The role of cryptic Mastomys rodents in the maintenance and transmission of encephalomyocarditis virus in southern Africa

Abstract

Historically, South Africa has only recorded two outbreaks of encephalomyocarditis (EMC); the first in pigs in KwaZulu-Natal in 1979 and the second in free-ranging African elephants (Loxodonta africana) in the Kruger National Park (KNP) in 1993/4, and each caused by genetically distinct viruses. During the latter outbreak, Mastomys rodents were implicated as the reservoir host of the EMC virus (EMCV) based on serology and RT-PCR assays, however, the virus in rodents was not isolated or sequenced. Moreover, as both members of this cryptic species complex, Mastomys natalensis and M. coucha exhibit sympatric distribution in the KNP, and as molecular identification of the rodents was not performed, the true reservoir remains to be identified. The main aim of this study was therefore to determine the role that members of the cryptic Mastomys species complex play in EMCV disease epidemiology by identifying the most probable reservoir host of this Cardiovirus. This was achieved using a combined virus characterisation and host species distribution mapping approach. The former entailed RT-PCR, Sanger sequencing and genetic characterisation of three historical viruses isolated from two Mastomys rodents and a Mastomys-associated mite. Phylogenetic analyses of the near complete (7,439 nucleotide) genome sequences of these virus strains revealed a sister relationship between the Mastomys and elephant viruses, with high levels of nodal support (99 – 100%) and 93.6% sequence identity. These results confirm the role of Mastomys rodents as the source of infection for the historical elephant outbreak strains. However, the host species identity of the Mastomys-associated viruses isolated from rodents sampled in 1961 at the Sizwe Hospital, Edenvale, Gauteng, remains unknown as genetic typing was not conducted at the time of trapping. To address this shortcoming, the Sizwe Hospital sampling site was reassessed using a combined host identification and EMCV screening approach. Rodents were trapped for three consecutive nights and Mastomys faecal and anal swab samples were respectively screened for EMCV presence and to confirm the rodent host species. Nucleic acids extractions, RT-PCRs and Sanger sequencing of a 248 nt region of the 5’UTR of the virus genome revealed the presence of an unclassified Cardiovirus most closely related to an unclassified Marmot Cardiovirus from China in four Mastomys rodents. Nucleotide sequence analysis of the cytochrome b gene region revealed that 24 of the 28 Mastomys rodents captured were M. coucha, confirming the dominance this species at the site. of the viral genome. Assuming that the current species composition at the Sizwe site reflects the historical composition, these results implicate M. coucha as the likely reservoir of the spillover event to elephants. Lastly, this study aimed to generate updated species distribution maps for each of the cryptic Mastomys species using only genetically identified specimen occurrence records from southern Africa. Models of predicted species distribution were generated using MaxEnt and a carefully selected combination of predictor variables. The models generated performed well (AUC = > 0.8) and the results of these models suggest that M. natalensis and M. coucha are driven by different climatic variables, being temperature for M. natalensis and rainfall for M. coucha and prefer different levels of climatic variability. Areas of predicted sympatry occur along the eastern escarpment of South Africa, the central and southern regions of Zimbabwe and the northern-central regions of Namibia. Furthermore, species distribution maps were overlapped with localities of EMCV incidence throughout southern Africa, demonstrating that most EMCV localities are in regions of M. coucha occurence (85%). However, only a subset of these viruses have been characterised. Based on the available virus genome and Mastomys host data, available evidence suggests that the virus strains originating from the KZN and KNP outbreaks are associated with different Mastomys hosts, namely M. natalensis and M. coucha, respectively. This study has provided the first clear link between Mastomys rodents and the outbreak in elephants and has refined southern African distribution maps for the two members of the Mastomys species complex in southern Africa. Additional studies to further clarify the role of each Mastomys species in EMCV transmission and infection in South Africa are needed and will require the demonstration of definitive virus-host links through simultaneous characterisation of EMC viruses and Mastomys.