The antiviral effect of jiadifenoic acids C against coxsackievirus B3

Abstract

Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0-6 h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.

Keywords: Antiviral activity; CAR, coxsackievirus and adenovirus receptor; CPE, cytopathic effect; CVB3; CVB3, coxsackievirus B type 3; CVBs, coxsackie B viruses; DAF, decay accelerating factor; DCM, dilated cardiomyopathy; IC50, 50% inhibitory concentration; IRES, internal ribosomal entry site; Jiadifenoic acids C; MOI, multiplicity of infection; NTR, non-translated region; RBV, ribavirin; RdRp, RNA-dependent RNA polymerase; SI, selectivity index; Vero, African green monkey kidney cells.

Graphical abstract

Figure 1

The chemical structure of jiadifenoic acids C.

Figure 2

Inhibition of CVB3-induced CPE by jiadifenoic acids C. Confluent Vero cultures were inoculated with 100 TCID₅₀ CVB3 (Nancy). After 1 h of adsorption at 37 °C, the monolayers were washed with PBS and incubated at 37 °C in the maintenance medium (MEM plus 2% FBS) with or without various concentrations of jiadifenoic acids C. The morphological changes in Vero cells were observed until the CPE of the control group cells reached 4.

Figure 3

Jiadifenoic acids C reduce CVB3 viral RNA synthesis in Vero cells. Vero cells (9×10⁵ cells/well) were plated into 6-well culture plates for incubation for 16 h. The medium was removed and cells were infected with 600 μL CVB3 of 100 TCID₅₀ for 1 h, after which various concentrations of jiadifenoic acids C were added immediately and the incubation continued for 24 h. The cells were harvested for one-step qRT-PCR assay. *P<0.001 vs control.

Figure 4

Jiadifenoic acids C reduces CVB3 viral proteins (VP1) synthesis in Vero cells by western blot assay. Vero cells (9×10⁵ cells/well) were plated into 6-well culture plates for incubation for 16 h. The medium was removed and cells were infected with 600 μL CVB3 (Nancy) of 100 TCID₅₀ for 1 h, after which various concentrations of jiadifenoic acids C were added and incubation continued for 36 h. The cells were harvested for western blot assay.

Figure 5

Time course assay of jiadifenoic acids C against CVB3. (a) An illustration of the treatment with jiadifenoic acids C. Confluent Vero cells were inoculated with CVB3 (Nancy) at a multiplicity of infection (MOI) of 1 for 1 h. Jiadifenoic acids C (80 µg/mL) were added at the designated times. (b) The morphological changes in Vero cells were observed until the CPE of the control group cells reached 4. The results show CPE induced by CVB3 infection with or without jiadifenoic acids C treatment, and the virus control group is set as 100%.

Figure 6

Jiadifenoic acids C reduce CVB3 viral proteins (VP1 and 3D) synthesis in Vero cells by western blot assay. Vero cells (9×10⁵ cells/well) were plated into 6-well culture plates for incubation for 16 h. The medium was removed and cells were transfected with 4 μg plasmids pEGFP-VP1, pEGFP-VP4-VP2-VP3 (pEGFP-VP4-3), pEGFP-2C, pEGFP-3A, pEGFP-3B, pEGFP-3C and pEGFP-3D. After 48 h cells were treated with various concentrations of jiadifenoic acids C and incubation continued for 36 h. The cells were harvested for western blot assay.