Virology

What prevents society (e.g., the scientific community) from achieving thesegoals/fulfilling these needs and acquiring an adequate knowledge base?

A lack of sufficient and interpretable data

Where are samples collected from?

Samples are collected from: soil, freshwater (e.g., lakes, rivers), saltwater (e.g., oceans, saltwater ponds), geothermal springs/pools/vapor, air, plant tissue, animal tissue, animal discharges (e.g., feces, saliva)

Sample Processing: Enrichment of Mixed Cultures (Liquid Culture)

Seed Cultures - different media Upscale Mixed Cultures - for plating on solid media Seed Culture - for upscale Seed Culture - contaminated Pure Culture - for single colony isolate

Sample storage and transportation

(1) Samples should be stored under conditions which mimic the natural environment (2) Sample storage and transport may require: buffering, RNA Later, rezasurin, VTM (3) Some sample processing may occur on-site, if possible to enhance lab-based culturing (4) Biosafety should be a consideration prior to and post-sampling

Koch's Postulates

1) Pathogen must be present in all disease cases 2) Isolate pathogen, cultivate in pure culture 3) Inoculate into susceptible animal, initiate disease symptoms 4) Re-isolate pathogen, confirm it's the same pathogen

Lytic cycle

1) Phage attaches to host cell and injects DNA 2. Phage DNA circulates and enters lytic cycle 3. New phage DNA and proteins are synthesized and assembled into virions 4. Cell lyses and release virions

Viral Protein and Genome Production

1. All viral RNAs must be translated by the host's cytoplasmic protein synthesis machinery in order to produce viral proteins 2. Viral infection often results in the modification of the host's translational apparatus so that viral mRNAs are selectively translated 3. Viral genomes are often copied by the cell's synthetic machinery in cooperation with viral proteins 4. Internal cellular compartmentalization must be managed; the cell provides the nucleotide substrates, energy, enzymes, and other proteins

Viral RNA Production

1. All viruses must copy genomes for packaging into progeny virus particles and must produce mRNA for synthesis of viral proteins. 2. RNA viruses all package their own polymerase to catalyze the synthesis of viral mRNAs and to copy the RNA genome 3. RNA viruses that employ reverse transcription package a polymerase that catalyzes the production of viral DNA from the packaged viral RNA template and then commandeers the host cell RNA polymerase (e.g., RNA polymerase II) to generate RNA genome copies and mRNA for translation of viral proteins 3. DNA viruses typically use the host cell's RNA polymerase for producing viral mRNA and viral DNA is produced by commandeering the host cell's DNA polymerase unless a special polymerase is packaged

Four fundamental bases for classifying viruses

1. DNA or RNA 2. Symmetry of the protein shell 3. Presence or absence of a lipid membrane 4. Dimensions of the virion or capsid

Integral Membrane Proteins

1. Either a single or multiple membrane spanning domains 2. have an extracellular domain, sometimes consisting of a receptor 3. Integral membrane proteins with multiple membrane spanning domains often form complexes that function to internalize nutrients, expel waste products, or move ions and water to maintain homeostasis

Properties of Environmental Samples

1. Heterogeneous populations of cells and viruses 2. May contain DNA viruses or RNA viruses or both 3. Label all sampling data (date, location, environment/species, pH, temperature, salinity, [O2]) 4. May need to repeat sampling from same location during different seasons

Virus Replication Cycle (Attachment & Entry)

1. Interactions between virus and host occur by chance in nature; greater the pop. of virus particles, the greater the chance for infection 2. Both promiscuous and highly specific virus-host interactions can occur via a viral protein 3. Receptors that are specific for select viruses, in part, determine a cell's susceptibility to virus infection. 4. An infected cell must also be permissive, amenable to viral replication 5. Successful infection depends upon a potential host being both susceptible and permissive. 6. Successful infection depends upon the ability for the virus to cross the plasma membrane and, in some cases, the nuclear membrane 7. Viruses often must disassemble so that the viral genome is accessible for trafficking to the correct cellular compartment for replication

Lysogenic cycle

1. Phage attaches to host cell and injects DNA 2. Phage DNA circulates and enters lysogenic cycle 3. Phage DNA integrated within bacterial chromosome by recombination to become a prophage 4. Lysogen bacterium reproduced normally 5. Prophage may excise and initiate lytic cycle

Origins of viruses hypotheses (3)

1. Reduction Hypothesis (Regressive Hypothesis or Degeneracy Hypothesis): contends viruses are reduced cells that once parasitized larger cells 2. Escape Hypothesis (Vagrancy Hypothesis): viruses evolved from bits of DNA or RNA that "escaped" from the genes of a larger organisms and may be of plasmid origin or transposon (jumping genes) origin 3. Coevolution Hypothesis (Virus-First Hypothesis): viruses may have evolved from complex molecules of protein and nucleic acid at the same time cells appeared on Earth.

Surface Proteins

1. Surface proteins can be either inner surface or outer surface proteins; 2. Do not span the lipid bilayer but instead are covalently bound via hydrocarbon chains to either integral membrane proteins or to charged sugars on glycolipids of either the inner leaflet or outer leaflet.

Viral Particle Assembly and Release

1. Various components of progeny virions may be synthesized in distinct cellular compartments so they need signals or tags to reach the virus particle assembly site 2. Primary sequences of viral proteins contain sufficient information to specify assembly thus 'self-assembly' is often used to describe viral assembly 3. Viral Pathogenesis is the process by which viruses cause disease in hosts. 4. The nature of viral disease depends upon the effects of viral replication on host cells, the responses of the host's defense systems, and the ability of the virus to spread in and among hosts

Three fundamental properties of all viruses

1. Viral genome packed into inside particles that mediate host-to-host transmission 2. Viral genome contains all information required for starting and completing an infection cycle in a susceptible and permissible host. 3. Infectious cycle includes attachment, entry, decoding of genome information, translation of viral mRNA by host ribosomes, genome replication, assembly of new virus particles, and release of virions. 4. Can invade the host for virus survival

Fundamental Properties of Viruses

All viral genomes are packaged inside particles that mediate their transmission from host to host The viral genome contains information for initiating and completing an infectious cycle within a susceptible, permissive cell An infectious cycle includes attachment, entry, decoding of genome information, translation of viral mRNA by host ribosomes, genome replication, assembly of new virus particles, and release of virions All successful viruses are able to establish themselves in a host population so that virus survival is ensured

Virus Propagation in Culture

Cell culture is the preferred method for propagating viruses; liquid Culture of blood cells or lymphocytes may also serve as a culture method Cytopathic effects may be used to monitor the progress of an infection and as a phenotypic indicator of mutant viruses

Why is virology important?

Ecosystem health, human health, food security, water quality, bioweapons protection, economic security

Virus Taxonomy and Classification

Order: (-virales) Family (-viridae) Subfamily (-virinae) Genus (-virus) Species (-virus) There are 96 families, 22 subfamilies, 420 genera, ~2600 species, and ~3000 remain unclassified

Sample Processing: Enrichment of Mixed Cultures (Liquid Culture)

Spot-On-Lawn (detection/phenotype) Colony Type (detection/phenotype) Streak Plate (single-colony isolate) Lawn (infection assay) Colony Growth (pure culture development)

Methods for Detecting Viral Infection and Viruses

TEM (visual) cryoEM (analytical/visual) SEM (analytical/physiological) growth assays/one-step growth curves (analytical/liquid culture) plaque assays (analytical/physiological) ESI/MS (analytical/physical) PCR/qPCR (analytical/genetic) Anti-body/ELISA (analytical/immunological) Fluorescence (analytical/immunological/dye-based) infection assays: LD50, TCID50 (analytical/physiological)

What is needed most in virology and virus ecology?

The ability to understand virus systems such that accurate forecasting can be done

What can ultra filtrate contain?

Ultrafiltrate from 0.22 mm filtration may also contain some larger eukaryotic viruses

VTM vs. Tris-HCL vs. HEPES

VTM: Tris-HCl: buffering agent (acidic buffer) commonly used by molecular biologists to adjust the pH of a solution or stabilize the pH HEPES:

Virus titer

Virus titer is either an underestimation or an over-estimation

infectious cycle

attachment and entry, translation, genome replication, assembly, release

Hershey-Chase Experiment

confirmed that DNA is the genetic material because only radiolabeled DNA could be found in bacteriophage-infected bacteria

Viral genome types:

dsDNA: Adenoviruses, Herpesviruses, and Poxviruses ssDNA(+): Parvoviruses dsRNA: Reoviruses ssRNA (+): Picornaviruses, Togaviruses ssRNA(-): Orthomyxoviruses ssRNA-RT(+): Retroviruses dsDNA-RT: Hepadnaviruses