Development of a novel plaque reduction neutralisation test for hantavirus infection

Abstract

In the Americas, hantaviruses cause severe cardiopulmonary syndrome (HCPS) with a high fatality rate. Hantavirus infection is commonly diagnosed using serologic techniques and reverse transcription-polymerase chain reaction. This paper presents a novel plaque reduction neutralisation test (PRNT) for detecting antibodies to Brazilian hantavirus. Using PRNT, plaque detection was enhanced by adding 0.6% of dimethyl sulfoxide into the overlay culture medium of the infected cells. This procedure facilitated clear visualisation of small plaques under the microscope and provided for easy and accurate plaque counting. The sera from 37 HCPS patients from the city of Ribeirão Preto, Brazil was evaluated for the Rio Mamoré virus (RIOMV) using PRNT. Six samples exhibited neutralising antibodies; these antibodies exhibited a low titre. The low level of seropositive samples may be due to fewer cross-reactions between two different hantavirus species; the patients were likely infected by Araraquara virus (a virus that has not been isolated) and RIOMV was used for the test. This assay offers a new approach to evaluating and measuring neutralising antibodies produced during hantavirus infections and it can be adapted to other hantaviruses, including viruses that will be isolated in the future.

An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection

“…The lack of cytopathic effects (CPEs) of hantaviruses in cultured cells also makes this method difficult to perform (McCaughey et al, 1999). Therefore, different methods have been developed to detect hantavirus replication, such as the improved PFT (Padua et al, 2015), the focus staining test (Tanishita et al, 1984), enzyme-linked immunosorbent assays (ELISAs) (Cheng et al, 2014), immunofluorescence assay (IFAs) (Yu et al, 2012), quantitative real-time PCR (qRT-PCR) (Jiang et al, 2013; Wei et al, 2013), flow cytometry (FCM) assays (Barriga et al, 2013), and in-cell western (ICW) assays (Ma et al, 2017). However, these methods have their own defects.…”

Development of a Comparative European Orthohantavirus Microneutralization Assay With Multi- Species Validation and Evaluation in a Human Diagnostic Cohort

“…Species determination of the infecting orthohantavirus can only be done by RT-PCR or comparative virus neutralization assays (VNT). To date, multiple variations of orthohantavirus neutralization assays have been described ( Tanishita et al., 1984 ; Lee et al., 1985 ; Takenaka et al., 1985 ; Niklasson et al., 1991 ; Horling et al., 1992 ; Chu et al., 1995 ; Heider et al., 2001 ; Maes et al., 2009 ; Padua et al., 2015 ; Li et al., 2017 ), however, the focus reduction neutralization test (FRNT) is generally viewed as the ‘Gold Standard’. Unfortunately, the orthohantavirus FRNT is rather laborious and time consuming and therefore not very suitable for assaying a large number of samples ( Horling et al., 1992 ; Li et al., 2017 ).…”

Experimental infection of Rio Mamore hantavirus in Sigmodontinae rodents

“…The rodents were infected with the RIOMV strain HTN-0007. The virus was cultivated for 14 days in Vero E6 cells (African green monkey kidney cells) ( de Pádua et al 2015 ). Culture supernatants were collected from the flasks of infected cells and aliquoted for use as the viral stock.…”