Based on phylogeny, pathogenicity and serological cross-reactivity, the lyssaviruses have been split into three phylogroups.^2,3,6 Phylogroup I consists of RABV, DUVV, EBLV1, EBLV2, ABLV, Aravan, Khujand and Irkut. Phylogroup II consists of MOKV and LBV and Phylogroup III is WCBV.⁴ All experimental evidence to date suggests that commercial vaccines protect against only the Phylogroup I lyssaviruses and not members of Phylogroups II and III.^3,7,8 Nevertheless, cross protection between some lyssavirus species is possible as a result of a similarity in glycoprotein antigenic sites towards which neutralising antibodies are directed.⁹

DUVV, for which there have been only five reported cases to date (Table 1), was first encountered in South Africa in 1970 following a human fatality that was associated with a contact exposure involving an unidentified bat.¹⁰ DUVV was later isolated from two insectivorous bats. One was suggested to be a member of the Miniopteridae (Miniopterus schreibersii) because of the wide distribution of this genus in the area in South Africa where the exposure occurred.¹¹ New classification now indicates that the bat species previously identified as M. schreibersii in Africa is now considered to be Miniopterus natalensis¹²; the other isolation was from Nycteris thebaica in Zimbabwe in 1986.¹³ DUVV was again identified 36 years later in two human fatalities: in South Africa in 2006 and a few months later in Kenya in 2007. In both of these recent cases, there was an exposure to a small bat (probably insectivorous) but the bats were never found or positively identified. These isolations support the notion that the incidence of African rabies-related lyssaviruses is underestimated as a result of poor surveillance influenced by the limited diagnostic capacities in African laboratories. Very few laboratories are capable of making a rabies diagnosis with the recommended fluorescent antibody test – which is, in any event, non-differentiating because of the broad spectrum lyssavirus conjugate used. Of these laboratories, only one national African laboratory regularly types positive specimens by monoclonal antibody typing or nucleotide sequencing procedures.

Various studies have shown that DUVV is most closely related to EBLV1, which, although exclusive to Europe, is also associated with insectivorous bat species.A study by Bourhy et al.¹⁴ showed that EBLV1 shares more epitopes with DUVV than with EBLV2. Also notable was the fact that EBLV1 and EBLV2 did not form a monophyletic group,¹⁵ as EBLV1 and DUVV were more closely related to each other than EBLV1 was to EBLV2. In an investigation to determine the evolution of EBLV,¹⁶ it was found that EBLV1 isolates have evolved into at least two genetically distinguishable groups, following geographical drift.^15,16 When investigating the host species involved with DUVV and EBLV1 infection, significant overlaps occurred with regard to geographic distribution and co-colonisation in roosts.Myotis myotis and M. schreibersii, for example, have been found to move between colonies and are also known to have direct contact with each other in these mixed colonies.¹⁷ The large roost sizes and high population densities of many of these bat species makes them well suited to the sustained transmission and exchange of RNA viruses,¹⁸ most likely through the transfer of infectious saliva during licking and biting.¹⁹

Several molecular epidemiological studies of RABV have been performed but only a few have been conducted on ABLV,²⁰ EBLV1 and EBLV2,^15,16 whilst there have been a limited number of studies focusing on the African lyssavirus.^21,22,23 With so little known about the molecular epidemiology of DUVV, and with the addition of two relatively recent viruses to the small pool of known DUVVs, the objective of this study was to investigate the relationship between these DUVV isolates relative to the diversity found in the Lyssavirus genus.

Analysis of genetic distances between all five DUVV isolates was carried out using a well-conserved 398-nt sequence from the nucleoprotein gene (nt 8–406). The intrinsic variation between DUVV isolates from southern Africa was low, with a 97.7% – 100% nt identity, even though these isolates were isolated several yearsapart (ranging from 1971 to 2006). Isolate DUVVZIM86 was found to be 100% identical to DUVVSA81 for this region of the genome, although they were isolated 5 years apart in different countries. The East African isolate, DUVVKenya, had a much lower sequence identity (88.9% – 89.7%) than the other DUVV isolates, which supports the phylogenetic analysis that suggested this isolate forms part of a different lineage. DUVVKenya was shown to be most similar to DUVVSA71 (89.7%) –the first DUVV to have been isolated.

FIGURE 1: Neighbour-joining tree of nt 8–406 of the nucleoprotein gene indicating the phylogeny of all the Duvenhage virus isolates (the rabies virus, RABV, was used as the outgroup).

As a result of a number of surveillance programmes for EBLV1, many isolates have been made over the past few decades, demonstrating the significant activityof EBLV1 in bat populations in Eurasia.¹⁶ EBLV1a shows phylogenetic homogeneity between isolates across geographical regions, possibly as a result of viral traffic amongst bat populations.¹⁸ For EBLV1b, however, geographic origin plays a significant role in phylogenetic clusters, as there is less contact between bat populations¹⁸; these observations may also hold true for DUVV. Despite the lack of widespread surveillance, our data suggest that the DUVV cycle is likely to be wellestablished and stable in insectivorous bat species. The relationship between genomic evolution in lyssaviruses and the host’s response to infection could explain the rarity of human or other terrestrial mammal encounters with this virus.

As DUVV is present in bat populations, persons interacting with these animals should follow appropriate precautions, including vaccination. Considering the global mobility of humans and animals – and the presence of potential vector species across the globe – African lyssaviruses can easily pose a threat to any continent. It is thus of importance that laboratory, veterinary and medical personnel understand and recognise the resultant public health implications. The demonstratedseparation between the Kenyan and the southern African DUVV isolates is indicative of significant spatial evolution that is clearly underestimated by current data, as it may be taken for granted that DUVV cycles are not restricted to South Africa, Zimbabwe and Kenya. It is imperative that more, active surveillance programmes are initiated in order to establish more information about the distribution, prevalence, genetic diversity and host species associated with DUVV.