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Principles in Infectious Diseases > Unit 2 > Flashcards

Unit 2 Flashcards

1
Q

What is a virus?

A

Obligate intracellular parasite

Needs a host cell to survive

Lacks organelles

Extremely small - filterable agents
- Range from 18-230

Need electron microscope to visualise

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2
Q

What is the size of a virus?

A

Around 10nm - 100nm

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3
Q

What is virus nomenclature?

A

Family name
- Ends in -viridae

Genus name
- Ends in -virus

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4
Q

What is virus species?

A

A group of viruses sharing the same genetic information and ecological niche

Can be further divided into types and subtypes

Example:
Orthomyxoviridae
Influenza virus A
Influenza virus A
H5N1 (avian influenza)
H1N1 (human influenza)
Influenza virus B…

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5
Q

Are viruses enveloped? What is the envelope?

A

Viruses can be unenveloped (naked) or enveloped

Virus envelope:
- Host derived lipid bilayer
- Virus encoded glycoproteins; often form spikes that protrude from the virus surface

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6
Q

How are viruses classified?

A

Nature of the viral genome:
- Genome composition
- Genome structure

Structure of the viral capsid

Presence (or absence) of an envelope

Morphology (virology practical)

Replication strategy (Baltimore classification)

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7
Q

What is Baltimore Classification (BRIEFLY)?

A

Based on genome type: It categorizes viruses into seven groups depending on the type of nucleic acid they possess: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA) positive sense, ssRNA negative sense, and retroviruses (RNA to DNA via reverse transcription).

Focus on mRNA production: The classification is primarily concerned with how viral mRNA is produced within the host cell.

Group I: dsDNA viruses: These viruses replicate their genomes in the nucleus using host DNA polymerase.

Group II: ssDNA viruses: They convert their single-stranded DNA genomes into double-stranded intermediates before transcription.

Group III: dsRNA viruses: These viruses possess a double-stranded RNA genome and replicate their genomes in the cytoplasm using RNA-dependent RNA polymerase.

Group IV: (+)ssRNA viruses: These viruses have a positive-sense single-stranded RNA genome and can directly serve as mRNA upon entering the host cell.

Group V: (-)ssRNA viruses: They carry a negative-sense single-stranded RNA genome and must first transcribe it into a positive-sense mRNA.

Group VI: Retroviruses: These viruses contain two identical copies of their single-stranded RNA genome, which are converted into DNA by reverse transcriptase upon entry into the host cell.

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8
Q

What is the nature of a viral genome?

A

Nucleic acid structure:

DNA:
- Double-stranded DNA viruses
- Single-stranded DNA viruses

RNA:
- Single-stranded RNA viruses
—–> Positive sense ss RNA, same sense as mRNA translated directly to protein
—–> Negative sense ss RNA, need to synthesis +ve sense RNA before viral proteins can be translated
- Double-stranded RNA viruses

Some Ds and Ss RNA can be segmented

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9
Q

What are some double-stranded DNA viruses?

A

Poxviridae
Asfarviridae
Herpesviridae
Papillomaviridae
Adenoviridae
Papovaviridae (circular and supercoiled)
Hepadnaviridae (partially ds circular and reverse transcribing)

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10
Q

What are some single-stranded DNA viruses?

A

Parvoviridae
Circoviridae (circular)

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11
Q

What do we know about viruses with a DNA genome?

A
  • All monopartite (all viral genes on a single segment)
  • Mostly double-stranded (except Parvo and circo)
  • Few are circular
  • Many linear DNA viruses have characteristics that enable them to adopt a circular configuration
  • Little diversity in structure
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12
Q

What do we know about RNA viruses?

A

They need an RNA polymerase to copy their RNA genome (no equivalent enzyme in the host)
- RNA dependent RNA polymerase

RNA polymerases are error prone
- No proof reading capability

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13
Q

What are the consequences of RNA polymerases being error-prone?

A

RNA viruses are more variable
- Within a species of virus are more subtypes/serotypes

Can evolve rapidly if needed
- If a virus jumps from one species to another, RNA viruses can more readily adapt
- Often zoonotic (jump from animals to humans)

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14
Q

What do we know about segmentation of RNA viruses?

A

Segmentation of RNA viruses allows the virus to increase its diversity very rapidly (reassortment)

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15
Q

What is the capsid?

A

The viral capsid is a protein shell that encloses and protects the viral genome (genetic material).

It’s composed of multiple protein subunits called capsomeres, which come together to form the capsid structure. The capsid plays a crucial role in protecting the viral genome from degradation and facilitating viral entry into host cells.

Additionally, it helps in the assembly and release of new virus particles during the replication cycle. The shape and arrangement of the capsid vary among different viruses and can influence viral stability, infectivity, and host specificity.

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16
Q

What are the three capsid types?

A

Icosahedral

Helical

Complex

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17
Q

What is the icosahedral capsid?

A
  • Twelve vertices
  • 20 triangular sides (facets)
  • Composed of capsomers (basic structural building block of the capsid)
    —> Penton capsomers
    —> Hexon capsomers
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18
Q

What are some non-enveloped icosahedral viruses?

A

Parvoviridae:
- 18-26nm in diameter
- The capsid consists of 12 capsomers (T=1 symmetry)
- 60 copies of a single protein (VP2)

Adenoviridae:
- Capsid is built up from 252 capsomers
—> 240 are hexavalent
—> 12 are pentavalent (situated at the apices) (T=25)
- Each capsomer contains 1-4 different proteins

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19
Q

What is the helical capsid?

A
  • Capsid protein are arranged in a ‘spiral’ configuration around a single axis
  • Structural unit is one capsid protein
  • Single capsid proteins are arranged as a helix around the genome

All animal viruses with helical symmetry are enveloped

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20
Q

What are some enveloped helical capsid viruses?

A

Paramyxoviridae:
- Helical nucleocapsid containing single-stranded RNA
- Roughly spherical (about 200nm in diameter)
- Can be much larger and more pleomorphic
- EXAMPLES: Measles, Nipah and Hendra

Rhabdoviridae:
- Approximately 180nm long and 75nm wide
- Bullet shaped vision
- Spike-like projections on surface
- Nucleoprotein encases the RNA genome
- EXAMPLE: Rabies

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21
Q

What is a complex capsid?

A

Some of the large viruses have capsid structures that are more complex

Poxviridae
- >100 proteins
- Neither helical or icosahedral structure
- Enveloped, brick shaped or ovoid virion
- Surface membrane displays surface tubules or surface filaments

EXAMPLES: Smallpox, mouse pox

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22
Q

Where is the virus envelope present (depending on capsids)?

A

Few viruses with icosahedral capsid

All viruses with helical capsid

Complex capsid

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23
Q

So a virus can be classified based on what?

A

Genomic composition (DNA or RNA) and its genome structure

Capsid structure (icosahedral, helical or complex)

Possession of an envelope (or not)

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24
Q

What can cause the biological properties of viruses to vary?

A

The biological properties of viruses can vary depending on:

  • Whether the virus has an envelope
  • The structure and composition of its genomic material
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25
Q

Enveloped vs unenveloped viruses:

A

Enveloped viruses:
- Acquire envelope as they bud through the host cell membrane
- Viral envelope contains host cell lipid bilayer as well as viral proteins
- Viral proteins contain receptors needed for virus entry

Unenveloped:
- Naked viruses are released by lysis of the infected cell
- Viral receptors are present on the capsid surface

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26
Q

What are the biological properties of enveloped viruses?

A
  • More fragile than viruses with just a capsid
  • More easily destroyed by
    Disinfectants
    Detergents
    Outside environment
  • If the envelope is destroyed, then the virus is not infectious
    –> destroys the receptors needed for entry
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27
Q

What are the differences in the biological properties of enveloped and unenveloped viruses?

  • Components
  • Properties
  • Consequences
A

UNENVELOPED VIRUSES
Components:
- Protein

Properties:
- Environmentally stable to:
–> Temperature
–> pH
–> Proteases
–> Detergents
–> Drying

Consequences:
- Resistant to detergents
- Can dry out and retain infectivity, are spread easily (aerosols)
- Can survive adverse conditions in the gut
- Lyse cell to release; therefore has to kill the cell. Usually cause acute infections

ENVELOPED VIRUSES
Components:
- Lipids, proteins, glycoproteins

Properties:
- Environmentally liable to be destroyed by:
–> Acid
–> Detergents
–> Drying
–> Heat

Consequences:
- Easily destroyed by detergents
- Must stay wet. Not easily spread (large droplets, secretions, transplants/transfusions)
- Cannot survive in the gastrointestinal tract
- Released by budding:
Does not need to kill the cell to spread
Can cause persistent infections

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28
Q

How does genome composition of a virus affect it’s biological properties?

A

DNA viruses are more stable, show very little variation

RNA viruses are more variable
- RNA polymerase is error-prone and has no proof reading
- Can adapt easily to new environments (jump species/zoonotic)

Some RNA viruses are segmented so can reassort or swap genes

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29
Q

What is the criteria used for classifying viruses?

A

Nature of the viral genome
- DNA or RNA; polarity of nucleic acid
- Structure of nucleic acid (ss or ds); linear or circular; segmentation

Structure and symmetry of the viral nucleoplasmid
- Icosahedral; helical; complex

Presence or absence of an envelope
- Size and morphology
- Genome organisation and different coding strategies
- Tissue and cell tropism
- Varying pathogenicity

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30
Q

What are some viral proteins?

A

STRUCTURAL PROTEINS:
- Capsid proteins
- Envelope proteins
- Matrix protein (layer inside the envelope and outside capsid)
- Virion associated enzymes

NON-STRUCTURAL PROTEINS:
- Proteins that are not structural components of the virus
- Often enzymes (but some enzymes can be structural)
- Also some viruses encode regulatory proteins, oncoproteins, etc.

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31
Q

What is the function of the virus capsid?

A

The structural component of the virus capsid (icosahedral and helical and some complex viruses)

Protect viral nucleic acid and deliver the viral nucleic acid to the cell

Capsids of naked viruses contain receptors that attach to the host membrane to allow entry

Contains sites that will induce an antibody response

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32
Q

What do virus envelope proteins do?

A

Contain receptors that allow the virus to attach and then enter the host cell

Are targets of the humoral and cellular immune response
- Antibodies will recognise these surface exposed viral proteins

Interact with the capsid during virus assembly

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33
Q

What are non-structural viral proteins?

A

Proteins that are NOT structural components of the virus particle

Made in the virus-infected cell following infection:
- Often enzymes involved in viral replication
—> Proteases
—> Helicases
—> Polymerase (can be a structural protein)
—> Protein primers for nucleic acid replication
- Can be proteins that help the virus avoid the host immune response
- Targets of the host cellular response (T cell epitopes)

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34
Q

How can naked viruses sometimes be transmitted?

A

They can be transmitted inside vesicles (so sometimes have an envelope!!)

Some RNA viruses can be transmitted as virus clusters inside vesicles

Rotavirus and noroviruses are transmitted as clusters

Vesicles remain intact (?) and they pass through the GI tract to the intestines

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35
Q

What is the ultimate goal of a virus?

A

To enter a host, enter and grow in the right cells for them

Produce more copies of itself

Spread and infect more cells/another host

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36
Q

What happens in viral infection?

A

Virus enters host cell and grows, replicates and spreads

May cause damage to cells and tissues
–> Some viruses do not cause disease

Pass onto new cells of the same or different type to produce the clinical effects we see

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37
Q

What is pathogenicity?

A

The ability of an organism to cause disease

Composed of two factors:

  • Infectivity (ability to infect and colonise a host)
    —> Measured as infectious dose (number of microbes necessary to initiate infection and cause disease)
    —> E.g. Ebola virus can cause disease if only a few virus particles enter, while other viruses may require 10,000 particles to cause disease
  • Virulence (ability to cause host cell damage)
    —> Occur along a spectrum
    —> Pathogenic organisms always cause disease while less virulent forms may not cause disease at all
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38
Q

What are the steps in the virus life cycle?

A
  1. Attachment
  2. Entry
    - Receptor-mediated endocytosis
    - Cell membrane fusion
  3. Uncoating
    - At the plasma membrane
    - In endosome by changes in pH
  4. Viral gene transcription
  5. Genome replication
  6. Translation
  7. Assembly
  8. Release
    - Non-enveloped viruses by cell lysis
    - Enveloped viruses by budding from the plasma membrane
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39
Q

What happens in virus attachment?

A

Attachment to the host cell is a highly specific process

  • Involves complimentary receptors on the surface of susceptible host cells - highly specific
  • Receptor can be protein or carbohydrate
  • Initial binding is reversible
  • May cause a conformational change that then allows binding to a co-receptor
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40
Q

What happens in virus entry?

A

Viruses must cross the plasma membrane to enter the host cell

Entry into the cell by either:
- Cell membrane fusion (non-endocytotic pathways)

  • Receptor-mediated endocytosis (endocytotic pathways)
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41
Q

What happens in cell membrane fusion?

A

Virus membrane fuses with plasma membrane and nucleocapsid is released into cytoplasm

Occurs at neutral pH (pH independent fusion)

Examples: HIV, Herpesvirus

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42
Q

What happens in receptor-mediated endocytosis?

A

Virus particle binds to host cell receptors

Enters cell in an endosome

Virus membrane fuses with the membrane of the end-some and nucleocapsid is released into the cytoplasm

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43
Q

Describe the process of the entry of herpes simplex virus:

A

Cell membrane fusion

  1. Initial binding gB or gC to heparin sulphate (a complex carbohydrate expressed on the surface of many cell types)
  2. Attachment of gD to:
    - HveA (lymphocytes, epithelial cells, fibroblasts)
    - Nectin 1 and 2 (neutrons, epithelial cells, fibroblasts)
  3. Fusion of the viral envelope with the cell membrane

Uses multiple types of spike glycoproteins to bind and enter different types of cells

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44
Q

Describe the process of the entry of human immunodeficiency virus (HIV):

A

Cell membrane fusion

  1. Binding of HIV gp120 to CD4+ T cells
    - Induces conformational change in gp120
  2. Enables binding of gp120 to CCR5 or CXCR4
    - Causes the gp120 trimer to break apart
    - Allows gp41 to be pulled towards the cell membrane
  3. Fusion of gp41 with cell membrane
    - Releases nucleocapsid into the cytoplasm
  4. Nucleoplasmids are targeted to the nucleus
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45
Q

Describe the process of the entry of influenza virus:

A

Receptor-mediated endocytosis:

  1. Binding of haemagglutinin (HA) to sialic acid receptor
  2. Internalisation in clathrin coated pit
  3. Movement into endocytotoic vacuole which fuse with lysosomes
  4. Low pH triggers conformational change in HA trimer
  5. Exposes fusion domain which allows the fusion of viral membrane and endosome membrane
  6. Release of nucleoplasmids into cytoplasm
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46
Q

What is uncoating?

A

The release of viral nucleic acid from viral capsid

Process is variable: For some viruses
- Nucleic acids may still be in a nucleoprotein complex
- The capsid is only partially disintegrated

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47
Q

What happens in virus replication for both DNA and RNA viruses?

A

DNA VIRUSES
- Ds DNA viruses use host machinery in the nucleus (except poxviruses) to make more ds DNA
- Ss DNA converted to ds DNA then replicates like ds DNA

RNA VIRUSES
- Replicate in the cytoplasm (except influenza and retroviruses)

All make viral mRNA which then migrates into the cytoplasm to synthesise viral proteins using the host ribosomes

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48
Q

What happens in virus assembly?

A

Translation of viral proteins in the cytoplasm

Assembly of virus capsids from newly synthesised components (de novo assembly)

Encapsidation of the viral nucleic acid

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49
Q

How are viruses released?
1. Enveloped viruses
2. Unenveloped viruses

A
  1. ENVELOPED VIRUSES
    Enveloped viruses are released by budding from the plasma membrane. Acquire the envelope on the way out from plasma membrane of internal membranes (nucleus, ER)
  2. Non-enveloped viruses released by cell lysis
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50
Q

What are the routes of virus entry and mechanisms of spread?

A

Viruses must overcome innate defences to enter the body
- Both physical and immunological

Many viruses enter via mucosal surfaces

Different viruses adopt different strategies
- Some viruses may use several entry routes
—> E.g. foot and mouth disease virus inhaled (most common) or ingested
- Entry route may differ in different host species
—> E.g. influenza: Faecal-oral in wild birds and respiratory in humans

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51
Q

How do viruses enter the body and initiate infections?

A
  • Skin
  • Respiratory tract
  • Alimentary tract (GI tract)
  • Urogenital tract
  • Eye

Viruses attach to cells at these locations by attaching to receptor molecules on certain cells

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52
Q

What are the innate defences from viruses in the skin?

A
  • The skin is an effective barrier (keratinised)
  • It must be breached by abrasions or bites
  • Macrophages, neutrophils, dendritic cells, natural killer cells
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53
Q

What are the mechanisms of spread of viruses?

A

Some viruses remain localised at the site of infection
- Influenza virus in the respiratory tract
- Rotavirus in the alimentary tract

Replication occurs in epithelium at initial infection site

Cell-to-cell spread occurs, but virus does not disseminate to other tissues

Usually acute (short incubation period, short duration)

Site of shedding = site of entry

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54
Q

What do we know about viruses in the respiratory tract?

  1. Defences
  2. Virus entry
  3. Examples
A
  1. DEFENCES
    Specialised ciliated epithelium and mucus: mucociliary escalator (in upper respiratory tract (URT) and bronchi)
    - Filters out large particles (particles <5um can enter terminal airways and alveolar)

Sneezing and coughing

Innate immunological defences (e.g. alveolar macrophages, complement, cytokines, natural killer cells)

  1. VIRUS ENTRY
    Via aerosolised droplets expelled by an infected individual
    - Spread by coughing or sneezing
    - Contact with saliva from an infected individual
  2. EXAMPLES OF VIRUSES ENTERING VIA RESPIRATORY ROUTE:
    Influenza
    Foot and mouth disease virus
    Rhinovirus (common cold)
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55
Q

What do we know about viruses in the gastrointestinal (GI) tract?

  1. Defences
  2. Virus entry
  3. Examples
A
  1. DEFENCES
    Low pH in stomach (denatures protein and kills most microorganisms)

Bile and proteolytic enzymes in intestines
- High pH in the duodenum (rapid change)

Mucous

  1. VIRUS ENTRY
    Oral route (ingestion)
  2. EXAMPLES OF VIRUSES ENTERING VIA THE GI TRACT
    Rotavirus
    Norovirus
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56
Q

What do we know about viruses spread via the bloodstream (viraemia) to other tissues?

A

Some viruses can spread to distant sites

Virus reaches blood via lymphatic system

Primary viraemia (clinically silent - increases virus levels allowing infection of distant organs)

Secondary viraemia (virus replication in other organs leads to high concentration of virus in circulation)

Infection spreads to more sites

Allows entry and exit routes from host to differ

Usually have longer incubation period (more severe pathology)

Greater involvement of adaptive immune responses and IgG

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57
Q

What do we know about viruses spread via the Central Nervous System?

A

Entry:
Bite of a rabid animal or contamination of scratch wounds by virus-infected saliva

Rabies replicate in peripheral tissues (striated or connective tissue) at the site of infection

Can remain at site of infection for days/weeks or longer

Virus enters peripheral nerves (infects unmyelinated nerve endings in muscle)

Spreads to CNS and enters the brain (causes behaviour changes)

Migrates to salivary glands (replicates) and excreted in saliva

No viraemia

Also allows rabies to evade the immune system

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58
Q

For virus infection to occur, the cell must be what?

A

Susceptible:
- Appropriate cell surface receptors for entry (susceptibility)

Permissive:
- Able to support replication of the virus
- May need particular cellular proteins to complete infection
- May need to be in a particular cell type
—> Eg. canine parvovirus needs rapidly dividing cells
- Specificity of a virus for a particular host, tissue or cell
- Determines the host range of virus

THIS IS VIRUS TROPISM

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59
Q

What are the factors affecting virus tropism?

A

Not just receptors that determine tropism

Cells need to be susceptible (able to support replication of the virus)

There are other factors:
- Cellular protease can activate fusion
- Protease cleavage by digestive enzymes
- Temperature of replication
- pH lability of viruses
- Anatomical barriers

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60
Q

How can cellular protease activate fusion?

A

Some enveloped virus require proteolytic cleavage of envelope glycoprotein for activation of fusion domain

  • Infectious virus is only found in cell types that contain proteases that cleave the glycoprotein
  • Eg. Influenza haemagglutinin (HA) spike protein
    —> Must be cleaved into HA1 and HA2 by cellular proteases
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61
Q

Describe protease cleavage by digestive enzymes:

A

Reoviruses are activated into infectious virions by cleavage with digestive enzymes

Cleavage of VP4 spike to VP8 and VP5

Conformational change permitting virus to bind to M cells in the gut

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62
Q

How does the temperature of replication affect tropism?

A

Most human viruses replicate at 37 degrees Celsius

Upper respiratory tract has a lower temperature - about 33 degrees Celsius

Rhinoviruses replicate efficiently at 33 degrees Celsius but poorly at 37 degrees Celsius

This limits their ability to spread beyond the upper respiratory tract

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63
Q

What is pH lability of viruses?

A

Gastrointestinal tract presents a harsh environment

  • Acid pH of stomach
  • Alkaline pH of intestine
  • Destructive effects of pancreatic enzymes

Not many viruses can survive this

  • Most respiratory viruses are inactivated if swallowed (the exception is adenovirus)
  • Rotavirus/caliciviruses can survive (unenveloped viruses)
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64
Q

How do anatomical barriers affect tropism?

A

Ability of virus to breach barriers such as blood brain barrier will limit their distribution

  • Poliovirus/West Nile virus
  • Sometimes (but not always) spread to CNS
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65
Q

How do we study pathogenesis?

A

Fenner’s pioneering experiments with mouse pox in the 1940s

Mousepox can cause severe skin rash in mice

Contact transmission

Is this skin-to-skin transmission?

How did Frank Fenner find this out?
–> First study of serial daily titration of virus content of various organs and tissues to trace where the virus went in the body

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66
Q

What is the experimental plan for study of mouse pox?

A

Inoculated groups of mice in the food pad with mouse pox (caused lesions in the skin)

At daily intervals looked for virus in:
- Inoculated foot pad
- lymph nodes
- Spleen
- Skin
- Blood

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67
Q

What is pathogenesis?

A

Pathogenesis is how a disease develops in an organism, involving interactions between the pathogen and the host’s immune system. It encompasses the steps from initial infection to symptom manifestation, influenced by factors like pathogen virulence and host immunity. Understanding pathogenesis is crucial for disease prevention and treatment strategies.

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68
Q

Why is pathogenesis important?

A

Scientific interest

Control of virus diseases
Diagnosis
Sources of infection/ transmission routes:
- Faecal-oral; respiratory
- Aid disease control by reducing/preventing transmission

Design effective vaccines:
- Stop viraemia
- No viraemia - many enteric viruses - vaccine design more difficult as injectable vaccines are not good at inducing IgA
- Rabies - stop virus getting to CNS

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69
Q

What are the methods for detecting virus, virus antigen and viral genomes?

Detection of virus or viral antigen:
1. In clinical samples
2. In virus-infected tissues

Viral genome detection

A

DETECTION OF VIRUS OR VIRAL ANTIGEN:
1. In clinical samples:
- Electron microscopy (virus particles)
- ELISA to detect viral antigens/virus
- Haemagglutination assay

  1. In virus-infected tissues (by taking swabs/biopsies/ aspirates)
    - Immunoperoxidase assay
    - Immunofluorescence assay

VIRAL GENOME DETECTION:
- RT-PCR / PCR
- Hybridisation

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70
Q

What does Electron Microscopy do?

A

Shows the morphology of viruses
–> can be used for characterisation and identification

Can be performed on specimens directly or viruses concentrated from:
- Faeces (rotaviruses, calicivirus)
- Vesicle fluid (herpes simplex)
- Skin scrapings (papillomavirus)

Fast - can be done in a few minutes

Excellent method for detecting rotaviruses, adenoviruses, astroviruses, caliciviruses

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71
Q

What are the cons of Electron microscopes?

A
  • Expensive
  • Need specialist equipment
  • Requires skilled personnel
  • Low sensitivity
  • Need concentrated virus samples (10^6 particles/ml)
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72
Q

What are the benefits of Immune electron microscopy?

A

Useful when numbers of virus particles is low

Sensitivity and specificity can be improved by using virus specific antibodies

Different viruses with similar morphologies can be identified

73
Q

What happens in Classical Immune EM?

A

The sample is mixed with antibody

Negative staining of the sample

Loaded onto EM grid and visualised

74
Q

What happens in Solid Phase Immune EM?

A

Grid is coated with antibody and used to capture virus particles

The virus sample is loaded onto antibody-coated grid

Negative staining etc

75
Q

What are the steps for an ELISA - Enzyme-linked immunosorbent assay?

A
  1. Antibody coated well (capture Antibody). Well must be “blocked” with XS protein
  2. Add sample containing Antigen
  3. Wash well. Add antibody that recognises the Antigen (detecting antibody)
  4. Wash well. Add conjugate labelled secondary antibody (and IgG peroxidase)
  5. Wash well. Add substrate
  6. IF POSITIVE: Colourless liquid –> coloured liquid
76
Q

Which virus families contain proteins that can bind to erythrocytes (red blood cells)?

A

Adenoviridae
Orthomyxoviridae
Paramyxoviridae

77
Q

What protein in influenza binds to erythrocytes (red blood cells)?

A

Influenza virus contains an envelope glycoprotein called haemagglutinin which binds to red blood cells

78
Q

What is the principle and protocol behind haemagglutination assay?

A

PRINCIPLE:
Red blood cells (RBC) –No virus–> RBC are not agglutinated and form a tight pellet at the bottom of the well

Red blood cells (RBC) and virus —> RBC bind to virus i.e. haemagglutination occurs and virus “cross-links” the RBC. Forms a shield

PROTOCOL:
1. Virus sample diluted in 2 fold dilutions across plate starting with 1/10 dilution
2. Red blood cells (RBC) added to each well including control well (C = no virus)
3. Incubation at room temperature for 1 hour
4. RBC in control well should not agglutinate but will form a button at bottom of well.
5. In presence of virus, RBC will bind to each other i.e. agglutinate forming diffuse lattices that coats the well.

79
Q

What happens in immunofluorescence?

A
  1. Cells from the clinical specimen are fixed onto glass slide
  2. Add virus specific
  3. Add a labelled antibody (fluorescent dye) that binds to the virus specific antibody
  4. View with fluorescent microscope

Used commonly for respiratory viruses in respiratory specimens

80
Q

What happens in immunoperoxidase?

A

Similar to immunofluorescence except that a peroxidase label is used.
1. Cells from the clinical specimen are fixed onto glass slide
2. Add virus specific
3. Add a labelled antibody (peroxidase label) that binds to the virus specific antibody
4. View with microscope

81
Q

What does a plaque assay do?

A

Virus quantification

Determine levels of virus (titre) in tissues by titration
- Virus spreads to adjacent cells causing damage and death
- Plaques are produced - regions with no cells, which can be seen with the naked eye after staining
- Virologists equivalent of bacterial colonies
- Used to quantify viruses
—> Virus titre = exact number of plaques
—> LD50 = dilution of virus that kills 50% of cells

82
Q

What happens in a plaque assay?

A

Monolayers of cultured cells are incubated with serial dilution of virus to allow adsorption to cells

After removal of the inoculum, the cells are overlaid with semi solid media (agar) which limits the spread of the virus to neighbouring cells

Hence, each infectious particle produces a circular zone of infected cells which damage or kill the cells resulting in a plaque.

Only viruses that cause visible damage can be assayed in this way

Stain with crystal violet (stains living cells) to enable counting the visible plaques

83
Q

How are plaque assays interpreted?

A
  1. Ten fold dilutions of the virus sample are made
  2. One ml of each dilution is added to columns 1 to 5 (4 replicates)
  3. The virus was grown for 4 days under medium containing agar
  4. After 4 days, the cells were stained using crystal violet and plaque growth visualised
  5. Virus titre in “plaque forming units” PFU per ml can be calculated from this:

Virus titre = number of plaques (10) / number of replicates (4) x dilution factor

= 2.5x10^7 PFU / ml

84
Q

What is the process for polymerase chain reaction (PCR)?

A
  1. Nucleic acid purification (RNA or DNA)
  2. Reverse transcription for RNA virus
  3. Polymerase chain reaction (PCR):
    —> Using virus-specific primers to amplify a specific target
    —> Repeated cycles of denaturation, annealing and elongation
  4. Agarose gel electrophoresis to visualise PCR products
85
Q

What are examples of indirect methods of virus detection?

A
  • Looking for signs of damage
  • Detection of antibody
  • Viral cytopathic effect (CPE) observed in vitro
  • Cell culture
86
Q

What is the process of looking for signs of damage in indirect methods of virus detection?

A

Look for signs of damage in:

  • Cells infected with virus in vitro
    —> cytopathic effects observed in virus-infected cells
    —> Type of damage caused by some viruses can be used by virologists for diagnosis
  • Experimental infection of animals
87
Q

How can the antibody be detected in indirect methods of virus detection?

A

Antibody can be detected by:
- ELISA
- Immunofluorescence/ Immunoperoxidase
- Haemagglutination inhibition assay
- Plaque inhibition assay

88
Q

What happens in Viral Cytopathic Effect (CPE) observed in vitro?

A

Grow cells in vitro and infect with viruses taken from clinical samples

Some viruses kill the cells they infect and as more cells are infected these changes can be visualised with a light microscope
–> Easier to see this than the viruses themselves (too small)

Range from massive damage to no visible damage (non-cytopathic)

If CPE is not easily visible, can look for viral antigens in cells by immunofluorescence or immunoperoxidase

89
Q

In what ways can we infect cell culture for CPE?

A

Sample from infected animal is used to infect cell culture in vitro:
- Primary cell culture
- Semi continuous
- Continuous cell lines

90
Q

What is the process for the preparation of Primary cell culture?

A
  1. Tissue fragments (cut with scissors/scalpel)
  2. Treated with trypsin or collagenase
  3. Cell suspension
  4. Addition of liquid media (Eagle’s and animal serum) Incubates in petri dish or tissue culture flask
  5. Cells attach to solid surface and start dividing
  6. Primary cell structure!
91
Q

Describe primary vs continuous cell lines:

A

PRIMARY CELL LINES:
- Prepped directly from animal tissue
- Subcultured only 1-2 times
- Technically more difficult
- Best cell culture system as it supports wide range of viruses
- Used when the state of cell differentiation is important
- Difficult to obtain a reliable supply
- Expensive

EXAMPLES
- Monkey kidney
- Human embryonic amnion

CONTINUOUS CELL LINES:
- Derived from tumours or by treatment of primary cell culture with a tumour virus
or mutagenic chemical
- Can be propagated indefinitely
- Most easy to handle
- Range of viruses that can be grown is limited
- Often do not resemble the original cell:
—> Less differentiated (lost morphology and biochemical features they possessed in the organ)
—> Often abnormal in chromosome morphology and number
—> Can be tumourgenic (cause tumours in mice when inoculated)

EXAMPLES:
- Hep-2; human epithelial
- HeLa; Henrietta lacks, Human cervical cancer. Used to propagate polio
- Vero; African Green Monkey Kidney cells

92
Q

What do you know about normal cell monolayers?

A

Confluent cell monolayer

Epithelial and fibroblastic cells attach to plastic surfaces to form a monolayer

Cells grown in a chemically defined medium

Double in 24-48 hrs

Retain viability after freezing at low temps (-70 to -176 degrees celsius)

Cell cultures vary greatly in their susceptibility to different viruses

93
Q

What is synctium?

A

Fused cells containing many nuclei

94
Q

What are inclusion bodies?

A

Virus factories in nucleus or cytoplasm

95
Q

What are the morphological alterations seen as cytopathic effects in different viruses?

A

Nuclear shrinking (pyknosis) in Picornaviruses

Proliferation of nuclear membrane in Alphaviruses and Herpesviruses

Vacuoles in cytoplasm in polyomaviruses

Synctia (cell fusion) in Paramyxoviruses and Coronaviruses

Margniation and breaking of chromosomes in Herpesviruses

Rounding up and clustering of cells (no shrinking, little detachment) in Adenoviruses

Rounding up and detachment of cultured cells in Herpesviruses, rhabdoviruses and picornaviruses

96
Q

What are the cytopathic effects (including inclusion bodies) on various viruses?

A

Virions in nucleus in Adenoviruses

Virions in cytoplasm (Negri Bodies) in Rabies Virus

“Factories” in the cytoplasm (Guarnieri bodies) in Pox viruses

Aggregation of inclusion bodies around ribosomes in Arenaviruses

Aggregation of inclusion bodies around chromatin in the nucleus in Herpesvirus

97
Q

What are the viral mechanisms of damage?

A

Inhibition of host cell biosynthetic machinery

Toxic viral proteins

Damage to cell membranes / cytoskeleton

Lysis - Exit of non-enveloped viruses

Apoptosis

98
Q

What are some questions we need to ask when looking at how viruses activate the immune system?

A

What is the innate immune system and what is its function?

What cells are involved in generating the innate immune responses?

What viral products are recognised?

What host receptors are involved?

What is/are activated and what do they do (in general terms)?

99
Q

What is the innate immune response?

A

The first line of defence against viral infection
—> Within hours of infection

Provides rapid protection from viruses until the adaptive immune responses are induced
- Broad specificity
- No antigen processing required
- No memory recall
- Short duration

Set the stage for virus-specific immune responses

100
Q

What are the major players in the innate response?

A

Neutrophils (phagocytose bacteria and other pathogens)

Antigen-presenting cells (APCs) (phagocytose the pathogen)
- Macrophages + dendritic cells

Natural killer cells (non-specifically kill virus-infected cells)

Eosinophils (for parasites)

Hold infection in check during the induction of specific acquired immunity

101
Q

What are neutrophils?

A

The first and predominant immune cell recruited in viral infection
- Depletion of neutrophils during influenza virus infection leads to a more severe disease in mice.

HSV infection results in neutrophil-depleted mice results in:
- Increased viral load
- Increased mortality (in mouse model)

102
Q

What are Natural Killer (NK) cells?

A

Cells that rapidly seek out and destroy virus-infected cells (not specific)

Identify markers of stress on the infected cells

Has two types of receptor:
- Activating receptor
- Inhibiting receptor (prevents NK cells from killing healthy cells)

103
Q

What happens upon activation of Natural Killer cells?

A

Capacity to kill virus-infected cells

Bind to infected cells:
- Release perforins
- Induce apoptosis
- Release interferon gamma

Non-specific
No memory
No MHC restriction

104
Q

What are antigen-presenting cells?

A

Involved early in the host response

Carry immunoglobulin Fc and C3b receptors (promote phagocytosis)

Professional antigen presenting cells

MHC restricted

Initiate the adaptive immune response

105
Q

How is a virus recognised as foreign?

A

Innate immune responses recognises pathogen-associated molecular patterns (PAMPs)

Un-methylated C-G dinucleotides (CpG motifs) on DNA viruses
–> High frequencies in viruses
–> Low frequency in mammalian cells

Double-stranded RNA (only RNA viruses produce this)

Uracil-rich, single-stranded RNA

106
Q

What host cell receptors are involved?

A

Pattern recognition receptors (PRRs)

  1. Toll-like receptors (TLRs)
    - Found on the surface of cells and within endoscopes of phagocytic cells
    - Detect extracellular viruses
  2. Cytoplasmic PRRs
    - Found in the cytoplasm
    - Rig-1-receptors (RIG-1 and MDA-5)
    - NOD-like receptors (NOD2)
    - Cytosolic DNA sensors

Bind to viral products and activate a signalling cascade that results in the synthesis of cytokines that block virus replication in the infected host

107
Q

What are Toll-like receptors?

A

Recognise extracellular viruses

Can be either:
- On the surface of cells (TLR-1,2,4,5 and 6)
- In the endosome (TLR-3,7,8,9)

TLR-3 binds ds RNA
TLR-7 binds uracil-rich ss RNA (e.g. HIV)
TLR-8 binds ss RNA
TLR-9 binds CpG motifs within viral DNA

Sense the presence of viral nucleic acid and other conserved molecular components of invading pathogens

108
Q

What are cellular pattern recognition receptors RIG-1 and MDA-5?

A

Cytoplasmic pattern recognition receptors

RIG-1
- Retinoic acid inducible gene-1
- Recognises ssRNA

MDA-5
- Melanoma differentiation association gene 5
- Recognises ds RNA

109
Q

What are interferons?

A

The most important of the broad host defences against viral infection

Protects adjacent cells from infection

Inhibition of viral replication

Helps activate T-cell mediated immunity:
- Activation of macrophages
- Up-regulated MHC receptors on virus-infected cells

110
Q

What is IFN alpha and IFN beta (Type 1)?

A

Produced by most cell types early in infection (innate response)

Activate genes that have antiviral activities
- dsRNA dep protein kinase R
- Rnase L

Helps stimulate MHC class I (enhance presentation of viral peptides to T cells)

Activates Natural Killer cells

Induces apoptosis

111
Q

What is IFNy (type 2, pro inflammatory)?

A

Involved in the regulation of nearly all phases of immune and inflammatory responses (both innate and adaptive)

Produced by natural killer cells and T lymphocytes

Enhances MHC expression on Antigen-presenting cells

More important as an immunoregulator than as an antiviral agent
- Enhances the cytotoxic activity of T cells, macrophages and natural killer cells

112
Q

What are the components of the adaptive immune response?

A

Humoral immune response (B lymphocytes)
- Days
- Antibody production

Cellular immune response (T lymphocytes)
- Days
- Cytotoxic T cells (CTLs) CD8+
—> Kill virus-infected cells
—> Cytokines that eliminated viral RNA
- T helper cells (Th) CD4+
—> Activate macrophages
—> Cytokines

Virus-specific immune response recognises viral proteins and carbohydrates.

113
Q

The adaptive immune response elicits several kinds of responses. What are these?

A

Eliminate virus

Destroy virus-infected cells

Prevent re-infection

114
Q

What are B lymphocytes?

A

Cells that are resident in the lymphatic tissue

Respond to antigenic stimulus by producing and secreting antibodies

Carry highly specific receptors that recognise viral epitopes

Recognise Antigens in their native form (no processing)

115
Q

How do B cells activate?

A
  1. Activates on binding to virus/antigen
  2. Requires a signal from T helper cells
  3. Clonal expansion of B cells producing a single antibody
  4. Initially IgM, class switches to IgG
  5. Some will become long-lived memory B cells

Antibodies early on are low affinity (poor binding).
B cells evolve by hyper-mutation in the V regions to produce high-affinity binding antibodies later on

116
Q

When are antibodies produced?

A

IgA and IgM are detectable within 10-14 days then they decline

IgG and IgA predominate in the secondary response

117
Q

What is IgG?

A

The main circulating antibody

Important for long term immunity

118
Q

What is IgM?

A

Circulating antibodies produced early in infection

Diagnostic marker?

119
Q

What is IgA?

A

Secretory antibody

Primary defence at mucosal surface

120
Q

What is the role of the antibody?

A

Antibody binds to circulating virus (outside of the cell)
- May prevent attachment to susceptible cells (neutralises virus)

Opsonisation
- Virus-coated with antibody activates complement
- Induces inflammatory response (attracts phagocytes)

Neutrophils and macrophages
- Phagocytose virus coated with antibody

Plays a major role in recovery following infection with viruses that cause viraemia

121
Q

What are T lymphocytes?

A

T-lymphocytes co-ordinate the activities of the cells involved in generating the immune response
- T helper cells (CD4+ T cells)
- Cytotoxic T cells (CD8+ T cells)

Carry highly specific receptors that recognise specific regions on a pathogen (single T cell recognises a single T cell epitope) in association with MHC proteins

On activation, T cells will divide and proliferate and secrete cytokines that regulate the immune response

Some T cells become long-lived memory T cells

122
Q

What do Cytotoxic T-cells (CD8+) (CTLs) do?

A

Recognise viral peptides on the surface of infected cells associated with MHC class 1

Kill virus-infected cells:
- Granules within the CTL polarise towards the target cell
- Perforin is released and creates pores in the cell membrane causing lysis

123
Q

What do helper T-cells (CD4+) do?

A

Recognise viral peptides on the surface of APCs associated with MHC class II

Plays essential role in initiation of B cell responses

Secrete cytokines that regulate the immune response

Main types are Th1 and Th2

124
Q

What are the differences between Th1 and Th2 T helper cells?

A

Th1:
- Secretes IL-2
- Inflammatory response. Augments immune response by attracting macrophages to site of infection
- Activates IFN y and TNF ß
- Promotes IgG2a production

Th2:
- Secrete IL-4, IL-5 and IL-6
- Provide help for antibody production
- Promotes switching of B cells (IgG2a to IgG1)

125
Q

What are the targets of the humoral immune response?

A

Surface exposed protein on virus particle:
- Envelope proteins
- Capsid proteins (of naked viruses)

Often spikes or loops that protrude from viral surface

Epitopes can be linear or ‘conformational’ (critical residues brought together by folding of the polypeptide chain)

126
Q

What are viral antigens that induce the adaptive immune response?

A

Surface proteins are recognised by antibodies

B cell epitopes are on surface exposed proteins and more likely to be “conformational”

Internal (non-structural) and surface proteins are recognised by T cells

T cell epitopes can be internal or external proteins
Epitopes are linear (continuous)

127
Q

What is a summary of the immune response to virus infection?

A

VIRUS:

Infects cell:
Stimulates innate response
Viral peptides are expressed on cell surface with MHC I
—> Activate CD8+ cytotoxic T cells –> Memory T cells

Taken up by APCs:
Macrophages and dendritic cells. Viral peptides expressed in association with MHC class II.
- Activates CD4+ T cells
—> Th1 cells activate macrophages (back to taken up by APCs)
—> Th2 cells. Helps B cells (goes to next part)

Drains to lymphatic tissue:

Lymph tissue in gut (GALT) and lungs (BALT) tonsils/Peyers patches
- Produce IgA

Lymph nodes/Spleen
- Receive virus from the blood circulation and lymph
- Produce IgM initially and then switches to IgG
—> Memory B cells

128
Q

What are some serological tests? What do they measure?

A

Immunofluorescence (IF)
ELISA
Virus neutralisation assay
Western Blotting (only rarely used)

Measures only the humoral immune responses (antibody)
Techniques for cellular immune responses are available for research, but are NOT routinely available or cost-effective for diagnostic testing.

129
Q

What do we know about ELISA + Pros/Cons?

A

Can be either:
- Qualitative (positive or negative)
- Quantitative (how much antibody)

PROS:
Rapid, cost-effective, easily scaled up
Often doesn’t require infectious virus (as recombinant proteins can be used)

CONS:
Species-specific (but can do competitive ELISA)

130
Q

How do we determine if a sample is positive or negative for ELISA?

A

Need to determine the cut off for the ELISA
- OD reading above which the sample is positive
- Need to run several negative samples

Cut off
Mean of the known negatives (non-infected) plus 2 (or 3) standard deviations
- Assumes distribution is normal

Twice the mean of the negatives (non-infected) samples

No consistent methods used
Method is decided for each ELISA (empirically)
Method that gives the best results with known positives and negatives is used

131
Q

How do you determine the concentration of antibody in a sample for ELISA?

A

Perform the ELISA with:
- Test samples
- Positive and negative controls
- Serial dilution of antibody standards (contain known concentration of antibody)
- Draw a standard curve (antibody concentration vs OD reading)

132
Q

How do we draw the Antibody standard curve and what does it show us?

A

Draw the standard curve:
- Plot Log10 Antibody concentration on the X axis
- Plot OD on the y axis

Determine the concentration of antibody in the test samples by reading from the graph

Above a threshold, the same will be considered positive
- Determined empirically for each disease

133
Q

What is the process for a western blot experiment?

A
  1. Run proteins on a SDA polyacrylamide gel
  2. Transfer separated proteins to a nitrocellulose/ nylon membrane
  3. Incubate membrane with antibody (from sample i.e. diluted serum sample). Wash, incubate with secondary antibody labelled with enzyme/tag that can be visually detected
  4. Detect/visualise proteins on addition of substrate
134
Q

What does Western blotting do?

A

Allows detection of antibodies to some or all of the viral proteins of a particular virus

Can be used to monitor the presence of antibodies to different antigens at different stages of infection

Not used routinely in diagnostic setting (not easily standardised or scaled up for automation)

Generally used as a confirmatory test

135
Q

What is the virus neutralisation test?

A

Loss of infectivity through reaction of the virus with specific antibody

Virus is incubated with serum sample (serial dilutions)

  • Using the constant concentration of virus
    –> Concentration where plaques are visible and beginning to decrease in number
  • Virus and antisera are then used to infect cell monolayers in a microtitre plate
  • Determine the highest dilution of antibody which when mixed with a standard virus concentration will prevent the formation of plaques
136
Q

What is the virus neutralisation protocol?

A

Fixed concentration of virus in all wells

Antibody is serially diluted across the plate

Neutralisation titre can be determined

137
Q

What does the virus neutralisation titre show?

A

Sometimes titres represent the serum dilution (or the reciprocal of the serum dilution) giving 100% neutralisation against CPE compared with uninfected cells

But different scientists can use different cut offs. Essentially the percentage plaque formation is calculated and plotted vs antibody dilution and then the titre is read off the graph

138
Q

What are the pros and cons of a virus neutralisation assay?

A

PROS:
Used for detection and quantification of virus specific antibodies

Gold standard in virology

Neutralising antibody correlates with protective antibody in vivo

When new virus emerges - such tests can be operational within weeks (take up to a year for an ELISA to be fully validated)

CONS:

Slow. Can take several days to get a result

Require infectious virus

Cost to maintain cell culture facilities

139
Q

What are the uses of serological tests?

A

Used when it is difficult to detect virus directly

Widely used when animals are imported and exported from countries
- Governments want certification to ensure animals are not bringing pathogens with them

Used to assess vaccine efficacy:
- Is vaccination being implemented correctly
- Eradication programmes

140
Q

What is influenza?
- The disease
- The location
- The host

A

DISEASE:

Respiratory disease in humans, horses and swine (and other mammals)

Enteric disease in birds

LOCATION:

Replicates in upper respiratory tract in humans

Replicates in the intestines and respiratory tract in birds

HOST:

Reservoir host is in birds

Human, horses, dogs, and swine (and bats)

Newly pathogenic strains emerge (increased tropism and jump species)

141
Q

What are influenza viruses?

A

Member of the orthomyxoviridae family

Enveloped virus (80-120nm)

Negative sense, ss, RNA genome

Segmented RNA genome (8)

Helical nucleocapsid

Most segments codes for a single protein (mostly!)
- Haemagglutinin (HA) spike
- Neuraminidase (NA) spike

142
Q

What does the influenza virus look like?

A

Inside the virus:
- NP (Nucleocapsid)
- PB1, PB2, PA (transcriptase complex)
- NS2

On the outer layer of the virus:
- M1 (Matrix protein)
- M2 (ion channel)
- Lipid bilayer

Protruding from the virus:
- NA (Neuraminidase)
- HA (Haemagglutinin)

Carry important type-specific antigens:
18 H types
11 N types

143
Q

What are some Influenza subtypes?

A

Haemagglutinin (H):
Human: 1,2,3
Equine: 3,7
Swine: 1,3
Avian: 1,2,3,4,5,6,7,8,9,10,11, 12,13,14,15,16

Neuraminidase (N):
Human: 1,2
Equine: 7,8
Swine: 1,2
Avian: 1,2,3,4,5,6,7,8,9

New H17 N10 and H18N11 subtype found in bats

144
Q

What is the influenza life cycle?

A
  1. Virus enters the host cell
  2. Virus replication occurs in the nucleus
    - Viral mRNA need to be capped at their 5’ end (5’C)
    - Some viral mRNAs are spliced

Capping and splicing enzymes are in the nucleus!!

  1. Neuraminidase aids release of the virus from the cell
  2. Spreads to other cells in the upper respiratory tract. Virus normally remains localised in the respiratory tract
145
Q

How are influenza viruses able to evolve?

A

Influenza viruses have the ability to evolve rapidly.

Antigenic shift:
- Complete change in subtype of HA or NA
- Change in HA can result in pandemics

Antigenic drift:
- Variation within the HA or NA subtype
- Drift in HA or NA results in epidemics and more rarely pandemics
- Can also allow influenza to “jump” species
- Can cause changes in virulence/tissue tropism (as seen with Avian influenza H5 and H7 subtypes)

Allow virus to change species specifically and to change virulence.

146
Q

What is reassortment, or antigenic shift?

A

Complete change in HA type

Virus acquires a different HA type from a different species as a result of re-assortment of the viral segments

Pigs generally act as mixing vessels as they can be infected with both human and avian viruses

Causes pandemics

Population are immunologically “naïve”

Host immune system has not seen this gene before

No protection

147
Q

What are some influenza pandemics in the 20th century and the subtypes through the years?

A

1918: SPANISH FLU
- 20-40 million deaths
H1N1 - Avian virus?

1957: ASIAN FLU
- 1 million deaths
H2N2 reassortment of human and avian viruses
Avian HA (H2)
Avian NA (N2)
Avian PB1

1968: HONG KONG FLU
- 1 million deaths
H3N2 - Reassortment of human and avian viruses
Avian HA (H3) and Avian PB1

1977: RUSSIAN FLU
H1N1
Re-emerging virus

2009: SWINE FLU
vH1N1

148
Q

How did swine flu emerge?

A

Classical H1N1 swine flu:
- Emerged in swine in 1918
- Circulating for decades in US
- Mutating slowly

Human H3N2:
- Emerged in humans 1968

Swine H3N2:
- Emerged in 1997

Swine H3N2 + Avian H1N1
= Swine H3N2
Emerged in 1999 spread rapidly in swine

+ Classical H1N1 Swine Flu

= Swine H1N2:
- Occasional transmission (11 cases) to humans.
- Usually severe

Swine H1N2 + Eurasian swine H1N1 (emerged in swine in 1979)

= New pandemic vH1N1

149
Q

What is H1N2v?

A

New swine influenza detected in a human patient

First human case of this kind in the UK

Identified as part of routine surveillance

Patient experienced mild illness and recovered

Source has not been identified:
- Patient had no contact with pigs
- Contact testing

150
Q

What is antigenic drift?

A

Minor variations in the sequence of HA and NA genes

Caused by mutation

Causes epidemics

151
Q

What does antigenic cause on the viral surface?

A

Variation in spike proteins present on the viral surface.

Produce viruses with slightly different amino acid compositions

Some may grow better in a particular host than others

Gradual accumulation of antigenic mutations

152
Q

What can amino acid changes caused by antigenic drift lead to?

A

Change in host (species specificity)

Change in tropism

153
Q

What is the haemagglutinin protein?

A

The most abundant protein on the viral surface and is the main target of immune response

Receptor binding site:
- Allows virus to bind to sialic acid on host cells
- Enables virus internalisation (within endosome)

Cleavage site:
- Cellular proteases cleave HA into two subunits (HA1 and HA2) to allow virus release from the endosome
- Cleavage aids fusion of the virus membrane with the membrane of the vesicle
- How well HA is cleaved by cellular proteases facilitates virus entry

154
Q

How does influenza enter the host cell?

A

Attachment:
- HA binds to sialic acid groups on membrane-bound proteins on the cell surface

Entry:
- Virus enters by receptor-mediated endocytosis

Release from endosome:

a) Acidic pH inside the endosome, causes a conformational change in HA, which exposes a fusion domain which promotes fusion of viral and endosomal membranes

b) Viral nucleocapsids to be released into the cytoplasm

155
Q

Mutations that lead to changes in cell tropism lead to increased pathogenicity. What is the difference between low pathogenic avian influenza and highly pathogenic avian influenza?

A

LOW PATHOGENIC AVIAN INFLUENZA:

  • Causes a wide range of symptoms in wild birds
  • Generally a mild illness
  • If the virus infects domestic birds, it can occasionally develop into a highly rapidly fatal disease or high pathogenic avian influenza

HIGHLY PATHOGENIC AVIAN INFLUENZA:

  • Results in severe epidemics
  • Sudden onset
  • Severe illness
  • Rapid death (within a few hours)
  • Mortality can be up to 100%
  • All pathogenic forms are caused by H5 and H7
  • Virus changes from a virus that is enteric to a virus that infects every cell in the body.
156
Q

Ease of haemagglutinin protein cleavage by cellular proteases can lead to changes in cell tropism. What are these in both low and highly-pathogenic avian influenza?

A

LOW PATHOGENIC AVIAN INFLUENZA:

  • HA1 and HA2 polypeptide chains linked by a single arginine residue
  • Cleaved by a few cellular proteases in a limited subset of cells
  • Therefore influenza normally can only replicate in a few cell types.

HIGHLY PATHOGENIC AVIAN INFLUENZA:

  • Insertional mutations add basic residues at HA cleavage site
  • HA1 and HA2 polypeptide chains linked by several basic amino acid residues
  • Cleaved by ubiquitously expressed cellular proteases (Turin and other subtilisin family proteases)
  • Virus can replicate throughout bird’s body producing necrotic foci in many places
157
Q

What are some changes in HA that can lead to a change in species specificity?

A

Influenza binds to sialic acid on the surface of host cells

Sialic acid is found on the end of polysaccharide chains

How sialic acid is bound to galactose determines its shape

158
Q

What are the two types of Sialic acid?

A

Sia(alpha)2, 3Gal
- Recognised by HA of avian influenza e.g. H5N1
- Long thin structure

Sia(alpha)2, 6Gal
- Kinked shape - slightly wider
- Recognised by HA of human influenza
- E.g. H1 and H3

159
Q

How do changes in receptor binding cause change in the species specificity?

A

Sia(alpha)2, 6Gal
- Present in the human upper respiratory tract
- Therefore human viruses can enter and cause infection

Sia(alpha)2, 3Gal
- Present in the human lower respiratory tract
- Therefore avian virus can enter and cause infection (but only when in close contact with avian viruses)

Therefore, under “normal” circumstances the avian virus is restricted to birds

160
Q

What are the differences in pathogenicity of H1 and H5 types?

A

H1N1:
Easily spread, rarely fatal

H5N1:
Spreads slowly, often fatal

161
Q

What changes in HA of H5N1 can cause a change in species specificity?

A

Several mutations have been implicated:
- Reduce binding to Sia(alpha)2-3Gal
- Increase binding to Sia(alpha)2-6Gal
- For H1/H3 - as few as 2 amino acids can switch human and avian receptor specificity

162
Q

How did influenza H5 adapt to replicate and transmit efficiently in ferrets?

A

Required just 4 mutations in HA
- three fro binding to Sia(alpha)2-6Gal (human HA receptor)
- One for stabilising HA

163
Q

What is the immune response to influenza?

A

Viral targets of humoral immunity
- HA and NA
- Antibodies to these proteins correlate with protection

Antibodies to one subtype are NOT cross-protective

Strain-specific immunity can be long-lasting

Infection does NOT induce life-long protection (WHY?)

164
Q

What is the immune response to Haemagglutinin?

A

Neutralising antibodies target the globular head of HA
- Neutralise virus by binding to or near the receptor for host cells
- Therefore blocks binding and entry of virus to cell

Are strain specific and lack cross neutralising antibody to different HA subtypes

Are epitopes on the stalk/stem region (these are hidden)
- Conserved amongst the different HA subtypes
- Can be broadly neutralising against many HA subtypes but titres are low during infection

165
Q

What is the immune response to neuraminidase (NA)?

A

Neuraminidase (NA) plays an important role in allowing release of influenza from the cell

After budding, NA cleaves sialic acid from the cellular receptors
- Destroys the receptor and prevents reinfection of the same cell

Antibodies to NA can block this enzymatic activity
- Therefore do not prevent infection but limit virus spread
- But do reduce replication by inhibiting the release of newly produced viral particles (reduce severity and length of infection)

166
Q

What is the cellular immune response to influenza?

A

CD4+ and CD8+ T cells are induced by:
- Envelope proteins (HA and NA)
- Internal proteins (NP, M1, PB1, PB2, PA and NS1)

CD8+ T cells:
- Eliminate virus-infected cells
—> Release perforin and granzyme
—> Release IFN gamma and TNF alpha

  • Epitopes are conserved within subtypes so may be cross-protective
167
Q

What is One Health?

A

One health is a comprehensive approach to health that focusses on:

  1. Improving health and wellbeing through the prevention of risks and the mitigation of effects of crises (e.g. emerging diseases) that originate at the interface between humans, animals and their environments
  2. Promoting multi- (cross-) sectoral collaborations and a “whole of society” treatment of health hazards, as a systemic change of perspective in the management of risk
168
Q

What are the origins of One Health?

A

Conference of Wildlife conservation society in New York introduced:
- The term “One World One Heath”
- 12 recommendations (Manhattan Principles) for preventing epidemic disease and maintaining ecosystem integrity for the benefit of people, domesticated animals and the foundation of biodiversity that supports us all.

169
Q

What is the Scope of One Health?

A

Zoonotic disease, emerging disease, multi-host pathogens, food safety, food security (farming practices), antimicrobial resistance, mental health, obesity, climate change, etc.

Applies across research, education, policy and practice

170
Q

What is One Health today?

A

Growing recognition of need for interdisciplinary collaboration to prevent and control emerging zoonoses. E.g. among doctors, veterinarians, wildlife specialists, anthropologists, economists

Berlin Principles (2020) update on Manhattan principles emphasising the need for action.

171
Q

Why the increasing interest in One Health?

A
  • COVID 19
  • Highly pathogenic avian influenza (H5N1); the 2009 swine-origin influenza pandemic
  • Emerging zoonoses/infections (e.g. 2014 Ebola epidemic)
  • Climate change
  • Population growth and urbanisation
  • Challenge of providing food
  • Encroachment and contact with wildlife
  • Antimicrobial resistance (human and animal overuse)
  • Globalisation of trade and animal movement.

It is estimated that 75% of recently emerging infectious diseases are of animal origin and 60% of all human pathogens are zoonotic. The class of pathogen most responsible for new human infections is viral because of their high replication and mutation rate.

172
Q

What is a zoonosis?

A

An infectious disease that transmits from a non-human animal to humans

173
Q

What is an anthroponosis?

A

An infectious disease that transmits from humans to non-human animals

174
Q

What is an emerging infection?

A

One that has either newly appeared in a population or that has a rapidly increasing incidence or expanding geographic range.

Emergence is associated with (previously unrecognised) cross-species transmission (host jumping)

175
Q

What do biodiversity surveys show?

A

Biodiversity surveys reveal a myriad of viruses at the interaction zones between humans and animals.

176
Q

What is the route to a pandemic for HIV (group M)?

A

Kinshasa’s railway helped make it one of Africa’s best connected cities

A perfect storm of population growth, sex trade and connectedness allowed HIV to spread during the 1920s

177
Q

What are some factors affecting pathogen emergence?

A
  • Human behaviour
  • Antimicrobial misuse
  • Globalisation, trade and travel
  • Conflict and political upheaval
  • Weak surveillance, control, health systems
  • Agricultural intensification
  • Water management
  • Climate change
  • Land use change
  • Population growth and urbanisation
178
Q

What are some take-home messages for One Health?

A

One Health has a broad scope: Zoonotic and emerging pathogens are one element

We live in a complex, inter-connected and altered world

A key feature of One Health is that it integrates multiple disciplines to solve complex challenges

Your biological and bioveterinary training is an excellent starting point for contributing to One Health

Principles in Infectious Diseases (3 decks)

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