Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
Loading...
Date
2021
Authors
Dimaculangan, Micah
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, taking into account the transcontinental transmission of the virus between South Africa and northern Europe by migratory birds. The conventional nested RT-PCR assay may be able to detect most alphaviruses due to minimal mismatches (0 – 1) detected between the primers and the partial nsP4 sequences of the alphavirus isolates, while the RT-HDA assay may not be well suited to detect other alphaviruses due to the many mismatches (>4) detected between the primers and the partial nsP4 sequences of the alphavirus isolates. Nevertheless, this shows that the RT-HDA is theoretically more specific that the conventional nested RT-PCR assay. The RT-HDA however failed to detect any alphaviruses in the 42 mosquito pools tested, which was not unexpected as the assay could only detect up to 4.8 x 105 copies of RNA. In contrast, the conventional nested RT-PCR assay was able to detect alphaviral RNA in five out of the 42 mosquito pools tested, and the nucleotide sequences were determined to identify the alphavirus species. SINV RNA was detected in three mosquito pools and Middelburg virus (MIDV) was detected in two pools. Phylogenetic analysis was subsequently performed to determine the genetic relationship of these isolates from the Free State with previously published/ reported SINV and MIDV isolates in South Africa, Africa, and around the world. The conventional nested RT-PCR assay developed in this study has shown to be a useful surveillance tool for the detection of mosquito-borne alphavirus infections. Given the low sensitivity determined for the RT-HDA assay, improvements, or alternative rapid and fieldable NAATs should be considered in the future for alphavirus surveillance applications in low resource settings.
Description
Dissertation (M.Sc. (Medical Microbiology and Virology))--University of the Free State, 2021
Keywords
Mosquito-borne alphaviruses, Prevention of diseases, Molecular nucleic acid, Venezuelan equine encephalitis virus