Poliovirus pathogenesis,prophylaxis, vaccine micro

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Poliovirus: Pathogenesis, Prophylaxis, and Vaccines

1. Classification and Structure

Poliovirus belongs to the family Picornaviridae, genus Enterovirus. There are 3 serotypes (types 1, 2, and 3). It is a non-enveloped, single-stranded positive-sense RNA virus with an icosahedral capsid composed of four viral proteins (VP1, VP2, VP3, VP4). The genome encodes a single polyprotein cleaved by viral proteases into structural and enzymatic components. The virus is:

Stable in acid (survives stomach), resistant to alcohol and detergents
Susceptible to chlorine-containing disinfectants
Can persist for days at room temperature
Sherris & Ryan's Medical Microbiology, 8th Ed., p. 463
Harrison's Principles of Internal Medicine, 22E, p. 1675

2. Pathogenesis

Enterovirus pathogenesis diagram showing route from oropharynx through viremia to target tissues including brain/motor neurons causing paralytic disease

Fig. 46.4 - Pathogenesis of enterovirus infection showing poliovirus target tissues (Medical Microbiology 9e)

Step-by-step sequence:

Step 1 - Entry and primary replication Virus enters via the fecal-oral route. After ingestion, it infects epithelial cells in the oropharyngeal mucosa, then passes to the gut. Virus binds to the poliovirus receptor (PVR) / CD155, an immunoglobulin-superfamily receptor expressed only in primates (explaining primate-limited tropism). Replication occurs in the mucosa and lymphoid tissue of the tonsils, Peyer's patches, and intestinal enterocytes. Virus is shed in oral secretions for up to 3 weeks and in stool for up to 12 weeks (>20 years in hypogammaglobulinemic patients).

Step 2 - Primary viremia Virus enters regional lymph nodes, then the bloodstream. Viremia is detectable 3-5 days after infection. The virus spreads to reticuloendothelial cells of the lymph nodes, spleen, and liver. If the host immune response is adequate, infection is contained here - this accounts for the majority (90%+) of subclinical infections.

Step 3 - Secondary viremia and CNS invasion In a subset of patients, a second round of replication drives secondary viremia. The CNS is reached via two routes:

Hematogenous - crosses the blood-CNS barrier, favored by reflex dilation of capillaries supplying motor centers of the anterior horn
Neural (axonal transport) - virus enters skeletal muscle at the neuromuscular junction (where PVR is expressed at the endplate), then travels retrogradely along peripheral nerve axons to the spinal cord - analogous to rabies virus spread. Sciatic nerve transaction in monkeys prevents CNS infection after IM injection, supporting this route.

Step 4 - Motor neuron destruction Poliovirus is cytolytic for anterior horn cells (lower motor neurons) of the spinal cord and brainstem motor nuclei. The virus shuts down host protein synthesis by cleaving eIF4-G (the cap-binding protein of ribosomes), preventing cellular mRNA translation. Up to 100,000 virions per cell are produced before lysis. The location and number of neurons destroyed determines the extent of paralysis.

Harrison's Principles of Internal Medicine, 22E, pp. 4455-4457
Sherris & Ryan's Medical Microbiology, 8th Ed., pp. 2958-2963
Medical Microbiology 9e, pp. 2922-2924

3. Clinical Manifestations

Presentation	Frequency	Features
Subclinical	~90%	No symptoms; still sheds virus
Abortive poliomyelitis	~5%	Fever, sore throat, myalgia, headache, GI symptoms; resolves in 2-3 days; no CNS involvement
Aseptic meningitis (nonparalytic)	~1%	Meningeal signs (stiff neck, back pain) + abortive symptoms; CSF: lymphocytic pleocytosis, normal glucose, normal/mildly elevated protein; recovers fully within days
Paralytic poliomyelitis	<2%	Asymmetric flaccid paralysis with NO sensory loss; maximum extent within days; recovery up to 6 months; permanent thereafter

Incubation period: 4-35 days (usually 7-14 days for paralytic disease; 3-6 days for minor illness).

Bulbar polio: brainstem involvement causes paralysis of cranial nerves and respiratory muscles - most life-threatening form.

Post-polio syndrome: decades after acute infection, previously affected patients develop new progressive muscle weakness, fatigue, and pain - thought to be due to loss of the enlarged motor units that had reinnervated muscle after original attack.

Sherris & Ryan's Medical Microbiology, 8th Ed., pp. 2974-2984
Harrison's Principles of Internal Medicine, 22E, pp. 4475-4477

4. Immunity

Poliovirus induces specific IgM (persists <6 months) and specific IgG (lifelong).
Capsid protein VP1 is the predominant neutralizing antibody target.
IgG eliminates viremia and prevents CNS spread.
Secretory IgA is key for eliminating virus from the GI tract and reducing fecal shedding.
Neutralizing antibody confers lifelong protection against the same serotype but does not prevent initial infection or shedding.
Patients with agammaglobulinemia develop severe disseminated disease - emphasizing the primacy of humoral immunity.
Breast milk IgA can protect neonates from infection.
Harrison's Principles of Internal Medicine, 22E, p. 4460

5. Vaccines (Prophylaxis)

Two vaccines exist, each containing all three serotypes (1, 2, 3):

A. Inactivated Polio Vaccine (IPV) - Salk Vaccine

Developed by Jonas Salk, introduced 1955
Killed (formalin-inactivated) virus; given by subcutaneous injection
Primarily stimulates IgG antibodies (systemic humoral immunity)
Eliminates virus during viremia, preventing CNS spread
4-dose schedule: ages 2 months, 4 months, 6-18 months, and 4-6 years - produces antibody in >98% of recipients
No risk of vaccine-associated paralytic polio (VAPP)
Currently used exclusively in the United States and most developed countries
Does NOT produce strong mucosal IgA; less effective at eliminating intestinal shedding

B. Oral Polio Vaccine (OPV) - Sabin Vaccine

Developed by Albert Sabin, licensed in the United States in 1963
Live, attenuated virus prepared by serial passage in cell cultures
Given orally at a lower dose than IPV (replicates in intestinal epithelium)
Stimulates IgA at mucosal surfaces (GI tract) AND systemic IgG + cell-mediated immunity (CD4, CD8)
Produces antibodies to all 3 serotypes in >95% of recipients
Key advantage: Like wild-type virus, OPV replicates and spreads - providing "contact immunity" to unvaccinated persons in the community
Key disadvantage: Rare vaccine-associated paralytic poliomyelitis (VAPP) - ~1 case per 2.4 million doses (higher risk with first dose and in immunocompromised recipients)
Vaccine-derived poliovirus (VDPV): Attenuated strains can revert to neurovirulence during replication, especially type 2 - now a major global concern (526 VDPV cases in 2023 vs. 12 wild-type cases)
WHO now recommends at least 1 IPV dose alongside OPV schedules

Comparison Table

Feature	IPV (Salk)	OPV (Sabin)
Type	Killed	Live attenuated
Route	Subcutaneous injection	Oral
Immunity induced	IgG (systemic)	IgA (mucosal) + IgG + CMI
Mucosal protection	Weak	Strong
Herd immunity via spread	No	Yes
VAPP risk	None	~1/2.4 million doses
VDPV risk	None	Yes (especially type 2)
Use in immunocompromised	Safe	Contraindicated
Current US use	Yes	No (discontinued 2000)

Sherris & Ryan's Medical Microbiology, 8th Ed., pp. 2994-3021
Harrison's Principles of Internal Medicine, 22E, pp. 4583-4631

6. Eradication Progress

1952: 57,879 cases in the US (peak)
1955: IPV introduced; cases plummeted
1979: Last endogenous US case
1991: Last paralytic case from wild-type virus in Western Hemisphere
2015: Wild-type type 2 declared eradicated globally
2019: Wild-type type 3 declared eradicated globally
2023: Only 12 wild-type cases globally (all type 1, in Afghanistan and Pakistan); 526 vaccine-derived cases in 21+ countries
Remaining challenges: Political instability, vaccine hesitancy, VDPV outbreaks, long-term poliovirus shedding by immunocompromised individuals (>25 years documented)
Harrison's Principles of Internal Medicine, 22E, p. 4587

7. Treatment

There is no specific antiviral treatment for established poliomyelitis. Management is supportive:

Respiratory support (ventilators for bulbar/respiratory paralysis)
IV immunoglobulin for chronic enterovirus infections in hypogammaglobulinemic patients
Glucocorticoids are contraindicated
Experimental agents: pocapavir (showed rapid resistance development), vapendavir - still in trials
Physiotherapy for paralytic sequelae

Quick Summary: Key Exam Points

Receptor: CD155 (PVR) - immunoglobulin superfamily - expressed only in primates
Virus shuts down host translation by cleaving eIF4-G
CNS tropism: hematogenous AND axonal (retrograde) transport
Paralysis: asymmetric flaccid, NO sensory loss, anterior horn cells (LMN lesion)
IPV = IgG = systemic; OPV = IgA = mucosal
VAPP is an OPV complication; IPV has NO risk
VDPV (especially type 2) now causes more cases than wild-type poliovirus globally
Agammaglobulinemia = severe polio risk (humoral immunity critical)
Poliovirus is the model used to understand all enterovirus pathogenesis

Orthomixo virus antigen shift and drift and lab diagnosis micro

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Orthomyxoviruses (Influenza): Antigenic Shift, Antigenic Drift, and Laboratory Diagnosis

1. Classification and Structure

Family: Orthomyxoviridae Types: Influenza A, B, C (and D - animals only)

Key structural features:

Enveloped, pleomorphic, 80-120 nm spherical particle
Single-stranded, negative-sense RNA in 8 separate gene segments (Influenza A & B); 7 segments in Influenza C (lacks neuraminidase gene)
The segmented genome is the molecular basis for antigenic shift
Nine structural proteins including:
- Hemagglutinin (HA) - surface spike glycoprotein, ~25% of viral protein; 18 subtypes (H1-H18)
- Neuraminidase (NA) - surface spike glycoprotein, ~5%; 11 subtypes (N1-N11)
- Nucleoprotein (NP) and Matrix (M) protein - internal; determine A/B/C typing
- PB1, PB2, PA - RNA polymerase complex
- M2 - ion channel (target of amantadine)

HA and NA are the two surface antigens that determine immune recognition, subtyping, vaccine composition, and the basis of antigenic variation.

Jawetz Melnick & Adelbergs Medical Microbiology 28E, pp. 587-590

2. Antigenic Drift vs. Antigenic Shift

Antigenic drift vs antigenic shift - drift shows stepwise HA/NA change over years while NP remains constant; shift shows abrupt change in one year

Fig. 39-3: Antigenic drift (gradual, stepwise change in HA/NA over years) vs. antigenic shift (abrupt, large change within one year). Internal proteins like NP remain constant. (Jawetz Melnick & Adelbergs, 28E)

Diagram: Antigenic drift - point mutations alter HA epitopes so antibodies no longer bind. Antigenic shift - RNA segment exchange between two viral strains in a secondary host produces virus with entirely novel HA that existing antibodies cannot recognize

Fig. 12.45: Mechanism of antigenic drift and shift in influenza A. (Janeway's Immunobiology 10e)

A. Antigenic Drift

Feature	Detail
Mechanism	Accumulation of point mutations in HA and/or NA genes
Result	Gradual amino acid changes at antigenic epitopes; virus "escapes" pre-existing antibody
Speed	Slow, stepwise over years
Viruses affected	All three types: A, B, and C
Epidemiologic consequence	Local/regional epidemics every 2-3 years (Influenza A); every 3-6 years (Influenza B)
Immune effect	Pre-existing antibody gives partial (reduced) protection - mild/modified disease, not complete failure
Minimum mutations	A variant must sustain 2 or more mutations before a new epidemiologically significant strain emerges

Key concept: The immune system does not cause antigenic drift - it acts as a selection force that allows new antigenic variants (which evade existing antibody) to expand in the population.

Jawetz Melnick & Adelbergs Medical Microbiology 28E, p. 590

B. Antigenic Shift

Feature	Detail
Mechanism	Genetic reassortment of RNA gene segments during co-infection of a single cell by two different influenza A strains
Result	Abrupt, drastic replacement of HA and/or NA with an entirely novel subtype
Speed	Sudden - can occur within one year
Viruses affected	Influenza A ONLY (not B or C)
Epidemiologic consequence	Pandemic - globally immunologically naive population
Animal reservoir	Aquatic birds are the natural reservoir of all 18 HA subtypes; pigs serve as "mixing vessels" for human + avian strains

Why only Influenza A? Influenza B and C are restricted to humans (no significant animal reservoir), so there are no related animal strains to reassort with. Influenza A circulates in birds, pigs, horses, seals, whales, and humans - providing the diversity needed for reassortment.

The "mixing vessel" (pig) model: Pig respiratory tract cells express receptors for both human influenza (SA α2,6 linkage) and avian influenza (SA α2,3 linkage). When a pig is simultaneously infected with a human and an avian strain, the 8 RNA segments from both viruses can randomly assort into progeny virions - generating a hybrid "reassortant" virus with a novel HA or NA.

Medical Microbiology 9e, p. 4733
Jawetz Melnick & Adelbergs Medical Microbiology 28E, pp. 590, 595

Comparison Table: Drift vs. Shift

Feature	Antigenic Drift	Antigenic Shift
Mechanism	Point mutations	Genetic reassortment
Speed	Gradual (years)	Abrupt (sudden)
Magnitude of change	Minor	Major (new subtype)
Virus types	A, B, C	A only
Animal reservoir involved	No	Yes (birds, pigs)
Epidemiologic outcome	Epidemic	Pandemic
Pre-existing immunity	Partial protection	Little to no protection
Frequency	Continuous	Every 10-40 years
Gene segments changed	HA or NA point mutations	Entire HA and/or NA segment swapped

Historical Pandemics (Antigenic Shift)

Year	Subtype	Common Name
1918	H1N1	Spanish flu (~50 million deaths)
1957	H2N2	Asian flu
1968	H3N2	Hong Kong flu
1977	H1N1	Russian flu
1997/2003	H5N1	Avian flu (China)
2009	H1N1	Swine flu (quadruple reassortant: human + avian + 2 swine lineages)

The 2009 H1N1 pandemic virus (A(H1N1)pdm09) was a quadruple reassortant containing gene segments from North American swine, Eurasian swine, avian, and human influenza viruses - a vivid example of multi-host reassortment.

Jawetz Melnick & Adelbergs Medical Microbiology 28E, pp. 595-596

3. Nomenclature of Influenza A Strains

Format: Type / Host of origin / Geographic site / Strain number / Year (HA subtype N subtype)

Example: A/duck/Alberta/35/76 (H1N1) = Influenza A, isolated from a duck in Alberta, 1976, with H1 hemagglutinin and N1 neuraminidase.

For human strains, the host designation is omitted: A/California/04/2009 (H1N1)

Influenza B strains: no HA/NA subtype designation (no antigenic shift) - e.g., B/Singapore/3/64.

4. Key Antigens and Their Functions

Protein	Function	Role in Immunity
HA (Hemagglutinin)	Binds sialic acid receptors on respiratory epithelium; mediates fusion; 18 subtypes	Primary target of neutralizing antibody; determines resistance to infection
NA (Neuraminidase)	Cleaves sialic acid; releases new virions; 11 subtypes	Antibody reduces severity and limits virus spread; target of oseltamivir/zanamivir
NP (Nucleoprotein)	Surrounds RNA; forms RNP complex	Determines A/B/C typing; no antigenic variation; target of cytotoxic T cells
M protein	Forms shell under lipid envelope	Determines A/B/C typing; no antigenic shift/drift
M2	Ion channel; required for uncoating	Target of amantadine/rimantadine (Influenza A only)

5. Laboratory Diagnosis

Specimens: Nasopharyngeal swabs, nasal aspirate, or nasal lavage - collected within 3 days of symptom onset for best yield.

A. Molecular Methods (Gold Standard)

RT-PCR (Reverse Transcriptase-PCR)

Method of choice - rapid (<1 day), highly sensitive, highly specific
Detects and distinguishes influenza A, B, and specific subtypes (e.g., H5N1, H3N2, H1N1pdm09)
Multiplex RT-PCR panels simultaneously detect influenza A, B, RSV, adenovirus, and other respiratory viruses in a single run
Can identify novel/pandemic strains and antiviral resistance mutations
Specimens: nasopharyngeal swabs, nasal aspirate, bronchoalveolar lavage

B. Rapid Antigen Detection Tests (RIDTs)

Results in <15-30 minutes at point of care
Detect influenza A and B antigens (usually nucleoprotein) by immunochromatography
Sensitivity: 50-70% (variable); Specificity: >90%
A negative result does NOT rule out influenza - PCR must confirm if clinical suspicion high
Useful for triage and infection control decisions; does not identify specific subtype

C. Viral Culture

Cell lines: Primary monkey kidney cells or Madin-Darby canine kidney (MDCK) cells (preferred)
Classic method: embryonated eggs (still used for vaccine production)
CPE (cytopathic effects): often indistinct/subtle; not reliable alone
Hemadsorption: Guinea pig erythrocytes added to infected cell monolayer will adhere to HA on infected cell surfaces - confirms virus presence even before CPE
Hemagglutination (HA test): Added RBCs agglutinate in supernatant fluid due to viral HA - indicates virus present
Culture takes 3-10 days; now largely replaced by molecular methods but still valuable for:
- Isolating strains for vaccine composition
- Antiviral susceptibility testing
- Definitive subtyping

D. Hemagglutination Inhibition (HI) Test

Most important serologic method for strain identification and antibody measurement
Principle: Specific antibody (in patient serum or reference antiserum) blocks HA from agglutinating RBCs
Uses: (1) Identify virus type and subtype using reference antisera; (2) Measure patient antibody response
Requires pretreatment of serum to destroy nonspecific mucoprotein inhibitors (receptor-destroying enzyme or kaolin)
Fourfold or greater rise in titer between acute and convalescent sera = diagnostic
HI titer ≥1:40 correlates with protective immunity

E. Immunofluorescence (DFA/IFA)

Direct/indirect fluorescent antibody staining of respiratory epithelial cells in nasal aspirate
Results in 2-4 hours; rapid but less sensitive than PCR
Distinguishes influenza A from B using monoclonal antibodies

F. ELISA (Enzyme-Linked Immunosorbent Assay)

Detects viral antigen in secretions or culture; more sensitive than HI for serology
Serology: paired acute + convalescent sera; fourfold rise = infection

G. Complement Fixation (CF)

Detects antibodies to internal proteins (NP, M) - type-specific but not subtype-specific
Largely replaced by ELISA

Summary Table: Lab Diagnosis of Influenza

Test	Detects	Time	Notes
RT-PCR	Viral RNA; type, subtype	<1 day	Gold standard; best sensitivity/specificity
Multiplex PCR	Multiple respiratory viruses	<1 day	Single specimen, broad panel
Rapid antigen test	HA/NP antigen	15-30 min	Low sensitivity (~50-70%); quick triage only
Cell culture (MDCK)	Live virus	3-10 days	Needed for vaccine strains; antiviral resistance
Hemadsorption	HA on infected cells	2-5 days	Confirms culture positivity
Hemagglutination inhibition	Antibody/strain identity	Hours-days	Strain typing; immune status
Immunofluorescence	Viral antigen in cells	2-4 hours	Moderate sensitivity
ELISA	Antigen or antibody	Hours	Sensitive serology
Complement fixation	Type-specific antibody	Days	Older method; type only

Medical Microbiology 9e (Murray), pp. 4859-4868
Jawetz Melnick & Adelbergs Medical Microbiology 28E, pp. 5358-5395

6. Treatment and Prophylaxis (Brief Overview)

Drug	Mechanism	Active Against
Oseltamivir (Tamiflu), Zanamivir, Peramivir	NA inhibitors - block virus release	Influenza A + B
Baloxavir marboxil	Inhibits PB2 cap-snatching polymerase	Influenza A + B
Amantadine, Rimantadine	M2 ion channel blockers	Influenza A only (widespread resistance; no longer recommended in US)

Vaccines are reformulated annually based on WHO surveillance of circulating drift variants.

Quick Exam Memory Points

Segmented genome (8 segments) = molecular basis of reassortment = antigenic shift
Drift = mutations (slow, all types A/B/C) → epidemics
Shift = reassortment (sudden, Type A ONLY) → pandemics
Pig = mixing vessel (has both α2,3 and α2,6 sialic acid receptors)
Birds = reservoir of all 18 HA subtypes
HA subtype determines resistance to infection; NA subtype determines severity
RT-PCR = gold standard for lab diagnosis
HI test = identifies strain, measures protective antibody; fourfold rise = diagnosis
Hemadsorption detects HA on culture before CPE develops
Rapid antigen tests = fast but low sensitivity; negative does NOT rule out flu
Original antigenic sin = anamnestic response to first-ever influenza subtype encountered, even when re-infected with a new drift variant

Orthomixo virus antigen shift and drift and lab diagnosis micro

This appears to be a duplicate of the previous question, which was just answered in full detail. Here is a concise summary of everything covered, for quick reference:

Orthomyxovirus: Antigenic Shift, Drift & Lab Diagnosis - Quick Reference

Structure Recap (Basis of Antigenic Variation)

Family Orthomyxoviridae; enveloped, negative-sense ssRNA
8 segmented gene segments (Influenza A & B) - segmentation is the molecular basis of antigenic shift
Key surface antigens: HA (Hemagglutinin) - 18 subtypes and NA (Neuraminidase) - 11 subtypes
Internal antigens NP and M protein - determine A/B/C typing; do not undergo antigenic variation

Antigenic Drift

Feature	Detail
Mechanism	Point mutations in HA or NA genes → amino acid changes at epitopes
Speed	Gradual, stepwise over years
Virus types	A, B, and C
Epidemiologic result	Local/regional epidemics (A: every 2-3 yrs; B: every 3-6 yrs)
Immune effect	Pre-existing antibody gives partial protection - mild/modified disease
Key point	Immune system acts as selection pressure - variants that escape antibody expand

Antigenic Shift

Feature	Detail
Mechanism	Genetic reassortment of RNA segments during co-infection with 2 different strains
Speed	Abrupt - can occur within one year
Virus types	Influenza A ONLY
Epidemiologic result	Pandemic - population has little/no pre-existing immunity
Animal reservoir	Aquatic birds (all 18 HA subtypes); pigs = "mixing vessel"

Why only Type A? Types B and C are restricted to humans - no animal strains to reassort with. Influenza A circulates in birds, pigs, horses, seals - providing genomic diversity for reassortment.

Antigenic drift (gradual stepwise HA/NA change over years) vs antigenic shift (abrupt single-step change in HA/NA within one year); NP remains constant

Fig. 39-3: Antigenic drift vs. shift - HA/NA change gradually with drift, abruptly with shift; NP (internal protein) never changes. (Jawetz Melnick & Adelbergs, 28E)

Drift: point mutations alter HA epitopes so existing antibodies no longer bind. Shift: RNA segment exchange between two strains in a secondary host produces a virus with novel HA - no cross-protective immunity

Fig. 12.45: Mechanism of antigenic drift and shift. (Janeway's Immunobiology 10e)

Drift vs. Shift - Comparison Table

Feature	Antigenic Drift	Antigenic Shift
Mechanism	Point mutations	Genetic reassortment
Speed	Gradual (years)	Sudden (months-year)
Change magnitude	Minor	Major (new subtype)
Virus types	A, B, C	A only
Animal reservoir	Not involved	Yes (birds, pigs)
Epidemic/Pandemic	Epidemics	Pandemics
Pre-existing immunity	Partial	None/minimal
Frequency	Continuous	Every 10-40 years

Historical Pandemics (All Due to Antigenic Shift)

Year	Subtype	Name
1918	H1N1	Spanish flu (~50M deaths)
1957	H2N2	Asian flu
1968	H3N2	Hong Kong flu
1977	H1N1	Russian flu
2009	H1N1	Swine flu (quadruple reassortant: human + avian + 2 swine lineages)

Laboratory Diagnosis

Specimen: Nasopharyngeal swab / nasal aspirate / nasal lavage - collected within 3 days of symptom onset

1. RT-PCR (Gold Standard)

Detects viral RNA; <1 day turnaround
Highly sensitive and specific
Identifies type (A/B), subtype (H1N1, H3N2, H5N1), and antiviral resistance mutations
Multiplex RT-PCR detects influenza + RSV + other respiratory viruses simultaneously
Method of choice in current practice

2. Rapid Antigen Detection Tests (RIDTs)

Results in 15-30 minutes
Detects HA or NP antigen by immunochromatography
Sensitivity: ~50-70%; Specificity: >90%
Negative result does NOT rule out influenza - confirm with PCR if clinically suspected
Useful for rapid triage and infection control

3. Viral Culture

Cell lines: MDCK (Madin-Darby canine kidney) or primary monkey kidney cells; historically embryonated eggs
Takes 3-10 days; CPE often indistinct
Confirmed by hemadsorption (guinea pig RBCs adhere to HA on infected cell surface)
or hemagglutination of supernatant fluid
Still essential for: vaccine strain selection, antiviral susceptibility testing, pandemic surveillance

4. Hemagglutination Inhibition (HI) Test

Most important serologic + strain-identification test
Specific antibody blocks HA from agglutinating RBCs
Uses: (1) Strain typing with reference antisera; (2) Measuring patient immune response
Requires pre-treatment of serum to remove nonspecific inhibitors (receptor-destroying enzyme/kaolin)
Diagnostic criterion: fourfold or greater rise in titer between acute and convalescent sera (collected 2-4 weeks apart)
HI titer ≥1:40 = protective immunity

5. Immunofluorescence (DFA/IFA)

Direct/indirect antibody staining of nasal epithelial cells
Results in 2-4 hours; moderate sensitivity
Distinguishes A from B; does not subtype

6. ELISA

Detects antigen in secretions or culture supernatant
More sensitive than HI for serology; widely used

7. Complement Fixation (CF)

Detects antibodies to internal proteins (NP, M) - type-specific only (not subtype)
Older method; largely replaced by ELISA and PCR

Lab Diagnosis Summary Table

Test	Time	Sensitivity	Identifies Subtype	Use
RT-PCR	<1 day	Highest	Yes	Gold standard
Rapid antigen test	15-30 min	Low (~50-70%)	No	Rapid triage
Viral culture (MDCK)	3-10 days	High	With HI	Strain isolation, antiviral testing
Hemadsorption	2-5 days	High	No (detects HA)	Culture confirmation
HI test	Hours	High	Yes	Strain ID, immunity testing
Immunofluorescence	2-4 hours	Moderate	A vs B only	Rapid antigen in cells
ELISA	Hours	High	No	Sensitive serology
Complement fixation	Days	Moderate	Type only	Epidemiology

Key Exam Memory Points

8 segments → reassortment → antigenic shift
Drift = point mutations → gradual → A, B, C → epidemics
Shift = reassortment → sudden → Type A ONLY → pandemics
Pig = mixing vessel (expresses both α2,3 avian and α2,6 human sialic acid receptors)
RT-PCR = gold standard lab diagnosis
HI test = identifies strain + measures antibody; fourfold rise = acute infection
Hemadsorption = guinea pig RBCs stick to HA on infected cells - detects culture before CPE
Rapid antigen tests - negative does NOT rule out flu
HA antibody → prevents infection; NA antibody → reduces severity
Original antigenic sin - anamnestic response biased toward first influenza subtype ever encountered

Jawetz Melnick & Adelbergs Medical Microbiology 28E
Medical Microbiology 9e (Murray)
Janeway's Immunobiology 10e

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