MICR3320 Viruses and Viral Diseases Exam Notes

Lectures

• Lecture 1 Introduction to Viruses 1 • Lecture 2 Virus Genomes and Genetics 2 • Lecture 3 Infection of a Susceptible Host 3 • Lecture 4 Acute Infections 5 • Lecture 5 Persistent and Latent Infections 7 • Lecture 6 Virus Attachment and Entry 9 • Lecture 7 Virus Assembly, Maturation and release 10 • Lecture 8 DNA Virus Replication 11 • Lecture 9 RNA Virus Replication 12 • Lecture 10 Retroviruses and Retrovirus Replication 14 • Lecture 11 Viral Diversity and Evolution 15 • Lecture 12 Emerging Viral Diseases 17 • Lecture 13 HIV and AIDS 18 • Lecture 14 Bloodborne Viral Infections 19 • Lecture 15 Vector-Borne Viral Diseases 21 • Lecture 16 Respiratory Viruses 22 • Lecture 17 Viral Infections of the Central Nervous System 24 • Lecture 18 Oncogenic Viruses 26 • Lecture 19 Virus-Host Interactions and Pathogenesis 27 • Lecture 20 Antiviral Agents 29 • Lecture 21 Antiretroviral Drug Resistance 30 • Lecture 22 Anti-Viral Vaccines 31

Lab Review

• Viruses 33 • Tests 33 o Virus Propagation 33

o Tissue Culture Infective Dose (TCID50) 33 o Virus Plaque Assay 34 o Plaque Reduction Neutralisation Test (PRNT) 34 o Molecular Analysis (RT-PCR) 34 o Particle Enumeration by Haemagglutination Assay 35 o Anti-Virus Antibody Detection by Dot-Blot Assay 35 • Materials 35

Lecture 1 Introduction to Viruses

In this lecture, you will be introduced to viruses, their properties, and some of the diseases they can cause. By the end of this lecture, you should be able to describe the basic components of virus morphology, structure, and taxonomy, and of the prion diseases.

• Viruses are obligate intracellular parasites that require host cells to replicate via assembly of pre-made components. • Viruses infect all living things. The host is determined by a host receptor that interacts with virus surface molecules required for attachment (susceptible) and host cell factors required for replication (permissive). • The main stages of virus replication are: (1) attachment; (2) entry of viral nucleocapsid; (3) translation and genome replication; (4) assembly and maturation; and (5) release of infectious virion.

• Viruses are classified into orders (-virales), families (-viridae), genus (-virus) and species (classes of viruses with many properties in common that constitutes a replicating lineage and occupies a particular ecological niche). • Family primary criteria include morphology, nucleic acid type and strategy of replication (Baltimore classification); Genus primary criteria include significant differences in genome sequence and minor differences in genome structure; separate species are associated with serological differences, genomic differences and structural and physical properties. • A type species is a virus which has all the properties characteristic of a certain genus. Variant species (subtypes, clades, strains, quasispecies) arise frequently as a result of minor mutations.

Virus Structure

• Viruses vary greatly in size and morphology. Acanthamoeba polyphaga mimivirus is the largest observed virus that resembled bacteria (mimicked Legionella pneumophila) within amoeba collected from water-cooling towers. Other large viruses include Pandoravirus salinus and dulcis, Pithovirus sibericum and Megavirus chilensis. • A virion is the complete, fully developed infectious viral particle composed of nucleic acid genome (DNA/RNA, ss/ds, linear/circular, segmented/non-segmented) surrounded by a protein coat. Some viruses are enveloped. • The capsid is composed of repeating single/several protein subunits (capsomeres) in an icosahedral (e.g. parvovirus, poliovirus, adenovirus) or helical (e.g. TMV, Ebola, Vesiculovirus) arrangement characteristic for a particular virus. Icosahedral symmetry can be described using the triangulation number, which is the number of asymmetric units per face of the icosahedron. Capsid structure can be regular (e.g. bacteriophages; components of both helical and icosahedral symmetry) or complex (e.g. Poxviruses; non-symmetrical capsid structure). • The envelope is a combination of lipids, proteins and carbohydrates derived from host cell plasma membranes, with some encoded by viruses (e.g. virally encoded glycoproteins). • Virus-like particles are self-aggregates of virus structural proteins that mimic the organisation and conformation of native viruses but lack the viral genome (non-infectious).

Prions

• Proteinaceous infectious particles (prions) are infectious, misfolded proteins that cause disease and lack instructional nucleic acid. Mutations in normal prion protein (PrPC; encoded by PRNP gene in neurons) associated with disease include single amino acid substitutions and truncation. Abnormal PrPSc can attach to PrPC and promote its transformation to PrPSc. • Transmissible spongiform encephalopathy is characterised by changes in memory, personality and behaviour, dementia and ataxia. Examples in humans include Kuru (first evidence), Creutzfeldt-Jakob disease (CJD), variant CJD, Gerstmann-Strässler-Scheinker disease, and fatal familial insomnia. Examples in animals include scrapie (sheep), bovine spongiform encephalopathy, and chronic wasting disease (deer).

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Lecture 2 Virus Genomes and Genetics

In this lecture, you will revise the structure and complexity of viral genomes, understand the Baltimore classification system and important features that contribute to viral evolution and diversity. By the end of this lecture, you should be able to name examples of viruses which replicate by the 7 replication strategies, and classify them based on the Baltimore system.

• All viral genomes must encode gene products and regulatory signals required for: (1) genome replication, (2) assembly and packaging of the genome, (3) expression and regulatory signals, and (4) modulation of cell defences and propagation to other cells. • However, viral genomes do not encode for complete protein synthesis, or proteins of energy metabolism or membrane biosynthesis.

Baltimore Classifications

• Viruses (families) are categorised based on the type of nucleic acid genome and replication strategy. All viruses MUST produce mRNA (+) that can be translated by cellular ribosomes to produce viral structural protein. Knowledge of strand polarity informs the steps to initiate replication and expression of the viral genome.

• Group 1 – dsDNA: viral genomes are copied by a DNA-dependent DNA polymerases (small viruses use cellular DNA pol; large viruses encode their own enzyme). mRNA is produced by a DNA-dependent RNA polymerase (DdRp; RNA polymerase may be host or viral). E.g. Herpes, Irido, Asfar, Pox, Polyoma, Papilloma, Adeno. • Group 2 – ssDNA: cellular DNA polymerase forms dsDNA (RNA requires dsDNA), then follows Group 1 mechanism; all replication occurs via cellular DNA polymerases. E.g. Parvo, Circo. • Group 3 – dsRNA: viral RNA-dependent RNA polymerase (RdRP) packaged into the nucleus is responsible for viral genome replication and or mRNA synthesis (RNA (–) template); REMEMBER eukaryotic cells have no RdRp for viral replication. E.g. Reo, Birna. • Group 4 – (+) ssRNA: viral genome replication occurs in 2 steps: (+) strand is copied into full-length (–) strand (template), then a full-length (+) strand is produced (viral RdRp is translated from the viral genome). REMEMBER (+) RNA can be translated directly into proteins by host ribosomes. E.g. Corona, Calici, Picorna, Flavi, Toga, Artesi, Astro, Nada, Hepe. • Group 5 – (–) ssRNA: same mechanism as Group 3. E.g. Borna, Bunya, Filo, Rhabdo, Arena, Orthomyxo, Paramyxo. • Group 6 – (+) RNA (RT): the viral genome is converted into a dsDNA intermediate by viral RNA-dependent DNA polymerase (reverse transcriptase (RT)). The viral dsDNA is integrated in the host genome (provirus) whereby cellular RNA polymerase transcribes proviral DNA into viral mRNA. Genomic viral (+) sense RNA is never used as mRNA. E.g. Retro. • Group 7 – dsDNA (RT): gapped DNA is repaired by cellular DNA polymerase to covalently closed circular DNA (cccDNA) and transcribed into pregenomic mRNA. pgRNA serves as a template for viral proteins and converted to DNA by viral RT. cccDNA genomes can persist in host cell nuclei for many years, acting as a minichromosome and supporting transcription of viral RNA. E.g. HBV.

• As eukaryotic cells do not have RdRp, All RNA viruses must encode either a RdRp or RT for efficient replication.

Evolution of RNA Viral Genomes

• RdRp and RT lack 3’ exonuclease proofreading activity (coronavirus, largest RNA virus, have some proofreading ability), with an average error frequency of 1 in 104-105 nucleotides incorporated. • Recombination is the exchange of nucleotide sequences among different genomic molecules, whereas reassortment is the exchange of entire RNA molecules between genetically related viruses with segmented genomes (e.g. IVA). • Accumulation of mutations during replication in combination with recombination events is responsible for all RNA viruses to exist as mixtures of genetic variants (quasispecies). Most mutations are inconsequential or detrimental, but certain mutations may allow a virus to escape a selective pressure.

• See Lecture 11 for further information on viral genetic diversity and evolution.

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Lecture 3 Infection of a Susceptible Host

By the end of this lecture you should understand: (i) the requirements for viral infection of a host, and the common routes of infection and dispersal; (ii) how transmission occurs between hosts, and that some viruses that infect humans also replicate in other animal species; (iii) how viruses may disseminate from the original sites of infection, to establish systemic infection in different organs, and how this is reflected in disease pathogenesis; (iv) that viral infection is commonly associated with typical rash; and (v) that viral infectiousness in a population is quantified by the R0 value.

• There are three requirements to ensure successful infection of a host: (1) there must be sufficient virions to initiate infection (dose); (2) the cells at the site of infection must be physically accessible to virions, susceptible (bear receptors for entry), and permissive (contain intracellular gene products needed for replication); and (3) local host antiviral immunity must be absent or insufficient. • The number of virus particles required to initiate and maintain infection depends on the: (1) particular virus, (2) site of infection, and (3) age and physiology of the host. Although a single virion can initiate infection, due to the complexity of the infectious cycle, the probability of a single virion to complete the cycle is not 100%. tissue infectivity may be determined by assessment of tissue culture infectious dose (TCID) and by plaque assay.

• Virus transmission is the spread of virus from one susceptible host to another, and follow one of two patterns: (1) perpetuation in one species, or (2) alternate infection between vertebrate and invertebrate hosts (zoonoses). • Types of viral transmission: (1) iatrogenic – activity of health care worker leads to infection of patient; (2) nosocomial – individual infected while in a hospital or healthcare facility; (3) vertical – transfer between parent and offspring; (4) horizontal – all other forms of transmission; and (5) germline – transmission as part of host genome (e.g. integrated proviral DNA).

• R0 (reproduction number) is a quantification of viral contagiousness as the average number of secondary cases generated by one primary case in a susceptible community, in the absence of control measures. • Viral infectiousness is also determined by the incubation period in the host. Viruses with high R0 and short incubation periods are significant public health problems (e.g. influenza), whereas those with low R0 and longer incubation periods allows time for the use of quarantine to stop an outbreak (e.g. SARS).

Viral Entry

• Mucosal surfaces are lined with living cells and are a major portal of entry. • The respiratory tract is the most common route of viral infection due to the lungs large absorptive area and high resting ventilation rate. Host defence mechanisms include: (1) mucociliary escalator (mucous-secreting goblet cells, subepithelial mucous-secreting glands, ciliated cells) that captures and expels foreign particles into the throat; and (2) resident alveolar macrophages that ingest foreign particles. • The alimentary tract is a common route of infection and dispersal, and is characteristically a hostile environment (acidic stomach, alkaline intestine, many digestive enzymes and bile detergents, intestine lined by mucous, luminal surfaces include antibodies and phagocytes). Some viruses (e.g. poliovirus) can replicate in low pH environments, whereby changes in the capsid are fully reversible (c.f. low pH induces irreversible disassembly of rhinovirus capsid). M cells of the intestinal wall can be manipulated to promote viral entry (e.g. reovirus). • Viral entry through the skin mostly occurs when integrity is breached (arthropods, hypodermic needle) as the dead outer layer cannot support viral replication. In contrast to strictly localised replication in the epidermis, infection in dermal or subdermal tissues can reach blood vessels, lymphatics and nerve cells. • The urogenital tract mucosal cells are protected by mucous and low pH. microabrasions allow viral entry and may produce local lesions or disseminate. • Transplacental infection involves transmission from a mother to her developing foetus.

Viral Spread

• Progeny virions may remain localised (contained within the epithelium by immune responses and physical nature of the tissue), or disseminate (spread beyond the primary site) and cause systemic infection. • Directional release of virions from polarised cells at a mucosal surface permits avoidance of local host immune response, and facilitates spread. Apical release establishes localised infection (underlying lymphatic and blood vessels

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are rarely invaded), whereas basolateral release promotes avoidance of host luminal defences, access to underlying tissue, and facilitate systemic spread.

• Haematogenous spread is a consequence of viral entry into the bloodstream by either: (1) direct invasion through capillaries, (2) endothelial replication, (3) inoculation by a vector, or (4) drainage of lymphatics into circulation. • Definitions: (1) viraemia – presence of infectious virus in blood; (2) active viraemia – presence of replicating virus in blood; (3) passive viraemia – introduction of virions into blood without replication at SOI; (4) primary viraemia – progeny virions released into blood after initial replication at SOI; and (5) secondary viraemia – delayed appearance of high concentration of infectious virions in blood following dissemination from initial SOI; passive → primary → secondary. • Neural spread is a consequence of viral entry into afferent/efferent fibres innervating the infected tissue, invading the PNS then to the CNS. Some neurotropic viruses become latent in peripheral neurons (e.g. HSV) and reactivate.

Organ Invasion

• There are 3 main types of blood vessel/tissue junctions that are routes for virion entry: (1) continuous endothelium and basement membrane (CNS, CT, muscle, skin, lung); (2) fenestrated epithelium (choroid plexus, intestinal villi, renal glomeruli, pancreas, endocrine glands); and (3) sinusoid, lined with reticuloendothelial macrophages (adrenal glands, liver, spleen, bone marrow). • Invasion of organs characterised by sinusoids are often a consequence of infection of macrophages lining the sinusoids by viruses present in blood, causing infection of underlying cells (e.g. HBV infection of Kupffer cells (macrophages) in liver cause infection of underlying hepatocytes (hepatitis)). • CNS, CT, and skeletal and cardiac muscle blood vessels re composed of continuous endothelium backed by a dense basement membrane (BM). In the CNS, the BM serves as a selectively permeable barrier (blood-brain barrier; BBB). Viruses may infect brain tissue by either: (1) directly pass through capillary endothelium and enter the stroma of the choroid plexus, then cross the epithelium into the CSF; or (2) cross the BBB within infected cells or via transcytosis. • Rashes are produced when virions leave the blood, and is common in many systemic viral infections. Maculopapular rash is characterised by macules (flat, small, non-elevated) and papules (small and swollen bumps on skin) that develop when infection occurs in the dermis with the lesion confined on or near the vascular bed (e.g. measles, Zika). Vesicular rash is characterised by vesicles and pustules that occur when the virus spreads to superficial skin layers (e.g. VZV). Rash may occur in mucosal tissue such as the mouth and throat (e.g. measles; Koplik’s spots).

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