Viral Pathogenesis

Question 1

What are the Six steps that viruses employ to propagate and survive?

Answer 1

1. Enter the cell and translocate the genome to the site of replication
2. Replicate its genome and produce mRNA
3. Generate viral proteins
4. Assemble progeny viruses and then emerge from the cell
5. Evade host defenses
6. Disperse and persist in the environment

Question 2

What are the Basic requirements for the initiation of infection?

Answer 2

1. Sufficient virus must be present
2. Cells at the site of infection must be accessible, susceptible, and permissive for the virus
3. Host antiviral defense must be absent or lacking

Question 3

How do viruses cross the hydrophobic, selectively permable lipid bilayer membrane of the host?

Answer 3

By hijacking one or more receptors on the host cell

Question 4

What receptors does HIV use to gain entry to the host cell?

Answer 4

1. Primary receptor bound first (CD4 - TH)
2. Secondary receptor (co-receptor)
   - Th: CXCR4

Question 5

What receptors does HSV use to gain entry to the host cell?

Answer 5

1. initially bind to glycosaminoglycans (GAG) sugar molecules on the cell surface
2. bind a protein receptor (nectin-1 or HVEM) to initiate entry

Question 6

What are some examples of viruses that can use multiple different receptors on a cell to gain entry?

Answer 6

Rabies and HSV too

Question 7

What are the benefits of a virus having the ability to use multiple different receptors to gain entry to a host?

Answer 7

The virus will have an expanded host range because there are multiple types of receptors it can use

Question 8

What glycoproteins does HSV have on its surface?

Answer 8

gB, gC, gD

Question 9

How does HSV gain entry to a cell?

Answer 9

1. gB and gC: binding to Glycosaminoglycans (GAG) which are sugars on surface of cell
2. gD: binds to nectin-1 (primary receptor) or HVEM (also works)
3. Virus enters cells (endocytosis or direct fusion depending on cell type)

Question 10

What is the relationship between viruses of the same family and receptors?

Answer 10

Many viruses from the same family evolve to use similar receptors

Question 11

What is EHV?

Answer 11

Enveloped virus w/ icosahedral capsid and large dsDNA genome

-no vaccines

Question 12

what is the pathogenesis of EHV-1?

Answer 12

1. Initial Respiratory Infection
   - most horses recover quickly but virus enters latent stage
2. Abortigenic Disease
   - causes viremia and infection of endometrium
3. Neurological Disease
   - can lead to myeloencephalopathy

Question 13

What did Dr. Frampton’s team know about EHV before beginning their experiment?

Answer 13

1. Glycoprotein D (gD) on surface of EHV-1 essential for entry into host cell
2. EHV-1 entry via direct fusion w/ host plasma membrane and then endocytosis
3. After entry EHV-1 travels along microtubules to reach host nucleus

Question 14

How did Dr. Frampton discover Glycoprotein D (gD) was essential for entry?

Answer 14

Knock out gD from EHV-1 by going into viral genome and making deletion

• they made a Glycoprotein D knockout virus
• put this gD knockout virus on cells it doesn’t get in

Question 15

What role do Glycoproteins B, C and D play in EHV-1 entry?

Answer 15

Other groups discovered:

• gB would bind Glycosaminoglycans (GAG)
• gC would also bind GAG

Dr. Frampton discovered:
-gD was important for entry but didn’t know what receptor it used

Question 16

When Dr. Frampton was beginning this experiment to discover what receptors EHV-1 uses. What was his first question?

Answer 16

EHV-1 is an alphaherpesvirus, Does EHV-1 utilize any of the known alphaherpesvirus entry receptors?

Question 17

What were the previously identfied alphaherpesvirus entry receptors?

Answer 17

HveA (HVEM) Co-receptor and HveC (nectin-1)

Question 18

What kinds of cells did Dr. Frampton use to test EHV-1 on in his study?

Answer 18

He used Chinese Hamster Ovary (CHO-K) cells because

1) They were resistant to alphaherpesvirus infection
- HSV-1, HSV-2, BHV-1
- EHV-1 unknown?

Question 19

CHO-K cell line is resistant to infection from HSV-1, HSV-2 and BHV-1. What did researchers do to manipulate this cell lines and allow infection?

Answer 19

Engineered this cell line to express entry receptors HveA (TNF-R) and HveC (nectin-1)

• CHO-A: Expression of HveA allowed infection by HSV-1 & HSV-2
• CHO-C: Expression of HveC allowed infection by HSV-1, HSV-2, and BHV-1

Question 20

Dr. Frampton tested Whether EHV-1 is mediated by HveA or HveC by testing it on CHO-K, CHO-A, and CHO-C. What were the resulsts? What was his control?

(sequenced genome of EHV-1, engineered virus to insert reporter gene that expressed beta-galactosidase. Now they could add a substrate and if virus was present in cell then it would light up blue)

Answer 20

His control was testing HPV-1 on all three cell lines
-result: CHO-K = no infection; CHO-A/CHO-C = infection

When testing EHV-1 on all three cell lines
-CHO-K (control) , CHO-A, CHO-C all became infected

Conclusion: EHV-1 must be using a different surface receptor that HveA or HveC

Question 21

How did Dr. Frampton go about finding the receptors that EHV-1 uses to gain entry into cells?

Answer 21

Need Resistant cells and Susceptible/Permissive cells

1. Got cDNA libary for permissive cell line, equine macrophages
   - somewhere in library is transcript for entry receptor
2. Got mouse melanoma cells that were resistant to infectino from EHV-1
   - don’t have receptor for virus
3. Separate the cDNA into pools and add all of cDNA’s from macrophage line and assay for infection
   - if infection occurs then the gene that codes for that receptor is somewhere in the pool of cDNA
   - further divide that pool
4. Amplified and sequenced the incorporated equine macrophage cDNA
5. Equus caballus MHC class I was the receptor

Question 22

How did Dr. Frampton confirm Equus caballus MHC class I
was in fact the receptor used by EHV-1?

Answer 22

Took the mouse melanoma cells that were resistant to EHV-1 and inserted Equus caballus MHC class I genes to see if susceptible to infection
-put fluroescent tag on Fc region of antibody that binds MHC I

To further prove it was MHC I he also loaded anti-MHC I antibodies on to host cell to see if it blocked EHV-1 from binding these receptors
-control: use another antibody to block cell surface receptor

Question 23

What cell types do viruses target after gaining entry?

Answer 23

1. Respiratory tract
2. Gastrointestinal tract
3. Genital tract
4. Conjunctiva (eyes)
5. Skin

Question 24

What routes of entry do viruses use to get into host?

Answer 24

1. Respiratory Tract
2. Alimentary Tract
3. Urogenital Tract
4. Eyes
5. Skin

Question 25

What are the host barriers to infection?

Answer 25

Respiratory tract Gastrointestinal tract Urogenital Tract Eyes Skin

Question 26

What is in the respiratory tract that blocks viral infection?

Answer 26

- mucociliary blanket (ciliated cells, mucous-secreting goblet cells) - macrophages

Question 27

What is in the Gastrointestinal tract that blocks viral infection?

Answer 27

- mucous-lined intestinal tract - alkaline conditions in intestines - acidic conditions in stomach - proteases - phagocytic cells

Question 28

What is in the urogenital tract that blocks viral infection?

Answer 28

- low pH | - mucous

Question 29

What is in the eyes that blocks viral infection?

Answer 29

- secretions | - sclera

Question 30

What is in the skin that blocks viral infection?

Answer 30

- many layers | - dead skin (surface layer) cannot support virus growth

Question 31

What is the most common route of entry for viruses? What are some examples?

Answer 31

viruses enter Repsiratory Tract as aerosolized droplets called fomites examples: Rhinovirus and Influenza virus

Question 32

What is a virus that enters via the Gastrointestinal tract? What kinds of symptoms does it produce?

Answer 32

Norovirus produces diarrhea, nausea, vomiting

Question 33

How do viruses that enter via Gastrointestinal tract spread?

Answer 33

Spread via fecaloral route

Question 34

What are the characteristics of viruses that enter via the gastrointestinal tract?

Answer 34

Viruses must be able to withstand harsh environment of intestines (alkaline) and stomach (acidic), digestive enzymes, mucous and microvilli - Viruses also must evade local antibody (IgA)

Question 35

How does transmission via the oralfecal route occur?

Answer 35

Human feces spreads to water or food that is consumed by people via the following vectors: -manure, untreated sewage, and unwashed hands

Question 36

What are some examples of viruses that enter via the Urogenital Tract (STDs)? What are their symptoms?

Answer 36

``` HIV = AIDS HSV-2 = Herpetic lesions of cervix and urethra ```

Question 37

What are viruses that enter via the Urogenital Tract?

Answer 37

They enter through skin abrasions caused during sexual intercourse

Question 38

What kinds of symptoms are caused by STDs?

Answer 38

Some enter and cause local lesions (papillomavirus – genital warts) Some cause systemic disease (HIV and hepatitis viruses) - Some cause a local infection followed by a latent (quiet) period (HSV)

Question 39

What are the characteristics of eye infections?

Answer 39

Eyes are usually fairly resistant to viral infections Infection may occur after protective layers of eye scratched providing a portal of entry for the virus Most infections localized and result in inflammation (conjunctivitis)

Question 40

What is an example of an eye infection?

Answer 40

conjunctivitis

Question 41

What are the characteristics of viruses that enter through the skin?

Answer 41

-skin is normally a very effective barrier against viral infections - Viruses enter when the barrier is compromised by: - insect bites - needle puncture, sexual contact - animal bite

Question 42

What are some examples of viruses that enter via the skin? What are their symptoms?

Answer 42

Rabies enters via animal bite and causes acute encephalitits West nile virus enters via mosquito bite and causes encephalitis

Question 43

What are some other rare routes of entry for viruses? What are some examples of these?

Answer 43

Transplants and blood transfusions offer a route of entry | -examples would be HIV, Hep B and Hep C

Question 44

What does latrogenic mean?

Answer 44

inadvertant exposure to a virus

Question 45

What are some examples of latrogenic viruses?

Answer 45

HIV, Hepatitis, Prions

Question 46

How are viruses classified based upon the outcome of infection?

Answer 46

Productive - progeny (new) viruses made Abortive - No progeny viruses made

Question 47

What are the different patterns of disease discussed in class?

Answer 47

1. Acute infections 2. Chronic (persistent) infections 3. Latent, reactivating infections 4. Slow infections (e.g TSEs)

Question 48

What is the general pattern of infection for acute viruses?

Answer 48

(Hit+Run): 1. cleared quickly 2. symptoms and amount of virus in host increase quickly and decrease quickly 3. rapid production of virus particles + transmission and then clearance

Question 49

What is the general pattern of infection for Persistent viruses?

Answer 49

Chronic Infection - May not see symptoms right away - symptoms (and even death) present late

Question 50

What is the general pattern of infection for latent infection viruses?

Answer 50

1. initial exposure = virus production 2. enters latent phase (“goes away”) 3. stress event causes reactivation event 4. symptoms not as severe usually second time around

Question 51

What is the general pattern of infection for slow infection viruses?

Answer 51

1. might not know you’re infected b/c the symptoms are very general (flu like) 2. good virus production initially but host immune system gets upper hand eventually 3. enter period of clinical latency 4. not true latency b/c the virus isn’t dormant/not replicating - viral load decreases but is never actually cleared - exhausted immune system allows for opportunistic pathogens => death

Question 52

What are some examples of viruses that cause acute infection?

Answer 52

Rhinovirus and influenza virus

Question 53

What are some examples of viruses that cause persistent infection?

Answer 53

Lymphocytic choriomeningitis

Question 54

What are some examples of viruses that cause latent infection?

Answer 54

Herpes Simplex Virus

Question 55

What are some examples of viruses that cause slow virus infection?

Answer 55

HIV

Question 56

What are the characteristics of acute infection?

Answer 56

1. occurs when a virus first infects a susceptible host - sudden onset of symptoms - usually self-limiting disease of short duration - immune response effectively clears the virus from the host 2. virus usually not infectious for the same host again as memory immune cells generated against the pathogen 3. often rapidly spread from person to person during asymptomatic “prodromal” period

Question 57

What are the characteristics of chronic infection?

Answer 57

1. continuation of infection beyond the time that the immune system should have cleared the virus - disease symptoms may persist or be intermittent - virus NOT cleared by the immune system - host is a reservoir for the virus (can act as a vehicle for transmission to other hosts)

Question 58

What is the definition of disease?

Answer 58

a harmful pathological consequence of infection

Question 59

What are the different forms viral disease can take?

Answer 59

1. direct cell and tissue destruction 2. induction of fever and pro-inflammatory cytokines 3. induction of malignant tumors/cancer

Question 60

What is virulence?

Answer 60

The relative capacity of a virus to cause disease

Question 61

What are the levels of virulence a virus can be?

Answer 61

highly virulent - cause severe disease in the host moderately virulent - cause less severe symptoms weakly or non-virulent (attenuated) - few clinical signs of disease or inapparent infections - many attenuated viruses used as vaccines

Question 62

What factors influence virulence?

Answer 62

The ability of the virus to: 1. Effectively bind to and enter susceptible cells 2. Replicate efficiently within the host 3. Evade the host immune response 4. Cause cell and tissue destruction

Question 63

Viral genetics are a key determinant of virulence. What are Genes that contribute to virulence called?

Answer 63

virulence genes or virulence determinants * All genes that are essential for virus replication are, by their very nature, virulence genes

Question 64

How do virologists study how a virus causes | disease (pathogenesis)?

Answer 64

Animal models are critically important for viral pathogenesis studies

Question 65

What are the types of animal models used to study human diseases caused by viruses?

Answer 65

1) A human virus is directly studied in infected animals - HSV, yellow fever, VEE, LCMV, poliovirus* in mice - Ebola, poliovirus, variola virus in primates 2) An animal virus that is related to the human virus is studied in an animal host (SIV in chimps)

Question 66

How can a virus that doesn't infect an animal model be studied in that animal?

Answer 66

Some viruses can be adapted to grow in non-human hosts - continual passaging of virus in the animal until adaptation achieved - genetic engineering (ex. making transgenic mice that express the poliovirus receptor*)

Question 67

How is virulence measured in an animal model?

Answer 67

1. Clinical signs 2. Severity of tissue pathology 3. Death of host 4. Growth/replication/yield of virus in infected tissues

Question 68

What are the clinical signs used to measure virulence in an animal model?

Answer 68

- weight loss - ataxia - loss of full control of bodily movements - labored or changes in respiration - ruffled fur

Question 69

What are the tissue pathology signs used to measure virulence in an animal model?

Answer 69

-infect animal then dissect animal to discover which organs are targeted

Question 70

How is the death of host used to measure virulence in an animal model?

Answer 70

- % survival of infected animals | - LD50 dose at which 50% of infected animals die

Question 71

How is the growth/replication of virus used to measure virulence in an animal model?

Answer 71

- virus measured by plaque assays, hemagglutination assays, PCR

Question 72

What were the strains being studies in the following study, when they wanted to assess whether glycoproteins I and E (gI and gE) are virulence factors for EHV-1 (ie. Do they contribute to disease caused by EHV-1 infection?)

Answer 72

They had a virulent (RacL11) strain and attenuated (KyA) strains of EHV-1 - KyA does not express gI and gE - RacL 11 expresses gI and gE

Question 73

What did they do when they assessed whether glycproteins I and E (gI and gE) are virulence factors for EHV-1?

Answer 73

* We genetically engineered KyA to now express gI and gE (KgIgE75) or just gE (KgE75) * We also removed gI and gE from the virulent RacL11 strain (L11ΔgIΔgE)

Question 74

What did Dr. Frampton hypothesize when they assess whether glycoproteins What did they do when they assessed whether glycproteins I and E (gI and gE) are virulence factors for EHV-1?

Answer 74

We hypothesized that KgIgE75 and KgE75 would exhibit increased virulence compared to the parental KyA strain and RacL11ΔgIΔgE would be more attenuated than the parental RacL11 strain in a mouse model of EHV-1 pathogenesis.

Question 75

What were the results of the gI and gE experiment for RacL11 and KyA?

Answer 75

Adding gI and gE to Kya made it more virulent -particularly mice infected w/ KgIgE75 had much more weight loss and recovered less Regular RacL11 killed the mice infected with it but removing gI and gE caused a similar weight loss but the mice recovered

Question 76

When Assessing brain pathology in mice infected with attenuated vs pathogenic EHV-1 (KyA vs RacL11 study) what was observed?

Answer 76

Brain sections day 5 post-infection. encephalitis observed in all cases except mock infected Brain sections day 9 post-infection. Encephalitis observed in KyA infected mice

Question 77

What was the survival rate for the EHV-1 (KyA vs RacL11 study)?

Answer 77

KyA had 100% survival KgI/gE/75 had 100% survival but remember they had weight loss KgE/75 same RacL11 was less deadly when gI and gE were removed

Question 78

When the growth/replication of EHV-1 was assayed (KyA vs RacL11 study) what was found?

Answer 78

Many RacL11 infected mice were dying around day 8 but at day 5 no virus was found in lungs (so it was cleared at this point) -Why were these mice still dying if the virus was cleared? The virus caused alot of damage from encephalitis (Look at pathology in slides) - Measured pro-inflammatory cytokines to measure immune response - Much more production of these cytokines in RacL11 and KgI/gE/75 than in Kya(attenuated) - Overaggressive immune response responsible

Question 79

What mechanism leads to changes in the virulence of a particular strain of virus?

Answer 79

Mutations are the mechanism for change in virulence

Question 80

Would you expect more changes in an RNA virus or a DNA virus or would the rate of change be about the same? Why?

Answer 80

RNA viruses make more errors and their polymerase doesn’t have proof reading capability

Question 81

How do we identify virulence genes?

Answer 81

Knockout gene and assess virulence in host. Gene sequencing and splice out the gene.

Question 82

How would you evaluate virulence?

Answer 82

Evaluate virulence using LD50 or other things listed in slide 40

Question 83

What are the possible consequences of point mutations within the coding sequence of a structural gene?

Answer 83

Silent -no change to amino acid produced Missense -changes one amino acid Nonsense -premature stop codon (very deleterious) Frameshift (very deleterious) -single addition/deletion causes different reading frame

Question 84

What are the types of infections that a virus can cause?

Answer 84

Localized infections: some viruses remain at site of initial infection Systemic infections: some viruses spread to target tissues beyond initial entry site

Question 85

What are the main ways that viruses are spread throughout a host?

Answer 85

1) hematogenous route: viremia = virus present in the blood 2) neouronal spread: spread of virus along nerves (HSV, VZV)

Question 86

Walk through the progression of systemic viruses?

Answer 86

1. Primary site of infection - entry 2. Virus enters nearest draining lymph node - replication and infection of immune cells 3. Primary Viremia - moves from lymph to blood 4. Target organs are infected - commonly the spleen or liver 5. Secondary Viremia - high quantity of virus in blood (most contagious) 6. Symptoms begin showing in target organ

Question 87

What are the steps of pathogenisis for polio? - routes of entry - sites of primary/secondary replication - means of spread throughout the body - target tissues infected - symptoms caused by infection - incubation period - is the virus cleared from the host or not

Answer 87

routes of entry -ingested sites of primary/secondary replication -small intestine and lymph node means of spread throughout the body -establish primary viremia target tissues infected -liver and spleen then secondary viremia symptoms caused by infection -paralysis incubation period -race against immune system, depends on how much virus in blood after secondary viremia ~6 days is the virus cleared from the host or not - cleared but may have lasting effects (CNS - motor neurons)

Question 88

Why do only a small fraction of poliovirus infections lead to paralysis?

Answer 88

The outcome of infection is a race between the virus and the immune system - Antibody made early enough to prevent spread to CNS = Immune System Wins - Virus gains access to CNS before antibody neutralizaion = Virus Wins

Question 89

What are the steps of pathogenisis for mousepox? - routes of entry - sites of primary/secondary replication - means of spread throughout the body - target tissues infected - symptoms caused by infection - incubation period - is the virus cleared from the host or not

Answer 89

routes of entry -through footpad of skin (smallpox is respiratory) sites of primary/secondary replication -at injection site, goes to draining lymph nodes and further replicates means of spread throughout the body -primary viremia target tissues infected Liver and spleen then establish secondary viremia symptoms caused by infection -skin infection (rash) and lesions incubation period -7-8 days is the virus cleared from the host or not -if you survive then virus cleared

Question 90

What are the steps of pathogenisis for varicella zoster virus (chicken pox)? - routes of entry - sites of primary/secondary replication - means of spread throughout the body - target tissues infected - symptoms caused by infection - incubation period - is the virus cleared from the host or not

Answer 90

routes of entry -oral or eye sites of primary/secondary replication -upper respiratory then draining lymph nodes means of spread throughout the body -primary viremia and T cells spread target tissues infected -liver and spleen then secondary viremia symptoms caused by infection -chicken pox rash incubation period -14 days is the virus cleared from the host or not - not cleared. hides out in neurons of host - reactivation = shingles

Question 91

How do viruses exit/shed?

Answer 91

Usually shed through routes of entry ``` Mucus Saliva Semen Feces Skin abrasions Breast milk Cervical secretions Urine Viremia—blood ```

Question 92

You are interested in studying the pathogenesis of virus X. You know that the virus is spread from person to person via the aerosol route and can cause encephalitis. Some researchers have shown that mice can be infected with this virus and also develop encephalitis You want to evaluate virus X pathogenesis in mice. 1) Where does the virus go after infection? What assays could you set up to examine this? 2) In what tissues does the virus replicate? 3) What is the incubation period (time from infection to symptoms)? - How could you examine this 4) How is the virus shed from the animal? Is it the same in mice as in the human host? When is it shed? 5) Is the virus cleared from the host? How do you assay this. The virus has been completely sequenced and we have many reagents available to help us study the virus including antibodies and PCR primers. Also, there is a tissue culture system that has been developed for virus X which is cytopathic (causes plaques) in cells.

Answer 92

1) Where does the virus go after infection? What assays could you set up to examine this? Hypothesis: mice that are infected w/ virus, the virus travels to CNS Assay: 50 mice, 25 control and 25 experimental. Every day take 5 mice and do tissue sample. Then do PCR to see which tissues contain the virus. 2) In what tissues does the virus replicate? Can use plaque assay to see which tissues contain active viruses 3) What is the incubation period (time from infection to symptoms)? - How could you examine this Animal model to see how long it takes for you to see symptoms present and graph the data for analysis 4) How is the virus shed from the animal? Is it the same in mice as in the human host? When is it shed? Cage control mice next to infected mice (not in contact) to see if virus spreads via aerosol route 5) Is the virus cleared from the host? How do you assay this. tissue samples+PCR to see if virus is still present in host or if it’s cleared