Viral culture principles and practice

Two main criteria for selection of specimen for viral cultures

• site of viral pathology

• site of viral shedding

Guidelines for specimen collection for virus isolation

• Timing

• Sterile sites and urine collected in sterile screw tops

• Non-sterile sires collected in VTM

Which types for viral isolation should be collected in VTM vs screw tops

VTM = non-sterile

Screw tops = sterile and urine

3 main purposes/reasons of using viral transport medium for virus culture samples

1. Prevent drying

2. maintain viability

3. inhibit growth of contaminants

four main components of viral transport medium

• Hanks balanced salt solution

  • keeps sample moist

• Proteinaceous supplement

  • cushions viruses

• Antimicrobials

  • inhibits growth

• pH indicator

  • signals out of range pH

Primary cell line

• fresh from tissue of origin

• passed 1-2 times

• indigenous contaminants

• ex: rabbit/mouse kidney cells

Diploid cell line

• derived from fetal tissue cells

• fibroblast cells

• limited passage ~50 times

• ex: fetal lung/fibroblast cells

Heteroploid cell line

• origin from carcinoma

• passed indefinitely

• ex: lung/epidermoid malignant cells

Location of the monolayer of living cells in a cell culture tube and a shell vial

Cell culture

• on the side of tube

Shell vial

• on the bottom

Rationale behind inoculation of more then one type of cell monolayer for most types of clinical specimens for viral culture

So you get multiple cells with the virus that can be tested and can determine the preferred cell line of that virus sample

Cell culture Incubation

• Temp = 35-37C

• Duration = 7-30 days

• Position = slanted

Shell vial incubation

• Temp = 35-37C

• Duration = 24-72 hours

• Position = upright

Cell culture media vs. viral transport media

Cell culture have the same as viral media but also

• amino acids

• glucose

• vitamins

Cytopathogenic effect (CPE)

degenerative changes in cell monolayer morphology

Syncytia

large multinucleated cell

Factors of predicting identity of virus

1. appearance of CPE

2. Time of appearance

3. Cell line

4. Source of sample

How to confirm viral identity

Direct immunofluorescence

basic steps of shell vial cell culture system

1. Inoculation

2. Centrifugation

3. Incubation

4. Viral detection

critical step in shell viral leading to increase of infectivity of viruses

Centrifugation for 1 hr on slow

greatest advantage for shell vial system over cell culture

rapid detection

R-mix shell vial

respiratory viruses

E-mix shell vial

Enteric viruses

H and V mix shell vial

HSV,CMV,VZV viruses

Polio virus

disease:

• poliomyelitis

replicates

• in cells of intestines

pathology manifested

• in CNS

Coxsakievirus A and B

Disease:

Symptoms:

Enterovirus

Disease:

Symptoms:

Polio virus

Disease:

Symptoms:

Rhinovirus

Disease:

Symptoms