Unit 6 - Introduction to Virology

Why do viruses require living cells to grow

Viruses cannot replicate independently and must use living host cells

Three types of cell lines used for viral propagation

Primary, low passage (finite), and continuous cell line

Primary cell lines

Diploid cells taken directly from animal tissue, few passages, most permissive expensive

Low passage (finite) cell lines

Diploid cells that can be passaged up to 50 times; become less sensitive to infection

Continuous cell lines

Aneuploid cells that divide indefinitely, least expensive and least permissive

Splitting (passage)

Removing, diluting, and transferring cultured cells into a new vessel

Examples of each cell line type

• Primary: PMK, RK.

• Finite: MRC-5, WI-38

• Continuous: HEp2, A549

Permissiveness

A cell line’s ability to support infection and growth of different viruses

What does cell culture growth medium contain

salts, glucose, serum (nutrients and growth factors), and antimicrobials

Why use several cell lines for one specimen

Different cell lines support different viruses, increasing the chance of recovery

What determines which cell culture vessel is used

The number of cells needed for viral propagation

Flasks

Incubated on their sides, large surface area allows growth of many cells

Shell vials

Contain a coverslip with a cell monolayer centrifuged and incubated after inoculation

Microtiter plates

Small wells allow multiple specimens to be tested in duplicate or triplicate

Microtiter plates

Small wells allow multiple specimens to be tested in duplicate or triplicate

Which method is the most rapid and sensitive for viral isolation

Shell vial culture

Incubation temperatures for viral cell cultures

33*C for respiratory viruses, 35-37*C for other viruses

Cytopathic effect (CPE)

Visible changes or damage to host cells caused by viral infection

Methods used to detect viruses after incubation

Microscopic examination for CPE, antigen detection, or nucleic acid amplification

What affects the time needed to detect a virus

virus type, inital viral titer (amount), and cell line used

How long does it take for viruses to produce CPE

• HSV: a few days

• Most viruses: up to one week

• Some never produce CPE

Which virus is difficult to grow in vitro

Hepatitis C virus (HCV), historically difficult to culture

Why is influenza difficult to detect using CPE

It often causes minimal host cell damage and little or no visible CPE

Hemagglutinin (H) protein

Influenza surface protein that binds red blood cells (RBCs)

Hemadsorption test

RBCs are added to infected cell cultures to detect viruses that cause RBC attachment

What indicates a positive hemadsorption test?

RBCs stick to infected cells or viral particles, suggesting influenza or a similar virus

Size of a Virus

very small (typically 20-300 nm, up to 1,000- 1,500 nm)

Characteristics of Viruses

• “Filterable virus” (small enough to pass through filter)

• Obligate intracellular parasites (needs an host to reproduce) (OIPs)

Why are viruses considered nonliving

Acellular (no cell structure), cannot self-replicate, and have no metabolism. However they can evolve

General Structure of Viruses

1. Helical

2. Capsid

3. Icosahedral

4. Complex

5. Envelope

Helical

capsid proteins arranged in a spiral around the viral genome

Example of helical

Tobacco mosaic virus

Icosahedral

prolyhedron (capsid) with 20 triangular sides (faces)

What does Icosahedral include

Capsomeres

Example of Icosahedral

Adenovirus

Complex

Structure that doesn’t fit a simple or icosahedral shape

Complex example

T4 bacteriophage, which has a head, tail, and tail fibers

bacteriophage

virus that infects bacteria, some have a complex structure

Nucleocapsid

Combination of

• Capsid

• Viral genome

Capsid

the protein coat that protects the viral genome

Viral genome

the genetic material which can be DNA or RNA

Which component are found in all viruses

• Viral genome

• Capsid (generally)

• Nucleocapsid (generally)

What is specific to certain viruses (not present in all of them)

• Viral enzymes

• Envelope

• Spikes

Viral enzymes

Help with replication or other processes

Envelope

Outer lipid membrane found in some viruses

Spikes

Help certain viruses attach to host cells

Components of the Envelope

Derived from host cell membrane: lipids, glycoproteins

Properties of Envelope

environmentally labile (meaning unstable, variable, or susceptible to changes)

What makes viral envelopes environmentally labile

• heat: can damage the envelope and viral proteins

• Drying (desiccation)

• Detergents and soap: break down the lipid membrane

• Alcohol and disinfectants: Can damage the envelope and inactivate the virus

• Extreme pH: can damage viral structures

Classification of Viruses

1. genome type

2. Number of strands in genome

3. Morphology

4. Enveloped vs naked virus

5. Baltimore classification

Genome type

A virus can have DNA or RNA

Number of stands in genome

a virus can be single-stranded (ss) or double-stranded (ds)

Morphology

Helical, icosahedral, complex

Baltimore classification

• mRNA synthesis

• 7 groups/ classes

Group 1

dsDNA, the mRNA is produced by DNA

Group 2

ssDNA, mRNA produced by ssDNA → dsDNA → mRNA

Group 3

dsRNA, mRNA produced: dsRNA -→ mRNA

Group 4

Positive-sense ssRNA, genome acts directly as mRNA

Group 5

Negative-sense ssRNA, must make complementary +RNA

Group 6

+ssRNA with reverse transcription, RNA → DNA → mRNA

Group 7

dsDNA with reverse transcription, DNA -→ RNA -→ mRNA during replication

steps of the lytic replication cycle of animal viruses

• attachment

• Penetration (entry)

• Uncoating

• Replication

• Assembly

• Release

Attachment absorption

• virus have adhesion molecules on their capsid or envelope

• These molecules recognize and bind to specific receptors on the host cell

Important about Attachment

No every virus can infect every cell because different viruses recognize different receptors.

Penetration (Entry)

Once the virus attaches, it must enter the host cell, there are 3 ways this can happen

Direct penetration/ injection

the virus introduces its genetic material into the host cell. Usually naked viruses, and bacteriophages

Membrane fusion

An enveloped virus’s lipid envelope fuses with the host cell membrane, allowing the viral contents to enter the cell. Requires the virus to have an envelope

Endocytosis

The host cell membrane surrounds the virus and brings it inside in a vesicle. Both enveloped and naked viruses can use this

Which viruses have a generally higher environmental stability

Naked viruses

Better resistance to drying

Naked viruses

Higher sensitivity to detergents

Enveloped viruses

Uncoating

Viral capsid (protein coat) is removed

Replication of viral genome and synthesis of viral proteins

• Early proteins - non-structural

• Late proteins structural

Assembly (Maturation)

• Capsid proteins aggregate

• Nucleocapsid forms

Release of mature virions

• Lysis - naked viruses

• Budding or exocytosis - enveloped viruses

Budding

A process where newly formed enveloped viruses leave the host cell by pushing through its membrane and acquiring a lipid envelope

Where does influenza gets its envelope

from the host cell membrane during budding

Viral budding

Enveloped viruses exit through host cell membranes, acquiring a lipid envelope

Influenza virus budding

Buds from the plasma membrane acquiring its lipid envelope from the host cell

Herpesvirus budding

Initially buds through the nuclear membrane, acquiring a temporary envelope

How does herpesvirus obtain its final envelope

loses its temporary nuclear envelope and gains a final envelope from internal membranes

Budding vs. lysis

Budding releases viruses without immediately rupturing the cell; lysis breaks the cell open

Effects of Viral Infection on Animal Cells

1. Cytopathic effect (CPE)

2. Lysogeny or Episomes

3. Oncoviruses

Vacuolation (vacuolization)

Formation of fluid-filled spaces (vacuoles) in the cytoplasm of infected cells

How does vacuolation appear under a microscope

Clear, bubble-like spaces inside infected cells; a possible sign of viral CPE

Inclusion bodies

Abnormal structures in infected cells, often containing viral proteins or particles; a type of CPE

Negri bodies

Inclusion bodies found in the cytoplasm of neurons infected with rabies virus

Why are Negri bodies important

They are a characteristic microscopic sign of rabies infection

Syncytium (cell fusion)

When multiple infected and neighboring cells fuse, forming one large cell with multiple nuclei

Which virus commonly causes syncytium formation

Respiratory syncytial virus (RSV)

Why is syncytium formation considered a CPE

It changes normal cell structure by creating large, multinucleated cells

Cell swelling and rounding (CPE)

Inected cells become enlarged and rounded instead of maintaining their normal shape

Cell clumping (CPE)

Virus-infected cells group together, forming visible clusters

Ghost cells

Dead or severely damaged cells that appear pale or empty under a microscope

Cell destruction (lysis)

Infected cells rupture and die, often releasing newly formed viruses

Viral latency

Viral genome persists in host cells with little or no production of new viruses

Provirus vs. episome

• Provirus: viral DNA integrated into host DNA.

• Episome: viral DNA maintained separately

Recurrent viral infection

A latent virus reactivates and begins producing new viruses, causing infection to return

Genetic transformation by viruses

Virus-induced changes in host cells that can contribute to uncontrolled growth and cancer

Oncoviruses

Viruses capable of contributing to cancer. Examples: HPV, EBV, HBV, and HCV