Diagnosis and Control of Viral Infection
Specimens ★★★
The deck opens by naming the three things a laboratory can look for, and it is the frame for the whole unit: ANTIBODY (IgM, IgG, titre), VIRAL PROTEIN (HBsAg, CMV-pp65, HCV core, adenovirus, RSV) and VIRAL GENOME (PCR, RT-PCR).
| Site of viral infection | Site of specimen collection |
|---|---|
| Upper respiratory tract | Pharyngonasal secretion |
| Lower respiratory tract | Sputum |
| Central nervous system | Cerebrospinal fluid |
| Gastrointestinal tract | Faeces |
| Purpose | Rule |
|---|---|
| For viral isolation | Take from the shedding site AS EARLY AS POSSIBLE. Inactivation and contamination must be prevented |
| For serological tests | PAIRED blood samples |
Examination by microscopy: inclusion bodies by light microscopy — such as the Negri body of rabies virus in an infected neuron (HE stain, intracytoplasmic) — and viral particles by electron microscopy, such as the wheel shape of HRV and the crown shape of SARS-CoV.
- Name the three diagnostic targets. → Antibody (IgM, IgG, titre), viral protein, viral genome
- Which specimen for each site? → URT — pharyngonasal secretion · LRT — sputum · CNS — CSF · GI tract — faeces
- Two rules for specimens? → For isolation, take from the shedding site as early as possible, avoiding inactivation and contamination; for serology, take PAIRED samples
- What is seen by light and by electron microscopy? → Inclusion bodies (e.g. Negri bodies) by LM; viral particles (HRV wheel, SARS-CoV crown) by EM
Viral isolation and cultivation ★★★
- Animal inoculation
- Embryonated chicken egg inoculation
- Tissue culture / cell culture
⭐ Indications of viral growth in cell cultures — four
| Indication | Detail |
|---|---|
| CPE | Including inclusion body formation — also rounding and clumping, and syncytia formation |
| Haemadsorption | Red cells adhere to the infected monolayer, because HA (haemagglutinin) has appeared on the cell membrane |
| Viral interference | Unit 19 §4 |
| Transformation | Loss of contact inhibition |
Some viruses — parainfluenza, mumps, and influenza in some systems — bud out of the cell without killing it. There is no CPE to see, so the culture looks negative.
But budding requires the virus to insert its spikes into the host cell membrane first (Unit 18 §5, release). One of those spikes is haemagglutinin. So the infected cell, though it looks normal, now has HA on its surface.
Add red cells and they stick — haemadsorption. An invisible infection made visible by exploiting the very molecule the virus needs in order to leave.
The same molecule underlies the haemagglutination test in §4: free virus particles bridge red cells into a lattice instead of sticking them to a monolayer. One spike protein, two assays.
- Name the three methods of viral cultivation. → Animal inoculation, embryonated chicken egg inoculation, tissue/cell culture
- Name the four indications of viral growth in cell culture. → CPE (including inclusion bodies) · haemadsorption · viral interference · transformation with loss of contact inhibition
- What is haemadsorption, and why does it work? → Red cells adhere to infected cells because haemagglutinin has been inserted into their membrane ready for budding
Measuring viral infectivity ★★★
Under overlay conditions, a small area of cell lysis (CPE) in a monolayer cell culture arising from infection by a SINGLE infectious virus. Expressed as PFU/ml.
50% tissue culture infectious dose / 50% infectious dose: the reciprocal of the highest dilution of virus that produces 50% experimental animal death, infection, or CPE in inoculated tissue culture.
The average number of infectious virus particles in each infected cell.
- Define PFU. → A small area of cell lysis in a monolayer under overlay, arising from a single infectious virus; expressed as PFU/ml
- Define TCID50. → The reciprocal of the highest virus dilution producing 50% animal death, infection or CPE in inoculated tissue culture
- Define MOI. → The average number of infectious virus particles per infected cell
Detecting viral components, serology and nucleic acid ★★★
| Component | Detection |
|---|---|
| Viral proteins | Haemagglutination — detecting haemagglutinin |
| Viral enzymes | Reverse transcriptase in serum indicates the presence of a retrovirus |
| Genetic material | Probes · PCR and RT-PCR — appropriate primers promote a million-fold amplification of a target sequence in a few hours; especially useful for detecting LATENT and INTEGRATED virus sequences, and viruses present in low concentration |
⭐ Serologic tests — two rules
- The detection of virus-specific IgM antibody, present during the first 2–3 weeks of a primary infection, generally indicates a RECENT PRIMARY infection
- Serological conversion is indicated by AT LEAST A FOURFOLD RISE in the antibody titre between the acute phase and the convalescent phase
Methods: HI (haemagglutination inhibition test) · ELISA (enzyme-linked immunosorbent assay) · RIA (radioimmunoassay) · Western blot analysis. The ELISA may be an antibody-capture assay (anti-IgM captures the patient's IgM, then antigen and enzyme-labelled antibody are added) or a competition assay (patient serum competes with a labelled antibody for antigen — so less signal means a positive result).
This is the reason §1 insisted on paired samples, and it is a favourite examination point.
A single positive antibody result tells you the patient met this virus at some point in their life — possibly twenty years ago, possibly as a vaccine. It does not say the present illness is caused by it.
Two ways round it:
IgM, which appears in the first 2–3 weeks and then disappears. Its presence means recent primary infection. One sample suffices.
A fourfold rise in titre between an acute and a convalescent sample — seroconversion. The titre is rising now, so the infection is now. Why fourfold and not double? Because titres are measured in doubling dilutions, and a single dilution's difference is within experimental error. Two dilutions — fourfold — is real.
This is also the rule behind the Mycoplasma pneumoniae diagnosis in Unit 14: a fourfold rise in IgG or IgM.
- What does serum reverse transcriptase indicate? → The presence of a retrovirus
- What is PCR especially useful for? → Detecting latent and integrated viral sequences, and viruses present in low concentration
- What does virus-specific IgM mean? → A recent primary infection — it is present in the first 2–3 weeks
- What defines seroconversion? → At least a fourfold rise in antibody titre between acute and convalescent samples
- Name four serological methods. → HI, ELISA, RIA, Western blot
⭐ Immunisation and antiviral drugs ★★★
| Active immunisation — vaccines | Example |
|---|---|
| Live attenuated vaccines | Sabin vaccine (polio) |
| Killed / inactivated vaccines | Salk vaccine (polio) |
| Subunit vaccines | |
| Genetic engineering vaccines | |
| ts strain vaccines | temperature-sensitive strains |
Passive immunisation: human immunoglobulin is used in emergencies, because it contains high titres of antibody.
⭐ Live attenuated versus killed vaccine
| Property | Live vaccine | Killed vaccine |
|---|---|---|
| Route of administration | Natural route or injection | Injection |
| Dose of virus, cost | Low | High |
| Number of doses | Single | Multiple |
| Need for adjuvant | No | Yes |
| Duration of immunity | Many years | Generally less |
| Antibody response | IgG and SECRETORY IgA | IgG and serum IgA |
| Cellular immune response | Good | Poor |
| Heat lability in the tropics | Yes | No |
| Interference | Occasional | No |
| Side effects | Occasional mild symptoms | Occasional sore arm |
| Reversion to virulence | Rarely | No |
Antiviral drugs — inhibiting viral replication at different stages
| Category | Examples |
|---|---|
| Nucleotide analogues | ACV (acyclovir), azidothymidine (AZT), ribavirin |
| Protease inhibitors | Saquinavir, indinavir, ritonavir |
| Uncoating and release inhibitors | Amantadine, rimantadine · zanamivir and oseltamivir (Tamiflu) |
| Interferon and interferon inducers | poly I:C |
| Antisense oligonucleotides | asON for mRNA, asODN for DNA |
Read the two columns as a single sentence: a live vaccine actually infects you, and a killed one does not.
Because it replicates, a live vaccine needs only a low dose and a single administration; it needs no adjuvant; it can be given by the natural route, so it induces secretory IgA at the mucosa where the real virus would arrive; it gives a good cellular response, because antigen is made inside cells and presented on MHC class I; and immunity lasts many years.
For the same reason it carries every disadvantage: it is heat-labile (the cold chain problem in the tropics), it can suffer interference (Unit 19 §4 — the three polio types), it can cause mild symptoms, and it can revert to virulence, rarely.
That last row is why Sabin's oral vaccine, superb for eradicating polio in a population through faecal spread of the vaccine strain, has been replaced by Salk's injected killed vaccine at the endgame — when the only remaining cases are vaccine-derived.
It is also why live vaccines are contraindicated in the immunosuppressed and in pregnancy: a genuine, if attenuated, infection is still an infection.
- Name the five types of active vaccine. → Live attenuated (Sabin), killed/inactivated (Salk), subunit, genetic engineering, ts strain
- When is passive immunisation used? → In emergencies, using human immunoglobulin with high antibody titres
- Give six differences between live and killed vaccine. → Natural route vs injection · low vs high dose and cost · single vs multiple doses · no vs yes adjuvant · many years vs less immunity · secretory IgA and good cellular response vs serum IgA and poor cellular response
- Which two disadvantages are unique to live vaccine? → Heat lability and possible reversion to virulence (plus occasional interference and mild symptoms)
- Name the five categories of antiviral drug with examples. → Nucleotide analogues (acyclovir, AZT, ribavirin) · protease inhibitors (saquinavir, indinavir, ritonavir) · amantadine/rimantadine and zanamivir/oseltamivir · interferon and inducers (poly I:C) · antisense oligonucleotides
⭐ The deck's four questions
Add its use: CPE is one of the four indications of viral growth in cell culture, and the basis of the plaque assay and TCID50.
A · Specimen collection — from the appropriate site (pharyngonasal secretion, sputum, CSF, faeces), taken from the shedding site as early as possible, avoiding inactivation and contamination; paired sera for serology.
B · Direct examination — inclusion bodies by light microscopy, viral particles by electron microscopy, and immunofluorescence.
C · Isolation and cultivation — animal inoculation, embryonated chicken eggs, or cell culture; recognised by CPE, haemadsorption, viral interference or transformation, and quantified as PFU, TCID50 or MOI.
D · Detection of viral components — viral proteins (haemagglutination), viral enzymes (reverse transcriptase indicating a retrovirus), and genome by probes, PCR and RT-PCR.
E · Serological tests — virus-specific IgM indicates recent primary infection; a fourfold rise in titre between acute and convalescent sera indicates seroconversion. Methods: HI, ELISA, RIA, Western blot.
Close with the trade-off in one sentence: a live vaccine replicates in the host, which is the source of all its advantages and all its risks.
Protease inhibitors — saquinavir, indinavir, ritonavir.
Amantadine and rimantadine; zanamivir and oseltamivir (Tamiflu).
Interferon and interferon inducers — poly I:C.
Antisense oligonucleotides — asON for mRNA, asODN for DNA.
The organising idea is the deck's opening line: they inhibit viral replication at different stages of the six-step cycle in Unit 18 §5.
Revision
The whole unit on one screen
| Question | Answer |
|---|---|
| Three diagnostic targets? | Antibody · viral protein · viral genome |
| Specimen sites? | URT pharyngonasal · LRT sputum · CNS CSF · GI faeces |
| Cultivation methods? | Animal · embryonated egg · cell culture |
| Growth indications? | CPE · haemadsorption · interference · transformation |
| PFU? | Area of lysis from a single infectious virus, per ml |
| TCID50? | Reciprocal of the highest dilution giving 50% death, infection or CPE |
| MOI? | Average infectious particles per infected cell |
| IgM means? | Recent primary infection (first 2–3 weeks) |
| Seroconversion? | ≥ fourfold rise in titre, acute to convalescent |
| ⭐ Live vaccine? | Natural route · low dose · single · no adjuvant · secretory IgA · good CMI · heat-labile, can revert |
| ⭐ Killed vaccine? | Injection · high dose · multiple · adjuvant needed · serum IgA · poor CMI · stable, no reversion |
| Sabin vs Salk? | Live vs killed polio vaccine |
- How would you confirm a viral diagnosis in the laboratory? → Specimen collection from the right site and time (paired sera) → direct LM/EM and immunofluorescence → isolation in animals, eggs or cell culture, recognised by CPE, haemadsorption, interference or transformation → detection of viral proteins, enzymes and genome by PCR → serology for IgM or a fourfold titre rise
- Compare live and killed vaccines. → See the eleven-row table — the trade-off is that a live vaccine replicates in the host
- Define CPE, PFU, TCID50 and MOI. → Morphological cell changes visible by LM · a plaque from a single infectious virus · the reciprocal of the dilution causing 50% infection or CPE · the average infectious particles per cell
- Name the five categories of antiviral drug. → Nucleotide analogues · protease inhibitors · amantadine/rimantadine and zanamivir/oseltamivir · interferon and inducers · antisense oligonucleotides