# Lecture 14: Virology I -- Structure and Classification
## Microbiology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Define viruses and describe their general properties
2. Describe the structural components of viruses: nucleic acid, capsid, envelope, and accessory proteins
3. Explain the principles of viral classification (Baltimore classification, ICTV taxonomy)
4. Differentiate between the major capsid symmetries: icosahedral, helical, and complex
5. Describe the methods used to cultivate, detect, and quantify viruses
6. Explain the concepts of viral tropism and host range
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## Lecture Content
### I. General Properties of Viruses
**Viruses are obligate intracellular parasites** that cannot replicate outside a host cell. They are not considered living organisms by most definitions, as they lack independent metabolism, ribosomes, and the capacity for energy generation. Most viruses range in size from approximately 20 to 300 nm, placing them below the resolution limit of light microscopy and requiring electron microscopy for visualization. The smallest viruses, the parvoviruses, measure roughly 20 nm, while the largest, such as Mimivirus, can reach approximately 750 nm.
A virus particle contains either DNA or RNA as its genetic material, but never both (with the exception of some rare giant viruses). Viruses possess no cell membrane, cell wall, or organelles of their own, although enveloped viruses do carry a lipid membrane derived from their host. The complete infectious particle is termed a **virion**. Viruses are extraordinarily abundant in nature, with an estimated 10^31 phage particles in the global ocean alone, and they infect all forms of life, including bacteria (as bacteriophages), archaea, protists, fungi, plants, and animals.
### II. Viral Structure
#### A. Nucleic Acid (Genome)
Viral genomes exhibit remarkable diversity. The nucleic acid can be **DNA or RNA**, **single-stranded (ss) or double-stranded (ds)**, **linear or circular**, and **segmented** (with the genome divided into multiple pieces) or non-segmented. RNA genomes introduce additional complexity: **positive-sense (+) RNA** can serve directly as mRNA, **negative-sense (-) RNA** is complementary to mRNA and must be transcribed first, and **ambisense RNA** contains both positive- and negative-sense regions on the same segment.
Genome sizes vary enormously across the virus world. The smallest viral genomes are approximately 1.7 kb, as seen in hepatitis D virus, which is viroid-like in its simplicity. Typical viral genomes range from 5 to 200 kb. At the extreme end, giant viruses such as Mimivirus carry genomes of approximately 1.2 Mb, which is larger than some bacterial genomes.
#### B. Capsid
The **capsid** is the protein shell that encloses and protects the viral nucleic acid. It is composed of protein subunits called **capsomeres**, which are themselves assembled from smaller units known as **protomers**. The capsid serves multiple functions: it protects the genome from nucleases and environmental damage, mediates attachment to host cells in non-enveloped viruses, and determines the antigenicity of the virion.
Three major capsid symmetries are recognized. **Icosahedral** capsids have 20 triangular faces and 12 vertices, giving them a roughly spherical appearance. This geometry is the most efficient structure for enclosing maximum volume with the minimum number of protein subunits. The complexity of icosahedral capsids is described by a triangulation number (T number). Examples include adenovirus, poliovirus, HPV, rhinovirus, and herpesvirus. **Helical** capsids consist of capsomeres arranged in a helix around the nucleic acid, producing a rod-shaped or filamentous particle. They can be rigid, as in tobacco mosaic virus (TMV), or flexible, as in the nucleocapsid of influenza virus. Other helical examples include Ebola and rabies viruses. **Complex** capsids do not fit neatly into either category. Poxviruses are brick-shaped or ovoid with a complex internal membrane and lateral bodies, while many bacteriophages, such as T4, combine an icosahedral head with a helical tail, tail fibers, and a baseplate.
#### C. Envelope
Many animal viruses are surrounded by an **envelope**, a lipid bilayer derived from host cell membranes -- whether the plasma membrane, nuclear membrane, ER, or Golgi apparatus. Enveloped viruses include influenza, HIV, herpesvirus, coronavirus, Ebola, rabies, and hepatitis B and C. Embedded within the envelope are **viral glycoproteins (spikes or peplomers)** that mediate attachment to host cell receptors and membrane fusion. These glycoproteins are major targets for neutralizing antibodies and vaccine development. Well-known examples include the hemagglutinin (HA) and neuraminidase (NA) of influenza, the spike (S) protein of SARS-CoV-2, and gp120/gp41 of HIV.
The presence or absence of an envelope has important practical consequences. Enveloped viruses are generally more sensitive to desiccation, detergents, heat, and disinfectants because the envelope is fragile, and they tend to be transmitted by close contact, respiratory droplets, blood, or bodily fluids rather than by the fecal-oral route. **Non-enveloped (naked) viruses**, by contrast, are more resistant to environmental conditions, can survive on fomites, in the gastrointestinal tract, and in sewage, and are commonly transmitted by the fecal-oral route, fomites, and respiratory droplets. Examples of non-enveloped viruses include norovirus, rotavirus, adenovirus, HPV, and poliovirus.
#### D. Other Components
Several additional components contribute to viral structure and function. **Matrix proteins**, found in some enveloped viruses, form a layer between the capsid or nucleocapsid and the envelope, providing structural support and playing roles in assembly. Examples include the M1 protein of influenza and the MA protein of HIV. Many viruses also package **viral enzymes** within the virion. Negative-sense RNA viruses such as influenza and Ebola carry an RNA-dependent RNA polymerase (RdRp) because their genomes cannot be directly translated. Retroviruses like HIV carry reverse transcriptase and integrase, hepadnaviruses like HBV carry reverse transcriptase, influenza virus has neuraminidase as a surface enzyme, and some bacteriophages include lysozyme.
A composite figure of viral structure. Panel A: Icosahedral virus (e.g., adenovirus) -- cutaway view showing the icosahedral capsid with triangular facets, capsomeres at vertices (pentons) and faces (hexons), fiber proteins projecting from vertices, and the linear dsDNA genome inside. Panel B: Helical virus (e.g., tobacco mosaic virus) -- longitudinal section showing helically arranged capsomeres around a central channel containing ssRNA, with dimensions labeled (300 nm length, 18 nm diameter). Panel C: Enveloped virus (e.g., influenza) -- cross-section showing the lipid bilayer envelope with HA and NA glycoprotein spikes, M1 matrix protein layer beneath, and eight helical ribonucleoprotein segments (ssRNA + NP + RdRp) inside. Panel D: Complex virus (bacteriophage T4) -- labeled icosahedral head containing dsDNA, collar, contractile tail sheath, baseplate, tail fibers, and tail pin.
### III. Viral Classification
#### A. Baltimore Classification (David Baltimore, 1971)
The **Baltimore classification** system organizes viruses into seven classes based on the relationship of the genome to mRNA synthesis, making the pathway to mRNA the central organizing principle.
**Class I (dsDNA)** viruses transcribe mRNA directly from their DNA using host or viral RNA polymerases. Examples include Herpesviridae, Adenoviridae, Poxviridae, and Papillomaviridae. **Class II (ssDNA)** viruses must first convert their single-stranded DNA to a double-stranded form before mRNA can be transcribed. The Parvoviridae (including B19) and Anelloviridae belong to this class. **Class III (dsRNA)** viruses use a viral RdRp to transcribe mRNA from each genome segment; Reoviridae (rotavirus) is the principal example.
**Class IV [(+)ssRNA]** viruses have genomes that serve directly as mRNA, allowing immediate translation upon entry into the cell. This large class includes the Picornaviridae (poliovirus, rhinovirus, HAV), Flaviviridae (dengue, Zika, HCV), Coronaviridae (SARS-CoV-2), and Togaviridae. **Class V [(-) ssRNA]** viruses carry genomes complementary to mRNA and must package an RdRp in the virion to transcribe their genomes. Examples include the Orthomyxoviridae (influenza), Paramyxoviridae (measles, mumps, RSV), Rhabdoviridae (rabies), and Filoviridae (Ebola, Marburg).
**Class VI [(+)ssRNA with reverse transcriptase]** encompasses the retroviruses, whose RNA genomes are reverse transcribed into dsDNA that integrates into the host genome before serving as a template for mRNA. HIV and HTLV are the key examples. **Class VII (dsDNA with reverse transcriptase)** includes the Hepadnaviridae (HBV), in which a dsDNA genome is transcribed to a pregenomic RNA that is then reverse transcribed back to dsDNA.
#### B. ICTV Taxonomy
The **International Committee on Taxonomy of Viruses (ICTV)** maintains a formal hierarchical classification system for viruses, organized as Realm > Kingdom > Phylum > Class > Order > Family > Subfamily > Genus > Species. By convention, family names end in **-viridae** and genus names end in **-virus**. Classification criteria include genome type, replication strategy, morphology, and host range.
A visual summary of the Baltimore classification system. Central element: mRNA (the reference point). Seven arrows radiating outward, each representing one class. Class I (dsDNA) with arrow to mRNA; Class II (ssDNA) through dsDNA intermediate to mRNA; Class III (dsRNA) to mRNA; Class IV (+ssRNA) directly serving as mRNA; Class V (-ssRNA) transcribed to mRNA by RdRp; Class VI (retrovirus +ssRNA) reverse transcribed to dsDNA then to mRNA; Class VII (dsDNA-RT) through RNA intermediate reverse transcribed back to dsDNA. Each class labeled with genome type, arrow direction, key enzymes involved, and 2-3 example virus families with icons or silhouettes.
### IV. Viral Cultivation, Detection, and Quantification
#### A. Cultivation
Because viruses require living cells to replicate, they cannot be grown on standard bacteriological media. **Cell culture** is the most common method of viral cultivation. Primary cell cultures are derived directly from animal tissue and have a limited lifespan. Diploid cell lines, such as WI-38 and MRC-5, can be passaged approximately 50 times and are used for vaccine production. Continuous (immortalized) cell lines, such as HeLa, Vero, and HEK-293, grow indefinitely and are widely used in research. Viral infection of cultured cells produces **cytopathic effects (CPE)** -- visible changes including cell rounding, lysis, syncytia (multinucleated cells formed by cell fusion), and inclusion bodies.
**Embryonated chicken eggs** remain important for influenza vaccine production, with the virus grown in the allantoic or amniotic cavity. **Animal inoculation** was used historically but has been largely replaced by cell culture and molecular methods.
#### B. Detection and Identification
Viruses can be detected and identified through several complementary approaches. **Electron microscopy** provides direct visualization of virus morphology. **Serological methods** detect viral antigens or host antibodies and include ELISA (enzyme-linked immunosorbent assay), immunofluorescence, hemagglutination inhibition (HAI) for influenza, and Western blot (used as a confirmatory test for HIV). **Molecular methods** have become increasingly central to viral diagnostics and include PCR, RT-PCR, and RT-qPCR for highly sensitive detection of viral nucleic acid, sequencing for genotyping, surveillance, and phylogenetics, and rapid antigen tests based on lateral flow immunoassays.
#### C. Quantification
Viral quantification employs several techniques. The **plaque assay** involves infecting a monolayer of cells and counting plaques (clear zones), where each plaque represents one plaque-forming unit (PFU), analogous to colony counting for bacteria. The **TCID50** (tissue culture infectious dose 50%) determines the dilution that infects 50% of cell culture wells. The **hemagglutination assay** quantifies viruses that agglutinate red blood cells, such as influenza and rubella, expressed as hemagglutination units (HAU). **Quantitative PCR (qPCR)** measures viral genome copies, providing a direct readout of viral load.
### V. Viral Tropism and Host Range
**Tropism** refers to the specificity of a virus for particular host cells, tissues, or organs. It is determined by the availability of appropriate host cell receptors (for example, CD4 for HIV, ACE2 for SARS-CoV-2, and sialic acid residues for influenza), the presence of intracellular factors required for replication such as specific transcription factors and polymerases, and the ability of the virus to evade host defenses in particular tissues.
**Host range** describes the spectrum of host species a virus can infect. Some viruses have a narrow host range: poliovirus and variola (smallpox) virus infect only humans. Others, like rabies virus, have a broad host range that includes all mammals, while influenza viruses can infect birds, pigs, humans, and horses. Zoonotic viruses can jump from animal reservoirs to humans, as demonstrated by Ebola, SARS-CoV-2, and Nipah virus.
### VI. Subviral Agents
Several infectious agents are even simpler than viruses. **Viroids** are small, circular, single-stranded RNA molecules of 200--400 nucleotides that lack any protein coat. They cause plant diseases, such as potato spindle tuber disease, and replicate in the nucleus using the host RNA polymerase. No animal viroids are known, although hepatitis D virus is viroid-like in some respects.
**Prions** are infectious protein particles that contain no nucleic acid whatsoever. A prion consists of a misfolded form (PrP^Sc) of a normal cellular protein (PrP^C). The misfolded PrP^Sc acts as a template, inducing conformational change in normal PrP^C molecules and thereby propagating the misfolding. Prions cause transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease (CJD), variant CJD (linked to bovine spongiform encephalopathy, or mad cow disease), kuru, scrapie in sheep, and chronic wasting disease in deer. Prions are exceptionally resistant to sterilization, withstanding standard autoclaving, UV radiation, ionizing radiation, and most chemical disinfectants. Effective inactivation requires prolonged autoclaving at 134 degrees C or treatment with NaOH or concentrated bleach.
**Satellite viruses** depend on a helper virus for their replication. The most clinically important example is **hepatitis D virus (HDV)**, which requires hepatitis B virus for its envelope and worsens hepatitis B outcomes when co-infection occurs.
A figure illustrating subviral agents. Panel A: Viroid -- circular ssRNA molecule shown as a hairpin-loop secondary structure; compared in size to a typical viral genome (drawn to scale). Panel B: Prion -- two protein conformations side by side: PrP^C (normal, predominantly alpha-helical, shown in blue) and PrP^Sc (misfolded, predominantly beta-sheet, shown in red); an arrow showing PrP^Sc templating the conversion of PrP^C; aggregated PrP^Sc fibrils accumulating as amyloid plaques. Panel C: Satellite virus (HDV) -- small circular RNA genome inside a coat provided by HBV surface antigen (HBsAg envelope), demonstrating dependence on the helper virus.