Basic Virology

Question 1

Types of specimens for different infections

Answer 1

Resp –> swab (throat/nasal), aspirate (NPA, tracheal)
GI –> stool
Neuro –> CSF
Genital –> urethral or endocervical swab
Vesicular rash –> skin scraping/ swab, vesicular fluid

Most will need serum sample as well

Question 2

Methods of Direct Detection in Viral Diagnosis: method, benefits, limitations

Answer 2

Immunofluorescence

• rapid point of care test (15-30 min)
• direct assay –> detect Ag using labelled Ab
• indirect assay –> serology (add Ab then labelled anti-IgG Ab)
• latex agglutination –> complex formation between Ag and Ab
• limitation: requires cell containing viral Ag – can’t use in serum, stool

Electron Microscopy

• low sensitivity (need 10^6 particles/ml to visualise)
• good for detection of virus in stool and skin (high titres)
• only detect morphology up to family level –> further use PCR or culture to differentiate
• benefits: rapid, high specificity
• limitations: technically demanding, low sensitivity

Histology/Cytology

• target disease tissue
• viral inclusion bodies: collections of replicating viral particles e.g. Owl’s eye in CMV
• limitations: low feasibility

Question 3

Molecular Detection: method, benefits, limitations, conventional vs real-time

Answer 3

Applicable to solids and fluids
Not dependent on viability of virus

Nucleic acid hybridisation using direct detection of viral nucleic acid by probes

PCR most widely used

• amplify viral nucleic acid with subsequent analysis
• EXTREMELY SENSITIVE –> prone to contamination
• difficult to interpret +ve results –> latent viruses can give positives (but no disease)
• benefits: fast, specific, sensitive
• limitations: contamination, pre-requisite knowledge for making appropriate primer

Conventional PCR = detect and quantify products at the very end, using post-PCR analysis e.g. gel electrophoresis (technically demanding)

Real Time PCR = detect signals after every cycle and measure threshold cycle – quantify based on comparison to standard with known amount of DNA – high precision
(use probe with reporter and quencher dye –> binds to PCR products and cleaved by Taq DNA polymerase cycle –> releases dye which is proportional to amount of PCR products)

Question 4

Viral culture: method, conventional vs rapid, benefits, limitations,

Answer 4

Inoculated and cultured cell lines (animal, tissue, embryonated egg)
- primary, semi-continuous and continuous cell cultures

Benefits: High sensitivity and specificity (growing live virus)
–> based on observation of cytopathic effect - changes in structure of cell due to viral invasion e.g. clumping, darkening, fusion of cells to form multinucleate cells, rounding into “grape-like” clusters

Conventional culture: 1-2 weeks (use in research)

Rapid culture: 2 days –> low speed centrifugation enhances infectivity of certain viruses e.g. CMV DEAFF (detection of early antigen fluorescent foci)

Limitations: conventional type time consuming, technically demanding

Question 5

Serology: class specific vs function specific, methods, ELISA

Answer 5

Detection of Ab against Ag

Class specific

• Enzyme immunoassay, radioimmunoassay, IF
• IgM for acute infection (but not all), transient
• IgG for immunity (takes longer to increase)
• take one blood sample >1 week after illness onset
• positive or negative result

Function specific

• haemaglutination inhibition test, complement fixation test, neutralisation
  concept: virus + patients serum would produce absence of cytopathic effect (neutralisation) or haemaglutination if there are virus neutralising Ab in serum
• take 2 samples for acute and convalescent sera 10-14 days apart (to look for increase in Ab levels)
• no single diagnostic level

ELISA

• detect Ag –> wells coated with Ab -> add serum -> add enzyme-linked Ab (to Ag) -> wash -> add substrate specific to enzyme -> colour change = Ag +ve
• detect Ab –> beads coated with Ag -> add serum -> add enzyme linked anti-human Ab -> wash -> substrate -> colour change = +ve

Question 6

Interpretation of Viral Diagnostics: sensitivities and specificities

Answer 6

Sensitivity

• serology: class>function
• viral detection: PCR > culture > others (PA > EM)

Specificity

• cross reaction e.g. CFT for HSV and VZV, IgM for Dengue and Japanese encephalitis
• non-specific reactions in unrelated infection
• timing of sampling - late first sample (already peaked), IgM too early, acute and convalescent sera too close together

Question 7

Viral Morphologies: sizes, characteristic features of reoviridae, adenoviridae, caliciviridae, hepadnaviridae, orthomyxoviridae, paramyxoviridae, herpesviridae, papovavirdae, poxviridae

Answer 7

Reoviridae – 70-75 nm, “wheel” like
Adenoviridae – 70-75 nm, hexagonal with triangular faces
Caliciviridae – 30-35 nm, “Star of David”
Hepadnaviridae – 40-45 nm for complete virion, Dane particles/ Spherical form/ Tubular form
Orthomyxoviridae – 80-120 nm, peripheral fringe, pleomorphic
Paramyxoviridae – 90-300nm, shorter and less obvious peripheral fringe, pleomorphic, “herringbone-like” tubular structure
Herpesviridae – 95-105 nm, “fried egg”
Papovaviridae – 45-55 nm, skew arrangement
Poxviridae – 200-250 nm, dimorphic (mulberry and capsule), capsule with threadlike structures over it

Question 8

Innate Immunity in Viral Infections: characteristics, mechanism of recognition, main 1st line immune response features, innate cells and their functions

Answer 8

Rapid, immediate, non-specific –> suppress viral replication early

PAMP e.g. dsRNA of nucleic acid is recognised by PRR (pattern recognition receptors) e.g. TLR on cell membrane or intracellularly –> activated cascade of events promoting innate immunity

1. increase IFN-1 (main innate immunity in viral infections)
   - released by plasmacytoid dendritic cells and macrophages
   - effects: antiviral by blocking viral replication, immunomodulation of T/B/NK and cytotoxic T cells, prodromal symptoms (fever, malaise, fatigue)
   - not virus specific but species specific and induces antiviral state in neighbouring non-infected cells too
2. inflammatory cytokines e.g. IL1, IL-6, TNF
   - cellular recruitment, phagocytosis, initiate adaptive immune response
3. complement system
4. acute phase proteins

Main cells in innate immunity:
- NK cells (cytotoxic, produces IFN-gamma to induce other antiviral responses), PDC (viral sensors, potent IFN-1 secretion), DC (Ag presenting, +ve adaptive), Macrophages (inflammation, high levels of IFN-1)

Question 9

Adaptive Immunity in Viral Infections: characteristics, humoral response effects, cellular response effects

Answer 9

Develops over time (1-2 weeks), eliminates infection, provides memory (augment response), specific

Humoral response:

• stimulated B cells –> plasma cells secreting Ab
• effects: neutralisation (agglutinate virions, prevent attachment), opsonisation, ADCC (Ag-Ab-complement to lyse cells), interfere uncoating

Cellular response:

• Treg cells (helper: suppressor in 2:1) – either promote or inhibit other T and B cells
• cytotoxic T cells attack and lyse infected cells

Rmb MHC presentation of Ag in order for successful recognition by T cells

Question 10

Clinical Applications of Immunity: immunisations and their features, examples of passive immunisation (3), other applications (2)

Answer 10

Active immunisation (vaccines)

• depend on active response from immune system
• takes a few weeks to develop
• lasts for years (have memory)

Passive immunisation

• enriched virus-specific Ab (pooled or specific Ab)
• no active response
• immediate protection but short lived (1-3mths)
• option for post-exposure prophylaxis

e.g. IVIG - pooled Ig from healthy donors with mixture of Ab against common infections
Specific Ig - high conc of specific Ig against certain virus from recovered donors e.g. Hep B IG, Zoster IG
Synthetic - using recombinant DNA tech e.g. monoclonal IgG against RSV for prevention in high-risk group

Other applications:

• recombinant interferons to augment immune response (less used nowadays)
• adjuvant in vaccines to augment response e.g. non-specific stimulation of TLR to produce higher levels of Ab

Question 11

Route of entry of viruses and examples, stages of viral infection (5)

Answer 11

Skin - abrasions, inoculations, insect/ animal bite
Mucous membranes - conjunctiva, respiratory tract, GI tract, genital tract

Skin as barrier to microbes –> some degree of trauma allows virus to reach basal cells and start infection
— percutaneous infections e.g. HBV, HIV, Dengue start in cell types other than skin

Mucous membrane –> direct access to receptors on mucosal epithelial cells
– site of entry not necessarily ultimate site of infection e.g. rubella, VZV via pharynx and disseminate in bloodstream/ enterovirus via GI affects CNS/ skin

Stages of viral infection:
- entry –> regional LN for viral replication –> primary viraemia –> disseminate to target organs –> released from target organ causing secondary viraemia OR shedding of virus when amount exceeds threshold (transmission)

Question 12

Patterns of Viral infection: incubation time, mechanism/ outcome, vaccination use, examples

Answer 12

Acute

• short incubation
• complete clearance and form memory
• vaccination useful
  e. g. Hep A, Influenza, rubella

Latent

• have immune escape strategies
• establish latency which is not cleared by antivirals –> inactive, not replicating (dormant), non-infectious
• reactivate upon immunosuppression or stimulation
• vaccinations less useful
  e. g. Herpesvirus family (HSV: neurons, VZV: dorsal root ganglia, EBV: B lymphocytes)

Chronic

• have immune escape strategies
• initially asymptomatic and may develop complications later
• continuous viral replication for years (active, infectious)
• only occurs in a proportion of infected subjects
  e. g. HBV, HCV, HIV

Persistent = latent + chronic

Question 13

Interactions between virus and host cells: pathology (4), autoimmunity and immunosuppression

Answer 13

Pathology:

• tropism (infect specific cells) e.g. HBV hepatotrophic
• cytopathic effects
• —- e.g. cell lysis - death and release of virions in non-enveloped virus (lytic infection) vs budding in enveloped virus (less damage to host)
• —- fusion of cells in RSV – multinucleated giant cells
• inclusion bodies (aggregation of viral proteins in nucleus or cytoplasm, altered staining) e.g. Owl’s eye in CMV
• immune mediated damage e.g. HBV specific T cells causing hepatocyte damage; IFN cause fever and malaise

Autoimmunity:
- molecular mimicry (post-infection or vaccination) e.g. GBS after influenza vaccine, post-infectious encephalitis after influenza

Immunosuppression:

• significant number of critical immune cells killed e.g. CD4 T cells in HIV
• alternation cytokine response in infected cells
• induction of Treg
• e.g. measles –> paradoxical immunosuppression for weeks increasing risk of secondary bacterial infection