Eilat virus (EILV) causes superinfection exclusion against West NILE virus (WNV) in a strain specific manner in Culex tarsalis mosquitoes

Abstract

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the United States. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and Culex tarsalis mosquitoes. The titers of both WNV strains—WN02-1956 and NY99—were suppressed by EILV in C6/36 cells as early as 48–72 h post superinfection at both multiplicity of infections (MOIs) tested in our study. The titers of WN02-1956 at both MOIs remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titers. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In Cx. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titers at 3 days post superinfection, but this effect disappeared at 7 days post superinfection. In contrast, WN02-1956 infection titers were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in Cx. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

AUTHOR SUMMARY

West Nile virus (WNV) is the main cause of mosquito-borne disease in the United States. In the absence of a human vaccine or WNV-specific antivirals, vector control is the key strategy to reduce WNV prevalence and transmission. The WNV mosquito vector, Culex tarsalis, is a competent host for the insect-specific virus Eilat virus (EILV). EILV and WNV potentially interact within the mosquito host, and EILV can be used as a safe tool to target WNV in mosquitoes. Here, we characterize the ability of EILV to cause superinfection exclusion (SIE) against two strains of WNV—WN02-1956 and NY99—in C6/36 cells and Cx. tarsalis mosquitoes. EILV suppressed both superinfecting WNV strains in C6/36 cells. However, in mosquitoes, EILV enhanced NY99 whole-body titers at 3 days post superinfection and suppressed WN02-1956 whole-body titers at 7 days post superinfection. Vector competence measures, including infection, dissemination, and transmission rates and transmission efficacy, as well as leg and saliva titers of both superinfecting WNV strains, were not affected by EILV at both timepoints. Our data show the importance of not only validating SIE in mosquito vectors but also testing multiple strains of viruses to determine the safety of this strategy as a control tool.

INTRODUCTION

West Nile virus (WNV) is a single-stranded, positive-sense RNA virus belonging to the genus Flavivirus (family Flaviviridae) (1) ; since its first report in the USA in New York City in 1999 (2,3), WNV has expanded its geographical prevalence throughout North America (4) and is now the predominant cause of mosquito-borne disease in the USA (5). WNV is maintained in nature by an enzootic cycle between Culex mosquitoes and birds; however, spillovers into dead-end hosts such as humans and horses frequently occur, triggering epidemics (1). The severity of symptoms in humans varies from asymptomatic WNV infection to neuroinvasive disease and death (5,6). Since 1999, the USA has had 50,000 confirmed WNV cases; 25,000 neuroinvasive cases; and 2000 deaths (5,6).

Since the first WNV outbreak in the USA in 1999 WNV has evolved, with the causative WNV genotype NY99 being displaced by the genotype WN02, which currently circulates in the US population (7,8). The WN02 genotype sequence differs from the NY99 genotype sequence by three nucleotides (7,8). Two of these mutations were in the envelope protein and one in the NS5 protein, but only one of these mutations was non-synonymous (7,8). The WN02 genotype is characterized by a single amino acid change in the envelope protein from valine to alanine at position 159 (VE159A). This mutation, VE159A, enhances the vector competence of the WN02 genotype in Culex mosquitoes, particularly in Cx. tarsalis (9)—the main WNV vector in rural areas (10). Cx. pipiens and Cx. quinquefasciatus are also key WNV vectors but mainly in urban settings (11). Currently, no human vaccines or WNV-specific antivirals are available, making vector control the primary strategy used to reduce WNV transmission (12).

The WNV vector Cx. tarsalis is also a competent host for Eilat virus (EILV) (13), an insect-specific virus (ISV) belonging to the genus Alphavirus (family Togaviridae) (13). EILV is a small, enveloped, single-stranded positive-sense RNA virus that is unable to infect vertebrate cells at both the attachment/entry and replication stages of the viral life cycle (14,15). ISVs, including EILV, interact and modulate the vector competence of human pathogenic viruses in their shared mosquito hosts (16–22).A deeper understanding of these interactions can potentially be used to develop these ISVs into safe tools to target pathogenic viruses in mosquitoes.

One such interaction of interest is superinfection exclusion (SIE), a phenomenon that occurs when a pre-existing viral infection in cells blocks or interferes with a secondary infection of the same virus (homologous interference) (23–25) or closely related virus (26–28), or even an unrelated virus (heterologous interference) (21,29,30). The precise mechanism(s) of SIE remains unknown. However, there is evidence suggesting that the primary virus impacts different stages of the virus life cycle of the challenge virus in cells, including attachment (31,32), penetration (24), and replication (33,34).

ISVs cause both homologous and heterologous interference in cell culture and in mosquitoes, although the results have been variable (19,20,22,33,35–37). For example, Palm Creek virus, an insect-specific flavivirus, when superinfected with WNV in C6/36 cells, caused heterologous interference against WNV (17) but Cx. flavivirus Izabal, another insect-specific flavivirus, had no effect on WNV under similar conditions (38). Previous studies have demonstrated that EILV causes homologous and heterologous interference against other alphaviruses in C7/C10 cells and Aedes aegypti mosquitoes (22). The superinfection of EILV-infected Aedes aegypti with Chikungunya virus (CHIKV) delayed CHIKV dissemination by 3 days (22). The mosquito species Cx. tarsalis is a more competent host for EILV than Aedes aegypti (13) and therefore, we propose to investigate the ability of EILV to cause heterologous interference against the unrelated flavivirus WNV in C6/36 cells and Cx. tarsalis mosquitoes.

MATERIALS AND METHODS

Cells and cell culture.

WNV- and EILV-EILV-susceptible Aedes albopictus mosquito cell line C6/36 was propagated in Roswell Park Memorial Institute (RPMI) medium—consisting of RPMI 1640 medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific), penicillin (100 U/ml; Gibco/Thermo Fisher Scientific), and streptomycin (100 µg/ml; Gibco/Thermo Fisher Scientific)—and maintained at 28°C with no CO₂.

Viral cDNA clone and virus propagation.

Eilat virus:

The EILV cDNA clone was obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch (Galveston, TX, USA) and used for all experiments. The EILV cDNA clone consists of EILV strain EO329 with an enhanced green fluorescent protein (eGFP) inserted into the nsp3 hypervariable region of the viral genome (EILV-eGFP). The EILV-eGFP virus was rescued as previously described (13).

West Nile virus:

WNV strain WN02-1956 (GenBank: AY590222) stocks were originally obtained from Dr. Gregory Ebel at the Center for Vector-Borne Infectious Diseases, Colorado State University (Fort Collins, CO, USA), whereas WNV strain NY99 (GenBank: AF196835.2) stocks were acquired from Dr. Laura Kramer at the Wadsworth Center, New York State Department of Health (Albany, NY, USA). WNV strains were amplified in C6/36 cells, and the virus-containing supernatant was collected at 7 days post infection (dpi). The collected supernatant was aliquoted and stored at −80°C until use. A focus-forming assay (FFA) was used to quantify both EILV and WNV titers (described below).

Superinfection fluorescent imaging.

EILV-WNV superinfection in C6/36 cells:

C6/36 cells were seeded in two T75 tissue culture flasks (Corning /Falcon) at a density of 6 × 10⁷ cells and incubated overnight without CO₂ at 28°C until they reached ~70%–80% confluency. Both T75 flasks were then washed with serum-free RPMI medium. One of the flasks was then inoculated with EILV-eGFP at a multiplicity of infection (MOI) of 10, whereas the other flask was mock inoculated with serum-free RPMI medium; the flasks were incubated at 28°C with no CO₂ for 1 h. After incubation, the remaining inoculum in the flasks were removed and replaced with complete RPMI, and the flasks were incubated at 28°C with no CO₂ for 48 h.

The EILV-eGFP and mock-infected C6/36 cells were detached using trypsin (Gibco/Thermo Fisher Scientific) after the 48 h incubation and seeded in a 24-well tissue culture plate (Greiner Bio-One) at a density of 5 × 10⁶ cells in triplicate and incubated overnight at 28°C with no CO₂. The cells in both groups were washed with serum-free RPMI medium, infected with the WNV strain WN02-1956 or NY99 at MOIs of 0.1 or 0.01, and incubated at 28°C with no CO₂ for 1 h. The cells were washed twice with complete RPMI medium after WNV infection to remove any unattached virus. Complete RPMI medium was then added to the cells, and the EILV–WN02-1956-superinfected or EILV–NY99-superinfected and the control WN02-1956-infected or NY99-infected cells at both MOIs were incubated at 28°C with no CO₂ for 72 h.

Superinfected cells were fixed 72 h post superinfection using 300 µl of 4% formaldehyde in 1× phosphate-buffered saline (PBS) for 30 min at room temperature (RT). Superinfected cells were washed twice with 500 µl of 1× PBS and then permeabilized by adding 300 µl of 0.2% triton-X in 1× PBS to each well for 15 min at RT. Then, the cells were washed twice with 1× PBS and blocked with 300 µl of 3% bovine serum albumin (BSA) in 1× PBS for 1 h at RT. The cells were washed again with 1× PBS twice; next, 250 µl of the primary antibody, monoclonal anti-flavivirus group antigen antibody (clone D1-4G2-4-15, diluted 1:500 in 3% BSA), was added to each well, and the cells were incubated overnight at 4°C. Superinfected cells were then washed twice with 1× PBS, and 250 µl of the secondary antibody, goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor Plus 594, diluted 1:1000 in 3% BSA), was added to each well. The plates were wrapped in aluminum foil and incubated overnight at 4°C. Finally, cells were washed twice and maintained in 300 µl of 1× PBS to prevent drying. EILV infection was observed under the fluorescein isothiocyanate (FITC) filter and WN02-1956 and NY99 infections under the tetramethylrhodamine isothiocyanate (TRITC) filter on the ECHO Revolve microscope. FITC and TRITC images were merged (all scale bars correspond to 180 µm).

In vitro superinfection assay.

EILV-eGFP-infected C6/36 cells were superinfected with WN02-1956 or NY99 at MOIs 0.1 and 0.01 in 24-well plates as described above. Superinfected and control WN02-1956-infected or NY99-infected cells at both MOIs were maintained at 28°C with no CO₂ for 96 h. At 12, 24, 48, 72, and 96 h post superinfection, 300 µl of supernatant was collected from each superinfection and control well and replaced with fresh medium. Samples were stored at −80°C until use, and WNV titers in the samples were quantified using FFA (described below). The in vitro superinfection assay was performed in duplicate using aliquots of the same EILV-eGFP and WNV stocks that underwent the same number of freeze-thaw cycles.

Viral attachment and internalization assay.

The viral attachment and internalization assay was performed as previously described with some modifications (25). For the viral attachment assay, a 24-well tissue culture plate with EILV-eGFP-infected (MOI 10) and mock-infected C6/36 cells seeded in duplicate was superinfected with WWN02-1956 or NY99 at an MOI of 0.1 as described above. Superinfected cells were then incubated at 4°C for 1 h to allow virus particles to attach but not enter the cells. Then, superinfected cells were washed with 1x PBS (Gibco/Thermo Fisher Scientific) and then washed twice with complete RPMI medium to remove any unattached WNV. Both groups of superinfected cells, with and without EILV-eGFP infection, were resuspended in 300 µl of TRIzol for RNA extraction.

Similarly, for the viral internalization assay, a 24-well plate with EILV-eGFP-infected and mock-infected C6/36 cells superinfected with WN02-1956 or NY99 at an MOI of 0.1 was incubated at 28°C with no CO₂ for 1.5 h to allow attachment and internalization of WNV. Superinfected cells were then washed with 1x PBS followed by a high-salt buffer containing 50 mM Na₂CO₃ (pH 9.5; Fisher chemicals, Thermo Fisher Scientific) and 1 M NaCl (Fisher chemicals, Thermo Fisher Scientific) for 3 min at RT. The high-salt buffer was removed, and the superinfected cells were resuspended in 300 µl of TRIzol for RNA extraction. RNA was extracted from all samples using the phenol/chloroform extraction method as previously described (39) and DNA contamination in the extracted RNA was removed from the samples using the TURBO DNA-free kit (Invitrogen, Thermo Fisher Scientific). Total RNA was quantified using NanoDrop ND-1000 (NanoDrop Technologies/Thermo Fisher Scientific), and WNV RNA in 60 ng of total RNA from each sample was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR; described below). The virus attachment and internalization assays were performed in duplicate using aliquots of the same EILV-eGFP and WNV stocks that underwent the same number of freeze-thaw cycles.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

WNV RNA was quantified using the Qiagen rotor gene Q-compatible qScript One-Step SYBR Green RT-qPCR kit (Quantabio). The RT-qPCR reaction was set up as per the manufacturer’s instructions, using WNV envelope protein-specific primers 5R-TTGCAAAGTTCCTATCTCGTCAG-3R and 5R-ACATGCCTCCGAACAGTGAG-3R for both reverse transcription and amplification of the target WNV sequence. The WNV polyprotein DNA fragment (1917–2338 bp) synthesized by IDT technologies served as standards for the RT-qPCR reaction, making a standard curve from 10⁶ to 10³ viral copies/µl. All samples and standards were run in duplicate, and WNV titers were determined using the standard curve.

Mosquitoes and mosquito rearing.

EILV-eGFP and WNV-competent mosquitoes, Cx. tarsalis (KNWR strain), were used in all experiments in our study. The KNWR strain was obtained from Dr. Christopher Barker at the University of California–Davis School of Veterinary Medicine (Davis, CA, USA). Uninfected mosquitoes were reared and maintained at the Millennium Sciences Complex (The Pennsylvania State University, University Park, PA, USA), whereas WNV-infected mosquitoes were maintained at the Eva J. Pell Biosafety Level 3 (BSL3) laboratory (The Pennsylvania State University, University Park, PA, USA), as previously described (13). Cx. tarsalis KNWR larvae were reared in 30 × 30 × 30 cm cages, and growth conditions for both KNWR adults and larvae were 25°C ± 1°C, 16:8 h light: dark diurnal cycle, and 80% relative humidity. Larvae were fed TetraMin (Tetra), and all adult mosquitoes were fed with 10% sucrose solution-soaked cotton balls ad libitum.

Mosquito superinfection assay.

Adult Cx. tarsalis KNWR female mosquitoes (3–5 days post emergence) were sugar-starved for 24 h and then fed an infectious blood meal consisting of 1:1 anonymous human blood (BioIVT) and 10⁷ FFU/ml of EILV-eGFP (EILV-eGFP-infected group) or RPMI medium (mock-infected group) at 37°C using a water-jacketed membrane feeder. Previous studies have shown that an EILV-eGFP dose of 10⁷ FFU/ml leads to a robust EILV infection in ~70% of the blood-fed Cx. tarsalis KNWR mosquitoes (13). After the infectious bloodmeal, mosquitoes from both groups were cold-anesthetized, and fully engorged female mosquitoes were counted and sorted into different cardboard cup cages with 10% sucrose solution-soaked cotton balls until further experimentation. At 5 dpi, mosquitoes in both groups were allowed to lay eggs in plastic cups filled with deionized water and the eggs were then discarded. Both EILV-eGFP-infected and mock-infected mosquitoes were moved to the Eva J. Pell BSL3 laboratory at 6 dpi and sugar-starved for 24 h. Mosquitoes in both groups were then fed again at 7 dpi with an infectious blood meal containing 1:1 anonymous human blood and WN02-1956 or NY99 at a dose of 10⁷ FFU/ml. Fully engorged females from the superinfected and single-infected control groups were cold-anesthetized and placed into cardboard cup cages with 10% sucrose solution-soaked cotton balls. These cages with WNV-infected mosquitoes were then double caged and maintained at 25°C ± 1°C, 16:8 light: dark diurnal cycle, and 80% relative humidity until assay timepoints at 3 and 7 days post superinfection.

Whole bodies from both superinfected and singly infected control groups were collected 3 days post superinfection and placed into 2 ml microcentrifuge tubes containing 300 µl mosquito diluent (20% FBS, 100 µg/ml of streptomycin, 100 µg /ml of penicillin, 50 µg/ml gentamicin [Gibco/Thermo Fisher Scientific], and 2.5 µg/ml amphotericin B [Gibco/Thermo Fisher Scientific] mixed in 1× PBS). Samples were then homogenized using a battery-operated tissue homogenizer (VWR) and disposable pestles (Fisher scientific/Thermo Fisher Scientific). Similarly, at 7 days post superinfection, mosquitoes from both groups were forced to salivate for 30 min at RT into a capillary glass tube containing a mixture of 1:1 FBS and 50% sucrose. After salivation, the saliva was expelled into a 2 ml microcentrifuge tube containing 100 µl mosquito diluent. Legs and bodies of superinfected and control mosquitoes were then collected in 2 ml microcentrifuge tubes containing 300 µl mosquito dilutant. The legs and bodies were homogenized using the battery-operated homogenizer and disposable pestles, and all samples were stored at −80°C until further processing. The WNV titers of all samples collected were quantified using FFA (described below). The mosquito superinfection assay was performed in duplicate, and aliquots of the same EILV-eGFP and WNV stocks that had undergone the same number of freeze-thaw cycles were used in both replicates.

Using the FFA results, the following vector competence parameters were determined. The infection rate (IR) was calculated as the proportion of WNV-infected mosquitoes among the total number of EILV-eGFP or mock-infected mosquitoes; the dissemination rate (DIR), as the proportion of WNV-infected mosquitoes with WNV-positive legs; the transmission rate (TR), as the proportion of mosquitoes with WNV-positive saliva from those with WNV-positive legs; and the transmission efficacy (TE), as the proportion of mosquitoes with WNV-positive saliva among the total number of EILV-eGFP or mock-infected mosquitoes.

Focus-forming assay.

We quantified EILV-eGFP titers using FFA as previously described (13). Similarly, WNV titers were quantified by FFA as previously described but with some modifications (21). 96-well plates were seeded with C6/36 cells at a density of 1 × 10⁵ cells/well and incubated at 28°C with no CO₂ overnight. The complete RPMI medium from the wells was then removed. Samples from the in vitro and in vivo superinfection assays were then serially diluted in serum-free RPMI medium from 10⁰ to 10⁻⁷ and 10⁰ to 10⁻³, respectively, and 30 µl of each diluted sample was added to the prepared C6/36 cells in duplicates. Mosquito saliva samples were not diluted, and 30 µl of each sample was added directly to the prepared cells. The cells were then incubated at 28°C with no CO₂ for 1 h, after which the samples were removed, and the cells were covered with 100 µl of RPMI containing 0.8% methylcellulose (Sigma-Aldrich). The WNV-infected cells were incubated at 28°C without CO₂ for 48 h. At 48 h post infection, the infected C6/36 cells were fixed using 50 µl of 4% formaldehyde (Sigma-Aldrich) in 1× PBS for 30 min at RT. Cells were washed twice with 100 µl 1× PBS and were then permeabilized with 30 µl of 0.2% triton-X in 1× PBS for 15 min at RT. The washing step was repeated, and the cells were blocked with 30 µl of 3% BSA in 1× PBS for 1 h at RT. The cells were washed again, and 30 µl of the primary antibody, monoclonal anti-flavivirus group antigen antibody (clone D1-4G2-4-15 [BEI resources], diluted 1:500 in 3% BSA), was added to each well. The cells were then incubated overnight at 4°C. Cells were then washed to remove any unattached primary antibody, and 30 µl of the secondary antibody goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor Plus 594 diluted 1:1000 in 3% BSA) was added to each well. The plates were wrapped in aluminum foil and incubated overnight at 4°C. After a final washing step, the cells were maintained in 100 µl of 1× PBS to prevent drying. Fluorescent foci of EILV-eGFP were counted using the FITC filter on the ECHO Revolve microscope, whereas WN02-1956 or NY99 foci were counted using the TRITC filter.

Statistical analysis.

A two-way ANOVA followed by the Tukey test was used to determine the difference between WNV replication kinetics under the superinfection and single infection conditions in C6/36 cells. The WNV viral titers from the post-attachment or internalization assay were compared using Mann-Whitney U tests. The WNV viral titers of the body, leg, and saliva samples from superinfected and singly infected Cx. tarsalis were also compared using Mann-Whitney U tests. The differences in the WNV IR, DIR, TR, and TE post superinfection in EILV-infected or mock-infected Cx. tarsalis mosquitoes were evaluated using Fisher’s exact test. GraphPad Prism version 9.0.4 was used to perform all statistical tests.

RESULTS

Eilat virus caused heterologous interference against West Nile virus strains WN02-1956 and NY99 in vitro.

To investigate the ability of EILV to cause SIE against WNV in cell culture, C6/36 cells were mock infected or infected with EILV-eGFP at an MOI of 10. After EILV infection was established at 72 h post infection, cells were superinfected with the WNV strain WN02-1956 or NY99 at MOIs of 0.1 and 0.01. Superinfected cells at both MOIs were also observed using fluorescent microscopy at 72 h post superinfection to determine whether EILV-eGFP-infected cells were resistant to WNV infection. Moreover, differences in WN02-1956 or NY99 replication kinetics under superinfection and single infection conditions at 12, 24, 48, 72, and 96 h post superinfection were determined to detect SIE in C6/36 cells.

We observed that C6/36 cells infected with EILV-eGFP, determined by eGFP expression by fluorescent microscopy, were susceptible to superinfection by WN02-1956 or NY99 virus, as indicated by Alexa Flour 594 fluorescence at both MOIs tested (Fig 1A). The viruses also co-localized in the cells, suggesting co-infection by EILV and WN02-1956 or NY99 (Fig 1A).

Figure 1.

Superinfection exclusion (SIE) against WNV strains, WN02-1956 and NY99 in C6/36 cells by EILV. (A) Superinfection of EILV-eGFP-infected C6/36 cells (multiplicity of infection [MOI] of 10) with WN02-1956 or NY99 at an MOI of 0.01 and 0.1 was visualized by fluorescent microscopy. Representative images show eGFP fluorescence for EILV-eGFP and Alexa Fluor 594 fluorescence for both WNV strains. All scale bars equal 180 µm. (B) Titers of WNV at 12, 24, 48, 72 and 96 h post-superinfection in EILV-eGFP-infected C6/36 cells (MOI 10) and mock-infected cells superinfected with WN02-1956 or NY99 at an MOI of 0.01 and 0.1 were determined by focus forming assay (FFA). Each point represents a single infection at each timepoint. Statistical significance was evaluated using two-way ANOVA followed by Tukey test. ** P< 0.01 and **** P< 0.0001.

we also found that the titers of WN02-1956, were statistically significantly lower at 72 and 96 h post superinfection in EILV-eGFP-infected C6/36 cells than in mock-infected cells at both MOIs tested (Fig 1B, two-way ANOVA with Tukey test, all P < 0.0001). A similar trend was observed in the second replicate of this assay, but with a significant suppression of WN02-1956 titers occurring earlier at 48 h in superinfected cells which continued at 72 and 96 h post superinfection at both MOI 0.1 (Fig S1A; all P < 0.0001) and MOI 0.01 (Fig S1A; P < 0.0001, P < 0.0001, and P ≤ 0.01 respectively).

In contrast, the titer of the WNV strain NY99 was significantly lower in superinfected cells at MOI 0.01 than in singly infected control cells only at 72 h post superinfection (Fig 1B; P < 0.0001), with the NY99 titers rebounding at 96 h post superinfection (Fig 1B, P > 0.05). The second replicate of the EILV–NY99 superinfection assay at MOI 0.01 in C6/36 cells differed from the first replicate, with significantly lower NY99 titers observed at 48, 72, and 96 h post superinfection (Fig S1A; all P < 0.0001). NY99 titers showed some but not significant recovery at 96 h post superinfection, unlike that observed in the first replicate. Similarly, superinfection of EILV-eGFP-infected C6/36 cells with NY99 at MOI 0.1 led to significantly lower NY99 titers at 72 and 96 h post superinfection (Fig S1A, all P < 0.0001) in superinfected cells than in singly infected control cells in both replicates. Overall, the WN02-1956 and NY99 titer reduction by EILV in C6/36 cells under superinfection conditions ranged between 10-fold and 100-fold.

Eilat virus interfered with the attachment of the superinfecting virus NY99 in C6/36 cells.

Having established that EILV causes SIE against WN02-1956 and NY99 in C6/36 cells, we next investigated how EILV causes heterologous interference against unrelated WNV in cells. To this end, we assessed whether EILV interferes with the attachment or internalization stage of the WN02-1956 or NY99 viral life cycle in C6/36 cells. EILV-eGFP-infected and mock-infected C6/36 cells were superinfected with WN02-1956 or NY99 at an MOI of 0.1. WNV particles were either allowed to only attach to their receptors or also internalize into the cells. WN02-1956 and NY99 RNA from superinfected and singly infected cells post attachment or internalization were quantified by RT-qPCR to determine whether EILV interferes with either of these stages of the WNV life cycle.

The viral titers of attached or internalized WN02-1956 in EILV-eGFP-infected C6/36 cells and in mock-infected C6/36 cells were not significantly different (Fig 2A and 2B; respectively, Mann-Whitney U tests, both P > 0.05). However, viral titers of attached NY99 were significantly lower in superinfected C6/36 cells than in singly infected cells (Fig 2A; P < 0.05). This difference between NY99 viral titers was not observed when comparing internalized virus particles in superinfected and singly infected cells (Fig 2B; P > 0.05).

Figure 2.

WNV attachment and internalization post-superinfection in EILV-eGFP-infected C6/36 cells. WNV titers at (A) attachment and (B) internalization stages of WNV life cycle in EILV-eGFP infected and mock-infected C6/36 cells superinfected with WN02-1956 or NY99 (MOI 0.1) were determined by RT-qPCR. Each vertical bar reflects the mean of duplicate infections, and error bars indicate standard deviation between replicates. Statistical significance was assessed using Mann-Whitney U tests. * P<0.05.

Eilat virus enhanced NY99 whole-body titers at an early timepoint post superinfection in Culex tarsalis mosquitoes.

We tested whether EILV causes heterologous interference against WNV strains in mosquitoes, as previously observed in C6/36 cells, by orally superinfecting EILV-eGFP-infected Cx. tarsalis KNWR mosquitoes with WN02-1956 or NY99. We examined the presence of EILV and WN02-1956 or NY99 using FFA in the bodies of 34 and 22 EILV-eGFP-challenged KNWR mosquitoes orally challenged with 10⁷ FFU/ml of WN02-1956 or NY99, respectively, at 3 days post superinfection. Simultaneously, for our controls, we examined the presence of WN02-1956 or NY99 using FFA at 3 days post superinfection in the bodies of 29 and 19 mock-infected KNWR mosquitoes orally challenged with 10⁷ FFU/ml of WN02-1956 or NY99, respectively. The IR for mosquitoes in our superinfected or mock-infected control groups was defined as the proportion of mosquitoes with WNV infection among the total number of EILV-infected or mock-infected mosquitoes or fully engorged females, respectively.

We observed that EILV-infected Cx. tarsalis were not refractory to a secondary WN02-1956 or NY99 infection at 3 days post superinfection (Table 1). At this early timepoint, the IR of WN02-1956- and NY99-superinfected mosquitoes did not significantly differ from the IR of mosquitoes singly infected with WN02-1956 or NY99 (Table 1; Fisher’s exact tests, both P > 0.05). However, NY99 whole-body titers were significantly higher in EILV-infected mosquitoes than in mock-infected mosquitoes at 3 dpi (Fig 3B; Mann-Whitney U tests, P ≤ 0.001). No significant difference in WN02-1956 whole-body titers was observed at this timepoint between the two groups (Fig 3A, P > 0.05).

Table 1.

Infection rate (IR) of Eilat virus (EILV)-enhanced green fluorescent protein (eGFP)-infected or mock-infected Culex tarsalis mosquitoes orally challenged with the West Nile virus (WNV) strain WN02-1956 or NY99

	3 days post superinfection
Superinfected mosquito group	IR (%) (nI /nT)
EILV-eGFP//WN02-1956	53.3 (8/15)
Mock-infected/WN02-1956	50 (11/22)
EILV-eGFP//NY99	92.8 (13/14)
Mock-infected/NY99	89.5 (17/19)

n_I, number of mosquitoes infected with WNV

n_T, total number of EILV-eGFP positive or mock-infected mosquitoes tested

Figure 3.

Whole-body WNV titers of superinfected and singly infected Culex tarsalis mosquitoes at 3 days post-superinfection. WNV Viral titers of whole-body samples from EILV-eGFP-infected (10⁷ FFU/ml) and mock infected Cx. tarsalis mosquitoes orally challenged with A) WN02-1956 and (B) NY99 (both 10⁷ FFU/ml) are plotted 3 days post-superinfection. Individual points represent a single mosquito sample, while group medians are depicted by horizontal bars. Significance was determined using Mann-Whitney U tests. *** P<0.001.

Eilat virus suppressed WN02-1956 body titers at 7 days post superinfection in Culex tarsalis mosquitoes.

We assessed the vector competence of WNV strains in EILV-infected and mock-infected Cx. tarsalis KNWR mosquitoes at a later timepoint to determine whether the ability of EILV to enhance or cause SIE changed with increasing time post superinfection. We determined the IR, DIR, TR, and TE of WN02-1956 in 37 EILV-infected and 36 mock-infected mosquitoes and of NY99 in 14 EILV-infected and 24 mock-infected mosquitoes at 7 days post superinfection. IR was calculated as previously defined; DIR was determined as the proportion of EILV- or mock-infected mosquitoes with WNV-positive legs, TR as the proportion of EILV- or mock-infected mosquitoes with WNV-positive saliva, and TE as the proportion of EILV- or mock-infected mosquitoes with WNV-positive saliva.

We found that the IR and DIR of WN02-1956 and NY99 in superinfected mosquitoes did not differ significantly from mock-infected mosquitoes at this timepoint (Table 2; Fisher’s exact test, P > 0.05). Both WNV strains disseminated past the midgut in EILV- and mock-infected mosquitoes, but WN02-1956 and NY99 were detected only in the saliva of mock-infected mosquitoes (Table 2, P > 0.05). However, the difference in the TR and TE of WN02-1956 and NY99 in EILV- and mock-infected mosquitoes was not statistically significant (Table 2, P > 0.05).

Table 2.

Infection rate (IR), dissemination rate (DIR), and transmission rate (TR) and transmission efficiency (TE) of Eilat virus (EILV)–enhanced green fluorescent protein (eGFP)-positive or mock-infected Culex tarsalis mosquito species orally challenged with the West Nile virus (WNV) strain WN02-1956 or NY99

	7 days post superinfection
Superinfected mosquito group	IR (%) (nI/nT)	DIR (%) (nL/nI)	TR (%) (nS/nL)	TE (%) (nS/nT)
EILV-eGFP/WN02-1956	84.2 (16/19)	25 (4/16)	0 (0/4)	0 (0/19)
Mock-infected-WN02-1956	87.5 (28/32)	28.6 (8/28)	12.5 (1/8)	3.1 (1/32)
EILV-eGFP/NY99	100 (14/14)	28.6 (4/14)	0 (0/4)	0 (0/14)
Mock-infected-NY99	100 (24/24)	50 (12/24)	8.3 (1/12)	4.2 (1/24)

n_I, number of mosquitoes infected with WNV

n_T, total number of EILV–eGFP-positive or mock-infected mosquitoes tested

n_L, number of mosquitoes with WNV-positive legs

n_s, number of mosquitoes with WNV-positive saliva

Moreover, we found that EILV significantly suppressed WN02-1956 body titers in superinfected mosquitoes (Fig 4, Mann-Whitney U tests, P ≤ 0.001). This suppression of WN02-1956 body titers did not result in an overall decrease in WN02-1956 titers in the legs and saliva of superinfected mosquitoes (Fig 4A, P > 0.05). Similarly, no difference was observed in the NY99 body, leg, or saliva titers between superinfected and singly infected mosquitoes (Fig 4B, P > 0.05).

Figure 4.

WNV titers of body, leg, and saliva samples from EILV-eGFP-infected and mock infected Culex tarsalis mosquitoes challenged with WN02-1956 or NY99. WNV titers in (A) body, (B) leg and (C) saliva samples from EILV-eGFP-infected (10⁷ FFU/ml) and mock infected Culex tarsalis mosquitoes challenged with WN02-1956 or NY99 (10⁷ FFU/ml) at 7 days post-superinfection are plotted. Individual points represent a single mosquito sample, while group medians are depicted by horizontal bars. Significance was determined using Mann-Whitney U tests. *** P<0.001.

DISCUSSION

WNV is a pathogenic arbovirus of public health importance, especially in the USA; however, there are currently no human vaccines or WNV-specific antivirals available against this pathogen (5,12). The main strategy to decrease the prevalence of this virus is vector control (5,12). One potential control strategy is the use of ISVs to target pathogenic viruses through SIE (16–22). The WNV vector Cx. tarsalis is a competent host for the ISV, EILV (13). Therefore, the primary goal of the present study was to determine whether EILV causes SIE against WNV— an unrelated flavivirus virus—in C6/36 cells and Cx. tarsalis mosquitoes. In the present study, we report that EILV suppresses the titers of both WNV strains WN02-1956 and NY99 in C6/36 cells. In contrast, EILV enhanced the viral infection titers of NY99 in Cx. tarsalis mosquitoes at 3 days post superinfection but suppressed WN02-1956 infection titers at 7 days post superinfection. Overall, EILV was found to cause SIE against WNV in both C6/36 cells and Cx. tarsalis mosquitoes but in a strain-specific manner

Previous studies on the interactions between ISVs and other viruses under superinfection conditions have reported variable results, ranging from interference (17,18,22,56,40–42)and enhancement (39,43) to no effect on the secondary infection (38,44). We found that the alphavirus EILV caused SIE against the flavivirus WNV in C6/36 cells, irrespective of the MOI of WNV tested in our study. Superinfection of EILV-infected C6/36 cells with either the WNV strain WN02-1956 or NY99 at an MOI of 0.1 or 0.01 suppressed WNV viral titers as early as 48–72 h post superinfection. Although NY99 titers at an MOI of 0.01 showed signs of recovery towards the final timepoint in our study, these findings are similar to previous findings indicating that ISVs cause heterologous interference in vitro against pathogenic viruses belonging to other families (30,35,45–47)The ability of EILV to cause SIE is also not limited to WNV; it causes SIE against itself and other alphaviruses, including Sindbis virus, western equine encephalitis virus, and CHIKV (22). This indicates that EILV provides a broad range of protection against secondary infection in cells.

The exact mechanism of SIE remains elusive, but interference at different stages of the life cycle of the secondary virus—including attachment (31,32), penetration (24), and replication (33,34)—by the primary virus can contribute to SIE. We showed that EILV significantly reduced the titer of attached NY99 under superinfection conditions in C6/36 cells. The ability of EILV to interfere with NY99 attachment can be explained by downregulation of WNV receptors or co-receptors after EILV infection in these cells, as previously observed regarding other superinfecting viruses (32,48,49). In contrast, the attachment of WN02-1956 was not altered by the presence of EILV in our study. The differences observed between the WNV strains are potentially owing to the mutations in the E and NS5 protein nucleotide sequences of the WN02 genotype compared the nucleotide sequences of these proteins in NY99 genotype (7,8). These mutations may help superinfecting WN02-1956 to escape or compensate for the interference caused by EILV at the attachment stage in C6/36 cells (9); however, further studies are needed to confirm the mechanism of strain-specific interference caused by EILV at the attachment stage. Although the interference at the attachment stage by EILV likely contributes to the mechanism of SIE, it is not the main cause because at a later stage in the viral life cycle—internalization—the titers of both superinfecting strains NY99 and WN02-1956 were similar in the presence or absence of EILV.

The RNAi defense pathway is another potential driver of SIE in mosquitoes and mosquito cell lines (50,51). However, our use of C6/36 cells excluded siRNA, the main antiviral defense pathway, as the root cause of the ability of EILV to suppress WNV titers in these cells because C6/36 cells lack a functional dicer-2 vital for siRNA activation (52). However, the PIWI-interacting RNA (piRNA) pathway remains active in these cells (52,53). A local pairwise comparison of the EILV genome with that of WN02-1956 or NY99 confirmed that these viruses do not share sequence similarities of 21–30 nucleotides in length (not shown), necessary for siRNA and piRNA pathways of cross-protection (52,54). The ability of CHIKV—another alphavirus—to cause heterologous interference was similarly independent of the RNAi pathway (30). Small RNA analysis of EILV and WNV superinfection in C6/36 cells may clarify the role of the RNAi pathway in the ability of EILV to suppress WNV infection in these cells.

Many superinfection studies have been performed in cell culture but not in mosquito vectors. In the present study, we found that EILV did not alter the vector competence of either WNV strain in Cx. tarsalis under superinfection conditions at any timepoint in our study. Although EILV did enhance the NY99 whole-body titers in Cx. tarsalis, the WN02-1956 body titers remained unaffected at 3 days post superinfection. Similarly, a previous study has reported that the ISV Culex flavivirus Izabal enhanced WNV transmission in Cx. quinquefasciatus (Honduras colony) mosquitoes when co-infected (38). A likely explanation for the enhanced NY99 titers in our study is that NY99 “piggybacks” and infects more cells or cell types with the help of EILV. This strategy is used by the human immunodeficiency virus during co-infection with viruses such as Epstein-Barr virus to expand its tissue tropism in its host (55). In the present study, at the later timepoint, 7 days post superinfection—NY99 infection titers were no longer enhanced, whereas WN02-1956 infection titers were suppressed. Previous studies have reported that other ISVs also suppress WNV, but these studies used only one WNV strain to determine SIE (19,38,40). The leg and saliva titers of both NY99 and WN02-1956 were not affected by EILV at this timepoint. NY99 and WN02-1956 are genetically distinct, and this genetic variation may explain the different results obtained in the present study (7–9). The mutations in the WN02 genotype enhance its dissemination in Cx. tarsalis compared with that of NY99 (9). The decrease in WN02-1956 whole-body titers may be attributed to a need for more shared resources with EILV at a faster rate than NY99 (9). Further investigation into the effect of individual mutations between NY99 and WN02-1956 or transcriptional differences between NY99 and WN02-1956 under superinfection conditions with EILV may elucidate the strain-specific effect of EILV on WNV in Cx. tarsalis.

A caveat of our study on EILV–WNV superinfection is the use of an acute EILV infection instead of a persistent infection as observed for most ISVs in nature (16,56,57). ISVs are thought to be vertically transmitted from mother to offspring in nature (40,56,57); however, although EILV infects the ovaries of Cx. tarsalis following an oral challenge, EILV does not exhibit vertical transmission in laboratory conditions (13). A persistent EILV infection may result in different outcomes when followed by superinfection with WNV. Alternatively, the intrathoracic route of infection mimics an ISV infection better than the oral route in mosquitoes (13). However, because EILV infection via the intrathoracic route did not result in the infection of Cx. tarsalis ovaries (13), we used the oral route of infection in the present study to maximize the likelihood of EILV–WNV interaction.

Overall, our study adds to the growing literature on ISVs and their ability to cause SIE against human pathogenic viruses (17,19,37,38,40). ISVs have been reported to be a potentially safe tool to target pathogenic viruses via SIE in nature (54), but our study demonstrates that although EILV caused heterologous interference against both WNV strains—NY99 and WN02-1956—in C6/36 cells, SIE in Cx. tarsalis was strain-specific. The WNV strain NY99 no longer circulates in the US population, but its enhancement in Cx. tarsalis by EILV is a dangerous outcome for a potential tool. Additionally, our results demonstrate the importance of testing all circulating virus strains to determine the safety and ability of an ISV to cause SIE. The suppression of WN02-1956 in Cx. tarsalis was modest, with no effect on the dissemination or transmission of the virus, indicating that SIE alone is inadequate as a control strategy. Paratransgenesis (47,58,59), a strategy by which genetically modified EILV expressing antiviral peptides such as vago (60) is used to target WNV in Cx. tarsalis, may potentially improve the ability of EILV to suppress WNV and increase its specificity.