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Viral agents: Cultivation and assay of viruses Why do viruses need to be isolate and cultivated? ○ For diagnosis Treatment options - for slow/persistent viruses Chemotherapy Passive immunization ○ R...

Viral agents: Cultivation and assay of viruses Why do viruses need to be isolate and cultivated? ○ For diagnosis Treatment options - for slow/persistent viruses Chemotherapy Passive immunization ○ Rabies takes weeks, so we can use vaccine (100% effective) - rare opportunity ○ Public health measures Blood bank screening for HIV, HBV, HCV Contact monitoring ○ Surveillance ○ Research Amplification needed Understanding replication, impact on cell function Many virus infections wane before clinical symptoms appear Virus stability ○ Viruses are sensitive to physical and chemical inactivation ○ Enveloped viruses do not survive well outside host cells ○ Naked viruses are resistant Norovirus has been found active after 3 years in the environment ○ To preserve infectivity for cultivation Low temperature For up to a day ○ 4 degrees Long term ○ Minus 80 Avoid repeated freezing and thawing Some viruses can be freeze dried ○ Used for some live-viral vaccine Buffered transfer medium Clinical samples ○ Respiratory infections: throat washing, nasal secretions ○ Enteric infection: fecal sample ○ Meningitis/encephalitis: CSF  ○ Vesicular rash: vesicle fluid ○ Systemic fever: blood ○ Sample is refrigerated or frozen in buffered transport medium Viral detection ○ Direct visualization by EM Allows for ID of virus family Crude purification of virus from heavily contaminated samples Can be visualised directly or combined with antibody to confirm specificity Negative stain  Confirms that antibody recognises virus -> identification ○ Assays Antigen capture assay Anti-viral antibody assay  Western blot assay Detergents are used to solubilise all the proteins in cells Proteins are separated by SDS-PAGE and transferred to filters Anti-viral antibody in patient serum binds to protein bands ○ High specificity Serological assays (e.g. ELISA) Generally slow Challenging for detecting related viruses ○ Cross-reactivity of antibodies to all flaviviruses (dengue, yellow fever, japanese encephalitis) ○ Can have a lot of false positives ○ High sensitivity, low specificity Viral protein detection assays ○ Can require very complex assays Reverse transcriptase assay Detecting host antibodies ○ Poor sensitivity - lots of false negatives Viral nucleic acid detection PCR for DNA ○ Cheap ○ Highly sensitive and specific ○ Prone to contamination ○ Fast ○ Automated ○ Easily adapted Reverse transcriptase PCR (RT-PCR) for RNA Southern (DNA) and northern blot (RNA) Sequencing  ViroChip ○ 36k sequences representing all known sequences as of Dec 2019 ○ Each viral microarray element is converted to a stripe ○ Stripes organised by viral family of origin ○ Red hybridization intensity plotted in linear scale Degrees of yellow ○ Infected with rhinoviruses ○ Very specific Viral DNA is loaded and bonds to complementary DNA and a laser activates dye to determine Comparison of assays Titres of a single virus preparation may vary depending on assay used For in vivo replication, it can be that only 1 in 100 particles assembled in a cell are infective For influenza: ○ Cultivation Gold standard for studying viruses Allows for production of reagents Antivirals Antibodies Nucleic acid  Some viruses have no culture systems (e.g. norovirus) Can be slow Result often does not impact on treatment Just because you can grow it doesn’t mean you can treat it Chicken egg cultivation Not widely used anymore Some flu vaccines are prepared this way Fertilised egg is inoculated Vaccinia virus produces pocks on the CAM of chick embryos ○ Visible leukocyte aggregation Mammalian cell culture Most commonly used method of virus cultivation Uses three different types of mammalian cells ○ Primary cells are prepared from animal tissue Only a handful of passages ○ Diploid cell lines have a limited lifespan in culture (up to 100 generations) ○ Transformed or continuous cell lines can be passaged indefinitely in vitro Aneuploid - abnormal chromosomes Cancerous cell lines Very convenient for virus growth Not used for human vaccines HeLa Routinely used in labs The immortal life of Henrietta Lacks - black woman Can also use insect cells  Plaque culture is virus amplified using cell monolayer Safety is needed To protect culture and operator, virus cultures have to be handled in class II biohazard cabinets (PC2 or PC3) Class IV isolation only for highly pathogenic viruses ○ Ebola, Nipa, H5N1 ○ Spacesuits Organoids Mini organ on plate Uses stem cells ○ Can add microbiota and other things  Important for growing lots of different viruses ○ Simulating infection ○ Antigen detection ○ Host serological response Antibodies ○ Viral gene detection Most widely used approach SARS Outbreak 2003 Started Nov 2002 11 Feb 2003: Reports to WHO of an outbreak of 305 cases 21 Feb 2003: Infected MD brought disease to Hong Kong hotel ○ 12 guests infected ○ Guest spread to Singapore 11-25th March: Doctor admitted to hospital where he infected 138 staff 26 March 2003: 31 deaths Late March: Community outbreak in Amoy Gardens, 329 infected residents April 2003: Peak of epidemic in Beijing and several parts of China 20th of April 2003: full cooperation with WHO ○ Strict control measures ○ Decline in cases 5,327 cases, 349 deaths Not as transmissible as SARS-CoV-2  Outbreak was hard to control due to porous border between Hong Kong and Southern China Severe flu symptoms Blood, sputum and respiratory washings were the samples collected, made available to virologists worldwide Steps in IDing new infectious agent ○ Epidemiology Isolated from civits, raccoon dogs and bats Australian scientist help ID bats as natural reservoir ○ Virus cultivation First done March 21st Vero cells and FLRK cells (Fresh rhesus monkey lung) Patient Ab binds to virus antigen on infected cells Infectious virus purified from cell lines ○ Electron microscopy  Coronavirus morphology No other coronavirus grows in Vero cells ○ Physical characterisation ○ Genome sequencing ○ Proving Koch’s postulates Took 2 years to ID HIV as AIDS cause ○ In 2003, WHO created extraordinary network of 13 laboratories in 10 countries which ID’d virus in 2 weeks ○ In 2019, COVID-19 Structure and composition Definitions Virion: particle Capsid: protein shell surrounding genome ○ Capsa - latin for box Nucleocapsid: Nucleic acid - protein assembly within virion Subunit: single folded polypeptide chain Structural unit: Unit from which capsids or nucleocapsids are built ○ One or more subunits ○ Protomer, capsomer Envelope: host derived lipid bilayer Energy & stability Must be  ○ Stable enough to protect nucleic acid ○ Must not be too stable - aim is to deliver genetic material Viral particles have not attained the minimum free energy conformation ○ They exist ready to release energy ○ There is an energy barrier that must be overcome - potential energy is used for disassembly ○ Target cells provide the proper trigger for this energy release and disassembly Purifying viral particles and identifying components Before characterising and classifying a virus, it must be biologically pure To obtain pure virus: ○ Plaque purification Like using a bacterial culture Select from single plaque to grow in stock ○ Limiting dilution of original sample Biological cloning ○ Generation from a molecular clone Capture viral genome in a plasmid Usual steps of getting viral particles purification process ○ Cell disruption Safest method - freeze and thaw 2-3 cycles Some non-ionic detergents will lyse cytoplasmic membrane but not nuclear membrane Keeps host genetic material separate Homogenizers chop up cells Rapidly rotating blades  ○ Shears cells and breaks membrane ○ Membrane fragments reform into vesicles If membrane is from the ER w ribosomes attached (RER), vesicles are called microsomes ○ Nuclei are broken and DNA/chromatin is released Results in viscous homogenate ○ Centrifugation Separates nuclei and large cell fragments Low speed centrifugation 15 min at 1000g g force = radius of rotor x rpm^2 Small organelles, ribosomes and viral particles are in supernatant fluid (SNF) Ultracentrifugation Spins tube 40-120,000 rpm Rotors are strong and sealed, run in a vacuum Temperature is controlled Balance is critical After high speed runs, particles should be in the pellet and SNF will contain smaller particles (ribosomes and soluble proteins) SNF discarded Pellet is not quite pure ○ Density gradient ultracentrifugation Viral particles have a unique combination of density and how fast they can move through a fluid  Rate zonal density gradient centrifugation Separation depends on the size and mass of what you need to purify ○ Sedimentation coefficient - size and mass Density of particles is greater than the density of the solvent Separation based on size - larger particles separate the fastest The amount of time the centrifuge runs is important in obtaining the correct particles ○ Gradient of sucrose, least to most dense ○ Generally, medium particles are virions Equilibrium (isopycnic) Depends on density only Solvent density encompasses density of particles Separation based on particle density ○ Centrifugation is carried out until equilibrium is reached All particles have banded at density corresponding to their own Can take a long time  ○ If you puncture a hole in the bottom of the tube and let the contents drip out, the density of plaque forming units usually peaks at 4 fractions Finding virus components ○ Disrupt the virus using SDS detergent Gel electrophoresis, immunoblotting (Western blotting)  SDS gel electrophoresis SDS: anionic, disrupts virions, binds to proteins Proteins containing bound SDS gain a negative charge and move towards anode (+) according to molecular weight Intensity of protein band directly correlates with protein mass/number of amino acids Virions are labelled with radioactivity for visualisation ○ Or with Silver Stain or Coomassie Blue Can tell us about relative amounts of protein ○ Mass spectrometry IDs every protein, including relative abundance and post-translational modification Identifying DNA vs RNA ○ Infect cells in the presence of 14C-thymidine and 3H-uracil ○ Purify virions produced in cells ○ Use a radioactivity detector (scintillation counter) to determine whether virus contains 3H (RNA) or 14C (DNA) Different rates/energy of decay SS vs DS RNA ○ Label viral RNA during growth ○ Extract nucleic acid from purified particles ○ Divide into two portions Add ribonuclease (RNase) to one and incubate RNase converts RNA polymer into free nucleotides RNaseA / T1 digests ssRNA but not dsRNA  ○ Use TCA to precipitate remaining radioactive RNA polymers from each sample Look at different between tube with RNase and without Icosahedral structures Structure: the virion ○ Created by the symmetrical arrangement of many identical proteins to provide maximal contact and non-covalent bonding Function: genome delivery ○ Structure is not permanently bonded together Minimum free energy state is the goal, genome release goes along with it Spherical shell is most economical (in terms of protein) to enclose the genome ○ Viruses make icosahedral capsids (5:3:2 cubic symmetry) ○ Identical protein subunits ○ Viruses from different families use different number of capsomers to form icosahedral capsids ○ 20 triangular faces, 12 vertices Adenovirus  ○ 12 pentons (proteins) extending outwards to engage with host cells ○ 20 faces ○ 240 hexons (capsomers) Triangulation ○ Way to describe how limited sets of small proteins make icosahedral particles with very different sizes ○ T1 = 60 units 20 faces Each face has 3 protein units 3 x 20 = 60 ○ T3 = 180 units 20 triangular faces Each face has 9 units 9 x 20 = 180 ○ Number of facets per triangular face of an icosahedron ○ Combining several triangular facets allows assembly of larger face from same structural unit Poliovirus ○ Poliomyelitis ○ Enteric infection Neurotropic Paralysis ○ Very small particles w icosahedral structure ○ Difficult to see capsomers with EM ○ Contains 60 capsomers T1?  Each capsomer has 1 copy of 4 viral proteins Equimolar amounts of these proteins ○ X-ray crystallography shows the subunit is icosahedral T = 3 - psuedo3 180 proteins 3 outer proteins x 60 triangles Icosahedral enveloped - Flavivirus ○ 50nm ○ T3, 60 copies of E trimers on the surface ○ Undergoes extensive rearrangements from exit to infection (dimers to trimers) ○ Equimolar amounts of capsomer proteins Helical structure Left or right handed helix Always enveloped Number of nucleocapid subunits per turn ○ Dictates shape Axial rise per subunit Pitch per turn Measles virus ○ Paramyxoviridae ○ MV structural proteins ○ Nucleoprotein is very important Most abundant protein in infected cells Self-assembles to form nucleocapsid (NC) Forms a ribonucleoprotein (RNP) complex (RNA/P/L) Most abundant protein of RNP Plays an important role in regulating replication Structural & non-structural proteins Structural: make up the virion ○ Present in purified virions ○ Genome is a structural viral component Non-structural: Additional proteins found in infected but not uninfected cels Because of some replication strategies, some viruses package replicative enzymes into their virions ○ Required for infection but not necessarily required for structure Coronavirus ○ Small part of LARGE genome is dedicated to structural components ○ RNA genome Rotavirus ○ Double capsid  ○ Attachment and entry Multiplicity of infection Viral infection is a random event ○ Brownian motion ○ No. of virions infection a cell is the multiplicity of infection ○ Can be calculated using a Poisson distribution  You need 4.6 infectious units per cell to get 99% of cell monolayer infected Virus infection Step 1: Adhere to cell surface ○ No specificity ○ Electrostatic interaction Step 2: Attach to specific receptor molecules ○ More than 1 receptor ○ Tropism Step 3: Transfer genome into the cell Tropism Presence of cell surface receptors determines susceptibility Presence of organelles and components/molecules required for virus replication determines whether a cell is permissive Tropism is determined by what cells are both susceptible and permissive Attachment Interaction between receptor and virus determines species and anatomical niche Receptors ○ Protein ICAM-1 for most rhinoviruses ○ Carbohydrate Sialic acid for influenza virus Terminal sugars on carbohydrates ○ Some viruses use 2 different receptors on the same host cell, one for initial attachment, then a coreceptor for closer attachment and entry  Plasma membrane receptors Influenza attachment Binds to sialic acid on cell glycoproteins ○ Haemagglutinin molecules bind to sialic acid ○ Neuraminidase releases sialic acid  Unknown what enables endocytosis of influenza virus Macromolecular uptake by cells Virions enter cells only by receptor mediated endocytosis or fusion ○ Not passive ○ Phagocytosis is possible but does not usually allow for successful viral replication Endocytosis ○ Cells take in virions using same pathways that other cargo uses ○ Invagination of a clathrin-coated pit ○ Forms an endosome ○ Endosome contents become acidic (in some cases) Causes conformational changes in virus capsid or envelope proteins Release Fusion and endocytosis strategies ○ Uncoating at the plasma membrane Genome directly enters cell Only enveloped viruses ○ Uncoating within endosomes ○ Uncoating at the nuclear membrane  Adenovirus Class I fusion proteins 3 identical protein subunits ○ Fold together into a six-helix bundle in the final post-fusion state Functional forms are generated via cleavage of units C-terminal end of one piece is anchored to viral membrane N-terminal has a characteristic stretch of 20 hydrophobic amino acids (fusion peptide) During fusion, 3 fusion peptides become exposed and are inserted into the target cell membrane ○ Can be triggered by lowering of pH  CoV spike protein ○ S1 subunit mediates ACE2 attachment ○ S2 subunit contains the fusion peptide ○ To be activated for fusion, the spike protein must be cleaved at 2 sites (S2 - TMPRSS2 and S1 - cathepsin/furin) directly at the cell membrane, through endosomes, or both ○ Class II fusion proteins Non-helical - beta-sheet (flat) Not cleaved during biosynthesis  The portion that inserts into the target membrane is thought to be an internal hydrophobic fusion loop pH plays a major role in the structural rearrangement of some class II proteins to expose the fusion loop ○ Usually occurs within the acidic environment of the endosome ○ Rearrangement is mediated via protonation of His residues Genome passes through pore into host cell Entry of non-enveloped viruses Stepwise uncoating of adenovirus ○ Penton fibre of adenovirus engages with cell adenovirus receptors (CAR) Integrin/immunoglobin-like molecules ○ Endocytosis ensues via clathrin-coated pits ○ During endosome acidification Uncoating leads to release of penton fibre which bursts the endosome Modified capsid is released ○ Capsid transported to nuclear pore along the cell microtubule network DNA viruses usually replicate inside nucleus  Polio ○ Binding to cellular receptor induces loss of VP4 ○ Loss of VP4 allows hydrophobic internal VP1 sequence to be exposed Buries into cell membrane, forms a pore ○ Viral RNA enters cytoplasm RNA viruses generally replicate in cytoplasm Fusion of enveloped viruses HIV-1 ○ Envelope glycoproteins bind to CD4 receptor Highly variable proteins ○ A gp160 envelope precursor is processed into a gp120 surface (SU) and a gp41 transmembrane (TM) protein Gp120 and gp141 are trimers ○ Attachment mediated via non-specific engagement with cell surface lectins (e.g. DC-SIGN) ○ HIV envelope glycoproteins (120/41) bind to CD4 receptor ○ Binding of gp120 SU to CD4 induces a conformational change in gp120 exposing a binding site for the chemokine coreceptor (CXCR4 or CCR5) ○ Binding of gp120 SU to CCR induces a conformational change in gp41 TM that leads to insertion of fusion peptide into membrane ○ HIV can bind to DCs to travel to lymphocytes to bind to cells with CD4 (T cells) ○ Fusion results in genome entering into the cytosol Endocytosis vs fusion To determine whether a virus enters via fusion or endocytosis ○ Cells are exposed to weak base (e.g. methylamine) during infection ○ This blocks infection via endocytosis by preventing acidification of endosomes Will not affect infection dependent on direct fusion at cell surface Pox virus Vaccinia virus utilise actin of filopodia of nerve cells to reach cell body Once at the cell body is induces membrane blebbing to facilitate uptake