Monoclonal antibodies to Cache Valley virus for serological diagnosis

Abstract

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

Fig 1. Virulence of CVV and anti-CVV specific Ab response in AG129 mice.

A) Mice (n = 6/group) were inoculated IP as described with 10-fold dilutions of CVV and monitored for signs of morbidity. 1000 PFU (open circle), 100 PFU (open triangle), 10 PFU (open square), 1 PFU (star), and 0.1 PFU (open diamond). B) and C) Binding of anti-CVV Ab from immunized mice to purified virus was determined by indirect ELISA with 4-fold dilutions of sera. Colors indicate day PI (day 35 (blue), day 66 (black), day 85 (green)). EC₅₀ values, shown as the reciprocal of dilution of sera, were determined by non-linear fit analysis, with the bottom of the curve constrained to 0, using GraphPad Prism software V6. EC₅₀ values were calculated for 35-, 66- and 85-days PI. IC₅₀ values for 85-days PI were determined by non-linear fit analysis, with the bottom and top of the curve constrained to 0 and 100, respectively, using GraphPad Prim software V6. > indicates no detectable binding reactivity or neutralizing activity at a 1:10 dilution of sera. D) CVV protein-specific Ab responses in AG129 mice 85-days PI. Lanes: N (negative control (PBS)), P (positive control (CVV MHIAF)), 1 (mouse 1), 2 (mouse 2), 3 (mouse 3), 4 (mouse 4), M (Bio-Rad Precision Plus Protein Dual Color Standard (Cat# 1610374)), Gc (glycoprotein C), Gn (glycoprotein N), N (nucleoprotein), kDa (Kilodaltons).

Fig 2. Binding, neutralization reactivity, and protein specificity of anti-CVV MAbs.

A) Binding of anti-CVV MAbs to purified virus was determined by indirect (blue) and capture (black) ELISA. B) EC₅₀ and IC₅₀ values (shown as MAb concentrations of ng/mL) in ELISA and PRNT were determined by non-linear fit analysis, with the bottom of the curve constrained to 0 for both analyses and the top of the curve constrained to 100 for neutralization analysis, using GraphPad Prism software V6. Dark blue: EC₅₀ values <100 ng/mL. Light blue: EC₅₀ values 100–10,000 ng/mL. > indicates no detectable reactivity at 10,000 ng/mL. C) Western blot of reduced purified CVV with anti-CVV MAbs was prepared as described in Materials and Methods. Lane nomenclature corresponds to MAb nomenclature. D) Western blot of non-reduced purified CVV with MAb CVV16, P (positive control (CVV MHIAF)), M (Bio-Rad Precision Plus Protein Dual Color Standard (Cat# 1610374)), Gc (glycoprotein C), Gn (glycoprotein N) N (nucleoprotein), kDa (Kilodaltons).

Fig 3. Cross-reactivity of anti-CVV MAbs with related orthobunyaviruses.

Vero cells were grown on glass coverslips in 24-well plates, infected with viruses in the Bunyamwera serogroup at an MOI 1.0 and fixed 24 hours post-infection. Cells were stained with anti-CVV MAbs and a goat anti-mouse FITC labeled conjugate (green). Nuclei of cells were stained with DAPI (blue).