Virology

Classification and Structure of Viruses

• Viral genomes are classified in the following ways:
  • Single- or double-stranded DNA
  • Single- or double-stranded RNA
    • Positive-sense (can directly encode protein) or negative-sense (needs to be transcribed first)
• Viral structures are organized in the following ways:
  • The most minimal viral unit is the nucleocapsid, which is composed of a viral capsid that encloses a core of nucleic acid associated with a nucleoprotein
    • The protein capsid, usually composed of multimers of capsomeres
    • Capsids have icosahedral or helical symmetry
  • Some viruses have an outer envelope composed of phospholipid, cholesterol, glycoprotein, and glycolipid that is derived from infected cell membranes
    • Matrix or tegument proteins fill the space between the nucleocapsid
    • Enveloped viruses are usually sensitive to lipid solvents or detergents, which dissolve the envelop, while unenveloped viruses are usually more resistant

Specific Viruses

• Understand the natural history and diagnosis, including pre-analytical, analytical, and post-analytical concepts, as well as epidemiology, pathogenicity, clinical characteristics, and treatment
• Respiratory viruses
  • Influenza A and B
  • Parainfluenza
  • Respiratory syncytial virus
  • Adenovirus
  • Rhinovirus/Enterovirus
  • Coronaviruses including MERS
  • Metapneumovirus
• Gastrointestinal
  • Enteroviruses
  • Rotavirus
  • Norovirus
  • Astrovirus
  • Adenovirus
• CNS
  • Herpes viruses
  • Enterovirus
  • Arboviruses
• Rash
  • Measles, mumps, and rubella
  • Parvovirus B19
  • HSV/VZV: either PCR or Tzanck smear
  • HHV 6 and 7
• Hepatitis
  • A, B, C, D, E
  • EBV and CMV
• Herpes viruses
  • Herpes simplex (HSV 1 and 2)
  • Varicella zoster
  • Epstein-Barr virus
  • Cytomegalovirus
  • HHV 6 and 7
  • HHV 8
• Arboviruses
  • Western and Eastern equine encephalitides
  • Venezuelan equine encephalitis
  • St. Louis, California encephalitis, Powassan
  • Japanese B encephalitis
  • Dengue, Yellow fever
  • West Nile virus
  • Chikungunya virus
  • Zika
• Retroviruses
  • HIV 1 and 2
  • HTLV 1 and 2
• Other
  • Rabies
  • HPV
  • Polyomaviruses (JC and BK)
  • Viral hemorrhagic fevers

Specimen Collection and Preparation

Specimen types

Specimen	Notes
Serology	Acute/convalescent collected at initial assessment and minimum of 2 to 3 weeks later
NP aspirate	Better than swabs for detecting viruses. Requires a mucous trap and vacuum source.
NP swab	More convenient than aspirate
Urine
Stool
Lesion swab	Unroof lesion and swab the base (rather than attempting to collect the vesicle fluid)
Biopsy
Urethral swab
Cervical swab
CSF

Transport media

• A transport medium is a solution that contains water, buffer, and salt (no nutrition)

Media	Used for…
Viral transport medium	VTM is ideal for diagnosis of viral infection. Ocular, respiratory and tissue swabs can be submitted in this medium. Fluid samples such as tracheal wash specimens or peritoneal fluid should be submitted as is, in sterile vials which prevent desiccation. In the absence of viral transport medium, submit swabs in sterile, sealed vials with several drops of saline added, to prevent desiccation. Cotton, plastic, wood-handled, and Dacron and other synthetic swabs are all acceptable. Calcium alginate swabs should be avoided. Bacterial transport medias are not appropriate for virology.
Universal transport medium	UTM is a VTM, and is used to viruses as well as atypical bacteria (Chlamydia, Mycoplasma, and Ureaplasma)
Plain/routine transport medium	For routine bacterial culture and sensitivity.

• Samples should be processed within 12 to 24 hours, and can be stored at 4ºC for up to 5 days (or -20 to -70 ºC for longer)

Specimen Processing

Method	Pros	Cons
Cell culture	Relatively sensitive, can detect many different viruses, and allows for further characterization (e.g. susceptibilities, serotyping, genotyping)	Many viruses are difficult to culture (slow or no growth), it's relatively expensive
Electron microscopy
Serology		Often cross-reacts with multiple infections (i.e. non-specific). May not be detectable at initial visit (so may not be helpful in guiding treatment decisions).
Nucleic acid testing	Sensitive, specific, rapid, and cheap	Usually no information about susceptibility. Needs specific primers/probes to be developed and validated.

Cell culture

• Three categories of cell cultures:
  • Primary cells: derived from tissue and have been propagated in vitro for the first time
  • Diploid/semi-continuous cells: develop during subculture of primary cells; 75-100% have the same karyotype as the progenitor cell
  • Heteroploid/continuous cells: a cell line that can survive indefinitely; less than 75% have diploid chromosome
• Culture in multiple cell lines are required to identify the virus in a given sample, since each is best at growing a small number of viruses

Culture type	Viruses
Primary
Monkey kidney	Influenza, parainfluenza, enteroviruses
Rabbit kidney	Herpes simplex
Human embryonic kidney	Enteroviruses, adenoviruses
Diploid
Fibroblast	CMV, VZV, HSV, adenoviruses, rhinoviruses, some enteroviruses
Continuous
HEp-2	RSV, adenoviruses, HSV, some enteroviruses
A549	Adenoviruses, HSV, some enteroviruses
LLC-MK2	Parainfluenza, HMPV
MDCK	Influenza
RD (rhabdomyosarcoma)	Echoviruses
Buffalo green monkey kidney	Coxsackieviruses

Electron microscopy

• Principles of EM for viruses
• Morphology of common viruses in skin lesions and stool

Antigen detection

• Principles of antigen detection, perform DFA, recgnoze positive specimen with typical pattern of
  • Resp: influenza, parainfluenza, RSV, adeno
  • Herpes: CMV, HSV, VZV
• Performance of antigen tests where commercial tests are available including rotavirus EIA, RSV antigen, influenza rapid antigen tests

Serology

• Types of serological tests available
  • Immunofluorescent assay (IFA): slide with organism, add serum and conjugated antibody (e.g. HSV, VZV, measles, mumps, parvovirus)
  • Enzyme immunoassay (EIA): particle coated with antigen, can be automated system (IMx) (e.g. HIV, MEIA for rubella)
  • Complement fixation (CF) with paired sera (historical) (e.g. influenza, RSV, adeno, measles, rubella)
  • Latex agglutination (LA): slide agglutination (e.g. CMV, rubella)

Nucleic acid testing

• Generally sensitive (from amplification) and specific (from hybridization), but lack information about serotyping and susceptibilities
• Some methods need NA extraction (e.g. PCR): release the NA from cells, stabilize the NA, remove inhibitors like herparin, concentrate the NA, and suspend in an aqueous solution
  • Currently automated
• Methods
  • Restriction fragment length polymerization (RFLP): encoducleases chop up the DNA, it gets run on a gel (pulse gel electrophoresis), and the pattern of bands can be compared to a known organism.
  • Probe hybridization: uses a probe to detect a complementary sequence in the DNA. E.g. FISH, Southern/Northern blots, microarrays, microtitre ELISA.
  • Amplification:
    • Non-isothermal (PCR): requires purified nucleic acid, and needs a reverse transcriptase step to process RNA. Outcome is usually the number of cycles to reach a prespecified threshold (CT). Can be used as an input for sequencing.
    • Isothermal:
      • Loop amplification (LAMP): runs at continuous 65ºC with a Bacillus stearothermophilus polymerase. Doesn't need nucleic acid purification. Uses 4 to 6 primers per target to create a very long unbroken chain of DNA, rather than replicating a single sequence. Cannot be used as an input for sequencing.
      • Others: strand-displacement amplification (SDA), transcription-mediated amplification or nucleic acid sequence-based amplification (TMA/NASBA), and rolling signal amplification (RCA)
    • Amplification signal may be turbidity, SYBR green, magnesium-related, or other. These are all designed to quantify double-stranded DNA. When paired with hybridization probes, multiple signals can be used, one for each target.
  • Sequencing: current techniques still fairly limited and expensive. e.g. PCR of bacterial 16S followed by sequencing for identification.

Viral Loads and Susceptibility Testing

• Understand the principle and availability of antiviral susceptibility tests for HSV, CMV, and influenza virus
• Understand the methods and limitations in testing viral load, including CMV, EBV, HIV, HBV, HCV
• Understand the clinical circumstances when viral susceptibility testing should be ordered
• Mechanism of resistance to antiviral agents and the clinical significance of specific mutations in the HIV rt-ase and protease for HIV resistance
• Mode of action of novel HIV antivirals including fusion inhibitors (enfuvurtide), CCR5, and integrase inhibitors
• Understand the principles of CMVC and HCV