Laboratory Diagnosis of Viral Diseases

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.

Cultivation of Viruses in the Laboratory

Viruses need a host system. Viruses can be grown in: Animals Embryonated eggs Tissue (cell) cultures (preferred method)

Laminar Flow Hoods

Virologists facility Laminar vertical flow hoods Contains HEPA filter

Removes 99.97% of particles of 0.3M or higher

Figure 5-3: Vertical laminar flow hood.

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

Inclusion body formation

Intracellular virus parts (replication or assembly)

Hemadsorption assays

Hemadsorption Test

Some viruses agglutinate RBCs Mumps, measles, influenza Hemagglutination

Clumps RBCs

Common Methods

Four methods:
Quantitative

assays

Plaque assays TCID50

Hemmagglutination

assays Transformation 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)

Figure 5.8: Plaque assays used to quantitate a viral stock.

Courtesy of Teri Shors

Quantitative Assays

Tissue Culture Infectious Dose: TCID50

Measure

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

Viral Diagnostics in the Clinical Laboratory

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.

Storage and Collection of Biological Specimens for Viral Testing

What

types of specimens are collected to diagnose?

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

Vesicular rash: vesicle fluid, skin scrapings

Maculopapular rash: throat, stool, and rectal swabs CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid Genital infections: vesicle fluid or swab Urinary tract infections: urine Bloodborne infections: blood

Three General Approaches for Laboratory Diagnosis of Viral Infections

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

tag bound to Fc region of Ab

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

Centrifugation Culture (Shell Vial Technique)

Used

a lot in clinical labs

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

Enzyme-Linked Immunosorbent Assays (ELISAs)

Enzyme

reacts with substrate to produce colored

product Very sensitive

HIV test

If positive twice, Western Blotting is performed next

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.

 Hank Morgan/Science Photo Library/Photo Researchers, Inc.

Figure 5.20: HIV ELISA test.

Viral Serology

Western Blotting
Viral

proteins are separated in SDS-PAGE gel Transferred to a nitrocellulose filter

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.

(b) Figure 5.21b: The structure of HIV-1.

(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

Courtesy of James Gathany/CDC

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

Figure 5.25b: A CDC researcher working on a BSL-4 infectious agent.

Courtesy of CDC

Figure 5.25c: A CDC scientist showers in a protective suit before leaving a BSL-4 laboratory.
Courtesy of CDC