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.
 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