Acellular Pathogens: Viruses, Viroids, Virusoids, and Prions

Introduction to Viruses and Epidemics

Rapid Global Spread: Viral epidemics spread as swiftly as human travel, especially due to air travel. The 2014 Ebola virus outbreak in West Africa exemplifies this rapid global dissemination. Viral epidemics have likely affected human populations since the origin of our species.
Historical Understanding (Pre-Electron Microscope):
- Viruses were known to affect humans long before they could be visualized, as electron microscopes, capable of seeing viruses, were only invented in the 1930s.
- Inoculation (Variolation): For centuries, this practice was used to protect against smallpox. Material from pustules of an infected person was introduced into superficial scratches of an uninfected person.
  - Approximately $1-2\%$ of inoculated individuals died.
  - In contrast, naturally acquired smallpox had a mortality rate of about $30\%$ .
- Vaccination (1700s): This process began with using materials from pustules caused by the much less severe cowpox (Latin: vacca = cow) to prevent smallpox disease, a significant advancement in preventing viral diseases.
Discovery of the First Virus – Tobacco Mosaic Virus (TMV):
- TMV was the first described virus, causing Tobacco Mosaic Disease (TMD) in plants.
- TMD was found to be caused by a "non-filterable element": an extract from an infected tobacco plant, when passed through a $0.1 \mu \text{m}$ pore-sized filter (too small for bacteria), still caused TMD in healthy plants, demonstrating the infectious agent was not bacterial.
- Acellular Nature: Viruses are considered acellular biological entities and are therefore not included in the primary phylogenetic tree of life.
- Evolutionary Concept: Despite being acellular, viruses possess the unifying concept of evolution, allowing them to be organized into similar taxonomic groups.

Characteristics of Viruses

Basic Properties:
- Infectious, acellular pathogens.
- Obligate intracellular parasites with high host and cell-type specificity (tropism).
- Possess a genome made of either DNA or RNA, but never both.
- The genome is encased within a protein capsid.
- Some viruses also feature a phospholipid membrane (envelope) studded with viral glycoproteins.
- Lack genes for many essential reproductive elements, forcing them to exploit their host cell's genomes and cellular machinery for reproduction.
Host Range and Outcomes:
- Viruses can infect any cell type: animals, plants, fungi, protists, bacteria, and archaea.
  - Bacteriophages (phages): Viruses that specifically infect bacteria.
- Most viruses exhibit a narrow host range, infecting cells of only one or a few species. Within multicellular hosts, they may infect only one or a few specific cell types.
- Viral infections can lead to various outcomes in infected cells:
  - Abnormal cell growth.
  - Cell death.
  - Alteration of the cell's genome.
  - Little to no discernible effect.
Modes of Transmission: Viruses can be transmitted through multiple mechanisms:
- Direct Contact: e.g., sexual contact, respiratory droplets.
- Indirect Contact: e.g., contaminated abiotic surfaces (fomites).
- Vectors: e.g., mosquitoes or other arthropods.
  - Mechanical Vectors: Carry viruses on the outside of their bodies and transmit them via physical contact.
  - Biological Vectors: Carry viruses within their bodies and transmit them via biting.
Size of Virions:
- Individual viral particles are called virions.
- Virions are significantly smaller than the cells they infect, which is crucial for their infectivity.
  - Viruses are often approximately $1,000$ times smaller in length than their host cells.
  - They can be around $1,000,000$ times smaller in volume than their host cells.
- Most viruses are only visible with electron microscopes.
- Virion sizes range from $20 \text{ nm}$ to $900 \text{ nm}$ .
- Some "giant" viruses can approach the size of bacterial cells.

Viral Structures

Genome: Composed of either DNA or RNA (never both).
Capsid: A proteinaceous shell surrounding the genome.
- Composed of protein subunits called capsomeres, which can be of one or more types.
- Contains only the genome and some enzymes; no cytosol.
Naked vs. Enveloped Viruses:
- Naked viruses: Consist only of their genome and capsid.
- Enveloped viruses: Their capsid is surrounded by a lipid layer called an envelope.
  - Envelopes are made of phospholipids derived from the host cell's cytoplasmic or intracellular membranes.
Spikes: Present on enveloped viruses (and some naked viruses), made of glycoproteins.
- Spikes allow viruses to attach to and enter host cells.
- Specific versions of spike proteins are used for viral classification (e.g., Influenza viruses are classified by their hemagglutinin "H" and neuraminidase "N" spike proteins, like $H1N1$ ).
Capsid Shapes: Viruses exhibit diverse capsid morphologies:
- Helical: Cylindrical or rod-shaped, with the genome fitting inside the length of the capsid. Can be naked or enveloped.
- Polyhedral: Often icosahedral (a $3 ext{D}$ , many-sided shape). Can be naked or enveloped.
- Complex: Possess features of both helical and polyhedral viruses, or unique structures.
  - T4 phages: Have a polyhedral head (containing the genome), a sheath, tail fibers, and tail pins.
  - Poxviruses: Can be brick-shaped with distinctive surface characteristics.

Viral Classification and Taxonomy

International Committee on Taxonomy of Viruses (ICTV): Refines and maintains viral taxonomies, which largely mirror the organizational scheme for life.
Classification Criteria: Viruses are classified by:
- Morphology.
- Nucleic acid type.
- Mode of replication.
- Host organism(s).
- Type of disease caused.
Binomial Nomenclature: As of 2021, viruses conform to a binomial nomenclature scheme, with old virus names updated to fit this system.
Taxonomic Hierarchy: Includes Realm, Kingdom, Phylum, Subphylum, Class, Order, Suborder, Family, Subfamily, Genus, Subgenus, and Species.
Baltimore Classification System: Defines seven groups based on a combination of nucleic acid type (DNA or RNA), strandedness (single-stranded or double-stranded), sense/antisense, and replication method.
- Class I: dsDNA viruses.
- Class II: ssDNA viruses ( $+\text{ strand / sense}$ ).
- Class III: dsRNA viruses.
- Class IV: $(+)$ ssRNA viruses ( $+\text{ strand / sense}$ ).
- Class V: $(-)$ ssRNA viruses ( $- \text{ strand / antisense}$ ).
- Class VI: ssRNA-RT viruses ( $+\text{ strand / sense}$ , RNA with a DNA intermediate in the life cycle).
- Class VII: dsDNA-RT viruses (DNA with an RNA intermediate in the life cycle).
Informal Groupings: Viruses are also informally grouped by shared characteristics:
- Naked vs. enveloped.
- Single-stranded (ss) vs. double-stranded (ds) genomes.
- DNA vs. RNA genomes.
- Segmented vs. nonsegmented genomes.
- Positive ( $+\text{ / sense}$ ) vs. negative ( $- \text{ / antisense}$ ) strand RNA.
- Examples: Herpesviruses are dsDNA enveloped viruses, while HIV is a $+$ ssRNA enveloped virus.
- Other features: Host specificity, tissue specificity, capsid shape, and specific genes or enzymes.

Common Pathogenic Viruses (Examples)

dsDNA, enveloped (Poxviridae, Herpesviridae): Orthopoxvirus (skin papules), Parapoxvirus (skin lesions), Simplexvirus (cold sores, genital herpes).
dsDNA, naked (Adenoviridae, Papillomaviridae): Atadenovirus (respiratory infection/common cold), Papillomavirus (genital warts, cervical/vulvar/vaginal cancer).
dsRNA, naked (Reoviridae): Reovirus (gastroenteritis/stomach flu), Rotavirus (gastroenteritis).
ssDNA, naked (Parvoviridae): Adeno-associated dependoparvovirus A & B (respiratory tract infection).
$+$ ssRNA, naked (Picornaviridae): Enterovirus C (Poliomyelitis), Rhinovirus (upper respiratory tract infection/common cold), Hepatovirus (Hepatitis).
$+$ ssRNA, enveloped (Togaviridae, Retroviridae): Alphavirus (encephalitis, hemorrhagic fever), Rubivirus (Rubella), Lentivirus (Acquired Immune Deficiency Syndrome/AIDS).
$-$ ssRNA, enveloped (Filoviridae, Orthomyxoviridae, Rhabdoviridae): Zaire Ebolavirus (Hemorrhagic fever), Influenzavirus A, B, C (Flu), Lyssavirus (Rabies).

Bacteriophage Life Cycles in Prokaryotic Hosts

Bacteriophages serve as a valuable model for understanding viral infection.
Virulent Phages: Typically cause the death of their host cells through cell lysis (lytic cycle).
Temperate Phages: Can either enter the lytic cycle or integrate into the host cell's chromosome (lysogenic cycle), replicating with the host genome until induced to produce new progeny viruses.
Lytic Cycle (Virulent Phage Infection): A five-stage process where the bacteriophage takes over the host bacteria cell, reproduces, and destroys the cell.
1. Attachment: The phage binds to specific bacterial surface receptors.
2. Penetration: The phage genome is injected into the bacterial cell.
3. Biosynthesis: Host cell machinery is hijacked to produce components of new virions.
4. Maturation: New virions are assembled within the host cell.
5. Lysis: Mature phage particles are released as the host cell is destroyed.
Lysogenic Cycle (Temperate Phage Infection): The viral genome enters the host cell (attachment and penetration), but instead of immediately killing the cell, it integrates into the bacterial chromosome.
- Prophage: The integrated phage genome.
- Lysogen: A bacterial host containing a prophage.
- Lysogeny: The process of bacterial infection by a temperate phage.
- The viral genome replicates with the host cell every time it divides.
- Lysogenic Conversion (Phage Conversion): The presence of the prophage can alter the host cell's phenotype by introducing extra genes (e.g., toxin genes).
- Induction: The process by which the prophage excises from the genome, typically leading to a lytic cycle.
  - Environmental stressors like starvation or toxic chemicals can trigger induction.

Transduction: Horizontal Gene Transfer via Phages

Definition: Transduction is the transfer of bacterial DNA from one bacterial cell to another via sequential phage infections.
Mechanism: During phage maturation, some bacterial DNA can be accidentally packaged into phage capsids along with phage DNA. When this phage infects a new bacterial cell, this bacterial DNA is injected, can recombine into the new host's genome, and potentially confer new genes and functions.
Generalized Transduction:
- Typically occurs with lytic phages.
- A random piece of bacterial DNA from anywhere on the chromosome is transferred.
- Result of a "mistake" in packaging, where bacterial DNA accidentally enters phage particles.
Specialized Transduction:
- Occurs with temperate phages.
- The phage DNA has a specific integration site in the host genome where it becomes a prophage during lysogeny.
- During induction, when the prophage de-integrates, a segment of bacterial DNA adjacent to the integration site remains attached to the viral DNA.
- The bacterial DNA packaged into these phage particles is not random; it is always the DNA adjacent to the prophage's original integration site.
- This bacterial DNA will integrate into the new bacterial host at the same phage integration site.

Animal Viruses: Tropism and Replication

Life Cycle Stages: Lytic animal viruses follow similar stages to bacteriophages: attachment, penetration, biosynthesis, maturation, and release.
Key Differences:
- Penetration: Animal viruses enter host cells via endocytosis or membrane fusion (for enveloped viruses).
- Tissue Tropism: Animal viruses are not only host-specific but also infect only specific types of cells within certain tissues. This specificity is called tissue tropism.
  - Poliovirus: Exhibits tropism for cells of the gastrointestinal (GI) tract, as well as brain and spinal cord tissues. This explains its fecal-oral spread and potential to cause paralysis.
  - Influenza virus: Has a primary tropism for the respiratory tract, leading to its spread through respiratory droplets and symptoms like sore throat and coughing.
Diverse Genome Strategies: The nature of the viral genome dictates its replication, transcription, and translation processes within the host cell.
- dsDNA: Replicated, transcribed, and translated akin to the host's genomic DNA.
- ssDNA: Requires host enzymes to synthesize the complementary second DNA strand.
- RNA Genomes (Three Types):
  1. dsRNA: Uses virus-encoded RNA-dependent RNA polymerase (RdRP) to create $+$ ssRNA (from the negative strand).
  2. $+$ ssRNA (sense RNA): Analogous to mRNA; it can be directly translated into protein by host ribosomes (it "makes sense" as a transcript).
  3. $-$ ssRNA (antisense RNA): Must first be replicated into $+$ ssRNA by a virus-encoded RNA-dependent RNA polymerase (RdRP) before ribosomes can "read" it. It is "antisense" because it needs this conversion.
Example: Influenza Virus ( $-$ ssRNA):
- A $-$ ssRNA virus with the unusual feature of replicating in the host cell's nucleus.
- Viral glycoproteins attach to host epithelial cells.
- The virus is engulfed via endocytosis.
- Viral RNA enters the nucleus, where it is replicated by RdRP.
- Viral RNA and proteins are synthesized and assembled into new virions, which are then released by budding.

Animal (Eukaryotic) Viruses: Retroviruses

Presence: Retroviruses are currently known to infect only eukaryotes, not bacteria or archaea.
Key Enzyme: Reverse Transcriptase: Retroviruses are $+$ ssRNA viruses that possess a unique enzyme called reverse transcriptase.
- Function: Reverse transcriptase uses the $+$ ssRNA genome as a template to synthesize double-stranded DNA (dsDNA).
  1. The $+$ ssRNA serves as a template to create a complementary single-stranded DNA (cDNA).
  2. The ssDNA then acts as a template to synthesize its complementary strand, forming a dsDNA molecule.
- The newly formed viral dsDNA can then be replicated, transcribed, and translated like the host's genome.
- Provirus Formation: Often, this viral dsDNA integrates into the host genome, becoming a provirus.
  - A provirus can remain in the host for extended periods, establishing a chronic infection.
  - Unlike a prophage, a provirus generally cannot undergo excision after integration into the genome.
- The host cell transcribes and translates the viral RNA and proteins, which assemble into new virions capable of infecting other host cells.
Example: HIV (Human Immunodeficiency Virus):
- An enveloped, icosahedral retrovirus.
- During infection, HIV attaches to a cell surface receptor on an immune cell and fuses with the cell membrane, releasing its contents into the cell.
- Reverse transcriptase converts the single-stranded RNA genome into DNA.
- Other viral enzymes incorporate the HIV dsDNA into the host genome.

Persistent Viral Infections

Definition: Occur when a host fails to completely eliminate a virus following an immune response.
States of Persistence:
- Silent: The virus is not actively replicating within the host.
- Productive: The virus is still replicating, but causing minimal to no damage to host cells at that stage.
Categories of Persistent Infections:
1. Latent Infections:
  - Caused by viruses such as herpes simplex virus, varicella-zoster virus (VZV), and Epstein-Barr virus.
  - Viruses remain hidden or dormant in host cells.
  - Latency may follow an initial, acute, symptomatic infection.
  - Latent viruses can exist as circular viral genome molecules outside the host chromosome or integrate into the host genome as proviruses.
  - During dormancy, they cause no disease symptoms and are often difficult to detect; a patient may be unaware they carry the virus.
  - Example: Varicella-Zoster Virus (VZV):
    - Causes chickenpox (varicella) during the initial acute infection (rash of blisters).
    - After symptoms resolve, the virus becomes dormant within nerve cell ganglia.
    - During dormancy, VZV does not kill nerve cells or continue replicating.
    - After decades, VZV can reactivate to cause shingles (zoster or herpes zoster), characterized by painful lesions typically on one side of the body.
2. Chronic Infections:
  - Diseases with symptoms that are recurrent or persist over a long period because the body is unable to eliminate the virus.
  - Example: HIV:
    - HIV infections often become chronic after a long period of latency.
    - Untreated patients may experience no symptoms for years despite detectable virus levels.
    - HIV maintains chronic persistence by interfering with the immune system's ability to detect and clear the virus, and by constantly changing antigens through mutation.
    - These immune evasion mechanisms are also seen in other chronically infecting viruses, like hepatitis C virus.

Non-Viral Acellular Pathogens

These particles self-propagate at the host's expense, consisting of only one type of biological molecule (either RNA or protein).

Viroids

Composition: Consist of a short strand of circular single-stranded RNA (ssRNA) capable of self-replication.
- The first discovered viroid caused potato tuber spindle disease, leading to deformities and slower sprouting in potato plants.
Replication: Like viruses, viroids hijack host machinery to replicate their RNA genome.
- Unlike viruses, viroids lack a protein coat to protect their genetic information.
- Viroid RNA does not encode any protein-coding genes; it directly instructs host cell machinery to copy it.
- Viroids act as ribozymes, capable of cleaving their own sequences.
Impact and Transmission:
- Can cause devastating losses in commercially important agricultural food crops.
- Dispersed mechanically (during crop maintenance/harvesting), via vegetative reproduction, seeds, and insects.

Virusoids

Composition: A type of pathogenic RNA, also infecting agricultural crops.
Dependence: Described as non-self-replicating ssRNAs that require a helper virus to enter a host cell.
- Once inside, the virusoid RNA directs the host to replicate it.
- Virusoid genomes are very small ( $\sim 200 - 400$ nucleotides long).
- Like viroids, virusoid RNA does not code for any protein; it only serves to replicate itself.
- Unlike viroids, virusoids cannot direct host cells to replicate them without helper virus assistance.
Mechanisms: Very few virusoids and their helper viruses are known.
- After the helper virus enters the host cell, virusoids are released into the plant cell cytoplasm, where they exhibit ribozyme activity.
- The helper virus undergoes typical viral replication independently of the virusoid's activity.
Satellite RNAs (satRNAs): Virusoids belong to this larger group of infectious RNAs found in animals.
- Unlike plant virusoids, satRNAs may encode proteins.
- Like plant virusoids, satRNAs must coinfect with a helper virus to replicate.
- Example: Hepatitis Delta Virus (HDV): A satRNA/virusoid that infects humans.
  - Larger than plant virusoids, with a $1,700$ nucleotide ssRNA genome capable of directing the biosynthesis of HDV-associated proteins.
  - Its helper virus is the Hepatitis B Virus (HBV).
  - Coinfection with HBV and HDV results in more severe liver damage, which led to HDV's discovery.

Prions

Composition: Proteinaceous infectious particles.
Misfolding Mechanism: A prion is a misfolded, rogue form of a normal cellular protein ( $\text{PrP}^{\text{C}}$ ).
- The misfolded prion protein ( $\text{PrP}^{\text{Sc}}$ ) is infectious, stimulating other normal $\text{PrP}^{\text{C}}$ proteins to become misfolded, forming plaques.
- The initial misfolding event (conversion of $\text{PrP}^{\text{C}}$ to $\text{PrP}^{\text{Sc}}$ ) can be caused by a genetic mutation or occur spontaneously.
Transmissible Spongiform Encephalopathies (TSEs): Prions cause various forms of TSEs in humans and animals, which are rare degenerative disorders affecting the brain and nervous system.
- Pathology: Accumulation of $\text{PrP}^{\text{Sc}}$ prions kills brain cells, leading to sponge-like lesions (holes) in brain tissue.
- Symptoms: This results in brain damage, loss of motor coordination, and dementia. Infected individuals experience mental impairment and progressively lose the ability to move or speak.
- Prognosis: There is no cure for TSEs. The disease progresses rapidly, typically leading to death within a few months or years.
Examples of TSEs:
- Humans: Kuru, Fatal Familial Insomnia, Creutzfeldt-Jakob disease (CJD).
- Animals: Mad cow disease, scrapie (in sheep and goats), chronic wasting disease (in elk and deer).
Transmission:
- Animal-to-animal and Animal-to-human: By eating contaminated meat or animal feed.
- Human-to-human: Can be hereditary or occur through contact with contaminated tissue (e.g., blood transfusion, organ transplant).
Resistance and Treatment:
- Prions are extremely difficult to destroy due to their resistance to heat, chemicals, and radiation. Even standard sterilization procedures are often ineffective.
- Currently, there is no treatment or cure for TSE diseases.
- Contaminated meats and infected animals must be handled according to strict federal guidelines to prevent transmission.
Mechanisms of Transmission for Human TSEs:
- Sporadic CJD (sCJD): Mechanism unknown; possibly spontaneous alteration of normal prion protein ( $\text{PrP}$ ) to the rogue form due to somatic mutation.
- Variant CJD (vCJD): Eating contaminated cattle products and by secondary bloodborne transmission.
- Familial CJD (fCJD): Mutation in germline $\text{PrP}$ gene.
- Iatrogenic CJD (iCJD): Contaminated neurosurgical instruments, corneal grafts, gonadotrophic hormone, and, secondarily, by blood transfusion.
- Kuru: Eating infected meat through ritualistic cannibalism.
- Gerstmann-Straussler-Scheinker disease (GSS): Mutation in germline $\text{PrP}$ gene.
- Fatal familial insomnia (FFI): Mutation in germline $\text{PrP}$ gene.