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1.1 Viro Intro

- Virus outbreaks present a major challenge to public health, ongoing viral diseases in society

- Use virus therapeutically: treatment for melanoma, CRISPR - HPV vaccine in Australia- significant drops in cervical cancer

Contact: tim.newsome@sydney.edu.au Discuss course content: piazza Assessment:

- prepare for pracs quiz: due 5pm day before - in class prac assessments - presentation - prac assessment - exam - need to attend all pracs (can’t miss more than 2)

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1.2 Intro to Viruses

Learning outcomes:

• Describe theories of the association of viruses with cellular life and their co-evolutionary history

• Describe the history of virology and landmark discoveries in the field • Define the key features and diversity of viruses that are unique within biology

Viruses are abundant, rapidly expanding field

- viruses are abundant in our ecosphere - large reservoir of genetic diversity - new sequencing technologies are rapidly expanding our knowledge of virus diversity.

Causing a shift in perspective from disease agents to broader impact on ecosystems - challenges in interpreting metagenomic sequence data include ascribing functionality

and matching newly discovered virus species with hosts - in comparison, bacterial genes are much more highly conserved: homology to known

genomes. Viruses drive cellular turnover

- estimated 10^23 stars, 10^30 viruses in ocean - viruses kill 20-40% of marine microbes every day, driving cellular turnover and carbon

depositions RNA world hypothesis Cellular life as we know it comes from RNA

- RNA found abundantly in all living cells today - RNA chains can replicate, evolve and interact with their environment, easily observed by

scientists - Random chains of RNA formed in hot springs – proteins and cells - GC’s and AU’s base pair on templates forming complementary strands - Mutations and evolution: Folded chain of RNA can make ribozymes, help their survival - RNA can catalyse reactions and store genetic information, whereas DNA and enzymes

not great at doing both

Virus and DNA: Virus First Hypothesis - Multiple competing RNA-based cell like organisms - One form adapted to a parasitic lifestyle - Host and parasite balance between cellular defence and invasion mechanisms - Host cell expresses enzymes that attack foreign nucleic acids - Restriction enzymes cut up/degrade viral phage genome

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- DNA may have been an invention of viruses to protect from the restriction enzymes degrading genetic material

- RNA virus precursor may have evolved DNA to protect its genome against the cellular defence system of RNA cells. DNA has enhanced chemical stability and offers many advantages over RNA as a genetic molecule.

Viral origin of the nucleus

- Recent obervation of assembly of a nucleus-like structure during viral replication in a bacterial cell

- The cell nucleus may have originated from a DNA virus that evolved and gradually took over cellular functions

- Conceptually similar as to how mitochondria and chloroplasts are thought to have origins as bacterial endosymbionts

Evidence for the antiquity of viruses

- Difficult as viruses don’t leave fossils, nucleic acids break down - Estimate divergence of related species based on molecular clocks and mutation

frequencies - Scenario 1: long association between virus and host, potentially following speciation

events. Typically milder pathogeneses or even asymptomatic. E.g herpes maintained in small isolated communities (tribes etc)

- Scenario 2: recent association between virus and host (sporadic zoonoses) typically virulent pathogeneses. Need much larger, interconnected population to maintain virus.

- Viral footprints in genomes: “Endogenous Viral Elements” Some viruses integrate into host chromosomes during replication. Roughly 5% of our DNA is believed to be attributed to retroviruses, mostly integrated into non-coding DNA. EVE’s impact expression of genes, turn genes on, mobile within genome. - Circumstantial evidence for viruses Some mummies have smallpox-like scars. Also clubbed foot of mummy indicative of poliomyeltis. Smallpox genome from 17th century mummified child in Lithuania. Rembrandt tulips highly prized in 17th century, caused by tulip virus infection.

Foundations of Virology:

- van Leeuwenhoek observed bacteria and protists using handmade microscope - Jenner used cowpox for smallpox vaccine - Pasteur germ theory of disease: attentuated form of rabies confer protection against

rabies. From dried spinal cord of infected animals. - Koch developed postulates to determine microbiological causes of infectious disease

(anthrax, TB) - Chamberland (colleague of Pasteur) developed porcelain filter, separated bacteria from

infectious broth

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- Ivanovsky Tobacco Mosaic Virus could pass through porcelain filter: still infectious - Beijerinck TMV self replenishing: not a toxin because you can dilute, filter, reinfect in a

cycle. - Reed/Finlay showed this with Yellow Fever virus - Franklin discovered helical nature of the TMV

Characteristics of Viruses:

- acellular, undergo de novo replication, almost always encode capsid proteins - often v. small - parasites, replicate in living cellular hosts Viruses don’t have active ribosomes so can’t make progeny. Also energy parasites as they are inert, no metabolism: no mitochondria or chloroplasts. - genome RNA or DNA

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Capsid: Capsids are a hard, outer shell which are hollow and carry viral genetic information. A virus uses the capsid to spread its genetic information from one cell to another. Acellular Replication

- Viral progeny are freshly synthesised ‘de novo’ during replication whereas with cellular division involves the partitioning of parental genetic and organelles into daughter cells

- Capsid structures often assemble spontaneously in the cytoplasm of infected cells. They fold randomly, using the lowest energy state to form a stable viral particle.

- Some mycoviruses (viruses which infect fungi) do not encode capsid proteins and spread via spores or anastomosis (touching to form channel)

Viruses are often small - recent discovery of Jumbo viruses e.g. Pandoravirus, larger than some tiny bacteria - there are also some virophage which infect viruses which are in a host cell

Viruses are intracellular parasites, some have a mutualistic relationship

- Viruses are parasites of host energy and translational machinery - Tropical panic grass with fungus infected with thermal tolerance virus means this grass

can grow in extremely hot temps. Some undergo extracellular morphogenesis

- example ATV virus at high temps will uncoil and change shape

Viruses vs Bacteria

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2.1 Classification of Viruses Learning Outcomes:

- Apply the classification schemes of Lwoff, Horne and Tournier, and Baltimore. - Classify infectious agents based on their physical properties and mode of replication to

categories such as viruses and subviral agents (satellite viruses, satellite nucleic acids and viroids).

- Define key terms used in virology Are viruses alive?

- not really but can fit into some definitions, e.g. “autonomous system with open-ended evolutionary capacity”

- not included in the tree of life, which share a single celled common ancestor - unsure of viruses’ origins at this stage

Characteristics:

- acellular, de novo replication - often small - parasites of host translation machinery and energy pathways

Challenges for comparing viruses based on molecular methodologies

- viruses often morphologically similar, despite different genomes - challenging to compare viruses. For example rna viruses mutate faster than dna viruses,

have to use different molecular clocks - viruses can be transmitted in different ways: horizontally, vertically - potentially multiple origins, unclear when groups diverged and how often - recombination and reassortment occurs at a v high frequency

Virion The mature, infectious form of a virus particle, cell free (released by an infected cell). Herpes virus

- that certain equine and human herpes viruses are more closely related than other human herpes viruses suggests a long association of viruses with their respective host, perhaps before divergence of human and horses

Mimivirus - over half of the Mimivirus genome has a eukaryotic origin

Varied practice in virus nomenclature

- associated disease (rabies) - replication (sputnik for satellite virus) - affected site of body (hepatitis due to liver inflammation) - site of discovery (Hendra)

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- discoverer (Epstein-Barr) Lwoff, Horne and Tournier classification scheme • Nature of the nucleic acid in the virion (DNA or RNA) • Symmetry of the protein shell • Presence or absence of the lipid membrane • Dimensions of the virion and capsid Used the Linnaean system of kingdom, phylum, class, order, family, genus, species Baltimore classification scheme Advances in molecular biology: DNA RNA Protein

- form of nucleic acid in the genome

- the relationship between the viral genome and the production of mrna (and therefore

protein)

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Baltimore Classes:

• form of nucleic acid the genome

• Pathway to make mRNA I dsDNA II ssDNA III dsRNA IV +ssRNA V -ssRNA VI ssRNA-RT VII dsDNA-RT

• Identified six classes (I-VI) and class VII added in the 1980s with the discovery of dsDNA-

RT viruses (Hepadnaviruses)

• arbitrary groups, not based on relatedness

• not adopted by plant or bacterial virologists Within Baltimore groups, genomic sequence comparison can be used to refine relationships between different virus isolates. Eg. a new variola virus isolate was identified as ancestral to all known strains.

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Viruses infecting Humans: variety, DNA and RNA Viruses infecting Bacteria: mostly DNA genome, no retroviruses Viruses infecting Plants: mostly RNA genome

Satellite viruses: Encodes genes, needs infection of Helper Virus in order to replicate. Small ss RNA molecules that do not encode genes for replication and replicate in the presence of a helper virus. So the Hepatitis B ‘Helper’ virus can infect a cell, then a Hepatitis D virus may come in and infect the cell, but it would not be able to do so without the HBV.

Virophage: Consensus not yet established whether virophage are a new category or part of the satellite viruses. Small virus that infects amoeba which is infected with Mimivirus. May be different to satellite virus as replication occurs within the replication centre of the giant Mimivirus, unlike the HDV. Interestingly the virophage attenuates the Mimivirus. Satellite nucleic acids: Similar to satellite viruses however no ORF’s: they do not encode genes. But they do replicate in the presence of the Helper virus. Viroids: RNA molecules: they do not encode genes (like satellite nucleic acids), but are not associated with a helper virus. Replicated by the host RNA polymerase II: usually would express plant genes from the plant genome, for viroids uses RNA template. Parasites of the host transcription machinery. Spread mechanically (through shears etc), through seeds, are not encapsidated.

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Unusual sub-viral infective agents: Prions Not viruses Infectious, protein-only disease agents (no nucleic acid genome) Cause Transmissible Spongiform Encephalopathies: fatal, rare, neurodegenerative diseases in humans and animal species.

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2.2 Structural Virology

Learning outcomes:

• Discuss the pros and cons of different imaging techniques used to image viruses. • Classify viruses according to the symmetry of their protein coat. • Describe the basic aspects of icosahedral symmetry and how protein subunits come

together to form icosahedral capsids. • Define the structural characteristics of virus particles and their essential features.

- Viruses are very small, to resolve structures requires electron microscopy, X ray crystallography rather than light microscopy

- Resolution is the ability to separate two objects at a certain distance and depends on a number of constraints such as:

- contrast, enhance by phase microscopy, dark field microscopy, staining - diffraction, dependent on wavelength (visible light, electrons, x rays smallest)

Light microscopy:

- lacks resolving power to discern structural features of virus particles - the picture above shows a Vaccinia virus (pox) which is relatively large - maintains tissue architecture, non invasive: live cell analysis - applications: study cytopathic effect and staining protocols (immunofluorescence

analysis) - super-resolution light microscopy: new techniques allow imaging at resoltions beyond

the diffraction limit: look at localisation of virus particles and stages of maturation Electron microscopy:

- EM uses beams of electrons instead of light, which are focused using electromagnetic lenses rather than glass ones

- Common uses include transmission EM, cryo EM, scanning EM - Biological samples have low contrast using EM, heavy metal dyes are used to resolve

virus structures.

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Transmission EM: Samples of viruses/virally infected cells are stained with heavy metal dyes to resolve virus structures and other viral compartments in infected cells (replication centres). Samples can be embedded in resin and then thing slices can be imaged.

- amenable with antibody labelling - antibody complexes are localised through the attachment of gold particles, which can

be multiplexed with different sized particles

- also use scanning electron micrograph, not great for structural resolution

- additionally, negative stain EM for resolving virions (cell-free virus) - heavy metal dye coated on surface, virus overlaid on the thin film of dye which is then

displaced, or the dye is overlaid onto a surface coated with virus - quite destructive to specimens (the adenovirus below usually has the little filaments

attached) but we can see structural resolutions.