Hepatitis B Virus (HBV) - Microbiology (5 Marks)

1. Classification & Morphology

• Family: Hepadnaviridae; the complete virion is called the Dane particle
• Size: 42 nm spherical enveloped virus (the nucleocapsid/core is 27 nm)
• Genome: Partially double-stranded (relaxed circular) DNA, ~3200 nucleotides - the smallest known human DNA virus
• Has 4 open reading frames (ORFs): S (surface), C (core), P (polymerase), X (HBx protein - transcriptional activator)
• Four major serotypes: adr, adw, ayr, ayw based on HBsAg determinants; 10 genotypes (A-J)

2. Structural Components & Antigens

Note: Aggregates of HBsAg in spherical (22 nm) and filamentous forms circulate in blood in huge numbers - up to 10¹⁰ particles/mL.

3. Replication Cycle (Unique Feature - Reverse Transcription)

1. Virion attaches to hepatocyte surface and is uncoated
2. Partially ds DNA is converted to covalently closed circular DNA (cccDNA) in the nucleus - this is the key replication template and persistence reservoir
3. cccDNA is transcribed into a 3.5 kb pregenomic RNA (pgRNA)
4. pgRNA is encapsidated with HBcAg
5. Viral reverse transcriptase copies pgRNA into negative-strand DNA (with RNase H degrading the RNA)
6. Positive-strand DNA synthesis begins but remains incomplete (hence "partial ds DNA" in mature virion)
7. Cores bud from pre-Golgi membranes acquiring HBsAg-containing envelope and exit the cell - OR are re-imported into the nucleus to amplify cccDNA pool

4. Pathogenesis

• Transmission: Parenteral (blood/blood products), sexual, vertical (mother-to-child) - NOT fecal-oral
• Incubation period: 60-150 days (average 90 days)
• Liver damage is immune-mediated - CD8+ cytotoxic T cells kill infected hepatocytes; circulating immune complexes cause extrahepatic manifestations (serum sickness, rash, arthritis, glomerulonephritis)
• Anti-HBs antibody = protective; correlates with recovery
• ~10% of adults develop chronic infection; >90% if perinatal transmission
• Chronic HBV → cirrhosis → Hepatocellular Carcinoma (HCC) (via HBx protein integration)

5. Serology - Interpretation of Markers

• Window period: Both HBsAg and Anti-HBs are negative; Anti-HBc IgM is the only positive marker - diagnoses acute HBV
• Pre-core mutant (e-minus mutant): Stop codon at nucleotide 1896 → no HBeAg production, but virus still replicates → HBeAg-negative chronic hepatitis; worse prognosis
• Chronic carrier: HBsAg positive for >6 months
• HBsAg is detectable 2-6 weeks before clinical jaundice appears

6. Stability & Treatment/Prevention

• Extremely stable: survives at 37°C for 60 min, dried at 25°C for 1 week; resistant to UV irradiation
• Destroyed by: 100°C for 1 min; sodium hypochlorite 0.5%
• Treatment (chronic): Pegylated interferon-alpha; nucleoside/nucleotide reverse transcriptase inhibitors - tenofovir (TDF/TAF), entecavir, lamivudine
• Vaccine: Recombinant HBsAg subunit vaccine - given at 0, 1, 6 months; first FDA-approved recombinant vaccine; provides long-term protection; anti-HBs >10 mIU/mL = protective
• HBIG (Hepatitis B Immunoglobulin) for post-exposure prophylaxis (e.g., needlestick, newborns of HBsAg+ mothers)

Laboratory Diagnosis of Hepatitis B

Specimens Used

• Serum - for antigens, antibodies, HBV DNA, liver enzymes
• Liver biopsy - for histology, HBcAg/HBsAg by immunofluorescence, cccDNA, and grading of fibrosis

A. Non-Specific Tests (Biochemical)

These are non-specific - raised in any hepatitis; used to assess severity, not diagnosis.

B. Specific Tests - Serological Markers (Most Important)

1. HBsAg (Hepatitis B Surface Antigen)

• First marker to appear in serum - detectable 2-6 weeks before jaundice
• Detected by ELISA (enzyme-linked immunosorbent assay) - method of choice
• Persistence >6 months = chronic infection
• Its disappearance signals recovery

2. Anti-HBc (Antibody to Core Antigen)

• IgM anti-HBc = marker of acute infection; appears at onset of symptoms; persists ~6 months; only positive marker in the window period
• IgG anti-HBc = past infection (persists for life); present in both resolved and chronic infection; NOT induced by vaccine
• Window period = HBsAg gone, anti-HBs not yet appeared → only IgM anti-HBc is positive - do NOT miss this!

3. HBeAg (Hepatitis B e Antigen)

• Appears shortly after HBsAg; marker of active viral replication and high infectivity
• Seen only when HBsAg is present
• Its replacement by anti-HBe = seroconversion = falling infectivity and start of resolution

4. Anti-HBs (Antibody to Surface Antigen)

• Appears after HBsAg clears (variable gap = window period)
• Protective antibody - correlates with recovery and immunity
• Present after: recovery from natural infection (+ anti-HBc positive) OR vaccination (anti-HBc negative; anti-HBs alone)
• Titer >10 mIU/mL = protective

5. Anti-HBe

• Appears as HBeAg disappears
• Signals start of resolution; declining infectivity
• Persists for years - useful epidemiological marker
• Not protective

C. Serological Course - Acute Self-Limiting Infection

• Incubation (4-12 wk): HBsAg and HBeAg rise; HBV DNA detectable
• Acute viremia (4-12 wk): HBsAg and HBeAg peak; Anti-HBc rises
• Convalescence (2-16 wk): HBsAg/HBeAg fall; Anti-HBe and Anti-HBs appear
• Healthy (years): Anti-HBc (IgG) + Anti-HBs persist

D. Interpretation of Serological Patterns

E. Molecular / Virological Tests

F. Histological Diagnosis (Liver Biopsy)

• "Ground glass" hepatocytes - smooth, eosinophilic cytoplasm packed with HBsAg; pathognomonic of chronic HBV
• Immunofluorescence: HBcAg in nucleus, HBsAg in cytoplasm of hepatocytes
• Grading of inflammation and staging of fibrosis (Metavir/Ishak scoring) guides treatment decisions
• cccDNA detected by Southern blot or PCR in tissue

G. Summary - Quick Diagnostic Approach

Differences Between Gram-Positive and Gram-Negative Bacteria

1. Gram Staining Result

The Gram stain was described in 1884 by Danish physician Hans Christian Gram. The decolorizer (acetone-alcohol) extracts the purple iodine-dye complex from Gram-negative cells, while Gram-positive bacteria retain it. An intact cell wall is necessary for a positive Gram reaction.

2. Cell Wall Structure (Most Important Difference)

3. Cell Wall Components in Detail

Gram-Positive Cell Wall Components:

• Peptidoglycan - multiple layers of glycan chains (GlcNAc + MurNAc) cross-linked by peptide bridges
• Teichoic acid - polyribitol or glycerol phosphate cross-linked to peptidoglycan; strengthens wall; sequesters calcium ions
• Lipoteichoic acid - lipid-anchored teichoic acid; activates innate host immunity (binds TLR-2)
• Surface proteins - bound to peptidoglycan or teichoic acid (e.g., Protein A of S. aureus)

Gram-Negative Cell Wall Components:

• Thin peptidoglycan - one to two layers only
• Periplasmic space - contains transport proteins, degradative enzymes (including beta-lactamases that destroy penicillin)
• Outer membrane - lipid bilayer; acts as barrier to many antibiotics
• LPS (Lipopolysaccharide) - consists of:
  • Lipid A (endotoxin - toxic component)
  • Core polysaccharide
  • O-antigen (outer polysaccharide chain - used for serotyping)
• Porin proteins - channel proteins (e.g., OmpC, OmpF) that allow passage of small hydrophilic molecules

4. Comprehensive Comparison Table

5. Clinical Relevance

Innate Immunity

Definition

Key Properties - Comparison with Adaptive Immunity

Components of Innate Immunity

1. Physical and Chemical Barriers (First Line)

2. Pattern Recognition Receptors (PRRs) - Key Concept

• PAMPs (Pathogen-Associated Molecular Patterns) - structures unique to microbes (e.g., LPS, peptidoglycan, flagellin, viral dsRNA, CpG DNA)
• DAMPs (Damage-Associated Molecular Patterns) - signals from stressed/dying host cells (e.g., HMGB1, ATP, uric acid crystals)

TLR signaling pathway: Ligand binding → TIR domain dimerization → MyD88 adaptor → IRAK → TRAF6 → NF-κB activation → pro-inflammatory cytokines (TNF, IL-1, IL-6, IL-12)

3. Cellular Components

A. Neutrophils (PMNs)

• First cells recruited to infection site (within minutes-hours)
• Phagocytose and kill bacteria via:
  • Oxidative burst (NADPH oxidase → superoxide, H₂O₂, HOCl)
  • Granule contents (elastase, myeloperoxidase, defensins, lactoferrin)
  • NETs (Neutrophil Extracellular Traps) - chromatin + granule proteins that trap bacteria

B. Macrophages

• Tissue-resident sentinels (Kupffer cells in liver, microglia in brain, alveolar macrophages in lungs)
• Key functions:
  • Phagocytosis and intracellular killing
  • Produce pro-inflammatory cytokines (TNF, IL-1, IL-6, IL-12)
  • Antigen presentation to T cells (link to adaptive immunity)
  • Produce reactive oxygen species (ROS) and nitric oxide (NO via iNOS)
  • Wound healing and tissue repair

C. Dendritic Cells (DCs)

• Present at epithelial surfaces and most tissues - act as "sentinels"
• Capture antigens → process → present to T cells via MHC II
• Critical bridge between innate and adaptive immunity
• Produce large amounts of Type I interferons (plasmacytoid DCs)

D. Natural Killer (NK) Cells

• Large granular lymphocytes; part of innate immunity but are lymphocytes
• Kill virus-infected cells and tumor cells WITHOUT prior sensitization
• Mechanism of recognition - "missing self" hypothesis:
  • Normal cells express MHC class I → inhibits NK killing
  • Virus-infected/tumor cells downregulate MHC I → NK cells activate and kill
• Kill via: Perforin-granzyme pathway + Fas-FasL interaction
• Produce IFN-γ which activates macrophages

E. Mast Cells and Basophils

• Tissue-resident mast cells (skin, GI, respiratory tract)
• Release histamine, prostaglandins, leukotrienes upon activation
• Important in allergy, parasitic infections, and early inflammation

F. Eosinophils

• Important against parasites (helminths)
• Release toxic granule proteins (MBP, ECP, EPO) that damage parasite membranes

G. Innate Lymphoid Cells (ILCs)

• ILC1 → IFN-γ (intracellular pathogens)
• ILC2 → IL-4, IL-5, IL-13 (helminths, allergy)
• ILC3 → IL-17, IL-22 (extracellular bacteria, fungi)

4. Humoral Components (Plasma Proteins)

A. Complement System

• Three activation pathways:
  • Classical pathway - activated by antigen-antibody complexes (C1q)
  • Lectin pathway - MBL (mannose-binding lectin) binds microbial mannose
  • Alternative pathway - spontaneous C3 hydrolysis on microbial surfaces
• All converge on C3 convertase → C3b (opsonin) → C5 convertase → MAC (membrane attack complex)
• Functions: Opsonization (C3b), Chemotaxis (C3a, C5a), Lysis (MAC/C5b-9), Anaphylatoxins (C3a, C5a cause mast cell degranulation)

B. Acute Phase Proteins

• Produced by liver in response to IL-6, IL-1, TNF
• CRP (C-reactive protein) - binds phosphocholine on bacteria → activates complement + opsonizes
• MBL (Mannose-binding lectin) - activates lectin pathway
• Serum amyloid A - opsonin
• Fibrinogen, ferritin - limit iron availability to bacteria

C. Cytokines and Chemokines

• TNF-α - fever, endothelial activation, septic shock at high levels
• IL-1 - fever ("endogenous pyrogen"), acute phase response
• IL-6 - fever, hepatic acute phase protein production
• IL-12 - activates NK cells; drives Th1 adaptive response
• Type I Interferons (IFN-α/β) - antiviral; induce ISGs (interferon-stimulated genes) in neighboring cells; upregulate MHC I; activate NK cells
• IFN-γ (from NK cells) - activates macrophages (classical activation)
• Chemokines (IL-8/CXCL8) - recruit neutrophils to infection site

5. Inflammation - The Innate Immune Response in Action

1. Microbe breaches barrier → recognized by tissue macrophages/DCs via PRRs
2. Pro-inflammatory cytokines (TNF, IL-1, IL-6) released
3. Endothelial cells upregulate selectins → neutrophil rolling
4. Chemokines (IL-8) → neutrophil chemotaxis and firm adhesion (ICAM-1/LFA-1)
5. Neutrophils transmigrate → phagocytose and kill microbes
6. Complement activated → opsonization, MAC formation
7. Resolution OR progression to adaptive immune response

Role of Innate Immunity in Activating Adaptive Immunity

• DCs activated by PRRs upregulate co-stimulatory molecules (B7/CD80/86) → present antigen to naive T cells
• Cytokine environment shapes T-cell differentiation:
  • IL-12 + IFN-γ → Th1 (cell-mediated, intracellular pathogens)
  • IL-4 → Th2 (antibody, extracellular/parasites)
  • IL-6 + TGF-β → Th17 (extracellular bacteria/fungi)
  • TGF-β → Tregs (suppression)
• Without innate activation, adaptive immunity is anergic (no co-stimulation = no T-cell activation)

Summary Diagram of Innate Immunity

MICROBE INVADES
      ↓
Barriers broken → PAMPs recognized by PRRs (TLRs, NLRs, RLRs)
      ↓
CELLULAR ARM                    HUMORAL ARM
  Neutrophils                     Complement (C3b opsonin, MAC)
  Macrophages  → Phagocytosis     Acute Phase Proteins (CRP, MBL)
  NK cells     → Kill infected    Cytokines (TNF, IL-1, IL-6)
  DCs          → APC              Interferons (antiviral)
      ↓
INFLAMMATION
      ↓
Bridge to ADAPTIVE IMMUNITY (via DCs + cytokines)