A novel procedure of quantitation of virus based on microflow cytometry analysis.

The accurate and fast titration of viruses is a critical step in research laboratories and biotechnology industries. Different approaches are commonly applied which either are time consuming (like the plaque and endpoint dilution assays) or do not ensure quantification of only infective particles (like quantitative real-time PCR). In the last decade, a methodology based on the analysis of infected cells by flow cytometry and fluorescence-activated cell sorting (FACS) has been reported as a fast and reliable test for the titration of some viruses. However, this technology needs expensive equipment and expert technicians to operate it. Recently, the "lab on a chip" integrated devices have brought about the miniaturization of this equipment, turning this technology into an affordable and easy-to-use alternative to traditional flow cytometry. In the present study, we have designed a microflow cytometry (µFC) procedure for the quantitation of viruses, using the infectious pancreatic necrosis virus (IPNV) as a model. The optimization of conditions and validation of the method are reported here.

Surveillance of viruses in wild fish populations in areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula).

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.