Development of PCR-based methods for detection of African lyssaviruses

The etiological agent of rabies encephalitis belongs to the genus Lyssavirus in the Rhabdoviridae family. Lyssaviruses are negative sense, single stranded RNA viruses and cause an estimated 55 000 human deaths per year with 44% of these deaths occurring in Africa (WHO, 2005). With intense research effort and increased sequence information it is becoming evident that the Lyssavirus genus is much more diverse than initially thought and therefore diagnostic methods need to be modified accordingly. The African continent sustains a diverse variety of lyssaviruses, however, most countries in Africa do not have active surveillance or necessary diagnostic tools and therefore rabies-related lyssaviruses are underreported. Previous studies have indicated that real-time PCR has improved sensitivity and rapidity over conventional molecular diagnostic methods with the added advantage of allowing accurate estimations of viral load in a wide variety of samples. Several realtime PCR assays have been developed; however, none were specifically aimed at detection of lyssaviruses present on the African continent. This study was therefore aimed at evaluating certain molecular diagnostic methods for the detection of African lyssaviruses. Furthermore, the application of real-time PCR for various fields in lyssavirus research i.e. diagnostics, surveillance and pathogenicity studies were evaluated. This study revealed two different hemi-nested PCR assays capable of detecting representatives of African lyssaviruses. A real-time PCR was developed that was successful for the detection of African lyssaviruses. In addition, a quantitative assay and internal control was successfully employed for confirming ante-mortem human rabies diagnosis as well as post-mortem animal rabies diagnosis in formalin fixed brain material. As such the real-time PCR assay developed in this study could therefore be routinely used for ante-mortem diagnosis and as a confirmatory test for post-mortem diagnosis. The ability of this assay to detect and quantify all currently known African lyssaviruses not only offers improved surveillance capacity, but offers unique potential as a sensitive tool to track virus movement in pathogenicity studies. These aspects are important in our search for a better understanding of the complex epidemiological and viral characteristics of African lyssaviruses. CopyrightDissertation (MSc)--University of Pretoria, 2010.Microbiology and Plant Pathologyunrestricte

Lagos bat virus, an under-reported rabies-related lyssavirus

Lagos bat virus (LBV), one of the 17 accepted viral species of the Lyssavirus genus, was the first rabies-related virus described in 1956. This virus is endemic to the African continent and is rarely encountered. There are currently four lineages, although the observed genetic diversity exceeds existing lyssavirus species demarcation criteria. Several exposures to rabid bats infected with LBV have been reported; however, no known human cases have been reported to date. This review provides the history of LBV and summarizes previous knowledge as well as new detections. Genetic diversity, pathogenesis and prevention are re-evaluated and discussed.This research is supported in part by the South African Research Chair Initiative (held by Wanda Markotter) of the Department of Science and Innovation and administered by the National Research Foundation of South Africa (UID: 98339). The National Research Foundation funded the equipment based at the DNA Sanger sequencing facility in the Faculty of Natural and Agricultural Sciences, University of Pretoria (UID: 78566).https://www.mdpi.com/journal/virusesdm2022Medical Virolog

Reverse transcription recombinase polymerase amplification assay for rapid detection of canine associated rabies virus in Africa

Rabies is a neglected disease mostly affecting the developing world. Accurate and reliable diagnostic and surveillance data forms the foundation for the formulation and monitoring of control strategies. Although various sensitive and specific tests are available for detection of rabies virus, implementation of these tests in low-resource settings are challenging and remains limited. In this study, we describe the developed of a reverse transcription recombinase polymerase amplification assay for the detection of rabies virus. The analytical sensitivity of this assay was determined to be 562 RNA copies and was performed in 20 minutes. The diagnostic sensitivity of the RT-RPA was 100% for detection of rabies virus in field samples. In conclusion, the RT-RPA assay allowed for very quick and sensitive detection of rabies virus and could be adapted for use in low-source settings.Supporting information: S1 Table. Details of rabies virus sequences used for primer and probe design for the RT-RPA assay.S2 Table. Evaluation of the mismatches between the RT-RPA primer and probe set binding regions to rabies-related lyssaviruses.The South African Research Chair initiative of the Department of Science and Technology and National Research Foundation of South Africa and the Poliomyelitis Research Foundation.http://www.plosone.orgpm2020BiochemistryGeneticsMedical VirologyMicrobiology and Plant Patholog

Development of a Pan-Filoviridae SYBR green qPCR assay for biosurveillance studies in bats

DATA AVAILABILITY: The data are contained within the article or supplementary materials.SUPPLEMENTARY MATERIALS : TABLE S1: Publically available sequences used for primer design; SUPPLEMENTARY FILE S1: Synthetic constructs sequences, TABLE S2: SYBR Green qPCR results for field samples.Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no panfilovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3–31.7 copies/reaction and was evaluated against the field collected samples. The assay’s performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.National Research Foundation (NRF) of South Africa.https://www.mdpi.com/journal/virusesMedical Virolog

Epidemiology of Rabies in Lesotho : the importance of routine surveillance and virus characterization

Rabies is widespread throughout Africa and Asia, despite the fact that the control and elimination of this disease has been proven to be feasible. Lesotho, a small landlocked country surrounded by South Africa, has been known to be endemic for rabies since the 1980s but the epidemiology of the disease remains poorly understood due to limited sample submission, constrained diagnostic capabilities, and a lack of molecular epidemiological data. Considering the existing challenges experienced in Lesotho, we aimed to evaluate the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis in Lesotho. Towards this aim, extensive training on the implementation and interpretation of the DRIT was hosted in Lesotho in April 2016 before both tests were applied to all samples subjected to routine rabies diagnosis at the Central Veterinary Laboratory (CVL). We found agreement between the DFA and DRIT assays in 90/96 samples (93.75%). The samples that produced inconsistent results (n = 6) were re-tested a further two times with both assays before being subjected to a real-time qPCR to confirm the diagnosis. Additionally, a statistically significant three-fold increase in the average number of samples submitted per month was observed after the DRIT implementation started, following continuous rabies awareness initiatives amongst the animal health professionals in the country over a 12-month period (p = 0.0279). Partial G-L intergenic regions of selected rabies-positive samples (n = 21) were amplified, sequenced, and subjected to phylogenetic analyses. Molecular epidemiological analyses, which included viruses from neighbouring provinces in South Africa, suggested that at least three independent rabies cycles within Lesotho were implicated in instances of cross-border transmission. This study has evaluated alternative methods for diagnosing and improving rabies surveillance in Lesotho, as well as providing new information that would be of importance in the planning of future disease intervention campaigns, not only in Lesotho, but also in neighbouring South Africa.Table S1. Panel of rabies viruses from Lesotho and neighbouring South African provinces included in the phylogenetic analysis performed in this study.This work is based on the research supported in part by grants from the World Animal Protection (WAP) (grant number: WAP/AF/IHC/GR/2016-01), the National Research Foundation (NRF) of South Africa (grant number 78566, NRF Research Infrastructure Support Programmes (RISP) grant for the ABI3500), and the Cooperative Agreement Number, [93.318 5 NU2GGH001874-02-00], funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centres for Disease Control and Prevention.http://www.mdpi.com/journal/tropicalmedam2018Forestry and Agricultural Biotechnology Institute (FABI)Medical VirologyMicrobiology and Plant Patholog

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

Utility of forensic detection of rabies virus in decomposed exhumed dog carcasses

This report describes four suspected rabies cases in domestic dogs that were involved in human exposures. In all these cases, the animals were buried for substantial times before rabies testing was performed. Animal rabies is endemic in South Africa and domestic dogs are the main vector for transmission to humans. Diagnosis of rabies in humans is complicated, and diagnosis in the animal vector can provide circumstantial evidence to support clinical diagnosis of rabies in humans. The gold standard diagnostic method, fluorescent antibody test (FAT), only delivers reliable results when performed on fresh brain material and therefore decomposed samples are rarely submitted for diagnostic testing. Severely decomposed brain material was tested for the presence of rabies virus genomic material using a quantitative real-time reverse transcription polymerase chain reaction (q-real-time RT-PCR) assay when conventional molecular methods were unsuccessful. This may be a useful tool in the investigation of cases where the opportunity to sample the suspected animals post mortem was forfeited and which would not be possible with conventional testing methodologies because of the decomposition of the material.The National Research Foundation (NRF) (South Africa) grant number UID 66187 and 78566.http://www.jsava.co.zaam201

Utility of forensic detection of rabies virus in decomposed exhumed dog carcasses

This report describes four suspected rabies cases in domestic dogs that were involved in human exposures. In all these cases, the animals were buried for substantial times before rabies testing was performed. Animal rabies is endemic in South Africa and domestic dogs are the main vector for transmission to humans. Diagnosis of rabies in humans is complicated, and diagnosis in the animal vector can provide circumstantial evidence to support clinical diagnosis of rabies in humans. The gold standard diagnostic method, fluorescent antibody test (FAT), only delivers reliable results when performed on fresh brain material and therefore decomposed samples are rarely submitted for diagnostic testing. Severely decomposed brain material was tested for the presence of rabies virus genomic material using a quantitative real-time reverse transcription polymerase chain reaction (q-real-time RT-PCR) assay when conventional molecular methods were unsuccessful. This may be a useful tool in the investigation of cases where the opportunity to sample the suspected animals post mortem was forfeited and which would not be possible with conventional testing methodologies because of the decomposition of the material.The National Research Foundation (NRF) (South Africa) grant number UID 66187 and 78566.http://www.jsava.co.zaam201

A case of human survival of rabies, South Africa

Human survival from rabies is exceptionally rare. We report a case of human survival (with severe neurological sequelae) in a child from South Africa. The patient was exposed to rabid dogs on two separate occasions and subjected to incomplete post-exposure prophylaxis for rabies.http://www.tandfonline.com/loi/ojid20am2017Microbiology and Plant Patholog