Establishment of a New Real-Time Molecular Assay for the Detection of Babanki Virus in Africa

Abstract

Babanki virus is a subtype of the Sindbis virus, a widespread arthropod-borne alphavirus circulating in Eurasia, Africa, and Oceania. Characterized by rashes and arthritis, clinical infections due to Sindbis were mainly reported in Africa, Australia, Asia, and Europe. However, its sub-type, Babanki virus, was reported in Northern Europe and Africa, where its epidemiology potential remains poorly understood. The diagnosis of alphaviruses is mainly based on serological testing and conventional PCR methods, which have considerable limits. In this study, we developed a real-time qRT-PCR assay for the detection of Babanki virus. The analytical sensitivity and specificity of the newly established assay were evaluated using in vitro standard RNA and related viruses relevant to the African context, respectively. In addition, its diagnostic sensitivity was assessed using a subset of Babanki virus-positive and -negative mosquito pools collected from the field. The new real-time qRT-PCR assay exhibited a 100% specificity, a 95% detection limit of 1 RNA molecule/reaction, and a diagnostic sensitivity of up to 120 pfu/reaction. This newly established assay could be useful not only for the detection of Babanki virus during epidemics but also in future experimental and surveillance studies focusing on their epidemiology and pathogenicity.

Keywords: Africa; Babanki virus; molecular diagnostic; qRT-PCR.

Figure 1

Analytical sensitivity of new qRT-PCR assay for BBKV. Linear regression analysis was performed by plotting qRT-PCR threshold cycle (Ct) values (A) against the number of RNA molecules per reaction detected in five replicates (5/5). Points represent mean values and error bars represent standard deviation. The qRT-PCR assay produced positive signals with dilutions from 10⁶ to 1 RNA molecule/reaction in five out of five analyses. Probit regression analysis was performed using data from the five qRT-PCR assays (B). Graphs were plotted using PRISM v10.2.2 (GraphPad Software Inc., San Diego, CA, USA), and the limit of detection with 95% probability is represented by the red triangle and is 1 RNA molecule/reaction.

Figure 2

Analytical sensitivity of the newly established qRT-PCR assay using 10-fold serial dilutions of BBKV in human serum (black curve) and 10% L-15 medium (gray curve). Dilutions were tested in triplicate (A). The qRT-PCR assay detected up to 120 pfu/reaction, corresponding to 120 RNA molecules/reaction (B) using the equation obtained in Figure 1A.

Figure 3

Vector competence of Aedes aegypti, Culex neavei, and Culex quinquefasciatus populations orally exposed to BBKV at 7 and 15 days post-infection (dpi). The (panel A) shows the percentage of infection in the body (red) and the percentage of dissemination in legs (green), midgut (blue), and salivary glands (purple) for the three mosquito species. The (panel B) shows the corresponding copy numbers detected by the newly established assay from the different organs of Aedes aegypti (in red), Culex neavei (in green), and Culex quinquefasciatus (in blue).