Structure of a Virus

1. Core Components

A. Viral Nucleic Acid (Genome)

• Every virus contains one type of nucleic acid only - either DNA or RNA, never both.
• The genome may be:
  • Single-stranded (ss) or double-stranded (ds)
  • Linear or circular
  • Segmented (multiple pieces, e.g., influenza) or nonsegmented (single piece)
• DNA genome size: 3.2 kbp (hepatitis B / hepadnaviruses) up to 375 kbp (poxviruses)
• RNA genome size: ~4 kb (picobirnaviruses) up to 32 kb (coronaviruses)
• Polarity matters:
  • Positive-sense RNA (e.g., picornaviruses, togaviruses) - the isolated RNA is itself infectious and acts directly as mRNA
  • Negative-sense RNA (e.g., orthomyxoviruses/influenza, rhabdoviruses) - isolated RNA is NOT infectious; requires a packaged RNA polymerase to first make mRNA copies

B. Capsid (Protein Coat)

• The capsid is a protein shell surrounding and protecting the genome from nucleases and the environment.
• Built from repeating protein subunits called capsomeres (and individual folded polypeptides called subunits).
• Functions:
  • Protects viral nucleic acid
  • Mediates attachment to host cell receptors
  • Determines antigenic characteristics (the immune response targets capsid/surface proteins)
• The nucleocapsid = nucleic acid + capsid together

C. Capsid Symmetry - Three Types

2. Viral Proteins

• Transfer the genome from one host cell to another
• Protect the genome against nucleases
• Attach to host cell receptors (e.g., influenza hemagglutinin binds sialic acid)
• Carry enzymatic activities - some virions package essential enzymes:
  • RNA polymerase (negative-sense RNA viruses - must make mRNA before replication begins)
  • Reverse transcriptase (retroviruses - converts RNA genome into DNA)
  • Transcriptional machinery (poxviruses - entire transcription system packaged inside the core)
• Antigenic determinants - the host immune system targets surface proteins/glycoproteins

3. Viral Lipid Envelope (Present in Some Viruses)

• The envelope is a lipid bilayer derived from the host cell membrane, acquired when the nucleocapsid buds out through the cell membrane during maturation.
• Budding occurs only where virus-specific proteins have been inserted into the host membrane.
• The phospholipid composition of the envelope reflects the host cell membrane (not virus-coded).
• Glycoproteins (virus-coded) are embedded in the envelope and project as spikes:
  • Examples: influenza hemagglutinin (HA) and neuraminidase (NA)
  • These are the key surface antigens recognized by the immune system

• Enveloped viruses are generally more fragile - they are inactivated by lipid solvents (ether, detergents)
• Non-enveloped viruses are hardier and can survive harsh environments (gut acid, desiccation)

4. Virus Size

5. Summary Diagram (Conceptual)

NON-ENVELOPED VIRUS           ENVELOPED VIRUS
+------------------+         +---------------------+
|   Capsid         |         | Envelope (lipid)    |
|  [Capsomeres]    |         |  + Glycoprotein      |
|                  |         |    spikes (HA, NA)  |
|  Nucleic Acid    |         |  Nucleocapsid inside|
|  (DNA or RNA)    |         |  (Nucleic acid +    |
|                  |         |   capsid proteins)  |
+------------------+         +---------------------+
e.g., Poliovirus              e.g., Influenza, HIV

The Virion = The Complete Virus Particle

"Virus" is the general biological concept (like saying "a dog"). "Virion" is the specific, complete physical particle (like saying "this particular dog standing in front of you").

"Virion: The complete virus particle... this structure serves to transfer the viral nucleic acid from one cell to another."

What the Virion Contains (Inside → Outside)

Two Types of Virions

[Nucleic acid] → [Capsid] = Virion
(the nucleocapsid alone IS the complete virion)

[Nucleic acid] → [Capsid] → [Envelope + spikes] = Virion
(all three layers together make the complete virion)

Where the Virion Exists

• Outside cell = Virion (complete, infectious particle)
• Inside cell = The virus has uncoated and the genome is actively replicating

Virion = Complete Virus Particle = (Usually) Infectious

1. Virion = Complete, but not always infectious

• Defective - structurally intact but carrying a mutated/incomplete genome, so they cannot replicate
• Inactivated - physically complete but their genome or surface proteins have been damaged (by heat, UV light, antibodies), making them non-infectious

2. Virus inside a cell is NOT called a virion

Simple Way to Remember It

Classification of Viruses

1. ICTV Taxonomic System - based on structure and genome
2. Baltimore Classification - based on how the virus makes mRNA

System 1 - ICTV Taxonomy (Structural Classification)

• Family: suffix -viridae (e.g., Herpesviridae)
• Subfamily: suffix -virinae (e.g., Herpesvirinae)
• Genus: suffix -virus (e.g., Herpesvirus)
• Species: common name (e.g., herpes simplex virus 1)

System 2 - Baltimore Classification (7 Groups)

Key rule: Positive-sense RNA (+ssRNA) acts directly as mRNA. Negative-sense RNA (-ssRNA) needs its own RNA polymerase packaged in the virion to first make mRNA.

Classification by Genome Type

RNA Viruses Infecting Humans

DNA Viruses Infecting Humans

Quick Summary - How to Remember

DNA Viruses (mnemonic: "Please Have Some Pretty Hot Popcorn"):
  Parvo → Hepadna → Polyoma → Papilloma → Adeno → Herpes → Pox

RNA Viruses (positive-sense = naked tend to be; enveloped tend to be negative/RT):
  Naked (+): Picorna, Calici, Reo, Astro, Hepe
  Enveloped (+): Flavi, Toga, Corona
  Enveloped (-): Paramyxo, Orthomyxo, Rhabdo, Filo, Bunya, Arena
  RT (Enveloped): Retro (RNA→DNA), Hepadna (DNA→RNA→DNA)

For MBBS Exams - Priority Guide

Baltimore Classification - Higher Priority

• Asked directly in exams: "Which group does HIV belong to?", "Classify HBV", "Positive-sense RNA viruses"
• Helps you understand replication - which is tested heavily in microbiology practicals and theory
• Only 7 groups - easy to memorize
• Explains WHY certain viruses behave differently (e.g., why HIV needs reverse transcriptase, why influenza needs its own RNA polymerase)
• Forms the conceptual backbone for understanding antiviral drugs (e.g., reverse transcriptase inhibitors for Group VI)

ICTV Classification - Also Required, but More as Reference

• You need to know family names (-viridae) and which viruses belong to which family
• Directly tested as: "Hepatitis B belongs to which family?" (Hepadnaviridae), "Rabies belongs to?" (Rhabdoviridae)
• The full table is not memorized - used as a reference, as Sherris & Ryan explicitly states: "The tables should not be memorized but rather used as a reference guide"
• Important for identifying structural features - naked vs enveloped, size, symmetry

What MBBS Students Actually Need to Know

Bottom Line

Learn Baltimore first - it gives you the logic. Then layer ICTV family names on top. Together they cover ~90% of virology questions in MBBS exams (1st Prof Microbiology, and clinical years).

Baltimore Classification - Full Explanation

The Core Idea

Every virus must produce mRNA. Why? Because viruses have no ribosomes. They MUST use the host cell's ribosomes to make proteins. Ribosomes only read mRNA. So no matter what genome a virus has - DNA or RNA - it must get to mRNA somehow.

The Concept of Polarity (+ and - Sense)

The 7 Baltimore Groups

GROUP I - Double-Stranded DNA (dsDNA)

dsDNA  →[transcription]→  mRNA  →[translation]→  Proteins

• Genome: Two strands of DNA (like our own chromosomes)
• Replication: Goes into the nucleus, uses host DNA-dependent RNA polymerase to make mRNA - just like normal cells do
• Highly dependent on host cell cycle - cell must be dividing for many of these
• Examples: Herpesviruses (HSV, VZV, CMV, EBV), Adenoviruses, Papillomaviruses (HPV), Polyomaviruses, Poxviruses (exception: replicates in cytoplasm, carries its own enzymes)

GROUP II - Single-Stranded DNA (ssDNA)

ssDNA  →[DNA polymerase]→  dsDNA  →[transcription]→  mRNA

• Genome: Only ONE strand of DNA
• Must first make a complementary strand to become dsDNA, THEN transcribe mRNA
• Smallest DNA viruses
• Examples: Parvoviruses (Parvovirus B19 - causes fifth disease / slapped cheek), Anelloviruses (TT virus)

GROUP III - Double-Stranded RNA (dsRNA)

dsRNA  →[viral RNA polymerase]→  mRNA  (positive strand)

• Genome: Two strands of RNA
• Cannot use host enzymes (host has no RNA-dependent RNA polymerase)
• Must carry their own RNA polymerase packaged inside the virion
• Replication occurs inside the capsid core in the cytoplasm (never in nucleus)
• Genome is usually segmented (multiple pieces)
• Examples: Reoviruses, Rotavirus (major cause of childhood diarrhea)

GROUP IV - Positive-Sense Single-Stranded RNA (+ssRNA)

(+)ssRNA  =  mRNA directly  →[translation]→  Proteins
             ↓[RNA-dependent RNA polymerase made]
           (-)ssRNA intermediate  →  more (+)ssRNA copies

• Genome IS the mRNA - ribosomes can read it immediately upon cell entry
• Most infectious isolated RNA (inject the RNA alone into a cell = infection)
• First thing that happens: genome gets translated into a polyprotein, which includes an RNA-dependent RNA polymerase (RdRp) for replication
• Largest group - most human RNA viruses are positive sense
• Examples:
  • Picornaviridae: Poliovirus, HAV, Rhinovirus (common cold), Coxsackievirus
  • Flaviviridae: Dengue, Zika, Yellow fever, West Nile, Hepatitis C (HCV)
  • Togaviridae: Rubella, Chikungunya
  • Coronaviridae: SARS-CoV-2, MERS, common cold coronaviruses
  • Caliciviridae: Norovirus

GROUP V - Negative-Sense Single-Stranded RNA (-ssRNA)

(-)ssRNA  →[viral RNA polymerase]→  mRNA (+)  →[translation]→  Proteins

• Genome CANNOT be read directly - it is the complement/mirror image of mRNA
• MUST carry RNA-dependent RNA polymerase (RdRp) packaged inside the virion - without it, the genome is useless
• Isolated RNA from these viruses is non-infectious
• Often have segmented genomes (influenza has 8 segments - key fact for exams!)
• Examples:
  • Orthomyxoviridae: Influenza A, B (8 segments - replicates in nucleus, unique among RNA viruses)
  • Paramyxoviridae: Measles, Mumps, RSV, Parainfluenza
  • Rhabdoviridae: Rabies (bullet-shaped)
  • Filoviridae: Ebola, Marburg (filamentous)
  • Bunyaviridae: Hantavirus, Crimean-Congo hemorrhagic fever

GROUP VI - ssRNA with Reverse Transcriptase (ssRNA-RT)

(+)ssRNA  →[Reverse Transcriptase]→  DNA  →[integrates into host genome]→  mRNA

• Retroviruses - unique because they go RNA → DNA (reverse of normal flow of genetic information)
• Carry reverse transcriptase (RT) and integrase in the virion
• After RT makes DNA copy, it integrates permanently into host chromosome as a provirus
• Host cell then transcribes it like its own gene → viral mRNA and proteins
• Genome is diploid (2 copies of RNA per virion)
• Examples: HIV-1, HIV-2 (causes AIDS), HTLV-1 (causes T-cell leukemia)
• Clinical importance: All anti-HIV drugs (NRTIs, NNRTIs) target reverse transcriptase

GROUP VII - dsDNA with Reverse Transcriptase (dsDNA-RT)

dsDNA  →[transcription]→  pregenomic RNA  →[Reverse Transcriptase]→  new dsDNA

• Most unusual group - DNA virus that uses reverse transcription in its replication cycle
• Goes DNA → RNA → DNA (uses an RNA intermediate called pregenomic RNA)
• Also carries reverse transcriptase
• Only one major human pathogen in this group
• Example: Hepatitis B virus (HBV) - Hepadnaviridae
• Clinical importance: Why HBV can integrate into the genome; why it can persist even after treatment; why drugs like tenofovir (an RT inhibitor) work against HBV

Summary Table - All 7 Groups

Memory Aid for MBBS

"Don't Stupid People Really Never Read Books"
 D     S    P     R     N    R    B
 I    II   IV    III    V   VI   VII
dsDNA ssDNA +RNA  dsRNA -RNA RT-RNA RT-DNA

• Groups I & II = DNA viruses
• Groups III, IV, V = Pure RNA viruses
• Groups VI & VII = Reverse transcriptase viruses (RNA↔DNA)

Most Commonly Tested in MBBS

1. HIV = Group VI (RT, integrase, diploid)
2. HBV = Group VII (dsDNA but uses RT - "DNA virus that behaves like a retrovirus")
3. Influenza = Group V (8 segments, carries RdRp, replicates in nucleus)
4. Poliovirus/Rotavirus/Rhinovirus = Group IV/III
5. Rabies = Group V (-ssRNA, bullet shape)
6. COVID-19 = Group IV (+ssRNA, largest RNA genome)

Baltimore Does NOT Classify Families

What Each System Actually Does

Think of It This Way

Baltimore = sorting people by blood group (A, B, AB, O) - a functional property, not physical appearance

ICTV = sorting people by nationality - based on origin, features, shared characteristics

So for MBBS - Use Both Together

• When asked "classify HIV" → Give BOTH: "HIV belongs to Baltimore Group VI (ssRNA-RT) and family Retroviridae"
• When asked "which family does influenza belong to?" → ICTV answer: Orthomyxoviridae
• When asked "classify influenza by genome" → Baltimore answer: Group V, negative-sense ssRNA

Positive Sense vs Negative Sense RNA

First - Understand mRNA

Positive Sense (+) RNA

Same sequence as mRNA

• The viral genome RNA reads exactly like mRNA
• Ribosomes can grab it and translate it directly into proteins the moment it enters the cell
• No extra step needed
• The genome itself IS the mRNA

Viral (+) RNA enters cell
        ↓
Ribosome reads it directly
        ↓
Viral proteins made immediately

• Isolated (+) RNA is infectious on its own - inject it into a cell, infection begins
• Does NOT need to carry an RNA polymerase in the virion (because no conversion needed at first)
• Fastest to start infection - proteins made immediately

Negative Sense (-) RNA

Complementary (mirror) to mRNA - opposite/antiparallel

• The viral genome RNA is the exact opposite of mRNA
• Ribosomes cannot read it - it is like a template strand, not a coding strand
• Must first be converted into positive-sense mRNA before anything can happen
• Needs an enzyme to do this conversion: RNA-dependent RNA polymerase (RdRp)
• This RdRp must be packaged inside the virion - because the host cell has no such enzyme

Viral (-) RNA enters cell
        ↓
RdRp (brought in the virion) converts it
        ↓
(+) mRNA produced
        ↓
Ribosome reads mRNA → Viral proteins

• Isolated (-) RNA is NOT infectious - without the packaged RdRp, it cannot be read
• Must carry RdRp in the virion - this is a defining feature
• Slightly slower to start infection (extra conversion step)

Side-by-Side Comparison

Simple Memory Trick

• (+) sense = the book is printed normally - you open it and read immediately
• (-) sense = the book is printed in mirror image - you need a mirror (RdRp) before you can read it

• (+) sense = the final photograph (what you see)
• (-) sense = the photographic negative (you need to develop/convert it first)

What About Ambisense?

Why This Matters Clinically

• Influenza (-ssRNA): Its RdRp is the target of baloxavir (a newer anti-influenza drug)
• HIV (+ssRNA): Goes RNA → DNA via reverse transcriptase - its RT is targeted by NRTIs and NNRTIs
• HCV (+ssRNA): Its RdRp is targeted by sofosbuvir (a direct-acting antiviral)
• Understanding polarity helps you predict which antivirals work on which virus

The Genetic Code - How Ribosomes Read mRNA

The Rule: Codons

• mRNA is made of 4 bases: A, U, G, C
• Every 3 bases = 1 codon
• Each codon codes for one specific amino acid
• This is the genetic code - fixed, universal, and non-overlapping

The Complete Genetic Code

The Fixed Rules of the Code

1. Start Codon - AUG

• Every mRNA must start with AUG (codes for Methionine)
• This is where the ribosome begins reading
• Without AUG, the ribosome cannot start

2. Stop Codons - UAA, UAG, UGA

• These three codons do not code for any amino acid
• When the ribosome hits one of these, it stops and releases the protein
• Called nonsense codons

3. Reading Frame

• The ribosome reads one codon at a time, left to right, without overlap
• The starting point (AUG) sets the reading frame - shift by even one base and the whole meaning changes

How Translation Works (Step by Step)

mRNA:  5'---AUG---UUU---CAG---UAA---3'
            MET   PHE   GLN   STOP

Ribosome reads left to right, 3 bases at a time
→ Makes protein: Methionine - Phenylalanine - Glutamine → Released

"The tRNA recognizes the proper spot to attach on the mRNA template because it has on its active end a set of three bases (anticodon) that are complementary to a set of three bases (codon) on the mRNA chain."

Now - Back to Positive vs Negative Sense

Properties of the Genetic Code (Important for Exams)

Why Viruses Exploit This

• A viral mRNA (or +sense RNA genome) placed inside a human cell gets translated perfectly
• The ribosome cannot distinguish "self" mRNA from "viral" mRNA - same code, same rules
• This is the fundamental trick of viral infection

Why Does HBV (a dsDNA Virus) Use Reverse Transcription?

First - Understand What Makes HBV Unusual

The Reason - It Is a Problem of Accuracy

"Due to lack of accurate DNA synthesis from the HBV DNA template, it utilizes the pregenomic RNA as a template to synthesize its genomic DNA through the action of reverse transcriptase."

The Full HBV Replication Cycle (Step by Step)

Step 1 - Entry and Repair

• HBV enters liver cells (hepatocytes) specifically
• The partially dsDNA genome is transported to the nucleus
• The incomplete (+) strand is repaired → forms cccDNA (covalently closed circular DNA)

Step 2 - Transcription (DNA → RNA)

• Host RNA polymerase transcribes the cccDNA into:
  • mRNAs - for making viral proteins (HBsAg, HBcAg, HBeAg, DNA polymerase)
  • Pregenomic RNA (pgRNA) - a full-length positive-sense RNA copy of the entire genome

Step 3 - Packaging

• The pgRNA + reverse transcriptase enzyme are packaged together inside the forming nucleocapsid core

Step 4 - Reverse Transcription (RNA → DNA) ← THE KEY STEP

• Inside the capsid, reverse transcriptase converts the pgRNA into (-) strand DNA
• The RNA template (pgRNA) is then degraded by the RNase H activity of the same reverse transcriptase enzyme
• The (+) strand DNA is then partially synthesized from the (-) strand DNA template

Step 5 - Maturation and Release

• The result is the characteristic partially dsDNA genome (one complete strand, one incomplete strand)
• Envelope (HBsAg) wraps around the nucleocapsid from the Golgi/ER
• Complete virion released by exocytosis

The Simplified Flow

NORMAL dsDNA virus (e.g., Herpes):
dsDNA → [transcription] → mRNA → proteins
dsDNA → [DNA polymerase] → new dsDNA copies

HBV (Group VII - dsDNA-RT):
Partial dsDNA → [repair] → cccDNA
cccDNA → [host RNA pol] → mRNA + pregenomic RNA (pgRNA)
pgRNA → [REVERSE TRANSCRIPTASE] → (-)DNA
(-)DNA → [DNA pol] → partial (+)DNA
= New partial dsDNA virion

Comparison: HBV vs HIV Reverse Transcription

Why This Matters Clinically

1. cccDNA persistence: The cccDNA sits in the nucleus like a mini-chromosome. Even if you kill all the circulating virus, the cccDNA remains. This is why HBV cannot be fully cured with current antivirals - only suppressed.
2. Antiviral targets: Drugs like tenofovir and entecavir inhibit HBV's reverse transcriptase - the same class of drugs used against HIV.
3. Hepatocellular carcinoma: HBV DNA can integrate into host chromosomes (especially in HCC patients), disrupting tumor suppressor genes.
4. HBeAg: The pregenomic RNA also encodes HBeAg - when HBeAg is detectable, active reverse transcription (active replication) is occurring. High infectivity.

RNA in HBV Replication - Always Single Stranded

What Gets Made: Pregenomic RNA (pgRNA)

• When cccDNA is transcribed by host RNA polymerase, it produces single-stranded mRNAs and single-stranded pregenomic RNA
• pgRNA is (+) ssRNA - it is a full-length, positive-sense single-stranded RNA copy of the entire genome
• It looks exactly like a regular mRNA - single strand, positive sense
• It serves two purposes simultaneously:
  1. Acts as mRNA - gets translated into viral proteins (core protein, reverse transcriptase)
  2. Acts as template for reverse transcription to make new DNA

Does dsRNA Ever Form?

• Technically, during reverse transcription inside the capsid, there is a brief moment where RNA and the new (-) DNA strand exist together as an RNA:DNA hybrid (not dsRNA - one strand is DNA, one is RNA)
• The RNA strand is then immediately degraded by RNase H activity of the reverse transcriptase
• So no stable dsRNA is formed at any point in HBV replication

Full Picture in One Flow

cccDNA (nucleus)
    ↓ [Host RNA polymerase]
(+) ssRNA  ←── This is the pregenomic RNA (pgRNA)
    ↓ [Packaged into capsid with RT]
    ↓ [Reverse transcriptase - RNA → DNA]
RNA:DNA hybrid  ←── Brief intermediate (NOT dsRNA)
    ↓ [RNase H degrades RNA strand]
(-) ssDNA
    ↓ [DNA polymerase activity of RT]
Partial dsDNA  ←── Final genome in virion

Summary

The Short Answer

The Key Distinction: What Goes INTO the New Virion?

Why Not Just Package the RNA and Skip the DNA Step?

1. Enter the cell
2. Go to the nucleus
3. Establish cccDNA - a stable, persistent DNA minichromosome that stays in the nucleus and keeps producing RNA long-term
4. From that cccDNA, keep transcribing mRNA and pgRNA forever

• The RNA would enter the cell
• It could make proteins once
• But RNA is unstable - it degrades quickly, cannot form cccDNA, cannot persist in the nucleus
• Infection would be short-lived, non-persistent
• HBV would lose its ability to cause chronic infection

The Analogy

cccDNA = Master blueprint (stored safely in nucleus, never used up)
pgRNA  = Working photocopy (used on the factory floor to make parts)
New DNA = Fresh copy of the master blueprint (sent to infect next cell)

Full Logic of the Cycle

CELL 1:
cccDNA (stable, nucleus)
  ↓ transcription
(+)ssRNA / pgRNA
  ↓ reverse transcription
New partial dsDNA  ← packed into virion

VIRION travels to CELL 2:
Partial dsDNA enters nucleus
  ↓ repaired
New cccDNA (stable again, persists for life of cell)
  ↓ transcription
pgRNA again...  → cycle repeats

Contrast With HIV (Group VI)

Laboratory Diagnosis of Viral Infections - MBBS Guide

Category 1 - Direct Detection of Virus

A. Electron Microscopy

• Visualizes the virus particle directly
• Used for: Rotavirus, Norovirus, Poxvirus
• Expensive, rarely used clinically now
• Exam point: Negative staining with potassium phosphotungstate

B. Light Microscopy - Inclusion Bodies

High-yield exam fact: Negri bodies in hippocampus (Ammon's horn) = pathognomonic of rabies

C. Antigen Detection Tests

• Fluorescent-labelled antibodies directly applied to specimen
• Fast, same day result
• Used for: Rabies (skin biopsy/brain), RSV, influenza, HSV

• Lateral flow immunoassay (like a pregnancy test strip)
• Used for: Influenza (rapid flu test), COVID-19, RSV, Rotavirus, Norovirus
• Advantage: Bedside/point-of-care, results in 15-30 minutes
• Disadvantage: Less sensitive than PCR - many false negatives

• Detects viral antigens in specimens
• Used for: HBsAg (Hepatitis B), HIV p24 antigen, Rotavirus in stool, Dengue NS1 antigen
• Most commonly used antigen test in clinical labs

Category 2 - Virus Isolation (Culture)

A. Cell Culture (Most Important)

• Inoculate patient specimen into live cell monolayers
• Virus infects the cells → produces Cytopathic Effect (CPE)
• CPE = visible changes in cells under light microscope

• Primary cell cultures (monkey kidney - for enteroviruses, influenza)
• Diploid cell strains (MRC-5 human fibroblasts - for CMV, VZV, HSV)
• Continuous cell lines (HEp-2 cells - for RSV; Vero cells - for many viruses)

B. Shell Vial Culture (Centrifugation-Enhanced)

• Faster than standard culture - results in 18-24 hours (vs 2-4 weeks for CMV)
• Specimen centrifuged onto cell monolayer on coverslip
• Detected with fluorescent monoclonal antibodies
• Used mainly for: CMV, VZV

C. Embryonated Eggs

• Old method, rarely used now
• Influenza was classically grown in eggs (still used for vaccine production)
• Inoculated into: amniotic cavity, allantoic cavity, chorioallantoic membrane (CAM)

D. Animal Inoculation

• Suckling mice for arboviruses, coxsackievirus A
• Rarely used clinically now - mainly research

Category 3 - Serology (Antibody Detection)

• IgM = recent/acute infection (appears first, disappears in weeks)
• IgG = past infection or immunity (persists for years)
• Rising titer = 4-fold rise in antibody between acute and convalescent sera (collected 2-4 weeks apart) = confirms active infection

Major Serological Tests:

Important Serology Exam Points:

Category 4 - Molecular Methods (Nucleic Acid Tests)

PCR (Polymerase Chain Reaction) - Gold Standard Now

• Detects viral DNA or RNA directly in specimen
• Most sensitive and specific test available
• Results in hours
• RT-PCR (Reverse Transcriptase PCR) - for RNA viruses (converts RNA → DNA first, then amplifies)
• Real-time PCR (qPCR) - quantifies viral load (copies/mL)

• HIV viral load monitoring
• HBV DNA and HCV RNA quantification (treatment monitoring)
• CMV and EBV in immunocompromised patients
• HSV encephalitis (CSF PCR - gold standard)
• COVID-19 diagnosis (RT-PCR from nasopharyngeal swab)
• Rabies, Dengue, Zika

Other Molecular Methods:

• NASBA (Nucleic Acid Sequence Based Amplification) - for RNA viruses
• Branched DNA (bDNA) - HIV, HCV, HBV viral load
• In-situ hybridization - detects viral nucleic acid in tissue sections
• Next Generation Sequencing (NGS) - virus identification and genotyping

Quick Reference - Which Test for Which Virus

Most Exam-Tested Points Summary

1. Negri bodies = Rabies (cytoplasmic, neurons)
2. Owl eye inclusions = CMV
3. Heterophile antibody / Monospot = EBV
4. HBsAg = Hepatitis B active infection; Anti-HBs = immunity
5. CPE = syncytia = RSV, measles; grape clusters = Adenovirus
6. PCR is gold standard for most viral diagnoses now
7. IgM = acute, IgG = past/immune
8. 4-fold rise in titer between acute and convalescent = confirms infection
9. Shell vial = faster CMV detection (18-24 hrs)
10. RT-PCR = used for RNA viruses (influenza, HIV, HCV, COVID)