Repurposing Cilnidipine as an Entry Inhibitor Against Crimean–Congo Haemorrhagic Fever Pseudovirus Infection

doi:10.21203/rs.3.rs-8320594/v1

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Repurposing Cilnidipine as an Entry Inhibitor Against Crimean–Congo Haemorrhagic Fever Pseudovirus Infection

https://doi.org/10.21203/rs.3.rs-8320594/v1

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Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne pathogen of the Nairoviridae family, is classified as a high-risk biosafety level-4 (BSL-4) agent, which restricts its handling and limits mechanistic investigations. To overcome these challenges, we developed a CCHFV pseudovirus as a safe and reliable surrogate for studying viral entry and evaluating antiviral candidates. Pseudovirus-based systems have proven valuable tools for elucidating viral infection mechanisms, conducting neutralization assays, and screening small-molecule inhibitors. In the present study, a lentiviral packaging system incorporating CCHFV envelope glycoproteins (Gn and Gc) was established in HEK293T cells to generate infectious pseudovirions. Viral infectivity was quantified using a luciferase reporter assay. Cilnidipine, a clinically approved dual L-type and N-type calcium channel blocker with established antihypertensive activity, was investigated for its potential to inhibit CCHFV pseudovirus entry in HEK293T and Huh-7 cells. Treatment with cilnidipine resulted in a marked, dose-dependent reduction in luciferase signal, suggesting potent inhibition of pseudovirus entry at micromolar concentrations. Co-treatment assays further confirmed its inhibitory action at the early stages of infection. Overall, this study highlights cilnidipine as a promising repurposed antiviral candidate targeting viral entry pathways. The findings provide a foundation for future mechanistic and preclinical evaluations aimed at developing effective therapeutics against CCHFV.

Crimean-Congo Haemorrhagic Fever Virus

Pseudovirus

Envelope Glycoproteins

entry inhibition

Cilnidipine

The Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-borne pathogen known to cause crimean-congo haemorrhagic fever. In 1944, CCHFV was first identified in Criema and its distribution crossed more than 31 countries throughout Africa, South east Asia, Southern and eastern Europe. Human CCHF infection is caused by the bite of infected tick or through contact with blood and tissues of infected animals. Hyalomma ticks have been identified as the primary reservoir for CCHF infection [1–3]. Studies have shown that CCHFV can also infect wild animal species such as hares, ostriches, small rodents, buffalo and even rhinoceroses and, importantly for human exposure, livestock without apparent disease. CCHFV can spread through human contact. According to European Centre for Disease Prevention and Control (ECDC), nearly 10,000-15000 cases have reported each year worldwide [3–4]. World Health Organization considers CCHFV as a potential source of future disease outbreaks.

CCHFV belongs to the Orthonairovirus genus in the Nairoviridae family of the Bunyavirales order [5]. CCHFV is an enveloped, multisegmented negative-sense RNA virus. The genome comprises of three RNA segments (total length ~ 19·2 kbp), as small (S), medium (M), and large (L) segments [1, 6]. The small segment (S) (~ 1·7 kbp) encodes for nucleocapsid protein (NP) and Non-structural protein (NSs), the medium (M) RNA segment (~ 5·7 kb) encodes for glycoproteins (Gn and Gc), and the large (L) segment (~ 12 kbp) encodes for RNA-dependent RNA polymerase (RdRp) [7–10]. On the basis of S segment, seven genetically distinct strains of CCHFV have been identified as Africa 1 (West Africa, Senegal), Africa 2 (Central, Democratic Republic of the Congo, South Africa), Africa 3 (South and West Africa), Europe 1, Europe 2 (Greece), Asia 1 (Middle East, Pakistan, Iran), and Asia 2 (China, Tajikistan, Kazakhstan) [11–13]. The M segment encoding glycoproteins are formed by processing of polyprotein precursor to form mature transmembrane glycoproteins (Gn and Gc). At N-terminal region GP160/85 is further proteolytically processed into GP-38, a mucin-like domain, a highly variable region and the M-segment non-structural (NSm) protein. The mature Gn (37 kDa) and Gc (70 kDa) forms a lipid envelope. Gn protein play crucial role in membrane fusion and Gc protein is involved in binding to cellular receptor such as low-density lipoprotein receptor (LDLR). The L-segment encoding RdRp is involved in cap snatching during viral replication [1, 5, 6, 8, 14]. The surface glycoproteins are essential for host attachment and entry, are the crucial target for developing neutralizing antibodies and entry inhibitors. In the absence of approved vaccination and antiviral strategies against CCHFV, rapid and accurate detection of the virus is a must for patient treatment and subsequent infection control. Current detection techniques range from conventional to quantitative real-time PCR. Indirect immunofluorescence assays and ELISAs are also employed for the detection of virus-specific IgM and IgG antibodies [15–17]. Isolation of live virus is crucial for identification of new strains, vaccine and antiviral development. CCHFV, is highly hazardous pathogen, requires state-of-the-art BSL-4 facility for its propagation [18]. The requirement of high-quality laboratories serves as a major setback in the development of vaccines & therapeutics against CCHFV. With advance technologies, development of pseudovirus-based CCHF virus, provided a safe alternative for studying the infectious pathogen in BSL-2 facility.

Pseudotype viral particles have been developed for the study of highly pathogenic BSL-4 viruses, like Influenza, Ebola virus (EBOV), Japanese encephalitis virus (JEV), Marburg virus (MARV), Lassa Virus and Hanta virus (HNTV) [19–26]. Vasmehjani. A. A et.al have reported the development of CCHFV pseudovirions using the lentivirus pLenti-III-mir-GFP backbone (PMID: 32189084). Ma Xue et.al have successfully demonstrated generation of Vesicular Stomatitis Virus-Based Crimean-Congo Haemorrhagic Fever Reporter Virus (PMID: 40539243). In addition to neutralization assay, pseudovirus have been used to study the entry inhibitors for developing the antivirals. Previous reports showed that Ribavirin, a synthetic nucleotide have been used against CCHFV, however its use is controversial due to its efficacy and safety standards [27]. In addition, many reports have exploited host directed calcium channel blockers (CCB’s) against EBOV, MARV, JEV, LASV and recently reported Influenza virus [28–31]. In this study, we have reported HIV-1 pNL-4.3-luc pseudotyped with CCHFV surface glycoprotein. In this study, we report the generation of an HIV-1–based pseudovirus (pNL4-3.luc) bearing the surface glycoproteins (Gn and Gc) of Crimean-Congo haemorrhagic fever virus (CCHFV). The pseudovirions were successfully produced in HEK293T cells, and their infectivity titres and stability were evaluated across multiple susceptible cell lines to assess host range and entry efficiency. Following the establishment of a robust and reproducible pseudovirus assay, we examined the antiviral efficacy of a known entry inhibitor, previously demonstrated to possess significant in vitro and in vivo activity against influenza virus (PMID: 40189517). Cilnidipine, a member of calcium channel blocker, have been reported as fusion inhibitor against influenza virus [31]. Calcium channel blockers inhibit virus infection by blocking the influx of intracellular calcium levels or by directing acting on the entry mechanism showing off target inhibition. Cilnidipine also showed effective inhibition in STSFV and HTNV replication [32, 33]. The compound was tested for its ability to block CCHFV pseudovirus entry, thereby assessing its potential as a repurposed broad-spectrum antiviral agent and found to be effective in blocking the CCHFV pseudovirus entry. Together, these efforts aim to establish a safe pseudovirus platform for studying CCHFV entry mechanisms and to identify potential broad-spectrum antivirals that can be rapidly repurposed for the management of high-risk viral infections.

Cells

Human Embryonic Kidney 293T cells (HEK293T), African Green Monkey kidney cells (Vero and Vero E6), Human Cervical Adenocarcinoma (HeLa), and Hepatocyte Derived Cellular Carcinoma Huh-7 cell lines were purchased from American Cell Culture Type ATCC).). All cells were cultured in Dulbecco’s modified Eagle’s medium ((DMEM, Gibco, Invitrogen, USA). supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Invitrogen, USA) and 1% (vol/vol) penicillin/streptomycin (Gibco, Invitrogen, USA), at 37ºC in a 5% CO₂.

Plasmids

The full-length sequence of CCHFV glycoprotein (CCHFV GP), gene Asia 1-genotype, strain Matin (GenBank Accession No: AF467769.2) was codon-optimized and cloned into the mammalian expression plasmid pcDNA3.1 vector (Thermo Fisher Scientific, USA) to generate the CCHFV envelope plasmid pcDNA3.1CCHFV GP. The packaging HIV-1 pNL4-3.Luc.R-E- plasmid was generously provided by Dr. Kalpana Luthra, AIIMS, India.

Compound

Cilnidipine was obtained from Merck (Catalogue no. C1493). The stock compound was dissolved in 100% DMSO and aliquots of 10mM cilnidipine stock were stored in -20 until use.

Plasmid confirmation by restriction digestion

The pcDNA3.1 CCHFV GP cloned plasmid was isolated using the QIAGEN Miniprep Kit. The purified plasmid was digested with BamHI and XhoI (Biolabs) restriction enzymes and the digested DNA fragments were separated on a 1% agarose gel in TAE buffer.

Immunofluorescence assay

To confirm the expression of pcDNA3.1CCHFV glycoprotein in HEK293T cells, an immunofluorescence assay was performed. HEK293T cells were seeded (70,000/well) in a 24-well plate at 37° in 5% CO₂. The next day, cells were transfected with pcDNA3.1CCHF GP using HD FuGENE transfecting reagent (Promega, E2311). Cells were fixed after 48 hrs of transfection with 4% paraformaldehyde (Sigma), permeabilized with 0.1% Triton-X 100 in PBS for 20 min at RT, and blocked with 4% FBS (Gibco) in 0.1% PBST for 1 h at 37ºC. Cells were incubated with primary monoclonal antibody (anti-Crimean-Congo Haemorrhagic fever virus pre-Gc glycoprotein, clone 11E7, NR-40277 (BEI Resources)) with 1:500 dilution overnight at 4ºC on rocker. Later, cells were washed 3 times for 5 mins with 1X PBS and incubated with Alexa Fluor-488 rabbit anti-mouse IgG (1:500) at room temperature for 1 h. Cells washed with 1X PBS, counterstained with Hoechst stain (D9542, Sigma-Aldrich, United States). Finally, cells were washed for 5 minutes each and imaged under fluorescence microscope (IX-71, Olympus) with magnification of 20X. Untransfected HEK293T cells were used as negative control.

Production and titration of pseudovirus

For CCHF pseudovirus production, HEK293T cells were seeded (10^6 cells/ml) in a 6-well plate. Cells were co-transfected with pcDNA3.1CCHFV GP (expressing plasmid) and HIV-1 pNL4-3.Luc.R-E plasmid (packaging vector) and pcDNA3.1GP as described previously (PMID: 37663753, PMID: 36583790) using HD FuGENE transfecting reagent (Promega, E2311). After 48 hours post transfection, supernatant containing CCHF pseudovirus was centrifuged and stored at 4ºC until use.

For titration of CCHF pseudovirus, the HEK293T cells (25000–35000 cells/well) were seeded in a 96-well plate in incomplete DMEM media (100 µl/well). Later, 100 µl of pseudovirus supernatant was added to each well (final volume 200µl/well) and incubated at 37ºC in a 5% CO₂. Post-incubation, 100 µl culture supernatant was gently aspirated leaving 100 µl in each well. Finally, 100 µl of luciferase substrate (Revvity) was added to each well. After 2 min of incubation at 37ºC, 100 µl of cell lysate was transferred to a white solid 96-well plate for the detection of luminescence using a microplate luminometer [34]. The experiments were performed in triplicates and atleast two times. The pseudovirus titration (RLU/ml) graph was prepared using GraphPad Prism version6.

Infectivity of Pseudovirus in different cell lines:

Different cell lines were procured for susceptibly of pseudovirus infection. Briefly, Vero (10,000/well), Vero E6 (10,000/well), HeLa (10,000/well), and Huh-7 (40,000/well) cells were seeded in 96-well plate in complete DMEM media (supplemented with 10% FBS) at 37°C in 5% CO₂. 100 µl of incomplete media added, followed by the addition of 100 µl pseudovirus in each well. After 48 hours of incubation, 100 µl of culture supernatant was replaced with 100 µl of luciferase substrate (Revvity) and incubated for 2 min at 37ºC and 100 µl of lysate was transferred to a white solid 96-well plate for the detection of luminescence using a microplate luminometer [34]. The experiments were performed in triplicates and repeated atleast two times. The pseudovirus titration in terms of Relative Light Units (RLU/ml) was calculated and graph was plotted using GraphPad Prism version6.

Pseudovirus stability

Pseudoviruses were produced as described above. CCHF pseudovirus titration was performed in HEK293T cells in 96-well plate at different time points (7 days, 14 days, and 21 days) and at different temperatures (4ºC, -20ºC, and − 80ºC). Post-pseudovirus harvesting, similar procedure for detection of pseudovirus was followed up as described above and graph was plotted using GraphPad Prism version6 [35, 36].

Neutralization Assay

To assess neutralization of CCHFV pseudovirus, pseudovirus was incubated with a serial dilution of the monoclonal antibody (anti-CCHF Gc Pre-Glycoprotein, Clone 11E7) (1:1000), for 1 h at 37°C. The virus–antibody mixture was added onto HEK293T cells and incubated for 48 h at 37°C. Following the incubation period, neutralization was quantified by measuring relative luciferase activity (Revvity substrate) using a luminometer. The neutralization efficiency was normalised with the virus control and graph plotted as neutralization (RLU/ml) vs dilution.

MTT Assay

The MTT assay was performed to examine the cytotoxicity of cilnidipine in the HEK293T and Huh-7 cell lines. HEK293T and Huh-7 cells were seeded in a 96-well plate using DMEM supplemented with 10% FBS at 37ºC in 5% CO₂. HEK293T and Huh-7 cells were treated with cilnidipine using two-fold serial dilution (50 µM to 3.125 µM) in a serum-free DMEM, followed by 48 hours of incubation at 37ºC. The supernatant was subsequently aspirated, and MTT solution (5 mg/mL) in serum-free DMEM was added to cells, followed by 2 hours incubation at 37°C in the dark. Finally, formazan crystals were observed and crystals were dissolved with 100µl DMSO. Absorbance readings were taken at 570 nm via a microplate reader. The experiments were run in triplicates and atleast twice. The graph was plotted as % cell viability vs log concentrations (µM) using non-linear regression in GraphPad prism verison10.

Dose-dependent assay

To analyse CCHF pseudovirus inhibition with cilnidipine, we performed dose-dependent assay with different concentration of cilnidipine using two-fold dilutions (50 µM to 3.125 µM) in a serum-free DMEM. Briefly, HEK293T and Huh-7 cells were treated with 100 µl of cilnidipine in each well and incubated at 37ºC for 2 hours The compounds were gently aspirated and replaced with 100 µl of CCHF pseudovirus in each well for 48 hours. After 48 hours incubation, 100 µl of the culture supernatant was aspirated gently leaving100 µl in each well. Then, 100 µl of luciferase substrate (Revvity) was added to each well. After 2 min incubation at 37ºC, 100 µl of lysate was transferred to a white solid 96-well plate for the detection of luminescence using a microplate luminometer. In each experiment, DMSO used as negative control was less than 1%.

Antiviral assay

After identifying non-cytotoxic doses, experiments were conducted using two different concentrations of cilnidipine (4 and 8 µM) in HEK293T cells and (25 and 12.5 µM) two treatment schedules— (a) Pre-treatment—HEK293T cells were pre-treated for 2hrs with cilnidipine (100 µl/well) using two concentrations (4 µM and 8 µM) at 37ºC in 5% CO₂. Later, the compound was gently aspirated and replaced with 100 µl of CCHF pseudovirus in each well for 48 hours. Post incubation, the culture supernatant was aspirated gently to leave 100 µl in each well and 100 µl of luciferase substrate (Revvity) was added to each well. After 2 min incubation at 37ºC, 100 µl of lysate was transferred to a white solid 96-well plate for the detection of luminescence using a microplate luminometer. Similar procedure was followed for Huh-7 cells with (25 and 12.5 µM).

(b) Co-treatment—HEK293T cells were co-treated with 100 µl of cilnidipine (4 µM and 8 µM) and 100 µl of CCHF pseudovirus for 48 hours at 37ºC in 5% CO₂. Following incubation, 100 µl of culture supernatant was replaced with 100 µl of luciferase substrate (Revvity). After 2 min incubation at 37ºC, 100 µl of lysate was transferred to a white solid 96-well plate for the detection of luminescence using a microplate luminometer. Similar procedure was followed for Huh-7 cells with (25 and 12.5 µM). Three independent experiments were performed in triplicates and graphs were plotted as % infection inhibition vs conc.

Virus entry assay

For virus entry assay a standardised protocol was followed with slight modifications. Briefly, HEK293T and Huh-7 cells were seeded in a 96-well plate. The next day, cells were pre-chilled at 4ºC for 1 h. Cells were infected with 100 µl of pseudovirus at 4ºC for 3 hrs, later, the supernatant was replaced with 100 µl of cilnidipine (20µM, 12.5µM, and 8µM for HEK293T cells; 50µM and 20µM for Huh-7 cells) at 37ºC for 3 h. After 3 h of incubation, drug-containing supernatant was replaced with 200 µl of incomplete DMEM at 37ºC for 48 hours. After 48 hrs incubation, detection of luminescence was performed as described previously [37].

Virus Attachment Assay

To evaluate the effect of cilnidipine on viral attachment, HEK293T and Huh-7 cells were seeded in 96-well plates. After 24 hours, cells were pre-chilled at 4°C for 1 hour. The cells were then co-incubated with 100 µL of pseudovirus and 100 µL of cilnidipine (final concentrations: 8 µM and 4 µM for HEK293T cells; 25 µM and 12.5 µM for Huh-7 cells) in a 1:1 ratio and maintained at 4°C for 3 hours to allow virus binding without internalization. Following incubation, the pseudovirus–drug mixture was removed, and cells were washed twice with 200 µL of ice-cold PBS to eliminate unbound virus. Subsequently, 200 µL of incomplete DMEM was added, and the cells were incubated at 37°C for 48 hours. After incubation, luciferase substrate added and detected as described above [37].

Statistical analysis

All data are expressed as mean ± SE. GraphPad Prism10 software was used to perform statistical analysis and prepare graphs. Statistical significance determined using one-way ANOVA for multiple comparisons with the Bonferroni correction. A P-value of less than 0.05 was considered statistically significant. NS = non-significant.

Generation and characterization of HIV-1–based pseudovirions bearing CCHFV Glycoprotein

The pcDNA3.1CCHFV GP, clone was confirmed by sequencing and restriction digestion. A full-length digested fragment of ~ 4925bp was obtained (Fig. 1A). The expression of pcDNA3.1 CCHFV GP was confirmed in HEK293T cells by immunofluorescence assay. Post transfection, cells were fixed and permeabilized followed by incubation with primary and secondary antibody. Nuclei were counterstained with Hoechst stain. The presence of green fluorescence in cells indicates the expression of pcDNA3.1 CCHFV GP protein (Fig. 1B). These results show the successful transfection of pcDNA3.1CCHFV GP in HEK293T cells. For production of pseudovirus, a schematic work flow showing the co-transfection of pcDNA3.1 CCHFV GP and pNL4-3.Luc.R-E was given and quantification performed by luciferase assay (Fig. 2A). To produce the pseudovirus, initially co-transfection optimization with different ratio of pcDNA3.1 CCHF GP and pNL4-3.Luc.R-E was done and optimization was performed. The pseudoviruses produced the titres of ~ 1.3 × 10⁵ RLU/ml and ~ 1.1 × 10⁵ RLU/ml respectively in different ratio of backbone and CCHFV glycoprotein (Fig. 2B). With the optimization, it was observed that the ratio of (4 µg pcDNA3.1 CCHF GP: 1 µg pNL4-3.Luc.R-E) yielded high titres and was selected for routine pseudovirus production, yielding significant titre of ~ 7.0 × 10⁵ RLU/ml (Fig. 2C). These results collectively confirm the successful cloning, expression, and production of functional CCHFV pseudovirions, establishing a reproducible system suitable for downstream applications such as entry mechanism studies and antiviral screening.

Evaluation of Cell Line Susceptibility and Stability Profile of CCHFV Pseudovirus

Following the successful production of CCHFV pseudovirus in HEK293T cells, the susceptibility of various mammalian cell lines to pseudovirus infection was evaluated to determine permissiveness and host range. For this analysis, Vero, Vero E6, HeLa, and Huh-7 cells were seeded at densities of 10,000 cells per well (Vero, Vero E6, and HeLa) and 40,000 cells per well (Huh-7) in 96-well plates and subsequently infected with equivalent volumes of CCHFV pseudovirus. Viral entry and replication competence were quantified 48 hours’ post-infection using a luciferase reporter assay. The results revealed measurable infectivity in all tested cell lines, with titres of approximately 1.42 × 10⁴ RLU/mL in Vero E6 cells, 1.0 × 10⁴ RLU/mL in HeLa cells, and a markedly higher titre of 3.2 × 10⁵ RLU/mL in Huh-7 cells. Among the tested lines, Huh-7 cells exhibited the highest susceptibility to pseudovirus infection, showing a nearly 20–30-fold increase in luciferase activity compared to Vero or HeLa cells and surpassing the levels observed in HEK293T producer cells (Fig. 3A). These findings indicate that both 293T and Huh-7 cells provide an efficient and reproducible system for evaluating CCHFV pseudovirus infectivity and can be employed for subsequent inhibitor screening and mechanistic studies.

Next determined the stability of pseudovirus. For this, the pseudoviruses stability was optimized at different temperatures (4ºC, -20ºC, and − 80ºC) and at different time intervals (7 days, 14 days, and 21 days). After production of pseudovirus, collected supernatants were stored at different temperature for 7, 14, 21 days. CCHF pseudovirus infected in HEK293T cell line and quantified by luciferase assay which showed a titre of about 4.5 x 10^5 RLU/ml, 1.6 x 10^5 RLU/ml, and 3.6 x 10^5 RLU/ml on 7th day at 4ºC, -20ºC, and − 80ºC respectively. Similarly, pseudovirus titres were found to be 2.4 x 10^5 RLU/ml, 6 x 10^4 RLU/ml, and 1.2 x 10^5 RLU/ml on 14th day at 4ºC, -20ºC, and − 80ºC respectively and 5.5 x 10^3 RLU/ml, 1.9 x 10^3 RLU/ml, 2.9 x 10^3 RLU/ml on 21st at 4ºC, -20ºC, and − 80ºC respectively. The titres obtained showed that CCHF pseudovirus is stable at 4ºC up to 21 days as compared to other storage conditions (Fig. 3B, C & D).

HIV-1 pseudotyped CCHFV GP pseudoviruses neutralizes the anti-CCHF Gc Pre- Glycoprotein, Clone 11E7 monoclonal antibody

To further validate the functionality and antigenic authenticity of the generated CCHFV pseudovirus, a neutralization assay was conducted using a monoclonal antibody targeting the viral Gc glycoprotein. The experimental workflow for the neutralization assay is illustrated in (Fig. 4A). Briefly, equal volumes of CCHFV pseudovirus were pre-incubated with serial dilutions of the anti-CCHFV Gc pre-glycoprotein monoclonal antibody (Clone 11E7) prior to infection of HEK293T target cells. After incubation, luciferase activity was measured to assess the degree of viral inhibition. The neutralization profile, presented as reduction in luminescence, demonstrated a decrease in signal intensity. At a 1:1000 antibody dilution, a substantial reduction in relative luminescence units (RLU) was observed compared to the virus-only control, indicating significant neutralizing activity and effective blockade of pseudovirus entry (Fig. 4B). These findings confirm that the CCHFV pseudovirus accurately displays functional surface glycoproteins that retain antigenic properties comparable to the native virus, thereby validating its utility for studying neutralization responses and antibody screening in a BSL-2 environment.

Evaluation of Antiviral Efficacy of Cilnidipine Against CCHFV Pseudovirus

In this study, the CCHF pseudovirus carrying the envelope glycoproteins are essential for virus entry and are crucial target for antivirals. Cilnidipine, a calcium channel inhibitor, has been reported as fusion inhibitor (off target inhibition) so we tested its effect on CCHF pseudoviruses infectivity. The chemical structure of cilnidipine was procured from MolView Software (Fig. 5A). We tested the effect of cilnidipine against CCHF pseudovirus in HEK293T and Huh-7 cells. We determined the cytotoxicity of Cilnidipine in HEK-293T and Huh-7 cells. Cilnidipine had no cytotoxic effect on cell viability up to 250 µM in HEK293T and 200 µM in Huh-7 cells even after 48 hours of treatment. The CC₅₀ values of cilnidipine on HEK-293T and Huh-7 were > 250 µM and > 200 µM respectively (Fig. 5B,(i) & (ii)).

Then, we proceeded for analysing Cilnidipine antiviral activity against CCHF pseudovirus in HEK293T and Huh.7 cells. The antiviral effect of Cilnidipine in HEK293T and Huh-7 cells showed dose-dependent inhibition (~ 100% inhibition with highest conc.). The half maximal effective concentration (EC₅₀) of cilnidipine was 6.57 µM and 14.45 µM in HEK293T and Huh-7 cells respectively (Fig. 5C, (i) &(ii)), and their selectivity index was found to be > 38 and > 14 respectively. The results indicates that Cilnidipine showed inhibition against CCHF pseudovirus more effectively in HEK293T cells followed by inhibition in HuH-7 cells as indicated with the EC₅₀ values. Based on these EC₅₀ values obtained, we have used the higher and lower concentrations of cilnidipine in further experiments.

Inhibitory effect of cilnidipine at different stages of virus life cycle

Virus entry is a crucial step in virus infection. To determine the antiviral potential of Cilnidipine at early stage of virus life cycle, we have performed pre-treatment and co-treatment assay with Cilnidipine against CCHF pseudovirus in HEK293T and Huh-7 cells. The schematic method of pre- and co-treatment given in (Fig. 6A, (i) &(ii)). During co-treatment of cilnidipine with CCHF pseudovirus in HEK 293T, we observed reduction in luciferase activity, indicating ~ 50% infection inhibition at 8µM as compared to pre-treatment (< 40% inhibition at 8 µM) (Fig. 6B & C). Similarly, pre-treatment and co-treatment assays were performed in Huh-7 cells that showed 80% infection inhibition during co-treatment assay at 25µM as compared to pre-treatment (Fig. 6D & E).

Since the co-treatment of cilnidipine indicates the significant inhibition activity against the CCHF pseudovirus but does not differentiate the stage at which virus inhibition is occurring, we hypothesized the inhibition by Cilnidipine at the early stages as represented in the schematic (Fig. 7A (i) & (ii)). To confirm whether Cilnidipine is inhibiting at the early entry stage, we further performed the attachment (virus binding occurs at 4°C but stops virus entry) and entry (internalization at 37°C) assay in HEK293T cells and Huh-7 cells. The HEK293T cells showed reduction in luciferase activity in the entry assay, indicating entry inhibition by 70% at 20µM (Fig. 7B). However, no significant inhibition observed during the attachment assay (data not shown). Unlike HEK293T cells, Huh-7 cells showed reduction of luciferase showing 90% inhibition at attachment stage and 50% inhibition at 50µM at the entry stage (Fig. 7C and D) suggesting that in Huh-7 cells, the infection is blocked at both the pre-entry (binding) and entry stage (internalization), though the effect is more pronounced in the entry stage.

Conclusively, these results suggest that Cilnidipine co-treatment with CCHF pseudovirus have antiviral potency at significant micromolar concentration in both cells. In addition, cilnidipine effectively inhibited at entry stages.

Pseudoviruses are efficient alternatives to study highly pathogenic BSL3 and BSL4 infectious viruses. Many studies have reported the development of pseudotype viruses and their application in vaccine development, neutralization and antivirals studies (PMID: 38164690, PMID: 38791226). In order to develop pseudovirus, various packaging system are available for example; human immunodeficiency virus (HIV-1) based lentiviral packaging, the murine leukaemia virus (MLV) based and the VSV packaging system [38]. In the present study, we have used the human immunodeficiency virus based lentiviral pNL4-3.Luc.R-E two plasmids packaging system (PMID: 32837985, PMID: 24731927, PMID: 29218769). Consistent with other reported studies, HEK293T cells were susceptible for producing HIV-1 pNL4-3.Luc.R-E pseudotyped CCHF GP expressing pseudoviruses ( Fig. 1) (PMID: 38548922). By optimising different ratios of pcDNA3.1 CCHF GP and pNL4-3.Luc.R-E CCHF pseudovirus of significant high titres were produced (Fig. 2)and the pseudoviruses were found to be highly stable at 4^oC for two weeks. Since pseudoviruses play significant role in immunization and antiviral studies, thus stability is an essential requirement to maintain its efficiency. In general, pseudoviruses are stored in -80ºC that allows long term storage to maintain its stability. However, several VSV based and lentivirus based pseudoviruses are stable at 4 ºC and − 80 ºC respectively [35, 36]. In this direction, we have performed a stability test for CCHF pseudovirus at different temperatures and time intervals. Interestingly, we observed that the CCHF pseudovirus produced were effectively stable at 4 ºC up to 21 days [Fig. 3]. This was an advantage in developing CCHF pseudovirus as it allowed easy handling and avoiding repeated freeze-thaw cycles. Further analyses of the infectivity of pseudovirus in other cell lines (Vero, Vero E6, HuH-7, and HeLa), however showed only HEK293T, HuH-7 susceptibility as compared to other cell lines (very low efficiency observed in Vero E6 and HeLa and no efficiency in Vero cells). The entry mechanism of CCHF is not well understood. Garrison. R.A et.al have demonstrated clathrin-mediated endocytosis and low pH–dependent fusion for CCHFV entry (PMID: 23791227). HeLa and Vero E6 cells have variable efficiency of clathrin-dependent endosomal trafficking and cathepsin-L activation, both crucial for Gn/Gc-mediated fusion. In contrast, Huh-7 cells possess robust endosomal acidification and protease activation, which might be facilitating efficient pseudovirus fusion and genome delivery. Additionally, luciferase reporter readout depends on intracellular transcriptional and translational activity. Huh-7 and HEK293T cells possess high metabolic and transcriptional rates, giving strong luciferase signals. HeLa and Vero E6 have lower basal expression capacity, leading to weaker luminescence even if some entry occurs. However, further investigations are warranted to validate these observations and elucidate the underlying mechanisms more clearly.

We further characterised the pseudovirus infectivity with neutralization assay using Gc specific glycoprotein monoclonal antibody (anti-CCHF Gc Pre- Glycoprotein, Clone 11E7, BEI resources, NR 40277) that showed effective neutralization with reduced RLU/ml at 1:1000 that explains the effective potency to neutralize the pseudovirus. However, the neutralization efficiency was found to be reduced showing increasing titres at 1:2000 dilution suggesting the neutralization effect diminishes if used higher dilutions (Fig. 4). Thus, using lower dilutions a standard can be generated to provide effective neutralization with CCHFV Gc specific antibody for further other studies. The 11E7 monoclonal antibody is a mouse monoclonal antibody specific to the Crimean-Congo haemorrhagic fever virus (CCHFV) pre-Gc glycoprotein and was purified from the clone 11E7 hybridoma supernatant using Protein G affinity chromatography. The antibody recognizes an epitope located within a highly conserved region of the viral M segment, spanning amino acid residues 1443 to 1566 of the IbAr10200 strain of CCHFV. The successful neutralization of the CCHFV pseudovirus by this antibody demonstrates that the HIV-1–pseudotyped CCHFV retains the native pre-fusion conformation of the Gc glycoprotein and preserves its antigenic integrity when expressed within the HIV-1 lentiviral backbone.

Pseudovirus are useful tools for screening entry inhibitors. Many studies suggest it as potential application in screening of entry inhibitors, since the pseudovirus carrying the envelope proteins. In this study, we have used Cilnidipine that might inhibit pseudovirus infectivity. Several studies have reported the use of Cilnidipine, a fourth-generation calcium channel blocker (CCB’s) as anti-hypersensitive drug and as fusion inhibitor [31–32, 39 ]. In context to this, according to our hypothesis Cilnidipine (reported as fusion) inhibitor may show off target inhibition i.e inhibiting at the entry stage against CCHFV glycoproteins, essential for entry in host cells. Thus, to check if this could inhibit the pseudovirus infectivity we have analysed the cytotoxicity (CC₅₀) of Cilnidipine in HEK and Huh-7 cells. No toxicity was observed up to > 250µM. Then, we have analysed the effective concentration (EC₅₀) that inhibited 50% infection in dose-dependent manner (Fig. 5).

When a virus infects, attachment and entry in to host cells occurs with the help of envelope proteins. The envelope proteins are temperature sensitive thus a shift in temperature conditions allow us to identify the stage for studying the attachment and entry/fusion stage. The attachment assay performed at 4°C allows only the binding while ceasing the entry functions (endocytosis, membrane fusion) whereas the shift to 37°C facilitates the entry mechanism that measure the inhibition at entry stage. Since Cilnidipine was found to be effective against CCHF pseudovirus, further validation was done with co-treatment and entry assay. In our study, we observed that HEK293T cells showed in the entry assay but not in attachment assay (Figs. 6 & 7). This observation strongly suggests that inhibition is specific at the post-binding and fusion stage which might be due to disruption of clathrin-mediated entry, a known mechanism of CCHFV entry in host cells [40]. Moreover, Cilnidipine as described previously, reported to disrupt the clathrin proteins during virus entry in influenza virus infection entry supporting our results [31]. Further, we have analysed that Cilnidipine effect in Huh-7 cells is showing slightly different mechanism i.e it showed inhibition at both the binding and entry stage suggesting the specific interference of Cilnidipine with its receptors. Conclusively, Cilnidipine, a calcium channel blocker, showing off target inhibition can i.e. blocking the entry pathway of CCHF pseudovirus. However, the study limits with the mechanistic insights of infection inhibition and including other calcium channel blockers reported in previous studies.

Overall, this platform provides a valuable surrogate model for studying CCHFV entry mechanisms and supports the discovery of novel therapeutic and vaccine strategies against this high-risk pathogen.

Acknowledgments: We sincerely thank Prof Ganesan Karthikeyan, Executive Director, iBRIC-THSTI, for the suggestions. We thank Dr. Manmohan Parida, Director DRDE and Paban kumar Dash, Division of Virology, Defence Research and Development Establishment, for the support and guidance.The following reagent was obtained from the Joel M. Dalrymple - Clarence J. Peters USAMRIID Antibody Collection through BEI Resources, NIAID, NIH: Monoclonal Anti-Crimean-Congo Hemorrhagic Fever Virus Pre-Gc Glycoprotein, Clone 11E7 (produced in vitro), NR-40277.

Author Contributions: S.S: Conceptualization, Study design, Formal analysis, Writing – review & editing, Funding acquisition, Supervision, S. A: Designing, Formal analysis, Writing – review & editing. S.G performed experiments, Writing – review & editing , L.G. data analysis, compilation, Writing – original draft, review & editing , B.A.B, and G.K, experiment designing, performed experiments, data compilation.

Funding Source

The works were supported by Translational Health Science and Technology Institute (THSTI) core funding and the INDCEPI project, the Translational Research Project (TRP) funded by the Biotechnology Research and Innovation Council (BRIC) of the Department of Biotechnology, Ministry of Science and Technology, and Defence Res. & Dev. Organisation, Life Sciences Research Board Sanction Letter No. LSRB/o1/15 001/LSRB-422 /BTB /2023, Govt. of India.

Data availability: All data supporting the findings of this study are available within the paper.

Conflict of Interest The authors declare that there are no conflicts of interest.

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Repurposing Cilnidipine as an Entry Inhibitor Against Crimean–Congo Haemorrhagic Fever Pseudovirus Infection

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Abstract

Figures

Introduction

Methods

Cells

Plasmids

Plasmid confirmation by restriction digestion

Immunofluorescence assay

Production and titration of pseudovirus

Infectivity of Pseudovirus in different cell lines:

Pseudovirus stability

Neutralization Assay

MTT Assay

Dose-dependent assay

Antiviral assay

Virus entry assay

Virus Attachment Assay

Statistical analysis

Results

Generation and characterization of HIV-1–based pseudovirions bearing CCHFV Glycoprotein

Evaluation of Cell Line Susceptibility and Stability Profile of CCHFV Pseudovirus

HIV-1 pseudotyped CCHFV GP pseudoviruses neutralizes the anti-CCHF Gc Pre- Glycoprotein, Clone 11E7 monoclonal antibody

Evaluation of Antiviral Efficacy of Cilnidipine Against CCHFV Pseudovirus

Inhibitory effect of cilnidipine at different stages of virus life cycle

Discussion

Declarations

References

Status:

Version 1