Clinical Virology: Part OneIntroduction

MLAB 2434 – MicrobiologyKeri Brophy-Martinez

General Characteristics

Obligate intracellular parasites Identified by either cell culture OR

rapid tests from clinical specimensEnzyme Immunoassay (EIA) ImmunofluorescencePCR/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

infectionsDirect detection of virus in clinical

specimenSerologic antibody assays to

detect viral antibodiesIsolation of virus in cultureNucleic 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 assaysDiagnosis of infections with

nonculturable organisms like hepatitisAbsence of viral sheddingLack of available nucleic acid testingDetermination of immune status (i.e.

rubella, etc.)Monitoring immunosuppressed or

transplant patientsEpidemiologic studies

Serologic Assays

Problems with serologic assaysMeasures host response rather

than detect virusAntibody-producing capabilities

of humans varyAntibody levels do not

necessarily correlate with acuteness of infection

Viral Isolation

Three methodsCell culture Animal inoculationEmbryonated 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.

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