection site only
rhino contained to upper resp tract
97
how can virus disseminate
breaching of physical and imm barriers
facilitated by virus (direct destruction of cells) or imm resp (inflammation causing leaky barriers)
98
directionality of virus release
apical vs basolateral
apical - facilitates dispersal to neighboring cells
basolateral - deeper tissues, blood stream, rest of body
99
hematogenous spread
Most effective, rapid, and common dissemination
Viruses can enter blood directly through capillaries, by replicating in endothelial cells, or through vector bite
Virus in extracellular fluid can be taken up by lymphatic capillaries (more permeable than circulatory capillaries), then spread to blood
Once in blood, virus has access to almost all tissues
Can occur cell-associated or as free virus particles
still dependent on tissue tropism
can atach to migratory cells (DCs, MOs, lymphocytes) or RBCs/platelets (w/o replicating)
100
1' vs 2' viremia
viremia - virus in bloodstream
1’ – virus rep at 1’ infection, then enters blood
2’ – reaches 2’ tissue via blood stream (from 1’), enters bloodstream AGAIN, much higher amount of virus than 1’
101
passive vs active viremia
Passive: virus introduced to blood w/o replication; rep AFTER it reaches target organ
(ie direct inoculation of arbovirus to blood from bite)
Active: virus replication occurs BEFORE viremic phase, then enters bloodstream (ie virus replicates in mucosal epithelium, then virus enters bloodstream)
102
neural spread
TRAVEL RETROGRADE
Axon > cell body > synpase with PREVIOUS neuron > axon etc
definitive characteristic of rabies and HSV
infrequent diversion in polio and reovirus
can also happen hematogenously
103
hematogenous neural spread
rare bc of BBB
can be thorugh transcytosis (virus travels through cell one side to other) or cell-associated (infected imm cell that enter CNS) - normally prevented by tight juncts (can be leaky from inflamm)
104
immunopathology (imm resp to viral infection)
greatest impact on outcome of infection
can be the major contributor of pathogology
typical there is clearance w/o symptoms
no clearance= infection and possible persistence
105
type 1 IFN
induces antiviral state
Cell infected > recog foreign molecules > upreg gene expression of IFN1 beta > IFN1b release binds to recptr on same or neighboring cells > upreg ISGs (IFN stimulated genes) > cell blocks virus
ISGs are antiviral, block viral rep, 100s of genes
106
virus evasion strategies
1. suppress IFN resp (via blocking IFN induction, signaling, or IFN-induced proteins), 2. block other cytokines, 3.suppress innate imm cell resp, 4. replicate in imm privileged sites, 5. interfere w Ag processing/presentation, 6. alter MHC trafficking, 7. direct cell-to-cell spread, 8. Ag variants (diff strains)
107
types of immunopathology
flu-like symptoms, over stimulation of innate imm resp, ADE, generation of immune complexes
108
vasoconstriction during fever
reduces heat loss through skin – person feels cold
109
why get fever when sick
certain imm cells work better at higher temps
110
what causes over stimulation of innate imm resp
high levels of pro inflamm cytokines
can cause immune suppression and cytokine storm
ex: ebola and dengue hemorrhagic fever
111
ADE
Ab dependent enchancement
happens in 2nd dengue infection with a different strain
1st infection, dengue infects MO, gets cleared
2nd infection, Ab from memory can bind the dengue but does not neutralize, MO takes up Ab through Fc receptor, transfers dengue directly into MO
112
generation of imm complexes
chronic infections result in constant Ab production and formation of imm complexes, deposited in kidney activation of complement and inflamm in kidney, damage renal filtration,
chronic ineffect BC stimulation can lead to BC cancers
113
types of virus transmission
horizontal (person to person via touch, saliva, sex etc)
vertical (partent to offspring via bodily fluid during birth or breast milk)
zoonotic (animal to human via bite, meat, contact etc)
114
congenital infection
infection caused during pregnancy that continues after child is born
115
wt lab vs natural strains
natural - mix of genomes
lab - consensus seq of natural, dubbed wt, "quasi-species"
116
selection vs screen in a mixed population
selection - conditions exist in which only the desired virus grows (ideal)
screen - both desired and unwanted virus grows (more common)
117
essential vs nonessential genes
describes phenotypes and depends on ASSAY CONDITIONS
mutations give advantage or disadvantages
118
attenuation via passaging
passaging a virus many times in specific conditions allows for evolution and selection for viruses more adapted for those conditions
if cultured away from normal target tissues, virus bc attenuated and cannot infect normal target
(basis for Sabin polio vaccine)
119
recombination
nucleic acid seq from two genotypically diff parental viruses are exchanged, progeny contains seq from both parents
in DNA viruses - homologous recombination
in nonsegmented RNA viruses - copy-choice recombination
120
copy choice recombination
nonsegmented RNA viruses
uses Rdrp
generates intermediate neg sense, jumps to neighboring genome, continues until it gets to end
pos RNA can be made from this strand to propagate the mutation
121
reassortment
segmented RNA genome
2 strains infect same cell, reassort genome segments
RARE, most segments will stay together
ex: influenza virus
122
types of mutations
spontaneous (classical)
induced (classical)
engineered (modern virology)
123
spontaneous mutations
spontaneous mutations from replication
much higher in RNA than DNA viruses bc of poor fidelity, faster replication, and no proofreading
DNA is more stable, RNA is more adaptible
124
laboratory consequence of spontaneous mutations
RNA viruses have higher rates of spontaneous mutations, more likely to have reversion mutations
125
induced mutations
inductions inc mutation frequency
goal of single nt change
chemical mutagens
physical means (UV crosslinking)
126
engineered mutations
consider genome size, what nucleic acids are infections (DNA, pos or neg RNA)
used most often in lab now
clone into plasmid, reintroduce in virus
127
forward vs reverse genetics
forward - phenotype to genotype (see an diff phenotype, analyze genes to see where change is)
reverse - genotype to phenotype (engineer specific mutation, see how it affects phenotype)
128
picornavirus general overview
small nonenveloped virus
pos sense RNA
4 capsid proteins
lacks 5' cap (uses IRES instead)
5' structural / 3' non structural
129
important picorna viruses
polio, hep A (HAV), foot-and-mouth disease virus (FMDV), rhino
130
picorna genome organization
pos ssRNA
single ORF --> makes polyprotein
polyprotein gets cleaved into diff segments (mostly by viral protease (3C) but also some host)
structural at 5', nonstructural at 3'
131
picorna 3C protein
protease, cleaves polyprotein
132
picorna 3CD protein
protease, cleaves polyprotein
133
picorna 2A protein
protease, cleaves elF4G
134
picorna P1 protein
structural, part of virion, cleaved into VP0, 1, and 3
135
picorna P2 and P3 proteins
nonstructural, enzymatic, genome regulation
136
picorna 3D
polymerase (RdRp),
efficient,
asymmetrically makes both neg and pos RNA (much more pos made than neg)
error prone (target for anti-viral drugs)
handle structure, palm = active site, RNA runs through tunnel
VPg uridylyation
137
picorna IRES
internal ribosomal entry site
long 5' untranslated region, initiates translation
138
picorna VPg
aka 3B
present instead of 5' cap, before IRES, protects RNA
primer for RNA replication
139
picorna capsid structure
60 subunits, 20 protomers
1 promoter = VP1 + VP2 + VP3
VP1-3 have no seq homology but same topology (wedge)
VP4 is internal (packaging), protect N terminus of VP3, seals and prevents pore
pH stability dependent on replication tropism (enteric resistant to low pH, respiratory are acid-labile)
140
picorna receptors
many receptors depending on virus, (Ig-like domains, adhesion, integrins, etc)
some share (CD55 aka DAF)
some use single receptor and some use co-receptors
141
picorna receptor binding site on virion
in canyon, not peak for polio and rhino (binds ICAM-1)
some picorna don't have canyons, bind on surface loops
142
picorna genome release from capsid
2 mechanisms:
1. pH dependent - receptor-mediated endocytosis followed by uncoating triggered by acidification in endosomes.
evidence: blocked by blocking acidification, Ab coated virus can bind Fc receptors and infect cells
2. receptor-induced cellular mem pore formation
evidence: capsids stable even at v low pH, blocking acidification does not inhibit, Ab coated virus cannot infect
143
picorna pore formation in HOST CELL
receptor binds in canyon > pocket factor dissociates > tighter binding of virus > VP1 inserts N terminus and VP4 inserts myristal group > unknown factor removes VP3 plug > release genome into cell
144
picorna hydrophobic pocket and WIN
Tunnel under canyon has pocket factor
need to release pocket factor
Block pocket factor from dissociating > virus cannot enter
WIN compounds blocks pocket factor, does not dissociate, virus cannot enter
145
picorna translation initiation
pos sense (translation read)
works via IRES
2' structure hairpin structure in 5' region
recruits host translation initiation factors > bring ribosomes
do not need all host translation factors (does not need elF4E, only needs C terminus of elF4G)
146
picorna translation: elF4E and 4G
elF4G is a scaffold for other elF's, one of which is elF4E
4E binds 5' cap of host RNA
picorna has no 5' cap so 4E is unnecessary (and therefore 5' end of 4G which is what recruits 4E)
virus has mech to cleave 4G to remove N terminus (5'), leaving C terminus (3') > host can only make virus proteins
147
picorna polyprotein cleavage
cleaved as it is translated, never detect full molecule
cleaved by 3C, 3CD, and 2A
3 main regions: P1, 2, 3
P1 contains VP0 (later 2 and 4), VP1, and VP3
148
advantages of polyproteins
smaller genome, many functions from a single RNA
timing control
quantity control
149
picorna RNA replication via VPg
VPg (aka 3B) is attached to 3A, 3AB inserts into membranes, at 5' end of RNA > 2C binds RNA genome (circular) > hydroxy group attaches to nt > uridylylated by 3D at CRE hairpin > Us templates to polyA tail > polymerizaton by 3D > 3C cleaves 3B and 3A, release primer and nascent RNA
additional 3B (VPg) gets uridylylated, pool of more primers
polymerase (3D) unwinds dsRNA and 2' structures
happens on membranes to sequester away (dsRNA = danger signal)
primes both pos and neg RNA synthesis
150
picorna assembly
P1 cleaved into VP0 (VP2+VP4), VP3, and VP1 > One copy ea of VP0, VP3, and VP1 > 5 protomers (protomer = VP1+3+0) assemble (5 fold vertex)
2 models: capsid first or simultaneously?
capsid first = capsid is formed, RNA is threaded into capsid via pore
simultaneous = pentamers form around genome (more likely)
unknown how VP0 gets cleaved, likely autocatalytic
151
where does picorna enter
respiratory (rhino, localized) or GI (enteric, systemic)
enteroviruses - enteric, intestinal track, drain to lymph node, travel to blood, viremia, can enter CNS
152
what type of virus is polio
picorna
153
polio pathogenesis
fecal-oral transmission
Viral rep in intestine epithelium, then lymph node, then viriemia
Then extraneural tissue (muscle and fat) - reaches high titer
Then CNS (retrograde axonal transport) > kill neurons
154
poliomyelitis
most infections asymptomatic
some abortive poliomyelitis (fever, headache, sore throat)
rare nonparalytic poliomyelitis (severe headache, neck stiffness)
very rare spinal paralytic poliomyelitis (weak lower limbs and respiratory muscles, incomplete recovery, fatality from asphyxiation)
very very rare bulbar paralytic poliomyelitis (cranial nerve paralysis, vasomotor and resp centers, fatality from asphyxiation)
155
polio vaccines: Salk vs Sabin
Salk = formalin-inactivated polio vaccine (IPV)
injected, weak mucosal immunity
used from 1955-1961 in U.S., reintroduced into U.S. in 2000 - present
Sabin = oral polio vaccine (OPV)
oral, can rep in intestine but not in neurons, shed for 30-60 days better mucosal immunity
introduced in 1962, was more effective bc of community protection - transmitted to unvaccinated children via fecal-oral route
used in U.S. from 1961-2000, eliminated poliovirus in U.S., discontinued due to viral reversion
156
polio eradication effort
Global Polio Eradication Initiative (GPEI)
why polio?
Species specific - no animal reservoir
Lifelong immunity after vacc
close to eradicated, only 2 polio-endemic countries left
Wild virus can spread to polio free countries
High risk countries need to continue oral vaccine
However risk of reverted vaccine polio
2022 - cause of paralytic polio in NY (cVDPV = reverted vaccine polio)
157
enterovirus D68
rare infections until recently
resp disease in children
rarely causes poplio-like symptoms (acute flaccid myelitis - AFM)
158
calici general overview
pos sense RNA
nonenveloped
1 capsid protein
translation via VPg cap substitute
5' nonstructural / 3' structural
broad host range w variety of symptoms
159
astro general overview
pos sense RNA
non enveloped
1 capsid protein (processed into at least 3)
translation via VPg cap substitute (maybe??)
5' nonstructural / 3' structural
160
calici capsid structure
icosahedral w 32 cup shaped depressions
nonenveloped
161
what type of virus is noro
calici
most significant calici for humans
162
why are noroviruses hard to treat
many many genotypes
hard to detect, treat, and immunize against
163
norovirus genome organization
ss pos RNA
5' nonstructural / 3' structural
uncapped but poly-A tail present
5' end covalently attached to VPg (but no IRES)
produces subgenomic RNA that encode structural proteins (also VPg linked)
polyprotein makes NS1-7 (nonstructural), sgRNA makes VP1 and 2 (2 ORFs)
164
noro NS5 protein
VPg
165
noro NS6 protein
only protease, 3CLpro, cleaves polyprotein cotranslationally (never see full polyprotein)
166
noro NS7
polymerase, RdRp, picorna 3D
in vitro replicaiton assays do not require polyA tail or U primer, maybe primer independent replication
167
what genes are on the sgRNA of norovirus
structural: VP1 and VP2
read by ORF4
stop codon exists between VP1 and 2
also has VPg at 5' end
168
noro p48
potential picorna 2AB activity
disrupts intracellular protein trafficking, localizes to golgi and disrupts golgi
possible scaffolding role for replication
169
noro NTPase
NS3
potential picorna 2C activity
170
noro p22 protein
potential picorna 3A activity
hydrophobic mem domain
inhibits host protein secretion and leads to golgi assembly
171
noro VPg
picorna 3B
linked to 5' ends of genomic and sg RNAs
translation initation
maybe protein primer for RNA synthesis
172
astrovirus genome organization
5' nonstructural, 3' structural
VPg at 5' end, has poly-A tail
2 polyproteins
makes structural proteins from subgenomic message
173
norovirus capsid structure
single capsid protein (VP1) that forms dimers, each dimer makes archlike protrusion
large hollows at 5 fold vertex
very stable at low pH, low Cl conc, high and low temp
does not need RNA to fold properly
174
noro VP1 structure
has S - P1 - P2 - P1 organization
S - conserved, core of VP1
P2 - variable, protrudes, binds receptor, targetted by imm resp
175
noro VP2 structure
inside the virus capsid
minor protein (1-8 copies)
basic, associates with capsid shell of VP1
required for replication
highly variable between strains
176
astro capsid structure
nonenvleoped
icosahedral
very stable (low pH, resistant to chloroform, detergents, etc)
star shape is rare
capsid protein cleaved into 3 smaller peptides
processing - caspase removes acidic domain, trypsin cleavage produces VP27 and VP25 extracellularly
177
calici receptor binding site
binds carbohydrates on cell surface, possible dual carbohydrate strategy
(most viruses that bind carbs needs a protein receptor, none known for calici)
178
calici viral entry
entry by proteases (no norovirus), acidicficaiton, or sialic acid (murine norovirus)
179
calici translation initiation
VPg at 5' end acts as cap and initiates - binds elF4E, forms initiation complex
start codon close to 5' end
VPg is not cleaved from incoming genomes
180
calici genome replication
NS7 = RdRp, makes neg compliment to pos strand
RNA flows through tunnel
how is sgRNA made? 2 theories:
1. premature termination - start 3' end, move towards 5' end, come into contact w stop codon, makes short transcript, Rdrp then makes pos strand
2. full length pos strand makes full length neg strand, internal initiation sites on neg strand, makes other initaition that forms sg sized RNA
181
noro VP2 translation
translation termination-reinitiation (TTR) on sgRNA
VPg recruits ribosome to ORF2 making VP1
ORF3 overlaps ORF2, short region of complementarity between to the 40S subunit of the ribosome and reinitiation motif
At low level, ribosome complements with subgenomic message, release of 60S, re-initiation
Don’t need true start codon
controls amount of protein made (much more VP1 than 2)
182
noro pathology
gastroenteritis, esp infants, young children, elderly, immunocompromised (dehydration)
leading cause of severe childhood gastroenteritis
all ages susceptible
short time course
183
noro transmission
fecal-oral
sick food handler, contaminated water on food, oysters (seafood) grown in contaminated water
stable - can exsist on surface for long time
person to person
associated w winter, long term care facilities, food, and cruise ships
184
noro virus intestinal entry
enters through M cells (used to sample environment), viruses use it to pass themselves
Virus can also infect macrophage that also samples the environment
185
noro virus strain / tissue tropism
MNV-1 (murine norovirus 1) prefers to infect Peyer’s patch (imm cells) over epithelial cells - cause more severe disease
MNV-CR6 infects tuft cells (rare and more specialized) - less severe disease
Genetically v similar
186
how does norovirus cause diarrhea
proinflamm cytokines
disruption of tight junctions
187
noro receptors
murine - CD300If, on muliple imm cells and tuft cells, there must be another determinant
human - attach to HBGAs (not receptor), present on RBCs, most pople also have on mucosal cells, polymorphism of HBGA means everyone is resistant to some norovirus
188
role of microbiota in noro infection
microbiota important for metabolism, immunity, and homeostasis
VIRUS BENEFITS FROM COMMENSAL BACTERIA
microbiota inc noro infectivity
189
flavi basic overview
pos RNA
enveloped
icosahedral
transmitted mostly by arthropods (arbovirus) (can also rep in them)
190
Toga basic overview
pos RNA
enveloped
icosahedral
transmitted mostly by arthropods (arbovirus) (can also rep in them)
191
what type of virus is dengue
flavi
192
what type of virus is west nile
flavi
193
what type of virus is yellow fever
flavi
194
what type of virus is hep C (HCV)
flavi
not arbo
195
what type of virus is Ross river
toga
196
what type of virus is chikungunya
toga
197
flavi genome organization
pos RNA
5' cap, no poly-A tail
single polyprotein w one ORF
5' structural / 3' nonstructural
some have IRES instead of 5' cap (HCV)
198
flavi C protein
capsid, associates w genome
acts as scaffold for envelope proteins and lipid bilayer
199
flavi prM protein
glycoproteins embedded in mem
interacts w E during assembly
200
201
flavi E protein
glycoprotein embedded in mem
binds cell surface and directs fusion
lays parallel to envelope (smooth structure)
202
flavi NS5 protein
methyltransferase-polymerase, used to give cap (normal RNA gets cap in nucleus, flavi never enters nucleus)
203
flavi NS3 protein
protease helicase
204
toga genome organization
pos RNA
5' cap and poly-A tail
5' nonstructural / 3' structural
uses sgRNA for structural proteins
nonstructural made first, then structural
205
toga nsP1 protein
RNA capping enzyme
206
toga nsP2
cysteine proteinase
207
toga nsP4
RNA pol (RdRp)
208
flavi virus structure
enveloped
icosahedral
180 glycoproteins (prM-E heterodimers) embedded in envelope
immature have spikes, mature is smoother (release of pr peptide)
209
toga virus structure
enveloped
icosahedral
80 flower-like structures ea w 3 subunits (petals)
ea petal is E1/E2 heterodimer
240 capsid protein molecules in contact w 240 envelope petals
identical symmetry (unusual for enveloped virus)
210
flavi receptor binding
bind attachment factors that concentrate virus on surface (glycans such as heparan sulfate)
then bind primary receptor (TIM family)
taken up by endocytosis
211
flavi entry
E dimer on virion surface > low pH induced E dimer dissociation, lifts stem, FP (fusion peptide) interacts w target mem > E protein trimerization > initiation of stem zippering - E come together and push towards target > hemifusion intermediate w fused other leaflets > postfusion hairpin-like E trimer w FPs and TM domains on same side of molecule
212
flavi translation
translated as one polyprotein
co and post translationally processed by NS2B/NS3 (viral) and host factors
10 mature proteins
213
flavi processing of polyprotein
co and post translationally processed by NS2B/NS3 (viral) and host factors
polyprotein as multiple TM domains, nonstructural on cytoplasmic side, structural (E and M) on ER luminal side
E and M have to be glycosylated via ER and golgi
exception: NS1 (imm evasion) in ER lumin
cleavage takes place on ER mem cytoplasmic cleavage by viral protease, ER luminal cleavage by host protease
214
flavi AncC protein
capsid, associates w RNA genome after rep, inefficient cleavage, doesn’t happen until later in life cycle
215
flavi RNA replication
viral replicase assmbled using NS proteins and genomic RNA and host factors > replicase associates w cellular mem > genome length neg template made > switch to synthesis of progeny genomes (pos sense)
216
how does flavi get an envelope
budding into ER and trafficking though golgi exocytosis
polyprotein insert in ER mem (M and E luminal side), AncC stuck on mem > AncC associates w genome to make nucleocapsid and M and E (have all components) > budding of immature virion into ER > glycosylation in golgi > fuse w plasma mem to release virion > right at budding, cleavage of prM by furin (keeps FP away so it doesn't fuse bud back into same cell)
217
how does toga get an envelope
bud at plasma mem
capsid protein and RNA remain cytoplasmic and move to plasma mem
C terminus folds to form protease that self cleaves capsid, N terminus binds RNA at packaging signal in NS region of genome (no sgRNA or neg strand RNAs) release from ER mem, travel to plasma mem
envelope inserts to ER mem and trafficked through golgi, glycosylation, inserted into plasma mem
capsid meets up with glycoproteins on plasma mem
218
flavi pathogenesis
humans are dead end (incidental) hosts
infect and cause disease but cannot spread to new host (human or insect)
219
west nile virus zoonosis
infection via mosquitoes (arbo)
birds harbor virus indefinitely
can infect human or horses, both dead end
220
what determines flavi virus disease
disease dictated by 2' tissue tropism
1' infection is blood
febrile illness (fever) with arthralgia (joint pain)
hemorrhagic fever
encephalitis (inflammation of parenchyma)
221
west nile virus encephalitis pathogenesis
deposit into tissue (skin) > replicate > spread to lymph nodes > replicate > enter blood > disseminate to spleen > CNS > damage neurons
222
how does WNV breach BBB
TNF induced cell permeability
breakdown of endothelial junctions (MMPs)
trojan horse via infected imm cells
direct axonal retrograde transport
223
hep C pathogenesis
sex, blood, iv drug transmission (not arbo)
persistant infection
silent virus - symptoms appear 10+ yrs later
cause chronic hepatitis
lead to cirrhosis and cancer
224
hep C treatment
treatment used to be long with significant side effects
sovaldi is new best drug, nt analog, inhibits RdRp
expensive
sovaldi + ledipasvir = harvoni
can cure HCV, but not accessible bc of cost
225
what type of virus is zika
flavi
226
zika pathogenesis
tansmission from mosquitoes (also sex, blood, and perinatal)
usually mild
cause microcephaly in fetus of pregnant woman
zika infects placental cells, disrupts barrier, inflamm resp to infection, cytokines cause microcephaly
ADE might happen in zika bc of close relation to dengue
227
alphavirses (toga) pathogenesis
natural host is usually wild mammals and birds
humans and horses are dead end hosts
228
chikungunya pathogenesis
mosquito vector
acute febrile illness, persistent arthralgic disease in some
rarely lethal
people are NOT dead end hosts, can spread back to mosquitos
infects osteoblasts (make bone), release inflamm cytokines, pro-osteoclastic (break down bone) molecules, both loss of bone formation and inc bone loss --> joint pain
229
what type of virus is rubella
toga
230
rubella pathogenesis
only non arbo togavirus, spread though air or contact
causes rash
congenital rubella causes fetal abnormalities and brith defects
in MMR vaccine
231
corona general overview
pos sense RNA
enveloped
large genome
unique replication w high recombination rate
mature virions bud at intracellular membranes
infect many species, cause wide range of disease
232
corona S protein
spike (all corona) glycoprotein
promotes binding and fusion
glycosylated
4 domains: S1 - globular head (variable, receptor binding), S2 - fusion promoting stalk (more conserved), trans mem domain, cytoplasmic domain
some coronas require proteolytic cleavage at one or two sites to have fusion activity (S1/S2 border or S2')
forms as a trimer on virion
some corona have additional HE spike protein (not SARS)
233
corona N protein
nucleocapsid, densely coats genome
forms helical ribonucleoprotein complexes
interacts w M to drive particle formation
only known particle to localize to nucleus
inhibits cytokines and IFN
234
corona M protein
membrane
forms inner core shell
short n terminal domain outside of envelope, spans mem 3 times, large c terminus inside envelope
thicker than most viruses
budding of virions, targeted to golgi
235
corona E protein
envelope
small, hydrophobic
required for budding, also localized to golgi
gained from budding intracellularlly
not entirely understood, has ion channel activity and induces apoptosis
236
corona genome organization
5' nonstructural / 3' structural and accessory
frameshift allows for ORF1a or ORF1b (includes 1a proteins) to be translated
5' cap and poly A tail
5' has leader (L) after cap
nested mRNA
237
corona nsp3 and nsp5 proteins
viral proteases
238
corona nsp12 protein
polymerase, Rdrp
239
corona nsp13 protein
helicase
240
corona nsp16 protein
methyltransferase
241
CoV-2 entry
S binds ACE2 > cleaved at S1/S2 boundary and S2' site by host > activated S2 ?fusion of viral and host membranes at plasma mem and endocytic vesicles
some coronas are pre-cleaved by furin (MERS)
242
corona nonstructural protein production
ORF1a and ORF1b translated from incoming genomic RNA
1ab is produced by a ribosomal frameshift (-1) mechanism involving pseudoknot in RNA
Both polyproteins are cis-cleaved into 16 active products
Once nonstructural proteins are made, they replicate the genome
243
corona replication
replication complex involves many viral proteins
pos > neg > pos sense RNA
if full length, packaged into nucleocapsid
if sgRNA, translation to proteins
244
corona sgRNAs
3' coterminal (all have same 3' end, variable 5' length)
only makes the 5' most coding region into protein even tho longer sgRNAs obviously also contain other proteins
monocistronic - one gene = one protein
all get leader and TRS
245
corona discontinuous transcription during neg-strand synthesis
only one leader at 5' end but all sgRNAs get it
multiple TRSs
TRS acts as pause, located at 5' end of a nested RNA
polymerase initatiates transcription at 3' end, when it hits TRS it pauses, can release RNA or continue
happens at ea TRS
hets leader from polymerase jumping - TRS repeats are identical, TRS on new RNA complements TRS on on template strand, repolymerizes, adds full leader to all sgRNAs
246
why does corona have high recombination rate
polymerase jumping promotes recombination between 2 diff genomes bc it can go from one TRS to diff viral TRS on diff nearby corona genome (trans polymerase switch)
247
corona cis vs trans polymerase switch
cis - polymerase jumping within a single genome, genome folds on itself
trans - polymerase jumping between 2 diff genomes, genome folding w another copy of the genome (from same or diff corona)
248
why does corona have high fidelity replication
most RdRps are error prone
corona has proofreading funct from the exonuclease (ExoN, nsp14)
low mutation rates compared to most RNA viruses
important bc of big genome, size exceeds error threshold of most Rdrps
249
corona assembly
N binds RNA = NC > M and E insert into ER membranes for glycosylation > S inserts into intracellular and plasma membranes > M interacts with N and S and initiates budding > Budding occurs into ERGIC (ER-golgi intermediate compartment) > Viral glycoproteins glycosylated in golgi after budding > Release occurs upon vesicle fusion with plasma mem (via exocytosis)
250
corona accessory proteins
at 3' end w structural proteins
nonessential
little to no homology
more important in vivo than in vitro (subverts imm resp)
some may be packaged
251
corona pathogenesis
diverse, infect mammals and birds
respiratory, GI, and CNS disease
respiratory disease is usually acute and self limiting, can sometimes cause chronic infection
replicates in alveolar lining, causes multi-nucleated giant cells (synctia), caused by S protein on plasma mem between 2 neighboring cells
infiltrate MO
efficient at evading IFN type 1 but induce high inflamm cytokine levels
infection thickens lining of lungs, thinning of air sacks
common cold via 229E and OC43 prototypes
SARS was first serious corona disease (2002-2003)
able to actively rep in GI but does not damage, how does it cause gastroenteritis
note: GI infection odd bc it is an enveloped virus, how does it survive pH??
can cause systemic infection in severe cases
252
SARS triphase pattern of disease
wk 1: prodromal (nonspecific) phase - fever, non-productive cough, sore throat, diarrhea
wk 2: shortness of breath, fever, diarrhea; respiratory decline in severe cases
wk 3: acute respiratory distress, likely due to cytokine storm
most disease is immunopathogenic
sheds in both resp and GI tract, high correlation between age and severity
253
SARS (2002-3 epidemic) cross species transfer
workers in markets w raccoon dogs and calm civet cat were infected, animals had related virus
probably not true reservoir: no wild animals w it (they did not bring it in from wild the picked it up at market), causes disease in these animals (reservoirs are usually asymmtomatic)
horseshoe bat are true reservoir; bat > civet > human
civet was intermediate host
254
SARS-CoV 2 receptor
ACE2
species specific -14 residues in receptor binding domain contact 18 residues on ACE2, only 2 differ between human and civet
changes: 1. pos charge on S repels ACE2 2. methyl group on S allows for tighter binding into groove
few changes lead to zoonotic jump
255
MERS corona pathogenesis
2012 epidemic
severe pneumonia
high mortality rate but also comorbidities
not efficient human to human transmission
widespread in dromedary camels in several Arbian countries, most MERS is linked to camels (others are hospital-acquired)
Bat > camel > human
256
measuring death toll of SARS-CoV 2
reported vs excess deaths do not line up, inaccurate records either purposefully or just poor reporting methods
diff qualifications: die in hospital/community? comorbidities? was there testing?
how to count?
satellite images of grave sites, reported numbers compared to computer models
257
SARS-CoV 2 pathogenesis
upper then lower resp tract
diff symptoms for ea strain
children and young adults asymptomatic, elderly more severe disease
5 day incubation, 8 day disease
highest shedding BEFORE symptoms, makes hard to control
258
Possible SARS-CoV 2 origins
1. direct jump bats to human
2. reservoir intermediate host to people
no intermediate identified yet
simliar viruses in pangolins and bats
3. lab escape (accidental or engineered)
259
why do people think SARS-CoV 2 was lab escaped
unique furin cleavage site: extra seq compared to closest relatives, only in human strain, inc in infectivity
does occur independently in other members of CoV family, maintained bc of inc in infectivity
codon usage in furin cleavage site: multiple codons for ea aa, specicies preferences, 6 possible codons for R, humans prefer 3, CGG is least common for corona but two of the R in furin cleavage site use it
Rare but it exists in all coronaviruses, roughly proportional to others in family, If artificially produced and corona didn’t like it, it could mutate to preferred seq, however despite all diff strains 99.8% use CGG
RRAR is rare furin cleavage and is suboptimal (researchers would not have picked it)
260
evidence for zoonotic transmission of SARS-CoV 2
75% early cases associated with market (none w Wuhan institute), evidence of animals at market (illegal), testing of surfaces in market, pos for SARS-CoV 2
261
SARS-CoV 2 vaccine
modified from 1st SARS vaccine development (already tested in animals and early human trials)
only had to change S protein for new vaccine
Spike cloned into RNA, introduced into host cells, translated by host, presented to imm sys, elicit imm resp
disparity over who is getting vaccine (low income countries)
262
influenza general overview
neg sense RNA
segmented genome
helical structure (all neg sense RNA viruses)
enveloped
replication in nucleus (odd for RNA viruses)
263
influenza genome organization
segmented (7-8 segments)
mostly monocistronic, exceptions: segments 7 and 8, ea have 2 proteins via alternative splicing
replication in nucleus
unusual twin helical conformation w central loop
coated w NP
both 5' and 3' ends complexed to PB1 (polymerase subunit)
PB1 binds PB2 and PA
264
influenza HA protein
hemagglutinin, glycoprotein, binds receptor and fuses w host mem
(HA and NA determine classification)
forms trimer in envelope
binds sialic acid-containing receptors at head
initiates receptor mediated endocytosis and fusion in endosomes
generated as fusion incompetent until cleavage by cellular proteases (fusion buried until it gets cleaved in endosome)
265
influenza NA protein
nuerominidase, glycoprotein, release of progeny from surface during budding
(HA and NA determine classification)
cleaves sialic acid
needed to release bound virions by mucoproteins in resp tract
266
influenza classification
influenza A causes most human disease, w occasional influenza B and rarely influenza C
HA and NA determine classification
A has many serotypes (differential Ab reactivity)
144 possible serotypes of A, only 19 observed
267
influenza virus structure
helical
enveloped
pleiomorphic - adopt diff conformations; can be spherical or filamentous
filamentous - better cell to cell (larger)
spherical - better person to person (smaller)
268
influenza NP protein
nucleocapsid protein, binds w segments of genome, 1 of ea segment per virion
269
influenza M2 protein
ion channel
forms tetramers to create pores in envelope, upon acidification in endosome - ion channels open > H+ cross > drives release of viral nucleocapsid into cytoplasm
anti-influenza drug amantadine blocks M2 ion channel
270
amantadine mode of action and efficacy
pits into M2 pore, prevents H+ exchange
not used anymore, most strains are imm to amantadine now, only a few aa changes for resistance
271
influenza M1 protein
matrix
272
influenza what coats the genome segments
nucleocapsid protein + polymerase complex proteins (PA, PB1, and PB2)
needs to have polymerase in virion bc of neg sense RNA, cannot directly translate upon entry into host
1 of ea segment per virion
273
influenza cytoplasm/nucleus shuttling
HA binding > endocytosis
gets shuttled between nucleus and cytoplasm
genome enters nucleus for transcription, mRNA goes to cytoplasm for translation
glycoproteins move through ER and golgi, all other proteins go back to nucelus to associate w genomes during replication, these then get exported to cytoplasm and directed to plasma mem > budding
274
influenza genome replication
neg > pos > neg RNA
intermediate pos is used as mRNA for translation
replication in nucleus:
segment enters nucleus > transcription > 5' cap and poly-A tail > cytoplasm > make viral proteins > proteins go back to nucleus to help w RNA rep > progeny exit nucleus to get packaged
275
what type of virus is influenza
orthomyxo
276
influenza cap stealing
most RNA viruses encode their own capping enzyme, influenza cap-steals
cellular pre-mRNA has cap, PB2 binds to it > PB1 cleaves mRNA downsream of A or G (allows UC repeat at 3' end to bp w opposite end) > PB2 bound cap acts as primer for transcription > PB1 is polymerase (Rdrp), initiates transcription 5' to 3'
all viral influenza mRNAs have some host at 5' end
277
influenza poly-A tail
All influenza genome segments contain a short poly-U stretch at their 5’ end, PB1 pauses and stutters here, reading through poly-U several times and repeatedly adding complementary A residues
PB1 eventually terminates at this position; thought to be due to presence of another PB1 molecule at the 5’ end of template RNA
278
influenza alternative splicing
M1 and NS1 mRNAs
M1 splicing = M2
NS1 splicing = NS2
low fraction (10%) is spliced (quantity regulation)
unspliced and spliced both transported to cytoplasm
279
influenza translation
in cytoplasm, some proteins shuttle to nucleus for rep
pos RNA template for translation and rep but process is distinct
genome replication requires free NP, made by translation, when there is a lot of NP it drives rep over translation
after neg RNA is made, complexes w M1 and NS2 (has NES) > exported
280
influenza NS1 protein
most abundant protein in infected cell
shuts down host cell
interferes w host poly-A tail formation (does not affect viral bc of stuttering mechanism), combined w cap stealing, most cellular pre-mRNA is degraded
suppresses IFN
NS1 mutations result in poor replication (unless IFN deficient cell)
281
influenza assembly
7/8 genome segments interact w M1 protein which interacts w cytoplasmic tails of HA and NA and ion channel (M2)
Cleavage at neck
NA needed to cleave from sialic acid
282
influenza A pathogenesis
severe acute disease
sore throat, cough, fever, headache, muscle ache
partially due to tissue damage from virus, mostly due to immunopathological levels of cytokines and chemokines (in severe cases can have cytokine storm)
annual epidemics, pandemics every 10-50 yrs
fatality usually only in juvenile, elderly, and immunocomp
influenza targets goblet cells, kills them, causes symptoms
lack of mucus allows for 2' infections
imm resp also causes disease (proinflamm cytokines and robust BC resp)
283
how can influenza kill its host
influenza usually does not - 2' bacterial infection can cause fatal pneumonia
influenza targets goblet cells, kills them, causes symptoms
lack of mucus allows for 2' infections
immunopathology as well
284
influenza Ag shift vs drift
drift - point mutations during error prone replication, Mutations in HA or NA can allow for imm evasion, subtype of strain DOES NOT CHANGE however diff can mean less protection for people already infected w original strain
Shift – reassortment of 2 diff flu strains together (ex: swine + human in same host)
Segments mix
NEW SUBTYPE GENERATED ex: H2 to H3
285
influenza pandemics: shift vs drift
shift (reassortment) more likely to cause pandemic bc of big diff in Ag/Abs
allows for zoonosis
often human w bird flu
sometimes swine (2009)
1918 was fully avian, might be exception - drift not shift, mutations in HA and RdRp
286
why was 1918 spanish flu so bad
high infectivity
global bc of war travel (and close contact)
severe symptoms (cytokine storm)
majority of deaths from 2' infection
targeted young adults (unusual for flu)
287
why did 1918 spanish flu target young adults
Originally thought imm resp was stronger, so worse immunopathology
Now - less protected from previous immunity:
Born before 1889 had some immunity to H1
Born during 1889 only exposed to H3
Born after 1900 had circulating H1 strain
People born between 1889 and 1900 had no prior immunity to H1
288
HPAI
Highly pathogenic avian influenza (HPAI) viruses
H5N1 and H7N9
2 strains that are avian, highly pathogenic in birds
Also highly pathogenic in human but rare human to human spread
Usually people in close contact w birds
Very concerned about H5N1 bc few mutations will allow may allow spread human to human, many many will die
H5 has been detected en masse in birds (domestic poultry and wild birds)
pos human infection and some wild mammals
289
influenza treatment
M2 inhibitors - amantadine, most/all strains are resistant now
NA inhibitors - temiflu
290
influenza prevention
vaccines
killed wt, live attenuated, recombinant HA protein made in insect cells
multivalent nature - WHO monitors and predicts which strains ea year (usually quadrivalent: influenza A H1N1, influenza A H3N2, and two influenza B viruses) - varied effectiveness
