First detection of European bat lyssavirus type 2 (EBLV-2) in Norway

Rabies is a fatal zoonotic neurological disease caused by RNA viruses belonging to the genus Lyssavirus in the family Rhabdoviridae. Rabies virus (RABV) is the prototype of the genus and causes approximately 59,000 deaths in humans yearly [1]. Red fox (Vulpes vulpes) is the main reservoir for RABV in Europe, where the disease has been eliminated in many member states of the European Union due to oral vaccination programs [2]. Rabies in non-flying mammals has never been diagnosed in mainland Norway [3]. However, in the Svalbard archipelago, which is under Norwegian jurisdiction, several occasional detections and two rabies outbreaks during the last 35 years have been reported and are related to arctic strains of RABV in Arctic fox (Vulpes lagopus), Svalbard reindeer (Rangifer tarandus platyrhyncus) and one ringed seal (Pusa hispida) [4–6].

The genus Lyssavirus consists of 14 recognized species [7], and all but two have been isolated from bats [8]. The different species display distinct features regarding geographical distribution and host specificity. In Europe, bat rabies is primarily attributed to European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) [9]. Most reported bat rabies cases are caused by EBLV-1 which is almost exclusively found in serotine bats (Eptesicus serotinus) and Isabelline serotine bats (E. isabellinus), while EBLV-2 is detected mainly in Daubenton’s bats (Myotis daubentonii) and to a lesser extent in pond bats (M. dasycneme) [9]. Evolutionary studies suggest that EBLV-2 diverged from other lyssaviruses more than 8000 years ago and that the current diversity of EBLV-2 has built up during the last 2000 years [10].

Hitherto, EBLV-2 has been detected in bats in the United Kingdom, the Netherlands, Germany, Switzerland, Finland and Denmark [11–16], while antibodies against EBLV-2 have been found in bats in Sweden [17]. Two cases of human rabies associated with EBLV-2 have been reported in Finland [18] and in Scotland [19]. To date, no cases of natural spill-over to other non-flying mammals have been reported, but EBLV-2 has successfully been transferred to sheep and foxes in experimental studies, even though the susceptibility seems to be low in these species [20, 21].

Among the 12 species of bats regularly reported in Norway, the northern bat (E. nilssonii), the soprano pipistrelle (Pipistrellus pygmaeus) and the Daubenton’s bat are the most common and widespread species [22–24]. The serotine bat has been recorded once and is not considered as a resident species [25]. The range of the Daubenton’s bat reaches to 63° N in Norway [24]. The results of ringing experiments in other countries suggest that this species does not migrate over long distances [26], which is likely also the case in Norway. The Daubenton’s bat is specialized in hunting insects and spiders, which it mainly catches with its feet from the water surface [27], but it is also found searching for food in forests [24]. Daubenton’s bats seldom occur above the timberline and roost mainly in tree cavities, but also use crevices in bridges and cliffs. Roosts in buildings are very rare [24, 28] and so are contacts with the general public [29].

There is no official surveillance program for lyssavirus in bats in Norway. According to the annals of the Norwegian Veterinary Institute (NVI), a total of 27 bats (swabs from 18 bats sampled alive and brain tissues from nine carcasses) were examined for lyssavirus in the period 1998–2015. Here, we report the first detection of EBLV-2 in Norway.

Here, we report the first detection of EBLV-2 in Norway. The detection of EBLV-2 in a Daubenton’s bat is in accordance with previous studies that have revealed that EBLV-2 is mainly found in this species [9]. As the bat was a subadult and ringing experiments suggest that this species does not migrate over long distances [26], it can be assumed that the bat was native to the area where it was found and that EBLV-2 is present in Norway. The latter is supported by the phylogenetic analyses, which indicate that the Norwegian isolate differs from other published isolates, further suggesting that the Norwegian isolate may have evolved separately from a common European ancestor [10]. Very few bats have been examined for rabies in Norway, and we have no knowledge of the prevalence and epidemiology of EBLV. In neighbouring countries, EBLV-2 was detected in diseased Daubenton’s bats in Finland in 2009 and 2016 [15, 44], while active surveillance has revealed viral RNA from mouth swabs from Daubenton’s bat in Denmark and seropositive Daubenton’s bats in both Sweden and Finland [16, 17, 45].

The FAT performed on brain impressions at the NVI was negative, and unfortunately, due to insufficient material, it could not be repeated at the Nancy OIE/WHO/EU Laboratory for Rabies and Wildlife (EURL). However, the EURL confirmed the presence of lyssavirus in the RTCIT. The negative result for FAT does not concur with findings in other Daubenton’s bats naturally infected with EBLV-2 [11, 13–15, 44, 46–51]. The annual proficiency tests for rabies diagnosis techniques organized by the EURL have demonstrated the difficulty for laboratories to reliably detect EBLV strains when using the FAT [52], with results depending on the rabies virus antibody conjugate and even the batch used [53]. However, the same batch used in the FAT for the present case has successfully been applied at the NVI for detection of this virus species in two proficiency tests (unpublished results). A polyclonal antibody was used to confirm the presence of lyssavirus in the RTCIT, and a difference between the antibodies regarding the ability to detect the current virus cannot be excluded.

The aspiration pneumonia might have been caused by a dysfunctional swallow reflex or as a result of the force-feeding. The bat had a low state of nutrition, displayed several signs of weakness and, in the end, an inability to fly. This could be caused by the EBLV-2 infection or due to general weakness as a consequence of either shortage of feed at the high altitude or inexperience in hunting since being a subadult. Also, a poor body condition could have made the bat more susceptible for EBLV-2 infection. Weight loss, the inability to fly and death within 14 days followed by detection of viral antigen in brain tissue is reported in Daubenton’s bats after intracerebral inoculation with EBLV-2 [54]. In that study the bats were fed ad libitum, so the weight loss could not be related to a decrease in hunting success. During a natural EBLV-2 infection, the course of the disease is probably longer than that found in the referred experiment, and thus the ability to hunt and eat could deteriorate over some time span, resulting in gradual loss of body condition. Poor body condition in bats can also be influenced by heavy parasite loads [55]. Other possible signs of the disease, like the inability to fly, were registered only up to 48 h before death in the experiment [54]. Most of the bats which were inoculated either intramuscularly or subdermally survived the study period of 123 days [54]. These findings highlight that there is a high intra-species barrier in transmission of EBLV-2 and that the incubation period under natural conditions is probably several weeks to months.

Two human cases of rabies caused by EBLV-2 have been reported, in Finland in 1985 and in Scotland in 2002 [18, 19]. Both persons were in close contact with bats over time and had been bitten several times without any history of immunization prior to exposure or post-exposure prophylaxis. Spill-over to other non-flying mammals under natural conditions has not been reported, and the detection of EBLV-2 does not pose an immediate risk to humans as Daubenton’s bats are rarely in contact with the public [29]. However, the possibility of human cases of bat-associated rabies acquired in Norway, which is considered free of rabies, cannot be neglected; hence adequate information should be given to the general public as well as to people and professionals who come into contact with bats.

All bat species in Norway are strictly protected under both national laws and international commitments to preserve bats [56, 57]. Culling bats for lyssavirus testing is therefore not a feasible strategy. Passive surveillance by testing bats found sick or dead is the most appropriate way of assessing the incidence of rabies as compared with active surveillance by testing swabs and/or sera from live bats from natural populations [58, 59]. Passive surveillance of bat rabies is therefore strongly recommended by international organizations [58, 60].

Monitoring of EBLV in bats has so far not been prioritized by the Norwegian authorities, probably due to few human cases of bat-associated rabies being reported in Europe. According to the annals of the NVI, only seven carcasses of bats including the EBLV-2 positive case were examined during the last three years. In 2014 and 2015, the NVI in collaboration with the NZS collected and tested nasopharyngeal and cloacal swabs from totally 16 free-ranging Norwegian bats for lyssaviruses with a negative result (unpublished data). The experience gained in this pilot study and from surveillance in other countries is important for future monitoring of bat rabies in Norway.

The authors believe that testing bats found sick or dead should get priority through a national rabies surveillance network in line with the recent recommendations of EUROBATS and EFSA [58, 60]. Additionally, an active surveillance program, over a limited number of years and in defined areas to obtain baseline data on the prevalence of bat rabies in Norway, is also recommended. Finally, we emphasize the importance of good cooperation between bat biologists and laboratory workers to ensure access to representative samples of good quality.