After inoculating cells with a new batch of fetal bovine serum, vacuoles appeared. How to investigate suspected virus contamination?

Don't jump to conclusions just yet. Vero cell spindle formation and vacuolation may also be related to mycoplasma, pH, osmolality, or batch-to-batch differences in serum composition. It is recommended to first rule out mycoplasma (PCR or Hoechst staining can be used), and at the same time send three samples to a third party: the new serum batch, the abnormal culture supernatant, and the old serum batch. For viruses, prioritize testing for BVDV, BPV, Reo-3, adenovirus, and parvovirus. Vero cells are not necessarily the most sensitive to BVDV, so MDBK/BT cells can be added for virus isolation. For sample submission, at least 10-20 mL per sample is recommended, and additional volume should be reserved for cell culture methods. qPCR plus cell culture is generally sufficient, but for cell culture it is best to perform 2-3 blind passages before judging a negative result. Rapid kits can only be used for internal preliminary screening and cannot serve as a formal conclusion.

If the old and new batches are of the same brand but show significant differences in results, it is recommended that the supplier provide the COAs for both batches and information on whether the testing methods have changed. The new batch of serum can first be filtered through 0.1 μm and then inoculated into MDBK, BT, and Vero cells, with 3 blind passages to observe CPE, while performing qPCR for two targets, BVDV 5'UTR and NS3, to avoid missed detection with a single target. For adenovirus and Reo-3, PCR screening followed by cell culture confirmation is also recommended. Samples should preferably be retained as 20–30 mL from the original unopened bottle, plus 10 mL each of pre- and post-filtration culture supernatant. If a rapid kit is used to test for BVDV antigen, it can serve as an internal trend reference but cannot replace confirmatory testing. If a third-party report is negative while cells still show abnormalities, it is also necessary to go back and check the culture medium, trypsin, and consumables for mycoplasma.

In this situation, it is recommended to first stop using and seal the new batch of serum, and restore the old batch in the cell room to avoid spreading contamination. Do not rush to discard the abnormal cells; collect the culture supernatant and cell pellet and aliquot them at -80°C. Keep at least 50 mL of the unopened new batch of serum, divided into 3–4 tubes, for nucleic acid testing, virus isolation, and arbitration re-testing, avoiding repeated freeze-thaw cycles. For testing, prioritize BVDV, BPV, Reo-3, and adenovirus; if bovine-derived material audits are involved later, it is recommended to also include parvovirus. Rapid test cards can be used for initial BVDV antigen screening, but their sensitivity is limited, and a negative result cannot rule it out; the final determination should be based on third-party cell culture methods plus qPCR.

First rule out mycoplasma, then send for a viral panel, and retain samples of both cells and serum.

Vacuolation is not necessarily caused by a virus; incorrect osmotic pressure or pH can also cause it. Check the basic parameters first.