Viral Pathogenesis I & II Flashcards
What is the primary method of virus detection from a patient sample (especially acute virus infections)?
Why is it used over other tests?
1) PCR for viral nucleic acids
2) Good sensitivity, specificity, & quick turn around time
Other methods for detection of a viral infection:
For Review
1 - Don’t worry about it! Exclude bacterial infection and support symptoms
2 - Detection by virus-induced changes in cellular morphology or evidence of cell killing (pathologists)
3 - Detect virus antigen(s) in sample by antibodies: ELISA, Western blot, Immunostaining of histology slides
4 - Electron microscopy
5 - PCR for viral nucleic acids
Methods 2-5 can be used directly on tissue, swab, blood, or exudate samples or can be done after the virus is replicated in tissue culture
6 - Detection of antibodies against the virus (serological tests): ELISA (indirect), Western blot
What type of infection is serological testing (detection of antibodies against the virus) useful for identifying? Acute or chronic?
Chronic
It is not useful for acute infections since a minimum of 2-16 weeks is needed for generating antibodies
What viral infections is serological testing useful for as an initial diagnostic tool?
Chronic viral infections such as HIV, Hepatitis C, CMV, or Hepatitis B
Between qualitative and quantitative (RT) PCR:
What steps are similar?
What steps are the same?
These steps are the same:
1 - If RNA virus, convert RNA to DNA with reverse transcriptase and primer
2 - Amplify DNA with specific primers for virus of interest (usually for 35/40 cycles)
This step differs:
Qualitative - 3 - run samples on an agarose gel with a molecular weight marker to verify presence of band and correct size of band
Quantitative - 3 - Production of amplified DNA product is detected by either a fluorescent reporter probe or fluorescent dye that intercalates between DNA
What are the major (2) causes of cell and tissue damage? Is all the damage done by the virus?
The major causes of damage are by the virus itself & the host defense functions, so no only part of the damage is done by the virus, itself. Either of these can alone cause damage, or they can both be at work
How do the two causes of cell and tissue damage correlate with the viral load?
- Virus replication - severity of disease directly correlates with quantities of virus produced
- Killing of infected cells, usually by T cell response, may not be highly correlated with level of virus replication or quantitatively
What is viremia?
Circulating virus in the blood
What is the pattern of infection and symptoms in an acute infection?
A high level of viral load correlates with symptoms. Then, the virus is cleared and symptoms will either resolve or may also manifest after drop in virus titers if too robust of an immune response is partly responsible for symptoms
What is an example of a virus that causes an acute infection?
Influenza virus
What is the pattern of infection and symptoms in an acute infection followed by latent infection with subsequent reactivation?
An acute infection (with symptoms) is followed by latency where there are no symptoms and no viral load. Then, a weakened immune system brings on a reactivation of infection with symptoms.
What is an example of a virus that causes an acute infection followed by latent infection with subsequent reactivation?
All herpes viruses
What is the pattern of infection and symptoms in an acute infection followed by chronic infection?
An acute infection (with symptoms) is followed by a consistent low level of virus where symptoms are seen later in the chronic infection
(severity does not always correlate with viral load)
What is an example of a virus that causes an acute infection followed by chronic infection?
HIV, Hepatitis B, Hepatitis C
What is the pattern of infection and symptoms in a slow chronic infection?
Slow build in viral load from very tiny levels with symptoms seen later in the chronic infection (after years)
What is an example of a virus that causes a slow chronic infection?
Not a virus - prions
What is the pattern of infection and symptoms in an immortalizing infection?
An acute infection without symptoms followed by either a chronic/persistent level of virus or only portions of the viral genome present. Symptoms present months to years later when malignancy occurs
What is an example of a virus that causes an immortalizing infection?
HPV
What is the pattern of infection and symptoms with a change in location of infection?
Early acute phase with no symptoms, then very low levels. Within days viral load increases significantly in a new location with the first occurrence of symptoms. Then, a latency period of months to years followed by reactivation with symptoms
What is an example of a virus that follows that pattern of latency and reactivation with a change in location of infection?
Varicella zoster (a herpes virus)
What is the pattern of infection and symptoms with varicella zoster?
Infection of respiratory mucosa with primary site of replication (respiratory epithelium, draining lymph nodes) -> primary viremia (at ~5 days)
Then, virus seeds other organs (spleen, liver, kidney, etc) -> secondary viremia (at ~12 days) with higher viral load and symptoms
Then, virus replicates in the target tissue (skin) and characteristic disease with rash occurs (at ~15 days)
After acute infection subsides, virus goes into latency in neural ganglia with no detectable virus replication
Years later virus is reactivated and infects epithelial cells along the dermatome of the neural ganglia
The virus can again become latent and reactivate
What is the iceberg concept of outcome of exposure to virus?
Most exposure to virus does not result in disease at all. Severe disease and death are relatively rare consequences of viral infection Most prevalent to least: Exposure without infection Infection without illness Mild disease Severe disease Death
What viruses are examples of the iceberg concept of disease outcomes?
Poliovirus, West Nile virus
What viruses exclusively infect humans and solely circulate in the human population?
Smallpox, polio, HIV
What viruses have broader species tropism with animal reservoirs but once introduced into a human population can spread person to person?
Influenza (animal reservoir - ducks), new Ebola virus outbreak (animal reservoir - bats)
What viruses have a significant human reservoir, but transmission NEVER occurs human to human but always thru a vector such as a mosquito?
Dengue virus, Yellow fever virus
What viruses are almost exclusively found in an animal or insect vector reservoirs and human infections are incidental. Little to no human to human transmission occurs nor are titers in humans high enough that there is transmission back to the vector?
West Nile virus, rabies virus
What is an example of a virus that becomes a systemic infections (circulate in bloodstream)?
Ebola virus (can be transmitted via open sores in skin, direct blood transmission, GI, respiratory, hand to eye but regardless of route of transmission, becomes systemic infection)
What are examples of viruses that remain limited to one organ system?
HPV only infects epithelial cells, limiting infection to the skin and/or mucosal epithelium
Influenza infection is limited to the respiratory tract (in humans)
What are the 3 most common routes (portals) of entry of a virus?
Upper respiratory tract
Oral
Sexual contact
What are the less common routes (portals) of entry of a virus?
(For review)
Fomites (clothes, tissues, etc) Blood transfusion Tissue transplant Zoonoses Maternal/neonatal
How does the viral structure (enveloped vs naked) affect the route of entry/transmission?
Enveloped viruses are sensitive to drying & may require close contact, body fluid, or droplet transmission
Naked viruses are more stable & may be picked up or ingested from surfaces. Many survive passage thru the stomach
What are the determinants of cell or tissue tropism of a virus?
1 - VAP-receptor interactions (presence of receptor on cells)
2 - Cellular permissiveness: stage of differentiation, mitotic activity, transcriptional permissivity
3 - Cellular restriction: type 1 interferon responses, viral restriction factors
How are viruses maintained in the human population?
For review
Other humans Animal/insect reservoirs Seasonality Immune status Susceptibility of human host: age
Contrast outbreak, epidemic, and pandemic:
Outbreak - really small (eg 2 dozen people)
Epidemic - multiple countries involved (eg ebola virus right now)
Pandemic - worldwide (eg influenza each year: starts in Asia, thru Europe, thru America, and then to South America)
What are the two forms of a noncytolytic infection?
Active replication of virus with little/no impact on physiology of the cell
Viral latency
What are the three mechanisms of cytolysis?
Altered cell morphology: Inclusion bodies, syncytium formation
Altered cell physiology: eg shut off translation of host messages
Activation of cell killing mechanisms
What are the 4 major cellular outcomes of viral infection?
Noncytolytic infection
Cytolysis
Transformation
Abortive infection
Define syncytium formation:
Induction of cell-cell fusion by viral entry proteins
End-product is large, multi-nucleated cell
What are the two direct methods for virus-induced tumors?
- DNA viruses usurp the cell cycle to make the cell ready for viral DNA replication
- Some viruses keep the cell dividing to prolong a short-lived latent infection in that cell’s descendants
What is the indirect method for virus-induced tumors?
Chronic virus replication leads to chronic inflammation. Chronic inflammation is tumorigenic
Which DNA viruses cause tumor formation?
Papillomaviruses (HPV)
Herpesviruses: Epstein-Barr virus (EBV) & Human herpesvirus 8 (HHV8)
Hepatitis B virus (HBV)
Adenovirus (not in humans)
Which RNA viruses cause tumor formation?
Human T cell leukemia virus (HTLV-1) - retrovirus
Hepatitis C virus - liver cancer
Many different animal retroviruses causes animal cancers but no human disease
What is innate immunity? Review
Memory?
Initial immune response to virus infections that are occurring for the first time
No memory
What are the basic steps of the innate responses? Review
What cells are involved
1) Recognizing pathogen with a PRR (pathogen recognition receptor) - PRRs present on most cells (macrophages, dendritic cells, fibroblasts, epithelial cells)
2) Phagocytosing foreign antigen and presenting peptide to adaptive immune cells - macrophages, dendritic cells
OR
1) Directly detecting problems with infected cell and killing it - NK cells
What do PRRs bind to?
PRRs (pathogen recognition receptors) bind to common molecules from potential pathogens (PAMPs - pathogen associated molecular patterns)
What molecules are encoded in the pathogen genome and not changed/modified in response to pathogen interactions?
PAMPs
What are the different types of PRRs?
TLRs - most common RIG I & related receptors NOD-like receptors C-type lectins others?
Which TLR is a dsRNA sensor?
TLR3
Which TLR is a ssRNA sensor?
TLR7/TLR8
Which TLR is a DNA (CpG) sensor?
TLR 9
What do TLRs 3, 7, 8, & 9 have in common?
They all bind to some part of viral genomes and RNAs
They promote production of type 1 interferon
They are all endosomally located
What do TLRs 2, 4, & 13 have in common?
They interact with some viral proteins
They promote type 1 interferon production
What is RIG I?
RIG I and related receptors are cytoplasmically located RNA helicases that bind dsRNA
This binding leads to type 1 interferon production
RIG I is more important than TLRs for what type of virus (DNA/RNA)?
RNA
What are NOD-like receptors?
Many detect bacterial wall products
Some form inflammasomes with caspase I
Generate pro-inflammatory cytokine (IL-1beta)
Result in strong pro-inflammatory cytokine production
What are C-type lectins?
Bind specific sugars in glycan chains
Believed to be immunomodulatory
What are the 2 type I interferons?
IFN alpha and beta
What cytokine is incredibly important for controlling viral infections?
Type 1 interferons
What cells produce inferferon alpha and beta?
IFN-alpha - produced by leukocytes upon stimulation by RIG-I or TLR-stimulated pathways
IFN-gamma - produced by other cells such as fibroblasts upon activation
When IFN-alpha is made synthetically as a medication. What does it treat?
Is the protein modified?
1) Some viral infections
Autoinflammatory diseases
2) Yes, pegylated to decrease rate of protein degradation
What steps occur once IFNs are released?
- Binding of IFN alpha/gamma receptors (IFNARs)
- Activation of cell signaling pathways that lead to expression of interferon stimulated genes (ISGs)
- Generation of antiviral state in cell & stimulate immune response
What are ISGs? What is their importance?
Interferon stimulated genes
There are over 300
They target different steps in viral life cycles to block virus infection/replication -> generate an antiviral state in the cell
How do type I IFNs stimulate immune responses?
- Stimulate production of chemokines to recruit and activate NK and CD8 T cells
- Upregulation of costimulatory molecules (CD40 & CD28) & MHC class II on antigen presenting cells as well as MHC class I on most cell types
- Block inflammasome formation inhibiting pro-inflammatory activity against some bacteria
Type I IFNs have clinical uses for what diseases?
Chronic hepatitis B Chronic hepatitis C Condyloma acuminatum (caused by HPV) AIDS-related Kaposi's sarcoma (caused by KSHV) HPV infections
What is another name for type 2 IFNs? What are their functions?
Type 2 IFN = IFN-gamma
Produced by immune (T-cells/NK) cells upon activation
Stimulates production of effector and memory cytotoxic T cells & stimulates adaptive immune responses
Also stimulates the production of some ISGs (interferon stimulates genes) and is an important antiviral cytokine
What innate immunity cells can undergo direct destruction of infected cells?
1) Natural killer cells - identify changes on the surface of infected cells and directly kill those cells; also important for control of tumor cells
2) Monocyte/macrophage - phagocytose dying cells removing infected cells from the site of infection, activating the macrophage, & eliciting cytokine and chemokine production
Adaptive immunity review:
What are the functions/is the importance of CD4+ T-cells?
CD4+ (T helper cells):
- critical for responding to intracellular pathogens
- recognize and respond to viral peptides that are presented in context of MHC class II on dendritic cells, macrophages, & B cells
- upon activation, produce and secrete proinflammatory cytokines and chemokines (regulate and stimulate B cell (antibodies) & CD8 T-cell responses)
Adaptive immunity review:
What are the functions/is the importance of CD8+ T-cells?
CD8+ (T cytotoxic cells - CTLs):
- kill foreign antigen-expressing cells by specific recognition of foreign peptides presented via MHC class I
- kill target cell via Fas pathway and perforin
- control non-cytoltic or persistent viruses
- produce cytokines that maintain appropriate T cell populations
- detects and kills cells expressing viral/foreign antigens
Which immune cell is thought to clear most of the virally infected cells?
CD8+ T cells
T/F
CD8+ T cells form memory cells that can be quickly activated and amplify to control the infection quickly
True
What are the 3 functions of secreted antibodies?
Neutralization
Opsonization
Cell lysis via complement action or ADCC (antibody dependent cell mediated cytolysis)
What is the pitfall of antibodies’ ability to control a viral infection?
Antibodies control viremia (free virus) only; cell associated virus is poorly controlled by antibodies
What is the principle protein that antibodies respond to on intact naked viruses?
the capsid
What is the principle protein that antibodies respond to on enveloped viruses?
glycoprotein
Adaptive immunity review:
What is the function of memory B cells?
Memory B cells to viral antigens circulate in the blood & can be rapidly activated to produce antibodies, leading to neutralization and/or opsonization
1) What are the predominate cytokines in the first 3 days of acute virus infection?
2) The predominate cells?
3) What is the virus titer?
1) INF-alpha, IFN-beta, TNF-alpha, & IL-12
2) NK cells
3) virus titer is relatively high
At 5 days of an acute virus infection what changes occur?
What does this do to the virus titer? When does it reach zero?
cytokines decline, NK cells decline, & antibody production becomes substantial (begins at ~3 days)
By the 6th day, the virus titer has gone down substantially and is zero by day 10
What are the three virus strategies against the immune system?
Elude detection
Damage immune cells
Interfere with immune function
What 5 ways do viruses elude immune detection?
1) restricted gene expression
2) antigenic variation
3) diminished cell surface expression of MHC molecules needed for T cell recognition
4) infection of sites less accessible to the immune system
5) immunologic tolerance (virus resembles host or eliminates epitopes readily seen by CD8 T cells)
What 2 ways do viruses damage the immune system?
1) destroy one or more arms of the adaptive immune system (ie HIV)
2) cause generalized immunosuppression that occurs following many viral infections (ie viral infection causing bacterial pneumonia)
What 3 ways do viruses interfere with immune function (counter-defenses)?
1) express proteins that block type 1 IFN or complement activity (many RNA viruses)
2) stimulate release of cytokines that favor viral spread (useful for viruses that infect immune cells) (poxviruses)
3) make own cytokines that inhibit immune response (pox & herpes viruses)
What virus genus blocks different aspects of type 1 IFN production and stimulation of SRGs?
flavirus
What virus downregulates MHC class I while displaying a MHC I mimic? How does this affect the interaction of the infected host cell with T-cells & NK cells?
Cytomegalovirus (HCMV)
NK cells are inhibited by binding the MHC I mimic molecule (inhibitory signal) as well as the activating receptor
T-cells do not bind to the MHC I mimic and are not activated
