West Nile Virus Infection in Human and Mouse Cornea Tissue

Abstract

The purpose of this study was to determine the in vitro and ex vivo susceptibility of human corneal cells to West Nile virus (WNV) infection and evaluate the ability of the virus to disseminate to the corneas of infected mice. Human corneal epithelial cells were challenged with WNV, incubated for 1-6 days, and tested for evidence of WNV infection. Viral RNA and antigen were detected at every time point, and the virus reached a peak titer of 2.5 × 10⁷ plaque-forming units (pfu)/mL at 3 days postinoculation (PI). Corneas procured from donors were incubated in culture dishes containing WNV for 1-5 days and tested for evidence of WNV. Viral RNA and antigen were detected, and the virus reached a mean peak titer of 4.9 × 10⁴ pfu/mL at 5 days PI. Mice were inoculated intraperitoneally with WNV, and their eyes were harvested at 2, 5, and 8 days PI and tested for evidence of WNV. Viral RNA was detected in corneas of four of nine systemically infected mice as early as 2 days PI. We conclude that human corneal cells support WNV replication in vitro and ex vivo, and WNV may disseminate into the corneas of experimentally infected mice. These findings indicate that corneal transmission cannot be ruled out as a novel mode of human-to-human WNV transmission and additional experiments should be conducted to assess this risk further.

Figure 1.

Detection of West Nile virus (WNV) RNA using reverse transcription polymerase chain reaction (RT-PCR) in virus-inoculated human corneal epithelial (HCE) cells in vitro. Subconfluent monolayers of (A-B) HCE cells and (C-D) Vero cells in 25-cm² flasks were inoculated with WNV at a multiplicity of infection of 0.1 (lanes 1–6) or they were inoculated with an equal amount of heat-inactivated virus (lanes 7–12). After 1 hour, the virus inocula were removed, and the cell monolayers were rinsed twice in phosphate-buffered saline and incubated in the appropriate cell culture media. Cells were homogenized in Trizol (Invitrogen) and total RNA was extracted at 1 (lanes 1 and 7), 2 (lanes 2 and 8), 3 (lanes 3 and 9), 4 (lanes 4 and 10), 5 (lanes 5 and 11), and 6 (lanes 6 and 12) days postinoculation. Equal amounts of total RNA (2 μg) were analyzed using RT-PCR with primers specific for (A–C) WNV, (B) human ribosomal protein RPL11, and (D) African green monkey β-actin.

Figure 2.

Detection of West Nile virus (WNV) antigen using western blot in virus-inoculated human corneal epithelial (HCE) cells in vitro. Subconfluent monolayers of HCE cells in 25-cm² flasks were inoculated with WNV at a multiplicity of infection of 0.1 (lanes 1–6) or an equal amount of heat-inactivated virus (lanes 7–12). After 1 hour, the virus inocula were removed, and the cells were rinsed twice in phosphate-buffered saline and incubated in the cell culture medium. Cell lysates were prepared at 1 (lanes 1 and 7), 2 (lanes 2 and 8), 3 (lanes 3 and 9), 4 (lanes 4 and 10), 5 (lanes 5 and 11), and 6 (lanes 6 and 12) days postinoculation. Equal amounts of total protein (8 μg) were resolved on 8–16% Tris-glycine gels and analyzed by western blot using (A) a pooled suspension of anti-WNV E protein monoclonal antibodies or (B) anti-human β-tubulin polyclonal antibody.

Figure 3.

In vitro growth kinetics and yields of West Nile virus (WNV) in human corneal epithelial (HCE) and Vero cells. Subconfluent monolayers of HCE and Vero cells in 25-cm² flasks were inoculated with WNV at a multiplicity of infection of 0.1 and incubated for 1–6 days. Supernatants were collected and viral titers were determined by plaque assay. The experiment was performed in triplicate, and each supernatant was tested six times. Data were used to calculate mean viral titers ±1 standard deviation.

Figure 4.

Detection of West Nile virus (WNV) RNA using reverse transcription polymerase chain reaction (RT-PCR) in virus-inoculated human corneal cells ex vivo. Intact human corneas were placed into individual 35-mm culture dishes that contained culture media and incubated for 4 days. Six corneas were inoculated with WNV at a multiplicity of infection of 15 (lanes 1–6). Another three corneas were inoculated with an equal amount of heat-inactivated virus (lanes 7–9). Corneas were incubated for 5 days and homogenized in Trizol (Invitrogen) using a mortar and pestle. Equal amounts of total RNA were subjected to RT-PCR using primers specific for (A) WNV or (B) human ribosomal protein RPL11.

Figure 5.

Ex vivo growth kinetics and yields of West Nile virus (WNV) in human corneal cells. Ten intact human corneas were placed into individual 35-mm culture dishes that contained culture media, incubated for 4 days, and inoculated with WNV at a multiplicity of infection of 15. Culture dishes that contained media in the absence of corneal cells were also inoculated with WNV to assess the longevity of the input virus. At 2, 3, 4, and 5 days postinoculation, an aliquot of each cell culture supernatant was collected from each culture dish, and virus titers were determined by plaque assay.