Characteristics of Nipah virus and Hendra virus replication in different cell lines and their suitability for antiviral screening

Abstract

We have recently described the development and validation of a high throughput screening assay suitable for henipavirus antiviral identification. While we are confident this assay is robust and effective, we wished to investigate assay performance in a range of alternative cell lines to determine if assay sensitivity and specificity could be improved. We evaluated ten different cell lines for their susceptibility to Hendra and Nipah virus infection and their sensitivity of detection of the effects of the broad spectrum antiviral, ribavirin and nine novel antivirals identified using our initial screening approach. Cell lines were grouped into three categories with respect to viral replication. Virus replicated best in Vero and BSR cells, followed by Hep-2, HeLa, BHK-21 and M17 cells. The lowest levels of RNA replication and viral protein expression were observed in BAEC, MMEC, A549 and ECV304 cells. Eight cell lines appeared to be similarly effective at discriminating the antiviral effects of ribavirin (<2.7-fold difference). The two cells lines most sensitive to the effect of ribavirin (ECV304 and BAEC) also displayed the lowest levels of viral replication while Vero cells were the least sensitive suggesting excess viral replication may limit drug efficacy and cell lines which limit viral replication may result in enhanced antiviral efficacy. However, there was no consistent trend observed with the other nine antivirals tested. While improvements in antiviral sensitivity in other cell lines may indicate an important role in future HTS assays, the slightly lower sensitivity to antiviral detection in Vero cells has inherent advantages in reducing the number of partially effective lead molecules identified during initial screens. Comparison of a panel of 54 novel antiviral compounds identified during routine screening of an in-house compound library in Vero, BHK-21 and BSR cells suggests no clear advantage of screening in either cell type.

Figure 1. Chemiluminescent immunodetection of henipavirus infection in various cell lines

Cell lines were infected with HeV (left panel) and NiV (right panel) and viral nucleoprotein was detected in using a previously described HTS format (Aljofan et al., 2008). ½ log dilutions of virus (100μl) were incubated for 24 hours at 37 °C with 20,000 cells per well. Monolayers were fixed with methanol, air dried and immunostained with anti-NiV-N polyclonal antisera (1:1000) followed by secondary antibodies (1:2000) of Horse Radish Peroxidase conjugate with chemiluminescent (HRP-CL) detection (n=4). Values are expressed as the Mean +/− S.E.

Figure 2. Quantitation of immunofluorescent detection of henipavirus infection in cell lines

HeV (left panel) and NiV (right panel) were inoculated onto a range of cells lines as described for Figure 1 and the number of syncytia expressing viral nucleoprotein were detected as previously described (Porotto et al., 2007) following overnight incubation. ½ log dilutions of virus (100μl) were incubated for 24 hours at 37 °C with 20,000 cells per well. Monolayers were fixed with methanol, air dried and immunostained with anti-NiV-N polyclonal antisera (1:1000) followed by detection with a fluorescent antibody Alexa-Fluor 488 conjugate (1:1000). Values are expressed as the Mean +/− S.E. (n=3).

Figure 3. Taqman PCR of henipavirus infection in various cell lines

HeV (left panel) and NiV (right panel) were inoculated onto a range of cells lines as described for Figure 1 and viral nucleoprotein RNA detected as previously described (Mungall et al., 2006, 2008). ½ log dilutions of virus (100μl) were incubated for 24 hours at 37 °C with 20,000 cells per well. Media was removed and cells were lysed with RLT buffer containing β-ME. RNA was extracted using the Qiagen RNeasy Mini kit and Taqman PCR was performed as described. Values are expressed as the Mean +/− S.E. (n=3).

Figure 4. Infectious titers following NiV infection in various cell lines

Cells were infected with 10,000 TCID₅₀/ml NiV in 100μl for 60 min at 37 °C. Inoculum was replaced with fresh EMEM-10 and cells were incubated overnight prior to titration of cell supernatants via serial ten-fold dilutions made in EMEM-10 in 96-well microtitre plates. Vero E6 cells in EMEM-10 were added (2 × 10⁴ cells/well) and plates were incubated at 37 °C for 5–7 days. Wells displaying cytopathic effect were scored as infected and virus titer was calculated using the Reed-Meunch method (Reed and Muench, 1938).

Figure 5. HTS screening of a small compound library against live NiV

Cells were treated with 10 μM of each compound (100μl) immediately prior to infection with 10,000 TCID₅₀/ml NiV in 100μl. Cells were incubated overnight at 37 °C. Monolayers were fixed with methanol, air dried and immunostained with anti-NiV-N polyclonal antisera as described above. 8,040 compounds were tested (n=3) with 54 compounds showing greater than 90% inhibition of NiV infection, while retest of freshly dissolved compound revealed 28 compounds with >90% inhibition. Dose response curves were generated for these 28 compounds (inset) showing a range of 50% inhibitory concentration (IC₅₀) values all less than 2 μM (n=3).

Figure 6. Effect of ribavirin on NiV replication in various cell lines and correlation with infectious virus titer

Cells were grown to 90% confluency in 96 well plates, log dilutions of ribavirin were added then cells infected with 10,000 TCID₅₀/ml NiV in 100μl. Cells were incubated overnight prior to harvesting of the cell supernatants for titration in Vero cells, or fixation of the monolayers in methanol and quantitation of viral nucleoprotein performed as described in Figure 1. Ribavirin inhibition was determined compared to untreated, infected wells and non-linear regression analysis was performed using GraphPad Prism software to determine the 50% inhibitory concentration (IC₅₀). Values are expressed as the Mean Log +/− S.E (n=3) ribavirin IC₅₀ and plotted against the infectious NiV titers recovered from untreated cell supernatants (n=3).

Figure 7. Correlation between NiV antiviral efficacy in Vero, BHK-21 and BSR cells

A panel of 54 putative NiV antiviral compounds were assayed as described for Figure 5 in Vero, BHK-21 and BSR cell lines (n=5). Non-linear regression analysis was performed using GraphPad Prism software to determine the 50% inhibitory concentration (IC₅₀). Average IC₅₀ values for each compound in each cell line are compared for each pair.