Clinical Virology: Part One Introduction MLAB 2434 – Microbiology Keri Brophy-Martinez

General Characteristics Obligate intracellular parasites Identified by either cell culture OR rapid tests from clinical specimens Enzyme Immunoassay (EIA) Immunofluorescence PCR/Nucleic Acid Probes

Structure of Viruses Contain a viral genome of either RNA OR DNA Genome can be double stranded (ds) OR single stranded (ss) Protein coat (capsid) Capsid + viral nuclei acid= nucleocapsid Genome + protein coat called a virion Some viruses have an envelope

Classification of Viruses DNA OR RNA Number of strands (ds or ss) Morphology Presence or absence of envelope

Viral Reproduction (Replication) Unique to viruses Virus attaches to surface of susceptible cell by specialized structures on specific receptors on the cell surface (ATTACHMENT) Virus enters cell by endocytosis (fusion of viral membrane & cell membrane) (PENETRATION)

Viral Reproduction (Replication) (cont’d) Inside the cell, virus loses protein coat, releasing DNA or RNA (UNCOATING) Viral genome directs host cell to make viral proteins and genome(ECLIPSE) Virus-coded proteins and genome re- assemble in host cell(ASSEMBLY) New virions released by host cell lysis OR budding from host cell membrane(RELEASE)

Viral Reproduction (Replication) (cont’d)

Specimen Collection and Transport Viral shedding greatest during early stages of infection, so specimens should be collected as early as possible Aspirates are best, but swabs are acceptable if dacron or nylon is used Calcium alginate/ cotton swabs inhibit growth of some viruses Commercial viral transport systems Provide moisture, prevent contamination, and preserve viral infectivity

Specimen Processing Optimal to process viral cultures immediately If impossible, store in refrigerator up to 48 hours If longer, freeze at -70° C -20° C will cause crystal formation, which disrupts host cells and results in significant loss of viruses

Methods in Diagnostic Virology Major methods to diagnose viral infections Direct detection of virus in clinical specimen Serologic antibody assays to detect viral antibodies Isolation of virus in culture Nucleic acid-based detection

Direct Detection Advantages Rapid diagnosis Detection of nonculturable viruses No need for culture Disadvantages Confined to specific virus Dependent of specimen adequacy and quality

Direct Detection (cont’d) Methods include Immunostaining/Immunofluorescence Enzyme Immunoassay Nucleic acid probes Gene amplification assays- PCR Electron microscopy looking for cell inclusions or cytopathic effects on cells

Serologic Assays Indications for serologic assays Diagnosis of infections with nonculturable organisms like hepatitis Absence of viral shedding Lack of available nucleic acid testing Determination of immune status (i.e. rubella, etc.) Monitoring immunosuppressed or transplant patients Epidemiologic studies

Serologic Assays Problems with serologic assays Measures host response rather than detect virus Antibody-producing capabilities of humans vary Antibody levels do not necessarily correlate with acuteness of infection

Viral Isolation Three methods Cell culture Animal inoculation Embryonated eggs Most cell cultures done for herpes and genital and respiratory viruses

Cell Cultures Once viruses are grown in cell culture, cells are examined microscopically for cytopathic effects (CPE) on cells Some viruses, such as influenza, do not cause CPE, so changes must be demonstrated with hemagglutionation or immunofluoresence tests

Types of Cell Culture Primary cell cultures Uses tissue from animals Seeded onto surface to form a monolayer Limited cell division Diploid cell cultures Cells can divide up to 50 times Human neonatal lung (HNL) is an example Continuous (heteroploid) cell cultures Cells are capable of unlimited cell division Derived from human cancer cells

Cell Cultures (cont’d) Advantages of cell culture Sensitive Can identify broad spectrum of viruses Disadvantages Time required for isolation and identification Viable organisms required Specialized resources and personnel needed

References Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach. Upper Saddle River, NJ: Pearson Education. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders