Surveillance for rabies-related lyssaviruses in South African insectivorous bat species

Abstract

Lyssaviruses are bullet shaped negative-sense RNA viruses that are all able to cause the fatal encephalitic disease known as rabies. The genus currently consists of 17 formally recognised viral species with one tentative species awaiting classification. The prototype virus for the Lyssavirus genus is the well-known rabies virus (RABV), while all other species in the genus are classified as rabies-related viruses. In South Africa specifically, RABV, Lagos bat virus (LBV), Duvenhage virus (DUVV), and Mokola virus (MOKV) are known to circulate, with RABV and DUVV associated with human fatalities. Active surveillance on rabies-related lyssaviruses in bats, specifically African insectivorous bat species, is either very sporadic or non-existing, providing an inaccurate overall representation of prevalence, diversity, and geographic distribution. Therefore, we conducted viral nucleic acid surveillance for lyssaviruses in different insectivorous bat species in South Africa. These samples were collected during routine field surveillance and included bats that were found dead, appeared to be displaying abnormal behavior or taken as vouchers specimens as part of bat taxonomic studies. A quantitative real-time reverse transcription polymerase chain reaction assay, capable of detecting the diversity of lyssaviruses were used to test extracted RNA. Three brain samples tested positive and were further characterized by conventional RT-PCR, DNA sequencing and phylogenetic analyses targeting the nucleoprotein gene. One of the positive brains was detected from a Common slit-faced bat (Nycteris thebaica) and the other two positive brains were detected from the Natal long-fingered bat (Miniopterus natalensis). Phylogenetic analysis of the nucleoprotein indicated one detection to be a Duvenhage lyssavirus with the other two detections showing a close relationship with the West Caucasian bat virus species, previously only detected in Eastern Europe. However, a more than 20 % nucleotide divergence indicated it to be a potentially new lyssavirus species, Matlo bat lyssavirus. The virus was successfully isolated using the mouse inoculation test followed by full genome next generation amplicon sequencing. The results of the full genome characterisation further supported the initial findings with concatenated coding regions nucleotide divergence ranging between 16% and 23.7% as well as consistent phylogenetic tree topology groupings identical to initial phylogenetic analyses using multiple evolutionary models. The identification of a putative new lyssavirus highlights the importance of routine lyssavirus surveillance to understand the diversity. Further investigation is required to determine the possible reservoir species since the Natal long-fingered bats are known to co-roost with different bat species in caves. The potential of spillover to humans and other animals is unknown but people often enter these bat roosts for traditional and recreational purposes and bats do come into contact with several animal species including humans during foraging.