Establishment of non-productive infection and activation of inf-alpha and -gamma gene expression in normal lymphomonocytes by sars-cov

Abstract number: 1134_02_136

Castilletti C., Bordi L., Lalle E., Rozera G., Poccia F., Agrati C., Abbate I., Capobianchi M.R.

Objectives:

Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV. During the early phases of the disease, the presence of the replicative intermediates of SARS-CoV has been shown in PBMC from patients. No information is available on the ability of SARS-CoV to stimulate IFN induction, although IFN-gamma may be activated in SARS patients. We investigate the capability of SARS-CoV to give rise to a productive infection in normal PBMC, in parallel with the ability to activate IFN-alpha and -gamma gene expression.

Methods:

Normal PBMC were infected with a MOI 0.1. Virus progeny formation was measured at subsequent time points, by back-titration of cell lysates on Vero cells. Cell mortality and apoptosis were detected by trypan blue and propidium iodide staining. In order to detect virus-specific plus- and minus-RNA strand, total RNA extracted from SARS-CoV-infected PBMC was retrotranscribed in the presence of the sense and antisense primers, respectively, targeting the replicase gene. Measurement of SARS-CoV genomic RNA was performed by quantitative Real Time RT-PCR. IFN induction was performed by exposing PBMC to SARS-CoV at different MOI, ranging from 0.1 to 10. Induction of IFN-alpha and -gamma was analysed by measuring their mRNA levels by limiting dilution RT-PCR. Sensitivity of SARS-CoV replication to exogenous IFN-alpha and -gamma was tested in Vero cells pre-exposed to the individual cytokines of to a combination of both.

Results:

In unstimulated PBMC, no infectious viral progeny formation was detected up to day 13 post-infection. Nevertheless, minus-strand and genomic SARS-CoV RNA peaked at 24 hours post-infection. Dose dependent induction of both IFN-alpha and IFN-gamma mRNA was observed, that was most evident at MOI 10. A combination of these two cytokines was shown to strongly inhibit SARS-CoV replication in Vero cells.

Conclusions:

Our results show that SARS-CoV is able to infect normal PBMC. However, this appears to be only a transient phenomenon, followed by a progressive disappearance of both virus-specific RNA strands, with no infectious virus progeny production. Moreover, SARS-CoV appears to have the intrinsic ability to activate dose-dependently both IFN-alpha and -gamma gene expression in PBMC cultures. Our results can be pathogenically relevant to the inflammatory events occurring in the diseased tissues that can be mediated by the endogenous activation of INF system.