Virology

In short

Virology is the study of viruses - obligate intracellular, acellular infectious agents made of a nucleic acid genome (DNA or RNA) in a protein capsid, sometimes with a lipid envelope, that replicate only inside a living host cell. It explains how viruses are classified, cultivated, titrated, diagnosed and controlled, and it underpins vaccine and diagnostic industries. For a B.Sc Microbiology student it is a heavily weighted unit in CBCS "Virology"/"Microbes" papers and a standard section in CSIR-UGC NET Life Sciences, GAT-B/DBT-BET, ICMR JRF and GATE Biotechnology (XL/BT). In India the knowledge is used daily at ICMR-NIV Pune and the VRDL network, NCDC Delhi, Serum Institute of India, Bharat Biotech, Biological E, Zydus, and in diagnostic chains (Dr Lal PathLabs, SRL, Metropolis) where RT-PCR, ELISA and cell-culture skills are hired for directly.

Explained

What a virus is - and is not

• Definition: a virus is a nucleic acid (DNA or RNA, never both in the virion) enclosed in a protein coat, incapable of independent metabolism, multiplying only by taking over a host cell's ribosomes, energy and precursors.
• Virion: the complete extracellular infectious particle. Virus: the replicating entity including its intracellular forms.
• Why not "living" by classical criteria: no ribosomes, no ATP generation, no growth by increase in size, no binary fission; they are replicated, not divided - the eclipse phase shows the virion is dismantled completely before progeny appear.
• Size range roughly 20-300 nm (parvovirus ~20 nm, poxvirus ~250-300 nm × 200 nm; "giant" mimivirus ~400 nm) - below the resolution of the light microscope, so electron microscopy, negative staining and cryo-EM are used.
• Filterability: classical definition - passes a 0.22 µm bacteria-retaining filter (Chamberland-Pasteur, Berkefeld). Historical landmarks: Ivanovsky (1892) and Beijerinck (1898) on tobacco mosaic disease, Twort and d'Herelle (1915-17) on bacteriophages, Stanley's TMV crystallisation (1935).
• Sub-viral agents: viroids - small naked circular RNA, plant pathogens (potato spindle tuber viroid); satellite viruses/RNAs - need a helper virus (hepatitis D needs HBV surface antigen); prions - infectious misfolded protein PrP^Sc, no nucleic acid, cause CJD, scrapie, BSE.

Structure and morphology

• Genome: dsDNA, ssDNA, dsRNA, ssRNA (+sense = mRNA-like; −sense; ambisense), linear or circular, segmented (influenza 8 segments, rotavirus 11) or non-segmented; size from ~1.7 kb (hepatitis D) to >1,000 kb (pandoravirus). Coronaviruses (~30 kb) have the largest RNA genomes.
• Capsid: built of repeating protomers → capsomeres; it protects the genome, carries attachment proteins and is an antigen. Nucleocapsid = capsid + genome.
• Symmetry:
  • Icosahedral - 20 triangular faces, 12 vertices, capsomeres as hexons and pentons; described by the triangulation number T (adenovirus T = 25, poliovirus pseudo-T = 3).
  • Helical - protein subunits spiral around the nucleic acid; rigid rod (TMV, 2,130 subunits) or flexible within an envelope (influenza, rabies, measles).
  • Complex - poxviruses (brick-shaped, biconcave core), bacteriophage T4 (icosahedral head + helical contractile tail + baseplate and tail fibres).
• Envelope: host-derived lipid bilayer acquired by budding, studded with viral glycoprotein spikes (peplomers) - influenza HA and NA, HIV gp120/gp41, SARS-CoV-2 spike; matrix protein lines it. Enveloped viruses are ether/lipid-solvent and detergent sensitive, heat labile and need a cold chain - the basis of soap and 70% alcohol working against SARS-CoV-2.
• Other virion components: enzymes carried in the particle (reverse transcriptase in retroviruses, RNA-dependent RNA polymerase in −ssRNA viruses, neuraminidase, lysozyme in phage tails).

Classification

• ICTV hierarchy: realm - kingdom - phylum - class - order - family (*-viridae*) - subfamily (*-virinae*) - genus (*-virus*) - species. ICTV has moved to binomial species names of the form *Genus* + species epithet (for example rabies virus is now the species *Lyssavirus rabies*); families relevant to the syllabus: Poxviridae, Herpesviridae, Adenoviridae, Papillomaviridae, Hepadnaviridae, Parvoviridae, Picornaviridae, Flaviviridae, Togaviridae, Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Reoviridae, Retroviridae.
• Baltimore classification - by genome and route to mRNA:

• Other useful bases: host range (animal, plant, bacterial, fungal, archaeal), nucleic acid type, symmetry, presence of envelope, mode of transmission (respiratory, faeco-oral, blood-borne, vector-borne, sexual, zoonotic).

Replication - the general cycle

1. Attachment (adsorption): specific binding of a virion protein to a host receptor - this determines tropism. CD4 + CCR5/CXCR4 for HIV, ACE2 for SARS-CoV-2, sialic acid for influenza HA, CD155 for poliovirus, lipopolysaccharide/pili for phages.
2. Penetration/entry: direct translocation, receptor-mediated endocytosis followed by low-pH fusion in the endosome, or envelope-plasma membrane fusion; phages inject only the genome and leave the capsid outside.
3. Uncoating: release of the genome; the eclipse period begins - no infectious virus is recoverable from the cell.
4. Macromolecular synthesis: early proteins (polymerases, inhibitors of host synthesis), genome replication (nucleus for most DNA viruses; cytoplasm for most RNA viruses, influenza being the nuclear exception), then late structural proteins. (+)ssRNA viruses translate a polyprotein cleaved by viral proteases.
5. Assembly/maturation: nucleocapsid formation; proteolytic maturation (HIV protease cleaving Gag - the target of protease inhibitors).
6. Release: budding (enveloped, often non-lytic, cell survives a while) or lysis (naked viruses, phage endolysin + holin); influenza needs neuraminidase to cut sialic acid and let virions detach - blocked by oseltamivir.
7. One-step growth experiment (phage): synchronous high-MOI infection, then dilution; the curve shows a flat latent period, a rise period and a plateau. Burst size = final PFU / initial infective centres. T4 on *E. coli*: latent ~22-25 min, burst ~100-200.

Bacteriophages, lysogeny and host interactions

• Virulent (lytic) phages: T4, T7 - always kill the host.
• Temperate phages: lambda - either lytic or lysogenic, where the genome integrates as a prophage and is replicated with the chromosome; the cI repressor maintains lysogeny, and UV or mitomycin C induces the lytic cycle. Lysogenic conversion gives the host new traits - diphtheria toxin (β phage), cholera toxin (CTXφ), botulinum and shiga toxins.
• Transduction: phage-mediated gene transfer - generalised (random host DNA, P22, P1) and specialised (genes flanking the prophage site, lambda).
• Plaque: a clear zone of lysis in a bacterial lawn in a soft-agar overlay; plaque morphology and efficiency of plating are standard restriction/host-range tests.
• Animal cell outcomes: lytic (polio), persistent/chronic (HBV, HCV), latent (herpes simplex in trigeminal ganglia, varicella-zoster → shingles), transforming (oncogenic), abortive.
• Cytopathic effects (CPE): rounding and detachment, syncytium formation (measles, RSV, HIV), inclusion bodies - Negri bodies in rabies (cytoplasmic), Cowdry type A in herpes (intranuclear), Guarnieri bodies in smallpox, Bollinger bodies in fowlpox.
• Interference and defence: interferons α/β induced by dsRNA act on neighbouring cells to induce PKR, 2′-5′ oligoadenylate synthetase/RNase L and Mx proteins; RNA interference in plants and invertebrates; CRISPR-Cas as the bacterial adaptive defence; restriction-modification systems.
• Genetic variation: antigenic drift (point mutations, seasonal influenza) versus antigenic shift (reassortment of segments, pandemic strains - the 2009 H1N1); recombination, complementation, phenotypic mixing, quasispecies due to error-prone RdRp (no proofreading, ~10⁻³-10⁻⁵ per base per replication).

Cultivation, titration and laboratory diagnosis

• Cultivation routes: (i) animal inoculation (mice for rabies - now largely replaced); (ii) embryonated hen's egg - chorioallantoic membrane (pox, herpes - pocks), allantoic cavity (influenza vaccine seed), amniotic cavity, yolk sac (chlamydiae, some arboviruses); (iii) cell culture - primary (monkey kidney), diploid (HDF, MRC-5, WI-38 - finite passages), continuous/heteroploid (HeLa, Vero, BHK-21, MDCK, HEp-2, HepG2/Huh-7). Vero cells made at the Serum Institute and Bharat Biotech are the workhorse for polio and the inactivated COVID-19 vaccine.
• Quantitative assays: plaque assay (PFU - infectious units), focus-forming assay, TCID₅₀/ID₅₀ by Reed-Muench or Spearman-Kärber (quantal, endpoint), transformation/pock counting.
• Physical/serological quantitation: electron-microscope particle count, haemagglutination (HA) titre and haemagglutination inhibition (HI) for influenza/Newcastle disease, protein or nucleic acid measurement, quantitative RT-PCR copy number. Physical counts always exceed infectious counts - the particle-to-PFU ratio (often 10:1 to 1,000:1) reflects defective particles and inefficient plating.
• Diagnosis in practice:
  • Direct antigen: rapid dengue NS1, influenza and SARS-CoV-2 rapid antigen tests, direct fluorescent antibody for rabies (corneal impression, brain smear).
  • Nucleic acid: RT-PCR, real-time PCR with Ct values, multiplex panels, loop-mediated isothermal amplification (LAMP), CRISPR-based (FELUDA, developed at CSIR-IGIB), next-generation sequencing for variants (INSACOG network).
  • Serology: IgM/IgG ELISA (dengue MAC-ELISA kits from NIV Pune), neutralisation (PRNT - gold standard for immunity), HI, complement fixation, western blot.
  • Sample integrity: viral transport medium, 2-8 °C for short transport, −70 °C or liquid nitrogen for storage; repeated freeze-thaw destroys titre.
• Biosafety and regulation in India: BSL-1 to BSL-4 containment; the only operational BSL-4 laboratories are at ICMR-NIV Pune and the NIHSAD, Bhopal (animal). Work with recombinant viruses is governed by the Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Micro-organisms/GEMOs, 1989 (Rule 1989) under the Environment (Protection) Act, 1986, administered through the institutional IBSC, RCGM (DBT) and GEAC; vaccines and diagnostics are licensed by CDSCO under the Drugs and Cosmetics Act, 1940 and the New Drugs and Clinical Trials Rules, 2019; outbreak response uses the Epidemic Diseases Act, 1897 (amended 2020) and the Disaster Management Act, 2005, with surveillance through IDSP/IHIP.

Control - vaccines, antivirals and public health

• Vaccine types: live attenuated (OPV, MMR, varicella, yellow fever, rotavirus), inactivated (IPV, rabies, hepatitis A, Covaxin), subunit/recombinant (hepatitis B surface antigen, HPV - Cervavac from SII), viral-vector (Covishield/ChAdOx1), mRNA (not the mainstay in India; Gemcovac-19 is an indigenous mRNA vaccine), toxoid-type not applicable to viruses.
• Live versus killed: live gives mucosal IgA and long immunity with one or two doses but can revert (vaccine-derived poliovirus) and is contraindicated in immunosuppression and pregnancy; killed is safe and stable but needs adjuvant and boosters.
• India's programmes: Universal Immunisation Programme with NTAGI advice; India certified polio-free in March 2014, bivalent OPV + injectable IPV now used; measles-rubella elimination drive; National Viral Hepatitis Control Programme; National AIDS and STD Control Programme (NACO); National Rabies Control Programme (post-exposure prophylaxis: wound washing, vaccine, and rabies immunoglobulin for category III).
• Antivirals by target: entry/fusion (enfuvirtide, maraviroc), uncoating (amantadine), nucleoside/nucleotide polymerase inhibitors (acyclovir - activated only by viral thymidine kinase; zidovudine, tenofovir, remdesivir, sofosbuvir), non-nucleoside RT inhibitors (nevirapine), protease inhibitors (ritonavir, nirmatrelvir), integrase inhibitors (dolutegravir - first line in Indian ART), neuraminidase inhibitors (oseltamivir), interferon-α (older HBV/HCV therapy). Resistance arises fast, hence combination therapy (HAART).
• Non-pharmaceutical control: vector control for dengue/chikungunya/JE, blood screening (NAT), sterilisation and disinfection (enveloped viruses inactivated by 70% ethanol, 1% sodium hypochlorite, autoclaving; naked viruses such as hepatitis A and polio are far more resistant).
• Emerging and re-emerging viruses in India: Nipah (Kerala, fruit-bat reservoir, BSL-4 handling), Kyasanur Forest disease (Karnataka, tick-borne), Crimean-Congo haemorrhagic fever (Gujarat, Rajasthan), Chandipura virus (AES in children, Gujarat/MP), Zika (Gujarat, Rajasthan, MP, Maharashtra, Kerala in various years), Japanese encephalitis, dengue 1-4, influenza A H1N1pdm09 and H3N2, measles outbreaks in under-vaccinated pockets.
• Oncogenic viruses: HPV 16/18 (cervical cancer - E6 degrades p53, E7 inactivates Rb), hepatitis B and C (hepatocellular carcinoma), Epstein-Barr virus (Burkitt lymphoma, nasopharyngeal carcinoma), HTLV-1 (adult T-cell leukaemia), Kaposi sarcoma herpesvirus, Merkel cell polyomavirus. Mechanisms: viral oncogene expression, insertional activation of host oncogenes, inactivation of tumour suppressors, chronic inflammation.

Key points

• Virus - acellular obligate intracellular parasite with one type of nucleic acid, no own metabolism, replicated by the host machinery.
• Virion - the complete infectious particle; nucleocapsid - genome plus capsid; peplomer - envelope glycoprotein spike.
• Capsid symmetry - icosahedral, helical or complex; T number describes icosahedral capsid size.
• Envelope - host lipid, so lipid-solvent sensitive, heat labile, needs a cold chain; naked viruses are hardier.
• Baltimore groups I-VII - classify by genome type and the route taken to make mRNA; group VI and VII use reverse transcriptase.
• Eclipse period - interval after uncoating when no infectious virus can be recovered; it lies inside the latent period (until release).
• Burst size - progeny virions released per infected cell; ~100-200 for T4.
• Lysogeny - prophage integration with repressor-maintained dormancy; lysogenic conversion adds toxin genes (diphtheria, cholera).
• Transduction - phage-mediated bacterial gene transfer, generalised or specialised.
• CPE and inclusion bodies - Negri (rabies), Cowdry A (herpes), syncytia (measles, RSV, HIV) - cytological clues to identity.
• MOI - infectious units per cell; infection distribution follows the Poisson law.
• PFU counts infectious particles; TCID₅₀ is a quantal endpoint; PFU ≈ 0.7 × TCID₅₀.
• Particle-to-PFU ratio - always >1 because of defective particles and plating inefficiency.
• Antigenic drift - gradual mutation; antigenic shift - segment reassortment causing pandemics.
• Interferon - host antiviral cytokine inducing PKR, OAS/RNase L; it protects cells, it does not kill virus directly.
• Acyclovir selectivity - phosphorylated only by viral thymidine kinase, so uninfected cells are spared.
• Indian regulatory frame - IBSC/RCGM/GEAC under Rules 1989, CDSCO licensing, New Drugs and Clinical Trials Rules 2019, Epidemic Diseases Act 1897 (amended 2020), IDSP/IHIP surveillance, BSL-4 at ICMR-NIV Pune.
• Sub-viral agents - viroids (naked RNA, plants), satellites (HDV), prions (protein only, no nucleic acid).

Formulas and examples

Example 1 - plaque assay titre. 0.1 mL of a 10⁻⁶ dilution of poliovirus is plated on Vero cells; 25 plaques appear.

• Titre = 25 / (0.1 × 10⁻⁶) = 25 × 10 × 10⁶ = 2.5 × 10⁸ PFU/mL.
• If electron microscopy gives 1.0 × 10¹¹ particles/mL, particle-to-PFU ratio = 10¹¹ / (2.5 × 10⁸) = 400 : 1.

Example 2 - MOI and Poisson. 2.0 × 10⁷ PFU of virus is added to a flask with 5.0 × 10⁶ Vero cells.

• MOI = 2.0 × 10⁷ / 5.0 × 10⁶ = 4.0.
• Uninfected fraction P(0) = e⁻⁴ = 0.0183 → 1.83% (about 91,500 of the 5 × 10⁶ cells escape).
• Cells with exactly one virion: P(1) = 4·e⁻⁴ = 0.0733 → 7.33%.
• At MOI 1: P(0) = e⁻¹ = 0.368, so 36.8% uninfected - this is why synchronous single-step work uses MOI ≥ 5 and plaque assays use MOI ≪ 1.

Example 3 - one-step growth and burst size. *E. coli* is infected with T4; after adsorption and dilution the infective-centre titre is 1.5 × 10⁵ PFU/mL. At 60 min the titre plateaus at 3.0 × 10⁷ PFU/mL.

• Burst size = 3.0 × 10⁷ / 1.5 × 10⁵ = 200 virions per cell.
• If the latent period is 25 min and the rise period 10 min, release averages 200/10 = 20 virions per cell per minute during the rise.

Example 4 - TCID₅₀ by Spearman-Kärber. Ten-fold dilutions, 8 wells each, 0.1 mL per well. Wells positive for CPE: 10⁻¹ 8/8, 10⁻² 8/8, 10⁻³ 8/8, 10⁻⁴ 6/8, 10⁻⁵ 3/8, 10⁻⁶ 1/8, 10⁻⁷ 0/8.

• Highest 100% dilution = 10⁻³. S = 1.00 (10⁻³) + 0.75 + 0.375 + 0.125 + 0 = 2.25.
• log₁₀ TCID₅₀ = −3 − (2.25 − 0.5)(1) = −4.75, i.e. 10^4.75 TCID₅₀ per 0.1 mL.
• Per mL = 10^4.75 × 10 = 10^5.75 ≈ 5.6 × 10⁵ TCID₅₀/mL; expected plaque titre ≈ 0.7 × 5.6 × 10⁵ ≈ 3.9 × 10⁵ PFU/mL.

Example 5 - qPCR copy number and herd immunity. A SARS-CoV-2 standard curve gives Ct = −3.32 log₁₀(copies) + 40.0.

• Efficiency = 10^1/3.32 − 1 = 10^0.3012 − 1 = 2.00 − 1 = 1.00 → 100% (the ideal doubling per cycle).
• A sample with Ct 25.0: log₁₀(copies) = (40.0 − 25.0)/3.32 = 4.52 → ≈ 3.3 × 10⁴ copies per reaction.
• For measles with R₀ ≈ 15: H = 1 − 1/15 = 0.933 → 93.3% coverage needed - which is why two MR doses are mandated in the UIP.

Where it is used

• Vaccine manufacturing: Serum Institute of India (Pune - measles-rubella, polio, HPV Cervavac), Bharat Biotech (Hyderabad - Rotavac, Covaxin, typhoid conjugate), Biological E, Zydus, Panacea; roles in upstream cell culture, virus seed banks, potency/titration QC and GMP documentation.
• Diagnostics and reagents: Mylab, Molbio (Truenat), Trivitron, HiMedia, Thermo Fisher India; molecular biologist and QC microbiologist roles running RT-PCR, ELISA and kit validation against CDSCO and ICMR evaluation requirements.
• Public-health and research laboratories: ICMR-NIV Pune and the ~160-laboratory VRDL network, NCDC Delhi, NIBMG/INSACOG sequencing hubs, CSIR-IGIB, state public-health laboratories - outbreak investigation of Nipah, dengue, Zika, Chandipura.
• Hospital and reference diagnostics: virology sections of AIIMS, PGIMER, CMC Vellore and of chains such as Dr Lal PathLabs, SRL, Metropolis - hepatitis B/C viral load, HIV CD4 and viral load under NACO, dengue NS1/IgM.
• Veterinary and agricultural virology: ICAR-IVRI Izatnagar and NIHSAD Bhopal (foot-and-mouth disease, avian influenza, lumpy skin disease), plant virology at ICAR institutes (tomato leaf curl, papaya ringspot) and ELISA-based seed certification.
• Biotechnology tools: phage therapy research, phage display, adeno-associated and lentiviral vectors for gene therapy, baculovirus expression systems, bacteriophage-based biocontrol in food processing.

Interview questions

• Q: Why are viruses called obligate intracellular parasites? A: They have no ribosomes, no energy-generating metabolism and no biosynthetic machinery, so they can replicate their genome and make proteins only inside a living host cell.
• Q: What is the difference between the eclipse period and the latent period? A: The eclipse period is the time after uncoating when no infectious virus can be recovered even from lysed cells; the latent period runs from infection to the first extracellular release, so it includes the eclipse.
• Q: How does the Baltimore system differ from the ICTV system? A: Baltimore groups viruses by genome type and the pathway used to produce mRNA (7 groups); ICTV is a formal taxonomic hierarchy using morphology, genome and phylogeny down to species.
• Q: Why does influenza cause pandemics but measles does not? A: Influenza has a segmented genome that can reassort between human and animal strains (antigenic shift), creating a subtype to which the population has no immunity; measles is non-segmented, antigenically stable and cannot shift.
• Q: What is the difference between PFU and TCID₅₀? A: PFU counts discrete infectious particles as plaques; TCID₅₀ is a statistical endpoint - the dilution infecting 50% of inoculated cultures - and roughly 1 PFU ≈ 1/0.7 TCID₅₀.
• Q: Why is the particle-to-PFU ratio always greater than one? A: Many particles are defective or incompletely assembled, some fail to attach or are inactivated, and plating efficiency is below 100% for most cell lines.
• Q: How is acyclovir selective for infected cells? A: It is a prodrug phosphorylated by herpesvirus thymidine kinase, so the active triphosphate accumulates mainly in infected cells, where it inhibits the viral DNA polymerase and terminates the chain.
• Q: Why is there still no vaccine for HIV? A: High mutation rate and quasispecies diversity, a heavily glycosylated shielded envelope, integration into the host genome creating a latent reservoir, and the lack of a clear correlate of protective immunity.
• Q: What does lysogenic conversion mean, with an example? A: A prophage gives its bacterial host a new phenotype - *Corynebacterium diphtheriae* produces diphtheria toxin only when carrying the β phage, and *Vibrio cholerae* produces cholera toxin only with CTXφ.
• Q: How would you confirm rabies in a suspected animal brain in an Indian laboratory? A: Direct fluorescent antibody test on brain impression smears (hippocampus, brainstem, cerebellum) is the reference method, supported by Negri body histology and RT-PCR on brain tissue or saliva - handled under appropriate containment.
• Q: Why do enveloped viruses need a cold chain while polio vaccine is comparatively robust? A: The lipid envelope and its glycoproteins denature with heat and drying, destroying infectivity or antigenicity; a naked capsid is a tightly packed protein shell that tolerates heat, acid and drying far better.
• Q: What containment level is needed for Nipah virus and where is it available in India? A: BSL-4 for virus isolation and animal work; the operational BSL-4 facilities are at ICMR-NIV Pune (human) and NIHSAD Bhopal (animal), with BSL-3 permitted for inactivated molecular diagnostics under laid-down protocols.

Common mistakes

• Writing that a virion contains both DNA and RNA, or that viruses have ribosomes or cell walls.
• Forgetting the dilution factor or the plated volume in a plaque-titre calculation, and reporting PFU per plate as PFU per mL.
• Treating MOI as the number of virions that actually enter every cell - the distribution is Poisson, and at MOI 1 nearly 37% of cells are uninfected.
• Confusing eclipse with latent period, and burst size with the plateau titre itself.
• Mixing up antigenic drift with shift, or claiming non-segmented viruses can reassort.
• Describing lysogeny as "the phage is destroyed" instead of integrated and repressed, and omitting induction.
• Saying antibiotics or an autoclave-only approach treats viral infections, or that interferon directly kills virus particles - it acts on cells to induce an antiviral state.
• Quoting titres without units or temperature/storage conditions, and ignoring that repeated freeze-thaw cycles and room-temperature transport silently lower infectivity before the assay.

Related topics

Bacterial genetics and gene transfer · Immunology: innate immunity, antibodies and vaccines · Medical microbiology and diagnostic methods · Molecular biology: replication, transcription and PCR · Microbial techniques: cell culture, sterilisation and biosafety · Epidemiology and public-health microbiology