Development and evaluation of a RT-qPCR assay for the detection of Orthoflavivirus wesselsbronense from formalin-fixed, paraffin-embedded tissues from sheep and cattle

Abstract

Wesselsbron disease (WSL) is a neglected zoonotic disease caused by Wesselsbron virus (WESSV) that belongs to the species Orthoflavivirus wesselsbronense in the family Flaviviridae, genus Orthoflavivirus. It is an economically and veterinary important viral mosquito-borne disease primarily affecting ruminants of young age. The most significant differential diagnosis for WSL is Rift Valley fever (RVF), also an important zoonotic disease. Both diseases have similar geographic distributions and cause similar clinical outcomes. However, WSL disease may be underreported during a large-scale RVF outbreak and human cases possibly also overlooked. This first aim of the study was to develop and optimize a reverse transcription quantitative real-time PCR (RT-qPCR) assay for nucleic acid detection of WESSV in formalin-fixed paraffin-embedded (FFPE) tissue samples. Virological and serological methods have been described for the definitive diagnosis of WSL, however, this study reports on the development of an RT-qPCR assay that is sensitive and specific for the detection of WESSV RNA extracted from FFPE tissue samples. This is because FFPE tissues are often the only sample available by the time diagnosis is suspected. An alternative test using immunohistochemistry (IHC) has been in use at the Faculty of Veterinary Science, University of Pretoria, since 1996. However, this test often yields what is termed ‘non-specific staining’ by pathologists. This is when cells or other material stain with a chromogen in a non-typical way. In the case of WSL, this is difficult to interpret and an alternative test was considered necessary. The newly developed test was used to test for WESSV from sheep (n = 100) and cattle (n = 99) tissue samples selected from the 2010 - 2011 RVF outbreak across different districts within South Africa. The purpose of the selected cases was to determine if WESSV was present during the 2010 - 2011 RVF outbreak. Three cases that were classified as having non-specific labelling using IHC were also tested. To achieve the first aim of the study, flavivirus sequences available on GenBank were downloaded and aligned. The assay was designed to avoid non-specific cross-reactions with other flaviviruses. It employed WESSV-specific primers targeting the conserved region of the WSL envelope (E) protein and a WESSV minor groove binding probe. FFPE-positive and negative controls were produced by spiking different virus titres (1 x 106, 3 x 106, 5 x 106) of WESSV in cell cultures, fixing the cells in formalin and subsequently embedding the cells in paraffin wax. Tissues from ten animals that were experimentally infected with Bluetongue virus (BTV) in a previous study, were included as negative controls. FFPE blocks containing tissues from natural field cases, that previously tested positive for WESSV, were included to confirm the amplification of the target RNA. Nucleic acid was purified using two methods in parallel: a modified method employing MagMAX™ CORE Nucleic Acid Purification Kit using Qiagen reagents to deparaffinise samples, and a MagMAX™ FFPE DNA/RNA Ultra Kit using xylene to deparaffinise samples. Results from the MagMAX™ FFPE DNA/RNA Ultra Kit data showed improvement in RNA quality extracted as it gave lower CT values with steeper amplification curves. Due to this, it was selected as the method of choice for RNA purification as it produced better PCR results. The optimal primer and probe concentrations of the assay were 400 nM and 150 nM respectively. VetMAX™-Plus One-Step RT-PCR Kit reagents were used to amplify the WESSV target with one-step RT-qPCR format on the Applied Biosystems StepOnePlus system. Results of tissue sections previously tested with antibodies to WESSV using immunohistochemistry (IHC) (n = 8) were compared with the results of the RT-qPCR assay and found to be congruent. The efficiency of the assay was calculated to be 87.41% by performing serial dilutions (10-1 to 10-7) of the WESSV RNA in triplicates. The established sensitivity of the assay was 88.89% using natural field cases which were previously determined IHC-positive for WESSV. The assay was confirmed as a highly specific method for identifying WESSV, given that no traces of the virus were found in any of the negative control FFPE tissue samples from sheep (n = 10) that were previously experimentally infected with BTV. The second aim of the study was to determine if WESSV can be detected in field cases that were sampled during the 2010-2011 RVF outbreak. Tissues from 100 sheep sampled during the outbreak were tested and only one case tested positive for WESSV (CT value = 34.66). Out of a total of 99 cattle tissue samples tested, 3 samples were positive (CT values = 20.58, 26.58, 31.88). Among these positive cases, one was a foetal brain FFPE sample. Additionally, one weak positive cattle case (CT value = 34.17) was detected. Although the limit of detection could not be determined, these findings from this study suggest that field tissue samples were either infected at levels that were too low to be detected by the developed assay, or they were not infected. If the latter, it might be possible that WSL disease occurred infrequently during the RVF outbreak in 2010-2011. However, more cases would need to be studied to determine the occurrence of WSL disease. Overall, the developed WESSV RT-qPCR assay was efficient, sensitive, and specific and is a useful additional test to confirm a diagnosis of WSL in cases where only FFPE samples are available and results for IHC are inconclusive.