Possible Emergence of West Caucasian Bat Virus in Africa

The prevalence of neutralizing antibody against West Caucasian bat virus (WCBV) in Miniopterus bats collected in Kenya ranged from 17% to 26%. Seropositive bats were detected in 4 of 5 locations sampled across the country. These findings provide evidence that WCBV, originally isolated in Europe, may emerge in other continents.

The prevalence of neutralizing antibody against West Caucasian bat virus (WCBV) in Miniopterus bats collected in Kenya ranged from 17% to 26%. Seropositive bats were detected in 4 of 5 locations sampled across the country. These fi ndings provide evidence that WCBV, originally isolated in Europe, may emerge in other continents.
B ats are reservoir hosts of several emerging zoonotic RNA viruses (1). In particular, bats host a range of lyssaviruses, as has been reported from different continents (2). Presently, 7 species are recognized within the genus RABV infection of bats is known in the Americas, but not in the Old World. Four lyssavirus species have been documented in Africa. Of these, RABV and MOKV have been isolated exclusively from terrestrial mammals, whereas LBV and DUVV are associated with bats and have been isolated only occasionally from other mammals (4). On the basis of this diversity and on the serologic cross-reactivity of MOKV with African non-lyssa rhabdoviruses, it has been hypothesized that Africa is the continent of the origin and initial evolution of members of the genus Lyssavirus (5). However, this hypothesis has been called into question by the isolation of WCBV in southeastern Europe. WCBV is the most divergent member of the genus Lyssavirus to date and has long genetic distances and lack of serologic cross-reactivity to other lyssaviruses (6,7). Our study objective was to enhance pathogen discovery for zoonotic agents in African bats, with particular focus on lyssavirus surveillance in Kenya.

The Study
During 2006-2007, bats of at least 30 species were collected from 25 locations in Kenya (Figure 1, panel A; online Appendix Table, available from www.cdc.gov/EID/ content/14/12/1887-appT.htm). The sample numbers and collection protocol were approved by the National Museum of Kenya and the Kenya Wildlife Service. Of the 1,221 bats collected, only 12 were sick or dead; the others appeared healthy. Captured animals were anesthetized by an intramuscular injection of ketamine hydrochloride (0.05-0.1 mg/g) and euthanized under sedation in compliance with a fi eld protocol approved by the Animal Institute Care and Use Committee of the Centers for Disease Control and Prevention. Bat size, sex, and species were identifi ed. When phenotypic species determination was not possible, DNA specimens were submitted for identifi cation to the University of Guelph (Ontario, Canada), where partial sequences of the cytochrome oxidase gene were generated and compared with those available from the Barcode of Life Data Systems (www.boldsystems.org). The brain and other organs of bats were collected into sterile plastic tubes. Oral swabs were collected and placed in tubes containing Minimum Essential Medium (MEM-10, Invitrogen, Grand Island, NY, USA) for virus isolation or in TRIzol (Invitrogen, Carlsbad, CA, USA) for RNA extraction. Serum was separated from the blood clot by centrifugation. All samples were transported on dry ice and stored at -80 o C.
The brains (n = 1,182) were subjected to the direct fl uorescent antibody test for lyssavirus antigen (8). In addition, the 277 brains that were collected in 2006 were homogenized and tested for virus isolation by the intracerebral mouse inoculation test as described elsewhere (9). Virus isolation was attempted for only a subset of brain samples (n = 210) from the specimens collected during 2007.
Total RNA was extracted from the oral swabs (n = 931) and subjected to nested reverse transcription-PCR, as described previously (10). We used primers designed for the nucleoprotein genes of LBV, MOKV, and WCBV.
The virus neutralizing antibodies in bat serum samples were determined by a modifi cation of the rapid fl uorescent focus inhibition test. We used 4-well (6-mm) Tefl oncoated glass slides (Cel-Line, Erie Scientifi c, Portsmouth, NH, USA) as described elsewhere (10) sults of this micromethod are comparable to those of the classical test in chamber slides. The neutralizing activity of each serum sample was determined against LBV, MOKV, DUVV, RABV, and WCBV. All samples were initially screened in dilutions of 1:10 and 1:25. Samples that showed reduced fl uorescence or no fl uorescence were subjected to additional titration in dilutions of 1:10 to 1:1,250. The 50% end-point neutralizing titers were calculated by the method of Reed and Muench (11). The samples that had a 50% endpoint neutralizing titer >1 log 10 were considered positive. Circulation of LBV was detected in fruit bats Eidolon helvum and Rousettus aegyptiacus as described previously (10). No other viruses were isolated during the study, and no lyssavirus RNA was identifi ed in oral swabs. However, virus-neutralizing activity against WCBV was detected in serum of Miniopterus insectivorous bats ( Figure 2) from 4 of the 5 locations where these species were collected (online Appendix Table). Among 76 serum samples with WCBV-neutralizing activity (Figure 1, panel B), only 1 sample additionally neutralized DUVV, but no cross-neutralization to other lyssaviruses was detected. This observation supported specifi city of the reaction and reliability of the selected cutoff threshold. The seroprevalence varied by roosts, 17% to 26% (95% confi dence interval 17-27). In general, seroprevalence among females (n = 201; seroprevalence 26%) was greater than that among males (n = 112; seroprevalence 19%). Although statistically insignificant (χ 2 = 2.38; p = 0.12), this difference was consistent across locations 1, 13, and 20. Only females were available from location 8. At all locations, Miniopterus bats shared caves with other species of insectivorous and fruit bats. However, no serologic activity against WCBV was detected in these other species. Of note, serum from fruit bats R. aegyptiacus that shared caves with Miniopterus bats neutralized LBV but not WCBV. Conversely, serum from Miniopterus bats neutralized WCBV but not LBV. This fi nding suggests that bats of different species, even those roosting in the same caves, do not readily expose each other to lyssaviruses.

Conclusions
We found WCBV-neutralizing antibodies in bats in Africa. Because limited serologic cross-reactivity between lyssaviruses and other rhabdoviruses has been demonstrated (12), the WCBV seroprevalence we detected may have been caused by some other serologically related virus. However, to date no other agent that could cross-neutralize WCBV is known (7).
We cannot explain why 1 WCBV-neutralizing sample additionally neutralized DUVV. This fi nding could indicate nonspecifi c virus inhibition, or it could be evidence of coexposure of the bat to several lyssaviruses. Our inability to isolate viruses in this study is not surprising because lyssavirus prevalence in bat populations is usually low (<1%), even when seroprevalence is as high as 40%-70% (10,13,14). Indeed, the seroprevalence may refl ect past exposures and peripheral virus activity rather than survival after clinical lyssavirus infection.
WCBV was fi rst isolated in 2002 in southeastern Europe from Miniopterus schreibersii (6)