SCH1111 Fundamental Biomedical Techniques - Viruses

Viruses: General Characteristics and Growth

• Viruses are too small to be seen with a light microscope.
• They are obligate intracellular parasites that infect living cells in:
  • Animals
  • Plants
  • Bacteria
• A key question is whether a virus is alive.

Properties of Viruses

• All viruses contain one type of nucleic acid:
  • RNA or DNA, but not both.
• Viruses do not grow by binary fission or any other form of cell division.
• Viruses lack organelles such as ribosomes.
• They do not possess any ATP-generating mechanisms.

Virus Structure

• A virion is a complete virus particle.
• Viruses are composed of nucleic acid (RNA or DNA) surrounded by a capsid, which is a protein coat, also referred to as a nucleocapsid.
• Capsids are made up of subunits called capsomeres.
• Capsid Shapes:
  • Helical: A ribbon-like structure.
  • Icosahedral: 20 faces.
  • Complex.
• Some viruses also have envelopes (acquired from the cells that they have infected).
• Enveloped viruses are more susceptible to inactivation by disinfectants.

Virus Classification

• Hierarchical Virus Classification System:
  1. Nature of the nucleic acid: RNA or DNA.
  2. Symmetry of the capsid.
  3. Presence or absence of an envelope.
  4. Dimensions of the virion and capsid.
• Baltimore Classification: Mechanism of viral genome replication.

Baltimore Classification Groups

• I: dsDNA viruses (e.g., Adeno, Herpes, Pox).
• II: ssDNA viruses (+ strand or "sense") DNA (e.g., Parvoviruses).
• III: dsRNA viruses (e.g., Reoviruses).
• IV: (+)ssRNA viruses (+ strand or sense) RNA (e.g., Picornaviruses, Togaviruses).
• V: (-)ssRNA viruses (- strand or antisense) RNA (e.g., Orthomyxoviruses, Rhabdoviruses).
• VI: ssRNA-RT viruses (+ strand or sense) RNA with DNA intermediate in life-cycle (e.g., Retroviruses).
• VII: dsDNA-RT viruses (e.g., Hepadnaviruses).
• All viruses must generate positive strand mRNAs from their genomes to produce proteins and replicate themselves. The various types of virus genomes require different basic strategies for their replication.

Requirements for Replication

1. A living cell.
2. A receptor to allow the virus to enter the cell.
3. A way to leave the cell.

Viral Replication

1. Attachment: Viruses attach to the cell membrane.
2. Penetration: Occurs by endocytosis or fusion.
3. Uncoating: Viral or host enzymes facilitate this process.
4. Biosynthesis: Production of nucleic acid and proteins.
5. Maturation: Nucleic acid and capsid proteins assemble.
6. Release: Budding (enveloped viruses) or rupture.

• Viral nucleic acid is replicated using the host cell processes.
• The replication method differs based on whether the virus contains DNA or RNA.
• Host proteins are also replicated before assembly takes place.
• Some viruses have different modes of replication, e.g., retroviruses, which integrate into chromosomal DNA and can remain dormant for many years.

Viral Pathogenesis

• Viral pathogenesis is the process by which a viral infection leads to disease and is an abnormal situation of no value to the virus.
• Outcomes include:
  • Recovery with no residue effects.
  • Recovery with residue effects (e.g., acute viral encephalitis).
  • Death.
  • Proceed to chronic infection.
• Acute vs. Chronic Infection:
  • Acute viral infection: Rapid onset of disease, a brief period of symptoms, and resolution within days; usually accompanied by early production of infectious virions and elimination of infection by the host immune system.
  • Chronic infection: A type of persistent infection that is eventually cleared, while latent or slow infections last the life of the host.
• Types of Chronic Infections:
  • Silent subclinical infection for life (e.g., cytomegalovirus).
  • A long silent period before disease (e.g., HIV).
  • Reactivation to cause acute disease (e.g., herpes and shingles).
  • Chronic disease with relapses (e.g., HBV, HCV).
  • Cancers (e.g., Epstein-Barr virus -> Burkitt's and Hodgkin's lymphomas, human papillomavirus -> cervical cancer).

Factors Influencing Viral Pathogenesis

1. Effects of viral infection on cells (cellular pathogenesis).
2. Entry into the host.
3. Course of infection (primary replication, systemic spread, secondary replication).
4. Cell/tissue tropism.
5. Cell/tissue damage.
6. Host immune response.
7. Virus clearance or persistence.
   • The consequences of viral infections depend on the interplay between a number of viral and host factors.

Cellular Pathogenesis

• Cells can respond to viral infections in 3 ways:
  1. No apparent change.
  2. Death.
  3. Transformation.
• Direct cell damage and death from viral infection may result from:
  • Diversion of the cell's energy.
  • Shut off of cell macromolecular synthesis.
  • Competition of viral mRNA for cellular ribosomes.
  • Competition of viral genes for cellular transcriptional machinery.
• Indirect cell damage can result from:
  • Integration of the viral genome.
  • Induction of mutations in the host genome, leading to malignant cell transformation.
  • Inflammation.
  • Host immune response.

Viral Entry

• Skin: Requires a breach in the physical integrity of this effective barrier (e.g., cuts or abrasions). Many viruses employ ‘carriers’ (e.g., ticks, mosquitoes) to breach this barrier.
• Conjunctiva and other mucous membranes: Rather exposed site and relatively unprotected.
• Respiratory tract: Sophisticated immune defense mechanisms, as well as non-specific inhibitory mechanisms (ciliated epithelium, mucus secretion, lower temperature) which viruses must overcome.
• Gastrointestinal tract: A hostile environment; gastric acid, bile salts, etc. Viruses that spread by the GI tract must be adapted to this hostile environment.
• Genitourinary tract: Relatively less hostile than the above, but less frequently exposed to extraneous viruses.

Routes of Infection

• Arboviruses: Infected through the bite of mosquitoes (e.g., Ross River virus, Dengue virus).
• Bloodborne: e.g., Hepatitis C, Hepatitis B, HIV.
• Sexually transmitted: e.g., HIV, Human Papilloma virus.
• Animal bite: e.g., Rabies virus.
• Zoonosis: e.g., Ebola virus, SARS, MERS.
• Ingestion (fecal/oral): e.g., Hepatitis A, polio.
• Inhalation: Respiratory viruses, measles.

Course of Viral Infection

• Primary Replication:
  • The place of primary replication is where the virus replicates after gaining initial entry into the host.
  • This frequently determines whether the infection will be localized at the site of entry or spread to become a systemic infection.
• Systemic Spread:
  • Apart from direct cell-to-cell contact, the virus may spread via the blood stream and the CNS.
• Secondary Replication:
  • Secondary replication takes place at susceptible organs/tissues following systemic spread.

Cell Tropism

• Tropism = viral affinity for specific body tissues.
• Determined by:
  • Cell receptors for virus.
  • Cell transcription factors that recognize viral promoters and enhancer sequences.
  • Ability of the cell to support virus replication.
  • Physical barriers.
  • Local temperature, pH, and oxygen tension, enzymes, and non-specific factors in body secretions.
  • Digestive enzymes and bile in the gastrointestinal tract that may inactivate some viruses.

Cell Damage

• Viruses may replicate widely throughout the body without any disease symptoms if they do not cause significant cell damage or death.
• Retroviruses do not generally cause cell death, being released from the cell by budding rather than by cell lysis and cause persistent infections.
• In contrast, some viruses cause lysis and death of the cells in which they replicate, leading to fever and increased mucus secretion in the case of Rhinoviruses, collagen digestion and immune triggered coagulation in the case of Ebola virus.

Immune Response

• In most cases, the virus is cleared completely from the body and results in complete recovery.
• In other infections, the immune response is unable to clear the virus completely and the virus persists.
• In a number of infections, the immune response plays a major pathological role in the disease.
• In general, cellular immunity (T cells – kill other cells) plays the major role in clearing virus infection, whereas humoral immunity (B cells – antibody production) protects against reinfection.

Viral Clearance or Persistence

• The majority of viral infections are cleared, but certain viruses may cause persistent infections.
• There are 2 types of chronic persistent infections:
  • True Latency: The virus remains completely latent following primary infection (e.g., HSV, VZV). Its genome may be integrated into the cellular genome.
  • Persistence: The virus replicates continuously in the body at a very low level (e.g., HIV, CMV, EBV).
    • HSV = human simplex virus; VZV = varicella-zoster virus; HIV = human immunodeficiency virus; CMV = cytomegalovirus; EBV = Epstein-Barr virus.
• After virus infection, viruses can:
  1. Remain localized (e.g., Rhinovirus remains in the respiratory epithelium).
  2. Spread to other tissues to involve more organs/sites of infection (e.g., Ebola virus).

Important Points to Remember

• Viruses grow only in living cells.
• Viruses cause disease by destroying or damaging the cells they infect, damaging the body's immune system, changing the genetic material (DNA) of the cells they infect, or causing inflammation which damages the organ.
• Viruses cause many diseases, such as human immunodeficiency virus (HIV), cold sores, SARS, chickenpox, measles, flu (influenza), Ebola, and cancer.

Virus Identification Methods

• A. Growth of virus in cell culture
• B. Nucleic acid testing (NATs)
• C. Serological tests – detecting antigens or antibodies
• D. Electron microscopy

Growing Viruses in the Laboratory

• Animal viruses can be grown in living animals (depending on the virus), in embryonated eggs, or in cell culture (using cells derived from specific organs or animals).

Cell Culture

• Cells are suspended in culture medium.
• Normal cells or primary cells grow in a monolayer across the glass or plastic container.
• Transformed cells or continuous cell cultures do not grow in a monolayer.
• A tissue is treated with enzymes to separate the cells.
• Making single cell suspensions – trypsin, collagenase.
• Observed effects: Syncytium, Cytomegaly, plaque formation.
• Growing viruses in culture can allow them to be “counted” – plaque assays.
• Calculation: Dilution \times number \ of \ plaques \rightarrow 1 \ mL
• Unit: Plaque forming units/mL
• 1 plaque is equivalent to one virus infecting a cell.

Detection of Viral Nucleic Acid – Nucleic Acid Testing

• Polymerase chain reaction (PCR).
• This method detects DNA – you can also modify the method to detect RNA.
• Samples can include serum, urine, and saliva.

Polymerase Chain Reaction (PCR)

• Steps include denaturation at 94°C, annealing with specific primers at 60°C, and extension at 72°C using Taq polymerase and dNTPs (ATCG).
• Repeat for 30 – 45 cycles.

Problems with PCR

• It is a method of detection – it gives no indication if the virus is replication competent.
• False positives are possible (though good primer design minimizes this risk).
• Cross-sample contamination is possible (so laboratory quality control must be carefully monitored).

Serological Tests

Enzyme-Linked Immunosorbent Assay (ELISA)

• Read in a spectrophotometer.
• Involves the addition of an antibody specific for human Ig (conjugate).

Detection using a Fluorescent Conjugate

• Slides with virus-infected cells adhered to wells.
• Incubated with serum with specific antibodies.
• Then incubated with a fluorescent conjugate.
• Antibodies adhere to cells and are visualized using a fluorescent microscope.

Flow Cytometry

• Principle:
  1. Cells are treated with an antibody that adheres a fluorophore to a cell component.
  2. Cells in suspension flow through the detector in single file.
  3. The optical system (laser) enables labeled cells to be counted.
  4. Cell sorters allow tagged cells to be filtered away from untagged cells.
• Example: Used to detect the number of CD4^+ T cells in blood using anti-CD4 and anti-CD3 antibodies.

Electron Microscopy (Direct)

• Requires 10^6 virus particles per mL for visualization.
• Normally uses 50,000 - 60,000 magnification.
• Viruses are 20-30nm small.
• Viruses may be detected in the following specimens:
  • Faeces: Rotavirus, Adenovirus.
  • Vesicle Fluid: Herpes simplex virus (HSV), varicella-zoster virus (VZV).
  • Skin scrapings: Human papillomavirus (HPV), molluscum contagiosum.

Control of Virus Infections

1. Understand the route of infection and break the chain of infection.
2. Develop a vaccine.
3. Develop an antiviral medication.

• Antibiotics cannot be used to treat virus infections. Why/Why not?

Breaking the Chain of Infection

• Sexually transmitted: Barrier protection.
• Fecal/oral: Wash hands after going to the toilet/changing nappies/wiping saliva from babies' mouths.
• Fomites: Use appropriate personal protective equipment.
• Foodborne: Cook food properly, avoidance, recall contaminated foods.
• Airborne: Isolation of infected people. Face masks give limited protection.

Vaccines

• Used to prevent viral diseases
• Refer to available resources for types of available vaccines and vaccine-preventable diseases.

Antiviral Medications

• Two main modes of action:
  1. Stop nucleic acid replication (e.g., acyclovir).
  2. Stop virus from entering cells in the first place (flu medications such as Relenza).
• Other treatments:
  • Interferon (stopping virus spreading to new cells).
  • Gamma globulin (pre-formed IgG).

Key Summary Points

• Describe the characteristics of viruses and how they are used for classification.
• Explain the virus life cycle.
• Explain the mechanism of viral pathogenesis.
• How would you test for a viral infection? Type of methods?
• What is an immunoassay? Give examples and how can they be used for viral detection.