Ch. 6 Viruses, Viroids, and Prions

Historical Discovery and Early Control Measures

• 1884: Charles Chamberland and Louis Pasteur created special filters ($0.1\,\mu\text{m}$ pores) to remove bacteria (which are bigger than or equal to $0.2\,\mu\text{m}$) from liquids.

• 1892: Dmitri Ivanovsky found that a liquid from infected tobacco plants could still cause disease even after passing through these filters. This led to the idea of a “filterable agent” – something smaller than bacteria causing illness.

• 1899: Martinus Beijerinck called this tiny infectious agent a “virus,” which means “poison” in Latin.

• Before we could even see viruses (electron microscopes came out in the late 1930s):

  • People in the 17th century used a method called variolation to protect against smallpox.

  • In 1796, Edward Jenner developed the cowpox vaccination, a safer way to prevent smallpox.

• 1935: Wendell Stanley was able to make crystals of the Tobacco Mosaic Virus (TMV), showing it was made of RNA and protein. He won a Nobel Prize in 1946 for this discovery.

Public-Health Context & Recent Epidemics

• Fast global travel helps viruses spread quickly, like the 2009 H1N1 flu or the 2014 Ebola outbreak in West Africa, even reaching cases in the U.S.

• Better hygiene and widespread vaccination have greatly reduced deaths from viral diseases in developed countries. However, new diseases spreading from animals to humans (zoonoses) and bacteria that resist many drugs (multi-drug resistance) are still big problems.

• Phage therapy, using viruses that infect bacteria, is being looked at again to fight these “superbugs.” Over $2\times10^6$ resistant infections and more than $23\,000$ deaths occur each year in the U.S. due to these superbugs.

General Characteristics of Viruses

• Viruses are not cells and can only reproduce inside other living cells (they are obligate intracellular parasites).

• Their genetic material (genome) is either DNA or RNA, but never both. This genetic material can be a single strand (ss) or a double strand (ds), and it can be in one long piece or several smaller segments.

• The core genome is protected by a protein shell called a capsid, made of smaller units called capsomeres. Some viruses also have an outer phospholipid envelope (a fatty layer) that they get from their host cell, which often has viral proteins (spikes) sticking out.

• Viruses lack many of the genes needed for their own energy and growth. Instead, they take over the machinery of the host cell to make copies of themselves.

• They produce new virus particles, called virions, which carry the viral genome to infect new cells.

Host Range, Tissue Tropism & Transmission

• Viruses can infect all types of living organisms: plants, animals, fungi, protists, bacteria (these are called bacteriophages), and archaea.

• Each virus usually infects only a narrow range of hosts and specific types of cells (tissue tropism):

  • Poliovirus mainly infects nerve cells in the brain and spinal cord.

  • Influenza virus infects cells in the respiratory system.

• Common ways viruses spread (transmission routes):

  1. Direct contact: Through body fluids (e.g., touching an infected person).

  2. Indirect contact: Through contaminated objects (fomites) like doorknobs.

  3. Vector-borne: Carried by arthropods (like mosquitoes or ticks), which can carry the virus externally (mechanical) or internally and spread it through biting (biological).

• Zoonoses are diseases that spread from animals to humans (like bird flu).

• Reverse zoonoses (human to animal spread) also happen.

Viral Structure & Morphology

• Viruses vary greatly in size: from tiny $20\,\text{nm}$ (parvoviruses) to larger typical viruses around $900\,\text{nm}$. Some giant viruses (like Pandoravirus, Pithovirus) can be as big as bacteria.

• Common shapes include:

  • Helical: Like a long rod or coil (e.g., TMV, Ebola).

  • Polyhedral/Icosahedral: Roughly spherical with many flat faces (e.g., poliovirus, rhinovirus).

  • Complex: More intricate structures (e.g., T4 bacteriophage with a head, tail, and fibers; poxviruses are brick-shaped).

• Envelopes are outer layers taken from the host cell's membrane when the new virus particles exit. These envelopes often have spikes made of viral proteins, like the HA (hemagglutinin) and NA (neuraminidase) proteins on the Influenza virus.

• Nomenclature: The different types of influenza are named based on their HA and NA spikes, such as H1N1 (seen in 1918 and 2009), H2N2 (1957), and H3N2 (1968).

Classification & Taxonomy

• The International Committee on Taxonomy of Viruses (ICTV) organizes viruses into 7 orders, 96 families (ending in -viridae), and 350 genera (ending in -virus).

• This classification is based on things like their genome type, shape, how they copy themselves, and what hosts they infect.

• The Baltimore system divides viruses into 7 groups based on their genetic material (DNA or RNA), whether it's single or double-stranded, its orientation, and if they use reverse transcription.

• ICD codes are specific codes that link viral diseases to medical records, insurance claims, and studies of disease spread (epidemiology).

Viral Genomes & Replication Strategies

• dsDNA viruses (double-stranded DNA): Most copy themselves in the cell's nucleus, except for large poxviruses which do it in the cytoplasm.

• ssDNA viruses (single-stranded DNA): Host cells first make a complementary DNA strand, turning the viral genome into double-stranded DNA before copying starts.

• +ssRNA viruses (positive-sense single-stranded RNA): Their RNA genome acts directly as messenger RNA (mRNA) and can be used immediately by host ribosomes to make viral proteins.

• -ssRNA (negative-sense single-stranded RNA) & dsRNA viruses (double-stranded RNA): These viruses need to bring their own special enzyme, called viral RNA-dependent RNA polymerase (RdRP), to first make a positive-sense RNA copy from their genome. This +ssRNA can then be translated into proteins.

• Retroviruses (+ssRNA): These viruses carry an enzyme called reverse transcriptase. This enzyme first converts their RNA genome into DNA (called cDNA), then into double-stranded DNA. This viral DNA then integrates into the host cell's own genetic material (becoming a provirus). This integration means the virus can stay hidden for life, potentially causing lifelong latent infections (e.g., HIV).

Bacteriophage Life Cycles

• Lytic (virulent) cycle: This is a destructive cycle. The phage (virus that infects bacteria) attaches to the bacterium, injects its DNA, takes over the cell to make more viruses, assembles new phage particles, and then causes the bacterial cell to burst open (lysis), releasing many new phages. T-even phages are an example.

• Lysogenic (temperate) cycle: In this cycle, the phage's genetic material (genome) integrates into the bacterial cell's DNA, becoming a prophage. The bacterial cell continues to live and divide, carrying the prophage with it. The prophage can remain silent until certain conditions trigger it to enter the lytic cycle. This can also give the bacteria new traits (lysogenic conversion), such as the toxins found in bacteria causing cholera (Vibrio cholerae) or botulism (Clostridium botulinum).

Transduction (Horizontal Gene Transfer)

This is how phages can accidentally transfer bacterial DNA from one bacterium to another.

• Generalized transduction: During the lytic cycle, pieces of the host bacterium's DNA are randomly packaged into new phage particles instead of viral DNA.

• Specialized transduction: This happens during the lysogenic cycle. When a prophage excises (cuts itself out) from the bacterial DNA, it sometimes takes specific bacterial genes right next to its integration site with it. These specific genes are then transferred when the phage infects a new cell.

• Summary: The lytic cycle is linked to generalized transduction, while the lysogenic cycle is linked to specialized transduction.

Animal Virus Replication

Here are the general steps for how animal viruses multiply:

1. Attachment: The virus attaches to specific receptor molecules on the surface of the host cell. This explains why viruses often target certain tissues (tissue tropism).

2. Penetration: The virus gets inside the cell. Non-enveloped and enveloped viruses can enter by endocytosis (the cell engulfs the virus). Enveloped viruses can also enter by membrane fusion (the viral envelope merges with the cell membrane).

3. Uncoating: The viral capsid (protein shell) is removed, releasing the viral genome into the host cell's cytoplasm.

4. Biosynthesis: The viral genome is copied, and viral proteins are made, using the host cell's machinery. (The specific strategy depends on the virus's genome type, as explained above).

5. Maturation/Assembly: New viral genomes and proteins are put together to form new virus particles.

6. Release: New virions leave the host cell. Enveloped viruses often leave by budding (pinching off from the cell membrane, which may not immediately kill the cell). Non-enveloped viruses usually cause the cell to lyse (burst open) or are released by exocytosis.

Persistent Infections

Some viral infections can stay in the body for a long time:

• Latent infections: The viral genome is present but silent, not actively producing new viruses. However, the virus can reactivate and cause symptoms later (e.g., Herpes Simplex Virus (HSV) which causes cold sores, or Varicella-Zoster Virus (VZV) which causes chickenpox and can reactivate as shingles).

• Chronic infections: The virus is continuously produced at low levels for a long period. The virus might hide from the immune system or change its surface proteins (antigenic variation) to avoid detection (e.g., HIV, Hepatitis C).

Plant Viruses

• Most plant viruses have +ssRNA genomes and can be either enveloped or naked (without an envelope).

• They are typically spread through:

  • Direct contact between infected and healthy plants.

  • Mechanical damage to plant tissues (e.g., by gardening tools).

  • Vectors like insects, fungi, or nematodes (worms) – often specific types of vectors for specific viruses.

• Once inside, they spread throughout the plant via its vascular tissues (like the phloem, which carries sugars).

• Examples include Citrus tristeza virus and Cucumber mosaic virus.

Viral Growth Curve (One-Step for Phage)

This describes how phages multiply in a controlled environment:

• Inoculation: Phages are added to a bacterial culture.

• Eclipse phase: The period shortly after infection where no new extracellular virus particles are detected because they are inside the host cells being assembled. It's like the virus is 'hidden'.

• Burst phase: After the phages multiply inside the bacteria, many new virions are released all at once as the cells burst. The burst size is the number of new virions produced per infected cell.

• Decline phase: If no more host cells are available, the number of active viruses will decrease.

Isolation, Cultivation & Quantification

How we find, grow, and count viruses:

• Filtration: Using filters with very small pores (equal to or less than $0.2\,\mu\text{m}$) allows viruses to pass through while trapping bacteria and larger particles.

• In vivo (in living organisms): Viruses can be grown in fertilized chicken eggs (in different parts like the chorioallantoic membrane, amniotic cavity, or yolk sac) or in live animals.

• In vitro (in lab cultures): Viruses are grown in cell cultures (cells grown in dishes in a lab):

  • Primary cell culture: Cells taken directly from tissues. They usually attach to a surface and stop growing when they touch each other (contact inhibition), so they need to be regularly transferred to new dishes (subcultured).

  • Continuous/immortal cell lines: These are cell lines, often from tumors (like HeLa cells), that can grow indefinitely without attaching to a surface or stopping due to contact inhibition.

• Plaque assay: Used to count bacteriophages. Clear spots (plaques) appear on a layer of bacteria where phages have infected and killed the cells. For animal viruses, scientists look for CPEs (Cytopathic Effects), which are visible changes in cells caused by viral infection, like cells clumping together (syncytia), forming internal structures (inclusion bodies), or becoming round.

Diagnostic & Serological Techniques

Methods to detect viruses or antibodies against them:

• Hemagglutination assay (direct): This test checks if a virus can clump red blood cells together.

• Hemagglutination-Inhibition (HAI) assay: This test detects if a person has antibodies specific to a virus. If their antibodies are present, they will prevent the virus from clumping red blood cells.

• Enzyme Immunoassay (EIA/ELISA): A test where antibodies or antigens bind, and an enzyme attached to one of them leads to a color change when a substrate is added, allowing detection.

• NAATs (Nucleic Acid Amplification Tests): These highly sensitive tests detect viral genetic material (DNA or RNA).

  • PCR (Polymerase Chain Reaction) is used for viral DNA (e.g., HIV DNA).

  • RT-PCR (Reverse Transcription-PCR) is used for RNA viruses, where the RNA is first converted to DNA (e.g., for rabies, HIV, influenza).

Alternative Infectious Agents

Besides typical viruses, there are other infectious particles:

• Viroids: These are tiny, circular single-stranded RNA molecules (about 250\unicode{x2013}400 nucleotides long) with no protein capsid. They mostly cause diseases in plants (e.g., Potato spindle tuber, Tomato planta macho, Avocado sun-blotch) and copy themselves using host plant enzymes.

• Virusoids (satellite RNAs): These are also small, non-self-replicating single-stranded RNA molecules (about 220\unicode{x2013}388 nucleotides long). They are like genetic parasites, needing a helper virus to copy themselves. An example is the subterranean clover mottle virus which needs its virusoid. In humans, the Hepatitis D virusoid (HDV) requires the Hepatitis B virus (HBV) to cause infection.

• Prions: These are unique infectious agents made entirely of misfolded proteins (called PrP$^{\text{Sc}}$). They don’t have any genetic material (DNA or RNA). These misfolded proteins can cause normal proteins (PrP$^{\text{C}}$) to also misfold into the infectious PrP$^{\text{Sc}}$ form, leading to disease. Prions are extremely difficult to destroy with heat, chemicals, or radiation.

  • They cause fatal brain diseases (transmissible spongiform encephalopathies):

    • In humans: Creutzfeldt-Jakob Disease (CJD), variant CJD (vCJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), Fatal Familial Insomnia, and Kuru.

    • In animals: Scrapie (sheep), Bovine Spongiform Encephalopathy (BSE or mad-cow disease), and Chronic Wasting Disease (deer/elk).

Clinical Focus Highlights

• Rabies case (David): A patient named David had rabies. Initial bacterial tests were negative. He was diagnosed with RT-PCR for rabies and confirmed by immunofluorescence from skin nerves and RT-PCR from saliva. He received human rabies immunoglobulin (HRIG) and a vaccine series, and remarkably, he fully recovered, which is very rare once rabies symptoms appear.

• HPV scare (Michelle): Michelle had an abnormal Pap smear (showing CPE from HPV) and an HPV NAAT confirmed the presence of the virus. She was advised to get vaccinated to prevent future HPV infections.

Ethical, Legal & Societal Issues

• Compassionate use of unregistered drugs: The use of experimental drugs like Z-Mapp during the 2014 Ebola outbreak brought up debates about who should get the limited supply (patients vs. healthcare workers) and balancing the urgency of treatment with unknown safety concerns.

• High-level containment: Handling very dangerous agents like Ebola requires specialized training and protective equipment (like full suits and labs with negative air pressure) in Biosafety Level 4 (BSL-4) facilities.

• HeLa cell line: These human cells, vital for much virology and vaccine research, were taken in 1951 from Henrietta Lacks without her informed consent. A 2023 settlement with her family highlights ongoing ethical concerns about patient consent, ownership of biological materials, and profits made from them.

Key Numerical & Statistical Data

• Virus sizes range from $20\,\text{nm}$ to $900\,\text{nm}$, with giant viruses exceeding $1\,\mu\text{m}$.

• Chamberland filter pore size: $0.1\,\mu\text{m}$.

• Burst size (new virions per cell) for phages generally varies, e.g., around 50\unicode{x2013}200 for some phages.

• In the U.S., there are more than $2\times10^6$ antibiotic-resistant infections and over $23\,000$ deaths each year.

• The 2014 Ebola outbreak had $24\,666$ suspected/confirmed cases and $10\,179$ deaths.

Connections & Implications

• Viral latency (virus goes dormant in animal cells) is similar to bacterial lysogeny (phage DNA integrates into bacterial DNA and goes dormant).

• Viral enzymes like RdRP (RNA-dependent RNA polymerase) and reverse transcriptase are key targets for antiviral drugs (e.g., AZT and remdesivir).

• Phage therapy (using viruses to kill bacteria) is relevant to protecting our natural microbial populations (microbiome) and using antibiotics wisely (antibiotic stewardship).

• The extreme resistance of prions to sterilization methods influences how surgical instruments are cleaned (e.g., requiring extended autoclaving, special chemical treatments with NaOH or bleach).

Exam-Type Reflection Points

• What are the differences between lytic, lysogenic, latent, and chronic viral infections?

• How does each Baltimore group's replication strategy relate to the enzymes it needs?

• Why do negative-sense ssRNA viruses have to carry their own RdRP, but positive-sense ssRNA viruses don't?

• How can the HAI test tell us if a person has antibodies against a virus or if the virus itself is present?

• What are the ethical pros and cons of using experimental antiviral drugs during major disease