Lecture Notes

Intro to Pathogenesis (3/3) - Wk 5

• Pathogenesis: The biological mechanisms that lead to disease

  • how viruses make us sick

• Common Symptoms - from Innate

  • Fever

  • Redness/swelling/rashes

  • mucus

  • body aches

• Immune system composed of:

  • Proteins (cytokines & antibodies)

  • Non-protein molecules ( histamines and leukotrienes)

  • Cells (macrophages, neutrophil, T cells)

  • Tissues (GALT)

  • Organs (bone marrow, thymus, spleen)

• Innate

  • non specific; born with it

  • antigens

  • minutes to hours

• Adaptive

  • specific defenses

  • Activated by innate

  • several days

Sections of Innate

• Chemical defense

• Cellular defense

• Pathogen recognition & phagocytosis

Chemical

• Acute phase proteins

• Cytokines

• Inflammation eliciting mediators

• antimicrobial peptides

• plasma protein mediators

• chemical & enzymatic mediators found in bodily fluids

Cytokines

• soluble proteins that act as communication

  • Autocrine - act on same cell

  • Paracrine - act on a nearby cell

  • Endocrine - travel through blood stream

• Important cytokines

  • IL-x

  • Chemokines

  • Interferons

Cellular Immunity

• Hematopoeisis

• PMNs

• Macrophages

• DCs

• Diapedesis or extravasion

PAMPS & PRRs

• once binded - signal transduction results in transcriptional changes

• recruit DCs - carry phagocytosed microbes to present to the adaptive immune system

• Innate immune system is very dependent on phagocytic cells that each serve a different purpose:

  • Macrophages - surveillance and recruitment

    • found in tissues and blood stream

  • Neutrophils - infection control w/in 30 min

  • DCs - APCs

Pathogenesis of Rhinovirus & Flu (3/4) - Wk 5

Rhinvirus

• ssRNA (+) genome

• Family Picornaviridae

• Naked, iscosahedral

• Have only 10-11 genes

• Use endosomal entry - use of ICAM - 1 host cell receptors

Rhinovirus Immune Activation

• Viral RNA is a PAMP

• Binds to PRRs in the endosome

• Results in transcription of cytokines by turning on transcription factors through signal transduction

  • cause a release of cytokines

Rhinovirus: The Uncommon Cold

• Individuals with and without asthma had roughly equal nasal viral loads (amount of viruses in their noses)

• P value indicate the likelihood that the two groups are not different from each other

• Individuals with asthma

  • worse upper respiratory symptoms

  • much worse lower respiratory symptoms

  • worse peak expiratory flow

    • how much free flow the pts have

  • By day four asthmatic pts are more symptomatic

  • Asthmatics have more cytokine release - more inflammation

• Common Principles

  • symptoms are often linked to spread

  • symptoms arise from immune response

    • PAMP + PRR = cytokine release = more symptoms and inflammation

  • evolutionarily successful viruses are often not very pathogenic

• Influenza

  • Symptoms: fever, runny nose, sore throat, muscle, pain, headache, coughing, and fatigue

  • Number of strains: 4

    • Four types: A, B, C, & D

  • Influenza A and B viruses cause seasonal epidemics of disease in humans almost every winter in the US (CDC, 2024)

  • Influenza A viruses are divided into subtypes based on two proteins on the surface: hemagglutinin (H) and neuraminidase (N)

  • There are 18 different hemagglutinin subtypes are 11 different neuaminidase subtypes ( H1 through H18 and N1 through N11, respectively)

  • So flue A = 130 different combinations in nature

  • But through reassotrment…many more!

• Reassortment

  • process by which influenza viruses swap gene segments

  • Reassortment can occur when two influenza viruses infect a host at the same time and swap genetic information

Pigs and birds - flu is not attaching onto respiratory cells

Koch’s Postulates and Viruses (3/6) - Wk 5

Common Principles

• Symptoms are often linked to spread

• Symptoms arise from immune response

• Evolutionarily successful viruses are often not very pathogenic

  • Immune system kills viruses - not very succesful

Vocab

• Infectious disease

  • disease caused by the direct effect of a pathogen

• Signs of disease

  • clinically measurable deviation for normal body functioning

    • fever, BP, HR

  • Symptoms of disease

    • things that are felt or experiences by a pt

      • nausea, pain

Koch’s Postulates

• How do you know a disease is infectious?

  • Rhinovirus: caused by weather? or caused by pathogen?

• Koch’s Postulates

  1. The suspected pathogen must be found in every case of disease and not found in healthy individuals

  2. The suspected pathogen can be isolated and grown in pure culture

  3. A healthy test subject infected with the suspected pathogen must develop must develop the same signs and symptoms of disease as seen in postulate 1

  4. The pathogen must be re-isolated from the new host and must be identical to the pathogen from postulate 2

  • Developed by Robert Koch, 1884 - before viruses were identified

Koch’s Postulates Updates

1. Some pathogens cause disease in some subjects but not others

   • this means that u can find a causative agent in some healthy subjects

   • Natural genetic and physiological variation in the host population

2. Some pathogens are not microorganisms that can be grown in pure culture

   1. Bacteria can grow on their own

   2. viruses need host cells!

• Could we research a respiratory disease like rhinovirus following Koch’s postulates?

  • No - not bc to inject a healthy subject, not able to isolate the virus

  • ethical issues - informed consent

Pathogenicity and Virulence

• Pathogenicity

  • The ability of a microbial agent to cause disease

• Virulence

  • the degree to which a microbial agent is pathogenic

    • how weak or strong

• How much of a viral pathogen does it take to cause a disease in host?

  • It varies

Infectious and lethal dose

• A single virion of most types of viruses is likely to cause disease

  • It could fail to find a cell to infect before falling apart

  • It could infect a single cell and the innate immune response shuts down replication before an infection can be established

• ID₅₀ (infectious dose) - number of virions that will cause disease in 50% of subjects

• LD₅₀ (lethal dose) - the number of virions that will cause lethal disease in 50% of subjects

• BE ABLE TO UNDERSTAND GRAPH

• X axis - ID
  Y axis - LD

Primary and Opportunistic Pathogens

• Multiple pathogen exposures can occur in one host individual simultaneously or concurrently

• Primary Pathogen - microorganism that initially causes disease

• Opportunistic Pathogen - microorganism that takes advantage of the opportunity presented by the first infection to also establish an infection and cause disease

  • Other viruses can hop on as well

  • Bacteria can hop on as well - even from own body

Bacterial Co-Infections with Respiratory Viruses

• Study that shows bactieral coinfection with influenza

Neurovirulence: West Nile Virus (3/10) - Wk 6

West Nile Virus

• An enveloped virus with an icosahedral capsid

• ssRNA (+) sense genome

• Member of the genus Flavivirus

  • other viruses in this genus - dengue virus, yellow fever virus, & Zika

• West Nile Viruses causes West Nile Fever in ~20% of infected humans

• WNV causes West Nile Encephalitis or other neurological disease in ~1% of people with West Nile Fever

• WNV is an Arbovirus

  • Arbovirus is not part of the formal order/family/genus classification

  • Arbovirus - ARthropod BOrn Virus

Disease Vectors

• Vectored Diseases do not spread directly from a host of a certain species to another host of that same species

• Vectored diseases spread using some sort of vector

  • Insects are common vectors

  • Rodents (not arbovirus vectors)

WNV Transmission Cycle

• Mammals are ‘dead end’ hosts for WNV

  • Titers of virus in the blood are too low in mammals to spread the virus back to mosquitoes

  • Birds are the amplifier host - natural transmission regularly occurs btw birds and mosquitoes

    • ramps up viral load - makes lots of virus

    • birds can spread to other birds

The Central Nervous System

• Brain & Spinal Cord

• Meninges - membranes surrounding brain

• Meningitis - inflammation of the meninges

• Meningitis symptoms

  • severe headache

  • stiff neck

  • fever

  • convulsions

  • confusion

• Encephalitis - inflammation of the brain tissue

  • Meningitis symptoms plus

    • Lethargy

    • Seizures

    • Personality changes

• Blood Brain Barrier - specialized endothelial barrier that makes crossing into the central nervous system very difficult

• Peripheral Nervous System

  • Nerves outside the brain and spinal cord

  • the PNS is less well protected from viruses than in the CNS

    • due to lack of barrier protection

Neurovirulence

• The degree to which it is able to cause a disease in the nervous system of its host

• Usually linked to neuroinvasiveness - the ability of a pathogen to enter the nervous system

• Neuroinvasion leads to inflammation within the CNS - the cause of many of the symptoms of meningitis and encephalitis

• Leads to damage of the CNS is a common feature of zoonotic viruses for which humans are not primary host

  • The CNS is not a great site from which to spread

  • Viruses that are better adapted to spread from human to human usually stay put of the CNS

WNV Dissemination

• Dissemination - moving from the original site of infection (the skin where you get bit by a mosquito) to the rest of the body

• Innate Immune Cells (DCs) at the site of infection become infected by WNV

• DCs travel to lymph nodes (immune organs)

• Also Provdies a new place for the virus to replicate

• From lymph nodes spreads to another immune system organ - the spleen

  • critical step

  • lymph and blood gets filtered to the spleen

• High levels of viral replication in immune organs leads to virus titers rising in blood

• Virus in blood allows for neuroinvasive strains of WNV to cross the BBB

• How does it get across the BBB?

  • “Trojan Horse” - an immune cell is allowed to cross the BBB - WNV rides along inside - hides with DCs

  • Inflammatory cytokines disrupt the BBB, allowing WNV through

  • The virus infects the endothelial cells that line the BBB and then get out the other (brain) side

• Once inside, WNV can infect astrocytes (a non-neuronal brain cell) and neurons, leading to symptoms of neuroinvasive disease

• Rabies virus

  • Rabies virus primarily enter the CNS by traveling from the site of infection to the CNS using neurons

  • A majority of rabies virus infections lead to neuroinvasive

• Arbovirus

  • neuroinvasive abroviruses largely enter the CNS by crossing the BBB

  • A small majority of arbovirus infections lead to neuroinvasive disease

Neurovirulent Viruses: Rabies, Herpes, Polio WNV - Wk 6

Rabies Virus (RABV)

• Enters mammals through saliva/mucous membranes → peripheral nerve → spinal cord → brain

• Rabies enters a neuron in the PNS by binding to a nerve growth factor receptor called p75, responsible for health neurons

• RABV is transported faster than NGF suggesting that RABV not only hijacks the transport machinery but can also manipulate it, gettin git into the neuron fast

• Once inside cell body neuron → directly path to the brain

• Slow, progressive zoonotic disease characterized by fatal encephalitis. Once symptoms appear, almost always fatal

• Two forms

  • Furious Rabies

    • agitation, disorientation, seizures, twitching

    • hydrophobia

  • Dumb rabies

    • pt paralyzed, disoriented, stuporous

  • Both progress to coma phase → lead to respiratory arrest

Rabies Transmission

• Reservoirs: wild mammals such as canine, skunks, raccoons, badgers, porcupines cats, & bats

  • Spread through bites and scratches, & inhalation of droplets

• Estimated 35,000 to 50,000 human cases

  • US: 6k to 7k animal cases per year

  • 2004: transmission of rabies through donated organs tissues

  • Transmission through cornea implants

Herpes (HSV) Encephalitis

• high restricted from CHNS (HSV-1 oral, HSV-2 genital)

• primarily causes by HSV-1

• Can be neonates-transferred through HSV+ mother

  • Can be treated with antiviral if caught early…BUT

  • within days: massive necrotic destruction of brain tissue, coma, & death

• You don’t have to have an outbreak before you get encephalitis

HSV

• Herpes simplex types I and II can cause encephalitis in newborns of HSV positive mothers

  • Virus is disseminated and progress is poor

• Older children and adults over 50 are also susceptible

  • Caused most commonly by HSV 1

  • Represents a reactivation of dormant HSC form the trigemminal ganglion

• HSV 1 most common cause of encephalitis

Neurovirulent Viruses

• Viruses that cause viral meningitis

  • Many viruses that do not normally cause neurovirulence can in rare cases cause viral meningitis

  • Echo and coxsachieviruses, herpesviruses, measles viruses, influenza, mumps, etc.

    • They most frequently occurs in immunocompromized individuals or very young infants

Polio

• Causes poliomyelitis in small subset of infections

• acute flaccid paralysis - affects PNS

• fecal to oral & repiratoy

• dangerous respiratory paralysis

• spread as a fecal oral pathogen

• (+)ssRNA, naked virus

MWMR Discussion

• Group One

  • 2813 cases total in 2018

  • caused by mosquitoes and ticks → other hosts → humans

    • mainly WNV, La Crosse, Jamestown, Powassan, EastEequineEnceph

  • Classified by sex, age, symptoms

• Group Two

  • WNV first reported in 2018

  • Clinical Syndromes: meningitis, encephalitis, acute flaccid paralysis

  • Powassan in Indiana: Likely caused by a blood transfusion from a donor in Wisconson

• Group Three

  • Limitations: Doesn’t require info abt clinical signs and symptoms, most are underreported

  • Information campaign abt household habits/community effors - get rid of standing water, control mosquitoes, considering arboviruses then properly reporting

Neurovirulence: TBEV (test heavy material) - Wk 6

Paper Discussion

• Tick Bonre Encephalitis Virus (TBEV)

  • Member of Flavivirdae

  • Arbovirus vectored by ticks

• TBEV causes Tick-Borne Encephalitis (TBE)

  • Generally two phases of disease

  • Phase I: Generic viral illness, no CNS involvement (most infections resolve here)

  • Phase II: CNS involvement

    • meningitis

    • encephalitis

• Focus Forming Units (FFU) - measure of how many infectious virus particles are in solution

• Named for a variation on a plaque assay

• Plaque Assays - experiments used to determine how many virus particles are in solution - BE ABLE TO EXPLAIN ON EXAM

  • Dilute the viruses until there are a countable number/unit of volume

    • Why dilute?: So that the amount of virus particles is measurable

  • Add that unit of volume to a petri dish that is 100% filled with a flat layer of cells that the virus can infect

  • After giving the virus enough time to perform several rounds of replication, look for ‘holes’ in the layer of cells

  • Each hole (plaque) = where one virus started an infection

• Focus Forming Assays - Plaque assays for viruses that don’t lyse their host cells - therefore going through budding

• First steps are identical, but instead of looking for holes, rely on detection of a ‘foci’ of viruses with an antibody

• Gene Expression Analysis

  • Not all genes in host cells are transcribed all the time

  • Certain genes only get turned on when needed for an immune response

  • How can you measure the expression of a gene?

    • Measure the amount of RNA transcript in a cell

  • How do you measure the amount of RNA transcript in a cell?

    • qRT-PCR

    • mRNA → complementary DNA (cDNA) → quantitative PCR (qPCR)

Kurhade ET Al

• Figure 1

  • B & C: More RNA expression in Toro → more viral load

    • You use spleen & lymph nodes bc of their filtration & greater immune response - you would see the viral load bc of the filtration presence

  • D,E,F,G,H,I - measuring cytokine response

    • D: GAPDH (spleen) - expressed more in spleen

    • E: IL6 - Toro spikes at day 9

    • F: CXCL10

    • G: TNFalpha (lymph node) - expressed more in the lymph nodes

    • H: IL6 - Toro spikes at day 9

    • I: CXCL10

    • Cytokine storm

• Figure 2

  • HB171/11 not present until 10 day

  • Toro present until around day 5

Concept Map

Hemorrhagic Fever Viruses (3/17) - Wk 7

Zoonotic Infections

• Zoonotic infections - caused by a pathogen that has ‘jumped” from one species to another

• Distinct from vectored infection

  • vectored infection - natural transmission cycle includes back ad forth btw two or more host species

• Vectored infections are a type of Zoonotic infection - many Zoonotic infections are not vectored

• Viruses with a long evolutionary history with a host species tend to be more benign

  • natural selection tends to favor lower virulence and higher contagiousness

    • the replication cycle - do not want to kill the host

• More severe viral diseases in humans tend to be recently ‘jumped” zoonoses

  • Recent: Last 20-30 years

    • HIV
      SARS/SARS-CoV-2

    • Ebola

• Pandemic potential

  • Epidemic but world-wide

    • host plasticity - ability to infect more than one species

      • rodents, monkey, bats

    • human-to-human

    • geographic distribution

• Reservoir - host species that is maintaining a pathogen pool

• Spillover - infection event where a pathogen that has historically circulated in one species infects a different host species

• What kinds of animals spark zoonotic events?

Zoonotic Infections + Bats

• Viral richness - number of viral species in a particular host (one individual organism)

• Proportion of zoonotic viruses

  • how many of those viruses have been seen before in human

• Bats, primates, and rodents - host a lot of zoonotic viral pathogens

  • a virus that has been found at least once in humans and once in another species

• Bats, primates, & rodents - also host a lot of viruses

• Many viral hemorrhagic fevers are caused by zoonotic viruses

  • Ebola

  • Hanta

  • Lassavirus

  • Rift Valley Fever Virus

Viral Hemorrhagic Fevers

• Viral hemorrhagic fecers

  • severe multisystem syndrome

  • vascular system extensively damaged

  • symptoms usually accompanied by hemorrhage (bleeding)

• Viral Causes of Hemorrhagic Fevers

  • all enveloped RNA viruses

  • All zoonotic infections

• Families

  • Arenavirdae

  • Bunyavirdae

  • Filoviridae

  • Falviviridae

Ebola Virus

• Ebola virus

  • causes a viral hemorrhagic fever

  • transmission human to human relies on direct contanct with bodily fluids

  • untreated mortality rate 50%-90%

• Ebolavirus is a genus

  • Zaire ebolavirus is the species that most commonly causes ebola virus disease

  • member of the family filoviridae

Pathogenesis of Ebolavirus

• Entry into the host depends on direct contact of infected bodily fluids with mucosal surface

  • At those mucosal surfaces Ebolaviruses encounter many potential host cells

    • resident macrophages and DCs

    • cells that make up the mucosal tissue

• Key to pathogenesis

  • very broad cell/tissue tropism

  • attachment and entry depend on Ebolavirus Glycoprotein (GP)

• Attachment and entry

• Does not rely on a specific host cell receptor

• Relies on non-specific host cell surface molecules that interact with phosphatidylerine and glycoproteins - non specifc cell surface markers

• Ebola v. SARS-CoV

  • Ebola infects more cells

  • Find only SARS-CoV in like respiratory cells

• Ebola disables and dysregulates many of the early innate immune mechanisms a host uses in attempt to halt infection

  • Blocks production of interferons, which are involved in preventing local spread of viral infections

  • causes ‘cytokine storm’

• Cytokine storm

• what is a cytokine?

• soluble proteins that act as communication signals btw cells

  • autocrine - act on same cell

  • paracrine - act on a nearby cell

  • endocrine - travel through bloodstream to act on remote cell

• Important cytokines

  • Interleukines (IL-x)

  • Chemokines

  • Interferons

• Cytokine storm - caused by uncontrolled release of excess cytokines

  • Cytokines ideally should result in targeted, lack immune response leading to pathogen clearance

  • Cytokine storms lead to systemic inflammatory response

    • immune response attacks organs can lead to multi organ failure, extremely high fever

• What causes hemorrhagic part?

  • Exact pathogenesis unknown

  • drop in platelets, clotting factors have been observed

  • injury of endothelial cells that line blood vessels by viral replication is implicated

  • not all pts develop the hemorrhage, can be fatal w/o it

• Viral replication in multi organ systems

  • liver (important in production of clotting factors)

  • spleen

Ebolavirus Outbreak

• 2014-2016

• west Africa started in guinea in late 2013

Ebolavirus (3/18) - Wk 7

Ebolavirus Outbreak of 2014-2016

• West Africa Ebolavirus outbreak started in Guinea in late 2013

• Likely started with spillover event form bats

• Total outbreak numbers

  • 28,600 cases

  • 11,000 deaths

• What ended it?

  • Public Health Measures

  • Vaccine Development

• December 2013

• 18 month old boy died from EbolaVirus Disease in rural Guinea

• Likely contracted by a bat

• March 2014

• Ebolavirus identified in the capital of Guinea, Conakry

• Outbreak declared by WHO with 49 cases and 29 deaths

• AUgust 2014

• Ebolavirus is spreading exponentially in capital cities of Guinea, Sierra, Leona, and Liberia

• During 2014-2016 there were 11 cases in the US

Using Genome Mutations to Track Viral Spread

• During infection of a single pt, an Ebolavirus will replicate millions of times

  • this is an opportunity for the accumulation of genetic mutations in the viral genome

Hemorrhagic Viruses - Marburg & Hanta - (3/24) Wk 8

Marburg Virus Disease (MVD)

• Cousin of Ebola, Filovirdae family

• MVD is severe, often fatal in humans

• The average MVD case fatality rate is around 50%. Case fatality rates have vareid from 24% to 88% in past outbreaks

• MVD initially detected in 1967 after two simultaneous outbreaks in marburg and Frankfurt in Germany, & in Belgrade, Serbia

• Traced to lab work using African Green monkeys imported from Uganda

• initial outbreak in labs where sera and vaccines were being made

• initial patients had direct contact with blood, organs, and cell cultures of African green monkeys

• Monkeys imported from Uganda were used for producing kidney cell cultures, critical for culturing polio vaccine

• Latest out break in 2024

• Natural host fruit bats

MVD Symptoms

• Fever, chills, headache muscle aches

• rash with both flat and raised bumps, often on torso

• chest pain, sore throat

• nausea, vomiting, & diarrhea

• as the disease advances, symptoms can become more severe, including liver failure, delirium, shock, bleeding, & multi organ failure

Recent Outbreaks

‘22 - Guinea

‘22 - Ghana

‘23 - Guinea

‘23 - Tanzania

‘24 - Rwanda

Diagnosing

• Can be difficult to diagnose

• Confirmations that symptoms are caused by Marbug virus infection are made using the following methods

  • ELISA or PCR

Treatment

• Rehydration

• no vaccines or antivirals that are approved

• monoclonal antibody and antivirals

Hanta Virus

• Causes hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS)

• Transmitted via rodents, not person-to-person

• (-)ssRNA virus

How Does it Spread?

• Virus is present in the rodent’s urine, feces, or saliva

• Inhaling viruses - the most likely form of transmission - when they become airborne from distributed rodent droppings or nesting materials

• Touching things contaminated with virus, such as nest, and then touching your mouth, eyes, or nose

• Being bitten or scratched by an infected rodent

Viral Replication

• Macrophages and vascular endothelial cells, particularly those in the lungs and kidneys, are targeted by Hantavirus

• Hantaviruses found in the Western Hemisphere including here in the US, can cause hantavirus pulmonary syndrome (HPS). The most common hantavirus that cause HPS in the US is spread via deer mice.

Symptoms of HPS

• Earl phase symptoms (1-8 wks after exposure)

  • Fever, fatigue

  • muscle aches

  • headaches, dizziness, chills

  • abdominal problems, like nausea, vomiting, diarrhea, and abdominal pain

• Late Phase symptoms (4-10 days have early phase)

  • coughing shortness of breathe

  • tightness in chest w/ fluid in lungs

HFRS Symptoms

• Hemorrhagic fever w/ renal syndrome (HFRS) - mostly in Europe & Asia

• Seoul Virus, a type of hantavirus that causes HFRS, is found worldwide, including US

• 1-2 wks after exposure

  • acute shcok

  • intense headaches

  • fever/chills

  • blurred vision

  • low BP
    lack of blood flow

  • internal bleeding (acute shock)

  • acute kidney failure

Treatment

• supportive care, rest, hydration, treatment

• pts with HFRS may need dialysis

• survival rate depends on the strain of the virus, but is relatively good

• using small interfering RNA (siRNA) and other immune based therapies to target specific gene segments

• drugs have shown efficacy in increasing survival rates during early stages of infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir, and vandetanib.

Oncoviruses (3/25) - Wk 8

Intro to Cancer

• Cancer - a group of related diseases
  Hallmarks

  • cells divide w/o stopping

  • Cells invade surrounding tissues

• Tumor - a growth of cells from inappropriate cell division

• Malignancy - the ability to invade other tissues

• Why do tumor cells divide without stopping?

  • Ignore cell death signals

  • inappropriate expression of telomerase

  • Broken cell cycle checkpoints

  • Cause blood vessel growth to feed themselves

  • evade the immune system

Cell Cycle Review

• Phases of the Cell Cycle

  • Interphase

    • G1, S, G2

  • M Phase

    • mitosis

    • cytokinesis

  • Checkpoint Control of Cell Cycle

Origin of Cancer

• Often, genetic changes

  • errors during DNA replication

  • Environmental exposures

  • chemicals (tobacco smoke)

  • Radiation (sun exposure)

Important Types of Genes in Cancer

• Proto-oncogenes

  • products are normally involved in cell growth and division

  • mutations that increase activity can lead to cancer

• Tumor Suppressor genes

  • products negatively regulate cell growth and division

  • mutations that decrease activity can lead to cancer

• DNA repair genes

  • products involved in repairing damaged or mutated DNA
    mutations that decrease activity can lead to failure to fix proto-oncogene or tumor suppressor gene mutations

Oncogenic Viruses

• 7 known viruses that cause cancer in human cells

  • HPV, EBV, Kaposi’s associated Herpesvirus, Hep B, Hep C, Human Adult T cell leukemia virus, Merkel Cell

• Discovered 1960s (EBV) - 2008 (MCV)

• Observations

  • very prevalent

  • about 90% of humans have EBV

  • In the US, ~1200 people/year are diagnosed with Burkitt’s lymphoma

• Discovery path

  • Study cancer → find virus

Viruses & Cancer

• What potential mechanisms can you image for viral oncogenesis

  • integration into host genome causing damage

  • viral proteins interfere with host cell cycle regulations

Human Papilomavirus

• Member of the Papillomavirdae family

• dsDNA genome

• Non-enveloped

• Causes warts

• some types cause sexually transmitted cancers, particularly cervical cancer

• Enters a cervical cell through endosomal entry

• uncoats through pH dependent mechanism

• Trafficked to the trans-golgi (the outgoing side)

  • ability to incorporate itself with the proteins

• How does it cause cancer from here?

  • When the nuclear membrane dissolved during mitosis, HPV is trafficked to the host chromosomes

  • In daughter cells of the initially infected cell, HPV is in the nucleus

• Steps to carcinogenesis:

  • integration of viral genome into host genome

  • ‘Normal” HPV is maintained as an episome

    • small piece of viral dna is incorporated into host cell dna

  • sometimes it is integrated

  • integration of viral genome into host genome

  • integration changes viral gene expression patterns, leads to increased expression of two viral oncogenes: E6 & E7

  • Viral oncogenes: genes whose products promote cancer formation

• p53 - tumor suppressor gene “guardian of the genome”

  • mutated in >50% of human cancers

  • transcription factor that changes transcriptional profile of the cell activated

    • activates DNA repair proteins

    • arrests cell growth at the G1/S checkpoint

    • intimates programmed cell death when needed

    • responds to short telomeres

• E6 from HPV causes p53 to be ubiquitanated and degraded by the proteasome

  • ubiquitin is a small regulatory protein found in most tissues of eukaryotic organisms

  • ubiquitination affects proteins in many ways: it can mark them for degradation via the proteasome

  • proteasome are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins

• Cancerous cells: no longer are the right type

  • become invasive

Pap Smear

• pap smears - tests that examine cervical samples for cancerous for pre-cancerous cells

Oncogenesis Themes

• access to the nucleus

• integration of viral genome into host genome

• long periods of time

• expression of viral oncogenes

Viral Epidemiology: HIV - Wk 9

Epidemiology of Pandemics

• HIV/AIDS pandemic: 1981 - present

• 1918 Influenza Pandemic: 1918-1919

• COVID-19 pandemic: 2019-2023

• Epidemiology - study of how disease originates and spreads throughout a population

• Epidemiology encompass:

  • geographic distribution + timing of a disease

  • the etiology (cause) of a disease

  • the transmission of a disease

• Endemic disease - constantly present in a particular region

  • HPV - Human Papillomavirus

• Epidemic: much larger than expected number of cases occur in short period of time in a particular reason

  • Annual Influenza Epidemics

• Outbreak: epidemic in a very limited geographic area

  • current cluster of Ebolavirus disease cases in Guineea: declared Feb. 14 2021 by WHO

• Pandemic: Worldwide epidemic

  • COVID 19

Pathogenesis of HIV

• Stage 1: Acute HIV Infection

  • Direct contact with bodily fluids introduces HIV to a new human host

  • 1-2 week “flu-like” illness

  • Resolves on its own

• Stage 2: Clinical (Classical) Latency

  • Virus is not completely latent, but no clinical signs of infections

  • usually lasts 2-15 years

• Stage 3: AIDS - Acquired Immunodeficiency Syndrome

  • Opportunistic Infections

  • Rare Cancers

    • Kaposi’s Sarcoma

    • Leads to skin lesions

  • Wasting Syndrome

  • Survival of ~3 years w/o treatment

• HIV exhibits cell tropism for CD4+ T cells

• CD4+ T Cells

  • Part of the adaptive immune system'

  • interact with DCs carrying an antigen from an infected site in the body

  • Become a “Helper” T cell that coordinates the rest of the adaptive immune response

• Succesful antiviral drugs for HIV have been developed

• HIV+ people can live full lives and expect to never progress to AIDS

Vocabulary of Epidemiology

• Morbidity - the state of being diseased

• Prevalence - number of diseased individuals within a population at a point of time

• Incidence - number of new cases within a population over a period of time

How do Epidemiologists Do Their Work?

• Tracking source of illness

  • determining etiology

• John Snow - 1813-1858

  • British physician

  • Investigated 1854 cholera outbreak in London

• Common source spread - a single source for all infected individuals

• Propagated spread - through person to person contact

• Tracking causes of mortality

  • Florence Nightingale (1820-1910)

  • English Nurse and statistician

  • Mapped causes of death in 1854 Crimean War

• Types of Modern epidemiological studies

  • Observational

    • No manipulation by researcher

    • measure associations

    • gather data

    • Descriptive epidemiology - gathers info abt an outbreak

    • Analytical Epidemiology - attempts to test hypotheses abt cause

  • Analytical Studies can be:

    • retrospective: gather past info abt present cases

    • Prospective: follow individuals over course of a study

  • Analytical studies can use:

    • Cohort method: group of individuals with a shared characteristic compared to some other group

    • Case Control Study Method: retrospective studies compare group of subjects with disease to matches ‘control’ group w/o the disease

    • Cross Sectional Method - randomly select individuals in a population and compare affected to unaffected individuals

Viral Epidemiology: 1918 Influenza (4/14) - Wk 10

Viral Life Cycle: Ex. Influenza Virus

• Flu is a respiratory virus

  • Fever

  • Runny nose

  • sore throat

  • aches/fatigue

  • cough

• Caused by influenza virus

  • enveloped virus with (-) ssRNA, segmented genome

• Attachment → penetration (endocytosis) → uncoating → replication → assembly → release

• Assembly of particles with segmented genome is more complicated

• influenza virus: example of concerted assembly - everything comes together at once to make a viral particle

Pathogenesis of Influenza

• Influenza exhibits cell tropism for cells that have Sialic Acid on their surface - including respiratory epithelial cells

  • infections begin through inhalation of droplets carrying infectious virus or contact with contaminated surfaces

• Influenza causes destruction of lung epithelium

• Influenza infectious causes immune response symptoms that are both systemic

  • fever/chills

  • aches

  • fatigue

• and local…

  • coughing

  • sore throat

  • congestion

Epidemiology of Influenza

• Influenza causes destruction of lung epithelial cells

  • difficulty breathing

  • open door for opportunistic infections

  • severe cases lead to pneumonia (inflamed air sacs, often includes fluid in lungs)

• Influenza is a seasonal illness

  • reasons??

    • humidity

• % of all deaths in the US due to influenza and pneumonia

• Why do people still get the flu w/ vaccines

  • different strains

Influenza: Zoonotic Infections and Reassortment

• Birds are a natural reservoir for influenza viruses

• Potential for zoonoses, and can cause disease in birds

• Ongoing highly pathogenic avian influenza outbreak 2022-2023

Reassortment

• Two different influenza viruses infect one cell

• during virion assembly, a mix of genome segments from both viruses go into one capsid

Influenza

• HxNx Nomenclature

  • H = hemagglutinin

  • N = neuraminidase

• Viral proteins found in envelope

• important antigens

• important to entry and infectivity of virus

• HxNx Nomenclature

  • 18 different known H versions and 11 known N versions

  • Reassortment can give rise to influenza epidemics or pandemics

Attachment and Entry

• Hemagglutinin binds to sialic acid

• neuaminidase cleaves silaic acid on the way out to allow exit

Viral Epidemiology: 1918 Influenza (4/17) - WK 10

Influenza Pandemic

• Worldwide 1918-1918

  • 1/3 world population infected

  • aprox. 10% mortality rate

• Caused H1N1 virus of avian origin

• unusual mortality characteristics - most deadly for 20-40 y/o

  • normal flu follow hourglass shape

    • 1918 flu was the inverse

Native American Boarding Schools

• schools often over crowded

• attendance was compulsory 1891-1978

• 1820s-1980 Government funded schools for native American children

Understanding the Virology

• How did it end?

  • decedent circulated un humans until 19509

  • genetic adaptations reducded virulence

  • the virus developed a more typical epidemic mortality curve

• Story of reconstruction

• Part One

  • Brevig Misson

    • Native American site

  • Dr. Johan Hultin

    • in charge of misson

  • Lucy

    • native american woman who was preserved in permafrost which lung tissue was taken from

  • Taubenberger et al.

    • article that inspirers group to go back

    • first sequence of 1918 flu

• Part Two

  • HA Gene

    • H entry piece

  • Antigenic drift

    • changes in surface proteins

  • Why 1918 flu different from other strains

    • didn’t have cleavage site mutation

    • activate w/o trypsin

  • Dr. Peter Palese

    • created plasmid to recreate 1918 flu

    • created first genetic map

• Part Three

  • Dr. Terrance Tumpey

    • in charge of reconstructing the 1918 flu

    • ended up reconstructing the virus

  • BSL Level?

    • biosecurity lvl 3

    • PAPR, scrubs, shower, gloves, shoe covers

  • Requirements/rules for reconstruction

    • one person to be granted access, lab access, and to reconstructed

    • required to take prescribed medicine

    • work mostly nights with no one else in building

  • Effects in mice

    • highly lethal in mice

    • 100x more lethal than recombinant viruses tested

    • spread to vital organs - brain, liver, heart, spleen

    • fluid filled lungs - serious pneumonia

• Part Four

  • What is different today

    • have had pandemics today

    • there is a flu vaccine now

  • What is 1918 flu occurred now?

    • not as severe affect now

Tumpey et. al

• Table 1

  • 1918 (2) takes a lot less to infect w/ and w/o trypsin

• Table 2

  • infecting eggs with LD and ID

  • 1918 had more of a death rate

• Fig. 1

  • A: leathlity

  • B: % survival tanked after day 4

• Fig. 3

  • how soon virus is released

Viral Epidemiology: COVID-19 pt. 1 (4/21) - Wk 11

Coronavirdae

• First CoV identified - in chickens, 1937s

• Pig CoV - millions of piglets died, 2014

• Feline, Canine CoVs - disease in domestic cats, dogs

• First human CoV cultured in 1960s from nasal cavities of people with common colds

• Categorized by 4 Greek letters: alpha, beta, delta, gamma

• Affects host immune responses, antagonistic for interferons

• Enveloped

• RNA recombination frequency in highest known of any RNA virus

• The cause of 15-25% of common colds

• Spreads like the flu and during cold/flu

SARS

• emerged in the human population in China 2002

• the virus most likely jumped form bats into Himalayan palm civets and then into humans

• Human to human transmission was by respiratory and fecal routes

• 8000 cases were reported worldwide, 26 countries

• 774 deaths occurred (~10% case mortality rate)

• Economic losses in Hong Kong were ~5.9 billion

• In July 2003, WHO reported that the last known human chain of transmission was broken

• Bats and birds are natural reservoirs of SARS-like viruses

• Laboratory-associated infections occurred in China

MERS

• MERS begins with coughing, fever, and breathing problems but may progress to pneumonia and kidney failure

• over 1600 human cases and the outbreak is ongoing

• Case fatality >30%

• sporadic, cant be linked to a single source

• countries most affected in Arabain peninsula

• most person-person transmission occurs in hospital setting

• many questions about the virus unanswered

• healthy camels have antibodies against

COVID-19 Pandemic

• first observations of COVID-19

• Cluster of pneumonia cases of unknown etiology in Wuhan China in Dec. 2019

• Epidemiological work identified Huanan Market as possible source of contagion

  • Later work suggests spread was already propagating at that point

Global Public Health

• WHO - World Health Organization

• Agency of the UN

• 3.4 billion annual budget proposed for 2024

  • For reference: The US annual spending for 2022 was 6.27 Trillion

• WHO

  • monitors and reports diseases

  • coordinates international responses during epidemics/pandemics

  • Designs and executes campaigns to reduce disease burdens globally

    • smallpox vaccination

    • other infectious disease control

• CDC - Centers for Disease Control and Prevention

  • part of the US department of Health and Human Services

  • formed in 1946

  • national public health agency

Viral epidemiology: COVID-19 pandemic pt. 2 - WK 11

COVID-19 Pathogenesis and Long COVID

• When an individual is exposed to SARS-CoV-2

  • the virus usually first infects cells in the nasopharynx, trachea, or nasal olfactory mucosa

    • some infections are cleared by the immune system

  • the virus then can move lower the respiratory tract

    • these infections can then develop into more critical cases

  • inflammation in the alveoli due to the viral replication and consequent immune responses can lead to dyspnea

    • SOB due to poor gas exchange in the alveoli due to inflammation and fluid (pneumonia)

    • leads to hypoxia

    • progressive respiratory failure from this point ca nlead to the development of ARDS

  • ARDS

    • inflammation in lungs

    • widespread lung damage

    • pulmonary vascular leakage

• Early control of SARS-CoV-2 by the immune system is critical to prevent later inflammatory mediated disease

• Post acute sequelae

• Myalgic encephalomyelitis/chronic fatigue syndrome

  • Long term illness

  • fatigue not improved with rest

  • sleep issues

  • brain fog/dizziness

COVID Pt. III (4/24) - WK 11

Hypothesis One

• Direct zoonosis/natural transmission

• SARS-CoV-2 evolves from a bat coronavirus

  • human contact with bats

  • immediate animal contact with bats then contact with humans

• ‘Lab Leak’

  • centers around work at Wuhan Institute of Virology

  • Does not imply engineering of virus

• Engineered Virus

  • could you ever definitely prove a virus wasn’t engineered by humans

  • Scientific process

    • always open to change with new evidence

    • extraordinary claims require extraordinary evidence

Pandemic

• virologists tend to favor the direct zoonosis hypothesis

• Podcast evidence for zoonotic hypothesis

  • came from seafood market

  • studies are preliminary

  • photographic evidence

    • raccoon dogs and flying foxes

    • can easily spread possible virus

    • near stall where scientists find SARS-CoV-2

      • found the virus on lots of surfaces - food machines, cages, etc.

    • shows that virus jumped from animal to animal

    • not evolved from human

    • genetics showed two diff. variants, which are not found in humans

Evolution

• Variant

  • mutations in the genome

• variants of concern

  • characteristics that put public health at risk

• alpha, beta, gamma, delta, ommicron

  • alpha, beta, gamma dominate population

  • delta and omicron

    • omicron is now dominant concern

    • argues that omicron should be strain and not a variant

• delta displayed increased disease severity

• omicron less disease severity but most dominant globally

  • transmission increased and increased immune invasiveness

Vaccines & Antivirals: Intro to Vaccination (4/28) - WK 12

Vaccines + Adaptive Immunity

• Vaccination relies on the adaptive immune system

• Two crucial components of adaptive immunity

  • specificity

  • memory

• Primary Immune Response is critical in generating memory

• Initial exposure → primary immune response → secondary exposure → secondary immune response

• During the primary immune response, some immune cells are programmed to remember a specific antigen

• These cells differentiate into memory

Adaptive Immunity

• Two crucial cell types for adaptive immunity

• T cells

  • mature in the thymus

• B cells

  • mature in the bone marrow

• T and B cells are generated by hematopoiesis from the same stem cells that innate immune cells are derived from

• T cells - responsible for cellular immunity

• T cells direct T cells and other immune cells to target and kill specific pathogens or pathogen infected cells

• B cells - responsible for humoral immunity

• Humors - bodily fluids

• Humoral immunity - mediated by macromolecules found in bodily fluids

  • mostly antibodies

• B cells - make and secrete antibodies

Adaptive Immunity and Vaccines

Both cellular and humoral immunity are important in their function of vaccines

Adaptive Immunity

• Where does specificity come from?

• Antigens -

  • name originally comes from antibody generating molecules

  • also stimulates cellular immunity

  • proteins are the best antigens

• Epitopes

  • small exposed regions on surface of antigens that T cell/B cell receptors or antibodies interact with

Antibody Structure

• Antibodies - glycoproteins produced and secreted by B cells

• Structure is critical to function and specificity

• Fab (variable) and Fc (constant)

• Five classes of antibodies are made by human B cells

  • Each has a heavy chain with a unique purpose

  • each has distinct purpose

  • IgG is the most abundant and ‘multifunctional’ antibody

Antibody Function

• neutralization - antibodies bind to antigens on target viruses, preventing cellular attachment and entry

• Opsonization- antibodies bind to pathogens

• The opposite end of the antibody binds to receptors on phagocytic innate immune cells, like macrophages, enhancing phagocytosis

Adaptive Immunity and Vaccines

• Immunity - the ability to resist infection with or pathogenesis of a pathogen

  • generally relies on adaptive immunity

• Immune memory can be acquired through the active function of the adaptive immune system

  • Option 1: Become ill with a pathogen, recover

  • Option 2: Introduce the adaptive immune system to relevant antigens in a lower risk setting (w/o giving the infectious pathogen)

• Natural

  • active: immunity from sickness

  • passive: breastmilk

• Artificial

  • active: vaccination

  • passive: serum transfusion

• Natural acquired immunity

  • requires natural infection with the pathogen against which you build a memory response

    • Ex: Smallpox

      • Viral Illness

      • eradicated in the 1970s

      • 20-30% mortality rate

• Smallpox

  • first infection with smallpox has 20-30% mortality rate!

  • but if you survive, you are protected

• Goal of vaccination - induce immune memory more rapidly and with less harm than natural infection

Intro to Vaccines

• Option One: Take virus or pieces of virus that cause an immune response but not enough to cause disease

• Option Two: engineered vaccine designed to raise immunity by mimicking a piece of virus

Vaccine Types to Focus On

• Live attenuated vaccines

• Inactivated

• Subunit Vaccines

  • purified from original virus

  • engineered into viral vector

  • ex: tetanus

• Nuclei Acid Vaccines

How Do Vaccines Get Approved?

• Three phases of clinical trails in people occur after tesing has occured in animal models

  • phase i: small group of participants, testing safety (maybe ~45)

  • phase ii: larger group of participants, testing safety and some immune response parameters (~600)

  • phase iii: largest group, safety and efficacy (30,000-40,000)

Smallpox & History of Vaccine (4/29) - WK 12

Smallpox vaccination

• smallpox is a disease caused by Variola virus

• Variolation

  • procedure developed by empirical methods

  • inoculation of healthy individual with a scab from a recently recovered smallpox individual

    • mortality rate of 2%

    • As compared to 20-30%

• Edward Jenner in 1796 noted that milkmaids who recovered from cowpox rarely got smallpox

  • developed an injection from a cowpox sore and gave it to a young boy

  • exposed the child to variola virus and never developed smallpox

    • 1801 published his work

• Modern smallpox vaccines still consist of a live Vaccinia virus

  • no longer produced by isolation from a sore on a milkmaids hand

What Is In a Vaccine?

• Whole viral particles, pieces of a virus, or nucleic acid from virus

• Adjuvant - enhances the reaction of the immune system

  • can cause allergic reactions

  • ex: formaldehyde (polio, diptheria), aluminum (DTAP, HPV, Hep B, pneumo), albumin (flu), and squalene (flu)

• Antibiotics

  • certain antibiotics may be used in some vaccine production to help prevent bacterial contamination during manufacturing

  • neomycin, polymyxin B, streptomycin, and gentamicin

Vaccine Clinical Trials (5/1) - WK 12

Development of COVID Vaccines

• Vaccine development was a critical part of the public health response to the COVID-19 pandemic

• First shots went into arms in clinical trials in the US on March 16, 2020

• Operation Warp Speed was announced on May 15, 2020

• $10 billion dollars in government funding to a public-private patnership to advance COVID-19 vaccine development

• Up to $1 Billion dollars by Oct. 2020

• Funded development of 6 different COVID vaccines by private companies

COVID MRNA Vaccines

• mRNA vaccines: mRNA delivered directly to human cells

• This is the technology used by Moderna and Pfizer vaccines

Clinical Trial Process

• IND Application - Investigational New Drug

• Phase I - small number of patients, monitors safety

• Phase II - are responses as expected? Larger group of pts, monitors safety

• Phase III - Will treatment produce desirable outcome, while monitoring safety

• marketing application submitted & FDA makes final decision

• Standard time: 8-15 years from IND submission

• Fast Track: 5 years or less (usually for drugs that treat highly contagious or aggressive diseases)

Antivirals (5/5) - WK 13

Antiviral Drugs

• Antibiotics target bacteria

  • bacteria are cellular life forms with their own replication machinery, enzymes, cellular structure, and ways of harvesting energy, that are potential drug targets

• Antivirals

  • viruses use host cell machinery for almost all functions

  • this severely limits drugability

• Acyclovir

  • antiviral used for treatment of many kinds of herpes virus infections

    • genital herpes

    • chickenpox/shingles

    • EBV (mono)

  • inhibits nucleic acid synthesis by herpes virus

Antiviral Drugs

• Acyclovir

  • nucleoside analog - mimics guanosine

  • specifically for virally infected cells - virus enzymes needed to add phosphate group to acyclovir

  • when acyclovir is incorporated by a DNA polymerase, elogation terminates

• Tamiflu (olsetamivir)

  • neuraminidase inhibitor

    • enzymes cleaves sialic acids and helps entry in to cells

• Highly Active Antiertroviral Therapy (HAART)

  • a combination of 3+ antiviral drugs

  • daily HAART successfully suppresses HIV load and prevents progression to AIDS

HAART

• Drug category 1

  • reverse transcriptase inhibitors

    • often work by being nucleoside analogs

    • same mechanism as acyclovir

  • Drug category 2

    • fusion inhibitors

    • stop binding of HIV to cell

  • Drug category 3

    • viral protease inhibitors

      • these are essential for replication

  • Drug category 4

    • integrase - viral enzyme responsible for inserting viral genome into host genome

• HAART generally consists of a combination of 2 RT inhibitors and at least one other drug

• importance of combination therapy - to prevent development of resistant viral variants

  • preventing replication = preventing mutation & variants

• Succesful HAART therapy

  • represses viral load

  • prevents transmission

    • because viral load is low

  • allows for maintenance of increased CD4

  • delays/prevents progression to AIDS death

PREP

• PrEP - PreExposure Prophylaxis

  • can you administer antiretroviral drugs prophylactically (a preventative) to individuals at high risk of infection with HIV?

COVID 19 & Antiviral Treatments (5/8) - WK 14

target

• inflammation

• thrombosis

• ARDS
  RAAS activation

Antivirals for COVID

• Remdesivir

  • nucleoside core

  • inhibits RNA replication

  • stop at RNA replication

• Paxlovid

  • nirmatrelvir boosted with liver modifier drug

  • Blocks proteolysis

Class Activity

• Pt I

• • paxlovid?

• Pt II

  • paxlovid - bc higher risk

• Pt III

  • paxlovid - for pregnant women

• Pt IV

  • nothing - healthy