Abstract
The reservoir host of Mokola virus (MOKV), a rabies-related lyssavirus species endemic to Africa, remains unknown. Only sporadic cases of MOKV have been reported since its first discovery in the late 1960s, which subsequently gave rise to various reservoir host hypotheses. One particular hypothesis focusing on non-volant small mammals (e.g. shrews, sengis and rodents) is buttressed by previous MOKV isolations from shrews (Crocidura sp.) and a single rodent (Lophuromys sikapusi). Although these cases were only once-off detections, it provided evidence of the first known lyssavirus species has an association with non-volant small mammals. To investigate further, retrospective surveillance was conducted in 575 small mammals collected from South Africa. Nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay of 329 brain samples did not detect any lyssavirus ribonucleic acid (RNA). Serological surveillance using a micro-neutralisation test of 246 serum samples identified 36 serum samples that were positive for the presence of MOKV neutralising antibodies (VNAs). These serum samples were all collected from Gerbilliscus leucogaster (Bushveld gerbils) rodents from Meletse in Limpopo province (South Africa). Mokola virus infections in Limpopo province have never been reported before, and the high MOKV seropositivity of 87.80% in these gerbils may indicate a potential rodent reservoir.
Keywords: Bushveld gerbil; lyssavirus; Mokola; non-volant small mammal; rabies-related; reservoir; rodent; surveillance.
The Mokola virus (MOKV), a rabies-related lyssavirus, represents one of 17 recognised species within the Lyssavirus genus, all capable of causing a fatal encephalitic disease (Walker et al. 2018). The Mokola virus is exclusively endemic in Africa with only 30 sporadic cases reported since its discovery more than 50 years ago (Figure 1; Table 1) (Coertse et al. 2017; Kgaladi et al. 2013). The reservoir host of MOKV is still unknown, with spillover dead-end hosts such as domestic cats (Felis catus) and dogs (Canis familiaris), most commonly reported to be infected with MOKV. This has led to the hypothesis that the reservoir of MOKV might be a prey species that interacts with domesticated animals via a prey-to-predator pathway (Kgaladi et al. 2013). Non-volant small mammals (i.e. shrews, sengis and rodents) have been suggested as possible reservoir hosts considering that previous MOKV isolations were in shrews (Crocidura spp.), four in Nigeria and one in Cameroon (Causey et al. 1969; Kemp et al. 1972; Le Gonidec et al. 1978), and a single reported case in a rodent (Lophuromys sikapusi) in the Central African Republic (Saluzzo et al. 1984). To investigate further, nucleic acid and serological surveillance were retrospectively conducted, targeting non-volant small mammals from specific locations in South Africa.
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FIGURE 1: Geographical distribution of all reported Mokola virus cases (n = 30) in the African continent. |
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| TABLE 1: Summary of all reported Mokola virus cases in Africa. |
Non-volant small mammals were captured and sampled in accordance with the field procedure guidelines of Sikes and Gannon (2011) during the period of 2015–2017 from two different sites in South Africa: Meletse area in Limpopo province (24.5914° S, 27.6258° E) and Secunda area in Mpumalanga Province (26.5158° S, 29.1914° E). All the species investigated were designated as of Least Concern by The International Union for Conservation of Nature Red List of Threatened Species. Morphological species identification followed classifications by Meester et al. (1986), Newbery (1999), as well as Monadjem et al. (2015). Following morphological identification, animals were anesthetised with Isofor (Safeline Pharmaceuticals, South Africa), after which blood was collected by cardiac puncture (1% – 3% volume/body mass) in 0.8 mL MiniCollect serum separator tubes (Greiner Bio-One, Austria). Serum was separated from whole blood by centrifugation (Centrifuge 5418, Eppendorf, Germany) at 4300 g for 5 min and transferred to 2.0 mL Sarstedt tubes (Sarstedt Inc.). Animals that were not collected as voucher specimens were marked with a unique tattoo number near the base of their tail, and released back to their respective capture sites. Voucher specimens were euthanised with an overdose of Isofor, after which their organs were harvested (i.e. brain, tongue, salivary glands, heart, kidney, lungs, pectoral muscle, spleen, intestines, rectum and bladder) in 2.0 mL Sarstedt tubes for a broader pathogen surveillance study and immediately stored in liquid nitrogen until storage at –80 °C. Carcasses were placed in a 3 L PathoPak (Intelsius Solutions, United Kingdom [UK]) containing 80% ethanol and were submitted to Ditsong National Museum of Natural History and the Natural History Collection for Public Health and Economics for voucher-based morphological identification, and museum archiving.
Total ribonucleic acids (RNAs) were extracted from brain samples (n = 329) (nine shrews, four sengis and 316 rodents) using TRIzol™ reagent (Invitrogen, United States [US]), followed by nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay as previously described (Coertse et al. 2019). Serum samples (n = 246) (three shrews, four sengis and 239 rodents) were subjected to serological surveillance using a micro-neutralisation test as previously described (Smith & Gilbert 2017), during which MOKV 12/458 (2012, Felis catus, Durban, KwaZulu-Natal, South Africa) (Coertse et al. 2017) was used as challenge virus. If a reduction or absence of fluorescence was observed at the 1:25 serum dilution during initial screening, the serum sample was subjected to follow-up screening (in duplicate) at 1:10, 1:50, 1:250 and 1:1250 serum dilutions. The 50% end-point (ED) neutralisation titre was calculated by the Reed and Muench method (1938) and considered positive for Mokola virus neutralising antibodies (MOKV VNAs) when they had a 50% ED neutralisation titre at a serum dilution of ≥ 25 (i.e. where ≤ 5 out of the 10 counted fields contain infected cells at the 1:25 serum dilution). If additional material was available, non-volant small mammals that tested positive for the presence of MOKV VNAs were subjected to genetic species identification with the Cytochrome B (CytB) barcoding PCR assay as previously described (Greenberg et al. 2012). Template deoxyribonucleic acid (DNA) required for the barcoding assay was extracted from various biological sample types (such as blood, kidney, heart and pectoral tissue) using the Quick-DNA™ Miniprep Plus Kit (Zymo Research, US).
All of the brain samples were negative for the presence of viral RNA with the pan-lyssavirus qRT-PCR assay (Appendix Table 1-A1). Negative results were expected as these animals were apparently healthy individuals and did not exhibit any visible signs of disease. An overall MOKV seropositivity of 87.80% (36 out of 41) was observed for the gerbils (Gerbilliscus leucogaster) tested from Meletse at the cut-off 1:25 serum dilution (Figure 2; Appendix Tables 1-A1, 2-A1, 3-A1 & 4-A1). The titre ranges for this rodent species were high when compared to another serological surveillance study conducted in Zimbabwe (Foggin 1988). Foggin identified MOKV VNAs in 5.63% (18 out of 320) of all rodents that were tested. An overall MOKV seropositivity of 17.57% (13 out of 74) was observed for gerbils which neutralised MOKV infection at various serum dilutions that ranged from 1:8, 1:16 to 1:32. None of the other MOKV serological surveillance studies have tested this rodent species for the presence of MOKV VNAs (Aghomo et al. 1990; Kemp et al. 1972; Nottidge, Omobowale & Oladiran 2007; Ogunkoya et al. 1990). Even though MOKV has been shown to cross-react in serological assays with other closely-related lyssaviruses (Kuzmin et al. 2008), cross-reactivity with other phylogroup II lyssaviruses was not investigated in this study.
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FIGURE 2: Graphical representation of the micro-neutralisation test results of the Gerbilliscus leucogaster serum samples from Meletse (n = 36). |
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Of the 36 gerbils showing MOKV seropositivity, only 28 were genetically identified with the CytB barcoding PCR assay (Table 2). The same identification was obtained from morphological examination of 24 voucher specimens (Table 2). Eight gerbils could not be identified to species level as they were released and no additional sample material was available. The Highveld gerbil, Gerbilliscus brantsii, is sympatric with G. leucogaster, however, based on known museum records, no G. brantsii has been caught at Meletse before (Rautenbach 1982) and these were, therefore, allocated to G. cf. leucogaster. The variability observed in the per cent identity (i.e. 83.78% – 100.00%) between the individual gerbils is expected since previous molecular characterisation assays performed on the Gerbilliscus genus have recorded intra-species genetic variation that range from 1% to 20% (Aghová et al. 2017; Colangelo et al. 2007).
| TABLE 2: Genetic and morphological species identification and voucher information for all Gerbilliscus leucogaster serum samples from Meletse, Limpopo province that were positive for the presence of Mokola virus neutralising antibodies. |
Members of the Gerbilliscus genus are nocturnal and terrestrial, exhibit no sexual dimorphism (Skinner & Chimimba 2005) and occupy simple to complex, deep burrows (i.e. warrens) (De Graaff 1981; Granjon & Dempster 2013). They are physiologically, morphologically and behaviourally adapted to live in arid climates (Granjon & Dempster 2013; Monadjem et al. 2015). Gerbilliscus leucogaster, however, is less arid adapted and can be found along rivers and drainage lines in open grasslands and wooded savannas (Dempster 2013; Monadjem et al. 2015). The breeding pattern and social organisation of G. leucogaster rodents are not well-understood, however, studies have reported a communal nature (De Graaff 1981; Smithers 1971) with burrows being occupied by a pair (Skinner & Chimimba 2005) and some warrens housing families or several adults (Choate 1972). The ecological nature of Bushveld gerbils may potentially be the reason why this specific rodent species are more likely to be MOKV seropositive compared to solitary rodent species belonging to the Steatomys and Rhabdomys genera occurring at Meletse.
More nucleic acid and serological surveillance studies in non-volant small mammal populations are required to obtain a better understanding of MOKV distribution, prevalence and its potential reservoir species. Brain and serum samples in this study were collected from seemingly healthy small mammals in areas that do not coincide with areas where previous MOKV cases have been reported in South Africa. Surveillance should be expanded to areas where MOKV spillover infections in cats and dogs have previously been reported. Furthermore, because lyssavirus distribution and dynamics might be influenced by seasonality, surveillance efforts should also include samples that were collected in different seasons and over multiple years. This expansion, together with representative sample sizes of certain non-volant small mammal species, will collectively increase the possibility of identifying more of these animals that are infected or that have previously been exposed to MOKV.
Acknowledgements
Competing interests
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
Authors’ contributions
W.C.M. performed all experiments associated with this study which forms part of his M.Sc. Medical Virology degree. J.C. and W.M. provided academic guidance and supervised the overall process and operations of this study. T.K., M.K. and L.H.S. assisted with non-volant small mammal sample collection and species identification in the field. T.K. provided museum information from the Ditsong National Museum of Natural History. All authors contributed equally to the construction of this research communication.
Ethical considerations
This study formed part of a larger surveillance programme of the Bio-surveillance and Ecology of Emerging Zoonoses Research Group in the Centre for Viral Zoonoses that focuses on zoonotic pathogens in bats and non-volant small mammals. The overall research had animal ethical clearance from the University of Pretoria’s Animal Ethics Committee (AEC) (principal investigator: W.M.; project reference number: EC071-15) and had permission to do research in terms of Section 20 of the Animal Diseases Act of 1984 (Act No. 35 of 1984) from the Department of Agriculture, Land Reform and Rural Development (DALRRD) (Project Name: Epidemiology of zoonotic pathogens in rodents, shrews and sengis in Southern Africa; project reference number: 12/11/1/1/8). Sampling permits were obtained from Limpopo’s Department of Economic Development, Environment and Tourism (ZA/LP/73972 [2016–2017] and ZA/LP/83642 [2017–2018]) and Mpumalanga’s Tourism and Parks Agency (MPB.5583 [2017]). The M.Sc. Committee from the University of Pretoria’s School of Medicine, Faculty of Health Sciences approved the protocol of this research project (Project Reference Number: 13057368). Individual animal ethical clearance (Principal Investigator: WM.; Project Reference Number: H008-18), as well as research ethical clearance was obtained from the University of Pretoria’s AEC and Research Ethics Committee (Project Reference Number: 426/2018).
Funding information
This study was funded by the South African Research Chair in Infectious Diseases of Animal (Zoonoses) from the National Research Foundation of the Department of Science and Innovation, W.M. (UID98339), as well as additional grants awarded to W.M. by the NRF (UID92524, UID85756 and UID91496). The National Research Foundation for funding the equipment based at the DNA Sanger Sequencing Facility in the Faculty of Natural of Agricultural Sciences, University of Pretoria (UID78566) and the Poliomyelitis Research Foundation.
Data availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
Disclaimer
The contents of this research communication are solely the responsibility of the authors. The opinions, findings and conclusions expressed do not necessarily reflect the official view of the National Research Foundation.
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Appendix 1
| TABLE 1-A1: Non-volant small mammal species included in the surveillance of Mokola virus in South Africa. |
| TABLE 2-A1: Sampling event details of collected Gerbilliscus leucogaster serum samples from Meletse, Limpopo province and their seropositivity. |
| TABLE 3-A1: The 50% end-point neutralisation titres of all Gerbilliscus leucogaster serum samples from Meletse, Limpopo province that were positive for the presence of Mokola virus neutralising antibodies. |
| TABLE 4-A1: Micro-neutralisation test results of all non-volant small mammal serum samples that tested negative for the presence of Mokola virus neutralising antibodies. |
Appendix 2
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