A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity

Abstract

Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.

In-vivo Activity of IFN-λ and IFN-α Against Bovine-Viral-Diarrhea Virus in a Mouse Model

“…We provided here two key contributions to the BVDV-BALB/c model-namely, (1) the use of the NS3-In-Cell ELISA to measure viremia by revealing virus infectivity directly on the MDBK infected cells using an antibody against NS3 and (2) the long-term follow-up of viremia. The NS3-In-Cell ELISA recently developed by our laboratory (25) can measure viral titers with a minimum amount (15 µl) of serum sample, enabling the daily bleeding of animals in accordance with standard animalwelfare regulations. Moreover, NS3 is a conserved non-structural protein used as a marker of viral replication for any BVDV strain, regardless their genotype or biotype.…”

“…We have recently reported the in-vitro antiviral activity of recombinant IFN-α and -λ with several BVDV strains of different biotype and genotype, including field strains (25). Human IFN-α has also prove to be effective against cp and ncp BVDVs strains (44).…”

“…In the instance where the virus has circulated during the pregnancy period, the pregnant animals can be treated with IFN-λ to prevent transmission, as innate mechanisms of immunity are active even during the fetal period (50). The possibility of expressing bovine IFNs as recombinant proteins (25,51) furthermore opens up a new scenario for preventing and treating bovine viral infections in the field. Studies in cattle are needed to confirm the activity of IFN-λ in the natural host and to evaluate the time-span of its biological activity, in order to define how long an IFN application can protect against infection in the field.…”

“…Viremia was determined in individual-or pooled-serum samples added to MDBK cells monolayers by revealing virus infectivity with a monoclonal antibody against NS3 (25). The protocol of this so-called "In-Cell ELISA, " has been described previously and was applied with the following modifications.…”

“…We have recently demonstrated that several BVDV strains are susceptible to bovine IFN-α and IFN-λ in vitro. We also developed a method to easily measure infectivity of BVDV by visualizing infected cells using a monoclonal antibody against a non-structural protein (NS3), considered a marker of virus replication (25). Recently, a mouse model of BVDV infection was set-up and tested with several strains showing that mice developed viremia without clinically manifest disease (26,27).…”