Table 3.
Examples of assays for the verification of replication competent virus presence
| Classification | Family | Virus (wild type or vaccine strain) | Assay for RCV | Results | Detection limit | References |
|---|---|---|---|---|---|---|
| Positive-sense nonsegmented | Flaviviridae | Kunjin virus | Microscopy (detection of E protein by immunofluorescence during 2 passages in Vero cells) | No fluorescence detected | Not described | 76 |
| Intracranial inoculation of suckling mice | No clinical signs upon inoculation with 4 × 106 IU | Not described | ||||
| Picornaviridae | Poliovirus | Intraspinal or intracranial inoculation of mice | No neurovirulence | Not described | 125,126 | |
| Togaviridae | SFV | Microscopy (infect BHK-21 cells and titrate supernatant in a PFU assay) | No PFU found in 4.6 × 109 particles from 13 independent SFV-lacZ recombinant stocks (frequency of recombination <4.6 × 109) | Not described | 127 | |
| VEEV | Microscopy (detection of syncytia upon 2 passages in Vero cells) | No RCV detected | 1 RCV/1 × 1010 gm-replicon particles | 128 | ||
| Microscopy (detection of syncytia upon 2 passages in Vero cells) | No RCV detected | <100 RCV/1 × 108 gm-replicon particles | 48 | |||
| Chimeric | SINV/VEEV | Microscopy (detection of syncytia upon 5 passages in BHK cells) | No RCV detected | Not described | 68 | |
| Reporter assay (test supernatant of 5 passages in BHK cells for β-galactosidase expression) | No RCV detected in 40 batches | Not described | ||||
| Negative-sense nonsegmented | Paramyxoviridae | Sendai virus | Microscopy (detection of syncytia) | Absence of syncytia | Not described | 37 |
| qPCR (detection of NP mRNA) | Negative | 2–3 Positive cells | ||||
| Fluorescence microscopy (detection of fluorescent-labeled antibodies to NP) | No fluorescence detected | Not described | ||||
| Negative-sense segmented | Phenuiviridae | Rift Valley fever virus | Microscopy (detection of fluorescence during 2 passages in BHK cells) | No fluorescence detected upon inoculation of cells at an m.o.i. of 1 | Not described | 72 |
| Inoculation of suckling mice | No clinical signs upon inoculation with 1.0 × 104 TCID50 | Not described | 129 |
BHK, baby hamster kidney; E, envelop; gm, genetically modified; IU, infectious units; m.o.i., multiplicity of infection; mRNA, messenger RNA; NP, nucleoprotein; PFU, plaque-forming units; qPCR, quantitative polymerase chain reaction; RCV, replication competent virus; TCID, tissue culture infectious dose.