Bat-borne viruses in Africa: a critical review.

In Africa, bat-borne zoonoses emerged in the past few decades resulting in large outbreaks or just sporadic spillovers. In addition, hundreds of more viruses are described without any information on zoonotic potential. We discuss important characteristics of bats including bat biology, evolution, distribution and ecology that not only make them unique among most mammals but also contribute to their potential as viral reservoirs. The detection of a virus in bats does not imply that spillover will occur and several biological, ecological and anthropogenic factors play a role in such an event. We summarize and critically analyse the current knowledge on African bats as reservoirs for corona-, filo-, paramyxo- and lyssaviruses. We highlight that important information on epidemiology, bat biology and ecology is often not available to make informed decisions on zoonotic spillover potential. Even if knowledge gaps exist, it is still important to recognize the role of bats in zoonotic disease outbreaks and implement mitigation strategies to prevent exposure to infectious agents including working safely with bats. Equally important is the crucial role of bats in various ecosystem services. This necessitates a multidisciplinary One Health approach to close knowledge gaps and ensure the development of responsible mitigation strategies to not only minimize risk of infection but also ensure conservation of the species.

Out of Africa: The origins of the protozoan blood parasites of the Trypanosoma cruzi clade found in bats from Africa.

Understanding geographic patterns of interaction between hosts and parasites can provide useful insight into the evolutionary history of the organisms involved. However, poor taxon sampling often hinders meaningful phylogenetic descriptions of groups of parasites. Trypanosome parasites that constitute the Trypanosoma cruzi clade are worldwide distributed infecting several mammalian species, especially bats. Diversity in this clade has been recently expanded by newly discovered species, but the common ancestor and geographical origins of this group of blood parasites are still debated. We present here results based on the molecular characterization of trypanosome isolates obtained from 1493 bats representing 74 species and sampled over 16 countries across four continents. After estimating the appropriate number of hypothetical species in our data set using GMYC models in combination with Poisson Tree Processes (mPTP) and ABGD, the 18S rRNA and gGAPDH genes were used for phylogenetic analyses to infer the major evolutionary relationships in the T. cruzi clade. Then, biogeographical processes influencing the distribution of this cosmopolitan group of parasites was inferred using BioGeoBEARS. Results revealed a large lineages diversity and the presence of trypanosomes in all sampled regions which infected 344 individuals from 31 bat species. We found eight Trypanosoma species, including: five previously known; one subspecies of Trypanosoma livingstonei (Trypanosoma cf. livingstonei); and two undescribed taxa (Trypanosoma sp. 1, Trypanosoma sp. 2), which were found exclusively in bats of the genus Miniopterus from Europe and Africa. The new taxa discovered have both an unexpected position in the global phylogeny of the T. cruzi clade. Trypanosoma sp. 1 is a sister lineage of T. livingstonei which is located at the base of the tree, whereas Trypanosoma sp. 2 is a sister lineage of the Shizotrypanum subclade that contains T. c. cruzi and T. dionisii. Ancestral areas reconstruction provided evidence that trypanosomes of the T. cruzi clade have radiated from Africa through several dispersion events across the world. We discuss the impact of these findings on the biogeography and taxonomy of this important clade of parasites and question the role played by bats, especially those from the genus Miniopterus, on the dispersal of these protozoan parasites between continents.

Spotted fever rickettsiosis in South Africa: Evaluation of laboratory diagnostic capacity and inter-laboratory comparison of serological testing.

BACKGROUND: Spotted fever rickettsiosis, also known as tick bite fever (TBF), is a common infectious disease in South Africa (SA). Although the diagnosis of TBF is often based on clinical grounds only, laboratory testing is important to confirm the diagnosis and can contribute to case management in the light of a myriad of differential diagnoses, and in complicated cases. OBJECTIVES: To report on the availability and scope of laboratory tests for investigating suspected cases of TBF in SA, and the outcome of an inter-laboratory comparison (ILC) conducted for serological tests. METHODS: A self-administered questionnaire was circulated to major pathology laboratories in SA to determine what TBF tests they offered for TBF investigation. In addition, a clinical panel was provided to willing laboratories in order to perform an ILC of the serological tests. RESULTS: Serological tests for TBF were available from five laboratories serving both the private and state medical sectors in SA. There was no standardised testing platform or result interpretation across the different laboratories. Polymerase chain reaction (PCR) tests were less frequently available, and not available to state-operated facilities. The outcome of the ILC indicated varied performance and interpretation of serological results for TBF. CONCLUSIONS: Laboratory investigation for TBF is routinely and widely available in SA. Both serological and PCR-based methods were varied, and the lack of standardisation and interpretation of tests needs to be addressed to improve the overall quality of TBF diagnosis in SA. The utility of ILC to identify problem areas in serological testing for TBF is highlighted, and laboratories in SA are encouraged to use it to improve the quality of testing.

Bat lyssaviruses.

Bats (order Chiroptera) are the principal reservoir host for 14 of the 16 officially recognised lyssavirus species. Rabies virus is the only lyssavirus that is well established in terrestrial carnivores (worldwide), as well as bats (but only in the Americas). The other bat lyssaviruses occur only outside the Americas. They have a distinct geographical distribution and association with specific bat species, with limited cross-species transmission to other animals and humans, resulting in deadend infections. The nucleoprotein gene is well conserved between all lyssavirus species. Therefore, gold-standard diagnostic techniques detect all lyssaviruses but do not discriminate between viral species. Lyssaviruses are divided into at least three phylogroups, based on their immunogenic and phylogenic properties. Owing to the diversity of glycoproteins among phylogroups, rabies vaccines and immunoglobulins only provide protection against phylogroup I, excluding several of the bat lyssaviruses. Africa hosts a high diversity of lyssaviruses, leading to the hypothesis that this region was the site of emergence; however, this has been challenged by more recent phylogenetic analysis, suggesting a Palearctic origin. Serological evidence indicates a more widespread and even higher diversity of lyssaviruses in bats, suggesting that the incidence of known lyssaviruses is underestimated and several new lyssavirus species are yet to be discovered. Most bats are, however, not able to transmit the virus and therefore pose a low risk to human and animal populations.

Les chauves-souris (ordre des Chiroptères) sont les principaux hôtes réservoirs pour 14 des 16 espèces de lyssavirus officiellement inventoriées. Parmi les lyssavirus, seul le virus de la rage affecte aussi bien les carnivores terrestres (distribution mondiale) que les chauves-souris (uniquement dans les Amériques). Les autres lyssavirus des chauves-souris circulent dans d'autres régions mais pas sur le continent américain. Leur distribution géographique est liée à celle des espèces hôtes de chiroptères, avec des cas limités de transmission interespèces à d'autres animaux ou à l'homme, non suivis d'une adaptation à ces hôtes et aboutissant donc à une impasse écologique. Le gène de la nucléoprotéine est présent dans chacune des espèces de lyssavirus. Par conséquent, les techniques de référence détectent tous les lyssavirus sans les différencier par espèces. Le genre des lyssavirus comporte trois groupes phylogénétiques qui se distinguent par leurs propriétés immunogènes et phylogéniques. En raison de la diversité des glycoprotéines au sein de ces groupes, la protection conférée par les vaccins et les immunoglobulines couvre uniquement le groupe phylogénétique I, ce qui exclut plusieurs virus des chiroptères. L'Afrique héberge un grand nombre de lyssavirus différents, d'où l'idée que cette région du monde était peut-être la source d'émergence des virus, hypothèse toutefois réfutée par de récentes analyses phylogénétiques qui ont mis en avant une probable origine paléarctique. Certains résultats sérologiques font état d'une distribution plus étendue et d'une diversité encore plus élevée des lyssavirus chez les chauves-souris, ce qui laisse supposer que l'incidence des lyssavirus connus est sous-estimée et que nombre d'espèces nouvelles du genre lyssavirus restent à découvrir. Néanmoins, la plupart des chauves-souris ne transmettent pas le virus et par conséquent ne représentent pas un risque significatif pour les populations humaines et animales.

Los murciélagos (orden de los quirópteros) son el principal reservorio de 14 de las 16 especies de Lyssavirus oficialmente descritas. El virus de la rabia es el único lisavirus que está bien implantado a la vez en carnívoros terrestres (en todo el mundo) y en murciélagos (solamente en las Américas). Ninguno de los demás lisavirus de los murciélagos está presente en las Américas. Cada uno presenta características muy definidas en cuanto a su distribución geográfica y su asociación con una u otra especie de murciélago, con escasa transmisión a otras especies animales o al ser humano, lo que se traduce en infecciones cerradas (dead-end). El gen de la nucleoproteína está bien conservado en todas las especies de lisavirus. De ahí que las técnicas de diagnóstico de referencia permitan detectar todos los lisavirus, pero no discriminar entre una u otra especie vírica. En función de sus propiedades inmunógenas y filogenéticas, los lisavirus se dividen en al menos tres filogrupos. Dada la gran variabilidad que presentan las gluproteínas entre esos distintos filogrupos, las vacunas e inmunoglobulinas antirrábicas solo confieren protección contra el filogrupo I, con exclusión de varios lisavirus del murciélago. África alberga muchos y diversos lisavirus, lo que permitió conjeturar que esta región es su lugar de origen, hipótesis que sin embargo parecen desmentir análisis filogenéticos más recientes, que apuntan a un origen paleártico. Los datos serológicos indican una mayor extensión e incluso mayor diversidad de los lisavirus en los murciélagos, lo que lleva a pensar que la incidencia de los lisavirus conocidos está subestimada y que aún quedan por descubrir varias nuevas especies. La mayoría de los murciélagos, sin embargo, son incapaces de transmitir el virus, por lo que entrañan poco riesgo para las poblaciones humanas y animales.

Human cases of Sindbis fever in South Africa, 2006-2010.

Sindbis virus (SINV), the prototype positive-sense RNA alphavirus, causes febrile arthritis and is present throughout Afro-Eurasia. Little is known of the epidemiology of Sindbis fever due to insufficient surveillance in most endemic countries. The epidemiological features of Sindbis fever in humans in South Africa are described here based on a retrospective study of suspected arbovirus cases submitted for laboratory investigation from 2006 to 2010. Cases were detected annually mostly during the late summer/early autumn months and an increase in cases was noted for 2010, coinciding with an outbreak of Rift Valley fever. Cases were reported most often from the central plateau of South Africa and involved mostly males. No severe or fatal cases were reported and cases were associated with febrile arthralgia as commonly reported for SINV infection. Further surveillance is required to reveal the true extent of the morbidity of Sindbis fever in South Africa.

Dog rabies in southern Africa: regional surveillance and phylogeographical analyses are an important component of control and elimination strategies.

In the resource-poor settings where dog rabies remains endemic, the demonstration of a need to divert scarce funds towards exhaustive surveillance activities is no easy task. Here, we investigate a recent case of human rabies in South Africa, which generated much public interest and wide media coverage. One of the factors contributing to the hype was an uncertainty about the geographical origin of the infection. This provided an opportunity to highlight the importance of increased regional surveillance and basic phylogeographical analyses in rabies control and elimination strategies. Our aim was to elucidate the origins of the virus responsible for this case, as the patient was from a well-vaccinated area that had been free from dog rabies cases for many years. The phylogeographical techniques that we applied would also be most useful in any end-stage infectious disease control programme, specifically in verifying the source of novel cases in order to rapidly respond towards maintaining the integrity of disease-free areas. The most likely origin of our case was shown to be from outside the disease-free area and indeed from outside the country of South Africa. We conclude that phylogeographical techniques can provide rapid and statistically rigorous answers to epidemiologically pertinent questions that impact on disease control strategies and resource allocation, but this will require coordinated regional surveillance practices.

A case study of rabies diagnosis from formalin-fixed brain material.

Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirm rabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.

Evolutionary history of African mongoose rabies.

Two biotypes or variants of rabies virus (RABV) occur in southern Africa. These variants are respectively adapted to hosts belonging to the Canidae family (the canid variant) and hosts belonging to the Herpestidae family (the mongoose variant). Due to the distinct host adaptation and differences in epidemiology and pathogenesis, it has been hypothesized that the two variants were introduced into Africa at different times. The objective of this study was to investigate the molecular phylogeny of representative RABV isolates of the mongoose variant towards a better understanding of the origins of this group. The study was based on an analysis of the full nucleoprotein and glycoprotein gene sequences of a panel of 27 viruses. Phylogenetic analysis of this dataset confirmed extended evolutionary adaptation of isolates in specific geographic areas. The evolutionary dynamics of this virus variant was investigated using Bayesian methodology, allowing for rate variation among viral lineages. Molecular clock analysis estimated the age of the African mongoose RABV to be approximately 200 years old, which is in concurrence with literature describing rabies in mongooses since the early 1800 s.

Lagos bat virus virulence in mice inoculated by the peripheral route.

Lagos bat virus (LBV) constitutes genotype (gt) 2 in the Lyssavirus genus. In contrast to the gt1 lyssavirus, rabies virus (RABV), LBV was reported to have markedly reduced levels of peripheral pathogenicity. However, this opinion was based on a study of one isolate of LBV only and the reduction in pathogenicity was essentially attributed to the amino-acid substitution at position 333 of glycoprotein ectodomain. In the present study we have demonstrated that peripheral pathogenicity of representatives of LBV in a murine model is as high as that of RABV. Comparison of amino-acid substitutions among the viral glycoproteins, demonstrated significant differences within two antigenic sites between different phylogenetic lineages of LBV. Such molecular variability potentially contributes to differences in peripheral pathogenicity of lyssaviruses.

Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material.

A rapid immunodiagnostic test kit was evaluated against a selection of isolates of lyssavirus genotypes occurring in Africa. The test was carried out in parallel comparison with the fluorescent antibody test (FAT) and isolates representing previously established phylogenetic groups from each genotype were included. The specificity of the rapid immunodiagnostic test compared favourably with the FAT and was found to detect all representatives of genotypes 1, 2, 3 and 4 in brain samples of either field cases or suckling mouse brain inoculates.

Epidemiology and pathogenicity of African bat lyssaviruses.

Lyssaviruses belonging to all four known African Lyssavirus genotypes (gts) have been reported and isolated from SouthAfrica over the past few decades. These are: (1) Duvenhage virus (gt4), isolated again in 2006 from a human fatality; (2) Mokola virus (gt3), isolated irregularly, mostly from cats; (3) Lagos bat virus (gt2) continually isolated over the past four years from Epomophorus fruit bats and from incidental terrestrial animals and (4) Rabies virus (gt1) - with two virus biotypes endemic in mongoose and in canid species (mostly domestic dogs, jackals and bat-eared foxes), respectively. Only two of these are associated with bats in Southern Africa, viz. Duvenhage virus and Lagos bat virus (gts 4 and 2). For both these genotypes the authors have embarked on a programme of comparative study of molecular epidemiology. Duvenhage virus nucleoprotein nucleotide sequence analysis indicated a very low nucleotide diversity even though isolates were isolated decades apart. In contrast, individual isolates of Lagos bat virus were found to differ significantly with respectto nucleoprotein gene nucleotide sequence diversity as well as in pathogenicity profiles.

Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy.

Lagos bat virus (LBV) belongs to genotype 2 of the Lyssavirus genus. The complete nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and glycoprotein (G) genes of 13 LBV isolates were sequenced and phylogenetically compared with other lyssavirus representatives. The results identified three different lineages of LBV. One of these lineages demonstrated sufficient sequence diversity to be considered a new lyssavirus genotype (Dakar bat lyssavirus). The suggested quantitative separation of lyssavirus genotypes using the N, P, M and G genes was also investigated using P-distances matrixes. Results indicated that the current criteria should be revised since overlaps between intergenotypic and intragenotypic variation occur.

Use of a molecular epidemiological database to track human rabies case histories in South Africa.

The KwaZulu Natal and Eastern Cape provinces of South Africa have experienced a serious dog rabies epidemic over the past three decades. Towards a better understanding of this epidemic, we have previously analysed nucleotide sequences of 142 rabies virus specimens that were obtained from these regions during 2003-2004 and provided a molecular description of the geographical distribution of rabies viral variants in the affected provinces. Here, as an extension, we studied five human cases that occurred during 2002-2003 and demonstrated the use of the sequence database in tracking unknown human rabies case histories. We were able to identify the geographical origin of viruses responsible for each human infection and in one case obtained evidence that suggested a non-bite transmission of rabies virus from an infected dog to a child. We argue for the value of this information in surveillance and epidemiological study and in the follow-up and management of potential exposures.

Segment specific inverted repeat sequences in bluetongue virus mRNA are required for interaction with the virus non structural protein NS2.

Computational secondary structure prediction of all 10 Bluetongue virus (BTV-10) RNA transcripts and mutant inverted repeat transcripts were performed. Transcripts with intact 5' and 3' inverted repeat sequences, all indicated base-pairing between the 5' and 3' ends when optimal folding parameters were applied. Secondary structure analysis of the mutant transcripts lacking the inverted repeat sequences indicated alterations in the secondary structures resulting from altered base-pairing. The importance of the inverted repeat sequences in RNA--protein binding was subsequently investigated. Deletion mutant clones lacking the 5' and/or 3' inverted repeat sequences have been constructed. A baculovirus recombinant expressing the BTV NS2 protein and radioactively synthesized RNA transcripts were subjected to nitrocellulose RNA--protein binding assays. The cumulative results suggested that the inverted repeat deletion mutants display weaker binding compared to BTV-10 segment 8 with intact 5' and 3' inverted repeat sequences. Inverted repeats may influence RNA--protein binding by altering the secondary structure of the RNA and consequently the specific NS2 protein-binding sites may no longer be available.