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Chapter 5
Kochs Postulates
1. Organism present only in diseased individuals 2. Organism cultivated in pure culture from diseased individual
Kochs Postulates
3. Organism causes disease when injected into healthy individuals 4. Organism re-isolated from infected individual from point 3.
Figure 1.15
Rivers Postulates
T.M. River, 1937 Modified from Kochs Postulates (proof of bacterial diseases) Isolate virus from diseased hosts. Cultivation of virus in host cells. Proof of filterability. Production of a comparable disease when the cultivated virus is used to infect experimental animals. Reisolation of the same virus from the infected experimental animal. Detection of a specific immune response to the virus.
1. 2. 3. 4. 5. 6.
Viruses need a host system. Viruses can be grown in: Animals Embryonated eggs Tissue (cell) cultures (preferred method)
Cytopathic Effects
Visible results of viral infection Cell death by Multiplying viruses Inhibition of DNA, RNA or protein synthesis Effects on permeability of membrane Cytopathic effects (CPEs) of infected cells can be observed with inverted light microscopes Rounding/detachment from plastic flask Syncytia/fusion Fusion of cells Shrinkage Increased refractility Aggregation Loss of adherence Cell lysis/death Common observations of CPEs
Hemadsorption assays
Hemadsorption Test
Clumps RBCs
Common Methods
Four methods:
Quantitative
assays
Quantitative Assays
Plaque assays Lytic viruses only Steps Serial dilution of virioncontaining solution Create tissue culture plates Spread diluted virus Overlay with agar prevents diffusion Count number of plaques Each plaque represents 1 PFU (Plaque Forming Unit)
Quantitative Assays
cytopathic effects other than lysis Concentration of virus it takes to produce cytopathic effect (CPE) in 50% of the dishes of cells infected with virus
TCID50 Assays
Transformation Assay
Also called focus assays For non-lytic, oncogenic viruses Immortalization of cells in culture Transformed cells are viral infected cells Transformed cells show other cytopathic effects Example: Loss of contact inhibition Rather than count plaques; count foci Focus: when cells pile up (tumor formation) Focus of infection = 1VIU
Over 60% of all infectious disease cases seen by a physician are due to viral infections. Quality of patient specimens and their transport to the laboratory is important.
Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum
Eye infections: throat and conjunctival swab/scraping Gastrointestinal tract infections: stool and rectal swabs
Direct detection
Microscopy
or staining
Virus Isolation
PCR
Serology
Antibodies
Direct Detection
Electron Microscopy
Examine
specimen for
viruses
Immuno-electron microscopy
Labeled
antibody
Immunoflourescence
Fluorescent
Virus Isolation
Nucleic
acid methods PCR (DNA), RT-PCR (RNA) Can be used to detect viruses that are noncultivatable Rapid identification (e.g. RT-PCR4 Corners outbreak of hantavirus or FRET in the field) Can be used to manage patients (e.g. HIV viral load)
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
Virus Isolation
Figure 5.17a: Tissue culture cells are grown on coverslips on the bottom of shell vials.
Reproduced from Athmanathan, S., S. R. Bandlapally, and G. N. Rao, BMC Clin. Pathol. 2 (2002): 1-5.
Figure 5.17b: Detection of Herpes Virus Simplex 1 using the shell vial technique and immunofluorescence.
Modified from J. H. Shelhamer, et. al., Ann. Intern. Med. 124 (1996): 585-599.
Viral Serology
Indirect
Primary
and secondary responses to viral infections IgM (1st exposure) IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.
Viral Serology
HIV test
ELISA Procedures
Figure 5-19a
Figure 5-19b
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000.
Viral Serology
Western Blotting
Viral
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.
Figure 5.21a: The basic principles behind the Western blotting procedure.
(c)
Image courtesy of Bio-Rad Laboratories
Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.
Laboratory Safety
Biosafety
Level (BSL) laboratories BSL-1 (minimum containment) for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans agents present minimal potential hazard to lab personnel and the environment. does not require a special containment equipment or facility design BSL-2 for work involving agents that pose moderate hazards restricted access to the lab procedures conducted in BSCs or other physical containment equipment if there is a potential for infectious aerosols or splashes
Safety requirements paraphrased from CDC publication: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm/
BSL-3
for
work performed with agents that may cause serious or potentially lethal disease through inhalation route exposure requires protective eye, face, clothing, gloves a series of two self-closing doors restricts access to the lab sealed windows, seams, floors, walls, and ceiling surfaces a closed air ventilation system HEPA filtration
Figure 5.25a: A CDC researchers working in a BSL-3 lab on highly lethal influenza strains.
BSL-4 (Suit Lab-maximum containment) for work with extremely dangerous and exotic agents that pose a high risk of life-threatening disease, aerosol transmission, or with unknown risk of transmission. special training and authorized entry for lab staff a time of entry and exit logbook lab is sealed completely; entry is through airlock fitted with airtight doors lab has clean-side and dirty-side clothing change and shower rooms personal clothing is removed in outer clothing change room and staff wears lab undergarments, pants, shirts, jumpsuits, shoes, and gloves lab staff wears positive pressure protective suits with redundant life support systems uses Class III biological safety cabinet to handle agents automatically-activated emergency power for the lab exhaust system, life support systems, alarms, lighting, entry and exit controls, BSCs, and door gaskets. multiple HEPA filters lab maintained at negative pressure to surrounding areas
Courtesy of CDC
Figure 5.25c: A CDC scientist showers in a protective suit before leaving a BSL-4 laboratory.
Courtesy of CDC