Summary auto-generated
This study describes persistent infection of MDCK cells with influenza virus A/PR/8/34 (H1N1). When semiconfluent MDCK cells were infected at high multiplicity, most cells survived and continued growing stably in medium containing 50% fetal calf serum, spontaneously shedding virus that gradually declined with passage number. Virus shedding increased significantly when cells were maintained in low-serum medium (0.2% bovine serum albumin). PCR analysis demonstrated that viral mRNAs for all eight influenza genes were constitutively transcribed within persistently infected cells (designated P-CK cells). However, viral proteins were undetectable by radioimmunoprecipitation assay, and viral genomic RNA was not detected by ribonuclease protection assay, suggesting very low levels of viral protein synthesis. The persistent viral genes could be induced to produce virus until day 40 post-infection when cells were maintained in antiserum-containing medium. The researchers propose that endogenous viral genes are regulated to express at levels sufficient for self-amplification and maintenance within dividing cells, but insufficient to cause cell lysis or be readily detectable by conventional assays. These findings suggest that host cell factors expressed during growth suppress viral gene function, allowing long-term persistence of influenza virus genome.
Key findings
- MDCK cells infected with influenza A/PR/8/34 at high multiplicity established persistent infection with stable cell growth and spontaneous virus shedding that declined over passages
- Viral mRNAs from all eight influenza genes were constitutively transcribed in persistently infected cells, but viral proteins and genomic RNA were not detectable by conventional assays
- Virus shedding was significantly enhanced when persistently infected cells were cultured in low-serum medium (0.2% BSA) compared to high-serum medium (50% FCS)
- Host factors expressed during cell growth appear to suppress viral gene function, allowing persistent viral genome maintenance without cell lysis
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Abstract
When MDCK cells in a semiconfluent monolayer were infected with 5 p.f.u. per cell of influenza virus A/PR/8/34 (H1N1), a majority of the cells continued to grow stably upon subsequent cultivation with a growth medium containing 50% foetal calf serum. While growing, the cells spontaneously excreted virus, the amount of which declined gradually as the passage number of the cells increased. The extent of virus shedding was significantly increased when the cells were subsequently maintained in a medium containing 0.2% bovine serum albumin. Within the cells, viral messenger RNAs for all eight genes of A/PR/8 were demonstrated by PCR indicating that endogenous viral genes were constitutively transcribed. However, viral proteins as well as viral genes were not demonstrable by radioimmunoprecipitation or ribonuclease protection assays, respectively.