EBOLA VIRUS - Detailed Notes for 2nd Year Medical Students

1. CLASSIFICATION AND TAXONOMY

Species of Ebolavirus (5 known)

1. Zaire ebolavirus - highest mortality (50-90%), named after Ebola River, DRC
2. Sudan ebolavirus - mortality ~50%
3. Bundibugyo ebolavirus
4. Tai Forest ebolavirus (Ivory Coast) - caused infection in one scientist from chimp autopsy, 1994
5. Reston ebolavirus - non-pathogenic in humans, causes disease in monkeys; isolated from Philippines-imported monkeys in USA, 1990

The four subtypes differ from one another by up to 40% at the nucleotide level but share some common epitopes.

• Jawetz Medical Microbiology, 28e

2. MORPHOLOGY AND STRUCTURE

Genomic Proteins (7 genes, 7 monocistronic mRNAs):

3. REPLICATION

1. GP binds to host cell receptors (NPC1 - Niemann-Pick C1 protein in endosomes)
2. Virus enters by endocytosis / macropinocytosis
3. Fusion of viral and endosomal membranes
4. Nucleocapsid released into cytoplasm
5. Replication occurs entirely in the cytoplasm using RNA-dependent RNA polymerase (L)
6. 7 monocistronic mRNAs synthesized from negative-sense genome
7. NP synthesis triggers switch from transcription to replication
8. New virions bud from plasma membrane (VP40-driven)

4. EPIDEMIOLOGY

Natural Reservoir

• Suspected: African fruit bats (Pteropus species) - not confirmed definitively
• Monkeys/apes are NOT reservoir hosts (they die too rapidly)
• Zoonotic: virus transmits from animals to humans, then spreads human-to-human

History of Outbreaks

5. TRANSMISSION

Routes:

1. Primary (animal-to-human): Contact with infected bats, primates (apes, monkeys), or bushmeat in endemic areas. Exact mechanism unclear.
2. Secondary (human-to-human):
   • Direct contact with blood or body fluids (urine, saliva, sweat, feces, vomit, breast milk, semen) of infected/sick/deceased persons
   • Via broken skin or mucous membranes (eyes, nose, mouth)
   • Contaminated objects: needles, syringes, bedding, clothing
   • Funeral rituals involving touching of corpses (major source in African outbreaks)
   • Sexual transmission (oral, vaginal, anal)
   • Nosocomial (healthcare setting) - major amplifier when PPE is inadequate

NOT transmitted by:

• Aerosol/airborne route between humans (no evidence)
• Asymptomatic individuals (infectivity parallels clinical severity)

Persistence:

• Ebola virus persists in semen for 3-9 months after recovery
• Also persists in: eye (aqueous humor), amniotic fluid, placenta, breast milk, CSF/CNS
• Risk of transmission during incubation period is negligible

Sexual transmission and contaminated needles have been shown. The incubation period is 2-21 days (average 4-10 days).

• Sherris & Ryan's Medical Microbiology, 8e

6. PATHOGENESIS

Primary Target Cells:

• Monocytes, macrophages, dendritic cells (first infected)
• Endothelial cells (especially microvascular)
• Hepatocytes, adrenal cortical cells
• Reticuloendothelial cells

Mechanism of Hemorrhage and Shock:

Why patients die:

• Failure to mount adequate immune response (fatal cases show impaired humoral immunity)
• DIC consuming clotting factors
• Multiorgan failure (liver, adrenal, kidneys, lungs)
• Hypotensive shock

Why some patients survive:

• Rapid generation of antibody response against GP
• Antibody titers against GP detectable in survivors
• Survivors retain protective immunity up to 10 years
• Exact mechanisms of recovery not fully understood

7. CLINICAL FEATURES

Incubation Period:

Phases of Disease:

Phase 1 - Prodrome/Febrile Phase (Days 1-5):

• Abrupt onset fever, chills
• Severe headache
• Myalgia, arthralgia
• Sore throat
• Malaise, anorexia

Phase 2 - GI Phase (Days 5-7):

• Nausea, vomiting (often severe)
• Profuse watery diarrhea (non-bloody initially)
• Abdominal pain
• Hiccups (characteristic of Ebola; pathogenesis unclear)

Phase 3 - Hemorrhagic Phase (Day 7 onwards in severe cases):

• Maculopapular rash (purplish-red), especially trunk
• Conjunctival injection/hemorrhage (without discharge - distinguishes from conjunctivitis)
• Internal hemorrhage: GI tract, lungs, liver
• External bleeding: from venipuncture sites, gums, nose
• DIC with widespread bleeding
• Shock, hypotension
• Multiorgan failure

Terminal Stage:

• Diffuse bleeding
• Hypotensive shock
• Multiorgan failure
• Death

Unique Clinical Signs:

• Hiccups - very characteristic of Ebola, distinguishes it from other VHFs
• Relative bradycardia (Faget sign) - may be seen
• Hepatosplenomegaly - common

Severity:

• Case fatality rate: Average 50%, ranges 25-90% depending on species and outbreak
• Zaire ebolavirus: 50-90% (highest)
• Sudan: ~50%
• Reston: 0% in humans

8. LABORATORY DIAGNOSIS

Warning: Work with Ebola samples requires Biosafety Level 4 (BSL-4) containment. Utmost care and strict isolation precautions are mandatory before any diagnostic attempts. Notify public health authorities immediately.

9. TREATMENT

Supportive Care (cornerstone):

• IV fluids and electrolyte correction
• Maintain renal function
• Treat coagulopathy
• Treat shock
• Antipyretics (avoid NSAIDs/aspirin due to bleeding risk)

FDA-Approved Specific Treatments (for Zaire ebolavirus only):

1. Inmazeb (2020) - Triple monoclonal antibody cocktail:

• Components: Atoltivimab + Miftivimab + Odesivimab
• All three are recombinant human IgG1 monoclonal antibodies
• Mechanism: All three target Zaire ebolavirus surface glycoprotein (GP) simultaneously; mediate ADCC (antibody-dependent cellular cytotoxicity) killing of infected cells
• From PALM trial: Reduced CFR to 35% (vs 50% for ZMapp control)

2. Ebanga (2020) - Single monoclonal antibody:

• Component: Ansuvimab (recombinant human IgG1κ mAb)
• Mechanism: Targets Zaire ebolavirus GP; inhibits attachment and membrane fusion
• From PALM trial: Reduced CFR to 34%

Both agents were evaluated in the PALM trial during the 2018-2020 DRC outbreak. Overall survival was much higher for patients receiving either treatment.

• Goodman & Gilman's, p. 1242

10. PREVENTION AND CONTROL

Vaccine:

• Ervebo (rVSV-ZEBOV) - FDA approved December 2019
• Single dose
• Recombinant vesicular stomatitis virus (VSV) - a rhabdovirus - engineered to express Ebola surface GP instead of its own VSV G protein
• Safe and protective
• Used in ring vaccination strategy (healthcare workers + contacts of confirmed cases) during DRC 2018 outbreak
• Only approved for Zaire ebolavirus

Infection Control Measures:

• BSL-4 laboratory containment for any work with live virus
• Strict barrier nursing (gowns, gloves, masks, face shields, full PPE)
• Isolation of confirmed/suspected cases
• Quarantine of contacts for 21 days (max incubation period)
• Safe burial practices (avoid touching corpses)
• No handling of bushmeat/primates in endemic areas
• Contact tracing and surveillance
• Decontamination: virus destroyed by:
  • Heating at 60°C for 30 minutes
  • UV irradiation, gamma irradiation
  • Lipid solvents (ether, chloroform)
  • Bleach (sodium hypochlorite)
  • Phenolic disinfectants

11. LONG-TERM COMPLICATIONS IN SURVIVORS

• Uveitis / Ocular complications (virus persists in aqueous humor)
• Joint pain, arthralgia
• Post-Ebola syndrome: fatigue, myalgia, headache, neurological sequelae
• Psychological trauma
• Sexual transmission risk persists for up to 9 months (semen)

12. QUICK SUMMARY TABLE

13. HIGH-YIELD EXAM POINTS

1. Family: Filoviridae - the only family with filamentous virions
2. Negative-sense RNA, non-segmented - unlike influenza (segmented), unlike HIV (positive-sense)
3. 5 species: Zaire (most deadly), Sudan, Bundibugyo, Tai Forest, Reston (only non-pathogenic in humans)
4. Named after Ebola River, DRC (then Zaire), 1976
5. GP is the key virulence factor - mediates entry, is the target of all approved therapies and vaccines
6. Transcriptional editing of GP gene - unique to Ebola, produces SGP primarily
7. BSL-4 required - highest containment level
8. Reservoir = fruit bats (suspected)
9. Semen persistence up to 9 months - reason for continued sexual transmission risk after recovery
10. Hiccups - characteristic clinical feature that helps distinguish Ebola from other VHFs
11. DIC - key pathological mechanism
12. No aerosol transmission between humans
13. Ervebo vaccine: rVSV vector + Ebola GP → approved 2019, single dose
14. Inmazeb + Ebanga: both target Zaire Ebola GP; FDA approved 2020
15. Outbreaks stopped by barrier nursing + quarantine + contact tracing

Laboratory Diagnosis of Diphtheria

Note: Apurba Sastry's Essentials of Medical Microbiology is not available in the digital library, but the following notes are compiled from the equivalent standard microbiology textbooks used in 2nd year MBBS curricula - Murray's Medical Microbiology 9e, Jawetz Melnick & Adelberg's Medical Microbiology 28e, Sherris & Ryan's Medical Microbiology 8e, and Henry's Clinical Diagnosis by Laboratory Methods - which cover the same content as Sastry.

IMPORTANT PRINCIPLE (Know This First!)

"The initial treatment of a patient with diphtheria is instituted on the basis of the clinical diagnosis, not laboratory results, because definitive results are not available for at least a week."

• Murray's Medical Microbiology 9e

SPECIMEN COLLECTION

Transport swabs promptly. Use cotton-tipped or polyester-tipped swabs.

STEP 1 - MICROSCOPY (Smear Examination)

Gram Stain:

• C. diphtheriae is a Gram-positive bacillus
• Pleomorphic rods, club-shaped (one or both ends swollen/clubbed)
• Arranged in characteristic "Chinese letter" / "V / L / Y / palisade" pattern (due to snapping division)
• Also called "cuneiform" arrangement

Special Stains for Metachromatic (Volutin) Granules:

Metachromatic granules = also called Babes-Ernst granules / volutin granules / polar bodies = composed of polymetaphosphate; represent stored energy. Their presence on Loeffler's serum slope after 6-8 hours is a rapid presumptive test.

Important: Direct smears are NOT reliable diagnostic tools (Sherris & Ryan). Metachromatic granules are not specific to C. diphtheriae alone. Microscopy is only presumptive.

STEP 2 - CULTURE

Media Used (in order of importance):

A. Loeffler's Coagulated Serum Slope (Primary/Enrichment Medium)

B. Potassium Tellurite Blood Agar (Selective/Differential Medium)

Colony Morphology on Tellurite Agar:

All three biotypes are pathogenic if they carry the tox gene.

Blood Agar (Non-selective):

• C. diphtheriae also grows on routine blood agar
• Corynebacteria grow as small colonies - useful as backup but not selective

STEP 3 - IDENTIFICATION (Biochemical)

Rapid presumptive ID: Cystinase positive + pyrazinamidase negative = presumptive C. diphtheriae. More extensive biochemical testing or gene sequencing needed for species-level confirmation.

STEP 4 - TOXIGENICITY TESTING (Most Important Step!)

All isolates of C. diphtheriae must be tested for exotoxin production. Identification alone is not enough - non-toxigenic strains exist and do NOT cause classic diphtheria.

A. Elek's Test (In Vitro Immunodiffusion) - Gold Standard

1. A strip of filter paper soaked in diphtheria antitoxin is placed in the centre of a special medium plate (Elek's gel/serum agar)
2. Test isolate + known toxigenic and non-toxigenic controls are streaked perpendicular to the antitoxin strip
3. Incubate 37°C for 24-48 hours
4. Examine for precipitin lines

• Positive (toxigenic): White precipitin line appears at 45° angle where toxin from the isolate meets antitoxin from the strip ("line of identity" with positive control)
• Negative (non-toxigenic): No precipitin line

B. PCR (tox Gene Detection) - Now Preferred Rapid Method

C. Rapid Immunochromatographic Strip (ICS) Test

• Newer method for rapid detection of diphtheria toxin protein
• Detects toxin directly
• Recently developed; not universally available

D. Virulence Test in Guinea Pig (Animal Inoculation) - Historical

• Guinea pig inoculation test: Toxigenic strains kill unprotected guinea pig but not the antitoxin-protected control
• Historical/reference method only - replaced by Elek and PCR in modern practice

SUMMARY FLOWCHART

CLINICAL SUSPICION OF DIPHTHERIA
         ↓
Notify Lab → Collect throat + nasopharyngeal swabs
         ↓
MICROSCOPY (Loeffler's serum slope 6-8h)
→ Albert's/Neisser's stain → metachromatic (polar) granules
→ Gram stain → Chinese-letter arrangement
         ↓
CULTURE (Tellurite media - Tinsdale/CTBA)
→ Grey-black colonies + brown halo → presumptive C. diphtheriae
         ↓
BIOCHEMICAL IDENTIFICATION
→ Cystinase (+), Pyrazinamidase (-) → confirmed C. diphtheriae
         ↓
TOXIGENICITY TESTING
→ Elek immunodiffusion (gold standard) OR PCR (tox gene)
         ↓
REPORT: Toxigenic / Non-toxigenic C. diphtheriae

SEROLOGY

• Schick Test (historical): In vivo test to assess host immunity to diphtheria toxin (not diagnostic)
  • Positive Schick = no immunity (susceptible)
  • Negative Schick = immune (has neutralizing antitoxin)
  • Now replaced by serum antitoxin assays
• Serum antitoxin levels: Protective level = ≥0.01 IU/mL

HIGH-YIELD EXAM TABLE