LABORATORY DIAGNOSIS OF VIRAL DISEASE Approaches: Cell Culture Microscopic identification Serologic Viral antigen Viral nucleic acid

acid Cultivation and Assay of Viruses Cultivation Viruses are obligate intracellular parasites, thus require living cells for propagation Cultivation of viruses ranges from simple (cell culture, embyonated eggs), difficult (whole animal) to impossible (unculturable) Cell culture methods Primary cell culture Uses freshly explanted tissues from animal Cells are dissociated from one another by trypsin a pancreatic protease cleaves at arginine and lysine residues does not cross cell membranes, thus remains extracellular

Primary cell cultures eventually undergo senescence (natural death)

Transformed cell lines Immortal Often easier to grow than primary lines Often aneuploid Cannot be used for vaccine production

Embyonated chicken eggs Specific pathogen free (SPF) live chicken eggs Inoculation of chorioallantoic membrane (CAM) or yolk

Live animal If virus cannot be propagated in cell culture or eggs, it must be grown in a living animal After virus replicates, the target organ must be collected, pureed and centrifuged to collect virus homogenate Cannot be used for vaccines (except vaccinia virus for smallpox)

Detection of viral infection Cytopathic effect (CPE) occurs with most viruses Cell death Irregularly-shaped cells Syncytia formation - fusion of cell membranes resulting in multinucleated giant cells Inclusion bodies - viral proteins aggregate within cells and become visible under the microscope Cytoplasmic or nuclear Virus factories

results in a single cell suspension Cells can be plated (or seeded) in a variety culture vessels Primary cells will grow to confluence (aka monolayer), then stop dividing (termed contact inhibition) After passage (or splitting) of cultures, cells will resume division Normal primary cells are sometimes referred to as diploid cells

Quantitation of viruses Immunological Enzyme-linked immunosorbant assay (ELISA) Hemagglutination/Hemagglutination inhibition assays

Remove liquid media and replace with media in a semi-solid form, such as agarose or methylcellulose Restricts virus infection from one cell to adjacent cells only

Allow infection to proceed for sufficient number of days Stain cells with formaldehyde containing crystal violet Select the dilution with 10 to 100 plaques and count Calculate mean of counts Calculate titer based upon log dilution and number of plaques

Molecular - Real-time polymerase chain reaction Biological (bioassay) Relies upon the ability of a virus to induce CPE in cell culture or embryonated eggs Uses limiting dilution (dilution to extinction) to determine virus numbers The dilution that infects (or kills) 50% of cells is termed tissue culture infectious dose-50 (TCID50) For eggs, EID50 For animals ID50 or LD50 (lethal dose)

Microscopic Identification Fluorescent Antibody Test (FAT) Radioimmunoassay (RAI) Enzyme-linked immunosorbent Assay(ELISA) Immunoelectron microscopy (IEM)

These assays all provide a titer of the virus Titer is the unit used to describe the greatest dilution of a substance (e.g., virus, antibody) that contains biological activity It is usually reported as the reciprocal of this dilution (10-6 activity has a titer of 6)

Light MICROSCOPY Inclusion bodies Multinucleated giant cells TZANCK SMEAR

UV MICROSCOPY ELECTRON MICROSCOPY Cell Culture Cytopathic effect(CPE) Hemadsorption Interference Decrease in acid production phenol red

Plaque Assay Method Some viruses lyse infected cells The plaque assay allows one to count plaque forming units (pfu) as a quantitative measure of infectious virus Basic protocol Allow susceptible cells to monolayer on 12 or 6 well plates Add 1 ml of log10 dilutions of virus to cells in duplicate and incubate 1 hour to allow virus to adsorb to cells

Complement Fixation Hemagglutination Inhibition Neutralization

SEROLOGIC DETECTION Serologic conversion Acute phase Convalescent phase (10-14 days) Heterophile antibody test

Determining copy number by real-time PCR In separate tubes, include plasmid standards with the target viral gene in known copy numbers (e.g. 102, 104, 106 copies) These provide data for generating a standard curve, which can then be used to quantify copy number in test samples using statistical algorithms, such as linear regression

Real-Time PCR Detection of Viral Nucleic Acids Some viruses are unculturable (i.e., can only be propagated in animals), so molecular techniques must be used for quantification Real-time PCR is one such method and relies upon fluorescence of DNA It requires known nucleic acid sequence for the virus For RNA viruses, the RNA must first be copied into cDNA It also requires gene-specific DNA primers

Purification and Identification of Viruses A common method of virus purification is precipitation with polyethylene glycol at 4 C overnight Once precipitated, the virus can be washed with a buffer to remove PEG

Identification of an unknown virus isolate Morphology by EM DNA or RNA? Enveloped? Reactivity against defined antisera CDC and other institutions have panels of neutralizing antisera raised against hundreds of known viruses These antibodies can be used to identify viruses in cell culture assays

Basic procedure Extract DNA or RNA from a virallyinfected tissue or blood If RNA, copy into cDNA using reverse transcriptase

Add DNA, forward and reverse primers, and real-time PCR mix (containing Taq polymerase, dNTPs and SYBR Green I dye) and conduct PCR reaction for 50 cycles SYBR Green 1 is a fluorescent dye that binds only to doublestranded DNA, such as PCR products

Laboratory Safety Laboratory safety is essential when working with pathogenic microbes Safety is based upon three levels of practice that ensure containment Proper technique Proper equipment Biosafety cabinets Centrifuge containment

After each cycle, the instrument reads the PCR tube for its fluorescence and records it. After 50 cycles there are 50 data points that are used to generate a graph of DNA abundance

Policies and procedures for preventing and managing breaks of containment

Aerosols and punctures are the principal threats Ingestion and splashes are less common

Biosafety practices Training in aseptic technique No mouth pipetting No eating, drinking, smoking or application of makeups or balms in the laboratory Use appropriate protective clothing and equipment (lab coat or gown, gloves, mask) Sterilization of infectious wastes Use of biosafety cabinets Immunization if necessary

Reaction to Physical and Chemical Agents Heat and cold - heat inactivates some viruses, while cold usually preserves them Stabilization by salts - unknown how this works pH - some viruses are resistant to dramatic changes in pH Radiation - damages nucleic acids; crosslinks viral proteins Photodynamic inactivation (e.g., neutral red) renders some viruses susceptible to visible light Ether susceptibility - ether is an organic solvent, thus damages envelop membranes of viruses Detergents - amphipathic, thus solublizes membranes and can dissociate noncovalent bonds between viral proteins Formaldehyde - cross-links nucleic acids and proteins