Co-circulation of three pathogenic hantaviruses: Puumala, Dobrava and Saaremaa in Hungary Angelina Plyusnina, Emöke Ferenczi, Gábor R. Rácz, Kirill Nemirov, Åke Lundkvist, Antti Vaheri, Olli Vapalahti, Alexander Plyusnin
To cite this version:
Angelina Plyusnina, Emöke Ferenczi, Gábor R. Rácz, Kirill Nemirov, Åke Lundkvist, et al.. Co- circulation of three pathogenic hantaviruses: Puumala, Dobrava and Saaremaa in Hungary. Journal of Medical Virology, Wiley-Blackwell, 2009, 81 (12), pp.2045. 10.1002/jmv.21635. hal-00531827
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Co-circulation of three pathogenic hantaviruses: Puumala, Dobrava and Saaremaa in Hungary
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Journal: Journal of Medical Virology
Manuscript ID: JMV-09-1405.R1
Wiley - Manuscript type: Research Article
Date Submitted by the 21-Jul-2009 Author:
Complete List of Authors: Plyusnina, Angelina; Haartman Inst, University of Helsinki, Virology Ferenczi, Emöke; B. Johan" National Center for Epidemiology Rácz, Gábor; Harold W. Manter Laboratory of Parasitology, University of Nebraska Nemirov, Kirill; Swedish Institute for Infectious Disease Control Lundkvist, Åke; Swedish Institute for Infectious Disease Control Vaheri, Antti; Haartman Inst, University of Helsinki, Virology Vapalahti, Olli; Haartman Inst, University of Helsinki, Virology Plyusnin, Alexander; Haartman Inst, University of Helsinki, Virology
Keywords: HFRS; hantavirus; Puumala virus, Dobrava virus, Saaremaa virus
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Fig. 1. Rodent collecting localities in Hungary (shown as full circles). Two localities (TR16 and TR17) 34 that are discussed in the article are marked. Stars indicate localities from where hantavirus sequences have been recovered earlier (Scharninghausen et al., 1999; Jakab et al., 2007a, 2008). 35 540x414mm (72 x 72 DPI) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Medical Virology Page 2 of 30
1 2 3 SNV 4 HTNV 5 TULV 6 100 Muju96-1 7 Muju0018 MUJV 8 Muju99-27 Muju99-28 9 100 Tobetsu 10 Kamiiso HOKV 90 11 Klippitztörl9 12 100 100 Ernstbrunn641 13 Hungary23 ALAD 14 100 Slovenia78 Hungary9 15 99 16 Slovenia65 Slovakia 17 MignovillardY02 18 For PeerBelgium13891 Review 19 100 CgErft 20 97 Couvin59 21 Momignies55 CE 22 Momignies47 23 Thuin33 24 Belgium14445 92 Belgium14444 25 Montbliart23 26 77 Sollefteå6 27 100 Eidsvoll24 S-SCA 28 Eidsvoll38 29 Virrat 30 Pallas63 31 Kolodozero Karhumäki117 32 100 Gomselga 33 Evo12 34 Evo15 FIN 35 99 Sotkamo 36 Puumala 37 100 OmskCg322 38 OmskCg144 89 39 OmskCg315 OmskCg168 72 40 100 Estonia205 41 99 Estonia49 100 42 Bashkiria1820 RUS 43 100 Bashkiria17 44 100 Udmurtia894 45 Udmurtia458 46 Kazan 100 Mellansel49 47 100 UmeaHu 48 VindelnL20 49 100 Hundberget N-SCA 50 Tavelsjo 51 "Vranica"/Hällnäs 52 100 Fyn19 DAN 53 Fyn131 54 PUUMALA Fyn47 55 56 0.1 57 58 59 60 John Wiley & Sons Page 3 of 30 Journal of Medical Virology
1 2 3 4 TULV 5 6 Fyn47 100 DAN 7 100 Fyn19 8 9 Fyn97 10 100 Belgium13891 11 CE 12 CgErft 13 88 14 Ernstbrunn641 15 Klippitztörl9 16 94 17 Hungary2 18 For Peer Review 19 Hungary9 87 20 100 94 Hungary23 21 ALAD 22 100 NovaGradiska430 23 Okucani421 24 89 25 Okucani387 26 100 27 Okucani416 76 28 NovaGradiska452 29 30 84 "Vranica"/Hällnäs 31 N-SCA 32 VindelnL20 33 100 Sundsvoll255 34 35 98 Sundsvoll26 36 Brandberget S-SCA 37 100 38 90 Sollefteå3 39 40 Lungvik72 41 Bashkiria1820 42 RUS 43 87 Kazan 44 45 82 Virrat 46 Sotkamo 47 48 98 Karhumäki117 49 50 Aitoo86 FIN 51 Längelmäki126 52 53 54 55 PUUMALA 56 57 58 0.1 59 60 John Wiley & Sons Journal of Medical Virology Page 4 of 30
1 2 3 4 SEOV 5 6 THAIV 7 100 8 9 HTNV 10 11 Greece-AP9 12 13 Hung27Af 14 15 Slovenia 16 17 Serbia-MontenegroMD02 18 For Peer Review DOBV 19 100 20 Slovakia400 21 22 GreeceEA 23 100 24 GreeceGA 25 96 26 GreeceHA 27 87 28 29 Saaremaa160 30 31 Denmark/Lolland 32 33 Hung1Aa SAAV 34 72 98 35 Slovakia862 36 37 Russia/Kurkino38 38 100 39 40 Russia/Kurkino44 41 42 43 0.1 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 5 of 30 Journal of Medical Virology
1 2 3 4 PUUV 5 6 HTNAH09 7 82 8 HTNFLi HTNV 9 100 10 HTNLR1 100 11 12 HTN76118 13 14 GOUV 15 100 16 SEOR22 17 100 SEOV 18 For PeerSEOSapporo Review 19 75 20 SEOZ37 21 22 THAI0017 23 100 24 THAIV THAI741 25 26 27 SANGV 28 Russia/Kurkino44 29 100 30 31 Russia/Kurkino53 32 96 33 HungAa1 34 99 99 35 Slovakia856 36 100 SAAV 37 Slovakia862 38 39 Denmark/Lolland 40 41 Estonia/Saaremaa160 100 42 100 43 Estonia/Saaremaa90 44 45 Russia/Ps1223Krasnodar 46 100 47 48 Russia/Ap1-GorKl 49 100 50 Slovenia/Dobrava DOBV 51 100 52 HungAf27 53 80 54 Greece-AP13 55 99 56 Greece-AP9 57 58 0.1 59 60 John Wiley & Sons Journal of Medical Virology Page 6 of 30
1 2 3 4 Denmark/Lolland 5 6 Russia/Kurkino44 100 7 8 Russia/Kurkino53 9 10 79 Slovakia862 11 99 12 13 Slovakia856 14 15 90 Taz ar2 SAAV 16 17 HungAa1 18 88 For Peer Review 19 20 Tazar8 21 22 93 Estonia/Saaremaa90 23 100 24 Estonia/Saaremaa160 25 26 Russia/Ps1223Krasnodar 27 100 28 29 Russia/Ap1-GorKl 30 99 31 Slovenia/Dobrava 32 33 88 DOBV 34 HungAf27 35 36 Greece-AP13 37 93 38 Greece-AP9 39 40 41 42 0.1 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 7 of 30 Journal of Medical Virology
1 2 3 4 Slovenia/Dobrava 5 100 Greece-AP13 6 74 7 Greece-AP9 8 74 9 10 HungHFRSPec 11 100 Slovenia/PrekmurjeAf10 DOBV 12 69 13 96 14 HungAf27 15 Russia/Ps1223Krasnodar 16 100 17 18 100 For PeerRussia/Ap1-GorKl Review 19 20 Denmark/Lolland 21 22 Estonia/Saaremaa160 23 100 24 Estonia/Saaremaa90 25 26 Russia/Kurkino44 27 100 28 Russia/Kurkino53 29 30 Slovakia856 31 84 100 SAAV 32 Slovakia862 33 34 100 Slovenia/PrekmurjeAa9 35 98 36 HungAa1 37 99 38 HungPecsAa77 39 66 40 41 100 HungPecsAa27 42 43 HungPecsAa31 44 45 0.1 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Medical Virology Page 8 of 30
1 2 1 3 4 5 2 6 7 3 Co-circulation of three pathogenic hantaviruses: Puumala, Dobrava 8 9 4 10 and Saaremaa in Hungary 11 12 5 13 6 (Short title: Hantaviruses in Hungary) 14 15 7 16 8 17 18 9 19 10 1 2 á 3 4 20 Angelina Plyusnina , EmökeFor Ferenczi Peer, Gábor R. R Reviewcz , Kirill Nemirov , Åke 21 11 Lundkvist 4, Antti Vaheri 1, Olli Vapalahti 1, 22 12 1 23 and Alexander Plyusnin 24 13 25 26 14 27 15 1Department of Virology, Infection Biology Research Program, Haartman Institute, 28 29 16 University of Helsinki, Finland 30
31 2 17 "B. Johan" National Center for Epidemiology, Budapest, Hungary 32 33 18 3 34 Harold W. Manter Laboratory of Parasitology, University of Nebraska Lincoln, W- 35 19 36 529 Nebraska Hall, Lincoln, NE, U.S.A. 37 20 4 38 Swedish Institute for Infectious Disease Control, Stockholm, Sweden 39 40 21 41 42 22 Correspondence to: 43 44 23 Alexander Plyusnin 45 46 24 Department of Virology, Haartman Institute 47 48 25 University of Helsinki P. O. Box 21 49 50 26 FIN-00014, Finland 51 52 53 54 55 56 57 58 59 60
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1 2 27 Phone: +358 9 19126486; Fax: +358 9 19126491 3 4 28 E-mail: [email protected] 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
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1 2 29 ABSTRACT 3 4 30 5 6 31 Hantaviruses ( Bunyaviridae ) cause hemorrhagic fever with renal syndrome (HFRS) in 7 8 32 Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS 9 10 33 11 is caused by Hantaan (HTNV), Seoul (SEOV), Dobrava (DOBV), Saaremaa (SAAV) 12 34 13 and Puumala (PUUV) viruses. Of those, only HTNV is not present in Europe. In 14 15 35 recent years, hantaviruses, described in other parts of Europe, were also detected at 16 17 36 various locations in Hungary. To study the genetic properties of Hungarian 18 19 37 hantaviruses in detail, sequences of the viral S and M segments were recovered from 20 For Peer Review 21 38 bank voles (Myodes glareolus ), yellow-necked mice ( Apodemus flavicollis ) and striped 22 23 39 field mice ( Apodemus agrarius ) trapped in the Transdanubian region. As expected, the 24 25 40 sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one 26 27 41 strain) and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV 28 29 42 strains located within the well-supported Alpe-Adrian (ALAD) genetic lineage that 30 31 43 included also Austrian, Slovenian and Croatian strains. Analysis of the Hungarian 32 33 44 SAAV and DOBV genetic variants showed host-specific clustering and also 34 35 45 geographical clustering within each of these hantavirus species. Hungarian SAAV 36 37 46 and DOBV strains were related most closely to strains from Slovenia (Prekmurje 38 39 47 40 region). This study confirms that multiple hantaviruses can co-circulate in the same 41 48 42 locality and can be maintained side-by-side in different rodent species. 43 49 44 45 46 50 47 48 51 Key words: HFRS; hantavirus; Puumala virus, Dobrava virus, Saaremaa virus. 49 50 51 52 53
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1 2 52 INTRODUCTION 3 4 53 5 6 54 Hantaviruses (genus Hantavirus, family Bunyaviridae ) cause hemorrhagic fever 7 8 55 with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary 9 10 56 11 syndrome (HCPS) in the Americas (for reviews, see Schmaljohn and Hjelle, 1997; 12 57 13 Vapalahti et al., 2003). HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), 14 15 58 Dobrava-Belgrade virus (DOBV), Saaremaa virus (SAAV) and Puumala virus (PUUV). 16 17 59 Sin Nombre virus (SNV), Andes virus and related hantaviruses are the main causative 18 19 60 agents of severe HCPS. Hantaviruses are host-specific, and thus their distribution 20 For Peer Review 21 61 correlates most often with the geographical distribution of the natural host species, 22 23 62 rodents and insectivores. Transmission of hantaviruses to humans occurs by 24 25 63 inhalation of infested aerosols of rodent excreta. Whether hantaviruses carried by 26 27 64 insectivores can infect humans and cause any disease remains to be seen. 28 29 65 Of HFRS-causing hantaviruses, only HTNV is not present in Europe [Vapalahti 30 31 66 et al., 2003]. PUUV, carried by bank voles (Myodes glareolus), is the most widely 32 33 67 distributed European hantavirus. It causes a mild form of HFRS (also called 34 35 68 nephropathia epidemica , NE) in Northern and Central Europe, European part of Russia 36 37 69 and in the Alpe-Adrian region. DOBV, harbored by yellow-necked field mice 38 39 70 40 (Apodemus flavicollis ), is the most severe European hantavirus pathogen; it is 41 71 42 associated with HFRS mostly in the Balkans [Papa et al., 1998; Avsic-Zupanc et al., 43 72 44 1999]. SAAV, carried by striped field mice ( Apodemus agrarius ), causes mild HFRS 45 46 73 resembling NE; the virus is found in the Baltics, Central Europe and European part 47 48 74 of Russia [Lundkvist et al., 1998; Plyusnin et al., 1999; Golovljova et al., 2000; Sibold 49 50 75 et al., 2001]. SEOV, carried by Norway rats ( Rattus norvegicus ), is so far found in 51 52 53
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1 2 76 France and Belgium [Heyman et al., 2004, 2009]. Apart from laboratory outbreaks 3 4 77 caused by SEOV-infected rats kept in captivity, no human cases in Europe have 5 6 78 been reported in connection to this hantavirus. 7 8 79 HFRS has been described in Hungary prior to the identification of the 9 10 80 11 causative agents, the hantaviruses [Trencsenyi and Keleti, 1981]. Until 1985, human 12 81 13 HFRS cases were diagnosed initially based on medical symptoms and 14 15 82 epidemiology; these were later confirmed using serological tests [Takenaka et al., 16 17 83 1985; Faludi and Ferenczi 1995]. With the advent of DNA and RNA sequencing 18 19 84 several hantaviruses have been identified in Hungary [Scharninghausen, 1999, 20 For Peer Review 21 85 Ferenczi 2003, Jakab 2007a, 2008]. 22 23 86 On average, there are 20-25 laboratory-confirmed human hantavirus infections 24 25 87 in Hungary each year. The fatality rate associated with clinical infections dropped 26 27 88 from approximately 6% in the 1950s to 2.5% in recent years. In case a physician 28 29 89 suspects HFRS, further serological tests are performed at the National Center for 30 31 90 Epidemiology (NCE). Serological diagnosis is based primarily on 32 33 91 immunofluorescence assay (IFA) and secondarily on enzyme-immunoassay (EIA). 34 35 92 Compared to the number of hospitalized patients, hantavirus seroprevalence is 36 37 93 much higher in Hungary, approximately 10% [Ferenczi et al., 2005]. These 38 39 94 40 controversial data might be explained by the low HFRS awareness of the medical 41 95 42 community and by the undiagnosed mild forms of the disease. 43 44 96 The aim of this study was to recover hantavirus sequences from tissue samples 45 46 97 of rodents known as hatural hosts for Puumala, Dobrava and Saaremaaa viruses, 47 48 98 perform their detailed (phylo)genetic characterization, assign them to proper taxa 49 50 99 and specify unequivocally the rodent hosts involved. 51 52 53
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1 2 101 MATERIALS AND METHODS 3 4 102 5 6 103 Trapping of rodents. Small mammals were collected as part of a larger 7 8 104 research project on studying the distribution of hantaviruses in Hungary. The 9 10 105 11 broader area, the southwestern part of Hungary, was selected based on the high 12 106 13 incidence rate of human hantavirus cases (Fig. 1). Zala county with only 3% of 14 15 107 Hungary’s total population gives 23% of HFRS cases in the country. In 2000, selected 16 17 108 trapping localities were chosen to survey wild mammals as reservoirs of 18 19 109 hantaviruses in forest habitats. The primary factor in selecting specific sites was the 20 For Peer Review 21 110 forest area, but several other characteristics of the plant community (e.g. disturbance 22 23 111 level and plant species composition) correlated with patch size. Rodents were 24 25 112 trapped using Sherman type live capture traps [Mills et al., 1995] set in the evening 26 27 113 and checked in the morning. At each locality trapping was continued until sufficient 28 29 114 numbers of animals (usually around 40-50) were captured. Animals were identified, 30 31 115 aged, sexed and their conditions were recorded. Euthanized rodents were dissected; 32 33 116 blood and tissue samples – lung, spleen, kidney, heart – were removed for further 34 35 117 analysis. Tissues were frozen immediately in liquid nitrogen. Upon arrival at the 36 37 118 Virology Department of the NCE tissue samples were transferred to -80°C freezers 38 39 119 40 for long-term storage. Skeletal materials of the collected animals were deposited as 41 120 42 voucher specimens at the Mammal Collection of the Museum of Southwestern 43 121 44 Biology, University of New Mexico. 45 46 122 Screening of rodent samples. Sera collected from rodents were screened 47 48 123 initially for the presence of antibodies to hantaviruses by commercially available 49 50 124 kits as described previously [Ferenczi et al., 2003]. Briefly, HTNV antigen-coated 51 52 53
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1 2 125 high density particle agglutination (HDPA) kit (Korean Green Cross) was used 3 4 126 according the protocol of the WHO Collaborating Center for Virus Reference and 5 6 127 Research (HFRS), Institute for Viral Diseases, Korea University, Seoul, South Korea. 7 8 128 9 To perform indirect immunofluorescent assay (IFA), HTNV-, PUUV- and SEOV- 10 129 11 specific tests (Progen Biotechnik, GMBH, Germany) with FITC-conjugated rabbit 12 13 130 anti-mouse immunoglobulin (DAKO, A/S, Denmark) were used. For the detection 14 15 131 of HTNV- and PUUV-specific antibodies by enzyme immunoassay (EIA), IgG and 16 17 132 IgM kits (Progen Biotechnik) were applied. 18 19 133 Twenty five out of 37 serum samples were tested additionally on EIA plates 20 For Peer Review 21 134 containing recombinant antigens for PUUV, DOBV and SEOV viruses [Elgh et al., 22 23 135 1997]. Lung tissue samples from seropositive rodents were screened further for 24 25 136 hantavirus nucleocapsid (N) protein antigen by immunoblotting as described earlier 26 27 137 28 [Plyusnin et al., 1995]. 29 138 30 Reverse transcription - polymerase chain reaction (RT-PCR) and sequencing. 31 32 139 RNA was extracted from lung tissue samples using the Tripure reagent (Boehringer 33 34 140 Mannheim) following recommendations of the manufacturer. RNA was then 35 36 141 subjected to RT-PCR to recover partial S segment and M segment sequences 37 38 142 (sequences of primers and other experimental details are available upon request). 39 40 143 PCR amplicons were gel-purified with QIAquick Gel Extraction -kit (QIAGEN) and 41 42 144 sequenced directly using ABI PRISM TM Dye Terminator sequencing kit (Perkin 43 44 145 Elmer/ABI, NJ). PUUV genome sequences described in this paper have been 45 46 146 deposited to the GenBank sequences database under accession numbers FN377821- 47 48 147 25. DOBV and SAAV genome sequences described in this paper have been deposited 49 50 148 51 to the GenBank sequences database under accession numbers FN377826-29. 52 53
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1 2 149 Mitochondrial DNA (mtDNA) analysis was performed as described earlier 3 4 150 [Nemirov et al., 2002]. Briefly, DNA was extracted from lung tissue samples using 5 6 151 the Tripure reagent. A 427 nt-long PCR-product from the D-loop-encoding region 7 8 152 was amplified with primers 5'-CCACCATCAGCACCCAAAGCTG-3' and 5'- 9 10 153 11 CTGAAGTAAGAACCAGATGTCTG-3'. The product was purified from the gel and 12 154 13 subjected to direct sequencing. For comparison, mtDNA sequences of A. agrarius and 14 15 155 A. flavicollis were retrieved from the GenBank nucleotide databases. 16 17 156 Phylogenetic analysis. Multiple nucleotide alignments were prepared 18 19 157 manually using SeqApp 1.9a169 sequence editing program. Phylogenetic analysis 20 For Peer Review 21 158 was performed using the PHYLIP program package [Felsenstein, 1993]. 500 22 23 159 bootstrap replicates (Seqboot program) were submitted to the distance matrice 24 25 160 algorithm (Dnadist program), with maximum likelihood model for nucleotide 26 27 161 substitutions); distance matrices were analysed with the Fitch-Margoliash tree-fitting 28 29 162 algorithm (Fitch program); the bootstrap support values were calculated with the 30 31 163 Consense program. Hantavirus sequences used for comparison were recovered from 32 33 164 the GenBank. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
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1 2 165 RESULTS 3 4 166 5 6 167 Prescreening of rodents. Rodent candidates for further RT-PCR and 7 8 168 sequencing were pre-screened for hantavirus antibodies by HDPA, IFA and EIA. 9 10 169 11 From more than 300 rodents, 37 were found positive or suspected positive: 8 bank 12 170 13 voles (Myodes glareolus ) and 29 Apodemus mice (18 A. agrarius , 2 A. flavicollis, 9 - 14 15 171 unspecified Apodemus spp.) . Positive M. glareolus were from the locality TR17, 16 17 172 positive Apodemus mice from localities TR16 and TR17 (Fig. 1). 18 19 173 Genetic analysis of PUUV strains. From eight pre-selected (Ab-positive) bank 20 For Peer Review 21 174 voles, six were found positive for PUUV N-Ag in immunoblotting. Of those, three 22 23 175 appeared also positive for PUUV genome when tested by RT-PCR; all three animals 24 25 176 were from the same locality, TR-17. Three wild-type PUUV strains (that were not 26 27 177 isolated) were designated PUUV/Mg2/HungaryTR17/00, 28 29 178 PUUV/Mg9/HungaryTR17/00, and PUUV/Mg23/HungaryTR17/00, or Hung2, 30 31 179 Hung9, and Hung23, for short. For two strains, Hung9 and Hung23, almost 32 33 180 complete sequences of the S segment have been recovered in a set of (semi)-nested 34 35 181 PCRs. For the strain Hung2, only partial sequence of the coding region (nt 191 to 36 37 182 1190) was recovered. It differed by two silent nucleotide substitutions from the 38 39 183 40 corresponding sequence of strain Hung9 and was excluded from further analyses. 41 184 42 Two other sequences included 5'-noncoding region (NCR) of 42 nt and the open 43 185 44 reading frame (ORF) for the N protein of 433 aa followed by 499 or 442 nt from 3'- 45 46 186 NCR (for strains Hung9 and Hung23, respectively). The 5'-NCR sequences were 47 48 187 identical (the first 22 nt originated from the primer sequence and thus were not 49 50 188 determined directly); the coding regions differed by 34 substitutions (identity of 51 52 53
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1 2 189 97.4%); the 3'-NCR sequences differed at 28 positions (identity of 95.9%). Deduced N 3 4 190 protein sequences differed by one substitution only (identity of 99.8%) and carried 5 6 191 signature residues V236 and P257 shared also by PUUV strains from neighbouring 7 8 192 Austria [Plyusnina et al., 2006] and Slovenia [Avsic-Zupanc et al., 2007]. Partial M 9 10 193 11 segment sequences (nt 2161-2570, primers excluded) of strains Hung2, Hung9, and 12 194 13 Hung23 were recovered as well; they appeared 94.4-96.1% identical to each other. 14 15 195 The encoded partial sequences of the Gc protein (136 aa long) were 97.8-99.3% 16 17 196 identical. Hungarian M segment/Gc protein sequences were most closely related to 18 19 197 the corresponding sequences from Austrian [Plyusnina et al., 2006] and Croatian 20 For Peer Review 21 198 [Cvetko et al., 2005] PUUV strains (corresponding sequences from Slovenian strains 22 23 199 are not available). 24 25 200 On the phylogenetic tree based on the complete S segment coding region, the 26 27 201 two Hungarian PUUV strains were located within the well-supported Alpe-Adrian 28 29 202 (ALAD) genetic lineage that included also Austrian and Slovenian strains (Fig. 2A). 30 31 203 Within this lineage, Hungarian and Slovenian strains shared the most recent 32 33 204 common ancestor (TMRCA) while Austrian strains formed a sister taxon to this 34 35 205 quartet. Six other PUUV lineages can be recognized: (1) Central European (CE); (2) 36 37 206 South Scandinavian (S-SCA); (3) Finnish (FIN); (4) Russian (RUS); (5) North 38 39 207 40 Scandinavian (N-SCA); and (6) Danish (DAN). PUUV shared more ancient common 41 208 42 ancestors (MACAs) with Hokkaido virus (HOKV) and then with Muju virus 43 44 209 (MUJV) associated with Myodes rufocanus and Myodes regulus, respectively. The 45 46 210 phylogenetic tree based on the partial M segment sequence showed similar topology 47 48 211 (Fig. 2B): Hungarian strains were located within the ALAD lineage which, in this 49 50 51 52 53
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1 2 212 case, included also strains from Austria and Croatia. Other PUUV lineages (CE, S- 3 4 213 SCA, FIN, RUS, N-SCA and DAN) were presented as well. 5 6 214 Genetic analysis of SAAV and DOBV strains. From 29 pre-screened 7 8 215 Apodemus mice (18 A. agrarius, 2 A. flavicollis, 9 - unspecified); six were found 9 10 216 11 positive for DOBV/SAAV N-Ag in immunoblotting. Of those, only two were 12 217 13 positive also in the RT-PCR test. Analysis of mtDNA sequences showed that one 14 15 218 mouse (#1, from locality TR-16) belonged to the species A. agrarius, another (#27 16 17 219 from locality TR-17) was A. flavicollis. Partial S and M segment sequences were 18 19 220 recovered from both rodents using DOBV/SAAV-specific primers and subjected to 20 For Peer Review 21 221 phylogenetic analysis. On phylogenetic trees (Fig. 3A-B), the sequence recovered 22 23 222 from A. agrarius mouse was placed with SAAV sequences (all recovered from 24 25 223 striped field mice) while the sequence recovered from A. flavicollis mouse co- 26 27 224 localized with DOBV sequences, originated from either yellow-necked mice or 28 29 225 human HFRS cases. Although the topologies of the trees based on partial M and S 30 31 226 segment sequences were quite similar, the M-tree showed a somewhat better 32 33 227 resolution, i.e. higher bootstrap support values for SAAV- and DOBV-clades. Based 34 35 228 on these data, novel Hungarian hantavirus strains were designated 36 37 229 SAAV/Aa1/Hungary TR16/00 and DOBV/Af27/HungaryTR17/00 or HungAa1 38 39 230 40 and HungAf27, for short. 41 231 42 New Hungarian SAAV and DOBV sequences were compared to previously 43 232 á 44 published ones recovered from A. agrarius trapped in Tasz r [spelled as “Tazar” in 45 46 233 the original publication], eastern Hungary [Scharninghausen et al., 1999]. The 47 48 234 analysis was restricted to the overlapping part of the S segment sequence recovered 49 50 235 in two studies: nt 707 to 935. This region of two Tasz ár strains appeared most 51 52 53
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1 2 236 closely related to the corresponding region of the SAAV strain HungAa1: only one 3 4 237 (silent) nucleotide substitution was observed between these sequences (identity of 5 6 238 99,6%). In contrast, the sequence of the DOBV strain HungAf27 differed from Tasz ár 7 8 239 sequences at 29-30 positions (identity of 86.9-87.3%). Phylogenetic analysis showed 9 10 240 á 11 TMRCA for the two Tasz r strains and the strain HungAa1 (Fig. 3C). This trio was 12 241 13 placed within the SAAV clade and shared MACA with SAAV strains from Slovakia. 14 242 á 15 These data suggested that the two Tasz r strains belong to SAAV. 16 17 243 New Hungarian SAAV and DOBV sequences were compared also to the 18 19 244 previously published sequences recovered from A. agrarius trapped in the Görcsöny 20 For Peer Review 21 245 and Sármellék areas and from an HFRS patient from the University hospital of Pécs 22 23 246 (all in the Transdanubian region) [Jakab et al., 2007a,b]. Since, in this case, the 24 25 247 analysis was restricted to shorter sequences of the S segment recovered by these 26 27 248 authors (nt 410 to 800), it became possible to include also partial SAAV and DOBV 28 29 249 sequences originated from Prekmurje region of neighbouring Slovenia [Avsic- 30 31 250 Zupanc et al., 2000]. 32 33 251 Direct sequence comparison showed that Hungarian SAAV strain HungAa1 34 35 252 was most closely related to Hungarian strain Pecs/77Aa from Sármellék (sequence 36 37 253 identity 99.5%) and to Slovenian SAAV strain Aa9 from Prekmurje (97.9%). 38 39 254 40 Sequence of our DOBV strain HungAf27 was most closely related to the 41 255 42 corresponding region of Slovenian DOBV strain from Prekmurje (99.2%) and then to 43 44 256 Hungarian HFRS-associated strain (92.0%). On the phylogenetic tree (Fig. 3D) all 45 46 257 four Hungarian A. agrarius originated sequences, as expected, were located within 47 48 258 the SAAV clade. The new strain HungAa1 shared TMRCA with the strain PecsAa77 49 50 259 and this couple shared the MACA with Slovenian strain Aa9 from Prekmurje. 51 52 53
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1 2 260 Similarly, our DOBV strain HungAf27 shared TMRCA with Slovenian DOBV strain 3 4 261 Af10 from Prekmurje; they, in turn, shared MACA with the Hungarian HFRS- 5 6 262 associated strain. These results demonstrated host-specific clustering of Hungarian 7 8 263 SAAV and DOBV genetic variants and, within each of these hantavirus species, also 9 10 264 11 geographical clustering. 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
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1 2 265 DISCUSSION 3 4 266 5 6 267 The results of this study demonstrate co-circulation of all three major 7 8 268 European hantavirus pathogens in Hungary: PUUV, DOBV and SAAV. They are in 9 10 269 11 line with earlier observations on human HFRS cases in this country [Ferenczi et al., 12 270 13 2005; Jakab et al., 2007a] and recovery of hantavirus sequences from rodents 14 15 271 [Scharninghausen et al., 1999; Ferenczi et al., 2003; Jakab et al., 2007a]. It should be 16 17 272 emphasized that while SAAV in Hungarian striped field mice has been already 18 19 273 reported (albeit under the name of Dobrava virus - see below), the first unequivocal 20 For Peer Review 21 274 evidence for PUUV and DOBV in Hungarian rodents, species M. glareolus and A. 22 23 275 flavicollis, respectively, is presented in this paper. 24 25 276 Rodents used in genetic studies were selected in two steps. First, pre-selection 26 27 277 by HDPA, EIA and IFA to detect hantavirus antibodies was performed. On that 28 29 278 stage, not only clearly Ab-positive but also borderline voles and mice were selected 30 31 279 for further analyses. This could explain the observed differences between the results 32 33 280 of antibody detection and the N-Ag detection, due to pre-selection not only clearly 34 35 281 Ab-positive but also voles and mice giving the borderline results. In turn, the 36 37 282 relatively low RT-PCR positivity of N-Ag-positive rodents could be due to partial 38 39 283 40 degradation of target RNA in tissue samples that were subjected to several thawing- 41 284 42 freezing cycles (to take material for other analyses) before RNA extraction. 43 285 44 Phylogenetic analysis of Hungarian PUUV strains placed them within the 45 46 286 ALAD genetic lineage that includes also strains from neighbouring Slovenia, Croatia 47 48 287 and Austria. This presents a good example of geographical clustering of PUUV 49 50 288 genetic variants, a phenomenon first discovered more than a decade ago [Plyusnin 51 52 53
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1 2 289 et al., 1994]. Phylogenies that included new Hungarian PUUV sequences confirmed 3 4 290 two interesting earlier observations [Plyusnina et al., 2006]. First, within the ALAD 5 6 291 lineage, two Austrian M-sequences are not monophyletic. One of the strains, Klipp9, 7 8 292 is clustering together with Croatian and Hungarian strains suggesting that M and S 9 10 293 11 segments of this strain might have different evolution history, i.e. this strain is a 12 294 13 reassortant one. Second, ALAD and CE lineages and RUS and FIN lineages share 14 15 295 MACAs thus suggesting somewhat closer relationships within these two pairs of 16 17 296 lineages that could be rooted to the last postglacial recolonization of these areas of 18 19 297 Europe. 20 For Peer Review 21 298 The analyses of Hungarian SAAV and DOBV sequences confirmed that genetic 22 23 299 variants of these hantavirus species show the host-specific clustering. Indeed, all 24 25 300 sequences recovered from A. agrarius belonged to SAAV while those from A. 26 27 301 flavicollis belonged to DOBV. Thus previously reported sequences from Hungarian 28 29 302 A. agrarius [Scharninghausen et al., 1999; Jakab et al., 2007a] should be re-classified 30 31 303 as SAAV strains. It seems that only the sequence recovered from human HFRS case 32 33 304 in Pécs [Jakab et al., 2007b] presents bona fide DOBV. As mentioned above, for 34 35 305 several years there was a controversy in taxonomy and hence terminology of SAAV. 36 37 306 When first discovered, this Dobrava-like virus was erroneously considered a genetic 38 39 307 40 variant of DOBV carried by the striped field mice [Plyusnin et al., 1997; Nemirov et 41 308 42 al., 1999]. It took several years to realize that this is in fact a distinct hantavirus 43 44 309 species [Brus-Sjölander et al., 2002; Nemirov et al., 2002], the view that is now 45 46 310 shared by the International Committtee for Virus Taxonomy [www.ictvonline.org]. 47 48 311 Another important finding of this study is that multiple hantavirus types can 49 50 312 be maintained at the same locality by their corresponding rodent hosts. The direct 51 52 53
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1 2 313 transmission mode and the high level of host specificity of the hantaviruses make 3 4 314 these temperate-zone viruses less affected by host biodiversity [Ruedas et al., 2004]. 5 6 315 7 Instead, the population density and size are the main factors driving the disease 8 9 316 temporal dynamics. 10 11 317 Of the four European hantavirus pathogens known so far, only SEOV has not 12 13 318 been found in Hungary, albeit serological evidence showed its causative role at least 14 15 319 in one case [Ferenczi, unpublished data]. Direct detection of this virus is rather 16 17 320 difficult because its primary hosts, Norway rats Rattus norvegicus , would not be 18 19 321 caught by the traps used. Similar observation have been made in neighbouring 20 For Peer Review 21 322 Slovenia [Avsic-Zupanc et al., 2002, 2007], Slovakia [Sibold et al., 2001] and Croatia 22 23 323 [Cvetko et al., 2004]. Our unpublished serological data on clinical cases confirm that 24 25 324 both PUUV-like and DOBV/SAAV-like HFRS cases occur in Hungary. Co- 26 27 325 circulation of three distinct hantaviruses that could cause HFRS of different severity, 28 29 326 including life-threatening infections, calls for improvements in both surveillance 30 31 327 and diagnostics. 32 33 328 34 35 329 36 Acknowledgements. The work was supported by The Academy of Finland 37 38 330 and Sigrid Juselius Foundation (Finland). Rodent survey was funded by grants from 39 40 331 Hungarian Ministry of Health (ETT 1998-1999. Rec. No.: 144/98), and Hungarian 41 42 332 Ministry of Environment (II. OKTKP id. No.:020837-01/2000). Field research 43 44 333 equipment was partially provided by funding from the Centers for Disease Control 45 46 334 and Prevention through a cooperative agreement with Dr. Terry L. Yates. The 47 48 335 authors are grateful to Borbala Juhasz, Marta Csire, Kaposi Tamasn é and Ann- 49 50 336 Christin “Anci” Verlemyr for excellent technical assistance. 51 52 53
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1 2 337 REFERENCES 3 4 338 5 6 339 Avsic-Zupanc T, Petrovec M, Furlan P, Kaps R, Elgh F, Lundkvist Å. 1999. Hemorrhagic 7 8 340 fever with renal syndrome in the Dolenjska region of Slovenia-A 10-year survey. Clin 9 341 Infect Dis 28: 860-865. 10 11 342 Avsic-Zupanc T, Nemirov K, Petrovec M, Trilar T, Poljak M, Vaheri A, Plyusnin A. 12 343 13 2000. Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two 14 344 distinct genetic lineages within the same natural focus. J Gen Virol 81:1747-1755. 15 Formatted: Bullets and Numbering 16 345 Avsic-Zupanc T, Petrovec M, Dun D, Plyusnina A, Lundkvist Å, Plyusnin A. 2007. 17 346 Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered 18 19 347 from patients and rodents. Virus Res 123: 204-210. 20 348 Brus-Sjölander K, GolovljovaFor I, Plyusnin Peer A, Lundkvi stReview Å. 2002. Serological divergence 21 22 349 of Dobrava and Saaremaa hantaviruses: evidence for two distinct serotypes. Epidemiol 23 24 350 & Infection 128:99-103. 25 351 Elgh F, Lundkvist A, Alexeyev OA, Stenlund H, Avsic-Zupanc T, Hjelle B, Lee HW, 26 27 352 Smith KJ, Vainionpää R, Wiger D, Wadell G, Juto P. 1997. Serological diagnosis of 28 353 hantavirus infections by an enzyme-linked immunosorbent assay based on detection of 29 30 354 immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral 31 355 serotypes. J Clin Microbiol 35:1122-1130. 32 33 356 Faludi G, Ferenczi E. 1995. Seriologically verified Hantavirus infections in Hungary, 34 357 35 Acta Microbiol Immunol Hung 42:419-426. 36 358 Felsenstein 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by 37 38 359 the author. Department of Genetics, University of Washington, Seattle. 39 360 Ferenczi E, Rácz G, Szekeres J, Balog K, Tóth E, Takács M, Csire M, Mezey I, Berencsi G, 40 41 361 Faludi G. 2003. [New data for the public health importance of hantaviruses in 42 362 Hungary]. [Article in Hungarian] Orv Hetil 144:467-474. 43 44 363 Ferenczi E, Rácz G, Faludi G, Czeglédi A, Mezey I, Berencsi G. 2005. Natural foci of 45 364 46 classical and emerging viral zoonoses in Hungary. In: Berencsi G, Khan AS, Halouska J, 47 365 editors. NATO Science Series: Emerging Biological Threat. IOS Press, p. 43-49. 48 49 50 51 52 53
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1 2 366 Golovljova I, Vasilenko V, Prukk T, Brus Sjolander K, Plyusnin A, Lundkvist Å. 2000. 3 4 367 Puumala and Dobrava hantaviruses causing hemorrhagic fever with renal syndrome in 5 368 Estonia. Eur J Clin Microbiol Infect Dis 19:968-969. 6 Formatted: Bullets and Numbering 7 369 Golovljova I, Brus Sjölander K, Lindegren G, Vene S, Vasilenko V, Plyusnin A, 8 9 370 Lundkvist Å. 2002. Hantaviruses in Estonia. J Med Virol 68:589-598. 10 371 Heyman P, Plyusnina A, Berny Ph, Cochez C, Artois M, Zizi M, Pirnay JP, Plyusnin A. 11 12 372 2004. Seoul hantavirus in Europe: first demonstration of the virus genome in wild R. 13 373 norvegicus captured in France. Eur J Clin Microbiol Infect Dis. 23:711-717. 14 15 374 Heyman P, Baert K, Plyusnina A, Cochez C, Lundkvist Å, Van Esbroeck M, Goossens E, 16 375 17 Vandenvelde C, Plyusnin A, Stuyck J. 2009. Serological and genetic evidence for the 18 376 presence of Seoul hantavirus in Rattus norvegicus in the Flanders, Belgium. Scand J 19 20 377 Infect Dis 41:51-56. For Peer Review 21 378 ICTV website: www.ictvonline.org 22 23 379 Jakab F, Horváth G, Ferenczi E, Sebok J, Varecza Z, Szucs G. 2007a. Detection of 24 380 25 Dobrava hantaviruses in Apodemus agrarius mice in the Transdanubian region of 26 381 Hungary. Virus Res 128:149-152. 27 382 28 Jakab F, Sebok J, Ferenczi E, Horváth G, Szucs G. 2007b. First detection of Dobrava 29 383 hantavirus from a patient with severe haemorrhagic fever with renal syndrome by 30 31 384 SYBR Green-based real time RT-PCR. Scand J Infect Dis 39:902-906. 32 385 Jakab F, Horváth, Ferenczi E, Sebok J, Szucs, G. 2008. First detection of Tula 33 34 386 hantaviruses in Microtus arvalis voles in Hungary. Arch Virol 153: 2093-2096. 35 387 Lundkvist Å, Vasilenko V, Golovljova I, Plyusnin A, Vaheri, A. 1998. Human Dobrava 36 37 388 hantavirus infections in Estonia. Lancet 352:369. 38 389 39 Mills JN, Yates TL, Childs JE, Parmenter RR, Ksiazek TG, Rollin PE, Peters CJ. 1995. 40 390 Guidelines for working with rodents potentially infected with hantavirus. J 41 42 391 Mammalogy 76:716-722. 43 392 Nemirov K, Vapalahti O, Lundkvist Å, Vasilenko V, Golovljova I, Plyusnina A, 44 45 393 Niemimaa J, Laakkonen J, Henttonen H, Vaheri A, Plyusnin A. 1999. Isolation and 46 394 characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus 47 48 395 agrarius) in Estonia. J Gen Virol 80:371-379. 49 50 51 52 53
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1 2 396 Nemirov K, Henttonen H, Vaheri A, Plyusnin A. 2002. Phylogenetic evidence for host 3 4 397 switching in the evolution of hantaviruses carried by Apodemus mice. Virus Res 90: 207- 5 398 215, 2002. (Corrigendum: Virus Res 92:125-126, 2003). 6 7 399 Papa A, Johnson AM, Stockton PC, Bowen MD, Spiropoulou CF, Alexiou-Daniel S, 8 400 9 Ksiazek TG, Nichol ST, Antoniadis A. 1998. Retrospective serologic and genetic study of 10 401 the distribution of hantaviruses in Greece. J Med Virol 55: 321-327. 11 12 402 Plyusnin A, Vapalahti O, Ulfves K, Lehväslaiho H, Apekina N, Gavrilovskaya I, Blinov 13 403 V, Vaheri A. 1994. Sequences of wild Puumala virus genes show a correlation of genetic 14 15 404 variation with geographic origin of the strains. J Gen Virol 75:405-409. 16 405 Plyusnin A, Cheng Y, Vapalahti O, Pejcoch M, Unar J, Jelinkova Z, Lehväslaiho H, 17 18 406 Lundkvist Å, Vaheri, A. 1995. Genetic variation in Tula hantaviruses:; sequence analysis 19 407 of the S and M segments of strains from Central Europe. Virus Res 39: 237-250. 20 For Peer Review 21 408 Plyusnin A,Vapalahti O, Vasilenko V, Henttonen H, Vaheri A. 1997. Dobrava 22 409 23 hantavirus in Estonia: does the virus exist throughout Europe? Lancet 349:1369-1370. 24 410 Plyusnin A, Nemirov K, Apekina N, Plyusnina A, Lundkvist Å, Vaheri A. 1999. 25 26 411 Dobrava hantavirus in Russia. Lancet 353:207. 27 412 Plyusnina A, Aberle S, Aberle J, Plyusnin A. 2006. Genetic analysis of Puumala 28 29 413 hantavirus strains from Austria. Scand J Infect Dis 38: 512-519. 30 414 Ruedas LA, Salazar–Bravo J, Tinnin DS, Armién B, Cáceres L, García A, Díaz MA, 31 32 415 Gracia F, Suzán G, Peters CJ, Yates TL, Mills, JM. 2004. Community ecology of small 33 416 34 mammal populations in Panamá following an outbreak of Hantavirus pulmonary 35 417 syndrome. J Vector Ecol 29:177-191. 36 37 418 Scharninghausen JJ, Meyer H, Pfeffer M, Davis DS, Honeycutt RL. 1999. Genetic 38 419 evidence of DOB virus in Apodemus agrarius in Hungary. Emerg Infect Dis 5:468-470. 39 40 420 Schmaljohn CS, Hjelle B. 1997. Hantaviruses: a global disease problem. Emerg Infect Dis 41 421 42 3:95-104. 43 422 Sibold C, Meisel H, Lundkvist Å, Schulz A, Cifire F, Ulrich R, Kozuch O, Labuda M, 44 45 423 Krüger DH. 1999. Short report: simultaneous occurrence of Dobrava, Puumala, and 46 424 Tula hantaviruses in Slovakia. Am J Trop Med Hyg 61:409-11 . 47 48 425 Sibold C, Ulrich R, Labuda M, Lundkvist Å, Martens H, Schutt M, Gerke P, Leitmeyer 49 426 K, Meisel H, Krüger DH. 2001. Dobrava hantavirus causes hemorrhagic fever with renal 50 51 52 53
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1 2 427 syndrome in central Europe and is carried by two different Apodemus mice species. J 3 4 428 Med Virol 63:158-167. 5 429 Takenaka A, Gibbs CJ, Gajdusek DC. 1985. Antiviral neutralizing antibody to Hantaan 6 7 430 virus as determined by plaque reduction technique, Arch Virol 84:197-206. 8 431 9 Trencséni T, Keleti B. 1971. Clinical aspects and epidemiology of Haemorrhagic fever 10 432 with renal syndrome. Analysis of the clinical and epidemiological experience in 11 12 433 Hungary. Akadémia Kiadó, Budapest , pp.247. 13 434 Vapalahti O, Mustonen J, Lundkvist Å, Henttonen H, Plyusnin A, Vaheri A. 2003. 14 15 435 Hantavirus infections in Europe (Review). Lancet Infect Dis 3:653-661. 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
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1 2 436 Legends to figures 3 4 437 5 438 Fig. 1. Rodent collecting localities in Hungary (shown as full circles). Two 6 7 439 localities (TR16 and TR17) that are discussed in the article are marked. Stars indicate 8 440 9 localities from where hantavirus sequences have been recovered earlier 10 441 (Scharninghausen et al., 1999; Jakab et al., 2007a, 2008). 11 12 442 13 443 Fig. 2. Phylogenetic trees of Puumala and related hantaviruses based on (A) 14 15 444 complete S segment coding sequences and (B) partial M segment sequences (nt 2161- 16 445 2570). Numbers show the bootstrap support values for the branching points (500 17 18 446 replicates). Only the values higher than 70% are shown. Abbreviations: SNV, Sin 19 447 20 Nombre virus, strain NM H10;For HTNV ,Peer Hantaan virus, Review strain 76-118; TULV , Tula virus, 21 448 strain Moravia02v; MUJV , Muju virus; HOKV, Hokkaido virus. Genetic lineages of 22 23 449 PUUV: ALAD , Alpe-Adrian; CE , Central European; S-SCA , South Scandinavian; FIN , 24 450 Finnish; RUS , Russian; N-SCA , North Scandinavian; DAN , Dannish. 25 26 451 27 452 Fig. 3. Phylogenetic trees of Dobrava, Saaremaa and related hantaviruses based on 28 29 453 (A) partial M segment sequences (nt 1704-1969), and partial S segment sequences: (B) nt 30 454 377-935; (C) nt 707-935; (D) nt 410-800. Numbers show the bootstrap support values for 31 32 455 the branching points (500 replicates). Only the values higher than 50% are shown. 33 456 34 Abbreviations: DOBV, Dobrava virus; SAAV, Saaremaa virus; HTNV , Hantaan virus, 35 457 strain 76-118 (if not specified otherwise); SEOV , Seoul virus, strain Sapporo rat (if not 36 37 458 specified otherwise); THAIV , Thailand virus, strain741; GOUV , Gou virus, strain 38 459 Nc167; SANGV , Sangassou virus strain SA14; PUUV, Puumala virus, strain Sotkamo. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
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