PMC

A 1×10⁶ PFU/ml stock of LCMV Arm was inactivated either by exposure to 1 Joule UV irradiation or by incubation at 57°C for 45 minutes. 3-fold serial dilutions were produced for inactivated samples as well as untreated LCMV Arm, incubated with Vero cells for 48 hours, and the percentage LCMV-NP positive cells was determined. Virus titers are represented in PFU/ml. All experiments were performed in duplicate and curves represent compilation of data for at least 3 individual experiments.

A 3-fold dilution series of a 1×10⁶ PFU/ml LCMV Arm virus stock was produced, incubated with Vero cells, and intracellular LCMV-NP levels were detected by FACS as described in Methods. (A) 48 hour virus exposure and FACS detection of LCMV-NP with the unconjugated antibody and a fluorescently conjugated goat anti-mouse secondary antibody. Shown are representative histograms for uninfected, 2×10³ PFU/ml and 5×10⁵ PFU/ml LCMV Arm. (B) Standard curves were produced from a 3-fold dilution series of LCMV Arm, where virus was incubated with Vero cells for 1 hour, 24 hours, or 48 hours with a total incubation time of 48 hours. Virus titers are represented in PFU/ml. All experiments were performed in duplicate and curves represent compilation of data for at least 3 individual experiments.

(A) The minimum virus exposure time required to detect LCMV-NP was ascertained by incubating 3-fold serial dilutions of a 1×10⁶ PFU/ml LCMV Arm stock with Vero cells for 2, 4, 6, 8, and 24 hours and subsequent determination of LCMV-NP expression by FACS analysis. (B) The effect of prolonged incubation of virus with Vero cells on LCMV-NP FACS assay sensitivity was determined by extending the 48 hour incubation of 1×10⁶ PFU/ml LCMV Arm stock serial dilutions by 24 hour increments; representative curves for 24, 48 and 72 hour incubations are shown. Virus titers are represented in PFU/ml. All experiments were performed in duplicate and curves represent compilation of data for at least 3 individual experiments.

(A) For validation of the AF647-conjugated LCMV-NP antibody, serum titers of LCMV cl13 infected mice (d.p.i) were analyzed by LCMV-NP FACS using both conjugated and unconjugated versions of the antibody; viral titers are represented in “derived PFU/ml”. (B) C57/BL6 mice were infected i.v. with 2×10⁶ PFU LCMV cl13, serum samples were collected and organ lysates prepared at 98 d.p.i., and viral titers were determined in parallel by LCMV-NP FACS using the AF647-conjugated LCMV-NP and standard plaque assay. Virus titers are represented in “derived PFU/ml” (FACS assay) or PFU/ml (plaque assay). (C) C57/BL6 mice were infected i.p. with 2×10⁵ PFU of LCMV Arm, serum samples were collected on 2, 4, 6, 8 d.p.i and organ lysates from kidney, spleen, liver and brain were produced on 8 d.p.i. Viral titers were determined for serum 2, 4, 6, 8 d.p.i. by both the LCMV-NP FACS using unconjugated anti-LCMV-NP, and standard plaque assay (left panel). Viral titers were determined on 4 and 8 d.p.i. in serum and in kidney, spleen, liver, and brain from 8 d.p.i. by both the LCMV-NP FACS using the AF647-conjugated anti-LCMV-NP and the standard plaque assay (right panel). Viral titers are represented as in panel B.

(A) A master standard curve was produced by combining 9 individual standard curves generated for separate experiments by incubation of 10 3-fold serial dilutions of the 1×10⁶ PFU/ml LCMV-Arm virus stock with Vero cells for 48 hours and subsequent determination of LCMV-NP expression by FACS. (B) C57/BL6 mice were infected i.v. with 2×10⁶ PFU LCMV cl13, serum samples were collected on 4, 6, 8, 15, 22, 35, 42 and 56 d.p.i., and viral titers were determined by both the LCMV-NP FACS assay (“derived PFU/ml”) and standard plaque assay (PFU/ml). “Derived PFU/ml” values were calculated with individual standard curves generated at the time of experiment (closed symbols) or the above master standard curve (open symbols) generated after completion of all experiments. The symbols represent 5 individual mice for 4, 6, 8 d.p.i. and 4 mice for 15, 22, 29, 35, 42 and 56 d.p.i.