1
Q
Acquired immune deficiency syndrome (AIDS) case study:
A
1981; LA, Ca; 5 men (29-36yo)
Hospitalized for pneumonia from Pneumocystis carinii (fungus); rare in healthy people
The men die
First documentation of AIDS (caused by HIV)
HIV infects immune system & causes it to decline so opportunistic pathogens can harm
Today, 36.9mill people live with HIV
2
Q
H5N1 Influenza virus case study:
A
Hong Kong; 3yo boy; sick poultry
Develops fever, sore throat, cough
Dies of acute respiratory distress syndrome (inflamed his lungs)
Previously found in birds only
New species (humans) never exposed to this virus before so no immunity built up
18 individuals infected (had direct contact with chickens at open-air markets)
20% of chick population infected (30% death rate)
- Government killed the rest (1.5mill)
3
Q
Norwalk virus case study:
A
Cruise ship from Washington to Florida; ~30% passengers first voyage; 305 passengers next 3 voyages
Patients got acute gastroenteritis (stomach illness)
Cruise ship was cleaned after 1st voyage
Had Norwalk virus (easily transmitted through food, water, air particles & don’t need many particles))
Need bleach to remove
Each year ~20mill cases occur in US
4
Q
Reasons to study viruses:
A
They are everywhere (present wherever there is life)
~3,000 documented viruses that infect living organisms
Thousands of strains & isolates within each species
5
Q
Where are viruses found?
A
Air Ocean Soil Streams Ponds
6
Q
What’s the difference between virus & virion?
A
Virion = the infectious virus package that is assembled; virus particle; extracellular form of the virus; released between cells Virus = Biological entity in all its stages; the general characteristics that differentiate it from another infectious entry
7
Q
How many virions are there?
A
10^31 total virions (10x more than bacteria)
Millions created & released in each cell
8
Q
What was one of the first major epidemics of viruses?
A
Poliovirus became more epidemic with growth/urbanization of cities
New York City; 1916
- 9,000 cases
- 2,343 deaths
1950s
- 20,000 cases of paralytic polio/year until 1955 (first polio vaccine)
9
Q
When was the first polio vaccine?
A
1955
10
Q
When was the first major pandemic?
A
1918 (originated in US)
Killed 20-50mill
Influenza strain (sever strain of influenza)
11
Q
What is the most effective way to prevent viruses/pandemics?
A
Surveillance
Vaccination
12
Q
Hershey & Chase’s experiment overview:
A
Verified that DNA is the molecule that encodes genetic information
Location: Cold Spring Harbor Laboratory, Long Island
Alfred D. Hershey & Martha Chase performed research with phage T2 (infects E. coli)
T2 attaches to E. coli cells & injects chemicals to create more T2
Tested if chemicals were composed of DNA or proteins
13
Q
What is a bacteriophage?
A
Viruses that infect bacteria
Bacteriophage means “bactera eater”
14
Q
How did Hershey & Chase’s experiment work?
A
Used radioactive phosphorus (32P) & radioactive sulfur (35S) isotypes
- P found in DNA only, S found in proteins only
- Grew two cultures of T2, one with P & one with S
- Infected bacteria with P culture & S culture (will go to DNA or protein of cell)
- Used centrifuge to agitate cells, shearing off any phage still attached
- S phage proteins remained outside cells, P phage DNA entered the cells
- Bacteriophages are composed of DNA surrounded by a protein coat
15
Q
Watson & Crick experiment
A
1953
Presented double-helix model of DNA structure
James Watson & Francis Crick
16
Q
What is phage therapy?
A
100 years ago
Felix d’Herelle (coined term bacteriophages)
Used bacteriophages to treat bacterial infections before antibiotics existed
- Declined after antibiotics then increased due to antibiotic-resistant bacteria
17
Q
What is gene therapy?
A
Delivery of DNA into cells to compensate for defective genes
Viruses are engineered to deliver a normal copy of the defective human gene
- Potential to cure genetic diseases, but procedural obstacles occur
18
Q
What is virology?
A
The study of viruses, how they replicated, and how they cause disease
19
Q
What are the 5 criteria of a living organism?
A
- Must have a GENOME or genetic material
- Has to be able to engage in METABOLIC ACTIVITIES (can obtain and use energy and raw materials from the environment
- Has to be able to REPRODUCE independently and GROW
- Must be able to compensate for changes in the external environment to MAINTAIN HOMEOSTASIS
- Populations of living organisms are able to adapt to their environments through EVOLUTION
20
Q
What criteria for life do viruses share?
A
- Genetic material (living organisms have only DNA genomes, viruses can have DNA or RNA genomes); have nucleic/amino acids
- Proteins translated by ribosomes (similar to living cells)
- Evolve (viruses evolve much quicker than organisms)
- Cycle energy & matter within ecosystems
Don’t: have metabolic activities, independent reproduction, homeostasis
21
Q
Why is quick viral mutation important?
A
Antiviral drugs become absolete (think yearly flue vaccine)
Great model for studying & observing evolutionary change
22
Q
What do viruses use instead of metabolism?
A
Host cells energy & machinery to synthesize new virus particles
23
Q
What is the basics of virus replication?
A
Virus enters a cell
Completely disassembles
Viral nucleic acid encodes the instructions & host cells machinery is used to make virions
24
Q
Why don’t viruses have homeostasis?
A
They are inert particles
Are unable to compensate for changes in their external environment
25
When did life on Earth begin?
About 3.5bya
26
What are the possible hypotheses on how viruses originated?
The precellular hypothesis (or virus-first hypothesis)
The escape hypothesis
The regressive hyptohesis
27
What is the pre-cellular hypothesis?
Basis: viruses existed before or alongside cells & contributed to development of life as we know it
- More likely they developed alongside cells because need cells to replicate
Life developed in an RNA world
- RNA viruses orginated in RNA world
- DNA was more stable so selected for and replaced RNA
The three domains of life each arose independently from the infection of cells with three distinct DNA viruses
Only theory to suggest viruses came before cells
28
What is an RNA world?
RNA, not DNA, was the first genetic material
Thought because:
- RNA is easier to create than DNA from precursor chemicals thought to exist on the early Earth
- In present-day cells, the sugar in DNA (deoxyribose) is made from the sugar found in RNA (ribose)
- RNA can encode genetic material (and sometimes catalyse reactions) like a protein enzyme does so RNA could have functioned as an enzyme that copied an early RNA genome (while DNA requires complex protein enzymes)
- Many important molecules in the cell include RNA or its parts (ATP or ribosomes): these are conserved remnants of cells in RNA world
29
In the pre-cellular hypothesis, where did DNA come from?
DNA first originated in RNA viruses, giving rise to DNA viruses
- Cells with DNA originated from infection of an RNA cell with a DNA virus OR
- Mechanisms used to create DNA within the virus could have been adopted by the cell
30
What do critics say about the pre-cellular hypothesis?
Viruses are parasitic and require a cellular host:
- Unlikely that viruses could exist before cells because would not have had reliable source of materials to replicate
Most viral genes are not found in cells
- Should see more similarities between cells & viruses if DNA virus was the origin of a cell's genetic material
31
What is the escape hypothesis?
Basis: Viruses are pieces of cells that broke away at one point in time & gained ability to travel from cell to cell
The viruses of the three domains of life may have arisen from distinct escape events within those three domains
Transposable elements helped increase popularity with this theory
- Since some transposons are similarities to retroviruses, think they originated from escaped transposons
- Restrovirus genomes can be found permanently integrated into cellular genomes as relics of past periods
32
What are transposable elements?
Also called transposons
Pieces of DNA that can physically move from one location to another in the genome of a living organism
Can be a few hundred to thousands of nucleotides long
Originally thought to be "junk DNA" with no function
Make up nearly half the human genome (many are no longer functional)
Can:
- Incorporate into the host's DNA when entering a cell (like retroviruses)
33
What do critics say about the escape hypothesis?
The majority of viral genes have no homologous cellular counterpart
- If viruses escaped from cells, should have more cellular genes in viruses
- Viruses have unique viral genes not found in cells
More likely retroviruses infected cells & integrated into their genomes
34
What is the regressive hypothesis?
Basis: Viruses were once independent intracellular organisms that regressed to less-advanced state where they were unable to replicate independently OR viruses were once living intracellular organisms that dissolved their membranes to facilitate easier access to cellular equipment & materials
The mitochondria and chloroplast are thought to originate in this manner
Thought because some bacteria (Chlamydia & Rickettsia) need intracellular environments of cells to replicate
The Mimivirus supports the regressive hypothesis
35
What is the Mimivirus and why does it support the regressive hypothesis?
Mimivirus = microbe mimicking virus
Giant (750nm in diamter) amoeba-infecting virus
First thought a small bacteria (largest virus of the time)
Has one of the largest viral genomes
- Several genes resemble genes for creating proteins meaning the virus may have been able to create its own proteins at one point
36
How do Mimiviruses replicate?
Infect the cell
Mimivirus & several large complex DNA viruses set up virus factories
- Made of cellular membranes
- Where replication & assembly occurs
- Contain enzymes necessary to copy the viral genome & contain it OR are in close proximity to ribosomes & mitochondria (for proteins & ATP)
This infection technique is similar to a Chlamydia reticulate body, a structure the bacteria forms within a cell to develop new bacteria
37
What do critics say about the regressive hypothesis?
The majority of mimivirus genes are unlike genes in bacteria or eukaryotic cells
- If once parasitic, more viral genes would show similarity to current genes
Instead, viral genes similar to cellular genes are stolen from the cells DNA at one point (horizontal gene transfer)
- Many viral genes are thought to originate this way
If viruses were once free-living parasites, why would they assemble completely from scratch instead of splitting in two (wat would cause such a major modification)
38
Who contributed to the identification of viruses as novel biological entitites?
```
Louis Pasteur: Germ theory
Robert Koch: Koch's postulates
Adolf Mayer: tobacco mosaic diease
Charles Chamberland: porcelain filter
Dimitri Ivanovksy: tobacco mosaic disease + porcelain filter
Matinus Beijerinck: the word "virus"
Friedrich Loeffler & Paul Frosch: foot-and-mouth disease virus (FMDV)
Thomas Rivers: first virology textbook
```
39
Who was Louis Pasteur?
Mid-1800s
Showed life doesn't arise through spontaneous generation
Experiments:
- Sterilized beef broth in a swan-neck flask (allowed air to enter but no dust or particulates)
- Broth only became contaminated if top of flask was broken & bacteria particulates could enter flask
Gained support for the germ theory
40
What does the germ theory state?
Infectious diseases are caused by microorganisms
| Increased popularity from Pasteur and discovery of other disease-causing bacteria (anthrax, cholera, tuberculosis)
41
Who was Robert Koch & what are Koch's postulates?
Koch was the first person to identify the causative agent of anthrax was bacteria
Called the father of bacteriology
Koch's Postulates are four criteria to demonstrate that a microbe is the causative agent of a disease:
42
What are the criteria for Koch's postulates?
1. The organism must be present in every case of the disease
2. The organisms must be isolated from the host with the disease & grown in pure culture
3. The disease must be reproduced when a healthy susceptible host is inoculated with the pure culture
4. The same organisms must again be recovered from the experimentally infected host
43
Who was Adolf Mayer?
1879
Investigated a disease that was affecting tobacco plants in the Netherlands (called the tobacco mosaic disease)
Discovered the disease can be transmitted between plants from sap
- Didn't find any microorganisms under the microscope but determined it must be bacterial anyways
- Wasn't able to complete Koch's 3rd postulate (needed cells to reproduce)
44
Who was Charles Chamberland?
In 1884, created a sterilizing filter made of unglazed porcelain
Pores in porcelain allowed water but no bacteria (too large)
Allowed solutions to be sterilized
45
Who was Dimitri Ivanovsky?
Determined that the infectious agent to the tobacco mosaic disease was able to pass through Chamberland's porcelain filter
- Realized the infectious agent was smaller than a bacterium
Couldn't complete Koch's postulates (couldn't culture the organism)
Thought the agent was a bacterial toxin small enough to pass through the filter
46
Who was Matinus Beijerinck?
Also found the infectious agent to be able to pass through Chamberland's filter
Discovered using the plant tissue was the only way to replicate the infectious agent (why Mayer & Ivanovsky couldn't culture)
Concluded the agent was a contagious living fluid called a virus (microbe at the time referred to bacteria)
47
Who were Friedrich Loeffler & Paul Frosch?
1898
Discovered the pathogen causing foot-and-mouth disease (FMD) was able to pass through Chamberland's filter
- Discovered first animal virus noted
Discovered the agent couldn't pass through a Kitasato filter
- Determined the virus was not a liquid (like Beijerinck thought)
48
What is the difference between a Chamberland filter and Kitasato filter?
Kitasato filter has smaller pores
49
What is the foot-and-mouth disease virus?
First animal virus discovered
Highly contagious respiratory virus
Infects cloven-hoofed animals
Causes blisters on hooves, mammary glands, tongue, lips, & mouth
Not lethal to adults but impacts growth and production of milk
Since very contagious, any animal is instantly culled to prevent the spread
50
What is the yellow fever virus?
First human virus discovered (1900)
Transmitted by mosquitoes
Found by the U.S. Army Yellow Fever Commission by Walter Reed
51
Who was Thomas Rivers?
American virologist
Published the first Virology textbook (1928)
Many viruses (and bacteriophages) had been discovered although they hadn't been seen yet (didn't see until the electron microscope)
52
What is the electron microscope?
Invented in 1933
Use a magnetic field to focus electrons to illuminate a specimen
- A more powerful magnifying system with better image resolution
First saw virions
Then could magnify 10x than light microscopes, now can magnify over 1mill times
53
What are viroids?
Small circular pieces of RNA only found in plants
Infectious
Have no protective protein coat
RNA does not have info to make proteins (enzymes copy the viroid RNA to transmit to the next plant)
Thought to be remnants of an "RNA world" & the precursor to some viruses
Infect tomato plants, citrus plants, apple trees, potato plants...
54
How do viroids cause damage?
Viroid RNA copying uses the enzymes of the host cell (no plant RNA made, no proteins made, harder to function)
Viroid RNA is cut up and acts as small interfering RNA (siRNA)
- Bind to complementary sequences of plant RNA, create dsRNA that the cell degrades, so the plant RNA can't make proteins
55
What are prions?
It is a normal mammalian protein whose shape becomes irreversibly modified
Normal prion = PrPc (cellular prion protein)
Abnormal prion = PRPSc (Scrapie-causing prion protein)
Causes spongiform encephalopathy (brain diseases)
- Scrapie (in sheep), bovine SE (in cows), CJD (in humans)
56
How are abnormal prions created?
Once one PrPSc is created, normal prion proteins can be transformed
Can't be removed from the tissue & build up in the brain
Caused by genetic mutation, eating food containing PrPSc, or (rarely) transmitted with improperly sterilized neurosurgical tools
Extremely difficult to destroy
57
What is the lengths of viruses?
20nm = smallest
100nm = normal (HIV, influenza)
20-200nm = avergae human virus size
400nm = large (poxviruses: variola virus, smallpox)
80nm extend to 1000nm = small (filoviruses: ebola, marburg)
Human cells are 100-1000x larger than viruses
Bacteria was usually 2000-3000nm but Mycovacteria can be 10x smaller = virus size
58
What viruses infect amoebas?
Giruses (giant viruses)
| Ex. Mimivirus: first girus isolated; infect through phagocytosis; encased in 125nm thick polysaccharide to entice amoeba
59
What does obligate intracellular parasite mean?
Completely dependent upon the internal environment of the cell to create new infectious particles (virions)
Use cell's energy & machinery for reproduction (from scratch)
Done by all viruses
60
What is de novo replication?
Virus genetic material (nucleic acid) encodes instructions for host proteins to assemble new virions
Done by all viruses
61
What is the average viral genome size?
7,000-20,000 base pairs (bp) (7-20 kilobase pairs (kb))
Smaller virions hold less nucleic acid
Large viruses don't necessarily have large genomes
dsDNA viruses usually have large genomes (herpesvirus has 120-200kb) (pandoravirus has up to 2.5 million bp)
* human cells have over 3billion nucleotides
62
What can viral genetic material be exposed to that will break it down?
Enzymes
Physical stresses (flow or air/liquid)
UV radiation
Radioactivity
63
What is a capsid?
Protein shell
Composed of 1+ types of proteins repeating to create a capsid (forms strong but flexible capsid)
Small size
Difficult to break open
64
What is a nucleocapsid?
The nucleic acid + capsid
| Capsid is made of only a few proteins because the small size of the nucleic acid can only make a few proteins
65
What is capsid self-assembly?
The spontaneous assembly of the capsid proteins
Held together by electrostatic and hydrophobic forces
Discovered by Fraenkel-Conrat & Williams in 1955
- Separated the RNA genome from the protein subunits in a TMV and put back together in a test tube
- Virions formed automatically
No need for additional info to assemble a virus
66
What is an envelope?
Lipid membrane derived from one of the cell's membranes to surround the capsid
Mostly from the plasma membrane
No envelope virus is called nonenveloped or naked (Ex. Totiviridae family)
Contain matrix proteins to connect the envelope to the capsid
67
What is a virus attachment protein?
Viral protein that facilitates the docking of the virus to the plasma membrane of the host cell
Found in the outer-most layer of the virus (capsid or envelope)
* SARS-CoV virions have petal-shaped spikes
68
What are the common capsid shapes?
Helical
Icosahedral
Complex
69
What is a helical capsid?
Spiral shape that curves cylindrically around the axis
Viral nucleic acid coils in between the capsid while the capsid proteins wind around the inside/outside of the nucleic acid, forming a long tube structure
The protein winding around the nucleic acid is called the nucleocapsid protein
Can be enveloped or naked
- Most plant viruses are helical & naked
- All helical animal viruses are enveloped (influenza, measles, mumps, rabies, Ebola)
70
What is the math for the helical capsid?
Amplitude: Diameter of the helix (tells us the width of the capsid)
Pitch: height/distance of one complete turn of the helix (subunits/turn x rise of each subunit)
71
What are the advantages of forming helical capsids?
One one type of capsid protein required
- Structure is simple and requires less free energy to assemble than a capsid composed of multiple proteins
Only one gene is required (due to one protein type)
- Reduces the length of the nucleic acid
Structure can continue indefinitely
- No restraints on how much NA can be packaged into the virion
72
What is a icosahedral capsid?
Genomes packaged competely within the icosahedral capsid that acts as a protein shell
20 sides (faces) composed of an equilateral triangle
- Also has edges and vertices
More prevalent than the helical architecture
Need at least 3 viral proteins to create a face (form a structural unit)
The number of structural units that create each side is called a triangulation number (T)
Can be naked or enveloped
73
What is the symmetry of the icosahedral capsid?
Has 2-3-5 symmetry (used to describe the possible ways that an icosahedron can rotate around the axis)
- 2-fold axis: when rotate along axis (edge), encounter starting structure twice in one revolution
- 3-fold axis: when rotate along axis (face), encounter initial view three times
- 5-fold axis: when rotating along axis (vertice), encounter starting structure 5 times
Used to indicate specific locations on the virus
74
Proteins in the icosahedral capsid?
Some form the pentamers (vertices) while others form the hexameres (faces) (*think soccerball)
Ex. cowpea mosaic virus
Capsomeres: morphological units that arise from the interaction of the proteins within the repeated structural units
75
What are the benefits of forming icosahedral viruses?
```
Requires less energy to form
Evolutionarily favored for this
- Human papillomavirus
- Rhinovirus
- HBV
- Herpesvirus
```
76
What is a complex capsid?
Rare
Doesn't conform to helical or icosahedral shape
Ex. Poxvirus, geminivirus, bacteriophages, HIV-1, baculoviruses
77
Complex capsid: conical and rod-shaped capsids
HIV-1 (conical) & baculovirus (rod-shaped)
Each virion contains a copy of the virus genome coated in a basic protein
Enveloped
78
Complex capsid: oval or brick-shaped
Poxviruses
Large (200-400nm long)
Inside contains dumbbell-shaped core with viral DNA surrounded by two "lateral bodies"
79
Complex capsid: geminiviruses
Geminiviruses contain two icosahedral heads joined together
(gemini = twins (two))
Plant-infecting virus
80
Complex capsid: alien spaceship
Bacteriophages (infect & replicate within bacteria)
Many have complex structure
Icosahedral head contains NA
Cylindrical sheath binds bacteriophage to cell
81
What are the two types of viral reproductive mechanisms of phages?
Lytic cycle (called virulent phage)
- Kills host
Lysogenic cycle
- Viral DNA is incorporated into host cell's chromosomes (called prophage)
- Environmental signal can trigger the virus genome to exit the host & switch to lytic mode
Lytic + lysogenic cycle = temperate phages
82
Importance of virus classification:
Scientists can compare new viruses to similar ones
| Scientists can study virus origin and evolution
83
What is the Baltimore classification system?
Categorizes viruses based on type of nucleic acid genome & replication strategy:
Class I: dsDNA viruses
Class II: ssDNA viruses
Class III: dsRNA viruses
Class IV: + sense ssRNA viruses
Class V: - sense ssRNA viruses
Class VI: RNA viruses that reverse transcribe
Class VII: DNA viruses that reverse transcribe
84
Why is it difficult to classify viruses?
Over 28000 species
| Different properties of each
85
What are postive-sense RNA viruses?
albe to be immediately translated into proteins
| Ex. mRNA in cell is positive strand
86
What are negative-sense RNA viruses?
RNA is not translatable into proteins
| - First needs to be transcribed into positive-sense RNA
87
Why is reverse transcription important for classification?
It is creating DNA from an RNA template
| Host cells can't do it
88
What is the International Committee on Taxonomy of Viruses (ICTV)
Sole body charged with classfiying viruses (since 1966)
Viruses aren't alive, so not part of tree of life
- Classified under Order, Family, Genus, Species
- There are 7 current orders & 103 families in them
- 77 families don't have an order (retroviruses, papillomavirus, poxviruses)
Parameters:
- Chemical & physical properties (type of NA, # of different proteins)
- Virions size
- Capsid shape
- (Non)enveloped
89
How are viruses named:
No names of people
Easy to use and meaningful
When directly referring to name (any classification taxon) italicize and capitalize name
Named after:
- Clinical conditions they cause ( HIV, HPV, Poxvirus)
- Location of discovery (Ebola virus, WN virus, Norwalk virus)
- After their properties (Coronavirus, Influenza virus, Poliovirus)
- After people (historically) (JC virus, Rous sarcoma virus)
90
How are the domains of life divided?
Presence or absence of a nucleus within the cells of the organism
Eukaryotes have nucleus in cells
Prokaryotes (bacteria, archaea) have no nucleus
91
What are the purpose of ribosomes in RER?
Make proteins after binding mRNA
Found on RER or free in the cytosol
On the rER, makes proteins transported into the lumen (inside) of ER
- Fold/modify proteins
- Modified with carbs = glycoproteins
- Modified with lipids = lipoproteins
After ER, proteins leave in vesicle to Gogli apparatus
92
What are lysosomes?
```
Specific vesicles on the Golgi apparatus
Lysosome enzymes digest molecules
Over 30 enzymes
Optimal at pH 5
- Cell pH is 7.2
```
93
Organelles vs viruses?
Viruses take over the organelles of cells to create viral proteins to create virions
- Nucleus -> rER -> Golgi apparatus
Viruses take over mitochondria to use the ATP of the cell
Viruses use cytoskeleton to be transported around the cell
- Microtubules, intermediate filaments, and microfilaments
94
What is the fluid mosaic model?
The current view of how the plasma membrane is assembled
Proposed by Singer & Nicolson
Contains proteins in the bilayer called integral proteins (includes glycoproteins) & phospholipids (includes cholesterol)
Proteins:
- Receptors for extraceullar substances
- Facilitate adhesion of one cell to another
Phospholids:
- Are involved in transmitting signals to interior of the cell
95
What is the difference between bulk-phase endocytosis & receptor-mediated endocytosis?
Bulk-phase: cell forms a vesicle that enguls the molecules present (nonspecific)
Receptor-mediated: initiated when specific ligands bind to receptors that are present on the cell surface (specific)
- Found in specific areas of the membrane called clathrin coated pits (from clathrin) or caveolae (from caveolin)
- Clathrin/caveolin coating is lost once in cell
Viruses use both
- Escape vesicles by changing configuration before vesicle fuses with lysosomes
96
What are the phases of the cell cycle?
Gap 1: growth
Gap 0: certain cells that don't continue to cell cycle (neurons)
Synthesis: replicate chromosomes
Gap 2: growth
Mitosis: Divide cell
Different viruses need different stages of the cell cycle to ensure infection of the cell
- Some viruses need enzymes only present during cell replication (mitosis)
- Some viruses can stop the cell cycle to increase their own replication efficiency
97
What is the central dogma of life?
DNA -> mRNA -> Proteins
Replication (copy DNA (nucleotides)) -> transcription (creating temporary mRNA (nucleotides) in nucleus) -> translation (creating proteins (amino acids) via ribosomes in cytosol)
98
How does strand polarity work in a growing strand of nucleic acid?
Phosphate group of nucleotide attaches to sugar at 5' carbon atom
New nucleotide is added to 3' carbon of the sugar
Called 5' -> 3'' (forward direction)
Since double helix is antiparallel, while one strand is going forward left-right, the other strand is going forward right-left
99
What is the DNA polymerase?
Enzymes that lays down the complementary nucleotides of the new strand of DNA (5'->3')
Cellular DNA polymerases are DNA-dependent DNA polymerases (synthesize DNA using DNA template)
100
What is primase?
Enzyme that lays down a short complementary RNA fragment onto the DNA strand, where the DNA polymerase can bind
101
What is ligase?
Enzyme that joins the short fragments together creating a continuous DNA strand
102
What enzymes are used during DNA replication?
```
DNA polymerase
Primase
Ligase
Viruses take advantage of them to replicate their own genomes
- Need to gain entry to nucleus first
```
103
How is transcription initiated?
Transcription factor proteins: bind DNA upstream of the gene starting site at the promoter or bind to enhancer regions
RNA polymerase II is between transcription factor proteins and DNA
104
What are RNA polymerases?
DNA-dependent RNA polymerases: (synthesize RNA based on DNA template)
Uses DNA template to create a strand of RNA (5'-3')
- Only one template used (RNA is single stranded)
Terminates transcription at end of gene
105
What happens during RNA processing?
Also called post-transcriptional modification
Changes precursor mRNA into mature mRNA via:
Add "cap" to 5' end (consists of a methylated guanine nucleotide)
- Protects the 5' end from unraveling
- Where ribosomes bind to begin translation
Add 3' poly(A) tail (consists of adenine nucleotides)
- Protect mRNA transcript
Remove introns via RNA splicing
- By changing where the introns are, different proteins can be created from a single mRNA (called alternative splicing)
- HIV undergoes RNA splicing
106
What are amino acids?
Building blocks of proteins
There are 20 different amino acids (64 codons)
- Since this is universal, viruses use same codons
Human proteins = 50-1000 AA in size
Sequence of mRNA determines how ribosomes will assemble the amino acids into the protein
3 AA = 1 codon
The start codon (AUG) in mRNA begins translation
- Found in the Kozak consensus sequence
- AUG encodes for methionine (all proteins begin with methionine)
- Begins the reading frame (the ribosome begins reading 3bp at a time)
The stop codon (UGA, UAA, UAG) causes the ribosome to fall off the mRNA, completing the protein
107
What is the Kozak consensus sequence?
Contain the AUG codon with other codons
| Sequence is generally GCCACCAUGG or GCCGCCAUGG
108
What are the different segments of mRNA called?
5' untranslated region (5' UTR): segment before translation
3' untranslated region (3' UTR): segment after translation
Open reading frame: translatable segment (between 5' UTR and 3' UTR)
109
What are transfer RNAs?
Act as adaptor between mRNA and ribosome
Brings amino acids to ribosome to join into a protein
Contains anticodon sequence complementary to mRNA codon
- Determines which codon encodes which amino acid
110
What are the three stages of translation?
Intiation: ribosomal small unit binds 5' cap of mRNA then scans until hit AUG
Elongation: tRNA contains corresponding amino acid and binds with mRNA
Termination: Release factor enters ribosome and translation ceases when stop codon is encountered
111
Polypeptide vs protein:
Protein: complete, functional entity consisting of 1+ polypeptide chain
Polypeptide: long chain of amino acids
112
What is monocistronic mRNA?
Means that any mRNA transcript codes for only one protein
Viruses depend upon their host cells for translation, so viral mRNA must conform to monocistronic mRNA
- Use tactics to ensure their own mRNA is chosen over hosts
113
What are the mechanisms to ensure viruses have successful transcription/translation?
Take adavatange of host splicing machinery to produce several mRNA transcripts from one precursor mRNA (ex. HIV-1)
Take 5'-caps from host mRNA to gain necessary cap for viral mRNA (ex. influenza)
Create mRNA that are translated into one long polyprotein then cleaved into several proteins after translation
Contain internal ribosome entry sites (IRES) to allow ribosomes to bind within mRNA sequence without 5'cap (ex. poliovirus)
Use frameshifting to encode several proteisn with just one portion of DNA (ex. HIV)
Use ribosomal skipping to release a protein with no stop codon, so ribosome continues to create multiple proteins
Use leaky scanning to create two viral proteisn from one viral mRNA
114
What techniques promote the synthesis of viral protein faster with less energy?
```
IRES
ribosomal frame shifting
Termination suppression
Ribosomal skipping
Leaky scanning
```
115
How do viruses interfere with transcription and translation of host proteins?
Interfer with host RNA polymerase II
Interfer with processing of precursor RNA
Interfer with export of mRNA transcript from nucleus
Cause degradation of host mRNA
Prevent translation of host mRNA
116
A PURple Apple Might Redden
```
The 7 stages of replication:
Attachment
Penetration
Uncoating
Replication
Assembly
Maturation
Release
```
117
What are glycosylated proteins?
They have been modified with sugars & carbohydrates
| Most of the plasma membrane proteins
118
Attachment of a virus:
Binds virus to host cell
Very specific:
- Virus attachment proteins adsorbs to cell surface receptor (usually glycoproteins or sugar/carb residue)
* Many antivirals target viral attachment proteins
Ex. Rhinovirus binds to intercellular adhesion molecule 1 (ICAM-1) protein
Ex. Influenza A binds to sialic acid sugars
Ex. HSV binds to glycosaminoglycans (GAGs) to bind to mediator protein or nectins
119
What are coreceptors?
Additional proteins needed to infect cells
Not all viruses need them
Ex. HIV binds to CD4 protein on T cells but needs a coreceptor protein to penetrate cell
- Some humans have modified version of one of the coreceptors, CCR5, which makes the cell resistant to HIV
120
What occurs between the virus attachment protein and cell surface receptor?
Electrostatic forces
| Doesn't require energy
121
Where are viral attachment proteins located?
On capsid or envelope (if one)
Extend from surface of virion or within "canyons" on capsids
Ex. 90% of rhinovirus serotypes bind to ICAM-1 on surface of cells from a deep canyon (formed by VP1, 2, and 3 proteins); 10% attach to very low-density lipoprotein (VLDL) receptors from vertices of the icosahedral capsid (formed by VP1 proteins)
- This shows that different strains of same species can use different cell surface receptors
122
What occurs with penetration of the cell?
Needs to quickly gain access (to avoid extracellular stresses (ex. flow of mucus))
This involves the crossing of the plasma membrane by the virus
Requires energy (via the host cell)
There are different mechanisms:
- Endocytosis (receptor-mediated (caveolin-mediated (ex. papillomaviruses) or clathrin-mediated (ex. HCV, dengue)) or buik-phase)
- Phagocytosis
- Fusion (used only by enveloped viruses)
123
What is tropism of a virus?
Specificity of a virus for a particular host cell or tissue
Viruses can only infect the cells that display the molecules to which their virus attachment proteins bind
Can cause narrow host range
Ex. poliovirus can only attach to CD155 protein, found exclusively in humans
124
What happens during uncoating?
Breakdown/removal or capsid, releasing the virus genome into the cell (to wherever genome replication will occur)
Achieved via different methods
125
How do rhinoviruses penetrate/uncoat?
Use clathrin-coated vesicles
| Expands (4%) and VP1 protein forms pores in the endosome to release RNA genome
126
How do influenza viruses penetrate/uncoat?
Hemagglutinin (HA) binds to sialic acid residues on the surface of respiratory epithelial cells to penetrate cell via receptor-mediated endocytosis
Low pH of endosome causes HA protein to change shape, revealing a fusion peptide to combine two membranes & fuse viral envelope to endosomal membrane, releasing genomes
Enter nucleus via nuclear pores
127
How does poliovirus penetrate/uncoat?
Capsid binds to cell surface receptor, causing conformational changes that create a pore in cell membrane
Viral RNA is released from pore into cytoplasm
128
How do reoviruses penetrate/uncoat?
Remain intact after penetration
| Don't uncoat in cytoplasm to allow a "home base" for genome replication
129
How do Herpesviruses uncoat?
Viral envelope fuses to plasma membrane
Nucleocapsid remains intact and is transported by microtubules to nucleus (proteins bind to dynein that walks them along the microtubules)
At nuclear pore:
- HSV capsid docks at nuclear pores and transports DNA inside nucleus
- HBV capsid can move through pore & uncoats in nucleus
130
What are replication strategies of viruses dependent on?
The type of nucleic acid genome it contains
The Baltimore classification system categorizes viruses into seven classes based on type of genome (replication techniques can be divided into the 7 classes)
131
How can genomes be diversely configured?
Linear or circular
| Nonsegmented (rabies) or segmented (influenza)
132
What happens during assembly?
New components of virion must be collected at particular site of cell to undergo assembly
- Can occur at different locations depending on virus (nucleus, plasma membrane, various intracellular membranes...)
- Most nonenveloped DNA viruses assemble nucleocapsid in nucleus (if too big to get through nuclear pores traverse double-membranes nuclear envelope or induce cell lysis/apoptosis)
- Viruses with envelope derived from plasma membrane usually assemble there
Assembly forms immature virus particle
133
What is the difference between assembly of different capsids?
NA genome in helical capsids is protected by repeating capsid proteins
- Can begin wrapping as soon as copied
NA genome in icosahedral capsids isn't added until assembly of capsid is almost complete
Complex capsids usually use scaffolding proteins (viral chaperone proteins) to assemble
- Large icosahedral viruses use scaffolding proteins (ex. herpesviruses)
134
What happens during maturation?
Needs to occur for virions to be able to initiation infection of new cells
Does the final changes within an immature virion, resulting in an infectious virus particle
135
What changes in maturation occur in influenza viruses?
Does structural capsid changes
- Mediated by host enzymes or virus-encoded enzymes
Influenza HA protein binds to cell's sialic acid after being glycosylated
- HA protein needs to be cleaved in HA1 & HA2 to become infectious (HA1 binds the cell surface receptor; HA2 fuses the viral envelope to endosomal membranes to release virus into cytoplasm)
- Cleaved via cell proteases
136
What changes in maturation occur in HIV?
Gag gene is translated into a polyprotein cleaved via the viral protease to form the:
- Capsid
- Matrix
- Nucleocapsid proteins
Occurs when the virion is in the extracellular fluid
137
What occurs during release of enveloped viruses?
Assemble on inside layer of plasma membrane, embedding their envelope proteins into plasma membrane
Membrane-associated viral proteins cause PM to curve around capsid
Called budding (can occur from any membrane system in cell)
If budding occurs in membrane other than PM, undergoes exocytosis to leave cell
138
What happens during release of nonenveloped viruses?
Three types:
- Exocytosis
- Lysis caused by lytic viruses (very common in naked viruses)
- Budding
139
What is viral pathogenesis?
How viruses cause disease within a host
Must overcome ___ to become successful:
- Sufficient number of virions must enter host
- Host cells must be accessible and susceptible (think of tropism)
- Cells must be permissive to infection (have proteins & molecules necessary for replication
- Must overcome mechanical, chemical, and microbiological barriers (immune system...)
140
What are the different portals of entry?
```
Respiratory tract
GI tract
Genital tract
Skin (direct contact or penetration into dermis/subcutaneous tissue)
Through placenta
Eye
Transplants (solid organs or blood)
```
141
What type of cells do most viruses interact with?
The host epithelium (layers of cells that line the outside surface or inner cavities of the body
Mucosal epithelium is covered in mucus and lines all internal surfaces of the body
Epithelium can be bypassed when viruses are delivered to internal sites through penetration of the skin
142
What happens in the respiratory tract?
Most common portal of entry for viruses
Most of the surface area is covered with mucus
Upper respiratory tract: nose, nasal passages, sinuses, pharynx, larynx
- Has many goblet cells & cilia (mucus contain IgA isotype)
Lower respiratory tract: trachea, bronchi (bronchioles & alveoli), lungs,
- Less goblet cells & no cilia in alveoli
143
What happens to viruses in the respiratory tract?
Viruses in large droplets end up in upper respiratory tract
- Because mucus trap inhaled particulate matter
- IgA isotypes bind to viruses
Viruses in smaller aerosolized particles/liquids are able to travel into lower respiratory tract
- Alveoli contain alveolar macrophages
Ciliated epithelial cells
- Contain receptors for respiratory viruses (influenza, rhinovirus)
- Virus must avoid mucus on epithelium & antibodies/macrophages
144
What happens in the GI tract?
Mouth: enzymes found in saliva break down food
Pharynx: has several mucus-secreting glands
Esophagus: numerous pits of cells produce mucus to protect cells from acidic gastric juices
Stomach: secrete acidic gastric juices
Small intestine: villi, microvilli, goblet cells, antibody-secreting lymphocytes (IgA), macrophages, other immune system cells, M cells (immune system cells)
- Viruses take advantage of additional surface area to infect
- Viruses exploit M cells to get past epithelium (ex. poliovirus, HIV)
Large intestine: Lost of goblet cells
145
Why is the GI tract hostile for viruses?
Flow of water, food, & saliva provide mechanical barriers
Mucus provides a physical barrier
Macrophages phagocytose virions, antibodies neutralize virions
Low pH of stomach acid & bile (most envelopes are disintegrated by bile)
146
What is the difference between acid-labile viruses & acid-resistant viruses?
Acid-labile: unable to withstand low pH of stomach (rhinovirus)
Acid-resistant: contain capsid proteins not denatured by low pH or denaturation is reversible (poliovirus)
147
How can viruses be transmitted in the GI tract?
Mother to child via breast milk (free virions or in cells)
- Ex. HIV, WNV
Fecal-oral route
- Ex. Norwalk virus, poliovirus
148
What happens with viruses the genital tract?
```
Organs involved in reproduction
Genital tract viruses are called sexually transmitted diseases
Viruses enter via:
- Breaks in genital epithelium
- Binding local cell receptors (HIV)
Need to overcome local barriers:
- Mucus
- Low pH of vagina
```
149
What happens in the skin?
Composed of 2 layers; epidermis & dermis (subcutaneous tissue beneath consists of fat & loose connective tissues)
150
What happens in the epidermis?
5 layers (strata) of keratin-producing cells
- Innermost layer (stratum basale) consists of living cells
- Outermost layer (stratum corneum) has dead cells
Has barriers to prevent infection:
- Flow of fluid/perspiration over skin to stop attachment
- Oil from sebaceous glands makes an acidic environment
- Stratum corneum has no living cells for viruses to replicate on
- Need small cuts/abrasions to get to lower stratums
Supports only localized viral infections
Most viruses bypass the epidermis via:
- Insect bites (to dermis or sub tissue)
- Needle punctures (to sub tissue)
151
What happens in the dermis & subcutaneous tissue?
Contains blood vessels & lympatic vessels
Viruses can enter bloodstream directly or through draining lymph that empties into bloodstream
Supports viral infections that travel
152
What happens in the eyes?
External layer of eye: sclera (tough white covering) & cornea (transparent)
Conjunctiva is epithelium that covers sclera & part of eyelid
Viral infections are rare (unless traumatic event occurs)
153
What happens during eye infections?
Cornea is infected via herpesvirus exposure (HSV)
- Causes corneal blindness
Conjunctiva is infected (more common) via adenoviruses
- Causes conjunctivitis (pink eye)
154
How do congenital infections occur?
When mother infects fetus before birth by crossing the placenta (ex. cytomegalovirus (herpesvirus) is most common (2.5% of births), rubella, measles, Zika...)
Via vertical transmission (goes between generations)
- Very uncommon mode of transmission (usually horizontal transmission)
- Don't need high rate of infection to sustain virus population (persist in child)
Effects include miscarriage, low birth weight, intellectual deficiencies, hearing loss, death of infant
155
What is intrapartum transmission?
Child is infected during the birthing process (from contact with mother's infected blood, secretions, or biological fluids)
Vertical transmission of HIV occurs during this usually (can come from breastfeeding but rare)
- More common during symptoms (high viral load)
- Less likely if C-section occurs
156
What happens during blood transplants?
Most commonly transplanted tissue
Before sensitive screening tests, transfusion-transmitted infections (TTIs) were possible (though not probable)
- Now, most viruses are tested for
- In US, before screening of HIV began (1985) over 50% of people with hemophilia got HIV from plasma & over 20k people got HIV from blood
- WNV was reported in 2002 and a test was quickly developed
Viruses that can be transmitted through blood are:
- Hep A
- Hep B
- Hep C
- HIV
- WNV
- Dengue virus
157
What happens during organ transplants?
Less frequent
Potent drugs to suppress the immune system are given to recipients to stop transplant rejection
- Becomes immunocompromised
Ex. Herpesvirus remains dormant in healthy hosts, can come back in transplant hosts (most common)
Ex. rabies, WNV, HIV, Hep viruses, respiratory viruses...
Reduced via:
- Risk questionnaires
- Screening of donor tissues
158
What is a localized infection?
Infect & replicate only within cells at the infection site
Ex. Rhinovirus infects epithelial cells of upper respiratory tract
Ex. Papillomarivus strains that infect skin epidermis
159
What is a systemic infection?
Viruses that initiate infection through one organ but spread to other sites within the body
Either replicate locally before spread or use infected local cells to travel to other locations within bod to replicate
Spread via:
- Hematogenous spread: spread through bloodstream
- Neurotropic spread: spread through the body using neurons
160
What happens during hematogenous spread?
Occurs through:
- Direct injection into the blood (animal or insect bites)
- Virions entering interstitial fluid (lymph) to lymph nodes where high virion conc. can allow some to escape & return to bloodstream
Viremia means that viruses are in the bloodstream
- Primary viremia: first time the virus is found in the bloodstream
- Secondary viremia: after replicating in the target organ, additional virions enter the bloodstream
161
What happens during neurotropic spread?
Rare to infect directly b/c difficult for viruses to access cells
- More common for viruses to replicate in cells at local site of infection & then infect neurons located nearby
First infect neurons of peripheral NS, then access CNS
- Ex. Once rabies reaches brain, causes death
162
How does the herpesvirus infect neurons?
First replicate within local epithelium then infect nerves associated with said tissue
dsDNA viruses (need nucleus to replicate)
- Virus first attaches to axon terminal (far from nucleus of neuron)
- Overcome this by binding to dynein (walks vesicles along microtubules to nucleus)
- At nucleus, capsid docks at nuclear pore & transport DNA into nucleus
163
What does shedding of viruses mean?
The release of infectious virions from a host
Localized infections: shed from primary site of infection
Modes of shedding:
- Skin: skin-skin contact
- Respiratory: cough, sneeze
- GI tract: aerosolized vomit or diarrhea
- Lungs, nasal cavity, salivary glands: saliva
- Genital: semen or vaginal secretions
- Viremia: blood
- Viruria: urine (systemic: measles, mumps) (local: JC polyomavirus, BK polyomavirus)
164
What is the stability of a virion outside a host?
```
Can remain infectious
Depends upon several factors:
- Biochemical characteristics of virion (type of NA, sensitivity or proteins to pH, (non)enveloped)
- Temperature
- Humidity
- Moisture content
- Sunlight
- pH
- Presence of organic matter
```
165
How do feces protect viruses?
Neutral pH
Organic matter buffers the chemical makeup & temp of environment
At high temp, inactivated
- Nonenveloped can remain infectious for days-months at ambient temps
166
What happens to airborne viruses?
Droplets spread short distances cause of size (about 20um)
Aerosolized particles travel farther (about 5um)
- Evaporate & allow particles to remain airborne for longer
Viruses are better protected from inactivation
Temp & humidity play key role:
- Enveloped respiratory viruses are better in lower temp & humidity (ex. influenza A, measles, SARS-CoV)
- Droplets fall out of air faster with higher humidity
- Nonenveloped viruses remain infectious longer in higher humidity environments (ex. rhinovirus, adenovirus)
167
What are the four patterns of infection?
```
Incubation period
Prodromal period
Illness period
Convalescence period
* Communicable (infectious) from end of incubation period to beginning of convalescence period
```
168
What happens during the incubation period?
Virus infects host
No symptoms appear
Can last hours to months
Ex. Rhinovirus = about 1-3 days
169
What happens during the prodromal period?
After incubation period
Symptoms first appear (nonspecific, mild symptoms)
- Low fever, muscles aches, malaise (faint)
Virus is quickly replicating
170
What happens during the illness period?
After prodromal period
Specific symptoms appear
Multiplying to high levels & immune system is activated
- Response takes time
In immunocompetent hosts, infected cells will be eliminated
171
What happens during convalescent period?
Symptoms subside
| Can last days or months (depends on severity of infection)
172
What is a carrier?
A reservoir that can transmit the virus with no symptoms
During incubation & convalescence period or in asymptomatic hosts
Communicability period varies depending on virus
Ex. Influenza is contagious from 1 day before symptoms arise to a week after becoming sick
Ex. Ebola virus is communicable in breast milk & semen weeks-months during convalescent period
173
What is an acute infection?
Virus replicates quickly and spreads to other individuals
Immune system clears virus (usually in 7-10 days)
Most epidemics are acute
Inapparent acute infection: produce no symptoms, but virus still replicated & activates immune system
- Not same as unsuccessful infection where virus can't successfully replicate in host & doesn't establish infection
- Not same as abortive infection where virus enters cells but can't complete replication due to no materials in cell to replicate with
174
What are persistent infections?
Last lifetimes usually
Done via:
- Viral proteins can modulate immune system & some viruses (HIV) can infect immune cells to stop proper functioning
- Production of defective interfering (DI) particles: incomplete or deleted genome virions that sequester antibodies & interfere with apoptosis of infected cells (more common in RNA viruses)
- Certain organs stop inflammation, which can protect cells against apoptosis and allow viruses to persist
- Viral latency, where virus remains dormant in shot cells
- Slow infections
175
What is viral latency?
Virus is dormant in host cells
Largely undetected by immune system
Can be reactivated later in life (ex. varicella zoster: chicken pox to shingles)
Ex. herpesviruses
176
What is a slow infection?
Can take years to reach symptomatic phase (if ever)
| Ex. HIV take years to arise and allow opportunistic infections
177
What are the different types of infection classifications based on their frequency?
Sporadic disease: infrequently with no consistent pattern
Endemic: usually presence of disease
Epidemic: More cases of disease in particular area than expected
Pandemic: epidemic spreading to several countries/the world
178
What is the epidemiologic triad?
Consists of three factors:
- External agent of pathogen
- Susceptible host
- Environment
Interventions between:
- Agent & host: protect, educate, alter exposures
- Host & environment: educate, change activity patterns, quarantine
- Environment & agent: remove breeding grounds, improve sanitation
179
What are the characteristics & interventions of the external agent?
```
Characteristics:
- Toxicity
- Virulence
- Infectivity
- Susceptibility to antibiotics
- Ability to survive outside body
Interventions:
- Eradicate
- Genetically modify
```
180
What are the characteristics & interventions of the environment?
```
Characteristics:
- Climate
- Physical structures
- Population density
- Social structure
Interventions:
- Housing quality
- Sanitation
- Preventive services
```
181
What are the characteristics & interventions of the host?
```
Characteristics:
- Age
- Prior exposure
- Susceptibility
- Co-infection
- Immune resposne
Interventions:
- Treat, isolate
- Immunize
- Nutrition
```
182
What is the difference between a vehicle & a vector?
Vehicle: nonliving physical substances that can indirectly transmit virions
Vectors: living intermediaries that can transmit viruses
- Arbovirus is used to denote arthropod-borne viruses
183
What step are control measures directed at usually:
Controlling/eliminated virus at the source
Preventing transmission
Protecting portals of entry
Increasing host defenses
184
What do PRRs do?
```
Called pattern recognition receptors
Part of immune cells that recognize pathogen-associated molecular patterns (PAMPs)
Can recognize:
- Viral NA in cells
- Virions attached to surface of cells
Two main classes:
- Toll-like receptors (TLRs)
- RIG-I-like recpeotrs (RLRs)
Induce production of cytokines
Found on macrophages
```
185
What are TLRs?
10 members of TLR family
- 6 recognize viral PAMPS: TLR2,3,4,7,8,9
- Located in cell at locations where viruses enter & replicate
- TLR2 & TLR 4 are transmembrane: interact with viral envelope proteins
- TLR3, TLR 7, TLR 8, & TLR9 sense viral NAs located in transmembrane endosomes: interact with NAs during uncoating
- TLR3: dsRNA
- TLR7 & TLR8: ssRNA
TLR9: viral DNA
186
What are RLRs?
Found within cytosol instead of membrane
Only recognize viral RNA (not bacterial)
- Recognize ssRNA or dsRNA of actively replicating viruses
187
What are cytokines?
Proteins that cause effects within target cells through cytokine receptors
- Code for proteins to activate immune system OR interfere directly with process of viral replication)
Main classes:
- Type 1 IFNs (interfere with influenza replication): reduce viral replication
- IFN-alpha
- IFN-beta
Plasmacytoid dendritic cells produce high amounts of IFN-alpha/beta after sensing viral NAs through TLRs
188
What are the 2 best pathways to an antiviral state induced by type 1 IFNs?
dsRNA-dependent protein kinase (PKR) pathway
- Cytoplasmic protein activated via dsRNA
- Binds & inactivates eukaryotic translation initiation factor 2alpha (eIF-2alpha)
- Stops synthesis of viral & host proteins by ribosomes
2'-5' oligoadenylate synthetase-ribo-nuclease L (OAS-RNase L) pathway
- Activated by dsRNA
- Synthesizes 2'-5' oligoadenylates that bind & activate cellular enzyme RNase L (degrades ssRNA)
- Viral mRNA & ssRNAs and host mRNA are cleaved
189
What are dendritic cells?
Professional antigen-presenting cells (APCs) pick up & process antigens
Antigens taken from:
- Phagocytosis of virion
- Phagocytosis of dead cell
- Endocytosis or pinocytosis of viral antigens in tissues
- Directly from viruses that have infected dendritic cells
Travel within lymph to lymph node to present antigens to T cells in lymph node & spleen
190
What are Natural killer cells?
NK cells possess lytic granules that induce apoptosis of infected target cells
Proteins delivered to target cell by NK cell set of cascade of events that activate enzymes called caspases
-Caspases fragment DNA in nucleus
Type 1 IFNs inc. the killing activity of NK cells
191
What is a T cell receptor (TCRs)?
Transmembrane receptor that specifically recognize antigens presented by dendritic cells
Antigens are presented on dendritic cells via MHCs
- CD8 (CTLs) recognize MHC I (located on all cells)
- CD4 (helper) recognize MHC II (located on certain immune cells: dendritic cells, macrophages, & B cells)
If MHC I are present, NK cells are inactivated
192
What are naive lymphocytes?
Lymphocytes that haven't previously encountered their antigen
193
What is the purpose of B cell?
Activated by helper T cells
- This differentiates the B cell into an antibody-producing plasma cell
Produce antibodies (immunoglobulins)
Contains B cell receptors (BCRs) that bind antigens
- Antibodies are same as BCRs except they leave the cell
194
What are the different antibody classes (isotypes)?
IgM: first antibody made; stays in bloodstream
IgG: have higher affinity; allows macrophages & NK cells to bind antibody-coated viruses; travels anywhere (including placenta)
- By differentiating between IgM & IgG, can tell if patient has recent infection (IgM only) or chronic infection/second exposure (IgG)
IgA: produced at mucosal epithelial surfaces & breast milk
- IgG & IgA are effective at neutralizing viruses by binding to external surfaces to prevent attachment
195
What is antibody-dependent cell cytotoxicity?
When the NK cells binds to antibody on a virus
196
What is cross reactivity?
When antibodies against one antigen recognize a similar antigen
Ex. vaccinia & variola viruses
197
What factors effect the duration of immunological memory?
Characteristics of the pathogen
| Design of the vaccine
198
What is the difference in immune system evasion by RNA viruses vs DNA viruses?
RNA viruses: have error-prone RNA polymerases b/c no proofreading so mutate quickly (no immunological memory)
DNA viruses: have large genomes that encode immune evasion proteins
199
What is severe combined immunodeficiency (SCID)
Genetic disorder that results in major defects of the immune system
Important immune cells are absent or nonfunctional
200
What are the ways for viruses to evade the immune system?
```
Antigenic variation
Antigenic drift
Reassortment
Antigenic shift
Latency
Virus-encoded evasion mechanisms
```
201
What is antigenic variation?
If viral mRNA has a mutation, different amino acid can be incorporated into the protein, leading to slightly different antigen created
Immune cells may not recognize new antigen
202
What is antigenic drift?
Constant acquisition of small mutations in viral proteins
| Frequent in influenza viruses & rhinovirus
203
What is reassortment?
Segmented viruses that reassort their genetic information creating new subtype of virus
204
What is antigenic shift?
The result of reassortment
New combination of antigens present, so immune cells have no memory
Ex. Influenza can create a human infection from an animal infection
- Can result in epidemics or pandemics
205
What is latency?
Remain dormant until immune system is weakened
No replication occurs, so no antigens produced
Ex. EBV
206
What are virus-encoded evasion mechanisms?
Genes whose protein products interfere with host immune response
More likely in DNA viruses
Types:
- Infect dendritic cells & prevent them from properly presenting antigen
- Interfere with loading of peptides into MHC I or presentation (so not recognized by CTLs)
- Interfere with various stages of host defense & inflammatory response (interfere with cytokines or antibodies)
Ex. Poxviruses and herpesviruses
207
How are vaccines tested?
```
Preclinical trials
- Drug approved for testing in humans
Phase 1: 20-80 people
Phase 2: 100-300 people
Phase 3: 1,000-3,000 people
- Drug submitted for FDA approval
FDA review
- Drug approved
Phase 4: 1,000+ people
```
208
What is variolation?
Pulverized dried smallpox scabs inhaled or injected
Led to milder form of disease
Resulted in 2-3% fatality
Used till 1700s
209
Who was Edward Jenner?
Farmer Jesty used cowpox lesions to prevent his family from contracting smallpox
Jenner tested this:
- He collected fluid from a cowpox sore on hand of milkmaid & injected into young boy
- Boy was protected from the virus
- Spread the technique & promoted his vaccine
210
Who was Louis Pasteur?
Created the second viral vaccine
Injected rabbits with rabies & removed their spinal cords, dried them, and used them to vaccinate dogs
- Tested against a boy who had been bitten by a rabid dog (worked)
211
Why are embryonated eggs used for vaccines?
Discovered by Alice Woodruff & Ernest Goodpasture
Support the growth of viruses
Used before cell growth
212
What do most vaccines rely upon to work?
The humoral response (production fo antibodies)
| Some need the cell-mediated response (T cells)
213
What are adjuvants?
Molecules that boost the immunogenicity of the vaccine
Can:
- Sequester antigens in the tissue for longer
- Stimulate pattern recognition receptors
214
What are the 5 types of viral vaccines?
```
Used in humans:
- Live attenuated
- Inactivated
- Recombinant subunit
Experimental:
- DNA vaccine
- Recombinant vector
```
215
What is a live attenuated virus vaccine?
Most common
Infectious, but weakened virus
Done by repeatedly growing virus in nonhuman cells or human cells at nonoptimal temperatures to create mutations to replicate in the new cell line rather than in the normal human cell targets
Ex. Varicella zoster virus vaccine against chicken pox was grown in nonhuman cells
Ex. The rubella virus vaccine against measles was adapted to replicate at 30C, while human body temp is 37C
Pros: stimulate strong T & B cell responses
Cons: need to be refrigerated to remain immunogenic, some viruses can't propagate in lab, can mutate
- Caused vaccine-associated paralytic polio (VAPP)
216
What is the inactivated virus vaccine?
Virus is completely inactivated via:
- High heat
- Low amounts of formaldehyde
Booster shots help maintain immunity
Ex. Poliovirus (can still be infected with wild poliovirus strains)
Pros: Won't mutate, don't need to be refridgerated
Cons: not all viruses are immunogenic after inactivation, some only produce short-lived immunity (mumps), some virues can't propagate in lab
217
What is used instead of live or inactivated virus vaccines?
Recombinant protein expression systems
| Don't need to propagate virus in lab
218
What is the recombinant subunits vaccine?
Uses recombinant DNA (DNA from one organism placed in another)
A viral gene is placed on a plasmid (called gene cloning)
Ex. HBV can't replicate in cell culture. It encodes 7 proteins, 1 is displayed on the surface (HB surface antigen (HBsAg)) so taken and placed in plasmid (of bacteria or yeast)
Ex. HPV
Ex. Yearly flu vaccine includes HA proteins from 3+ influenza strains
Pros: Doesn't need to be propagated
Cons: determining which & how many proteins needed to ensure adequate immunity, needs booster shots, expensive
219
What are DNA vaccines?
Experimental
Deliver viral DNA directly into the cells
- DNA is transcribed & translated so host immune system responds
Delivered via:
- Into muscle
- Gene gun: coats bits of gold with DAn & uses compressed air to shoot into skin cells (called particle bombardment) (needs direct contact with tissue of interest)
- Needle-free injector using compressed air
- Electroporation: electric pulse creating temporary pores in PM
Pros: don't need to propagate, less expensive than recombinant subunit vaccine
Cons: need to understand immunological response, which promoter should be used, can't generate protective immune response on their own
Ex. WNV in clinical trials for humans, used in horses
- Inserted premembrane & envelope genes
220
What is a prime-boost strategy?
DNA vaccine is followed by a vaccine that uses a virus to deliver the same or different antigens
221
What is a recombinant vector vaccine?
Uses viral vectors as carriers to deliver DNA
Vector DNA of an attenuated virus is modified to include DNA that encodes the viral antigen of interest
Used in wildlife vaccines as ingested
Ex. Rabies
Pros: Prevent generation of additional infectious vector virus, have been used in humans before so they are safe
Cons: Expensive, proteins are immunogenic so immune system responds to vector virus & vaccine antigen
222
What are the types of passive immunity?
Natural: from mother to baby
Artificial: antibodies acquired from an immune serum medicine
- IVIG therapy: immediately neutralizes virions to prevent continued infection (used against HBV, rabies virus, vaccinia virus)
223
How do antivirals work?
By blocking one of the seven stages of the viral life cycle (except assembly)
Most inhibit viral enzymes involved at 1+ stages or prevent genome replication
224
What's the difference between antivirals & antibiotics?
Antivirals are limited, can't cure the infection, affect host cells, specific
Antibiotics kill teh bacteria present, can be targeted, can be wide range
225
What is highly active antiretroviral therapy (HAART)?
Uses multiple antiviral medications to attack the virus
| Due to fast mutation
226
What antivirals inhibit entry?
Immunoglobulins: bind to virus & prevent attachment
Maraviroc (inhibits HIV-1): inhibits binding to corecpetor, CCR5 (doesn't work against strains that use CXCR4)
- CCR5 is not only protein that carries out that activity, so blocking it does not harm cell
Enfuvirtide (inhibits HIV): peptide that binds HIV protein that fuses the envelope to the PM
Docosanol (inhibits HSV-1): same as Enfuvirtide
227
What antivirals inhibit uncoating?
Amantadine & rimantadine (influenza): small molecule drug that inhibits M2 protein (forms pores in viral envelope) so endosomal fusion & uncoating are inhibited
228
How do nucleoside analogs inhibit genome replication?
Most antivirals inhibit replication (mostly those that reverse transcribe)
Nucleoside analogs: small molecule drug that terminates reverse transcription; compete with normal nucleosides & are converted into nucleotide analogs that are incorporated into strand that can't bond to subsequent nucleotides, terminating the growing strand
- Called nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) when they use viral polymerases (HIV or HBV)
- Ex. HCV uses sofosbuvir (nucleoside inhibitor) to copy uracil (U)
- Ex. Herpesvirus use enzymes to inc. nucleotides such as TK, which doesn't act upon purine (what the analog is made of)
229
What is a nucleoside?
DNA or RNA nucleotide without the phosphate group (nitrogen base + sugar)
Converted into nucleotides during DNA/RNA synthesis put into the replicating genome
230
What antivirals inhibit maturation?
Protease inhibitors: block HIV protease that cleaves the gag gene (stops virion from becoming infectious)
231
What antivirals inhibit release?
Neuraminidase inhibitors: inhibits influenza A virus neuraminidase, which cleaves the sialic acid from the cell surface; prevents attachment of virions to the cell
Ex. Oseltamivir, zanamivir, peramivir
232
What antivirals inhibit genome replication?
Nucleoside analogs
Foscarnet: inhibits binding site of herpesvirus DNA polymerase
Ledipasvir: interferes with HCV RNA polymerase (this + sofosbuvir cures over 90% of chronic HCV)
Nonnucleoside reverse transcriptase inhibitors (NNRTIs): small molecule drugs that bind to active site of HIV-1 reverse transcriptase, disruptive RNA-dependent DNA-dependent DNA polymerase
Integrase inhibitors: block the active site of integrase to stop HIV from integrating into a host chromosome
233
What is the human immunodeficiency virus (HIV)?
Originated in Africa in 1920s from cross species infections (human & simian)
Causes acquired immune deficiency syndrome (AIDS) first in 1981
Infects helper CD4 T cells, causing them to decline
Now able to manage for years with antivirals
Today, about 1.5 mill people worldwide die/year (6th most common death)
Two species: HIV-1 & HIV-2
In Retroviridae family (replicate through reverse transcription of +ssRNA genome into cDNA, which permanently integrates into host genome); Orthoretrovirinae subfamily, Lentivirus genus
234
What is the difference between HIV-1 & HIV-2?
Cause clinically indistinguishable conditions
| HIV-2: Viral loads are lower; less easily transmitted; progression to AIDs is slower (if it occurs)
235
What are the classes of HIV-1?
Group M (main group)
- Cause the pandemic (worldwide)
- Responsible for over 90% of HIV infections
- Subtypes A,B,C,D,F,G,H,J,K
- Subtype C is responsible for 50% of total infections (worldwide)
Group O (outlier)
- Responsible for 1% of HIV-1 infections (in Cameroon, Gabon, & neighboring countries: Central Africa)
Group (not M or O)
- Only 15 cases documented (in Cameroon only)
Group P (new)
- Only 2 cases documented (in Cameroon only)
236
What are the classes of HIV-2?
```
Group A
- Most prevalent (with Group B); others are rare
- Found in Western Africa
Group B
- Most prevalent (with Group A); others are rare
- Found in Cote d'Ivoire (Central Africa)
Group C
Group D
Group E
Group F
Group G
Group H
```
237
What is the percentage of infectious diseases that come from wildlife?
70%
| This includes HIV
238
What is Simian immunodeficiency virus (SIV)?
Every HIV-1 & HIV-2 group results from different cross-species infection from primates
- All HIV-2 groups originated from SIVmm (SIV of mangabey monkey)
- HIV-1 M & N originated from SIVcpz from Cameroon
- HIV-2 O & P originated from SIVgor from Cameroon
Crossed to humans from blood
- From hunting
Designated using lowercase letters that describe the species (Ex. SIVcpz = chimpanzee)
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How is HIV transmitted?
Sexual contact that exposes mucosal epithelium to semen, vaginal secretions, rectal secretions or blood
- Using condom stops 80% infections)
Mother to child (transplacentally, during birth, from breast milk)
- Depends on mother viral load at time of birth
- 40% of infants are infected at birth
- Reduce this by 70% through C-section
Through blood (needles, infectious materials)
- Less likely
240
What are the clinical stages of HIV infection?
Acute
Asymptomatic (clinical latency)
AIDS
241
What happens during the acute stage of HIV?
Occurs with 2-4 weeks of infection for 50% of infected
Develop flu-like symptoms
After a week of symptoms, HIV replicates to highest levels (10^6-10^7 viral RNA/ml of plasma)
- Associated with inc. in CD8 T cells & reduction in CD4 T cells
Last 2-4 weeks
242
What is seroconversion in HIV?
Occurs after 2-4 weeks of acute stage
CD4 T cells recover
Goes into asymptomatic stage
243
What happens during the asymptomatic stage of HIV?
HIV slowly replicates, causing the gradual decline of CD4 T cells
Last about 10 years
- The actual progression can be predicted based on the viral load & CD4 T cell count
- Min is 2-3 years in rapid progressors (10-15% of infected))
- Long-term nonprogressors (LTNPs) are 5% of infected that keep normal CD4 T cell counts & low HIV (AIDs still occurs, just a lot slower (decades))
- Elite controllers are subset of LTNPs that keep viral loads below limits of detection
244
What happens during the AIDS stage of HIV?
CD4 T cells fall below 500cells/uL
- This increases opportunistic infection chances
Herpesvirus reactivation causes EBV or KSHV in HIV+ people
- Before antiviral therapy (ART), 20% of patients with AIDs got Kaposi's sarcoma from KSHV
Cervical & anal cancers from human papillomavirus
Antiviral drugs, infection of kidney cells by HIV, & antigen-antibody immune complexes lodging in the kidneys increases the chance of kidney disease
Neuronal damage occurs in 50% of HIV people
- Encephalitis (inflammation of brain)
- Meningitis (inflammation of covering of brain & spinal cord)
- Dementia in 40-60% of individuals before ART
245
What does the HIV virion capsid look like?
Enveloped retrovirus with complex capsid
Capsid is built from one protein, capsid protein (CA)
- Forms a cone or bullet-shaped core
Pseudodiploid cause only one copy of the RNA is used for reverse transcription
Nucleocapsid protein (NC) coats the RNA & the viral enzymes required for replication:
- Reverse transcriptase (RT)
- Integrase (IN)
Protease (PR)
246
What does the HIV virion envelope look like?
120nm
The matrix protein (MA) attaches to the inner surface, keeping its shape
Contains trimer envelope (ENV) glycoproteins containing gp120 (surface subunit) & gp41 (transmembrane subunit)
- Cleaved from gp160
247
How does HIV effect dendritic cells?
Virions gain entry by bypassing mucosal cells (epithelium) & tight-junctions or going through tears in epithelium
HIV binds to a receptor, DC-SIGN, on dendritic cells that triggers the cells to endocytose the viruses
When the dendritic cell comes in contact with CD4 T cells, virological synapse occurs (HIV is exocytosed & comes in contact with T cell)
- Think of the dendritic cell as a trojan horse
248
What coreceptors do HIV virions use?
CCR5 or CXCR4 (depending on variations in the gp120 molecule)
- CCR5 is primary (macrophages & memory CD4 T cells)
- CXCR4 arises later in infection (naive T cells)
Switching occurs due to progression into AIDS
249
How does HIV attach & penetrate?
Bind gp120 to CD4
Causes a change to reveal a coreceptor
gp120 binds to coreceptor, causing gp41 to insert into the cell membrane
- Creates a fusion pore
The nucleocapsid is release into the cytoplasm
250
How does uncoating of HIV occur?
Don't know
251
What are the redundant sequences on the HIV genome?
On both ends of the genome (5' (called U5) & 3' (called U3))
| Form long terminal repeats (LTRs) on teh cDNA used to integrate HIV cDNA into host's DNA
252
What happens during HIV reverse transcriptase?
Reverse transcriptase enzymes are targets for antivirals
- Unique because it can do several functions (DNA polymerase (proofreading), RNase H activity)
Steps:
- Cellular tRNA binds to primer-binding site (PBS)
- RT binds & adds DNA nucleotides, forming negative-strand strong-stop cDNA
- RT RNase H degrade the RNA of the RNA:DNA section
- Stop cDNA is transferred to RNA & binds to R site
- Synthesis occurs, creating -sense DNA strand
- RNase H degrades RNA template (except polypurine tract (PPT))
- PPT acts as primer for synthesis of +sense DNA strand
- +strand strong-stop cDNA is transferred to complete replication, creating LTRs
253
How does HIV get into the nucleus?
HIV cDNA crosses with assistance of proteins (cause its a lentivirus)
- Able to replicate in nondividing cells
The preintegration complex (PIC) is the cDNA & proteins taht allow cDNA into the nucleus
- The structural proteins (CA, NC, & MA), enzymes (RT & IN), and accessory proteins (Vpr) interact with the nucleoporin proteins
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Why are the LTRs on the resulting HIV DNA important?
Important during integration of proviral DNA into genome of host cell
Integrase (IN) carries out integration
- Removes two bp from each end of proviral DNA, creates a nick in host chromatin, & join proviral DNA & host DNA
- HIV is now called a provirus
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What major genes do all retroviruses contain?
gag, pol, env
| Translated into polyproteins later cleaved to form the structural proteins & enzymes
256
How does HIV assemble?
Gag precursor polyprotein
MA
Env subunits gp120 & gp41
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How is HIV released?
Buds from surface of cell (gets an envelope)
| Viral protease cleaves several sites with Gag & Pol precursors to mature
Eukaryotic Cells & Viruses
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