Tentative novel lyssavirus in a bat in Finland

Summary A tentative novel member of the genus Lyssavirus , designated as Kotalahti bat lyssavirus , was detected in a Brandt ’ s bat ( Myotis brandtii ) in Finland. Based on phylogenetic analysis, the virus differs from other known lyssaviruses, being closely related to Khujand virus, Aravan virus, Bokeloh bat lyssavirus and European bat lyssavirus 2.

In Finland, EBLV-2 has been isolated twice and antibodies have been detected in Daubenton's bats, and it is therefore considered to be endemic in the Daubenton's bat population (Jakava-Viljanen, Lil- Brandt's bat is mainly sedentary, and the seasonal migration is usually less than 40 km. Compared to other species, it is not so often found near human settlements. During summer, it roosts in tree holes and trunk cracks, whilst, in winter, it chooses to hibernate in caves and mines. Nursery colonies usually comprise 20-60 females, and colonies mixed with Myotis mystacinus and Myotis daubentonii have been observed. In winter roosts, M. brandtii/mystacinus usually hibernate clustered up to 13 individuals. (Dietz & Kiefer, 2016;Siivonen & Wermundsen, 2008). It has the longest known lifespan of all bat species, exceeding 40 years (Podlutsky, Khritankov, Ovodov, & Austad, 2005).

| MATERIALS AND METHODS
In August 2017, a dead bat was found outside a vacation home in Eastern Finland in the municipality of Lepp€ avirta (62°29 0 30″N, 027°47 0 15″E), in the village of Kotalahti. The bat was sent to the Finnish Food Safety Authority Evira for autopsy. The bat was a male bat weighing 3.5 g and it was quite autolysed on arrival, with maggots inside the corpse. It was identified as a Brandt's bat by PCR method amplifying ND1 mitochondrial gene (Boston et al., 2011).
Smears prepared from brain tissue for the fluorescent antibody test (FAT) were fixed in high-grade cold acetone, air dried and then stained with specific conjugate (FITC anti-rabies monoclonal globulin, Fujirebio Diagnostics, and Rabies anti-nucleocapsid conjugate, Bio-Rad). FAT slides were examined for specific fluorescence using a fluorescence microscope.
A 10% suspension was made from the brain sample. The suspension was then inoculated in mouse neuroblastoma (MNA) cells according to a rabies tissue culture infectious test (RTCIT) procedure described in the OIE manual (OIE, 2016).
RNA was extracted from the brain suspension with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The OneStep RT-PCR kit (Qiagen) was used to amplify two partially overlapping fragments of 612 nt and 1,026 nt of the nucleoprotein (N) gene. The reaction volume was 25 ll and the temperature profile of cDNA synthesis and amplification was 30 min at 50°C, 15 min at 94°C for reverse transcriptase inactivation and DNA polymerase activation, followed by 30 amplification cycles of 1 min at 94°C, 1 min at 50°C and 1 min at 72°C. The primers were published by Davis et al., (2005). The PCR products were separated on a 2% agarose gel stained with ethidium bromide. PCR products that produced bands of expected sizes on the agarose gel were purified from the gel using the Qiaquick Gel Extraction Kit (Qiagen). The reaction products were purified using the DyeEx 2.0 Spin Kit (Qiagen). The PCR products were sequenced using an ABI 3100 Avant Genetic Analyzer (Applied Biosystems) with the primers used in the PCR and a Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems). The sequences were analysed using DNASTAR Lasergene 10 (DNASTAR, Inc), BioEdit (http:/www.mbio.ncsu.edu) and MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) software and compared with previously published lyssavirus sequences in NCBI GenBank using BLASTN program. A phylogenetic tree was constructed using maximum likelihood method, with general time-reversible correction for multiple substitutions, as implemented in MEGA software.

| RESULTS AND DISCUSSION
The   previously unknown virus might not grow in MNA cell culture as well as other bat lyssaviruses or our cell culture procedure might not be sensitive enough. The FAT-negative result with the other conjugate used raises concern over whether FAT alone or FAT together with virus isolation in cell culture is sufficient for bat samples. The sensitivities of both the FAT and RTCIT have been shown to depend on the quality of the specimen (Fooks et al., 2012;Wacharapluesadee & Hemachudha, 2010). McElhinney, Marston, Brookes, and Fooks (2014) detected viral RNA from a carcass decomposing at 25°C for 48 days, whilst virus was isolated using the RTCIT on day 3 and the FAT detected viral antigen on day 12.
It is advisable that bat samples should undergo viral genome analysis when decomposed samples are likely to be submitted. As the bat was found dead, there is no information on whether it displayed clinical signs of rabies. Bats are the natural reservoir of lyssaviruses. The detection of seropositive bats without clinical signs suggests that bats can be infected and clear the infection. Lyssavirus infection can also cause clinical disease in bats. Spillover events from Chiroptera to other mammals are rare but have occurred with RABV (Daoust, Wandeler, & Casey, 1996;Leslie et al., 2006), EBLV-1 (Dacheux et al., 2009;M€ uller et al., 2004;Tjørnehøj, Fooks, Agerholm, & Rønsholt, 2006), MOKV (Sabeta et al., 2007), LBV (Markotter et al., 2006) and ABLV (Shinwari et al., 2014), but these infections are usually dead-end events. In addition to RABV, also ABLV, EBLV-1, EBLV-2, IRKV, DUVV and MOKV (reviewed in Johnson et al., 2010) have been responsible for human deaths, even though only in few cases. Therefore, we can assume that humans might also be susceptible to Kotalahti bat lyssavirus.
Based on the close relationship with phylogroup I viruses, vaccines available for animals and humans will probably also offer some level of cross-protection against Kotalahti bat lyssavirus, even though the sequence alone cannot provide a reliable interpretation of antigenic differences, and further studies are needed. Brandt's bats were analysed for rabies 1998-2014, all with negative results (Schatz et al., 2014). Many bats have not been identified to species level, but, nevertheless, it seems that low numbers of Brandt's bat have been tested for the presence of lyssaviruses in Europe.

ACKNOWLEDG EMENTS
We thank Marja Isomursu, Riikka Holopainen and Tiina Peltonen for their contribution. This work was funded by Finnish Food Safety Authority Evira.

CONFLI CT OF INTEREST
None declared.