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BMC Veterinary Research

Open Access

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

BMC Veterinary ResearchBMC series – open, inclusive and trusted201713:37

https://doi.org/10.1186/s12917-017-0948-0

Received: 9 August 2016

Accepted: 12 January 2017

Published: 31 January 2017

Abstract

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.

Keywords

Mokola virusLyssavirusRabies-relatedSouth Africa

Background

The Lyssavirus genus currently consists of 14 recognized species all capable of causing rabies, a fatal encephalitic disease. The prototype species of this genus is Rabies lyssavirus and the rest are known as the rabies-related lyssaviruses [1]. Mokola virus (MOKV), a rabies-related lyssavirus, was first isolated from shrews in the Mokola forest in Nigeria in the late 1960s [2, 3]. Shortly thereafter, MOKV was reported as the causative agent of a neurological disease in two children from Nigeria [4, 5]. However, these reports remain controversial for a variety of reasons. The first isolation was made from the cerebrospinal fluid of a 3.5-year-old girl in 1968. The girl recovered without any neurological sequelae while no virus neutralizing antibodies (VNAs) were detected [4]. This is unexpected as reports of patients surviving rabies are exceptionally rare. In the majority of survivor cases the patient is burdened with moderate to severe neurological sequelae [6, 7] with VNAs regarded as a primary mechanism of viral clearance [6, 8]. The second human isolate was obtained from the brain tissue of a 6-year-old girl in 1971 that died from suspected meningitis or encephalitis. In both these human cases clinical symptoms were atypical for classical rabies virus (RABV) infection [5]. It is doubtful that these isolates are still in existence and no genetic information is available [9].

Up until 2013, only 18 confirmed MOKV isolations, from a variety of mammalian host species including shrews, cats, dogs and a rodent (Lophuromys sikapusi), were known to exist [9]. MOKV was first encountered in South Africa in 1970, when the virus was isolated from a domestic cat in the KwaZulu-Natal (KZN) province [10]. In 1981, it became evident that the available rabies vaccines did not confer protection against MOKV when this virus was isolated from vaccinated cats and a dog in Zimbabwe [11]. It appears that MOKV is exclusive to the African continent and all isolations for the last 20 years have been made from southern Africa. Little is known about the epidemiology of this lyssavirus and the problem is compounded by an unknown reservoir and limited surveillance throughout the continent. In the few instances where rabies diagnostic facilities are available and operational in Africa, only the fluorescent antibody test (FAT) is used. The FAT relies on the use of a polyclonal fluorescein isothiocyanate conjugated anti-lyssavirus globulin that is capable of detecting all known lyssavirus species but cannot distinguish between them. As a result, positive cases are reported as rabies but the actual causative lyssavirus species is rarely identified. In South Africa, where molecular characterization of FAT-positive samples is frequently done, the majority of MOKV isolations have been from the KZN (n=4) and Eastern Cape (EC) provinces (n=5) with a single isolation reported from the Mpumalanga province. These isolates differ by between 0–5.7% and 0–2% at the nucleotide and amino acid levels of the nucleoprotein (N) gene. However, comparison of the N-gene of all known MOKV isolates demonstrated variations of up to 15% and 6.4% on the nucleotide and amino acids levels respectively [9].

Rabies virus (RABV) vaccines do not protect against MOKV [9, 11, 12] and it should be appreciated that the domestic cat is the most commonly MOKV-infected host species. The frequent contact between cats and their owners suggests a potential risk of spill-over infection to humans. Considering this situation, it is clear that a better understanding of the incidence and ecology of MOKV ecology is of utmost importance. Here we report the isolation and characterization of three MOKV isolations from cats from KZN, South Africa, 4 years after the last isolation of this virus in 2008 from South Africa. The first case was identified in January 2014 and subsequently a small retrospective study on selected samples was undertaken, identifying an additional two cases from 2012.

Methods

The study

In January 2014, a private veterinarian submitted brain material from a domestic cat that had died of suspected rabies in Pietermaritzburg, KZN, South Africa (laboratory reference number: 14/024) to the Allerton Provincial Veterinary Laboratory. The FAT was performed by staining acetone-fixed impression smears of the brain material with a polyclonal fluorescein isothiocyanate conjugated anti-lyssavirus globulin (OIE Rabies Reference Laboratory of the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI), South Africa) [13]. Lyssavirus antigen was observed, but in this specific case the staining was of dull fluorescence clearly atypical of rabies virus positive samples. Such atypical staining which has previously been noted in MOKV infections prompted further investigation of this case. The positive FAT result was confirmed at the OIE Rabies Reference Laboratory at Onderstepoort (ARC-OVI, South Africa) and the sample genetically characterized at the University of Pretoria. Following the subsequent identification of sample 14/024 as MOKV, it was decided to investigate and molecularly characterize other FAT positive rabies samples from domestic animals from throughout KZN. Selected archived samples (n=36), were characterized by nucleotide sequencing resulting in the identification of an additional two MOKV cases.

RNA extraction, RT-PCR and phylogenetic analysis

Total RNA was isolated from brain material using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The complete N-, phosphoprotein- (P), matrix protein- (M) and glycoprotein (G) genes were sequenced using different primer combinations and cycling conditions (Additional file 1: Table S1) for all MOKV samples. Briefly, reverse transcription was performed for all samples using the following protocol: 10 pmol of forward primer was added to 5 μl total RNA and incubated at 94 °C for 1 min. These reactions were cooled on ice for 5 min followed by reverse transcription for 90 min at 42 °C in a final volume of 20 μl containing 1 x reverse transcriptase buffer (containing 250 mM Tris-HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithiothreithol, Roche), 2.2 μl dNTP mix (10 mM, Promega), 8 U Avian myeloblastosis virus reverse transcriptase (Roche) and 16 U Recombinant RNasin Ribonuclease inhibitor (Promega). The genes were subsequently amplified using 20 μl cDNA in a final volume of 100 μl containing 1 x DreamTaq Buffer (containing KCl, (NH4)2SO4 and 20 mM MgCl2, Thermo Scientific), 10 pmol of forward primer (Additional file 1: Table S1), 12.5 pmol reverse primer (Additional file 1: Table S1) and 1.25 U DreamTaq DNA polymerase (Thermo Scientific). Analysis and sequencing of PCR products were performed as described previously [14]. Nucleotide sequences were edited and assembled using BioEdit Sequence Alignment Editor Version 7 [15]. The partial N-gene for RABV sequences (Additional file 2: Table S2) or concatenated and individual genes for MOKV sequences (Additional file 3: Table S3) was analyzed with jModeltest 2 [16] for each dataset to determine the most appropriate substitution model. Data was analyzed using BEAST v.1.8 [17] using a random starting tree with a strict clock for each dataset and assuming an exponential population growth with Markov Chain Monte Carlo (MCMC) chains of 50 million generations.

Virus isolation

Virus was isolated from MOKV samples on murine neuroblastoma cells as described previously [18].

Results

The partial N-gene sequences (using primers 001lys/550B, Additional file 1: Table S1) [19] of the 36 samples were determined and subsequent to the analyses two more MOKV cases were identified i.e. 12/458 and 12/604 (Table 1). Based on partial N-gene sequences, the remaining 33 archival samples were determined to be the canid variant of RABV. Bayesian analysis indicated that all new RABV sequences group with other RABV sequences from the same time period (Fig. 1).
Table 1

Information of domestic animals from the KwaZulu-Natal province submitted for molecular characterization

Laboratory reference number

Host

Collection area

Date

Genbank accession number (gene)

07/30

Felis catus (feline)

Empangeni

11/01/2007

KP994621

07/76

Felis catus (feline)

Melmoth

29/01/2007

KP994616

08/105

Felis catus (feline)

Kwadukuza

18/02/2008

KP994617

08/426

Felis catus (feline)

Jozini

03/07/2008

KP994618

08/642

Felis catus (feline)

Exodondakukuska

15/10/2008

KP994619

09/353

Felis catus (feline)

Nkambanana

07/08/2009

KP994620

10/268

Canis familiaris (canine)

Umdoni

20/05/2010

KJ744302

10/274

Canis familiaris (canine)

Hibiscus coast

24/05/2010

KJ744308

10/387

Canis familiaris (canine)

Umzumbe

23/08/2010

KJ744303

10/458

Capra aegagrus hircus (goat)

Umzimkulu

29/09/2010

KJ744309

10/509

Canis familiaris (canine)

Mkhambathini

22/10/2010

KJ744304

10/598

Felis catus (feline)

Dundee

16/11/2010

KP994606

11/28

Canis familiaris (canine)

Richmond

14/01/2011

KP994597

11/185

Canis familiaris (canine)

Mkahambathini

24/03/2011

KJ744305

11/195

Canis familiaris (canine)

Mkhambathini

28/03/2011

KP944598

11/217

Canis familiaris (canine)

Umdoni

12/04/2011

KJ744310

11/300

Canis familiaris (canine)

Richmond

30/05/2011

KJ744307

11/419

Felis catus (feline)

Ethekweni

16/08/2011

KP994607

12/069

Felis catus (feline)

Okhalamba

31/01/2012

KP994610

12/458

Felis catus (feline)

Durban

13/06/2012

KP899610(N), KP899619(P), KP899613(M), KP899616(G)

12/604

Felis catus (feline)

Durban

08/07/2012

KP899611(N), KP899620(P), KP899614(M), KP899617(G)

12/696

Felis catus (feline)

Ethekweni

27/07/2012

KP994608

12/903

Felis catus (feline)

Okhahlamba

02/10/2013

KP994609

13/058

Felis catus (feline)

Ethekweni

25/01/2013

KP994611

13/079

Canis familiaris (canine)

Umlazi

01/02/2013

KP994601

13/104

Canis familiaris (canine)

Amanzimtoti

14/02/2013

KP994599

13/107

Canis familiaris (canine)

Westville

18/02/2013

KP994602

13/167

Canis familiaris (canine)

Adams mission

18/03/2013

KP994612

13/256

Canis familiaris (canine)

Amanzimtoti

09/05/2013

KP994613

13/310

Canis familiaris (canine)

Umkomaas

10/06/2013

KP994603

13/339

Canis familiaris (canine)

Umlazi

21/06/2013

KP994614

13/355

Canis familiaris (canine)

Amanzimtoti

28/06/2013

KP994615

13/522

Canis familiaris (canine)

Athlone Park

27/09/2013

KP994604

13/525

Canis familiaris (canine)

Lewis Drive

30/09/2013

KP994605

13/589

Canis familiaris (canine)

Uthukela

29/10/2013

KP994600

14/024

Felis catus (feline)

Pietermaritzburg

09/01/2014

KP899612(N), KP899621(P), KP899615(M), KP899618(G)

Fig. 1

Bayesian analysis of the partial N-gene sequences (540 bp) of the 33 archival samples and other rabies virus sequences from South Africa (Additional file 2: Table S2) 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 (EC SA: Eastern Cape province, South Africa; FS SA: Free State province, South Africa; GP SA: Gauteng province, South Africa; KZN SA: Kwa-Zulu Natal province, South Africa; LP SA: Limpopo province, South Africa; MP SA: Mpumalanga province, South Africa; NW SA: North West province, South Africa) and year. All rabies virus sequences determined in this study are indicated in blue

Details and clinical history of the cats that tested positive for MOKV are summarized in Table 2. For all the MOKV cases, the complete sequences of four structural genes (i.e. N-, P-, M- and G genes) were determined.
Table 2

Details and clinical history of cats from the KwaZulu-Natal province, South Africa that tested positive for Mokola virus

 

Laboratory reference number

 

14/024

12/458

12/604

Location

Pietermaritzburg

Durban

Durban

Host details

Male, 4-year-old

Female, 1-year-old

Female, 1-year-old

Clinical signs

Fever (>40 °C), ataxia convulsions

Fever (39.6 °C), ataxia, decreased appetite

Growling, aggression, ataxia

Clinical duration

8-9 January 2014

10-13 June 2012

6-8 July 2012

Clinical outcome

Died of the disease

Euthanized after collapsing and being non-responsive

Euthanized after being non-responsive

For all the MOKV cases, the complete coding region sequence of four structural genes (i.e. N-, P-, M-, and G-genes) were determined. Each dataset (concatenated or individual genes) were analysed using a Bayesian approach. The tree topology (Fig. 2) observed was similar irrespective of the gene used for analysis (Additional file 4: Figure S1, Additional file 5: Figure S2, Additional file 6: Figure S3, Additional file 7: Figure S4) with the exception of the M-gene. For the M-gene dataset (Additional file 6: Figure S3), isolate RV4 (from Nigeria) grouped with an isolate from Zimbabwe (posterior probability = 0.8144) and not with the isolate from the Central African Republic as observed for the other datasets. This support findings from previous studies that MOKV phylogeny is strongly influenced by geographical derivation [9]. For the isolates from South Africa, two separate clusters were found to represent isolates from the two provinces viz. KZN and EC. Within the KZN cluster, the new MOKV isolates form a well-supported new clade. Nucleotide divergence of all known MOKV isolates (Additional file 8: Table S4, Additional file 9: Table S5, Additional file 10: Table S6 and Additional file 11: Table S7) ranged from 0.1-13.9%, 0-25.2%, 0-17.8% and 0.2-19.6% for the N-, P-, M- and G-genes respectively.
Fig. 2

Bayesian analysis of the concatenated coding region of the N-, P-, M- and G-genes of all Mokola virus isolates (Additional file 3: Table S3) 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

Discussion

There is no active surveillance for rabies-related lyssaviruses in Africa and subsequently the epidemiology of these viruses remains obscure. Rabies, caused by RABV, is endemic in KZN, South Africa and has been tackled through mass vaccination campaigns [20]. As a result, the annual number of suspected and confirmed rabies cases in dogs has decreased due to an enhanced awareness about the disease. This in turn has led to veterinary laboratories and veterinarians identifying unusual cases of animals displaying rabies symptoms. More cases (and isolations) of rabies-related lyssaviruses should occur in future – like the cases described here. Identification of infections caused by rabies-related viruses is important and any additional information may improve our understanding of the epidemiology of these viruses. However, the recommended diagnostic technique for rabies, the FAT, cannot distinguish between the lyssavirus species. A broad-spectrum polyclonal fluorescein isothiocyanate conjugated anti-lyssavirus globulin, capable of detecting all lyssavirus species, is used in countries with laboratory diagnostics. In some MOKV cases atypical staining, i.e. the inclusions stain less intensely than is usually seen with rabies virus, has been observed [21]; as described for sample 14/024. The identification of a MOKV infection prompted a small retrospective study to determine if other MOKV cases, which did not produce atypical staining with the FAT, were overlooked. Subsequently, 35 samples collected over a 7 year period were tested and two additional MOKV cases from 2012 were identified. The remaining 33 samples were shown to belong to the canid variant of RABV.

In southern Africa, two variants of RABV circulate i.e. the canid variant infecting members of the Canidae family and the mongoose variant infecting members of the Herpestidae [22]. The conserved N-gene used in the Bayesian analysis is not ideally suited to distinguish between closely related viral variants. Nevertheless, the RABV sequences determined in this study all grouped with other canid variant RABV sequences from the same time period which is indicative of the continued rabies epidemical cycle in domestic dogs that was established in 1976 [23].

Bayesian analysis including all known MOKV isolates demonstrated a pattern of grouping of viruses according to geographical origin, irrespective of the gene(s) used; in agreement with previous reports [9]. The South African isolates grouped according to province – KZN and EC cluster. All previous isolates from KZN collected over a 28 year period (1970–1998) displayed a variation of 2.3% and 0.7% at the nucleotide and amino acid levels of the N-gene respectively. When comparing the new isolates from 2012–2014 (14 years after the last isolation from KZN) to the previous isolates from KZN, variation of 3% and 1.1% is observed at the nucleotide and amino acid levels of the N-gene respectively. Although the monophyletic grouping of South African MOKV isolates is indicative of the continual presence and stability of MOKV [9], the new isolates form a unique and well-supported clade (posterior probability = 1) within the KZN cluster. These new cases provide yet further confirmation that MOKV cycles are well established and that cases are underreported given the lack of capability/capacity to routinely characterize rabies positive samples and the lack of rabies surveillance in most developing countries. While the majority of MOKV cases are reported in cats, the identity of the reservoir species is unknown. It has been suggested that the reservoir might be a species that interacts with cats [9, 21], but there is little evidence to support this notion. MOKV has been isolated from shrews on three occasions and once from a rodent (Lophuromys sikapusi) and in conjunction with limited pathogenicity studies demonstrating significant amounts of virus in the saliva of shrews and rodents sufficient for transmission, invite speculation that these animals could be possible reservoir hosts [12, 24]. The majority of the lyssaviruses have a strong association with bats, with the exceptions of MOKV and Ikoma lyssavirus [25]. Given the diversity observed for bat lyssaviruses, it has been suggested that bats constitute the main evolutionary hosts [26]. Although MOKV has never been isolated from bats, the possibility of an African bat(s) reservoir cannot be excluded. Nevertheless, the identity of the reservoir host(s) for MOKV remains speculative. The close association of humans with domestic cats, an unknown reservoir and the lack of MOKV-protective vaccines [9, 11, 12] collectively support the argument that more research into the epidemiological aspects of MOKV is warranted.

Conclusion

Considering the close contact of domestic cats with humans and the lack of protection from RABV based commercial vaccines, the risk to public and veterinary health is highlighted. These cases emphasize the lack of surveillance for rabies-related lyssaviruses and as such, the true incidence may be underestimated and is a major contributor to our incomplete understanding of the epidemiology and ecology of MOKV.

Abbreviations

ARC-OVI: 

Agricultural research council-onderstepoort veterinary institute

EC: 

Eastern Cape province

FAT: 

Fluorescent antibody test

G-gene: 

Glycoprotein gene

KZN: 

KwaZulu-Natal province

MCMC: 

Markov Chain Monte Carlo

M-gene: 

Matrix protein gene

MOKV: 

Mokola virus

N-gene: 

Nucleoprotein gene

P-gene: 

Phosphoprotein gene

RABV: 

Rabies virus

VNAs: 

Virus neutralizing antibodies

Declarations

Acknowledgments

Not applicable.

Funding

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. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Availability of data and materials

All sequencing data generated during this study are available in the Genbank repository, http://www.ncbi.nlm.nih.gov. Accession numbers are included in this published article [and its Additional files].

Authors’ contributions

J.C., W.M. and L.H.N. conceived and designed the experiments, K.L.R. and D.S. collected samples for molecular characterization and compiled case histories, C.T.S. confirmed FAT results and assisted in obtaining samples for molecular characterization, J.C. performed the experiments and analyzed the data, J.C., W.M., and L.H.N. wrote the paper. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable. Rabies is a controlled animal disease in South Africa. According to legislature all suspected rabies cases must be reported and investigated. Therefore, veterinarians are legally obligated to notify the responsible authorities and collect and submit samples for laboratory investigation.

Ethics approval and consent to participate

Ethical approval for this study was granted by the University of Pretoria Animal Ethics Committee (EC055-14).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Center for Viral Zoonoses, Department of Medical Virology, School of Medicine, Faculty of Health Sciences, University of Pretoria
(2)
Allerton Provincial Veterinary Laboratory
(3)
Department of Agriculture and Environmental Affairs, KwaZulu-Natal Rabies Project
(4)
Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI)
(5)
Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria

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Copyright

© The Author(s). 2017

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