1. Hantaviruses - still surprising!!!
Petra Strakova, MSc Masaryk University, Brno, Czech Republic Institute of Vertebrate Biology, Academy of Sciences, Valtice, Czech Republic Veterinary Research Institute, Brno, Czech Republic Friedrich-Loeffler-Institut, Insel Riems, Greifswald, Germany

2. Members of family Bunyaviridae
RNA virus, spherical, enveloped virions; trisegmented genome
More than 24 hantaviruses („Old and New World“)
Cause of hemorrhagic fever with renal syndrome (HFRS), milder form nephropathia epidemica (NE) and hantavirus cardiopulmonary syndrome (HCPS) diseases
Hosts (reservoirs): „robovirus“, specific - rodents, now: also insectivores and bats; longlife persistent infection

3. Central Europe – hot spot?
Bank vole (Myodes glareolus)
- host of Puumala virus (PUUV)
Common shrew (Sorex araneus)
- host of Seewis virus (SWSV)
FH07JUN_STOMOL_01.JPG
Common vole (Microtus arvalis)
- host of Tula virus (TULV)
Common mole (Talpa europaea)
- host of Nova virus (NVAV)
Bats (Chiroptera) ?
Striped field mouse (Apodemus agrarius) and
Yellow necked mouse (Apodemus flavicollis)
-host of Dobrava-Belgrade virus (DOBV)
And of course many more…
(Klempa et al. 2013)

4. Why surprise? More and more hantaviruses described
New mammal species found as hosts
Our own projects:
Unknown virus in Microtus oeconomus
from Lithuania
2) Bat hantavirus from Brno,
Czech Republic
„virus hunting“ using NGS 

5. Project 1 - Lithuania
Objective: to get evidence of the presence of hantaviruses in Lithuanian rodent population
341 lung samples, RT-PCR for S (PUUV, TULV) and L (DOBV) segment
Out of 135 bank voles – 5 RT-PCR positive (PUUV)(Sandra Jagdmann, article already published); no A. agrarius was positive, out of 71 field and root/tundra voles – 6 RT-PCR positive (TULV)
BLAST – Topografov virus (approx. 78%, AJ011646) in all positive animals

6. Project 1 - Lithuania
Root/tundra vole (M. oeconomus) – globally distributed, holartic species (north Europe extend to Germany, Poland, Belarus and central Europe, North America, Siberia, China, Mongol Altai); tundra, taiga, forest-steppe, riverbanks even flooded shores; they feed on green vegetation
Poland
Germany
Sweden
our samples
established
Poland
Poland
Lithuania
Norway
our samples
Poland
Map of Lithuania. (Straková et al. 2017)
Phylogenetic tree based on cytb gene of European tundra voles.

7. Project 1 – Lithuania, „TOPV“ results
PUUV
TULV
DOBV
Fig. Phylogenetic tree of partial S segment.

8. Project 1 – Lithuania, „TOPV“ results
Latvia-My.glareolus
Japan-My.rufocanus
China-My.rufocanus
Mi.fortis – far east Russia
Mi.maximomowiczii - China
Mi.californicus - USA
Mi.arvalis - CR
Fig. Phylogenetic tree of partial S segment.

9. Project 1 – Lithuania, „TOPV“ results
Fig. Phylogenetic tree based on sequences of TULV RT-PCR positive animals from Germany, France and the Czech Republic (Maximum Likelihood Tree, Jukes-Cantor model, bootstrap value 1000). For this tree, sequences of 340 bp were used. Identical sequences are not included.

10. Project 1 – Lithuania, „TOPV“ results - NGS
STD
NGS
1951
NGS
361
1040
1186
1303
129
376
FW2+REV2
FW3+REV3
NCRS+REV1
Progress: now 1450 nt (78% Puumala Kamiiso, 79% Topografov) M
NGS
NGS
NGS
NGS 1
3728
790
1023
1534
1875
1922
2215
3326
3438
NGS
NCRS+REV1
FW2+REV2
1746
1892
FW4+REV4
FW3+REV3
Progress: now 1273 nt (77% Yuanjiang, 77% Khabarovsk, 76% Topografov) L 1
STD
6700
6187
6527
NGS
2956
3362
6142
6328
From NGS = 9 reads 
Designing primers to fill gaps
NGS

11. Project 1 – Lithuania, „TOPV“ results - NGS
- S segment – cds – 1323 nt, 441 aa
New
Topografov
Khabarovsk
Yakeshi
Vladivostok
Fusong
Kamiiso
Isla vista
Lodz
Tula
New virus
0,88
0,87
0,59
0,80
0,85
0,86
0,81
0,79
Topografov virus
0,95
0,83
0,82
Khabarovsk virus
0,77
0,60
Yakeshi virus
0,62
0,53
0,58
0,57
Vladivostok virus
0,71
0,73
0,74
0,55
0,78
0,76
Puumala virus Fusong
0,70
Puumala virus Kamiiso
Isla vista virus
0,75
0,69
0,84
Hantavirus Lodz
0,61
0,72
0,97
Tula virus
0,68
- Sequence identity – down left – nucleotide level, up right – amino acid level

12. Project 2 – bat hantavirus
Several bat hantaviruses already described (Africa – Magboi, Makokou; Asia – Laibin, Longquan, Xuan Son; South America – serological evidence)
Objective: what about Europe ?
In Brno – out of 53 bats, 2 RT-PCR positive (Nyctalus noctula) – liver, kidneys (Klempa et al – panHanta primers) – just 364 bp – too short to publish 
Dr. Bernd Hoffmann designed primers for Real-Time RT-PCR, used for screening 1000 bats (positive bats also in Germany )

13. Project 2 – bat hantavirus – Brno virus
at IVD FLI - NGS approach – after 2 runs – 559 reads
S segment – (approx. 72%, Longquan virus)
M segment – (approx. 69%, Longquan virus)
L segment – (approx. 70%, Laibin virus)
(Straková et al. 2017)

14. Project 2 – bat hantavirus – Brno virus
S segment/N
M segment/GPC
L segment/RdRp
Host
Virus strain
Country
1272 nt
424 aa
3411 nt
1137 aa
6435 nt
2145 aa
Bats
Longquan virus
China
65.9 65
66.3
62.5
78.3*
80.5*
Laibin virus
58.7
56.3
55.4
45.6 66
66.7
Huangpi virus
65.6*
64* -
71.7*
81.7*
Xuan Son virus
Vietnam
58.5
54.3
61.2*
54.4*
70*
75.1*
Mouyassue virus
Côte d´Ivoire
73.4*
78.9*
Magboi virus
Sierra Leone
74.2*
75.7*
Makokou virus
Gabon
66.8*
67.6*
Quezon virus
Philippines
59.2
55.5
54.7
44.5
65.4
66.6
Shrews
Uluguru virus
Tanzania
50.9
40.9
54.8*
42.9*
64.5
62.7
Altai virus
Russia
56.9*
52.6*
48.9*
63.3
62.3
Cao Bang virus
57.1
51.7
51.9
40.4
63.7
62.1
Seewis virus
Switzerland
57.8
49.4
54.2*
46.9*
60.7*
60.3*
Thottapalayam virus 54
46.5
50.1
38.9
62.8
62.2
Moles
Nova virus
Belgium
57.5
54.6
44.2
Asama virus
Japan
55.6 51
52.2*
40.3*
64.8*
64.1*
Rodents
Puumala virus
Finland
58.3
40.1
63.2
60.3
Sin Nombre virus
USA
58.8 53
52.5
41.6
63.4
61.3
Seoul virus
Korea
48.8
51.6
39.1
60.7
Hantaan virus
56.6 40
60.6
Dobrava-Belgrade virus
Greece
55.7
49.8
50.4
61.1
Tula virus
Czech Republic
57.2
52.9
63.1
(Straková et al. 2017)

15. Project 2 – bat hantavirus – Brno virus
EF Cao Bang virus
EU Asama virus
EF Seewis virus
NC Hantaan virus
NC Seoul virus
NC DOBV
NC Sin Nombre virus
NC Puumala virus
NC Tula virus
JX Uluguru virus
NC Thotta.virus
KT Nova virus
JX Huangpi virus
KX Brno virus
JX Longquan virus
KM Laibin virus
KF Xuan son virus
KU Quezon virus
KM Altai virus
100 99 55 90 87 84 92 94 95 72 56 38 26
0.10
NC Hantaan virus
NC DOBV
NC Seoul virus
EF Cao Bang virus
EU Asama virus
NC Sin Nombre virus
NC Puumala virus
NC Tula virus
NC Thotta.virus
KX Brno virus
JX Longquan virus
KU Quezon virus
KM Laibin virus
KT Nova virus 53
100 98
0.2
100
100
100
100
100
100
100 98
Phylogenetic tree based on the partial nucleocapsid protein-encoding
sequences (S segment, approximately 1000 bp).
Phylogenetic tree based on the partial glycoprotein-encoding
sequences (M segment, approximately 3100 bp).
(Straková et al. 2017)

16. Conclusion - current situation and outlook
Unknown virus – „new“ virus in population of M. oeconomus in Lithuania
- finishing M and S segment with termini
- L segment – NGS again?
- publish this result
2) Bat hantavirus – article is published (Novel hantavirus identified in European bat species Nyctalus noctula, Straková et al. 2017, infection, Genetics and Evolution)
- investigation of bats from Germany
- another bats from Czech Republic (attempt to isolate this virus )

17. Thank you for your attention…
FLI – INNT – Dr. Rainer Ulrich & his team
Chao Wen
David Kohlhause
IVD – Dr. Dirk Höper
Dr. Bernd Hoffmann
Florian Pfaff
Department of Virology
Dr. Daniel Růžek
Dr. Lucie Dufková
Dr. Jiří Salát
Dr. Jana Širmanová
Dr. Boris Klempa
Nature Research Centre, Vilnius, Lithuania
Laima Balčiauskienė, Linas Balčiauskas
Dr. Tomáš Bartonička