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Clinical Virology: Part One Introduction

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.

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Clinical Virology: Part One Introduction

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  1. Clinical Virology: Part OneIntroduction MLAB 2434 – Microbiology Keri Brophy-Martinez

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

  3. 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

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

  5. 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)

  6. 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)

  7. Viral Reproduction (Replication) (cont’d)

  8. 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

  9. 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

  10. 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

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

  12. 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

  13. 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

  14. 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

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

  16. 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

  17. 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

  18. 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

  19. 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. • http://www.fifthdisease.org/general.html • http://www.idph.state.il.us/about/immunepics/measles.htm • http://www.idph.state.il.us/about/immunepics/mumps.htm • http://www.mc3cb.com/viruses.html

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