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    Localization of rodent trapping sites. ▴, trapping site; ▴, site with positive samples; ♦, Haidian district, where the first patient in Beijing was found; *, Beijing West Railway Station, origin of a Rattus norvegicus from which the first genetic evidence of SEOV infection in Beijing was observed. S1, Yanqing, N 40°33.455′, E 115°54.118′; Forest, S2, Changping, N 40°13.507′, E 116°10.674′; Exurb, S3, Haidiant, N 40°03.672′, E 115°57.682′; Suburb, S4, Haidian, N 39°56.578′, E 116°15.517′; Outskirt, S5, Fangshan, N39°38.964′, E 115°54.714′; Exurb, S6, Fengtai, N39°49.290′E 116°20.125′; Outskirt, S7, Fengtai, N 39°47.92′, E 116°22.96′; Outskirt, S8, Dongcheng, N 39°56.277′, E 116°25.254′; Urban, S9, Huairou, N 40°18.528′, E 116°37.71′; Forest, S10, Mengtougou, N 39°56.233′, E 115°30.196′; Forest, S11, Shunyi, N 39°56.568′, E 116°37.67′; Outskirt. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Phylogenetic tree of hantaviruses based on 356-nt M-segment sequences. The tree was calculated by neighbor-joining method using PHYLIP software. Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. HTNV, Hantaan virus; SEOV, Seoul virus; THAIV, Thailand virus; strain Thai749; NYV, New York virus, strain NY-1; BAYV, Bayou virus; TULV, Tula virus; PUUV, Puumala virus, strain puu/kazan.

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    Phylogenetic tree based on 350-nt partial S segment sequences (595–944 bp) of BJFT01, BJHD01, and some previous characterized SEOV and HTNV. The tree was calculated by the neighbor-joining method using PHYLIP software (version 3.63). Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. HTNV, Hantaan virus; SEOV, Seoul virus; THAIV, Thailand virus, strain Thai749.

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Prevalence and Genetic Diversities of Hantaviruses in Rodents in Beijing, China

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  • 1 Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, Beijing, China; Haidian Center for Disease Control and Prevention of Beijing, Beijing, China; Dong Cheng Center for Disease Control and Prevention of Beijing, Beijing, China; Department of Public Health, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

A total of 835 rodents captured in Beijing, China, were tested for hantavirus infection. Fifty-five (6.6%) were positive for viral RNA when lung tissue samples were examined by reverse transcriptase-polymerase chain reaction. Of 666 sera collected from the above rodents, 50 (7.5%) were positive for IgG antibody by ELISA. Among the 50 seropositive rodents, 37 were positive for viral RNA. In addition, five rodents were positive for viral RNA but negative for IgG antibody. The infection rates among study sites (χ2 = 28.93, df = 8, P = 0.001) and habitats (χ2 = 22.88,df = 7, P = 0.02) were significantly different. The sequences of partial M-segment of hantaviruses detected in 11 representative rodents had 0.1–8.2% divergence. Phylogenetic analysis showed that our hantavirus sequences fell into three different lineages regardless of geographical origin or rodent species. A strain detected from a trading center of agricultural products, which might be imported from other provinces, was genetically different from other strains of Beijing.

INTRODUCTION

Hantaviruses, the causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are the rodent-borne tri-segmented negative sense single-stranded RNA viruses belonging to the family Bunyaviridae. The three segments are designated as L, M, and S, which encode the L protein, glycoprotein envelope precursor, and the nucleocapsid protein, respectively.1 Recently, the International Committee on Taxonomy confirmed 22 species of hantaviruses,2 each of which is predominately associated with one certain or a few related rodents species as its primary natural reservoirs.3 These hantaviruses, classified into arvicolinae, sigmodotoninae, and Murinae-associated viruses, have been proposed to co-evolve with the distinct rodent species that carry them.3 Humans usually acquire hantavirus infection by contact or inhalation of aerosols and secretions from infected rodents. Hantavirus infections including HFRS and HPS have become a significant global health concern.4

In China, Hantaan virus (HTNV) and Seoul virus (SEOV) are the causative agents of HFRS, which cause a case fatality ~10% and 1%, respectively. In 2003, Puumala-like hantavirus was detected in bank voles (Clethrionomys rufocanus) from northeastern China,5 where the first HFRS outbreak was recorded (Heilongjiang province, about N 45°, E 126°) in the 1930s. The disease is currently one of the significant public health problems in mainland China. The reported HFRS cases account for nearly 90% of cases recorded in the world.68 The vaccination program has been initiated in the endemic two decades ago and has effectively prevented transmission from animal hosts to humans. However, the overall number of patients in the country has not been decreased, because new foci have continuously emerged in recent years, especially in urban areas.8

In Beijing, HFRS was first reported in 1986, when a patient in the Haidian district was diagnosed. Thereafter, a few sporadic cases occurred from 1987 to 1995.9 In 1996, the first genetic evidence for SEOV infection was discovered in a brow home rat (Rattus norvegicus) collected from a container at the railway station.10 Since then, the incidence of HFRS has increased annually.

Previous studies detected hantavirus-RNA in domestic rodents in some areas of Beijing.9 However, little additional information is available concerning hantavirus infection of rodents and hantavirus diversity among reservoirs in Beijing. Lack of such knowledge limits our understanding of the ecology, epidemiology, and potential threats of this pathogen to humans. In this study, 11 study sites were selected from the downtown, outskirts, suburbs, exurbs, and countrysides of Beijing to investigate the prevalence of hantavirus infection in rodents and to characterize the agents using molecular biologic approaches.

MATERIALS AND METHODS

Survey sites and sample collection.

A total of 11 study sites were selected, and their geographic locations and habitat characteristics are indicated in Figure 1. Four sites (S1, S5, S9, and S10) were in the exurbs, three sites (S2, S3, and S11) were in the suburbs, three sites (S4, S6, and S7) were in the outskirts connected to the suburbs, and one site (S8) was in the downtown of the Dongcheng district. Rodents were captured at each site with snap-traps from September 2002 to May 2005. In two study sites (S2 and S6), rodents were captured monthly for a year. The others were selected for the cross-sectional surveys in spring or autumn. A total of 100–150 traps per patch with fresh peanuts as baits were placed for two to three consecutive nights. After identification of species and sex, blood samples were collected, and lung tissues were removed from the captured rodents and stored in liquid nitrogen until tested. For unidentified species in the field, the craniums were brought to the laboratory for further identification.

Serologic test.

Sera were tested for specific IgG antibodies against respective HTNV (strain Z10) and SEOV (strain Z37) using enzyme-linked immunosorbent assays (ELISA). Sera samples diluted in TSTA (50 mmol/L Tris pH = 7, 142 mmol/L NaCl, 0.05% Tween 20, and 2% calf albumin Fraction V) were incubated for 45 minutes, followed by horseradish peroxidase–labeled goat anti-mouse IgG (Dingguo, Shanghai, China). TMB substrate was added, and the reaction was stopped with 1 N H2SO4. Plates were washed three times between each step with washing buffer (phosphate-buffered saline with 0.1% Tween 20) in ImmunoWash (1575; Bio-Rad, Hercules, CA). For all sera, the optical density (OD) was determined at 450 nm. The positive controls used in the serologic assays were sera from immunized laboratory BALB/c mice with the inactivated gerbi kidney cell vaccine, and the negative controls were sera from non-immunized BALB/c mice. The cut-off value of antibody was defined as two times the negative control absorbance.

RNA extraction and nested reverse transcriptase-polymerase chain reaction.

Total RNA was extracted from lung tissues of rodents with TRIZOL reagent (Invitrogen, Life Technologies, Rockville, MD) following the manufacturer’s recommendations, and reverse transcription (RT) was implemented using AMVase (Promega, Madison, WI) and random hexamers as specified by the manufacturer. A nested polymerase chain reaction (PCR) was performed to amplify the partial M-segment of the hantavirus gene. The primers HTV-MFO1910 and HTV-MRO2373, previously described by Wang and others,11 were applied for the primary amplification. In nested PCR, SEOV-MF1936 and SEOV-MR2318 and HMF1958 and HMR2331 were used for specific amplification of SEOV and HTNV. To further identify the positive samples with variation in the M-segment sequence, another nested PCR was performed to amplify the 400-bp partial S-segment using primers KS1, KS2, KS3, and KS4, described previously by Kim and others12 and Pan and Bai.13 In the primary amplification, a 30-μL reaction mixture contained 1 U of Taq polymerase (Sangon Corp., Shanghai, China), first-round primers (MRO/MFO for M segment and KS1/KS4 for S segment, 10 pmol each), 3 nmol of each dNTP, and 3 μL of the cDNA template mixture. For the nested PCR reaction, 1/10 of the primary amplification product (3 μL) was added to another 30 μL of reaction mixtures containing identical components except for the second-round primers (SEOV-MF/SEOV-MR, and HMR/HMF for M segment and KS2/KS3 for S segment). Each amplification was carried out under the following protocol: an initial denaturation at 95°C for 3 minutes; followed by 35 cycles at 95°C for 20 seconds, 54°C for 30 seconds, and 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. The PCR products were separated by electrophoresis on a 1.2% agarose gel stained with ethidium bromide and visualized under UV light. To avoid contamination, DNA extraction, the reagent setup, amplification, and agarose gel electrophoresis were performed in separate rooms. In parallel with each amplification, a positive control (a plasmid containing M cDNA gene of hantavirus agent, kindly provided by Professor Zhongduo Li, Department of Virology at our institute) and a negative control (distilled water) were included.

Sequencing and phylogenetic analyses.

The specific amplicons were purified using a QIAquick Gel Ex kit (BioDeu Corp., China). The purified products were sequenced with automated DNA sequence (ABI PRISM 373; Perkin-Elmer, Norwalk, CT). The sequences obtained in this study were compared with the previously published sequences deposited in GenBank using the BLAST program from the National Center for Biotechnology Information website. The representative strains were included in multiple sequence alignment with Clustal X, version 1.83 (http://www.bio-soft.net/fomat.html). Pairwise genetic distances were calculated with MegAlign module of Lasergene software package (DNASTAR, Madison, WI).14 Phylogenetic analysis was performed with PHYLIP software package (version 3.63).15,16 The support value for tree topology was estimated by bootstrap analysis with 1,000 pseudo-replicate datasets generated by the SEQBOOT program. The Jukes-Cantor distances matrix was calculated for phylogenetic inference with DNADIST program, whereas a set of 1,000 phylogenetic trees based on the matrix was calculated using NEIGHBOR program. The CONSENCE program was used to create the consensus tree. The tree was imported into the TreeView program (version 1.52) for text editing and printing purposes. Ratios of the non-synonymous (amino acid-changing; dN) to the synonymous (silent; dS) substitutions in the nucleotide sequences were calculated to assess the evolution process and the selective pressure at the protein level.

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this study were deposited into Gen-Bank with accession numbers listed in Table 1.

Statistical analyses.

The differences in proportions were analyzed using the χ2 test or Fisher exact test. P < 0.05 was considered significant.

RESULTS

A total of 835 rodents, belonging to seven species of three families, were captured at the 11 study sites. Fifty-five rodents were positive for hantavirus infection by RT-PCR of partial M-segment, with an overall positive rate of 6.6%. The difference in infection rates among species was statistically significant (χ2 = 14.843, df = 6, P = 0.049). Only two dominant species, R. norvegicus and Mus musculus, were found to be infected, with positive rates of 8.9% (52/590) and 1.6% (3/186), respectively. The infection rates among various habitats were significantly different (χ2 = 22.88, df = 7, P = 0.02; Table 2). Serum samples were collected from 666 of 835 rodents and examined for IgG antibodies by ELISA. Fifty were positive for a specific antibody against SEOV, with a seroprevalence of 7.5%. Among the 50 seropositive rodents, 37 were positive in the above RT-PCR assay. In addition, five rodents were positive for viral RNA by RT-PCR but negative for IgG antibody by ELISA (Table 3). The difference in positive rates between the two assays was not statistically significant (McNemar test, P = 0.096). Concordance of the results was high between these two assays (measure of agreement, κ = 0.79, P < 0.001; Table 3). The results of both of the assays by districts are summarized in Table 4. The difference in positive rates of viral RNA among districts was statistically significant (χ2 = 28.93, df = 8, P = 0.001), as was the sero-positive rate (χ = 18.84, P = 0.01).

The 356-nt fragment corresponding to position 1958–2313 of the M segment gene of the positive specimens was sequenced. Considering sequences of some positive samples from the same study site were nearly identical, 11 representative sequences of the positive samples from S1, S2, S3, S4, S6, S8, and S11 (Figure 1) were used for alignment and phylogenetic analysis. The identities of the nucleotide sequence ranged from 91.8% to 99.9%. All virus sequences except that of the BJFT01 strain were closely related to Z37 (SEOV strain) isolated from the Zhejiang province. BJFT01 detected from R. norvegicus in a large trade center of agricultural products in Fengtai district was divergent from the others of this study but close to K24-e7 SEOV from the Zhejiang province with 96.0% identity.

The phylogenetic tree based on the alignment of partial M segment showed that, except for BJFT01, all other viruses detected in this study clustered in two closely related sub-lineages (designated as clusters 3 and 4) within cluster 2, regardless of either geographical origin or rodent species (Figure 2). It was noticed that dc501, dc1105, and cp211, which were first recovered in M. musculus in Beijing, were distributed in different sub-lineages. BJFT01, which was different from other Beijing strains, belonged to another distinct lineage, together with K24-e7, L99, HB55, and R22 SEOV, respectively, from Zhejiang, Jiangxi, and Henan provinces (Figure 2).

Table 5 shows the single nucleotide polymorphism of the 356-nt partial M segment of SEOV. BJFT01 had mutations in 15 loci in comparison with master sequences, and most mutations were different compared with other samples in this study, with only one non-synonymous change that caused valine to substitute for alanine at position 44, with a dN /dS value of 0.067. There were several A → G (5/15) and T → C (4/15) transitional mutations. For the samples from R. norvegicus in Changping county (cp1107 and cp1116), two unique variations (at positions 73 and 116) caused the changes of amino acids from valine to isoleucine and from serine to tyrosine, respectively. As for HD403, there were two unique mutations at positions 259 and 284, which caused the non-synonymous changes from arginine to glycine and from histidine to arginine. YQK2 had three non-synonymous changes: methionine to lysine, asparagines to aspartic, and cysteine to tryptophan. The dc1208 also had three unique variations of eight nucleotide mutations and caused the amino change from asparagine to tyrosine or asparticacid and from proline to serine. Among the sequences from M. musculus, all nucleotide sequence mutations were synonymous changes, and only dc1105 had two unique variations.

A further comparison of the partial S segment showed that BJFT01 and BJHD01 had an identity of 96.4% for nucleotides and 99.2% for amino acid sequences. The phylogenetic tree based on alignment of the 350-nt partial S segment sequences showed that BJFT01 was most closely related to the 80–39 strain detected in R. norvegicus from South Korea and was in the same clade with BJHD01 from the same rodent species in Haidian, Beijing, but genetically distinct from K24-e7 and other SEOVs (Figure 3). The unique presence of ala-nine at position (aa)289 was special for BJFT01 and distinct from Z37, L99, R22, K24-e7, and BJHD01, which present serine in the corresponding position.

DISCUSSION

The previous surveillance data of hantavirus infection among animals in Beijing during 1982–1990 showed that the hantavirus-reactive antibody in M. musculus (1.62%, 10/616) was higher than in R. norvegicus (0.41%, 6/1434).17 Since the first evidence of the virus was detected in R. norvegicus in 1996, the prevalence is increasingly appreciated, and more patients with mild clinic symptoms have been reported in recent years in Beijing. This study presented the prevalence of SEOV in rodents in Beijing. R. norvegicus was identified as the predominant rodent species and main carrier of hantavirus. In our previous work, a SEOV strain was isolated from R. norvegicus captured in Haidian.18 We also found that male R. norvegicus in the reproduction periods and those with wounds were more likely to be infected with hantavirus than others.19 This study found that hantavirus infection rates were higher in the suburbs than in downtown areas. Natural foci were mainly located in (peri-)domestic habitats where the ecologic and hygienic conditions were possibly favorable for R. norvegicus. In the field, no positive rodent was detected. There is a wide distribution of infected R. norvegicus according to a previous report9 and the findings of this study. The wide distribution of infected R. norvegicus is probably the important factor for increased HFRS incidence in this region.

Three partial M segments of hantaviruses were firstly recovered from M. musculus in Beijing. The positive M. musculus were captured from the outskirts (cp211), downtown areas (dc1105), and a railway station (dc501). The sequences of hantavirus in M. musculus had no significant difference with those from R. norvegicu (Figure 2). The nucleotide sequences of dc501 also was highly similar (96.3%) to those of Jakarta 37 and France 90 isolated from R. norvegicus in Indonesia and France, respectively.20,21 These findings indicate that hantaviruses carried by the two rodent species did not show host-related specificity. The sequence heterogeneity observed in this study might be caused by different geographic origins of the rodent carriers rather than the host species itself. The SEOV infection of M. musculus probably originates from multiple spill-over infections rather than a host switch event, just as Puumala virus (PUUV) and Dobrava spill-over infection in M. musculus.22,23 However, it should be pointed out that there was a high density of M. musculus in the free market at downtown, and both the overall hantavirus seroprevalence and PCR-positive rate of M. musculus were higher than those of R. norvegicus in urban areas (Table 4). Further study is needed to understand the dynamics of the M. musculus populations. Because M. musculus usually adapts to a variety of environments, the density of M. musculus is often higher than that of R. norvegicus in urban areas. The findings of this study suggest that M. musculus might play a potential role in HFRS incidence of Beijing.

Most SEOV strains in the world showed unclear clustering of genetic variants. This is likely to reflect the dominant host of SEOV, R. norvegicus, which often migrates across far distances by ships and vehicles and is much more mobile than the hosts of other hantaviruses. In Europe, after a few reports of hantavirus infection in laboratory workers,24 the first confirmed SEOV in wild R. norvegicus, France 90,21 had a high similarity to the Jakartal 37 from Indonesia.20 Our study indicates that at least three distinct lineages of SEOV are maintained in Beijing. The SEOVs from the same rodent species (R. norvegicus), regardless of the geographic origin, were similar. Little difference was observed among SEOV sequences from different rodent species at the same study site. However, the partial M segment of BJFT01 was quite different from the other strains of Beijing and more closely related to the strains from places distant from southern China. BJFT01 was detected from R. norvegicus captured in a free market of Fengtai district to which the agricultural products from different places around China were transported for trading. In addition, our previous studies showed that randomly amplified polymorphizm DNA (RAPD) maker of the rodent host of BJFT01 was different from those of other R. norvegicus populations captured in other study sites.25 These findings suggest that BJFT01 is possibly imported through the infected rodents carried by vehicles from other provinces.

The frequent of A → G and T → C transitional mutations and a few amino acid mutations of BJFT01 provided an indication of hantavirus persistence in rodent hosts over time as described by Feuer and others for Sin Nombre virus in Peromyscus maniculatus.26 It has been reported that similar mutations can caused decreased virulence of some HTNV strains.27 These mutations are possibly associated with virus virulence and persistence in rodent hosts. BJFT01 may be a special adventitious virus strain that is adapting to the hosts in the novel endemic region.

In conclusion, a comparison of our findings to previous surveillance data suggests that there is a higher and perhaps increased hantavirus prevalence in the study sites. The increase may be responsible for the increased occurrence of human cases. The predominant host, R. norvegicus, plays a vital role, and M. musculus plays a potential role in HFRS prevalence in Beijing.

Table 1

Novel and reference hantavirus strains used in the study

Accession no. in GenBank
HantavirusStrainHost origin*Geographic origin (site)†MS
* Strain in this study (except BJHD01), together with Hebei4, HB55, and SD227, was directly from rodent or patients. The other sequences were obtained from culture-adapted viruses.
† Study sites shown in Figure 1.
‡ Novel sequences identified in this study.
HTNVZ10HFRS patientZhejiang ProvinceAF184987NC006433
A9Apodemus agrariusJiangsu ProvinceAF035831AF329390
SEOVSR-11Rattus norvegicusJapan, SapporoM34882M34881
Gou3R. rattusZhejiang ProvinceAB027521AF184988
Z37R. norvegicusZhejiang ProvinceAF190119AF187082
80-39R. norvegicusSouth KoreaS47716AY273791
K24-e7R. norvegicusZhejiang ProvinceAF288653AF288653
Hebei4R. norvegicusHebei ProvinceAB027089
HB55HFRS patientHenan ProvinceAF035832
SD227R. norvegicusShandong ProvinceAB027091
L99R. loseaJiangxi ProvinceAF035833AF488708
R22R. norvegicusHenan ProvinceS68035AF488707
IR461‡Laboratory-acquired infectionUnited KingdomAF458104AF329388
BjHD01‡R. norvegicusBeijing, Haidian (S4)AY627049AY627049
BjFT01‡R. norvegicusBeijing, Fengtai (S6)AY645717DQ519033
YQ-K2‡R. norvegicusBeijing, Yanqing (S1)AY725998
CP1107‡R. norvegicusBeijing, Changping (S2)AY725991
CP1116‡R. norvegicusBeijing, Changping (S2)AY725992
CP211‡Mus musculusBeijing, Changping (S2)AY725993
HD403‡R. norvegicusBeijing, Haidian (S3)AY725997
dc1208‡R. norvegicusBeijing, Dongcheng (S8)AY725995
dc1105‡Mus musculusBeijing, Dongcheng (S8)DQ246440
dc501‡M. musculusBeijing, Dongcheng (S8)AY725996
shy17‡R. norvegicusBeijing, Shunyi (S11)DQ246441
THAIVThai 749Bandicota indicaThailandL08756U00471
NYVNY-1Peromyscus leucopusNorth AmericaU36802
BAYVBayouOryzomys palustrisSoutheast AmericaL36930
TULVTulaMicrotus arvalisRussiaZ69993
PUUVKazanClethrionomys glareolusRussiaZ84205
Table 2

Results of RT-PCR for hantavirus infection of rodents by habitats

Number positive/number tested
HabitatsR. norvegicusM. musculusA. peninsulaeA. agrariusOther species*Total
* Includes Niviventer confucianus, Sciurotamias davidianus, and Cricetus triton.
† Includes cultivation, shrub, and forest areas.
‡ Includes a railway station, airport, and a large trade center of agricultural products.
Breeding sites19/1501/3420/184
Refuse dumps10/730/240/310/100
Construction sites6/596/59
Domestic6/1860/260/20/16/215
Vegetable gardens9/720/199/91
Field†0/110/280/40/140/57
Food markets0/221/561/78
Centers of communication and transportations‡2/281/160/73/51
Total52/5903/1860/280/160/1555/835
Table 3

Comparison of RT-PCR and ELISA for hantavirus infection in rodents

RT-PCR
+-Total
McNemar-Test, P = 0.096; measure of agreement, κ = 0.79, P < 0.001.
+371350
ELISA-5611616
Total42624666
Table 4

Results of RT-PCR and ELISA for hantavirus infection of rodents by districts

IgG antibody (no. positive/no. tested)RT-PCR (no. positive/no. tested)
District/countyR. norvegicusM. musclusOther species*TotalR. norvegicusM. musclusOther species*Total
* Includes Apodemus peninsulae, A. agrarius, Niviventer confucianus, Sciurotamias davidianus, and Cricetus triton.
Changping32/2191/410/533/26532/2421/430/533/290
Dongcheng6/1714/610/010/2321/1712/610/03/232
Fengtai2/290/90/12/391/290/90/11/39
Yanqing0/40/20/220/281/40/20/221/28
Haidian4/290/160/04/4516/1360/360/016/172
Fangshan0/41/190/31/260/40/190/30/26
Mentougou0/30/60/70/160/30/60/70/16
Huairou0/00/10/140/150/00/10/140/15
Shunyi0/00/00/00/01/10/90/71/17
Total44/4596/1550/5250/66652/5903/1860/5955/835
Table 5

Comparison of partial M gene sequences of hantaviruses from Beijing rodents with known representative sequences of different origins

Nucleotide difference at position*
StrainOrigin4448515861738996102116123126129138142147150156160169172201222231
Gou3Rattus rattusAG
L99Rattus loseaGGC
HN71R. norvegicusAGCCGC
K24-e7R. norvegicusGCCGCC
BjFT01†R. norvegicusCAGCCGGC
CP1107†R. norvegicusA
CP1116†R. norvegicusA
HD403†R. norvegicus
YQ-K2†R. norvegicusAAGG
dc1208†R. norvegicusATGGT
Shy17†R. norvegicusGA
BjHD01†R. norvegicus
Z37R. norvegicusC
Jakart37R. norvegicusCCTCCC
SD227R. norvegicusA
Hebei4R. norvegicusC
CP211†Mus musculus
dc1105†M. musculusGA
dc501†M. musculusA
MasterTTCAAGTATCATTTAAATAAGATT
Nucleotide difference at position*
Strain234237243259261264265267270273276282284288291297301309315316324327TotaldN /dS
The nucleotide bases in italic are unique in corresponding strain.
* The nucleotide in the blank is the same as that of the master in each corresponding position.
† The novel sequence of this study.
‡ The non-synonymous changes.
Gou3AG
L99AG
HN71CGGTT
K24-e7CGAGCTT
BjFT01†AGGTATC150.067
CP1107†GCGC30.67
CP1116†CGC30.67
HD403†GCGC40.50
YQ-K2†GGC70.43
dc1208†TTT80.37
Shy17†T30.0
BjHD01†CC20.0
Z37GGCTCC
Jakart37TT
SD227
Hebei4CT
CP211†CC20.0
dc1105†CGC50.0
dc501†T20.0
MasterTTAAAGATAACTACTCACTCCT54
Figure 1.
Figure 1.

Localization of rodent trapping sites. ▴, trapping site; ▴, site with positive samples; ♦, Haidian district, where the first patient in Beijing was found; *, Beijing West Railway Station, origin of a Rattus norvegicus from which the first genetic evidence of SEOV infection in Beijing was observed. S1, Yanqing, N 40°33.455′, E 115°54.118′; Forest, S2, Changping, N 40°13.507′, E 116°10.674′; Exurb, S3, Haidiant, N 40°03.672′, E 115°57.682′; Suburb, S4, Haidian, N 39°56.578′, E 116°15.517′; Outskirt, S5, Fangshan, N39°38.964′, E 115°54.714′; Exurb, S6, Fengtai, N39°49.290′E 116°20.125′; Outskirt, S7, Fengtai, N 39°47.92′, E 116°22.96′; Outskirt, S8, Dongcheng, N 39°56.277′, E 116°25.254′; Urban, S9, Huairou, N 40°18.528′, E 116°37.71′; Forest, S10, Mengtougou, N 39°56.233′, E 115°30.196′; Forest, S11, Shunyi, N 39°56.568′, E 116°37.67′; Outskirt. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.98

Figure 2.
Figure 2.

Phylogenetic tree of hantaviruses based on 356-nt M-segment sequences. The tree was calculated by neighbor-joining method using PHYLIP software. Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. HTNV, Hantaan virus; SEOV, Seoul virus; THAIV, Thailand virus; strain Thai749; NYV, New York virus, strain NY-1; BAYV, Bayou virus; TULV, Tula virus; PUUV, Puumala virus, strain puu/kazan.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.98

Figure 3.
Figure 3.

Phylogenetic tree based on 350-nt partial S segment sequences (595–944 bp) of BJFT01, BJHD01, and some previous characterized SEOV and HTNV. The tree was calculated by the neighbor-joining method using PHYLIP software (version 3.63). Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. HTNV, Hantaan virus; SEOV, Seoul virus; THAIV, Thailand virus, strain Thai749.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.98

*

Address correspondence to Wu-Chun Cao, 20 Dong-Da Street, Fengtai District, Beijing 10071, China. E-mail: caowc@nic.bmi.ac.cn

Authors’ addresses: Jia-Fu Jiang, Shu-Qing Zuo, Wen-Yi Zhang, Xiao-Ming Wu, Fang Tang, Wen-Juan Zhao, Pan-He Zhang, and Wu-Chun Cao, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, 20 Dong-Da Street, Fengtai District, Beijing 100071, China. Sake J De Vlas, Department of Public Health, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Zhe Dun, Haidian Center for Disease Control and Prevention, Beijing 100082, China. Ri-Ming Wang, Dong Cheng Center for Disease Control and Prevention, Beijing 100054, China.

Financial support: This work was funded by Nature Science Foundation of China (30590374) and the Beijing Nature Science Foundation (7021004).

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Author Notes

Reprint requests: Wu-Chun Cao, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, 20 Dong-Da-Jie Street, Fengtai District, Beijing 100071, China, Telephone: 86-10-63896082, Fax: 86-10-63896082, E-mail: caowc@nic.bmi.ac.cn or caowc2000@yahoo.com.cn.
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