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