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

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 http://www.nature.com/nrmicro/journal/v11/n8/images/nrmicro3066-f1.jpg

Central Europe – hot spot? Bank vole (Myodes glareolus) - host of Puumala virus (PUUV) Common shrew (Sorex araneus) - host of Seewis virus (SWSV) http://hostedmedia.reimanpub.com/TFH/Step-By-Step/ 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)

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 

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

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.

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

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.

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.

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

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

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. 2006 – 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 )

Project 2 – bat hantavirus – Brno virus at IVD FLI - NGS approach – after 2 runs – 559 reads S segment – 1- 1441(approx. 72%, Longquan virus) M segment – 1-3575(approx. 69%, Longquan virus) L segment – 1- 6528 (approx. 70%, Laibin virus) (Straková et al. 2017)

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)

Project 2 – bat hantavirus – Brno virus EF543524 Cao Bang virus EU929072 Asama virus EF636024 Seewis virus NC 005218 Hantaan virus NC 005236 Seoul virus NC 005232 DOBV NC 005216 Sin Nombre virus NC 005224 Puumala virus NC 005227 Tula virus JX193695 Uluguru virus NC 010704 Thotta.virus KT004445 Nova virus JX473273 Huangpi virus KX845678 Brno virus JX465415 Longquan virus KM102247 Laibin virus KF704710 Xuan son virus KU950713 Quezon virus KM361048 Altai virus 100 99 55 90 87 84 92 94 95 72 56 38 26 0.10 NC 005219 Hantaan virus NC 005234 DOBV NC 005237 Seoul virus EF543526 Cao Bang virus EU929075 Asama virus NC 005215 Sin Nombre virus NC 005223 Puumala virus NC 005228 Tula virus NC 010708 Thotta.virus KX845679 Brno virus JX465397 Longquan virus KU950714 Quezon virus KM102248 Laibin virus KT004446 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)

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 )

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