Aspects of the molecular epidemiology of rabies in Zimbabwe and South Africa

Rabies, one of the oldest recognized viral zoonotic diseases, is a fatal encephalomyelitis transmitted to man via contact with infected animals. Evan today, rabies still is a disease of public health concern with many potentially preventable deaths occurring mainly in Asia, Africa and Latin America. Rabies and rabies-related viruses are members of the Lyssavirus genus, which comprises the rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssaviruses 1 and 2 (genotypes 5 and 6) and the Australian bat lyssavirus (genotype 7). Antigenic and genetic studies have shown that rabies virus strains circulating in particular host species tend to undergo genetic adaptation and evolve into distinct biotypes that differ in antigenicity and pathogenicity. Two biotypes of rabies virus are recognized in southern Africa. The first called the canid viruses, infect carnivores of the family Canidae (dogs, jackals and bat-eared foxes) and the second, the viverrid viruses, infect carnivores of the family Herpestidae (the yellow mongoose Cynictis penicil!ata and the slender mongoose Galerella sanguinea). In an endeavour to better understand the molecular epidemiology of lyssaviruses in Zimbabwe and South Africa, we analysed nucleotide sequences of the glycoprotein and the G-L intergenic region (rabies viruses) and the nucleoprotein gene (Mokola viruses). The main aim of the studies described in this thesis was to characterise lyssaviruses (genotypes I and 3) from Zimbabwe and compare them to those present in South Africa. In addition, we wanted to establish the role of the various rabies variants in rabies epizootics in the southern African subcontinent. It could be shown from this study that all the southern African canid viruses were closely related, with no general distinction between viruses from any of the canid species. Despite the general overall similarity between the canid viruses, certain phylogenetic groupings were apparent and by association with host species, geography and year of isolation, certain groups could be identified as particular epidemiological cycles. A high genetic diversity was evident amongst viverrid rabies viruses, the opposite of our observation for canid viruses. The viverrid virus groups corresponded to geographical pockets that were independent of host species. Mokola viruses from Zimbabwe were shown to be different from those from South Africa and phylogenetic relationships of these viruses were related to their geographical location of origin. This study has demonstrated the value of multinational surveillance and investigation in understanding the epidemiology of lyssaviruses in southern Africa and elsewhere in Africa. The results presented here will serve as basis for future studies on lyssaviruses in Africa and will contribute to the improved surveillance and control programs of rabies and Mokola viruses in the region.Thesis (PhD (Microbiology))--University of Pretoria, 2006.Microbiology and Plant Pathologyunrestricte

Antigenic characterisation of lyssaviruses in South Africa

There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and Mokola virus (0.5%). Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.This work was partly funded by the Rabies Diagnostic Project, Onderstepoort Veterinary Research Institute (OVI 15/4/P001) and the European Virus Archive (EVA) (04/17/C215).http://www.ojvr.orgam201

Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.National Research Foundation (NRF; Grant number: 66187), Poliomyelitis Research Foundation (PRF; Grant number: 12/38 [MSc]) and the European Virus Archive project (EVA; Grant number: 04/17/c215).http://www.plosntds.orghb201

New isolations of the rabies-related Mokola virus from South Africa

BACKGROUND : Mokola virus (MOKV) is a rabies-related lyssavirus and appears to be exclusive to the African continent. Only 24 cases of MOKV, which includes two human cases, have been reported since its identification in 1968. MOKV has an unknown reservoir host and current commercial vaccines do not confer protection against MOKV. RESULTS : We describe three new isolations of MOKV from domestic cats in South Africa. Two cases were retrospectively identified from 2012 and an additional one in 2014. CONCLUSIONS : These cases emphasize the generally poor surveillance for rabies-related lyssaviruses and our inadequate comprehension of the epidemiology and ecology of Mokola lyssavirus per se.Additional file 1: Table S1. Primers and PCR conditions for amplification of the Nucleoprotein-, Phosphoprotein-, Matrix protein- and Glycoprotein genes of Mokola virus isolates.Additional file 2: Table S2. Details of sequences used for the Bayesian analysis of the rabies virus positive samples.Additional file 3: Table S3. Details of sequences used for the Bayesian analysis of the new Mokola virus isolates.Additional file 4: Figure S1. Bayesian analysis of the coding region of the Nucleoprotein gene (1353 bp) of all Mokola virus isolates (Additional file 3: Table S3) applying the general time reversible substitution model with invariable sites. Laboratory reference numbers are shown for all sequences, followed by the host species, country of origin (KZN SA: KwaZulu-Natal province, South Africa; EC SA: Eastern Cape province South Africa; ZIM: Zimbabwe; CAR: Central African Republic; NIG: Nigeria) and year of isolation.Additional file 5: Figure S2. Bayesian analysis of the coding region of the Phosphoprotein gene (913 bp) applying the general time reversible substitution model with gamma distribution. Laboratory reference numbers are shown for all sequences, followed by the host species, country of origin (KZN SA: KwaZulu-Natal province, South Africa; EC SA: Eastern Cape province South Africa; ZIM: Zimbabwe; CAR: Central African Republic; NIG: Nigeria) and year of isolation.Additional file 6: Figure S3. Bayesian analysis of the coding region of the Matrix protein gene (609 bp) applying the general time reversible substitution model with gamma distribution. Laboratory reference numbers are shown for all sequences, followed by the host species, country of origin (KZN SA: KwaZulu-Natal province, South Africa; EC SA: Eastern Cape province South Africa; ZIM: Zimbabwe; CAR: Central African Republic; NIG: Nigeria) and year of isolation.Additional file 7: Figure S4. Bayesian analysis of the coding region of the Glycoprotein gene (1569 bp) applying the general time reversible substitution model with gamma distribution and invariable sites. Laboratory reference numbers are shown for all sequences, followed by the host species, country of origin (KZN SA: KwaZulu-Natal province, South Africa; EC SA: Eastern Cape province South Africa; ZIM: Zimbabwe; CAR: Central African Republic; NIG: Nigeria) and year of isolation.Additional file 8: Table S4. Nucleotide identity of the Nucleoprotein gene of all Mokola virus isolates.Additional file 9: Table S5. Nucleotide identity of the Phosphoprotein gene of all Mokola virus isolates.Additional file 10: Table S6. Nucleotide identity of the Matrix protein gene of all Mokola virus isolates.Additional file 11: Table S7. Nucleotide identity of the Glycoprotein gene of all Mokola virus isolates.This work was partially funded by the National Research Foundation (Grant UID 92524 & RISP grant UID78566), the Poliomyelitis Research Foundation (Grant no. 10/40, 12/14) and the Animal and Zoonotic Diseases Institutional Research Theme of the University of Pretoria.http://www.biomedcentral.com/bmcvetresam2017Medical VirologyMicrobiology and Plant Patholog

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 confirmrabies 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.http://www.journals.co.za/ej/ejour_savet.htmlnf201

Rabies outbreak in black-backed jackals (Canis mesomelas), South Africa, 2016

Rabies, a fatal and vaccine-preventable disease, is endemic throughout Africa. In 2016, a rabies outbreak occurred in black-backed jackals (Canis mesomelas) along the western boundary of Gauteng Province, South Africa. We investigated the possible drivers of the 2016 outbreak and established its origin. Using spatio-temporal locations of cases, we applied logistic regression and Geographic Information System techniques to investigate environmental covariates driving occurrences of emerging rabies cases in Gauteng Province. About 53.8% of laboratory-confirmed lyssaviruses in Gauteng Province in 2016 originated from jackals. Phylogenetic trees reconstructed from a partial region of the glycoprotein gene of these and historical rabies viruses (RABVs) demonstrated the lyssaviruses to be of canid origin with 97.7% nucleotide sequence similarity. The major cluster comprised jackal RABVs from the 2012 KwaZulu/Natal outbreak and the 2016 outbreak in Gauteng Province. The second cluster was composed of both jackal and dog RABVs. Both clusters correlated with independent RABV introductions into Gauteng by dogs and jackals, respectively. This study demonstrated an expansion of a jackal rabies cycle from north-west Province into Gauteng Province during the 2016 dry period, as jackals ranged widely in search for food resources leading to increased jackal-dog interactions, reminiscent of the intricate links of domestic and wildlife rabies cycles in South Africa.EVAglobalhttp://journals.cambridge.org/action/displayJournal?jid=HYGdm2022Veterinary Tropical Disease

Molecular detection of rabies lyssaviruses from dogs in Southeastern Nigeria : evidence of transboundary transmission of rabies in West Africa

Despite being the first country to register confirmed cases of Mokola and Lagos bat lyssaviruses (two very distant lyssaviruses), knowledge gaps, particularly on the molecular epidemiology of lyssaviruses, still exist in Nigeria. A total of 278 specimens were collected from dogs in southeastern Nigeria between October 2015 and July 2016, and 23 (8.3%) of these tested positive for lyssaviruses with the direct fluorescent antibody test (DFA). The lyssaviruses were genetically characterized by amplifying the highly conserved nucleoprotein (N) gene of the rabies lyssaviruses (RABVs) of the viral genome. Phylogenetic analyses of the nucleotide sequences showed that all the RABV sequences in this study were of the Africa-2 lineage. Our results demonstrated that transboundary transmission of rabies lyssavirus is a key event, given that one of the RABV sequences (MN196576) clustered with rabies variants from neighboring Niger Republic. Furthermore, three RABVs from dogs from Anambra State clustered separately forming a novel and distinct group. Our results demonstrated that transboundary transmission of RABLVs is a key driver in the spread of rabies in West Africa. In order for the successful control of this zoonotic disease, a multinational stepwise surveillance and elimination of rabies in Africa by 2030 is probably the solution for regional elimination.The Tertiary Educational Trust Fund (TETFund) of the Nigerian government through University of Nigeria IBR and Bench Space Intervention (TETFUND/DESS/UNI/NSUKKA/RP/VOL.V) and also the ARC-OVI National Assets [P10000029] Onderstepoort Veterinary Institute, South Africa.http://www.mdpi.com/journal/virusesam2021Veterinary Tropical Disease

Enhanced diagnosis of rabies and molecular evidence for the transboundary spread of the disease in Mozambique

Rabies is a neglected zoonotic disease with veterinary and public health significance, particularly in Africa and Asia. The current knowledge of the epidemiology of rabies in Mozambique is limited because of inadequate sample submission, constrained diagnostic capabilities and a lack of molecular epidemiological research. We wanted to consider the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) for rabies diagnosis at the diagnostic laboratory of the Central Veterinary Laboratory (CVL), Directorate of Animal Science, Maputo, Mozambique. Towards this aim, as a training exercise at the World Organisation for Animal Health (OIE) Rabies Reference Laboratory in South Africa, we performed the DRIT on 29 rabies samples from across Mozambique. With the use of the DRIT, we found 15 of the 29 samples (52%) to be negative. The DRIT-negative samples were retested by DFA at the OIE Rabies Reference Laboratory, as well as with an established real-time Polymerase chain reaction, confirming the DRIT-negative results. The DRIT-positive results (14/29) were retested with the DFA and subsequently amplified, sequenced and subjected to phylogenetic analyses, confirming the presence of rabies RNA. Molecular epidemiological analyses that included viruses from neighbouring countries suggested that rabies cycles within Mozambique might be implicated in multiple instances of cross-border transmission. In this regard, our study has provided new insights that should be helpful in informing the next steps required to better diagnose, control and hopefully eliminate rabies in Mozambique.The National Research Foundation (NRF) of South Africahttp://www.jsava.co.zaam2017Microbiology and Plant Patholog

Rabies of canid biotype in wild dog (Lycaon pictus) and spotted hyaena (Crocuta crocuta) in Madikwe Game Reserve, South Africa in 2014–2015 : diagnosis, possible origins and implications for control

Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (Cynictis penicillata), the rest are from other wild carnivores including the highly endangered wild dog (Lycaon pictus). Lyssavirus infection was confirmed in two wild dogs and a spotted hyaena (Crocuta crocuta) in the Madikwe Game Reserve, North West province in South Africa, in 2014 and 2015, using a direct fluorescent antibody test and immunohistochemistry. There had been no new wild dog introductions to the Madikwe Game Reserve for many years and the wild dogs were last vaccinated against rabies approximately 11 years prior to the incident. The first euthanised wild dog was the last surviving of a break-away pack of 6, and the second was the last of a larger pack of 18, the rest of which died with no carcasses being found or carcasses too decomposed for sampling. Subsequent antigenic typing of the lyssaviruses indicated that they were canid RABVs. The RABVs originating from 22 wild carnivore species, 7 dogs, and a caprine, mostly from the North West province, were genetically characterised by targeting a partial region of the nucleoprotein gene. The nucleotide sequence analyses of these viruses and two previously characterised RABVs confirmed that the outbreak viruses were also canid rabies, phylogenetically clustering with virus isolates originating from black-backed jackals recovered between 2012 and 2015 from the North West province, and domestic dogs from neighbouring communal areas. The source(s) of the mortalities and possible reservoir host(s) for the virus could only be speculated upon from data on specific predator numbers, movements and behaviour, kills, park management and the changing environmental ecology, which were monitored closely in Madikwe over several years. The most likely rabies sources were from boundary fence contacts between wild carnivores within the park, with domestic dogs or cats and/or naturally occurring wild carnivores outside the park. The associated risk of zoonotic infection and threat to important and endangered predators may be mitigated through regional rabies control primarily in domestic dogs and cats, as well as by preventative vaccination of at-risk park employees and their pets. The importance of ongoing prophylactic rabies protection by regular vaccination of highly endangered wildlife carnivores and the submission of carcasses for rabies diagnosis of any wild or domestic animals behaving uncharacteristically or found dead is emphasised.http://www.jsava.co.zaParaclinical Science

Rabies vaccination of 6‐week‐old puppies born to immunized mothers: a randomized controlled trial in a high‐mortality population of owned, free‐roaming dogs

To achieve global elimination of human rabies from dogs by 2030, evidence-based strategies for effective dog vaccination are needed. Current guidelines recommend inclusion of dogs younger than 3 months in mass rabies vaccination campaigns, although available vaccines are only recommended for use by manufacturers in older dogs, ostensibly due to concerns over interference of maternally-acquired immunity with immune response to the vaccine. Adverse effects of vaccination in this age group of dogs have also not been adequately assessed under field conditions. In a single-site, owner-blinded, randomized, placebo-controlled trial in puppies born to mothers vaccinated within the previous 18 months in a high-mortality population of owned, free-roaming dogs in South Africa, we assessed immunogenicity and effect on survival to all causes of mortality of a single dose of rabies vaccine administered at 6 weeks of age. We found that puppies did not have appreciable levels of maternally-derived antibodies at 6 weeks of age (geometric mean titer 0.065 IU/mL, 95% CI 0.061–0.069; n = 346), and that 88% (95% CI 80.7–93.3) of puppies vaccinated at 6 weeks had titers ≥0.5 IU/mL 21 days later (n = 117). Although the average effect of vaccination on survival was not statistically significant (hazard ratio [HR] 1.35, 95% CI 0.83–2.18), this effect was modified by sex (p = 0.02), with the HR in females 3.09 (95% CI 1.24–7.69) and the HR in males 0.79 (95% CI 0.41–1.53). We speculate that this effect is related to the observed survival advantage that females had over males in the unvaccinated group (HR 0.27; 95% CI 0.11–0.70), with vaccination eroding this advantage through as-yet-unknown mechanisms.Supplementary Materials: Table S1. Results of sensitivity analysis for survival analysis (6 to 13 weeks of age), considering subjects reported as lost or stolen by owners as dead (n = 22); Table S2. Results of sensitivity analysis for survival analysis (6 to 13 weeks of age), censoring subjects that reportedly died from accidents (n = 5).http://www.mdpi.com/journal/tropicalmedhj2021Companion Animal Clinical StudiesProduction Animal StudiesVeterinary Tropical Disease