Virus/Host

What is a virus

1. Extremely small (20-400 nm)

2. Nonliving

3. Acellular

4. Obligate intracellular pathogens

Compare and contrast viruses and bacteria

Viruses:

• Acellular

• No “cells”

• Nonliving

• Hijack their host’s cell system to replicate themselves

• DO NOT exhibit metabolism

• Made of RNA

• Filterable due to size

• Has protein capsid coating and nucleic acid (bacteria have cells w/o nuclei or membrane-bound organelles)

• Virus can be composed of both DNA and RNA (bacteria w/DNA)

Describe a viral structure

A virion is a single, infectious virus particle (virus in the singular)

• Has a capsid (protective exterior protein)

• Has genetic material (DNA or RNA)

• SOME have outer lipid envelope

• Has spikes (protruding from capsid or outer lipid envelope)

Helical capsid

Looks like a hollow tube

Icosahedral capsid

Looks like a 3-D polygon

Complex capsid

Deviation from the other 2 possible structures of capsids (helical or icosahedral)

List 4 characteristics or features that are used to name and classify viruses?

1. Type of nucleic acid present (RNA or DNA)

2. Capsid symmetry (helical, icosahedral, or complex)

3. Presence or absence of an envelope (enveloped or naked)

4. Genome architecture (ssDNA, ssRNA, etc.)

Why are bacteriophages medically important

They do not infect humans, but they can serve as a means for bacteria to evolve.

Bacteriophages facilitate transduction, which enables bacteria to develop new genetic combinations despite their inability to reproduce sexually

Describe the Lytic and Lysogenic cycles of bacteriophages

Lytic pathway: Replication of bacteriophages (bacterial viruses) that kill the host cells

Lysogenic pathway: Replication of temperate phages, a type of bacteriophage that has lambda

Attachment phase (adsorption)

A phage binds to bacteria

• Bacteriophage tail fibers help the virus adhere to specific proteins on the bacterial cell wall surface.

Release phase

Phage encodes and releases lysozyme to break host cell walls (causing bacterial lysis) once phages are mature

Assembly (maturation) phase

Phage parts are replicated

• Genome is packed into capsid

• Phage parts are assembled to complete virions

Replication phase (synthesis)

Phage commandeers host cell factors to transcribe and translate viral genes

• DNAases (DNA-degrading enzymes) are formed

• Proteins to build new phage particles

Penetration phase

Phage injects its genome into the host

• Empty capsid remains outside the cell

Lytic pathway steps

1. Attachment

2. Penetration

3. Replication (synthesis)

4. Assembly (maturation)

5. Release

Lysogenic pathway steps

1. Attachment

2. Penetration → phophage or lysogenic replication occurs

3. Replication

4. Assembly

5. Release

What happens during lysogenic replication

1. Phage DNA integrates into the host genome, forming a prophage

2. As the bacterial cell divides, it copies its own genome and the prophage.

3. The phage may enter the lytic pathway if the host is stressed, called the “abandon ship” approach.

   • This allows for bacteriophages to replicate and find a new host in case their current host dies

Describe a prophage?

The combination of bacteriophage genomes with that of their host cell genome.

• Medically important because prophages allow bacterial cells to gain new pathogenic properties, a process called phage conversion (e.g., toxins)

What about a provirus?

Prophages that gave bacteria the ability to make toxins

C. diphtheria→ causes diphtheria toxins

C. Botulinum → causes botulinum toxins

Compare bacteriophage and animal viral multiplication

They both have the same 5 steps, but animal viral multiplication has “uncoating” as an additional step

Attachment of animal viral multiplication

1. A naked virus attaches to a host cell’s membranes through capsid proteins

2. Enveloped viruses (and naked viruses) bind via spikes

• Knowing this makes the attachment proteins of the virus a target for drug therapy to limit or prevent infection

What drug blocks HIV’s attachment to host cell proteins

Maraviroc (Selzentry)

Penetration of animal viral multiplication

Naked and enveloped viruses enter via Endocytosis or membrane fusion.

• Membrane fusion→ Viral envelope blend w/cell’s plasma membrane

• Endocytosis →Virus binds to host cell surface receptors, triggering uptake of virus into vesicles

Uncoating of animal viral multiplication

Animal virus capsids DO enter the host cells and are then entirely or partially broken down, releasing the genome.

• A virus that entered via membrane fusion may have its capsid degraded in the cell cytoplasm,

• A virus entering via endocytosis may have its capsid digested away by enzymes within the vesicle

• Poliovirus is an exception and spits out its genome via a pore on the capsid and endocytic vesicle

• Viruses’ w/DNA may not let go of their genome until they have reached the nucleus

Replication (synthesis) of animal viral multiplication

The genome is replicated, and viral proteins are made

• Viruses’ w/DNA do replication and transcribe in the nucleus

• RNA replication and protein synthesis occur in the cytoplasm

What viruses are an exception and replicate their RNA within the nucleus during animal viral multiplication

• Orthomyxoviruses (which cause influenza)

• Retroviruses such as HIV

Assembly of animal viral multiplication

New virions are formed

• Capsids usually build around the genome

• Sometimes capsids even have the genome inserted before they are even finished or sealed

Release of animal viral multiplication

1. Enveloped virus: Before it is enveloped, viral proteins are embedded in the host cell plasma membrane, and virions usually take a portion of the cell’s plasma membrane, enriched with the viral surface proteins, making it enveloped.

2. Naked virus: Naked viruses rupture the host cell during release, usually killing the cell

Acute infections

Viruses infect cells and reproduce immediately. Usually cleared by host immune system

• E.g. common cold and influenza

Persistent infections

Viruses have replication strategies that allow them to avoid immune system clearance

• Chronic (e.g. HIV)

  • Continuous release of virions over time and slow progression of the disease

• Latent (E.g. Herpes and Cancer)

  • Flare-ups w/intermittent periods of dormancy

  • During flare-ups, virions are shed, and the person experiences symptoms

  • Flare-ups can be triggered by stress (fever, sunburn, hormones)

Human papilloma viruses (HPVs)

Integrates w/host genome

Has a DNA viral genome

Uncontrolled cell division

• Cervical, oropharyngeal, anal, and vaginal/penile cancer

Human herpes virus-8

DOESNT integrate w/host genome

Has DNA viral genome

Uncontrolled cell division

• Kaposis sarcoma

Epstein-Barr virus

DOESNT integrate w/host genome

Has DNA viral genome

Uncontrolled cell division

• B cell and T cell lymphomas and Hodgkin’s disease

Human T lymphotropic viruses (HTLVs)

Integrates w/host genome

Has RNA viral genome

Uncontrolled cell division

• Adult T-cell leukemia

Hepatitis B virus

DOESNT integrate w/host genome

Has a DNA viral genome

Chronic inflammation triggers host cell DNA damage and mutations

• Liver cancer

• Hepadnaviridae family

Hepatitis C virus

DOESNT integrate w/host genome

Has RNA viral genome

Chronic inflammation triggers host cell DNA damage and mutations

• Liver cancer

• Flaviviridae family

How does oncogenic viruses cause cancer

They stimulate uncontrolled host cell division and decrease host cell responsiveness to death signals

• HPVs and HLTVs are some examples

Briefly describe the replication of HIV virus

1. Attachment → HIV attaches to a host cell and enters by fusion

2. Uncoating

3. Reverse transcription → HIV genome is reverse transcribed by viral reverse transcriptase to make DNA

4. Viral integration → DNA version of the virus integrates into the host cell’s genome to make a provirus

5. Replication → Provirus is transcribed to make viral genome and translated to make viral proteins

6. Assembly

7. Release → HIV provirus directs production of new virions, which bud from the host cell

Herpesviridae family

HSV-1 → cold sores

HSV-2 → genital herpes

HHV-3 → Chickenpox and shingles

What is reverse transcriptase?

An enzyme used in HIV replication to transcribe the RNA genome of HIV into DNA to be read by the nucleus of the host cell and translated into viral genome again for multiplication of the virus

How are viruses cultured?

Bacteriophages use the plaque assay technique

Plaque assay

1. initial virus stock is diluted sequentially

2. A portion of the diluted samples is mixed w/bacteria and melted agar and poured into a Petri dish

3. Clear zones or plaques form where viruses kill host cells

4. After incubation, plaques are counted, and the initial viral titer is calculated and presented as PFU/ml

   1. Viral titer→ Quantity of virus present in a given volume sample. Determines how much of a given virus is present

   2. PFU →Quantity of bacteriophages in an initial volume of sample that will form a plaque

HeLa cells – what is the historical importance of these cells?

Cancer cells derived from a patient named Henrietta Lacks are among the most common commercially available human cells used in research today.

• They may not always support the growth of a virus bc they get used to being outside of the animal they come from and lose the ability to make specific surface factors that the virus requires for attachment

• Primary cell lined like white blood cells can instead be used along w/embryonated eggs

Can you use embryonated eggs to grow viruses

Embryonated eggs are useful for growing certain viruses.

• Once the egg is injected w/viruses, the egg is incubated for viral replication. The injection site depends on which virus is grown.

• Once the virus is grown, they are purified from the eggs

• A minuscule amount of egg protein may remain and is the reason why patients are asked about egg allergies

Differentiate between Bacteriophages and animal viruses in how they are cultured.

Animal viruses are grown using tissue culture techniques

• Live hosts may be required to support growth of some viruses

• Embryonated eggs (fertilized eggs) are also useful for growing animal viruses

Cell tissue culture

1. A tissue is treated w/enzymes to separate the cells

2. Cells are suspended in culture medium

3. Normal cells grown in a monolayer across the glass/plastic container. Transformed cells or continuous cell cultures do not grow in a monolayer

What is a plaque forming unit?

The quantity of bacteriophages (viruses) in an initial volume of sample that eventually form a plaque

List and explain 3 diagnostic tests used to identify viruses

• Agglutination tests

• ELISAs

• Latex agglutination test

What are agglutination tests

• Purified antibodies linked to tiny latex beads (coated w/viral proteins)

  • Specific antibodies are used to coat latex beads

  • These beads are exposed to a sample containing antigens

  • If beads clump (agglutinate), then it indicates that the antigens bound to the antibodies

  • Used to screen bodily fluids like urine, cerebrospinal fluid, blood for viruses, etc

Latex agglutination test

• Viral antigens/proteins are linked to tiny latex beads

• Mixed with the sample that has antibodies

• If the patient has antibodies that bind to the viral antigen, then the beads will agglutinate when exposed to the patient’s serum

ELISA

Can detect either antigens or antibodies in a sample

• Instead of clumping beads, ELISAs adhere the antigen or antibody to A SURFACE and usually change color if there is binding

  • Use of polyclonal antibodies (primary and secondary)

  • Antibodies bind to antigen

  • Enzyme-linked antibody interacts w/substrate

  • Change of color indicates binding

Explain the difference between specificity and sensitivity with regards to diagnostic tests.

Specificity → Identifies only the virus of interest (no false positives)

Sensitivity → Test detects the lowest levels of the target (no false negatives)

• Sometimes antibodies bind to viral antigens as well

List 4 DNA oncogenic viruses and 1 RNA oncogenic viruses

• HPVs, Human herpes virus-8, epstein-barr virus, and hepatitis B have DNA

• HTLVs + Hep C have RNA

Limitations to agglutination tests and ELISA

• The sample must be a liquid, and the antigens being detected must be fairly well characterized to ensure detection specificity

• IF there is an antigenic shift, it may no longer be detectable

• Tests are futile during early infections because it takes a couple of weeks for antibodies to develop, a time period called seroconversion window

Mechanism of action of selected antiviral drugs

• Blocked attachment

• Blocked penetration

• Blocked Uncoating

• Blocked replication

• Blocked release

• Several antiviral drugs prevent viral entry into cells by blocking the above

Antisense antivirals

Short sequences of nucleotides complementary to the viral DNA

• Bind to viral RNA, inhibiting translation

• Targeted RNA is destroyed by cellular enzymes

• Vitravene

What is a Prion?

Infectious proteins w/no genetic materials

• DO NOT replicate

• Cause transmissible spongiform encephalopathies (TSEs)

How does a Prion cause a disease?

Spongiform encephalopathy can be inherited or acquired by

• Gerstmann-Sträussler-Scheinker syndrome

• Fatal familial insomnia

  • Both inherited

• Creuzfeldt-Jakob disease (CJD)

List human and animal diseases associated with prions.

Human:

• CJD

• Gestmann-Strassler-Schenker syndrome

• Fatal familial insomnia

• Kuru

Animal:

• Scrapie

• Transmissible mink encephalopathy

• Chronic wasting disease

• Bovine spongiform encephalopathy