Purpose: Coronaviruses are RNA viruses encompassing four genera. The alpha- and betacoronaviruses commonly cause mild disease in humans. However, outbreaks of severe respiratory disease in 2002 and 2012 led to the identification of highly pathogenic human betacoronaviruses, SARS- and MERS-CoV, respectively. Bats are believed to be the reservoir host from which all mammalian coronaviruses emerged. Few studies have been published on South African bat coronaviruses; only 16 bat alphacoronavirus and two betacoronavirus sequences have been reported. Phylogenetic inference shows that the betacoronavirus sequences belong to the same viral species as MERS-CoV. Through a One Health approach, this study aimed to describe coronavirus diversity within South African bat populations as well as factors that might influence bat-coronavirus ecology. Methods & Materials: During a general surveillance effort, 404 bat faecal pellets were screened using PCR assays targeting conserved regions of the coronavirus genome. An additional 183 faecal pellets, collected from Neoromicia capensis bats, were screened as part of a species-specific surveillance study. Using mixed effects logistic regression analyses, collected ecological sampling data were collated with screening results to identify possible predictors of coronavirus infection in N. capensis bats. Results: Based on putative coronavirus species classification criteria, the general surveillance effort detected nine coronavirus species, eight alphacoronaviruses and one MERS-related betacoronavirus, from eight different bat species. The species-specific surveillance detected three coronavirus species, including MERS-related betacoronaviruses, and identified several instances of coinfection with two different coronaviruses. The mixed effects logistic regression analyses indicated that female N. capensis bats and bats trapped at low altitude sites with low body condition scores were most likely to be coronavirus positive. Conclusion: This study demonstrates that diverse coronaviruses are present in different South African bat species and lends additional support to an ongoing circulation of MERS-related betacoronaviruses in this region. The observed cases of coinfection indicate the potential for recombination that could lead to the emergence of a new coronavirus that might have zoonotic potential. The collation of ecological data with screening results revealed that both host and environmental factors may influence coronavirus ecology. These findings could assist the development of improved wildlife surveillance sampling strategies for better detection of novel bat coronaviruses.

Arenaviruses are feared as agents that cause viral hemorrhagic fevers. We report the identification, isolation, and genetic characterization of 2 novel arenaviruses from Namaqua rock mice in Namibia. These findings extend knowledge of the distribution and diversity of arenaviruses in Africa.

ABSTRACT The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes lethal respiratory infections mainly on the Arabian Peninsula. The evolutionary origins of MERS-CoV are unknown. We determined the full genome sequence of a CoV directly from fecal material obtained from a South African Neoromicia capensis bat (NeoCoV). NeoCoV shared essential details of genome architecture with MERS-CoV. Eighty-five percent of the NeoCoV genome was identical to MERS-CoV at the nucleotide level. Based on taxonomic criteria, NeoCoV and MERS-CoV belonged to one viral species. The presence of a genetically divergent S1 subunit within the NeoCoV spike gene indicated that intraspike recombination events may have been involved in the emergence of MERS-CoV. NeoCoV constitutes a sister taxon of MERS-CoV, placing the MERS-CoV root between a recently described virus from African camels and all other viruses. This suggests a higher level of viral diversity in camels than in humans. Together with serologic evidence for widespread MERS-CoV infection in camelids sampled up to 20 years ago in Africa and the Arabian Peninsula, the genetic data indicate that camels act as sources of virus for humans rather than vice versa. The majority of camels on the Arabian Peninsula is imported from the Greater Horn of Africa, where several Neoromicia species occur. The acquisition of MERS-CoV by camels from bats might have taken place in sub-Saharan Africa. Camelids may represent mixing vessels for MERS-CoV and other mammalian CoVs. IMPORTANCE It is unclear how, when, and where the highly pathogenic MERS-CoV emerged. We characterized the full genome of an African bat virus closely related to MERS-CoV and show that human, camel, and bat viruses belong to the same viral species. The bat virus roots the phylogenetic tree of MERS-CoV, providing evidence for an evolution of MERS-CoV in camels that preceded that in humans. The revised tree suggests that humans are infected by camels rather than vice versa. Although MERS-CoV cases occur mainly on the Arabian Peninsula, the data from this study together with serologic and molecular investigations of African camels indicate that the initial host switch from bats may have taken place in Africa. The emergence of MERS-CoV likely involved exchanges of genetic elements between different viral ancestors. These exchanges may have taken place either in bat ancestors or in camels acting as mixing vessels for viruses from different hosts.

Background: Hantaviruses are hosted by rodents from different subfamilies and shrews from the Crocidurinae subfamily. Hantaviruses are responsible for two types of disease in human beings: Haemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. As the first African hantavirus, Sangassou virus was identified from the rodent Hylomyscus simus in Guinea in 2006, followed by Tanganya virus in Guinea and Azagny virus in Cote d'Ivoire, both from shrews. There is evidence of human infections occurring. Our study aims to identify hantaviruses in Southern African small mammals and detect evidence of infection in human beings. Methods & Materials: Total RNA was extracted from the lungs of rodents and shrews trapped in the Western and Northern Cape Provinces between 2007 and 2012 and screened for hantavirus RNA by RT-PCR using Pan-hantavirus primers. Human sera left over after routine biomedical laboratory testing at four laboratories in the Western Cape province were screened for hantavirus IgG antibodies by in-house ELISA using recombinant nucleocapsid proteins from Dobrava and Puumala viruses. Reactive specimens underwent a confirmatory algorithm using more specific assays: mAb-capture IgG and IgM ELISA, Western blot, indirect immunofluorescence test and focus reduction neutralisation test (FRNT). Results: Of 2544 small mammal specimens screened for hantavirus genome by RT-PCR so far all had negative results. Of 1442 human sera screened for hantavirus IgG antibodies so far, 210 were reactive and underwent confirmatory testing through which fourteen samples were confirmed true-positive, resulting in a prevalence of 1% in the tested population. Conclusion: Our inability so far to identify a hantavirus does not mean that these viruses do not occur in South Africa. Apart from their host species specificity, highly localised distribution patterns and enormous seasonal and interannual fluctuations are well-known phenomena. A greater diversity of species from different biomes will be screened. The seroprevalence of 1% in humans is comparable to prevalences reported inform known endemic areas in Europe. Sera will be collected from rural laboratories to give a more complete picture of human hantavirus antibody prevalence.

This paper summarizes the progress in the search for hantaviruses and hantavirus infections in Africa. After having collected molecular evidence of an indigenous African hantavirus in 2006, an intensive investigation for new hantaviruses has been started in small mammals. Various novel hantaviruses have been molecularly identified not only in rodents but also in shrews and bats. In addition, the first African hantavirus, Sangassou virus, has been isolated and functionally characterized in cell culture. Less is known about the ability of these hantaviruses to infect humans and to cause diseases. To date, no hantavirus genetic material could be amplified from patients' specimens collected in Africa. Serological studies in West Africa, based on a battery of screening and confirmatory assays, led to the detection of hantavirus antibodies in the human population and in patients with putative hantavirus disease. In addition to this overview, we present original data from seroepidemiological and field studies conducted in the Southern part of Africa. A human seroprevalence rate of 1.0% (n = 1442) was detected in the South African Cape Region whereas no molecular evidence for the presence of hantavirus was found in 2500 small animals trapped in South Africa and Namibia.

Background: Arenaviruses are hosted by Murid rodents in the Murinae and Sigmondontinae subfamilies. They are divided into two serocomplexes; Tacaribe containing viruses hosted by New World rodents, and the Lassa-Lymphocytic choriomeningitis complex containing various arenaviruses hosted by African rodents, the ubiquitous LCMV and Dandenong virus from Australia. While non-pathogenic in rodents, some arenaviruses are highly pathogenic for human beings: In Africa, these are Lassa fever virus hosted by Mastomys natalensis and endemic to West Africa and Lujo virus with an unknown host that emerged in Zambia and caused a nosocomial outbreak in South Africa. Methods & Materials: In this study South African rodents were screened for arenaviral RNA sequences. A total of 1648 small mammals representing 16 different genera were trapped at various locations in 8 provinces in South Africa between 2008 and 2012. RNA extracted from lung tissue was tested using broadly reactive RT-PCR assays targeting the L gene. The positive samples were amplified using PCR assays targeting the GPC and NP genes, which were then used for phylogenetic analysis. Results: Thirteen novel arenavirus sequences were identified in four different rodent species indigenous to Southern Africa. Phylogenetic analysis showed that these arenaviruses belong to different clades; 3 outliers of the Lassa-Mopeia clade and 10 forming a monophyletic clade with Merino Walk virus, only very recently described from Myotomys unisulcatus from the Eastern Cape. Conclusion: A surprisingly high diversity of arenaviruses was detected in this study and further characterisation efforts are ongoing. The identification of arenaviruses by our group and others in Southern Africa suggests that they are more widely distributed than previously thought. The distribution and pathogenicity (or not) for humans is yet to be defined. The outbreak of Lujo virus in 2008 is a reminder that there may be horrible surprises out there.

To the Editor: The severe acute respiratory syndrome (SARS) outbreak of 2002–03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (CoV), prompted numerous studies of bats and the viruses they harbor. A novel clade 2c betacoronavirus, termed Middle East respiratory syndrome (MERS)–CoV, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the Arabian Peninsula (1). Extending on previous work (2), we described European Pipistrellus bat–derived CoVs that are closely related to MERS-CoV (3). We now report the identification of a South Africa bat derived CoV that has an even closer phylogenetic relationship with MERS-CoV. During 2011–2012, fecal pellets were collected from 62 bats representing 13 different species in the KwaZulu-Natal and Western Cape Provinces of South Africa and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA). Details about the bat sample are available in the Technical Appendix. RNA was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Screening for CoVs was done by nested reverse transcription PCR using broadly reactive oligonucleotide primers targeting a conserved region in the RNA-dependent RNA polymerase (RdRp) gene (online Technical Appendix). PCR results were positive for 5 (8%) of the 62 specimens. PCR amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from South Africa (4). The other positive specimen, termed PML/2011, was from an adult female Neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KC869678,term_id:666386896}}KC869678). Technical Appendix Figure 1 shows the distribution of this bat species. To obtain better phylogenetic resolution, we extended the 398-nt RdRp fragment generated by the screening PCR to 816 nt, as described (5). PML/2011 differed from MERS-CoV by only 1 aa exchange (0.3%) in the translated 816-nt RdRp gene fragment. Thus, PML/2011 was much more related to MERS-CoV than any other known virus. The amino acid sequence of the next closest known relatives of MERS-CoV, from European Pipistrellus bats (3), differed from MERS-CoV by 1.8%. The amino acid sequences of viruses from Nycteris bats in Ghana (3) and the 2c prototype bat CoVs, HKU4 and HKU5, from China (6) differed by 5.5%–7.7% from MERS-CoV. The smaller 152- to 396-nt RdRp fragments of 2c bat CoVs from a Hypsugo savii bat in Spain (7), bat guano in Thailand (8), and a Nyctinomops bat in Mexico (9) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. However, in their respective RdRp fragments, these CoVs yielded amino acid sequence distances of 3.5%–8.0% and were thus probably more distant from MERS-CoV than the virus described here. A Bayesian phylogenetic analysis of the 816-nt RdRp sequence confirmed the close relationship between PML/2011 and MERS-CoV (Figure). Their phylogenetic relatedness was as close as that of SARS-CoV and the most closely related bat coronavirus known, Rs672 from a Rhinolophus sinicus bat (Figure). Like PML/2011 and MERS-CoV, Rs672 and SARS-CoV showed only 1 aa exchange in the translated 816-nt RdRp fragment. To confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for PML/2011 (oligonucleotide primers available upon request from the authors). A partial spike gene–based phylogeny using this sequence yielded the same topology as that using the partial RdRp sequence (Technical Appendix Figure 2). Again, PML/2011 was most closely related to MERS-CoV, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for CoV attachment and cellular entry. This distance was less than the 13.3% aa sequence distance between MERS-CoV and the European Pipistrellus CoVs (3) and less than the 20.5%–27.3% aa sequence distance between MERS-CoV and HKU5 and between MERS-CoV and HKU4 (6) in the same sequence fragment. Figure Partial RNA-dependent RNA polymerase (RdRp) gene phylogeny, including the novel betacoronavirus from a Neoromicia zuluensis bat in South Africa (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KC869678,term_id:666386896}}KC869678 ... Our results further support the hypothesis that, like human CoV-229E and SARS-CoV, ancestors of MERS-CoV might exist in Old World insectivorous bats belonging to the family Vespertilionidae, to which the genera Neoromicia and Pipistrellus belong (3). Knowledge of the close relatedness of PML/2011 and MERS-CoV, which contrasts with the more distant relatedness of CoVs in bats from the Americas and Asia, enables speculations of an African origin for bat reservoir hosts of MERS-CoV ancestors. This hypothesis is limited by a global sampling bias, the small sample size, and the single clade 2c betacoronavirus detection in this study. Still, a putative transfer of MERS-CoV ancestors from Africa to the Arabian Peninsula would parallel the transfer of other viruses (e.g., the exportation of Rift Valley fever virus from East Africa, which led to a severe outbreak in Saudi Arabia in 2000) (10). Studies of Vespertilionidae bats and potential intermediate hosts (e.g., carnivores and ungulates, such as camels) are urgently needed to elucidate the emergence of MERS-CoV. Such studies should focus on the Arabian Peninsula and Africa. Technical Appendix: Description of bat sampling, screened bat species, distribution of Neoromicia zuluensis bats, and spike gene phylogeny of the 2c betacoronavirus clade. Click here to view.(326K, pdf)