Zwiesel bat banyangvirus, a potentially zoonotic Huaiyangshan banyangvirus (Formerly known as SFTS)–like banyangvirus in Northern bats from Germany

Bats are reservoir hosts for several emerging and re-emerging viral pathogens causing morbidity and mortality in wildlife, animal stocks and humans. Various viruses within the family Phenuiviridae have been detected in bats, including the highly pathogenic Rift Valley fever virus and Malsoor virus, a novel Banyangvirus with close genetic relation to Huaiyangshan banyangvirus (BHAV)(former known as Severe fever with thrombocytopenia syndrome virus, SFTSV) and Heartland virus (HRTV), both of which have caused severe disease with fatal casualties in humans. In this study we present the whole genome of a novel Banyangvirus, named Zwiesel bat banyangvirus, revealed through deep sequencing of the Eptesicus nilssonii bat virome. The detection of the novel bat banyangvirus, which is in close phylogenetic relationship with the pathogenic HRTV and BHAV, underlines the possible impact of emerging phenuiviruses on public health.

pusillus and Hipposideros caffer bats in Guinea 10 . Malsoor virus, a fourth novel banyangvirus within the genus Banyangvirus, was recently isolated from a Rousettus leschenaultii bat in India 11 .
Bats may be the source of many more yet unknown viruses which can cause morbidity and mortality in wildlife, animal stocks and humans 12 . Novel sequencing technologies allow massive parallel sequencing of all organisms in one sample, also allowing the detection of viruses with low sequence similarity to already known viruses 13,14 .
In this study, organs of microchiropteran bats, which were moribund or found dead, with potentially virus-related histopathological changes were used for deep sequencing of the bat virome. A novel banyangvirus, named Zwiesel bat banyangvirus (ZbbV) after the town of bat origin in Germany, was detected in the virome of several Northern bats (Eptesicus nilssonii). Phylogenetic analysis of the ZbbV polymerase protein showed that the closest relationship was to Malsoor virus, HRTV and BHAV.

Results
The combination of sequences obtained from virome data and gap-filling PCR revealed the partial genome sequence of the novel ZbbV with 6,277, 3,434 and 1,793 nt for the L, M and S segment, respectively. The L segment open reading frame encodes for the RNA polymerase, the M segment for the two glycoproteins. Two open reading frames in ambisense orientation were determined on the S segment, which code the N protein (sense) and the nonstructural NSs protein (antisense).
Using specifically designed primers for the ZbbV L gene and subsequent sequencing of the PCR products, ZbbV was detected and quantified by qPCR in liver, lung, brain, spleen and intestine of 5 out of 12 Eptesicus nilssonii bats used in this study (Table 1). qPCR screening of pooled organs from the remaining 363 bats, including 3 additional Eptesicus nilssonii bats, did not reveal any additional ZbbV sequences.
The sequences of the L, M and S segments of the novel virus were deposited in GenBank with the accession numbers MN823639, MN823640, and MN823641, respectively. The novel virus was named Zwiesel bat banyangvirus (ZbbV), after the town of Zwiesel in Bavaria, where one of the Eptesicus nilssonii bats was found positive.
To obtain the virus isolate of the novel banyangvirus, Vero and Paki cell lines were inoculated with pooled organs of all 12 Eptesicus nilssonii bats. After 3 passages, no cytopathic effect was observed and cell culture supernatants were tested negative for ZbbV.

Discussion
In this study we identified a novel banyangvirus, named Zwiesel bat banyangvirus, in the virome of Eptesicus nilssonii bats. Genetic characterization showed that the virus is related to the tick-borne zoonotic phenuiviruses BHAV and HRTV, responsible for severe disease and death in humans, as well as to Malsoor virus which was recently isolated from a Rousettus bat in India.
Since similar viruses have been detected in far apart countries such as North America, India, China, Japan, South Korea and Australia and in ticks of different genera, numerous zoonotic phenuiviruses may be widely distributed in different parts of the world. So far, ZbbV is the first banyangvirus detected in bats of northern Europe. Grouping of ZbbV within the tick-transmitted genus Banyangvirus suggests ticks to be the vector of the novel virus. Additionally, we have not found any evidence that ZbbV does encode for a NSm protein on segment M, this is corresponding with all other members of the genus Banyangvirus. As shown before, NSm is important for infectivity in Aedes aegypti mosquitoes 15 . However, although Haemaphysalis longicornis ticks are supposed to be the vector of BHAV, most infected patients did not report tick bites prior to disease onset 1 . Moreover, investigation of a cluster of BHAV cases suggests evidence of person-to-person transmission 15 , making the further study of BHAV-and BHAV-like viruses imperative regarding human public health.
It is tempting to suggest that viruses discovered in insectivorous bats are only remnants of the bats' diet. Although we found sequences of ZbbV in the intestines of the bats, we also detected the virus in bat liver, lung, brain and spleen using specific PCR assays.
Considering the close relationship of ZbbV to BHAV and HRTV as well as the broad distribution of zoonotic phenuiviruses and Eptesicus nilssonii bats, which are the most abundant bats in northern Eurasia, the potential zoonotic impact of emerging banyangviruses on public health should be further investigated.

Methods
Study. In  . We herewith further confirm that all experiments were performed in accordance with relevant guidelines and regulations. All bats in a broad study on diseases of native bats in Germany (n = 375) were found dead or moribund with subsequent euthanization and were kindly provided by bat researchers and bat rehabilitation centers 16 . 12 Eptesicus nilssonii bats collected throughout urban and suburban areas of Bavaria, showed potentially virus-related histopathological changes and have been further examined in this study.  www.nature.com/scientificreports www.nature.com/scientificreports/ Methods. All work was performed at Biosafety level-2 conditions with appropriate precautions. After obtaining the result that Zwiesel virus is related to SFTS virus (rated BSL-4 in Germany), further work on the original material was restricted to BSL-3. Homogenized organs [n = 31] with potentially virus-related histopathological changes (data available on request) were pooled (Table 1), followed by purification and enrichment of viruses directly from virus-infected tissue and subsequent deep sequencing of the virome 17 . Deep sequencing was performed using the Illumina HiSeq. 1500 (Illumina Inc.) technology according to the Illumina sequencing protocol. Raw reads were trimmed for primers, adapters, low-quality bases and length (>30 bp) 18 . Sequences that mapped to RefSeq sequences of eukaryotes, fungi and bacteria using Bowtie 2 version 2.2.6 with default parameters were discarded. Remaining reads were de novo assembled using Velvet assembler 1.2.10 in default mode 19,20 . Contigs and remaining sequences were aligned to the NCBI RefSeq viral protein database using DIAMOND v0.7.1 21 . All contigs and sequences with similarity to members of the genus Banyangvirus were assembled into larger contigs. The complete genome sequences of the L, M and S segments were obtained by using newly designed gap-filling PCR primers (sequence information available on request). Reagent concentrations were as follows: ClustalW by aligning to annotated phenuivirus genomes as a ref. 22,23 . Accession numbers of the L, M and S segments used for are indicated in the respective phylogenetic trees Figs. 1, 2 and 3). Specific qPCR primers were designed to determine the viral copy numbers within the organs tested positive and to screen pooled organ tissues of an additional 363 microchiropteran bats, moribund or found dead, from the broad study (BatBanyang F: 5′ACTTCCAAAGCCACAAAAGATGGTA′3; BatBanyang R: 5′CTGAAATCGCATTAAATGTTCCATTC′3; BatBanyang MGB: 5′FAM CCTCCTAAGAAGAAATACT MGB′3 16