Lecture - 2Virology_2_w2025

Historical Background of Virology

• The study of virology began with experiments involving the Tobacco Mosaic Virus (TMV) in the late 1800s by scientists Ivanovsky and Beijerinck.

  • Method: Crush TMV infected leaves and filter to observe that an agent in the filtrate could infect new leaves.

  • Key Observation: The agent did not “grow” on its own unlike bacteria, which do not pass through filters.

Lecture Objectives

• Main Objective: Understanding viral infection cycles and methods of virology.

  • Elements Covered:

    • Overview of infectious cycles

    • Basic viral replication kinetics

    • Lab hosts used for studying cycles

    • Assessment methods for infectious cycles

    • Cytopathic effects and infectivity

    • Physical measurement techniques

Major Themes in Virology

• Key survival strategies for viruses:

  1. Packaging their genome inside a particle

  2. Transferring their genome from host to host

  3. Ensuring the genome has the information for initiating and completing the infectious cycle

  4. Establishing themselves in hosts for long-term survival

• Key Concept: The Virus Genome is crucial.

• during attachement and entry the protective shell will be lost allowing viral infection to enter cell and take over

  • expose geome and use ribosome…. reassemble on their own to form new virions, which can then exit the host cell to infect additional cells, perpetuating the cycle of infection.

• Coronavirus

  • will attach on outside of cell make way inside loose shell… nucleic acid is now exposed… will find ribosome machinery and make proteins…. reform virions bud outside of cell and repeat with new host cell

Overview of the Viral Infectious Cycle

• The virus genome orchestrates all necessary components for replication and transmission.

• Virions are critical for transmitting the virus to new hosts.

• Susceptible cells

  • must be available for the virus to successfully enter and initiate the infection process.

  • has a receptor that allows entry to certain viruses, such as the CD4 receptor for HIV, enabling the virus to attach and penetrate the host cell membrane.

  • enables entry

  • not always permissive; various factors can influence whether a virus can successfully infect a particular cell type, including the presence of specific co-receptors and the overall health and state of the host cell.

• Permissive Cells:

  • These are cells that support the replication of the virus once it has entered.

  • They provide the necessary cellular machinery and resources for viral propagation, allowing the virus to multiply and spread within the host.

  • enables steps of infectious cycle to completely occur

• Resistant Cells:

  • These cells do not support viral replication, either due to the absence of specific receptors or the presence of antiviral defenses that inhibit the virus's ability to replicate.

  • if virus doesn’t find receptor…. the host cell is resistant to that specific virus, preventing the infection from establishing and allowing the immune system to effectively respond to potential threats.

The Viral Infectious Cycle for Coronaviruses

1. Binding and Viral Entry:

   • Entry happens via membrane fusion or endocytosis.

2. Translation:

   • The viral RNA (+sense) is translated into proteins.

3. Replication:

   • Subgenomic and genomic RNA replication occurs in convoluted membranes inside the cell.

4. Formation of Virion:

   • Nucleocapsid buds into vesicles composed of structural proteins for exocytosis.

Host Cell Requirement for Viral Replication

• Viral replication relies on the following host cell machinery:

  • Nucleus

  • Mitochondria (for energy)

  • Cytoplasmic transport vesicles

  • Protein synthesis machinery

Cell Susceptibility and Permissiveness

• Susceptible Cell: Contains a functional receptor for the virus.

• Permissive Cell: Allows for viral replication to occur.

• Resistant Cell: Lacks receptors and does not permit viral infection.

Viral Hosts in Laboratory Settings

• Viruses depend entirely on hosts for replication.

• Common laboratory hosts:

  • Whole animal hosts: Often costly.

  • Fertilized Chicken Eggs: Commonly used for replicating influenza viruses for vaccine production.

    • influenza can replicate in chicken eggs

    • how glu vaccine is made

    • used as host to replicate influenza virus allowing us to make vaccines. This process involves injecting the virus into the eggs, where it can grow and multiply, subsequently enabling the extraction of the virus for further purification and formulation into a vaccine.

• How and when viruses are infecting host cells

  • look at what effect virus has on the host cell

Methods of Assessing Virus Effects on Cells

1. Cytopathic Effects (CPEs):

   • Visual cues that you can understand to see if virus is affecting the host cells include cell lysis, changes in cell morphology, and the formation of syncytia.

   • longer cells infected by virus can exhibit elongated shapes or unusual clustering, indicating alterations in their normal growth patterns.

   • cells decrease in system when infected… darker areas are lacking cells

   • Changes in infected cells due to viral activity. Examples:

     • Cell lysis (breakdown)

       • membrane looses integrity

     • Syncytia formation (fusion of cells)

       • fusion of adjacent membranes can lead to the formation of multinucleated giant cells, which can enhance viral spread and infection within the host.

       • early sign that infection has taken course

     • Cellular transformation (uncontrolled growth)

       • cells won't stay flat… cells start to clump

   • can never actually see what a virus is doing to its cell

2. Measuring Infectivity:

   • Plaque Assay:

     • measure of infectivity

     • used with bacteriophage…. virus that infects bacteria…. host cell = bacteria

     • plate has host cells (susceptible and permissive) that will be infected by virus

     • Used to quantify viral infectivity by counting plaques formed in a bacterial lawn.

       • area that is darker = area where the virus has successfully infected and lysed the bacterial cells, resulting in a visible clearing known as a plaque.

       • lost the host cells

     • Involves diluting samples and counting resultant plaques to establish plaque forming units (pfu/ml).

     • adding agar on top spread is halted, allowing for the isolation of individual viral particles, which can then be quantified based on the plaques formed.

   • Examples of dilutions and calculations are performed to determine infectious particles.

     • dilution calms down virus

     • final dilution = 10^8

     • diluted virus is applied to susceptible and permissive cells… repeated multiple times

     • purple = active host cell

     • non purple area… host cells ah==have been obliterated

3. Particle-to-PFU Ratio:

   • A concept to determine the ratio of total viral particles to those that are infectious, highlighting the fact that not all particles are infectious.

   • particle can be considered as anything

   • pfu is indication of how many of particles will form a plaque

   • when values are small, particle is very infectious

   • if number gets bigger…. less infectious

4. Transformation Assay:

   • Some viruses do not form plaques but instead create foci such as Rous Sarcoma Virus (RSV).

   • Resulting foci can be quantified for foci-forming units.

   • host doesn’t form mono layer instead they form mountains on top of eachother

Physical Measurement Techniques

1. Hemagglutination Assay:

   • Measurement based on viral protein binding to red blood cells, resulting in a visual lattice formation.

   • no longer assessing if virus has gone through all steps of viral cycle

   • is a viral protein present…. does this viral protein attach on host cell

   • red blood cell is susceptible cell

   • if virus is not present , hemagglutinin protein is not present and fails to react w RBD. This indicates that the virus is unable to bind effectively to the host cell, preventing successful entry and subsequent infection.

   • will bind receptor on RBC

   • samples that express H more fuxxy… no H, only dot is present

2. Viral Enzyme Activity:

   • Certain viruses, like retroviruses, contain active enzymes which can be measured.

   • certain viruses have very specific enzymes

   • reverse transcriptase con revert RNA to DNA

   • specific antibodies can colourfully image viral components, allowing for precise localization within infected cells.

3. Immunostaining & Immunoblotting:

   • Techniques used for identifying and quantifying viral proteins using specific antibodies.

4. Sequencing:

   • PCR cycles to amplify viral DNA, useful for low abundance genes, employing specific temperature protocols for denaturation and elongation.

5. Use of Fluorescent Proteins:

   • Employed for the visualization of viral components in experimental setups.

   • if we artificailly attaches sequences that sequence fluorescent proteins they can be used to track the expression and localization of viral proteins within host cells, providing insights into viral behavior and interactions.