Retrovirus Detection

Three techniques are recommended to test samples for retroviruses:

  • Infectivity assays
  • Transmission Electron Microscopy (TEM)
  • Product-Enhanced Reverse Transcriptase (PERT) assays

As specified in the “Points to Consider on the Characterization of Cell Lines Used to Produce Biologicals” and in the international “Note for guidance on Quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin” (CPMP/ICH/295/95).

Infectivity assays

The major focus of infectivity assays is on the use of murine cell lines or hybrid cell lines containing a murine component. These cell lines are inherently capable of producing infectious mouse retroviruses.

Texcell provides sensitive protocols for detecting a wide range of retroviruses such as ecotropic, xenotropic, amphotropic and Mink Cell Focus-forming (MCF) retroviruses.

For the detection of retroviruses, two kinds of protocol exist:

  • The direct focus assays–using one detector cell line–enable direct detection of retrovirus particles.
  • Extended assays use an initial cell line to amplify the retrovirus with three to five passages, followed by a direct focus assay.

In the table below, the detector cell line is modified with sarcoma-positive (S+) and leukaemia-negative (L-) isotypes in order to obtain foci showing the presence of the retrovirus in the cells during the focus assay.

Detector Origin Assay Retrovirus Susceptibility
Fg10 S+L- Murine Direct focus assay Murine ecotropic retroviruses (including Mo-MuLv)
PG4 S+L- Feline Direct focus assay Murine xenotropic/amphotropic/MCF retroviruses
NIH3T3*
Fg10 S+L-
Murine
Murine
Extended assay with three passages followed by a focus assay Murine ecotropic retroviruses (including Mo-MuLv)s
Mus dunni*
PG4 S+L-
Murine
Feline
Extended assay with three passages followed by a focus assay Murine xenotropic/amphotropic/MCF retroviruses
NIH3T3*
Fg10 S+L-
Murine
Murine
Extended assay with five passages followed by a focus assay Murine ecotropic retroviruses (including Mo-MuLv)s
Mus dunni*
PG4 S+L-
Murine
Feline
Extended assay with five passages followed by a focus assay Murine xenotropic/amphotropic/MCF retroviruses

Transmission electron microscopy

Transmission Electron Microscopy (TEM) is commonly used to detect retroviruses in test articles and to type retrovirus particles (A, B, C or D) in cell lines.

TEM can also be used to characterize cell lines and to detect other contaminants such as Mycoplasma, bacteria, fungiyeast and other viruses.

Moreover, TEM is an indispensable to check on the health of the cells.

To perfectly fits with your study, Texcell provides cutom-made protocols in which an indicator cell line is inoculated with the test product.

F-PERT assay

When TEM and infectivity assay results are negative, test samples should be confirmed to be free of retrovirus particles by the absence of reverse transcriptase activity.