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Notes for Virology Review (complete) ...

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Unit C: Virology Contents: Topic 21: Introduction to Virology Topic 22: Viral Infectious Topic 23: Viral Genomes Topic 24: Viral Structure Topic 25: Viral Entry Topic 26: Viral Pathogenesis Topic 27: HIV Pathogenesis Topic 28: Koala Retroviruses Topic 29: Virology Review

Topic 21: Introduction to Virology Lecture Objectives: 1. Impact for society 2. Where can we find viruses? 3. All viruses are not evil 4. Major concepts in virology 5. History of Virology 6. Seeing viruses Why is Virology important? - Pro-MED = Program for Monitoring Emerging Diseases o Promotes communication amongst the international infectious disease community o Ex) Dec. 30, 2019 – Wuhan Municipal Health Community issues urgent notice for treatment of pneumonia of unknown cause  Now a global pandemic Case Study: How my trip resulted in an outbreak (Measles) - Transmitted in aerosols – coughing & sneezing - Vaccination has made disease less prevalent in Canada - Still major problem for part of South America & Africa Analysis of Outbreak - Measles virus highly contagious - 94% of infected individuals unvaccinated - patients who were unvaccinated die to gear of adverse events from vaccine In Canada: - routine 1-dose MMR vaccine introduced (1983) - routine 2-dose MMR vaccine introduced (1996-97) H1N1 Influenza outbreak in Alberta (2013) - Influenza virus causing the flu (~10 deaths) - No age discrimination Ebola Virus Outbreak in western Africa (2014) - 3 western African countries afflicted w/ severe outbreak Zika Virus in Latin/South America - transmitted via placenta (pregnant women) o links to microcephaly (= developed mental delays) Where can we find Viruses? - Viruses are everywhere - 80-85% of all the globe infected w/ herpes virus for life (2 different types) - Viruses infect all living things – they are useless on their own unless they find a living host Our own genetic code contains viral genes (or elements) - Viral genetic sequences integrated into our own genetic material o ~10% of genetic material of viral origin - Ex) Endogenous retroviruses (such as HERVs) which are different from Exogenous retroviruses such as HIV o HERV = human endogenous retroviruses

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These endogenous viral sequences are remnants from infections that occurred millions of years ago but are no longer infectious

3 Levels of Defense Mechanisms

All Viruses are not Evil Example 1: Good relationship (virus & host) – Polydnaviruses, wasps, and caterpillars - Within wasp’s genome, the wasp expresses viral genes o Integrated viral gene sequences that originate from the polydnavirus - Wasps lay their eggs inside a living insect larva - When female wasp deposits eggs inside a caterpillar she also deposits her polydnavirus genome sequences - Innate immune system of larva would normal kill the egg, preventing its development o Wasp genes carried by the polydnavirus genome suppress this innate immune response - Symbiogenic Relationship = positive relationship between the virus and the wasp Example 2: A virus that helps a fungus - Dichanthelium lanuginosum (plant) grows in extreme temperatures o Resists over 50 C for plant to grow - Fungus Curvularia protuberate permits plant survival o However, fungus cannot grow at T>50 C - Fungal thermotolerance is mediated by the curvularia thermal tolerance virus (CThTV) o CThTV infects the fungus o Result in the fungus colonizing the plant for it to grow Example 3: A virus that makes nicer flowers - Tulipomania (first documented economic crisis) o Holland in the 1600s o Broken tulip (tuple w/ many colors) rare & expensive o Tulip market crashed in mid 1600s - A potyvirus (Tulip breaking virus – TBV) o Studies determined that TBV results in the pattern of a broken tulip  TBV interferes with the synthesis of pigments in the flowers

Major Themes in Virology  In order for viruses to survive they must: o Package their genome inside a particle o Use this particle to transfer their genome from host to host o Genome contains information to initiate and complete the viral infectious cycle o Genomes establish themselves in host ensuring long term viral survival Major Themes in Virology 2  Viral genomes are obligate molecular parasites o They can only function after they replicate in a host cell  Viruses must make mRNA that can be translated by host ribosomes o They use host protein synthesis machinery to make viral proteins  NO VIRUS CAN TRANSLATE PROTEINS FROM mRNA ON THEIR OWN  Thus, we study viral-host interactions to increase our knowledge of how the host functions Viruses are not smart (they cannot think) – they just replicate and make a lot of everything - N.B. Terminology: Viruses do not grow, they replicate - Creation of mutant & some of these mutants survive in host Darwin: Survival of the fittest - Recall: Viruses need host in order to survive - Q: What could happen if viruses always killed their hosts? o Most viruses don’t kill host b/c they need hosts to survive - Q: What would happen if the virus did not evade the host defense system? o There would not be any viruses Viruses in Time - Viruses existed in the dinosaur age o Viruses have most likely existed for over 250 million years o Molecular clock sequencing techniques have enabled such affirmations  Sequencing = establishing the order of nucleotides of a genome  Specific order of nucleotides allows us to define what that genome is  Gain a sense of where some viruses originated from Documented Beginning - Ancient Greeks: Described an ailment that they termed “Herpes” o Creeping/crawling lesions that appear on the skin - Ancient Romans: Emperor Tiberius banned kissing in public to avoid transmission of herpes Actual Experiments

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Tobacco mosaic virus (TMV infects tobacco plant)

Applying fundamentals of virology to Ivanovsky/Beijerinck experiments - Viral genomes are obligate molecular parasites o They can only function after they replicate in a host cell - Viruses must make mRNA that can be translated by host ribosomes o They use the host protein synthesis machinery to make viral proteins o No virus can translate proteins from mRNA

First Animal Virus - Discovered in 1898 by Loeffler & Frosch - Discovered as an agent causing foot & mouth disease AND being filterable

How can we see viruses? - Viruses are ~0.001 the size of E. coli o Bacteria can be seen using light microscope - Electron microscopy and modern structural biology has allowed virologists to observe the complexity of viruses Exception: the Mimivirus - Mimiviruses can be seen under a light microscope - Mimiviruses do not pass through a 0.2-micron filter

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Host is Amoeba

Summary: A virus is a very small, infectious, obligate intracellular parasite that needs a cellular host

Topic 22: Viral Infections Viral infectious cycle and virology methods Main Objective: - Understand the basics of an infection cycle and virological methods utilized to study it Elements we will study to understand this objective: - Infectious cycle overview - Basic viral replication kinetics - Lab hosts utilized to study infectious cycle - How to assess infectious cycle o Cytopathic effects o Infectivity o Physical measurements Recap: Major themes in Virology - In order for viruses to survive they must: o Package their genome inside a particle o Use this particle to transfer their genome from host to host o Genome contains information to initiate and complete the viral infectious cycle o Genomes establish themselves in host ensuring long term viral survival

Step 1: - Virus needs to make its way inside the cell - Attachment of virus to an element on outside surface of the cell (recognition event) o Recognize a “door” on surface of cell - Virus enters host cell via endocytosis Step 2: - Outer shell protecting viral genome is lost - Genome will be exposed & will go to specific parts inside the cell to start making viral proteins (using host ribosome machinery) o Genome copy itself to make more genome Step 3:

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Viral -

Virus assemble replicated pieces through process of assembly to make a new infectious virion Virion = when all the pieces of a virus have come together and are ready to infect another cell

infectious cycle requires the host cell machinery Virus uses transport vesicles of cell to move around inside the cell Virus uses energy of the cell Virus uses protein translation machinery to make viral proteins

A cell that takes up a virus and allows the virus to replicate is susceptible and permissive - Susceptible cell = functional receptor for virus - Permissive cell = allows the virus to replicate - Resistant cell = no receptor for virus Hosts for Virus Replication (in the lab) - Whole animal hosts o Ex. Non-human primates, mice (measles) - Animal hosts can get costly – Allowing viruses to replicate in fertilized chicken eggs

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Allowing viruses to replicate in cell culture o Demonstrates how cells are growing o Media = food o Cells form monolayer on bottom of the plastic flask

How to tell what viruses are doing to cells? - Cytopathic effects = the different changes that a virus induces inside a cell o Ex. Cell lysis (bursting), Syncytia, Transformation

Syncytia: - Groups of cell bunch together (ex. HIV) Transformation: - Inhibit cell being on monolayer on its own - More dense and darker areas (mountain effect) Measures of Infectivity - Plaque assay (host = bacterial cell) - Phage plaques = area where bacterial phage has obliterated the host bacteria o Bacteria are replicating virus - Infectivity determined by # of plaque forming units o More phage plaques formed = Higher infectivity

Measure Infectivity: Plaque Assay - General idea: Add virus to cells

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o Overlay cells with a gel like substance (agar) o When infected cells release progeny; spread is halted by gel (agar) Host cell deposited on the surface of plate = lawn Virus added to the cells Overlay whole system with agar = stop spread & release from initially infected cell to neighboring cell o Needed or entire lawn of cells will be gone (viral cycle)

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Must be diluted in order to get discrete and smaller plaques (not too much infection)

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Plaque assay where Crystal Violet will stain cells that are alive o Donuts in middle = parts obliterated by the virus

Measure Infectivity: Are all viral particles infectious Concept of Particle-to-PFU Ratio: # of virus particles / number of infectious particles - Closer value is to 1 = imply that all particles are infectious - Measure # particles required to get an infection o Ex. If 200 particles and PFU = 1, most particles would be non-infectious Measure of Infectivity: Transformation assay - Certain viruses do not form plaques but form foci - Rous Sarcoma Virus (RSV) forms foci - Can count foci and get foci forming units per mL

Measure of Viral Particles (not infectivity but #s) - Sometimes we cannot measure infectivity using assays or transformation assays - One still needs to determine if cells are infected Physical Measurement: Hemaglutination assay - Certain viruses contain proteins that bind to red blood cells o Hemagglutinin inside the virus interacts with RBCs and form lattice - If a sample contains viruses, they will bind RBC & form a distinct Lattice which coats the side of the tube - Lack of virus = TBCs forming a “dot” - Different dilutions are done => more dilute the sample gives less interaction o Diluting out the hemagglutinin & viral proteins = less lattice structure formed - The hemaglutination assay detects interaction of hemagglutinin (viral protein) with host cell

Physical Measurement: Viral Enzyme Activity - Retroviruses (HIV) contain active enzymes such as Reverse Transcriptase (RT) - Measuring the activity of a viral enzyme Physical Measurement: Immunostaining - Viruses can express viral proteins - Different types of antibodies to stain the viral proteins o Detecting fluorescent tagged viral protein in sample

Physical Measurement: Immunoblotting - Take infected cells & lyse them to separate the proteins using SDS-page electrophoresis (denaturing system) - Use antibodies to detect for the presence of viral proteins within cells

Physical Measurement: Sequencing - Expand viral nucleic acid (using polymerase chain reaction – PCR) o Useful for low viral abundance genes o Expand & get more of initial gene - Amplify genome of cell & potential viral genomes within them o Get sequence = order of nucleotides = determine presence of viral genome in cell - Exponential growth of gene product Physical Measurement: Presence of Fluorescent Proteins - Insert sequences next to viral genes that will light up under a fluorescent microscope - Insert tag expressing fluorescent protein = light up infected cell Take -

Home Message: Basic kinetic of viral cycle Lab viral hosts Distinguish infectivity from physical assays Plaque assay = very important

Topic 23: Viral Genomes Review: Physical measurement, Fluorescent proteins - Viral genome makes viral proteins - Manipulate viral genome & insert sequences adjacent to coding protein sequences o Result in viral protein being expressed fused to fluorescent tag o Green cells contain virus

Learning Objectives Main Objective: - Understand the various types of genomes found in viruses & how these genomes function Elements: - Background on importance of genome - Viral classification scheme using the Baltimore method - How DNA based genomes get their message across - How RNA based genomes get their message across Hershey-Chase Blender Experiment (Proof the Genome is key) - Q: Is viral genome or shell transmitted to other cells o Bacteriophage infects bacterial host o Red = radioactive (shell of virus labelled) o Genome (nucleic acid) also radioactively labelled - Blender = remove bacteriophages on surface of the host cell - Separation via centrifugation o The cellular pellet goes to bottom of tube & liquid stays on top - Radioactive phosphorous detected in next generation of bacteriophage o No sulfur detected in cells w/ shell labelled Modern-Day Hershey-Chase Experiment - Bacteriophage mixed w/ cyanine dye (binds viral genomes) - Upon infection into bacterial host => Dye leaves bacteriophage & enters host interior o Transfer of fluorescence from phage capsid to cell interior

Viral Classification using the Baltimore Classification Scheme

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Classifying viruses according to their genomes Viral genomes must make mRNA o This mRNA must be read by host ribosomes o mRNA is read in 5’ – 3’ direction

Review of Genes & Proteins - viral genomes must make mRNA & this mRNA must be read by host ribosomes - viruses do not do this in a linear fashion (no dogma) o starting material different depending on virus Goal: Production of mRNA to be translated by host cell machinery Baltimore Classification System - mRNA = by convention the positive (+) strand & is ready to be translated - Positive (+) DNA strand = equivalent polarity to + RNA strands - RNA or DNA complements of Positive (+) strands = Negative (-) strands The 7 Types of Genomes: - Genomes are DNA or RNA (but not both) RNA - Single-stranded + RNAx - Single-stranded – RNA - Double-stranded RNA - Single-stranded + RNA with DNA intermediate DNA - Single-stranded DNA - Double-stranded DNA - Gapped ds DNA DNA Genomes – How the various genomes function Double stranded (ds) DNA genomes - DNA-dependent RNA polymerase transcription o DNA-dependent b/c starting material is DNA o RNA polymerase b/c we are making RNA - Make + ssRNA to make proteins - Similar to the Dogma

Where does DNA-dependent RNA polymerase originate from? – host or viral protein DNA-dependent RNA polymerase from host - Small genomes (5 Kbp) – Polyomaviridae o JC virus – example of Polyomaviridae o 80% of NA are infected – immune system takes care of virus

o immunocompromised patients (AIDS) will develop degenerative brain diseases due to JCV - In double-stranded DNA families, the smaller the genomes are the more dependent on using host enzymes DNA-dependent RNA polymerase encoded in viral genome - Larger genomes (100-370 Kbp) – Poxviridae o Variola virus (smallpox) – example of Poxviridae - Larger genomes = have its own enzymes to convert ds DNA to +ssRNA Gapped Double-stranded (ds) DNA genomes - Double stranded DNA with gap (not fully hybridized together) o Need to make double stranded DNA molecule - Viral-associated DNA polymerase fills in gaps - Followed by host DNA-dependent RNA polymerase transcription - Example: Hepadnaviruses (Hepatitis B viruses) o Hep B – now treatable but can cuase liver damage o Spread through sexual contact o 100X more infectious than HIV-1

Single-stranded DNA Genomes - single strands cannot be copied to mRNA - host DNA polymerase makes ds DNA - host RNA polymerase makes mRNA

Example: Parvoviruses - Mostly known for dog/cat infections - Infect cells of intestine, hematopoietic system & fetus - Canine parvoviruses infect & kill puppies

RNA Genomes – How the various genomes function Viral RNA Genomes - Host does not have an enzyme that is an RNA-dependent RNA polymerase - Negative (-) RNA strands require a virally encoded RNA-dependent RNA polymerase to make mRNA readable by host translation machinery Double stranded (ds) RNA genomes - Double stranded RNA cannot get translated (hybridized on each other) - Make (+) RNA strand using viral RNA-dependent polymerase and (–) RNA strand as template - Newly made (+) RNA strand becomes mRNA

Example: Reoviridae (virus family) – Rotavirus - 4/5 children develop in first 5 years of life - most important cause of gastroenteritis worldwide & infant mortality in developing world - dehydration is main contributor to mortality - fever, diarrhea, abdominal pain Single stranded (ss) + RNA genomes

- no need for a virally encoded RNA-dependent polymerase Example: Picornaviridae – Poliovirus - small RNA virus - poliovirus causes paralytic poliomyelitis: spinal cord & brain stem - disease: disabling paralysis Example: SARS-2 CoV-2 (Family: Coronaviridae) Single stranded (ss) + RNA genomes with DNA intermediate - reverse transcriptase makes DNA from RNA o ssDNA cannot be transcribed - cellular DNA polymerase makes ds DNA

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DNA-dependent RNA polymerase makes + ssRNA o DNA transcription to mRNA Example: HIV (Family: Retroviridae) -

Single Stranded (ss) – RNA genomes - (-) RNA needs to be copied by viral polymerase to make (+) RNA - Examples: Paramyxoviridae, Orthomyxoviridae

Orthomyxoviridae: Influenza Virus - Respiratory disease (can be deadly) Filoviridae: Ebola Virus (Ebola Genome Basics) - Need to make + mRNA - Need the viral RNA-dependent RNA polymerase o Copy single RNA strand to make (+) RNA strand - L-protein = function to serve as viral RNA dependent RNA polymerase to make positive RNA strand Viruses will eventually require more viral genome for replication Take Home Message:

Topic 24: Viral Structure Main Objective: - Understand the different pieces that make a virus Overview: - Techniques in viral structure - Building Virions - Helical Symmetry - Polyhedral Symmetry - Envelopes - Assembly Overview of Virus Structure: A Generic Virus - Capsid = Viral container or shell - Virion = a complete infectious particle - Nucleocapsid = Situation where the capsid also contains the genome - Envelope = lipid bilayer membranes enclosing nucleocapsids (Is host derived) o hosts give viruses their envelopes – not all viruses have envelopes Protein Functions: Overview of virus structure - Key Concept: The virion must be both stable & unstable. The stability varies during different steps of infectious cycle - Discussion: At what points during the infectious cycle are virions unstable/stable? 1. Protection of the Genome - Assembly of a stable protective shell - Specific recognition & packaging of the nucleic acid genome - Interaction with host cell membrane to form the envelope (in certain cases) 2. Delivery of the G...