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Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia. (Research).

In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.


Currently, at least 20 serotypes and genotypes of the Hantavirus genus (family: Bunyaviridae) have been identified worldwide. Rodents are the natural reservoir for hantaviruses, although one virus strain has been isolated from the house shrew (Suncus murinus), an insectivore (1). A unique characteristic of hantaviruses is the close association between the virus type and its natural reservoir (2).

Hantaviruses cause two forms of human disease, hemorrhagic fever with renal syndrome (HFRS), and hantavirus pulmonary syndrome (HPS); human infection occurs after the inhalation of aerosolized rodent excreta. HFRS is manifested as high fever, renal dysfunction, and hemorrhage; HPS is characterized by an acute progressive pulmonary edema and a fatality rate of about 40%. Among the hantaviruses that cause HFRS in Eurasia are Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), and Dobrava-Belgrade virus (DOBV) (3), which are carried by the striped field mouse (Apodemus agrarius), Norway rat (Rattus norvegicus) and black rat (R. rattus), bank vole (Clethrionomys glareolus), and yellow-necked field mouse (A. flavicollis), respectively. DOBV was also found in A. agrarius in Europe (4,5). Sin Nombre virus (SNV), New York virus (NYV), Black Creek Canal virus (BCCV), Bayou virus (BAYV), Andes virus (ANDV), and other related viruses cause HPS in the New World and are carried by the deer mouse (Peromyscus maniculatus), white-footed mouse (P. leucopus), cotton rat (Sigmodon hispidus), marsh rice rat (Oryzomys palustris), and Oligoryzomys longicaudatus, respectively (2,6). Although the known genotypes and serotypes have increased in number with advances in the knowledge of epidemiology and epizootiology of hantavirus infection (2), some still-unidentified hantaviruses carried by specific rodent hosts may exist. HFRS is generally known to be endemic to Far East Russia. However, the genetics of hantaviruses that are pathogenic for humans are not well defined. Reed voles (Microtus fortis) in Far East Russia were found to harbor two novel hantaviruses, Khabarovsk virus (KHAB) and Vladivostok virus (7,8). Another hantavirus, Topografov virus (TOPV), was isolated from brown lemmings (Lemmus sibiricus). The correlation between these three viruses and their pathogenicity for humans are not yet known (9).

A recent study reported two novel hantaviruses, designated as Amur (AMR) and Far East (FE), that were identified from HFRS patients in Far East Russia (10). The natural reservoir of AMR genotype seems to be A. peninsulae, according to a recent study on nucleotide sequence comparisons by Yashina et al. (11).

In 1999, we carried out an epizootiologic survey in a suburb of Vladivostok, Russia, to determine the characteristics of hantaviruses circulating in Far East Russia and to examine the possibility that A. peninsulae is a carrier of pathogenic hantaviruses. We detected antibodies to hantaviruses in A. peninsulae, and the viral genome characteristics were extremely similar to the newly identified genotype, AMR (10). Using phylogenetic analysis to characterize the sequences of viruses identified from HFRS patients and A. peninsulae, we were able to corroborate the assumption of Yashina et al. (11). We also found that A. peninsulae-related viruses are pathogenic for humans and are distributed over a large area of east Asia that includes Far East Russia.

Materials and Methods

We collected sera and organs from wild rodents captured during 1999. We also collected sera and autopsy materials from HFRS patients in two rural villages in the Primorye region of Russia, located 400 km and 600 km from Vladivostok.

Rodent sera were screened for antibodies to HTNV and PUUV or both by indirect immunofluorescent antibody assay (IFA). Vero E6 cells infected with the Hantaan 76-118 strain of HTNV or the Sotkamo strain of PUUV were used as antigen slides. Diluted sera (1:16 and 1:64) were spotted onto the antigen slides and incubated at 37[degrees]C for 1 h. After three washes with phosphate-buffered saline (PBS), protein G-conjugated fluorescein isothiocyanate (FITC) (Zymed Laboratories, Inc., San Francisco, CA) was spotted onto the slides. After incubation at 37[degrees]C for 1 h, the slides were washed and observed by fluorescence microscopy. Scattered, granular fluorescence in the cytoplasm of infected Vero E6 cells was considered a positive reaction. Antibodies in HFRS patient sera were detected by the same protocol, except for the substitution of FITC-conjugated antihuman immunoglobulin (Ig) G (ICN Pharmaceuticals, Inc., Aurora, OH).

Total RNA was extracted from lung tissues of seropositive A. peninsulae with Isogen (Nippon Gene Co., Ltd., Osaka, Japan), which is based on the acid guanidium-phenol-chloroform technique, according to manufacturer's instructions. Similarly, total RNA was extracted from lung, liver, kidney, spleen, and brain tissues of HFRS patients. Reverse transcription (RT) was carried out at 42[degrees]C for 30 min by using Superscript II and random primer (Gibco-BRL, Rockville, MD). Full-length S segments were amplified with Platinum Taq (Gibco-BRL) and HTNV-full S primer for 30 polymerase chain reaction (PCR) cycles of denaturation at 94[degrees]C for 30 s, annealing at 55[degrees]C for 30 s, and extension at 68[degrees]C for 2 min. Amplification of M segments was identical to that of S segments, except for the use of M genome-specific primers (Table 1). Part of the M segment (232 nucleotides) and the entire S segment (except for the 5' and 3' ends) were sequenced with primers specific for HTNV or SEOV or both. Amplification of the partial M segment was achieved only with nested PCR. The PCR-amplified products were separated by using a Rapid Gel Extraction kit (Gibco-BRL) according to the manufacturer's instructions. Purified DNA fragments were cloned into the PCR 2.1 vector provided in the TA cloning kit (Invitrogen Corporation, Carlsbad, CA). The ligated products were transformed into Top 10 competent cells (Invitrogen Corporation) and purified with a. Miniprep kit (QIAGEN GmbH, Hilden, Germany). DNA sequencing was performed with the ABIPRISM Dye Terminator Sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 373-A genetic analyzer.

We used the ClustalX program package (version 1.81; available from: URL: to generate the phylogenetic trees by using the neighbor-joining method with 1000 bootstrap replicates. Hantavirus sequences used in the comparisons were obtained from GenBank. The S and M genome sequences used in this study are listed in Table 2.

Formalin-fixed lung, liver, kidney, and brain tissues from an HFRS patient who died of acute renal failure were observed under light microscopy and subjected to immunohistochemical analysis with monoclonal antibodies against Hantaan virus.


We carried out the epizootiologic survey on 122 rodents captured in a suburb of Vladivostok; results of serologic screening of rodent sera by IFA are shown in Table 3. Identified rodent species included (70) A. peninsulae, (39) A. agrarius, (8) C rufocanus, (3) M. fortis, and (2) Tamias sibiricus. Screening by IFA showed that one A. agrarius (2.5%), four A. peninsulae (5.7%), and one C. rufocanus (12.5%) had antibodies to HTNV or PUUV or both. HTNV-antibody titers ranged from 1:32 to 1:512. All the seropositive rodents, except for C. rufocanus, lacked antibody against PUUV (Table 4). Lung tissues from seropositive A. peninsulae were subjected to RT-PCR to amplify the virus genomes. Two of the four rodents with high IFA titers to HTNV (1:256 and 1:512) were positive by PCR for both the S and M segments of hantavirus.

We obtained the clinical histories of two fatal cases of HFRS in the Primorye region. The patients, who lived in villages 400 km and 600 km from Vladivostok, died 8-13 days after the onset of illness; gastrointestinal bleeding and acute renal failure were the causes of death. Serologic screening showed that both patients were positive for hantaviral antibodies. Antibody titers to HTNV and SEOV were apparently higher than to PUUV. We used lung, liver, kidney, spleen, and brain tissues of these HFRS patients for RT-PCR analysis; the lung and kidney tissues of patient no. 1 and the spleen tissue of patient no. 2 were positive for hantaviral M segment.

To examine the histopathologic changes in HFRS patients, we used light microscopy to examine sections of formalin-fixed lung, liver, kidney, spleen, and brain tissues from patient no. 2, who had died of acute renal failure (Figure 1). The kidney was the only tissue that showed the recognizable histopathologic changes. Salient changes included interstitial edema with mild infiltration of mononuclear cells (Figure 1, small arrow) and degeneration of renal tubules (Figure 1, large arrow) in the cortex (Figure 1, A). Although proteinaceous casts and exudates were observed in the lumina of renal tubules (Figure 1, arrowhead), there were no apparent glomerular changes. In addition, a prominent well-defined necrotic lesion (Figure 1, asterisk) was noted in the medulla (Figure 1, B). Viral antigens were not detected in these specimens by using monoclonal antibodies to HTNV.


The entire S segments of the viruses from two seropositive A. peninsulae were amplified and sequenced. We designated these segments as Solovey/AP61/1999 and Solovey/AP63/ 1999 based on the name of the village closest to the survey point, the rodent species from which the sample was taken, and the year in which the epizootiologic survey was done. We compared the coding regions of these sequences with those of other hantaviruses (Table 5). The S segments of the two Solovey sequences had 99.0% and 98.8% identities in nucleotide and amino acid sequences, respectively. Solovey sequences and Hantaan viruses had 78.2%-84.5% nucleotide sequence identity and 86.7%-93.3% amino acid sequence identity, regardless of their source or geographical origin. Lower nucleotide sequence identities were seen than in Solovey sequences and other viruses: DOBV (73.6%), SEOV (73.9%), and SNV (63.9%).

To explore the genetic diversity of hantaviruses identified in A. peninsulae in more detail, we sequenced the partial M segment of the G2 region (232 nt). We also sequenced the partial M segments of genetic lineages identified in the two HFRS patients from the Primorye region, designated as Primorye/H1/ 2000 and Primorye/H2/2000. The M segment of Solovey and Primorye sequences were compared with those of other hantaviruses (Table 6). Nucleotide sequence identities among these sequences were between 92.2% and 98.2%; amino acid sequence identities were almost identical (98.7%-100%). We also compared the M segment sequences of Solovey and Primorye with those of AMR genetic lineage, recently identified in HFRS patients and A. peninsulae in Far East Russia (10,11). The nucleotide and amino acid identities between Solovey, Primorye, and AMR lineages were 91.3%-98.3% and 93.5%-98.7%, respectively. The M segment sequences of Solovey, Primorye, and AMR lineages were compared with that of H8205, isolated from an HFRS patient in China. In this case, the nucleotide sequence identities were 93.5%-96.1%, and the amino acid sequence identities were 94.8%-100%. Lower nucleotide identities were seen with HTNV (78.8%-86.2%), SEOV (79.3%-81.4%), and DOBV (75.8%-77.1%). This high level of sequence identity among Solovey, Primorye, AMR, and H8205 sequences suggests that some patients acquired the infection from the Korean field mouse (A. peninsulae) in Far East Russia and China. Our results also suggest that this genetic lineage is widely distributed throughout east Asia.

The M segments of Solovey, Primorye, and AMR sequences formed a common phylogenetic lineage with high bootstrap support values, regardless of viral origin (Figure 2, A). Furthermore, H8205 shared a common lineage with Solovey and Primorye sequences. Another phylogenetic analysis, based on a different region of the M segment, showed that Chinese virus isolates (H8205, H3, H5, and B78) formed a distinct lineage within the Hantaan clade (Figure 2, B). The phylogenetic tree constructed for the S sequences (Figure 3) showed that Solovey sequences formed a single cluster, together with Maajil (a Korean isolate) and B78, in a common lineage with high bootstrap support values within the Hantaan clade.


To identify signature amino acids for each virus type, we compared the deduced partial amino acid sequences of their G2 regions using ClustalX multiple-sequence alignment (Figure 4). The presence of leucine or isoleucine at amino acid position (aa) 903 was unique to HTNV except for AMR lineage. The signature amino acids for SEOV were leucine at aa 918 and valine, isoleucine, and serine at aa 955-957. The signature amino acids for AMR lineage were methionine at aa 932 and aspartic acid at aa 967.



Each hantavirus serotype or genotype is generally associated with a specific rodent host, and various rodent species act as reservoir animals and sources of human infection. Since contact between rodents and humans occurs frequently during agricultural and forestry activities, most infections have been reported in rural areas. However, an urban epidemic of HFRS caused by SEOV has also been reported (26). A large number of rodent species may serve as reservoir animals for pathogenic hantaviruses. For example, few researchers suspected that P. maniculatus could transmit highly virulent hantavirus to humans until SNV was identified (27,28). Later studies showed that the other viral agents of HPS such as NYV, BCCV, BAYV, and ANDV, were carried by P. leucopus, S. hispidus (29), O. palustris (30), and O. longicaudatus (31), respectively. We emphasize the importance of discovering the characteristics of hantaviruses found in endemic areas and identifying the primary hosts.

Although Far East Russia has long been considered an HFRS-endemic area,, few reports describe the hantaviral sequences in this region, and information on reservoir animals carrying pathogenic hantaviruses is limited. Our studies therefore focused on determining the genetic characteristics of hantaviruses circulating in this geographic area. We identified A. peninsulae as the natural reservoir rodent for a hantavirus pathogenic for humans in Far East Russia. We

also identified hantavirus sequences designated as Solovey and Primorye in A. peninsulae and HFRS patients, respectively; genetic analysis showed that these sequences were very closely related to each other. This information and the pathological findings from the HFRS case in which Primorye sequence was identified strongly suggest that the virus of Solovey sequence is the causative agent of HFRS. The nucleotide sequence and phylogenetic analysis also showed that Solovey and Primorye sequences were most closely related to AMR and H8205 sequences from patients in Russia and China, but were clearly distinguishable from the prototype of Hantaan virus. Genetic and phylogenetic analysis indicated that Solovey and Primorye sequences were closely related to AMR, Maajil, H8205, and B78 sequences, viruses derived from distant areas. While Solovey sequences were identified in a suburb of Vladivostok and PRI sequences in two villages 400 km and 600 km from Vladivostok, the H8205 and B78 viruses were derived in China, and Maajil was isolated in Korea. A. peninsulae is distributed in the same region where g. agrarius is prevalent in Korea (PW Lee, pets. comm.). Recently, AMR sequences were found in both HFRS patients and A. peninsulae (11). We suggest that some of the viruses circulating in the area of this study cause severe HFRS and are carried by the same host species, g. peninsulae. Comparison of the deduced hantaviral amino acid sequences showed that aspartic acid and methionine represented signature amino acids for AMR genetic lineage, regardless of the region in which the virus was identified or its origin (Figure 4). These signature amino acids may be used to distinguish AMR genetic lineage from other hantaviruses. We conclude from our results that A. peninsulae carries a hantavirus that is pathogenic for humans. Since A. peninsulae is widely distributed in Far East Russia, China, Korea, and Japan, this hantavirus and associated cases of HFRS may also be widely distributed.

In the kidney tissue of one HFRS patient (no. 2) from Primorye region, we detected pathologic changes typical of severe HFRS caused by hantavirus infection (32-35). We also detected and sequenced the partial M segment in the spleen of the same patient. However, we could not detect the viral antigen in the kidney samples, possibly because of low levels of the virus in the kidneys of this patient. Nested PCR allowed the amplification of viral M segments from the spleen, but not from kidney, of this patient.

Through epizootiologic, clinical, pathologic, and sequencing studies, we identified a hantavirus carried by A. peninsulae as one of the causative agents of HFRS. We think that this information may be helpful in preventing human infections in East Asia. Controversy persists over whether A. peninsulae carries a distinct virus type or a subtype of HTNV. A similar question arises with Dobrava/Slovenia and Dobrava/Saaremaa, which are carried by A. flavicollis and A. agrarius, respectively. The S segment identities between Dobrava/Slovenia and Dobrava/Saaremaa (both obtained from GenBank for comparison purposes) were 87.8% (nucleotide) and 92.7% (amino acid). Similarly, the nucleotide and amino acid sequence identities of the S segments of Solovey sequences and HTN 76-118 were 82.7% and 92.2%, respectively. We suggest that Solovey sequences belong to a sublineage within the HTNV clade.
Table 1. Primers used for reverse transcription-polymerase chain
reaction and/or sequencing of S and M genome segments of

Gene Primer name Primer sequence (5'-3') Position

S segment M13 Fw ctggccgtcgttttac
 PEN 215 S Fw gaattgaaagacaattggc 215-233
 KPS3 (a) tc(a/c)agcatgaaggc 592-703
 PEN 780 SFw acagaggcaggcagctttag 780-799
 PEN 1042 S Fw gcaggatatgcggaatacaa 1042-1061
 HTNV 1390 S Fw attgcactattattatcagg 1390-1409
 HTNV Full S ttctgcagtagtagtag(t)a
 PEN 180 S Rv ttccctgtctgttaatgctc 180-199
 PEN 585 S Rv tgggcaaggacacatagaga 585-604
 PEN 946 Rv atgatggtgactcgatgtct 946-965
 PEN 1160 S Rv gttgtattcccattgactgt 1160-1179
 HTNV 1493 SRv cacccacaacggattaactg 1493-1512
 M13 Rv caggaaacagctatgac
M segment HS1 (a) ac(a/c)tgtca(c/a)tttgg 2636-2655
 HS2 (a) tcaca(g/a)gcctttattga(g/t)gt 3072-3091
 HS3 (a) t(tc)aggaa(ga)aaatg 2715-2736
 HS4 (a) acacc(a/t)gaaccccaggc(a/c)cc 3000-3019
 M13 Fw ctggccgtcgttttac
 M13 Rv caggaaacagctatgac

(a) Primers designed by Yashina et al.
Table 2. Hantavirus sequences used in this study (a)


Virus type Strain Source Region

HTNV SL/AP61/1999 Apodemus peninsulae Far East Russia
 SL/AP63/1999 A.peninsulae Far East Russia
 PRI/H1/2000 Human Primorye
 PRI/H2/2000 Human Primorye
 AMR/680 A.peninsulae Far East Russia
 AMR/1166 A.peninsulae Far East Russia
 AMR/1169 A.peninsulae Far East Russia
 AMR/4234 Human Far East Russia
 AMR/4309 Human Far East Russia
 AMR/4313 Human Far East Russia
 H8205 Human China
 HTNV261 -- China
 Z10 Human China
 Chen4 Human China
 Maaji1 A. agrarius Korea
 Maaji-2 Human Korea
 HTN 76-118 A. agrarius South Korea
 Q32 -- China
 HV114 A. agrarius China
 A9 A. agrarius China
 Hojo Human South Korea
 FE/7866 Human Far East Russia
 NC167 Niviventer confucianus China
 H3 Human China
 H5 Human China
 A3 A. agrarius China
 B78 Human China
 Q36 A. agrarius China
 Q7 A. agrarius China
 Q20 A. agrarius China
 Niongxia-A A. agrarius China
 Q10 A. agrarius China
 A16 A. agrarius China
 Q37 A. agrarius China
 Q33 A. agrarius China
 Bao9 A. agrarius China
 Jiang13 A. agrarius China
 Bao14 A. agrarius China
 Bao10 A. agrarius China
 Lee Human South Korea
 62HTNV -- --
 6B -- --
HTNV Vaccine -- --
 H2 -- North Korea
 HN26-L A. agrarius China
 Luyao Human China
 B659 Human China
 Hu Human China
 Q83 -- --
 B256 -- --
 Thailand Bandicota indica Thailand
 Topografov Lemmus sibericus Far East Russia
SEOV L99 Rattus losea China
 SR11 R. norvegicus Japan
 Gou3 R. rattus China
 NM39 R. norvegicus China
 HB55 Human China
 Wan Human China
 J12 Human China
 Henan94 R. norvegicus China
 Shanxi -- --
 HN71-L R. norvegicus China
 Guang199 -- --
 Beijing-Rn R. norvegicus China
 c3 Human China
 Hebei4 Cricetulus barabensis China
 SD227 -- China
 SD10 R. norvegicus China
 Hbei1 Human China
 Seoul R. norvegicus South Korea
 Tchoupitoulas R. norvegicus North America
 B-1 R. norvegicus Japan
 Girard Point R. norvegicus North America
DOBV DOB/SLOV A. flavicollis Slovenia
 DOB/SAA A. agrarius Estonia
SNV SNV Peromyscus maniculatus North America
PUUV PUU/Sot Clethrionomys glareolus Finland
 Kamiiso C. rufocanus Japan
KHAB Khabarovsk Microtis fortis Far East Russia

 Country Accession nos. References

Virus type Location M S

HTNV Solovey AB071185 AB071183 This report
 Solovey AB071186 AB071184 This report
 Cavalerovo AB071187 -- (b) This report
 Cavalerovo AB071188 -- This report
 Khabarvosk AF332571 -- 11
 Khabarvosk AF332569 -- 11
 Khabarvosk AF332570 -- 11
 Amursk AF172422 -- 10
 Amursk AF172423 -- 10
 Korphovsky AF172424 -- 10
 -- AB030232 -- --
 Heilongjiang -- AF252259 --
 Zhejiang AB027076 AB027108 12
 Anhui -- AB027101 12
 -- -- AF321094 Lee PW (c)
 -- -- AF321095 Lee PW (c)
 -- M14627 M14626 13,14
 Guizhou -- AB027097 12
 Hubei L08753 AB027110 12,15
 Jiangsu AF035831 -- 16
 -- D00376 -- 17
 Razdolnoye AF172439 -- 10
 Anhui AB027115 AB027523 12
 Hubei -- -- 18
 Heilongjiang -- -- 18
 Zhejiang AB027055 -- 12
 Shandong AB027056 AB027093 12
 Guizhou AB027057 AB027094 12
 Guizhou AB02058 AB027095 12
 Guizhou AB027059 AB027096 12
 Niongxia AB027060 -- 12
 Guizhou AB027062 AB027098 12
 Sanxi AB027063 AB027099 12
 Guizhou AB027064 AB027100 12
 Guizhou AB027065 AB027102 12
 Heilongjiang AB027066 AB027103 12
 Heilongjiang AB027067 AB027104 12
 Heilongjiang AB027068 AB027105 12
 Heilongjiang AB027069 AB027106 12
 -- D00377 -- 17
 -- AB027070 -- 12
 -- AB027071 -- 12
HTNV -- AB027072 -- 12
 -- AB027073 AB027107 12
 Hainan AB027074 -- 12
 Shandong -- AB027109 12
 Shandong S72339 -- 18
 Hubei AB027077 AB027111 12
 Guizhou AB027078 -- 12
 -- AB027079 AB027112 12
 -- L08756 -- --
 Siberia AJ011647 -- 9
SEOV Jiangxi AF035833 AF288299 --
 Sapporo M34882 M34881 19
 Zhejiang AB027521 AB027522 12
 Neimeng AB027080 -- 12
 Henan AF035832 -- 17
 Jiangsu AB027081 -- 12
 Jieling AB027082 -- 12
 Henan AB027083 -- 12
 -- AB027084 -- 12
 Hainan AB027085 -- 12
 -- AB027086 -- 12
 Beijing AB027087 -- 12
 Hebei AB027088 -- 12
 Hebei AB027090 -- 12
 Shangdong AB027091 -- 12
 Shangdong AB027092 -- 12
 Hubei S72343 -- 17
 -- S47716 -- 20
 -- U00473 -- 21
 -- X53861 -- 22
 -- U00464 -- --
DOBV -- L33685 L41916 23
 -- AJ009774 AJ009773 4
SNV -- L25783 L25784 24
PUUV -- X61034 -- 25
 Kamiiso AB011631 -- 8
KHAB Khabarvosk AJ011648 -- 9

(a) Abbreviations used: HTNV and HTN, Haantan virus; SL, Solovey;
PRI, Primorye; AMR, Amur; SEOV, Seoul virus; DOB and DOBV,
Dobrava-Belgrade virus; SLOV, Slovenia; SAA, Saarema; SNV, Sin
Nombre virus; PUUV, Puumala virus; and KHAB, Khabarovsk virus.

(b) --, not reported/not used in this study.

(c) Pers. comm.
Table 3. Serologic screening by immunofluorescent antibody assay for
Haantan virus and Puumala virus antibodies in rodents, Vladivostok,
Russia (a)

 by IFA (%)

Rodent species No. of sera tested HTNV PUUV

Apodemus peninsulae 70 4(5.7) 0
A. agrarius 39 1(2.5) 0
Clethrionomys rufocanus 8 1(12.5 1(12.5)
Microtus fortis 3 0 0
Tamias sibiricus 2 0 0
Total 122 6(4.9) 1(0.8)

(a) Abbreviations used: IFA, immunofluorescent antibody assay; HTNV,
Haantan virus; PUUV, Puumala virus.
Table 4. Haantan virus and Puumala virus antibody titers determined
by immunofluorescent antibody assay and polymerase chain reaction


Species Sample number HTNV PUUV PCR

Apodemus peninsulae 47 256 <16 - (b)
A. peninsulae 61 512 <16 + (c)
A. peninsulae 63 256 <16 +
A. peninsulae 74 64 <16 -
A. agrarius 10 32 <16 NA
Clethrionomys rufocanus 32 256 256 ND

(a) Abbreviations used: HTNV, Haantan virus; PUUV, Puumala virus;
IFA, immunofluorescent antibody assay; PCR, polymerase chain
reaction; NA, not available; ND, not done.

(b) -, negative.

(c) +, positive.
Table 5. Comparison of nucleotide (open reading frame) and amino
acid of S genome between those from Apodemus peninsulae and other
hantaviruses (a)

 Nucleotide and amino acid identities % (b)

 SL/AP61 SL/AP63 HTNV261 Z10 Chen4 Maaji-1

SL/AP61 99.0 84.5 83.5 83.4 82.9
SL/AP63 98.8 84.2 83.5 83.4 82.9
HTNV261 91.9 91.5 85.6 85.7 83.0
Z10 91.9 91.5 92.9 89.1 83.6
Chen4 93.0 92.5 93.2 96.2 82.8
Maaji-1 91.5 90.8 90.8 91.3 93.0
HTNV76-118 92.2 91.5 94.9 92.9 93.7 91.0
Q32 92.7 92.3 93.7 94.4 96.0 91.8
NC167 87.2 86.7 85.3 85.8 85.3 84.8
SR11 75.0 74.5 74.1 73.9 74.6 74.3
GOU3 75.7 75.5 75.0 74.8 76.2 74.3
Dob/Slo 76.4 76.4 76.8 75.7 77.6 76.6

 Nucleotide and amino acid identities % (b)

 HTNV 76-118 Q32 NC1167 SR11 GOU3 Dob/Slo

SL/AP61 82.7 82.3 78.3 73.7 73.7 72.9
SL/AP63 82.8 81.5 78.2 73.9 73.8 72.2
HTNV261 88.6 84.7 78.9 74.1 73.6 72.6
Z10 85.9 87.5 79.8 75.3 74.2 73.3
Chen4 85.8 90.3 78.7 73.2 74.2 73.4
Maaji-1 82.9 82.1 78.2 74.2 73.0 74.2
HTNV76-118 84.4 78.2 74.6 73.8 74.0
Q32 93.2 79.1 73.1 74.3 73.8
NC167 86.9 85.1 75.3 73.6 72.7
SR11 74.8 74.1 77.2 87.8 73.7
GOU3 74.8 76.7 76.7 91.5 73.1
Dob/Slo 75.5 77.2 76.0 73.1 73.1

(a) Values in bold show the close identities between the two
Solovey sequences. Abbreviations used: SL, Solovey; HTNV,
Haantan virus; Dob, Dobrova; Slo, Slovenia.

(b) Values above the diagonal and to the right show nucleotide
identities; those below the diagonal and to the left show
amino acid identities.
Table 6. Comparison of nucleotide (bases 2737-2969) (a) and amino
acid of M genome between those from Primorye patients, Apodemus
peninsulae, and other hantaviruses

 Nucleotide and amino
 acid identities % (b)

 SL/ SL/ AMR/ PRI/ PRI/ H8205
 AP61 AP63 1169 H1 H2

SL/AP61 (c) 99.5 97.8 96.1 98.2 94.8
SL/AP63 100 97.8 92.2 94.3 94.8
AMR/1169 94.8 94.8 96.5 98.7 95.6
PRI/H1 100 100 94.8 96.9 93.5
PRI/H2 98.7 98.7 93.5 98.7 94.8
H8205 100 100 94.8 100 98.7
AMR/4313 98.7 98.7 93.4 98.7 97.4 98.7
HV114 93.5 93.5 88.3 93.5 92.2 93.5
A9 93.5 93.5 88.3 93.5 92.2 93.5
HTNV76118 94.8 94.8 89.6 94.8 93.5 94.8
Hojo 94.8 94.8 89.6 94.8 93.5 94.8
FE 92.2 92.2 87.0 92.2 90.9 92.2
NC167 86.8 86.8 80.5 86.8 85.5 86.8
DOB/Slo 88.3 88.3 83.1 88.3 87.0 88.3
SR11 83.1 83.1 79.2 83.1 81.8 83.1
PUUV 53.2 53.2 53.2 53.2 51.9 53.2

 Nucleotide and amino
 acid identities % (b)

 AMR/ HV114 A9 HTNV Hojo FE
 4313 76-118

SL/AP61 (c) 94.3 86.2 85.7 84.4 82.7 82.7
SL/AP63 94.3 85.7 85.3 84.0 82.3 83.1
AMR/1169 95.6 86.6 86.2 84.9 83.1 81.4
PRI/H1 92.2 84.0 83.6 83.1 82.3 80.6
PRI/H2 94.3 85.7 85.3 84.0 82.3 81.4
H8205 91.3 83.6 83.1 85.3 84.9 80.6
AMR/4313 85.7 85.3 83.6 81.8 82.7
HV114 92.2 99.5 86.6 84.4 87.9
A9 92.2 98.9 86.2 84.0 87.5
HTNV76118 93.5 97.4 96.1 94.6 88.7
Hojo 93.5 97.4 96.1 100 87.9
FE 90.9 87.9 87.5 97.4 98.7
NC167 85.5 89.5 88.2 90.8 90.8 88.2
DOB/Slo 87.0 88.3 87.0 87.0 87.0 84.4
SR11 81.8 83.1 81.8 81.8 81.8 83.1
PUUV 51.9 62.9 62.5 51.9 61.6 53.2

 Nucleotide and amino
 acid identities % (b)

 Slo 11

SL/AP61 (c) 79.3 79.3 79.7 60.3
SL/AP63 78.8 80.1 81.4 60.7
AMR/1169 79.7 80.1 79.3 60.3
PRI/H1 79.3 78.8 79.3 60.3
PRI/H2 78.8 79.3 78.8 59.4
H8205 77.1 79.3 77.1 60.7
AMR/4313 78.0 78.0 78.8 59.9
HV114 78.4 75.8 83.1 51.9
A9 78.0 75.4 81.8 50.6
HTNV76118 79.7 78.4 76.7 59.9
Hojo 78.0 78.8 76.7 51.5
FE 75.8 73.7 78.4 59.9
NC167 75.4 77.5 49.3
DOB/Slo 81.6 75.0 59.9
SR11 80.3 80.5 56.0
PUUV 61.6 49.4 61.2

(a) Based on Haantan 76-118.

(b) Values above the diagonal and the right show nucleotide
identities; those below the diagonal and to the left show
amino acid identities.

(c) Values in bold show the close identities between those
sequences. Abbreviations used: SL, Solovey; AMR, Amur; PRI,
Primorye; HTNV, Haantan virus; FE, Far East virus; DOB,
Dobrova; Slo, Slovenia; PUUV, Puumala virus


We thank Kimiyuki Tsuchiya of Miyazaki Medical College and Masahiro Iwasa and Hitoshi Suzuki of Hokkaido University for providing rodent information.

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sport, Japan (projects 1357529 and 13660311) and by Health Science Grants for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor, and Welfare, Japan.


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Ms. Lokugamage is a doctoral candidate studying the epidemiology of hantaviruses at the Laboratory of Public Health, Graduate School of Veterinary Medicine of Hokkaido University, Sapporo, Japan. She has also served as a faculty lecturer at the School of Veterinary Medicine, University of Peradeniya, Sri Lanka.

Address for correspondence: Hiroaki Kariwa, Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan; fax: 81-11-706-5213; e-mail:

Kumari Lokugamage, * Hiroaki Kariwa, * Daisuke Hayasaka, * Bai Zhong Cui, * Takuya Iwasaki, ([dagger]) Nandadeva Lokugamage, * Leonid I. Ivanov, ([double dagger]) Vladimir I. Volkov, ([double dagger]) Vladimir A. Demenev, ([section]) Raisa Slonova, ([paragraph]) Galina Kompanets, ([paragraph]) Tatyana Kushnaryova, ([paragraph)] Takeshi Kurata, ([dagger]) Kenji Maeda, * Koichi Araki, * Tetsuya Mizutani, * Kumiko Yoshimatsu, (#) Jiro Arikawa, (#) and Ikuo Takashima *

* Hokkaido University, Sapporo, Japan; ([dagger]) National Institute of Infectious Diseases, Tokyo, Japan; ([double dagger]) Plague Control Station, Khabarovsk, Russia; ([section]) Far Eastern Medical Association, Khabarovsk, Russia; ([paragraph]) Russian Academy of Medical Sciences, Vladivostok, Russia; and (#) Hokkaido University School of Medicine, Sapporo, Japan
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Author:Takashima, Ikuo
Publication:Emerging Infectious Diseases
Article Type:Statistical Data Included
Geographic Code:4EXRU
Date:Aug 1, 2002
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