Lecture 4 - The Organisms - Viruses

What are viruses?

• Molecular parasites

  • Infect all biota

  • Not metabolism of their own

• Filterable agents

  • 20-200nm in diameter or 2-60X smaller than gram+ bacteria

• Differ based on composition, morphology, genome organization

What are the components of conventional viruses?

• Nucleic acid genome

  • DNA or RNA; single or double stranded; contagious or segmented

  • Codes all virus-specific macromolecules

• Capsid

  • Protein shell; proteins self assemble; icosahedral, helical, or other symmetry; packages, protects and delivers the genome

• Envelope

  • Some are surrounded by a lipid envelop derived from the host cell; membranes contain virus-derived glycoproteins (spikes) have a role in viral tropism

What are the 3 virus shapes?

• Helical

• Polyhedral

• Complex

What are the 8 steps in viral infection of a eukaryotic cell?

1. Virion adsorption to the cell surface

• Cell receptors or co-receptor proteins; this interaction determines tropism, neutralizing antibodies produced by the immune system block this interaction

2. Viral penetration of the cell surface

• Membrane fusion or endocytosis

3. Uncoating of the viral genome

• pH chnages in endocytosis

• Capsid is degraded

4. Primary Transcription (gene expression)

• Many viruses start expression right after entry by using a viral polymerases or string transcription activators carried with them

5. Replication of Viral Genome

• Viral proteins - targets for antiviral development

6. Secondary Transcription

• Expression of progeny genomes, early and late proteins

7. Packaging of progeny genomes

• Capsid proteins self-assemble, and the nucleic acid is inserted

8. Release of progeny virons

• Enveloped viruses acquire lipid bilayer

What is field or street isolate?

Isolate obtained directly from the natural host

What is co-infection of viruses?

• Infection by more than one virus

• For new viruses with segmented genomes this can lead to re-assortment and new biologic/antigenic properties

What are the virus mutation rates?

• DNA: 10^-8 to 10^-11 per base per generation

• RNA: 10^-3 to 10^-4 per base per generation (polymerases lacking proof reading)

What is phenotypic mixing or pseudotype formation?

Viruses switching coats

How can you control a viral infection?

Vaccination

• Eliminated smallpox and polio transmission

• Effective against SARS-CoV-2

• Historically reduced transmission of measles, mumps, and rubella

What are the different vaccine types and antiviral drugs?

• Vaccine

  • Killed, live-attenuated, subunit

  • RNA and DNA-based

• Antiviral

  • Influenza targets

  • Evolving field, historically targeted viral proteins

What are human infecting DNA viruses?

• Families based on morphology, genome, genome organization, gene expression, antigenic properties, host range, etc.

• Transcription in the nucleus of the host is completed by host RNA polymerases

• Transcription in the cytoplasm of the host is completed by viral RNA polymerases

• Usually dsDNA, but some ssDNA

What are some dsDNA virus families?

• Genome is a template for transcription

• Papovaviridae

  • Circular genome; icosahedral symmetry

• Poxviridae

  • Large enveloped viruses; linear genome; complex viral capsid symmetry

• Adenoviridae

  • Linear genome, icosahedral symmetry

• Herpesviridae

  • Linear genome, icosahedral symmetry

• Hepadnaviridae

  • Very small circular genome, partially ds, icosahedral symmetry; replicates using viral encoded reverse transcriptase

What are some ssDNA virus families?

• Must be converted to dsDNA before transcription and translation

• Parvoviridae

  • Small linear genomes; non-segmented; icosahedral symmetry

• Anelloviridae

  • Circular genome, non-segmented, icosahedral symmetry

What are RNA viruses?

• dsRNA

• ssRNA positive sense (+; mRNA sense)

• ssRNA negative sense (-; complementary to mRNA)

• Ambisense (both + and -)

• Segmented or non-segmented

What are segmented RNA viruses?

• RNA viruses that contain two or more unique segments of nucleic acid, negative-sense, ambisense or ds

• Rapid evolution due to re-assortment of genomic segments

What are positive-sense RNA viruses?

• 8 families that are fully infectious when delivered to the cell in the absence of viral proteins

• 3 subgroups based on gene expression

  • Single large polyprotein that is proteolytically cleaved

  • Two or more polyproteins encoded in the genome and subgenomic RNAs produced during infection

  • Production of numerous protein-coding subgenomic RNAs from a large genome

What are the ss +sense RNA virus families?

• Picornaviridae (icosahedral)

  • Small stable virus, most of which can survive severe conditions

  • Eg. Rhinovirus, Poliovirus

• Flavivridae (icosahedral)

  • Primarily spread by insect vectors

  • Eg. Dengue virus, West Nile virus, Hep C

• Togaviridae (icosahedral)

  • Anthropod-borne encephalitis

  • Eg. Rubella

• Astroviridae & Caliciviridae (icosahedral)

  • Icosahedral symmetry

  • Eg. Astroviruses

• Coronaviridae (helical)

  • Large genomes; helical symmetry

  • Common colds, upper and lower respiratory tract infections

• Arteriviridae (icosahedral)

  • Arterivirus caused vascular lesions, edema, fever, etc.

  • Eg. Equine arteritis virus

• Hepeviridae (icosahedral)

  • Eg. Hep E virus

What are the -sense RNA viruses?

• 6 families each containing viruses with helical nucleocapsids in an enveloped particle

• Gene expression governed by:

  • Viral polymerase initiates transcription only at one end of the genome

  • Transcription or replication determined by level of nucleocapsid protein

• Recombinant virus production possible

What are the ss -sense RNA virus families?

• Paramyxoviridae

  • Upper respiratory tract infections

  • Eg. Measles, Mumps

• Rhabdoviridae

  • Protracted time to cross the neuromuscular junction; therefore, it is the only virus for which post-exposure immunization is helpful

  • Eg. Rabies

• Filoviridae

  • Eg. Ebola

• Bornaviridae

  • Unique in that they replicate in the nucleus to take advantage of splicing machinery

  • Eg. Borna disease

• Orthomyxoviridae

  • Segmented genome

  • Antiviral block uncoating and neuraminidase activity

  • Eg. Influenza A/B/C/D

• Bunyaviridae

  • Eg. Hantavirus

What are the dsRNA virus families?

• Reo

  • ds genome

  • Eg. Rotavirus

• Retro

  • Two identical strands of +sense mRNA

  • During infection, RNA is converted to DNA by reverse transcriptase, which integrates into the cellular genome

  • Cellular DNA-dependent RNA polymerases are responsible for transcription

  • RNA → DNA → mRNA → protein

  • Eg. AIDS HIV

How do you isolate viruses?

• Sites for viral isolation

  • Obtain samples early and from multiple locations

  • Blood, skin, body fluid swabs, manifestations of infection

• Must be labile

  • Avoid extreme pH, direct sunlight and freezing temperatures to preserve the material, generally on ice, analyzed ASAP

• Cultured in animals, grow at various temperatures; observe morphological changes

• Some viruses cannot be grown in cell lines and may require injection into animals

What is the cytopathic effect (CPE)? 

• When a viral infection causes structural changes in a host cell

• Animal cells grow in a monolayer, and when viruses are cultured within these cells, CPE is possible

What does CPE look like?

Rounded and enlarged cells, cells growing in grape-like clusters, cell fusion and subsequent detachment from the substrate, formation of large multi-nucleated cells or cells with reduced nuclei and proliferation of membranes

What is electron microscopy (EM)?

• Uses a beam of electrons to enable magnification up to 1000000X to observe particle morphology

• Visualization can be enhanced by negative staining or ultrathin sectioning

What are the morphology for some common viruses?

• Rabies → Ribbion-like

• Measles → Rod-shaped

• Polio → Spherical

• Ebola → Filamentous

• Covid → Sphere with spikes

What are the 4 different types of serological assays?

• Neutralization Assay

• Hemagglutination Assay

• Immunostaining Assay

• Enzyme-linked Immunosorbent Assay (ELISA)

What are Neutralization Assays?

• Dilutions of neutralizing antibodies are mixed with the virus, and the mixture is assayed for infectivity

• Antibodies that recognized the virus bind to it and prevent cell infection

• Virus can be placed in serogroups depending upon which antibodies are effective in neutralizing the infection

What are Hemagglutination Assays?

• Erythrocyte suspension mixed with viruses and added to a V-bottom microtitre plate

• Single erythrocytes sink to the bottom forming a red dot, aggregated red blood cells uniformly coat the well

• Viruses that produce proteins that bing erythrocytes readily detected

What are Immunostaining Assays?

Polyclonal antisera and monoclonal antisera linked to a fluorescent dye or enzyme enable detection of interacting molecules by microscopy

What is ELISA?

• Most common

• Involves capturing virus antigens or virus specific antibodies on a solid surface then exposing these to a substrate that allows detection

• ELISAa specofoc for IgM and IgG allow for distiction of previous exposure from recent infection

• Increase in IgG antibodies 2x to 4x over 2 weeks indicated seroconversion, diagnostic of active primary infection

• ELISAs to screen populations for immunization efficacy or previous exposure to newly identified virus