Lecture 5_ Ch 29.7-29.8 General Properties of Viruses.pdf

Chapter 29.7-29.8: General Properties of Viruses Twenty-Eighth Edition Jawetz, Melnick, & Adelberg’s Medical Microbiology...

Chapter 29.7-29.8: General Properties of Viruses Twenty-Eighth Edition Jawetz, Melnick, & Adelberg’s Medical Microbiology Twenty-Eighth Edition Stefan Riedel Slides by Dr. Jose Sapien Jawetz, Melnick, & Adelberg’s Medical Microbiology, Twenty-Eighth Edition  29.7: Cultivation and Detection 29.8: Purification and Identification of Viruses 29.10: Reaction To Physical and Chemical Agents © McGraw-Hill Education, 2019 29.7: Cultivation and Detection Cultivation of Viruses The basics: Possible to grow in cell culture or fertile eggs with highly controlled conditions. Using animals to isolate certain viruses is common for learning about pathogenesis and oncogenesis. In vitro grown cells can be useful for characterization of viruses. Techniques of Virus Cultivation (microbiologyinfo.com) © McGraw-Hill Education, 2019 29.7: Cultivation and Detection Cultivation methods Primary cultures- dispersing cells with trypsin from freshly obtained host tissue (can only grow a few sub-cultures for limited time) Ex: used for primary isolation of virus for vaccines Diploid cell lines- have had changes made to allow limited culture (about 50 sub-cultures) but keep normal chromosome pattern as parent cells. Continuous cell lines- can have an indefinite amount of growth. They have been derived from diploid or malignant tissue (irregular numbers of chromosomes). Come from cancer cells and need to be kept very cold, but not useful for vaccine production. The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus. Techniques of Virus Cultivation (microbiologyinfo.com) © McGraw-Hill Education, 2019 From: https://facellitate.com/what-are-primary-cells-advantages-and-limitations/ © McGraw-Hill Education, 2019 From: https://www.ptglab.com/support/cell-culture-protocol/cell-types-culture-characteristics/ © McGraw-Hill Education, 2019 29.7: Cultivation and Detection A. Detection of Virus-Infected Cells Multiplication of a virus can be monitored in a variety of ways: 1. Development of cytopathic effects. 2. Appearance of a virus-encoded protein. 3. Detection of virus-specific nucleic acid. 4. Adsorption of erythrocytes to infected cells. 5. Viral growth in an embryonated chick egg. © McGraw-Hill Education, 2019 29.7: Cultivation and Detection A Enterovirus B Herpesvirus A. Detection of Virus- Infected Cells 1.- Cytopathic effects (ex: morphological changes to cell). Includes: cell lysis or necrosis, inclusion body formation, giant cell formation, and cytoplasmatic vacualization. A : Enterovirus—rapid rounding of cells progressing to complete cell destruction. B: Herpesvirus—focal areas of swollen, rounded cells. © McGraw-Hill Education, 2019 © McGraw-Hill Education, 2019 Varicella Zoster virus infection. A-B Tzanck Smear preparation. A: Multinucleated cells, with ballooned nuclei. Note the nuclear and cytoplasmic inclusions. Diff Quick. C: Epithelial hyperplasia and hyperkeratosis. H&E. D: " smudge cells " , peripheral chromatin (Arrow). Infected Keratinocytes with nuclear and cytoplasmic inclusions, vacuolization of the cells (H&E). Flora, Y, et al., (2016) © McGraw-Hill Education, 2019 29.7: Cultivation and Detection A. Detection of Virus-Infected Cells 2.- Appearance of a virus-encoded protein (ex: hemagglutinin). Can use antisera to detect the making of viral proteins in infected cells. © McGraw-Hill Education, 2019 29.7: Cultivation and Detection A. Detection of Virus-Infected Cells 3.- Detecting a virus-specific nucleic acid. PCR provide rapid, sensitive, and specific methods of detection. Douedi, S., et al., (2020) © McGraw-Hill Education, 2019 29.7: Cultivation and Detection A. Detection of Virus-Infected Cells 4.- Adsorption of erythrocytes (hemadsorption), caused by the presence of virus-encoded hemagglutinin (parainfluenza, influenza) in cellular membranes. This reaction becomes positive before cytopathic changes are visible and in some cases occurs in the absence of cytopathic effects © McGraw-Hill Education, 2019 A. Detection of Virus-Infected Cells 5.- Viral growth can cause death to the embryo if cultured in embryonic egg. Can produce plaques or develop hemagglutinins in the embryonic fluid © McGraw-Hill Education, 2019 29.7: Cultivation and Detection B. Inclusion Body Formation In the course of viral multiplication within cells, virus-specific structures called inclusion bodies may be produced. May be situated in nucleus, cytoplasm or both. They become far larger than the virion. Ex: Herpesvirus inclusion body located in the nucleus Affinity for acid dyes Depending on the stain and tissue, inclusion body appearance may vary From: https://www.stepwards.com/?page_id=678 © McGraw-Hill Education, 2019 © McGraw-Hill Education, 2019 29.7: Cultivation and Detection Quantitation of Viruses A.- Physical methods: Quantitative nucleic acid-based assay, such as: PCR can be used to determine the number of viral genome copies  Infectious and non-infectious genomes get detected Serological tests: many tests cannot distinguish between infectious and non-infectious  EX: Radioimmunoassay and immunosorbent assay to quantify the amount of virus in sample. May also, incorrectly, detect the unassembled proteins. © McGraw-Hill Education, 2019 From: https://embryo.asu.edu/pages/radioimmunoassay-image By: Madeleine Howell-Moroney Published: 2022-01-24 © McGraw-Hill Education, 2019 29.7: Cultivation and Detection Hemagglutinin assays may provide ability to quantify and separate infectious vs non-infectious particles of these viruses. Electron microscope, virus particles can be counted, but need a large amount and cannot distinguish between particle types. © McGraw-Hill Education, 2019 29.7: Cultivation and Detection B. Biologic Methods End point biologic assays: use dilutions of the virus from the tissue culture. 1. Titer is expressed as 50% infectious does (ID 50) (reciprocal dilution of virus that makes infection in 50% of cells inoculated. © McGraw-Hill Education, 2019 29.7: Cultivation and Detection B. Biologic Methods Plaque Assays Used for viruses that grow well in tissue culture. Monolayers of host cell are inoculated and overlaid with medium made of agar to prevent virus spreading. After several days virus will be produced in initial cells and spread to surrounding. Several rounds of killing and replicating causes plaques. The plaque formations vary depending on virus replication cycle. Faster method: immunofluorescence with a viral antigen to “tag” the infected cells © McGraw-Hill Education, 2019  29.8: Purification and Identification of Viruses Purification of Virus Particles 1. Starting material: large volume of tissue culture medium 2. Body fluids or infected cells 3. Concentration of virus by precipitation (sulfate, ethanol, etc) or by ultrafiltration 4. Virus can be separated from host material by differential centrifugation, density gradient centrifugation, column chromatography, electrophoresis 5. Usually need more than one step for good purification. Note final step always involves the density gradient centrifugation © McGraw-Hill Education, 2019 Purification process example A simplified purification protocol for recombinant adeno-associated virus vectors - PMC (nih.gov) © McGraw-Hill Education, 2019 29.8: Purification and Identification of Viruses Facts to keep in mind: Icosahedral viruses are easier to purify than enveloped ones Enveloped viruses are heterogeneous in size and density Very difficult to purify viruses completely © McGraw-Hill Education, 2019 29.8: Purification and Identification of Viruses Identification of a Particle as a Virus Criteria when identifying it: 1. Particle only obtained from infected cells or tissues. 2. Particles are identical regardless of cellular origin in which virus is grown. 3. Particle contains (DNA or RNA) and the sequence is not the same as host. 4. Degree of infective activity of preparation varies with number of particles present. 5. Destruction of the particle by chemicals or physical means is associated with loss of viral activity. © McGraw-Hill Education, 2019  29.8: Purification and Identification of Viruses Criteria when identifying a virus particle continued… 6. Infectivity properties must be shown identical- sedimentation behaviour, ultracentrifuge or pH curve within the particles. 7. Antisera (blood serum with antibodies against specific antigens) should react with characteristic particle and vise versa. 8. The particles should be able to induce the characteristic disease in vivo (if such experiments are feasible). 9. Passage of particles in tissue culture should cause ‘offspring’ with antigenic properties of virus. © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Heat and Cold Large variability in heat stability of viruses Icosahedral: tend to be stable even at 37°C for long periods Enveloped: much more impacted, titer drops rapidly at 37°C. Most viruses’ infectivity is lost at 50-60°C for 30 min (except hep. B and polymaviruses) Can be stored at subfreezing temperatures Some enveloped viruses lose infectivity at -80°C if prolonged storage © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Stabilization of Viruses by Salts and pH impacts Can be stabilized with salts to resist heat inactivation (need for making vaccines). Example: Polio vaccine can maintain stability for weeks at varying temperatures if stabilized with salts. pH Most viruses stable between pH of 5.0-9.0 Exception: enteroviruses can be resistant at acidic conditions All viruses are destroyed by alkaline conditions. © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Radiation Ultraviolet, x-ray and high-energy particles will inactivate viruses Dose varies depending on virus Infectivity is the most sensitive to radiation because replication requires the entire genetic contents to be expressed Irradiated particles cannot replicate, but may express some function in host cells. © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Ether Susceptibility Ether is used to distinguish if a virus does or does not have an envelope Detergents Nonionic detergents solubilize constituents of viral membranes Viral proteins in envelope get released Anionic detergents solubilize viral envelopes and disturb capsids into polypeptide pieces © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Formaldehyde Destroys viral infectivity by interfering with the nucleic acid Single strand viruses are more easily inactivated this way Only minimal effects on antigenicity of proteins, good for vaccine production © McGraw-Hill Education, 2019  29.10: Reaction To Physical and Chemical Agents Photodynamic Inactivation Dyes can penetrate viruses by varying degrees (toluidine blue, neutral red, etc) Dyes bind to viral nucleic acid and can become inactive by visible light Adding Antibodies To Enhance Photodynamic Therapy for Viral and Bacterial Disease - AIP Publishing LLC © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Antibiotics and Other Antibacterial Agents Antibacterial and sulfonamides do not have any effect on viruses. Will cover antivirals next chapter! Alcohols/Iodine are relatively ineffective to kill viruses, and large concentrations of chlorine treatments must be used. © McGraw-Hill Education, 2019 29.10: Reaction To Physical and Chemical Agents Common Methods of Inactivating Viruses for Various Purposes Variety of methods used to kill or inactivate viruses depending on the surface, material, etc. (safe for skin, lab equipment, drinking water) Need for creating vaccines with inactivated viruses Sterilization: steam pressure, dry heat, ethylene oxide, γ-irradiation Surface disinfectants: sodium hypochlorite, glutaraldehyde, formaldehyde, and peracetic acid. Vaccine production: formaldehyde, psoralene + UV irradiation or detergents. Skin: chlorhexidine, 70% ethanol and iodophors © McGraw-Hill Education, 2019 Questions? :D © McGraw-Hill Education, 2019