Molecular Epidemiology and Genetic Diversity of Orthohantaviruses in Small Mammals in Western Poland

Abstract

Orthohantaviruses are negative-sense, single-stranded RNA viruses harbored by multiple small mammals. Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) cause hemorrhagic fever with renal syndrome (HFRS) in Europe. In Poland, serological surveys have demonstrated antibodies against DOBV and PUUV in patients with HFRS. Molecular evidence of DOBV and PUUV has been found in Apodemus flavicollis and Myodes glareolus, respectively, in southeastern Poland, and Seewis virus (SWSV) has been reported in Sorex araneus in central Poland. However, data on the geographic distribution and phylogeny of orthohantaviruses are unavailable for other regions in Poland. To ascertain the prevalence and genetic diversity of orthohantaviruses in western and northern Poland, lung tissues from 106 small mammals were analyzed for the presence of orthohantavirus RNA. DOBV and SWSV were detected in two of 42 (4.8%) Apodemus agrarius and in three of 10 (30%) S. araneus, respectively. Phylogenetic analyses of partial L- and S-segment sequences of DOBV indicated a shared genetic lineage with the Kurkino genotype from Slovakia, Russia, and Hungary, whereas the partial M segment of DOBV clustered with the Kurkino genotype from Germany. Phylogenetic relationships of the SWSV L and S segments showed a geographic lineage with SWSV strains from central Poland, Czech Republic, and Germany. In conclusion, the study provides insights into the molecular prevalence, phylogenetic diversity, and evolutionary relationship of DOBV in A. agrarius and SWSV in S. araneus. This report increases awareness among physicians for HFRS outbreaks in western Poland.

Figure 1.

Geographic location of small mammal trapping sites in Poland. Trapping sites for small mammals are indicated as circles. Red circles represent the location where the genomic sequence of Dobrava-Belgrade virus and Seewis virus was obtained. Dobrava-Belgrade virus was identified in the Milicz Ponds and Ślęża Landscape Park. Seewis virus was discovered in Osobowice. (The map was generated using Adobe illustrator CS6.)

Figure 2.

Phylogenetic trees of Dobrava-Belgrade virus (DOBV) L, M, and S segments. Phylogenetic trees were generated by the maximum likelihood method using the T92+G, T92+G, and GTR+G model, based on 326 nt (position 2,997-3,312) of the L segment (A), 419 nt (position 2,046-2,464) of the M segment (B), and 1,290 nt (position 36-1,325) of the entire coding region of the S segment (C), respectively. The numbers at each node are bootstrap probabilities (expressed as percentages), as determined for 1,000 iterations. Dobrava-Belgrade virus strains Aa15-14 (L: MH598462, M: MH598464, and S: MH598465), Aa15-42 (L: MH59846), SK/Aa (L: GU904039, M: AY961616, and S: AY961615), Mu08_0118 (L: KJ182938), Mu07_0293 (L: KJ182937), Mu05_0239 (L: KJ425422), Saaremaa/160V (L: AJ410618, M: AJ009774, and S: AJ009773), Ano-Poroia/Af19 (L: NC005235 and M: NC005234), Slo/Af-BER (L: GU904042, M: GU904035, and S: GU904029), LT-RS1 (L KF425495), LT-RS2 (L: KF425496), LT-RS3 (L: KF425497), 10636/Ap (L: KP878308), 10645/Ap (L: KP878309), Ap/Sochi/43 (L: KM192209 and S: JF920151), Ap/Sochi/79 (L: KM192208 and S: JF920152), GER/08/131/Af (M: GQ205413 and S: GQ205408), GER/07/607/Af (M: GQ205410), GER/07/293/Aa (M: GQ205409), GER/08/118/Aa (M: GQ205412), Aa1854/Lipetsk-02 (M: EU188453 and S: EU188452), Saaremaa/90/Aa/97 (M: AJ009777), Dobrava 3970/87 (M: L33685 and S: L41916), Ap/Sochi/hu (M: JF920149), Ap1584/Sochi-01 (M: EU188450 and S: EU188449), Kurkino/53Aa/98 (S: AJ131673), Esl/81Aa/01 (S: AY533120), Esl/856/Aa (S: AJ269549), Sarmellek/77Aa06 (S: KC848501), Pecs/27Aa/06 (S: KC848499), Lolland/Aa1403 (S: AJ009773), GER/07/1064/Aa (S: GQ205404), GER/05/239/Aa (S: GQ205405), GER/05/477/Af (S: GQ205406), Gola/242Af/07 (S: KC848496), Gola/235Af/07 (S: KC848495), Croatia_Zutica/Af813 (S: KC676602), Esl/400Af/98 (S: AY168576), and Ano-Poroia/13Af/99 (S: AJ410619). Seoul virus strain 80-39 (L: NC005238, M: NC005237, and S: NC005236), Hantaan virus strain 76-118 (L: NC005222, M: NC005219, and S: M14626), PUUV strain Sotkamo (L: NC005225, M: NC005223, and S: NC005224), Sin Nombre virus strain NM-H10 (L: NC005217, M: L25783, and S: L25784). The newly detected DOBV strains in Poland are shown in bold red letters and designated by red circles. The scale bar indicates the number of nucleotide substitutions per site. The genomic sequences of other DOBV genotypes (Kurkino, Saaremaa, Dobrava, and Sochi) and other hantaviruses were analyzed together as out-groups.

Figure 3.

Phylogenetic trees of Seewis virus (SWSV) L, M, and S segments. Phylogenetic trees were generated by the maximum likelihood method using the GTR+G, T92+G, and TN93+G model, based on 325 nt (position 2,968–3,292) of the L segment (A), 794 nt (position 446–1,239) of the M segment (B), and 732 nt (position 407–1,138) of the S segment (C), respectively. The numbers at each node are bootstrap probabilities (expressed as percentages), as determined for 1,000 iterations. The GenBank accession numbers for virus strains in the L, M, and S segment trees are as follows: SWSV strains mp70 (L: EF636026, M: EF636025, and S: EF636024), 10-1 (L: MK402015 and S: MK402016), 10-5 (L: MK402017, M: MK402018, and S: MK402019), 10-6 (L: MK402020, M: MK402021, and S: MK402022), 2121 (L: KC537794), 2049 (L: JX990941 and S: KM394269), 2063 (L: JX990942), 2080 (L: JX990943), 2049 (L: 990945 and S: KM394271), 3334 (S: KM43419 and S: KM434198), 3338 (L: KM394264), 3343 (L: KM394265 and S: KM434199), 3500 (L: KM394266), 3504 (L: KM394267), 3505 (L: KM394268), Beskydy400 (L: JQ425336), Beskydy402 (S: JQ425302), CeskeBudejovice145 (L: JQ425313 and S: JQ425267), CeskeBudejovice146 (S: JQ425268), Drahany313 (L: JQ425318 and S: JQ425273), Drahany322 (S: JQ425274), Drahany323 (L: JQ425320 and S: JQ425275), Drahany330 (L: JQ425322 and S: JQ425277), Kacov362 (L: JQ425329 and S: JQ425287), Ratkov234 (S: JQ425269), Ratkov237 (L: JQ425316 and S: JQ425271), Dreetz08144 (L: JQ425310 and S: JQ425261), Gotha08503 (S: JQ425263), Gotha102749 (L: JQ425312 and S: JQ425266), Horst092292 (L: JQ425311 and S: JQ425265), Koednitz08068 (L: JQ425309 and S: JQ425260), DGR18226 (L: GQ293102), 13 Kuopio (S: KJ136615 ), 21 Muonio (M: KJ136604 and S: KJ136610), 48 Lappeenranta (L: KJ136619, M: KJ136603, and S: KJ136609), EWS1 Hanko (L: KJ136629, M: KJ136607, and S: KJ136612), EWS13 Lohja (M: KJ136605 and S: KJ136613), EWS25 Tammela (M: KJ136608 and S: KJ136614), PJ12 Mounio (L: KY651079) PJ14 Mounio (L: KY651023), PJ23 Mounio (L: KY651024), SR154 Kannus (L: KY651025 and S: KY651077), PJ2 Mounio (L: KY651026 and S: KY651080), PJ39 Mounio (L: KY651027), PJ46 Mounio (L: KY651028, M: KY651067, and S: KY651078), PJ63 Mounio (L: KY651029), PJ98 Mounio (L: KY651030), SR17 Kuusamo (L: KY651052 and S: KY651076), SR18 Kuusamo (L: KY651054 and S: KY651075), SR20 Puolanka (L: KY651055, M: KY651065, and S: KY651073), SR21 Puolanka (L: KY651056, M: KY651069, and S: KY651074), SR42 Virolahti (L: KY651057), SR55 Tohmaiarvi (L: KY651058), SR68 Leiksa (L: KY651059), SR69 Leiksa (L: KY651060), SR72 Tammela (L: KY651061), SR77 Leiksa (L: KY651062), SR87 Puukaharju (L: KY651048), SR91 Tammela (L: KY651044), SR101 Mikkeli (L: KY651032), SR102 Puukaharju (L: KY651033), SR106 Suonenjoki (L: KY651034), SR107 Suonenjoki (L: KY651035), SR109 Suonenjoki (L: KY651036 and M: KY651064), SR115 Suonenjoki (L: KY651037 and M: KY651066), SR131 Parkano (L: KY651038, M: KY651063, and S: KY651082), SR133 Karvia (L: KY651039), SR139 Karvia (L: KY651040 and S: KY651072), SR140 Karvia (L: KY651041 and S: KY651081), SR141 Karvia (L: KY651042), SR145 Puolanka (L: KY651043 and S: KY651083), SR148 Leiksa (L: KY651049), SR160 Tammela (L: KY651045), SR161 Mikkeli (L: KY651031), SR163 Parikkala (L: KY651046 and S: KY651071), SR164 Leiksa (L: KY651047), SR167 Puukaharju (L: KY651050), SR168 Puukaharju (L: KY651051, M: KY651068, and S: KY651070), SR173 Mikkeli (L: KY651053), Novosib-Sa374 (L: GQ267804), Irkutsk St456 (L: GQ267805 and S: GQ284572), Irkutsk St475 (L: GQ267806 and S: GQ284573), Irkutsk St489 (L: GQ267807 and S: GQ284574), Irkutsk Sa56 (L: GQ267809 and S: GQ284586), Parnaya-Sa1220 (L: GQ267810 and S: GQ284580), Krasn-Sa5 (L: GQ267811 and S: GQ284584), Krasn-Sa6 (S: GQ284583), Kemerovo-Sa65 (L: GQ267812 and S: GQ284582), Novosib-Sa374 (S: GQ284581), Telet-Sa300 (S: GQ284578), Telet-Sa301 (S: GQ284577), Telet-Sa313 (S: GQ284579), Teletskoye 500 (L: EU424338), Kosice260 (L: JQ425317 and S: JQ425272), Notr-Kras_395_90 (L: KF060922). Dobrava-Belgrade virus strain DOBV SK-Aa (L: GU904039, M: AY961616, and S: AY961615), Hantaan virus strain 76-118 (L: KT885047, M: Y00386, and S: M14626), Jeju virus strain 10-11 (L: HQ834697, M: HQ834696, and S: HQ834695), Thottapalayam virus strain TPMV VRC66412 (L: EU001330, M: EU001329, and S: AY526097), and Imjin virus strain MJNV 04-55 (L: EF641807, M: EF641799, and S: EF641805). The newly detected SWSV strains in Poland are shown in bold red lettering and designated by red circles. The scale bar indicates the number of nucleotide substitutions per site.