Animal models: virology II Flashcards
What is the percentage of results obtained in mice models that are somehow translated to humans?
~ 8%
Why do we want fewer animals?
3E’s –> Ethics, Economics and Efficacy
What is needed to replace animal models? (3)
- Legislation
- Mindset
- Science
How can horizontal virus transmission between rodents occur? (3)
- Saliva
- Urine
- Faeces
What are Orthohantavirus transmission risk factors? (3)
- Virus in the environment
- Human infection
- Rodent infection
What are risk factors for orthohantaviruses to be in the environment? (3)
- Rodent density
- Host infection phase
- Temperature, moisture, UV-radiation
What are the risk factors for rodent infection? (9)
- Seasonality
- Age
- Sex
- Maternal antibodies
- Co-infections
- Resistance genes
- Rodent density
- Species diversity
- Habitat type
What are the risk factors for human infection? (5)
- Rodent density
- Occupation/recreation
- Environmental conditions
- Season
- Resistance genes
Why does each strain of Orthohantavirus have a specific rodent host?
Strains have co-evolved with specific rodent species
Why is there a clear geographical distribution of Orthohantavirus strains?
Strains have co-evolved with specific rodent species –> live in certain location
Rodents carrying Orthohantaviruses are mostly symptomatic/asymptomatic
Asymptomatic
What symptoms do Orthohantaviruses often cause?
Haemorrhagic fever with renal syndrome (HFRS)
Which Orthohantaviruses are found in rodents in Europe? (4)
- Puumala virus
- Dobrava virus
- Seoul virus
- Tula virus
On what factors is a Orthohantavirus infection diagnosis based?
Clinical signs and serology
Serological assays to confirm Orthohantavirus infection are based on?
Cross-reactive antigens
What are the three different clinical manifestations of Orthohantavirus infections?
- Haemorrhagic fever with renal syndrome (HFRS)
- Nephropathia epidemics (NE)
- Hantavirus cardiopulmonary syndrome (HCPS)
Which Orthohantavirus causes nephropathia epidemica (NE)?
Puumala virus
What are the structural proteins of Orthohantaviruses? (3)
- Membrane glycoproteins Gn and Gc
- Polymerase L
- Nucleocapsid N
How do Orthohantaviruses attach?
Interaction with Gn/Gc (cell surface receptors)
Which process is initiated by the attachment of Gn/Gc proteins to integrin?
Endocytosis
Which integrins are bound by pathogenic orthohantaviruses?
B3-integrins
Which integrins are bound by NON-pathogenic orthohantaviruses?
B1-receptors
Integrins are present on which cell types?
Endothelial cells, macrophages and platelets
Integrins are involved in…(3)
- Regulation of endothelial cell adhesion
- Platelet aggregation
- Extracellular matrix interactions
Which process plays a central role in the pathogenesis of Orthohantavirus infections?
Vascular barrier loss
Orthohantaviruses: what causes disruption of vascular integrity?
Binding of Orthohantavirus glycoproteins to B3-integrin
What encompasses the disruption of vascular integrity caused by Orthohantaviruses? (3)
- Capillaries more permeable
- Arteriole vasoconstriction & vasodilation disrupted
- Binding to platelet receptor affects clotting and platelet function
Why do different Orthohantaviruses have different pathogenesis?
Different viruses bind endothelium in different locations
Where do HFRS bind to the endothelium?
Lungs, kidneys, spleen
Where do HCPS bind to the endothelium?
Lungs, liver, heart, spleen
Which immune response is mounted in humans to combat Orthohantaviruses?
Virus epitopes expressed on surface of host cells –> CD8+ T cell attack on host tissue
Why does the immune response against Orthohantaviruses that is mounted in human not occur in rodents?
Downregulation by Tregs
Describe the kinetics of Orthohantavirus infection in rodents (2)
- After acute infection: virus disappears from blood
- As infection continues: long-lasting antibody response are produced
Why do rodents not get sick from hantavirus infection
Local increases of Tregs, and decreases of CD8+ T cells at primary sites of replication
What does the term ‘neuro-invasion’ mean?
The ability of a virus enter either the PNS or CNS
How is neuro-invasion described in the case of respiratory viruses?
The ability to travel from the respiratory tract to the CNS
What are the routes of virus spread into the CNS? (4)
- Peripheral nerves
- BB barrier
- Cranial nerves
- Blood-CSF barrier
Virus spread: which kind of transport is used via the peripheral nerves?
Retrogade transport
Virus spread: which kind of transport is used via the BB barrier?
Intracellular (Trojan Horse)
What does the term neurotropism mean?
The ability of a virus to infect and replicate in cells of the nervous system
What does the term neurovirulence mean?
The ability of a virus infection to cause lesions in the CNS that contribute to the development of clinical disease of the nervous system independently of its neuroinvasiveness or neurotropism
Which route of neuro-invasion occurs in SARS-CoV2 infection?
Mostly olfactory nerve
Describe the preferred neurotropism in SARS-CoV2 infection (2)
- Glomerular layer neural cells
- Cortical neurons
Describe the neurovirulence in SARS-CoV2 infection (4)
- Lasting immune activation -> cognitive impairments
- Loss of oligodendrocytes -> memory impairments
- Shrinkage of specific brain regions
- Increased risk for depression, anxiety, dementia, psychosis
What categories of pathological changes due to virus infections exist? (5)
- Necrosis
- Inflammation
- Hyperplasia/neoplasia
- Hypoplasia
- Atrophy
What is the difference between hypoplasia and atrophy?
Hypoplasia = something is not formed which would usually be formed (lack of growth)
Atrophy = loss of tissue
Name an example of necrosis caused by viral infection
Herpesviruses cause necrosis of epithelial cells
Name an example of inflammation caused by viral infection
Pneumonia from H5N1 influenza –> infection of alveolar epithelial incited marker immune response from host
Name an example of hyperplasia/neoplasia caused by viral infection
Pock from poxvirus –> hyperplasia of epidermal cells
Name an example of hypoplasia caused by viral infection
Cerebellar hypoplasia from ZIKV
Name an example of atrophy caused by viral infection
Villus atrophy from coronaviruses (in animals)
What are the pathological mechanisms of viruses? (3)
- Direct damage to host cell
- Induction of host immune responses
- Transformation of infected host cell
Direct damage to infected host cell, can be through… (4)
- Inhibition of host cell DNA/RNA/protein synthesis
- Damage to host cell integrity
- Lysis of host cells
- Induction of apoptosis/necroptosis
Why can inhibition of host cell DNA/RNA/protein synthesis cause direct damage to infected host cells?
Viruses take over the replication machinery to produce virus particles
What causes damage to host cell integrity by viral infection?
Viral replication processes
What is often the purpose of lysis of host cells caused by viral infection?
Release of viral particles
Induction of apoptosis/necroptosis is often not a viral strategy, what is it?
Usually a side effect of cellular replication
What are the main components of the innate immune response against viral infection? (5)
- Infected cells
- Sentinel cells
- Effector cells
- Cytokines
- Complement
What is the main effector cytokine during viral infection?
IFN –> triggers neighboring cells to prevent further infection
Describe the events (in order) of the innate immune response against viral infections (3)
- PRRs on sentinel cells recognize virus motifs
- Infected/sentinel cells produce cytokines
- Complement activation
What are the typical presentations of innate immune responses against viral infections? (5)
- Calor
- Rubor
- Tumor
- Dolor
- Functio laesa
What is Calor?
Increased blood flow
What is Rubor?
Increased blood flow (by cytokines) -> dilutes pathogen, extravasation of inflammatory cells
What is Tumor?
Increased blood vessel permeability (oedema) -> dilutes pathogen, extravasation of inflammatory cells
What is Dolor?
Inflammatory processes
What are examples of lesions associated with the adaptive immune response? (B-cells) (2)
- Virus-antibody complexes
- Non-protective antibodies
Name an example of virus-antibody complexes in viral infection
LCMV infection in mice, hepatitis B infection in humans
Name an example of non-protective antibodies in viral infection
Dengue virus infections in humans
Name two examples of viruses that have caused systemic inflammatory response syndrome
- H5N1 influenza
- SARS-CoV2
What are the mechanisms of transformation of the infected host cell caused by viral infections? (3)
- Expression virus-encoded oncogenes
- Insertional mutagenesis
- Anti-apoptotic
Name an example of the expression of virus-encoded oncogenes
HPV production of E6 and E7
Name an example of insertional mutagenesis
HPV viral genes inserted into host genome
Name an example of anti-apoptotic measures
EBV -> immortalization and proliferation of B cells
What are the possible evolutionary benefits/selective advantages causing disease due to viral infections? (3)
- Virulence is coincidental byproduct
- Virulence is beneficial for the virus
- Co-evolution of virus and host
What are the characteristics of ‘virulence is a coincidental byproduct’? (2)
- No advantage to virus
- Often in zoonotic viruses
What are the two main hypotheses of ‘virulence is beneficial for the virus’?
A: intermediate virulence hypothesis
B: short-sighted evolution hypothesis
Describe the intermediate virulence hypothesis
- Virulence = cost to virus -> lower host survival
- Virulence = benefit -> higher transmission
- Intermediate level of virulence = favoured
Describe the short-sighted evolution hypothesis
- Within host competition among virus genotypes
- Genotype with higher virulence favoured without enhanced transmission
What are the three scenarios of co-evolution of virus and host?
- A: Slow host evolution
- B: Fast host evolution
- C: Balance in evolution
How does slow host evolution work?
Virus has upper hand -> outcome = high virulence
How does fast host evolution work?
Host has upper hand -> outcome = low virulence
How does balance in evolution of virus and host work?
Both host and virus evolve -> outcome = intermediate virulence
What are possible complications in understanding evolution of host-virus interaction? (3)
- Host usually infected by multiple pathogens
- Some viruses can infect several host species
- Environment involved
Which Rabiesvirus structural proteins are involved in blocking of the interferon/antiviral response? What do they inhibit? (3)
- P-protein -> IRF3/STAT1,2 signaling
- N-protein -> RIG-I activation
- M-protein -> NF-kB activation
Which Rabiesvirus structural protein is involved in the blocking of inflammatory responses?
G-protein -> inhibition of inflammatory responses by macrophages
How does the P-protein of Rabiesvirus inhibit IFR3 signaling?
Blocks phosphorylation of IRF3 -> no dimerization and translocation to nucleus
How does the N-protein of Rabiesvirus inhibit RIG-I activation?
Inhibits RIG-I’s ability to recruit factors to phosphorylate IRF3
How does Rabiesvirus block NF-kB activation?
No translocation to nucleus of p50-p65
How does Rabiesvirus block interferon-stimulated genes (ISGs)?
P-protein blocks translocation to nucleus & binding of the complex to ISGs tf’s
Dog bites normally recruit strong immune responses due to high bacteria numbers and tissue damage. Which pathway prevents this during Rabiesvirus infection?
Cholinergic anti-inflammatory pathway
What are commonly used in vivo (invertebrate) models that replace animal models? (3)
- Drosophila melanogaster
- Nematode
- Galleria mellonella = greater wax moth larvae
What is a con of using G.Mellonella as a model?
Relatively new model -> no techniques to genetically manipulate this organism
What are the pro’s of using G.Mellonella as a model? (5)
- Drug discovery
- Tissue recovery
- Phagocytosis
- Hyphae evaluation
- Mammalian temperatures
What kind of infection models are used using G.Mellonella as a model? (4)
- Gram-negative bacteria
- Gram-positive bacteria
- Fungi
- Virus
Why is G.Mellonella a relatively cheap model?
Larvae can be brought or grown on site
What can be said about the temperature at which you keep the G.Mellonella larvae? Why is this advantageous?
Can be kept at 37C -> not possible for some other invertebrate model systems
What are types of experiments that can be performed using G.Mellonella? (5)
- Survival/virulence
- Fungal load
- Histology
- Host responses
- Efficacy of treatments
The immune system of G.Mellonella has similarities/no similarities to that of humans
Similarities
How do you perform a survival experiment using G.Mellonella?
Infect larvae using injection in pro-legs -> monitor survival
How do you monitor survival of G.Mellonella larvae?
Melanisation -> immune system uses melanin -> blacker larvae = sicker larvae
How can you analyse the survival data of G.Mellonella larvae? (2)
- Kaplan-Meier plots -> survival for various doses
- LD50 -> concentration at which 50% of larvae die
Which two techniques can be used to determine the fungal load in G.Mellonella larvae?
- Harvest hemolymph and plate out
- Homogenize complete larvae
How do you perform a histological examination of infection of G.Mellonella larvae? (2)
- Fix larvae in formalin
- Process for histology
G.Mellonella only has an innate immune system, consisting of two components… (2)
- Cellular immune response: haemocytes
- Humoral immune response
What are haemocytes? Why?
They are like neutrophils -> receptors, signaling pathways and defense mechanisms are similar
What encompasses the humoral immune response of G.Mellonella? (3)
- Opsonins
- Antimicrobial peptides
- Melanisation
How is the severity of the immune response measured in G.Mellonella?
Haemolymph melanisation -> measuring concentrations
What types of experiments are possible using G.Mellonella as a drug discovery model? (3)
- Toxicity
- PK/PD
- Survival analysis
How do you perform a PK/PD experiment using G.Mellonella?
Harvesting hemolymph at different timepoints after injection -> how fast is compound broken down?
What is a difficulty in antifungal therapy studies using G.Mellonella?
Consecutive injection impossible due to small larvae size (max. 3 injections)
