PMC:3537549 / 39802-46367 JSONTXT

{"project":"2_test","denotations":[{"id":"22906110-4308914-143530983","span":{"begin":1628,"end":1630},"obj":"4308914"},{"id":"22906110-7515537-143530984","span":{"begin":1632,"end":1634},"obj":"7515537"},{"id":"22906110-7515537-143530985","span":{"begin":6295,"end":6297},"obj":"7515537"}],"text":"ODN 2216 inhibit replication of common feline viruses in vitro\nFelids are frequently affected by four viruses of different families: the feline herpesvirus (FHV), calicivirus (FCV), parvovirus (FPV) and coronavirus (FCoV). Although these viruses cannot productively infect purified PBMCs in vitro, they share the ability to induce cytopathic effects (CPE) in CrFK and fcwf-4 cells. These feline cell lines, however, do not alter their mRNA levels of genes selected as markers for innate immunity upon direct treatment with ODN 2216 (Figure3A and B and data not shown), preventing from assessing the potential of this molecule to inhibit viral replication directly in these cells. Thus, CrFK and fcwf-4 cells were incubated, prior to their inoculation, with the cell-free supernatants of PBMCs mentioned above: Sup 2216, Sup 2243 and Sup Neg. To this aim, supernatants derived from PBMCs treated for 24 h were selected, as Sup 2216 produced by the cells of several adult cats were estimated to contain optimal type I IFN amounts at this time point, according to the induction of Mx transcription measured directly in these immune cells (Figure4A and4B). CrFK and fcwf-4 target cells were then incubated with the supernatants for 24 h before inoculation, as this time span had indicated highest induction of antiviral mechanisms (Figure4F). The antiviral effects of the supernatants were initially tested on vesicular stomatitis virus (VSV) as control, as this virus is widely recognized for both its potential to induce CPE in cell lines of multiple species and for its particularly high sensitivity to the effects of type I IFN[65, 66]. When the Sup 2216 derived from the PBMCs of eight adult cats that had broadly responded to in vitro ODN 2216 stimulation (Figure2B and C, groups 2 and 3) were incubated with fcwf-4 cells prior to their inoculation, significant inhibition of VSV replication was observed (p = 0.0039) (Figure5A). The replication of this virus was also to some degree repressed by Sup 2243 (p = 0.0078), an observation reminiscent of the slight induction of Mx in target cells incubated with these supernatants (Figure4C and D). In turn, the propagation of FCV, FCoV, FHV and FPV on fcwf-4 cells was also significantly suppressed by the Sup 2216 when compared to Sup 2243 (p = 0.0039, p = 0.0039, p = 0.0078 and p = 0.0039 respectively) and Sup Neg (p = 0.0039), however with expected lower sensitivity than VSV (Figure5B-E and Table1). Both Sup 2243 and Sup Neg failed to inhibit replication of this heterogeneous group of feline viruses, underlining the essential role of the 2216 molecule in conferring the observed effects. Importantly, cells stimulated with ODN 2216 directly did not indicate any resistance to viral replication, in concordance to their impaired response to this molecule already measured on a genetic level (Figure3A and B and data not shown). With respect to the younger cats, the Sup 2216 of those 2 kittens whose cells responded to ODN 2216 stimulation (Figure2A, group 1) could also inhibit both VSV and FCV on fcwf-4 cells, while the supernatants derived from the PBMCs of the other 2 kittens indicated no inhibition potential on these viruses (Figure5K and L). Altogether, the viral suppression potential of Sup 2216 from all the cats could be compared to that conferred by treatment of the cells with 10 to 100U rfeIFNα, a quantity determined in titration experiments of rfeIFNα conducted together with the viral inhibition assays (data not shown).\nFigure 5 Supernatants derived from ODN 2216 stimulated PBMCs inhibit viral replication in target cells. (A-E) fcwf-4 cells were incubated for 24 h with the indicated supernatants derived from PBMCs of eight adult cats (groups 2 and 3) or medium only as control before inoculation with the indicated viruses. Each dot represents mean optical density (OD) values from spectrophotometric readings of viral inhibition assays conducted on duplicate wells treated with supernatants from an individual cat. (F-J) Correlation of individual inhibition ratios of each virus with Mx mRNA expression induced in fcwf-4 cells incubated with supernatants of ODN 2216 stimulated PBMCs from the eight cats of groups 2 and 3. Note the different scale on the x-axis for each graph indicating the differences in the inhibitory effects of these supernatants on the different viruses. (K, L) fcwf-4 cells were incubated for 24 h with the indicated supernatants derived from PBMCs of 4 kittens (group 1) or medium only as control before inoculation with VSV or FCV. Each dot represents mean OD values from spectrophotometric readings of viral inhibition assays conducted on duplicate wells treated with supernatants from one cat. **p \u003c 0.01. VSV = vesicular stomatitis virus, FCV = feline calicivirus, FPV = feline parvovirus, FCoV = feline coronavirus, FHV = feline herpes virus.\nTable 1 Means of viral inhibition rates measured in fcwf-4 cells after treatment with supernatants derived from stimulated PBMCs of 8 adult cats Depicted are mean viral inhibition rates for 8 cats. Viral inhibition rates for each cat were calculated with the following formula:\nMean optical densityODvalues of duplicate wells treated with SupernatantMean OD values of quadruplicate wells treated with medium alone.\nAbbreviations: VSV Vesicular Stomatitis Virus, FCV Feline Calicivirus, FCoV Feline Coronavirus, FHV Feline Herpesvirus, FPV Feline Parvovirus. The differential inhibition of the viruses tested by the same supernatants is reflected by the distinct viral inhibition ratios observed (Figure5B-E and Table1). Sensitivity of each virus to rfeIFNα correlated with sensitivity to the Sup 2216 and induction of Mx in target cells by the supernatants highly correlated with the inhibition of all viruses (Figure5F-I). Finally, Sup 2216 derived from PBMCs of cats c08, whose cells had indicated strong responsiveness to stimulation with ODN 2216 in previous experiments, most efficiently inhibited the replication of all viruses.\nSimilar results were notably obtained when the supernatants of PBMCs derived from all cats were incubated with CrFK cells prior to their inoculation with all the above-mentioned viruses (data not shown). This cell line has previously indicated less sensitivity to the antiviral effects of type I IFN[66] and generally 10-fold higher amounts of rfeIFNα were found in titration experiments to be required for the inhibition of all five viruses. Concordantly, average fold viral inhibition in CrFK cells by the Sup 2216 was approximately half that observed in fcwf-4 cells."}