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Genetic analysis of hantaviruses and their rodent hosts in central-south China
Virus Research 163 (2012) 439–447
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Genetic analysis of hantaviruses and their rodent hosts in central-south China Jing Liu a,1, Dong-Ying Liu a,b,1, Wen Chen a,1, Jin-Lin Li a, Fan Luo a, Qing Li c, Jia-Xin Ling a, Yuan-Yuan Liu a, Hai-Rong Xiong a , Xiao-Hua Ding a , Wei Hou a , Yun Zhang d , Shi-Yue Li e , Jie Wang d , Zhan-Qiu Yang a,∗ a
State Key Laboratory of Virology, Institute of Medical Virology, School of Medicine, Wuhan University, Wuhan 430071, PR China Department of Microbiology, School of Medicine, Wuhan University, Wuhan 430071, PR China Department of Basic Medicine, Xiamen University Medical College, Xiamen 361005, PR China d Institute of Military Medical Sciences, Nanjing Command, Nanjing 210002, PR China e School of Public Health, Wuhan University, Wuhan 430071, PR China b c
a r t i c l e
i n f o
Article history: Received 26 August 2011 Received in revised form 2 November 2011 Accepted 4 November 2011 Available online 17 November 2011 Keywords: Hantavirus Phylogeny Rodents
a b s t r a c t Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped viruses with a negative-sense RNA genome organized into three segments: L (large), M (medium) and S (small) (Plyusnin, 2002). The L segment encodes an RNA-dependent RNA polymerase and shows the highest homology among sequences (Khaiboullina et al., 2005). The M segment encodes two envelope glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid (N) protein (Plyusnin et al., 1996). Variations in the M and S segments may alter the antigenicity and virulence of hantaviruses (Zhang et al., 2007). Two human diseases are associated with hantavirus infections: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas (Jonsson et al., 2010). Unlike the rest of the family Bunyaviridae, hantaviruses are not arthropod-borne but rather are transmitted by rodents and insectivores (Plyusnin et al., 1996). The first hantavirus described,
∗ Corresponding author at: State Key Laboratory of Virology, Institute of Medical Virology, School of Medicine, Wuhan University, 185 Donghu Road, Wuhan 430071, PR China. Tel.: +86 27 68759136; fax: +86 27 68758766. E-mail addresses: [email protected], [email protected] (Z.-Q. Yang). 1 These authors contributed equally to this article.
Hantaan virus (HTNV), was isolated from striped field mouse (Apodemus agrarius) in 1976 (Lee and Lee., 1976). This landmark study launched the discovery of many different sero/genotypes of hantaviruses, such as Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV) and Puumala virus (PUUV) in Eurasia; Sin Nombre virus (SNV), Andes virus (ANDV), and related viruses in the American continents (Schmaljohn and Hjelle, 1997). Each rodentborne hantavirus species appears to be primarily associated with one (or a few closely related) specific rodent species. The substantial similarity in the phylogenetic relationships of hantaviruses and their reservoir hosts have been observed and explained by a long-standing history of hantavirus-host co-evolution and codivergence (Morzunov et al., 1998; Plyusnin and Morzunov, 2001). However, the applicability of the co-divergence theory has recently been questioned and preferential host switching and local adaptation has been suggested as an alternative explanation for the convergent phylogenies (Ramsden et al., 2009). The ecological and phylogenetic relationships between some hantaviruses (PUUV, Dekonenko et al., 2003; Nemirov et al., 2010; SAAV, Nemirov et al., 2002; Tula virus, Schmidt-Chanasit et al., 2010; SNV-like viruses, Morzunov et al., 1998; and ANDV Torres-Perez et al., 2010) and their rodent hosts have been studied, suggesting that the distribution and natural history of the reservoir hosts play important roles in the geographic distribution and epidemiology of hantavirusrelated diseases.
0168-1702/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.11.006
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J. Liu et al. / Virus Research 163 (2012) 439–447
HTNV and SEOV, mainly carried by striped field mice (A. agrarius) and Brown Norway rats (Rattus norvegicus), respectively, are two major agents of HFRS (Chen and Qiu, 1993; Wang et al., 2000; Zou et al., 2008c). HFRS is highly endemic in China accounting for 90% of the total cases in the world (Fang et al., 2010). Hubei province, which is located along the Yangtze River in the centralsouth China, had been one of the most severe epidemic areas of HFRS, with the highest annual incidence 49.68 cases/100,000 individuals in 1983 (Cohen et al., 1981; Wang et al., 1990; Xiao et al., 1993). A SEOV strain (Hubei-1) highly identical with strain 80-39, and a HTNV strain (HV114) closely related with strain A9 were isolated from HFRS patients in 1970s and 1980s, respectively (Lee et al., 1980; Liang et al., 1994; Xiao et al., 1993; Xu et al., 2004, 2007). Our previous study on variability of Gn gene of hantaviruses in HFRS patients revealed four types of restriction fragment length polymorphism (RFLP) pattern of HTNV and one RFLP pattern of SEOV (Li et al., 2005b), suggesting the presence of uncharacterized genetic variants of hantaviruses in Hubei. However, the evolution and prevalence of hantaviruses in their natural reservoirs in Hubei have not been fully assessed. In the present study, we sought to investigate the phylogenetic relationships, genetic diversity and geographic distribution of HTNVs and SEOVs in rodent reservoirs in Hubei. The existence of novel genetic variants of HTNV and/or SEOV was expected. The study is valuable for a better understanding of the evolution of hantaviruses, and control of hemorrhagic fever with renal syndrome. In this study, rodents were trapped and screened for the presence of hantaviruses in the period from 2000 to 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene of rodents were identified from lung tissues. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analyses revealed several novel genetic lineages of SEOV and one of HTNV in Hubei, which showed geographic clustering. Probable inter-lineage reassortment and spillover infection of SEOV were observed. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs. 2. Materials and methods 2.1. Rodent samples From 2000 to 2009, rodents were captured with snap-traps, which were generally set at five meters and baited with peanuts in both residential areas and fields. Trapping was conducted at five location in four geographically diverse HFRS epidemic areas in Hubei province, China: including Dangyang (southwest mountains), Nanzhang (central hills), Xinzhou and Jiangxia (Jianghan plain), and Qichun (southeast low hills) (Fig. 1). Trapped animals were identified as described previously (Chen et al., 1986; Zhang et al., 2010b). All the animal research was conducted in accordance with the internationally accepted principles and guidelines for Care and Use of Laboratory Animals of Wuhan University. Lung tissues were collected and stored at −80 ◦ C until use. 2.2. Detection of hantavirus antigens Hantavirus-specific antigens in rodent lungs were detected by indirect immunofluorescence assay (IFA), as described previously (Zou et al., 2008b). Scattered, granular fluorescence in the cytoplasm was considered a positive reaction. 2.3. RT-PCR and sequencing Total RNA was extracted from rodent lung tissues using the RNAprep Tissue Kit (Qiagen, Germany). First-strand cDNA
Fig. 1. Map showing the trapping sites for the rodents in Hubei province, China.
was generated from total RNA using random primers and moloney murine leukemia virus reverse transcriptase (Promega, USA). The primers for S- and M-segments were designed (Supplementary Table 1) according to HTNV and SEOV sequences obtained through GenBank. Partial S segments (nt 588–1147 for SEOV, nt 615–1141 for HTNV) were amplified using three pairs of primers: HTN-S558F/HTN-S830R, HTN-S774F/S1007R, and S927F/S1168R for HTNVs; SEO-S567F/SEO-S870R, SEOS802F/S1007R, and S927F/S1168R for SEOVs. Partial M segments (nt 53–490 for SEOV, nt 61–496 for HTNV) were amplified using two pairs of primers: HTN-M40F/HTN-M282R and HTN-M255F/HTN-M517R for HTNVs; SEO-M32F/SEO-M343R and SEO-M238F/SEO-M511R for SEOVs. The total volume of each PCR reaction was 50 l and contained 5 l of cDNA, 0.2 M of the forward and reverse primers, 200 M of dNTPs, 1 unit Taq DNA polymerase (Tiangen, China), 10 mM Tris/HCl (pH 8.3), 50 mM KCl and 1.5 mM MgCl2 . The PCR reaction was run under the following conditions: 5 min at 94 ◦ C; 30 cycles, each cycle consisting of 45 s at 94 ◦ C, 45 s at 55 ◦ C, and 45 s at 72 ◦ C; followed by a final extension step of 5 min at 72 ◦ C. RT-PCR products were gel-purified, and sequenced with an ABI 3730 automatic sequencer. 2.4. PCR and sequencing of mitochondrial (mt) DNA D-loop sequences Total DNA was extracted from hantavirus-positive lung tissues using the DNAprep Tissue Kit (Qiagen, Germany). The entire 899nucleotide region of the D-loop of mtDNA was amplified by PCR, using primers DF (5 -GTCAACTCCCAAAGCTGAAATTC-3 ) and DR
J. Liu et al. / Virus Research 163 (2012) 439–447
(5 -TCTCGAGATTTTCAGTGTCTTGCTTT-3 ). PCR was performed in 50 l reaction mixtures, containing 0.5 g of DNA template, 0.1 M of each primer, 200 M of dNTPs, and 2 units Taq DNA polymerase. Cycling conditions consisted of an initial denaturation of 3 min at 94 ◦ C; 35 cycles, each cycle consisting of 1 min at 94 ◦ C, 1 min at 50 ◦ C, and 1 min at 72 ◦ C; followed by a final extension step at 72 ◦ C for 10 min. Amplified DNA was gel-purified and then sequenced with an ABI 3730 automatic sequencer. 2.5. Phylogenetic analyses Multiple sequence alignment was carried out using ClustalX 2.0 (Larkin et al., 2007) with default parameters and revised with BioEdit 7.0 (Hall, 1999) manually. The occurrence of recombination events among hantaviruses sequences were evaluated using multiple recombination-detection methods, including RDP, GENECONV, MaxChi, Chimaero and 3seq implemented in the RDP3 Beta 4.9 program (Martin et al., 2005). The best substitution models were estimated as the GTR + I + G, GTR + G and HKY + G for partial S-, M-segments, and mtDNA D-loop sequences, respectively, as selected by using jModelTest version 0.1 (Posada, 2008). Phylogenetic trees were constructed using maximum-likelihood (ML) and neighbor-joining (NJ) analyses in PAUP* 4.0 (Swofford, 2002). Node support was evaluated by bootstrap analysis of 1000 iterations, using ML and NJ trees in PAUP* 4.0. We considered clades with bootstrap values ≥95% as highly supported, and 70–95% as moderately supported (Alfaro et al., 2003; Hillis and Bull, 1993). For the hantaviruses sequences, Sin Nombre virus strain NM-H10 was used as the outgroup. For the mtDNA D-loop sequences, Microtus kikuchii and Peromyscus maniculatus pallescens belonging to the family Cricetidae were used as the outgroup to root the phylogenetic tree. 2.6. Molecular diversity analysis For the partial S- and M-segment sequences of hantavirus variants, estimates of genetic divergence were obtained by pairwise analysis using MEGA 5.0 (Tamura et al., 2011). Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated from the dataset. The number of alleles, allele diversity, and nucleotide diversity were calculated with DnaSP 5.00 (Librado and Rozas, 2009). 2.7. Nucleotide sequence accession numbers The partial S- and and mtDNA D-loop have been deposited HQ655891–HQ655992. used in the study were Table 2).
M-segment sequences of hantaviruses, sequences described in this study in GenBank with accession numbers Other previously published sequences obtained from GenBank (Supplementary
3. Results 3.1. Hantavirus infection in rodents Between 2000 and 2009, a total of 687 rodents including 148 (21.54%) A. agrarius, 507 (73.80%) R. norvegicus and 32 (4.66%) Rattus tanezumi were captured from five trapping sites in Hubei province (Fig. 1). Lung tissues were screened using IFA to detect hantavirus antigens, and 46 (6.70%) samples, including 11/148 (7.43%) A. agrarius, 35/507 (6.90%) R. norvegicus and no R. tanezumi samples were identified positive. The numbers of trapped and hantavirus-antigen positive animals at each location are summarized in Table 1. The total percentages of positive rodents were similar among all the
441
Table 1 Detection of hantavirus antigen in rodents trapped at different sites in Hubei, China. Site
No. of hantavirus antigen positive rodents/no. of trapped rodents (%) A. agrarius
R. norvegicus
R. tanezumi
Total
Nanzhang Dangyang Jiangxia Qichun Xinzhou
0/10 2/28 (7.14) 1/23 (4.35) 0/6 8/81 (9.88)
8/77 (10.39) 5/73 (6.85) 8/125 (6.40) 7/75 (9.21) 7/157 (4.46)
0/18 0 0 0/14 0
8/105 (7.62) 7/101 (6.93) 9/148 (6.08) 7/95 (7.37) 15/238 (6.30)
Total
11/148 (7.43)
35/507 (6.90)
0/32
46/876 (6.70)
locations, ranging from 6.30% to 7.62%, although, the positive percentage rates of A. agrarius and R. norvegicus varied among different sampling sites. In summary, hantavirus antigens were detected in A. agrarius and R. norvegicus, with the latter as the predominant rodent species. 3.2. RT-PCR and sequence analysis Partial S-segment sequence analysis indicated that among the 46 hantavirus-positive samples, 15.22% (7/46) were HTNV, and 84.78% (39/46) were SEOV variants. For 16 samples (13 SEOVs, 3 HTNV), only amplifications with primers S927F/S1168R (product length 242 bp) were successful, and these sequences were not included in further analysis because of their short lengths. For 30 samples, including 26 SEOV and 4 HTNV variants, partial S-segment covered nucleotide (nt) 588–1147 (560 bp) for SEOV (numbering as for strain Z37, AF187082) and nt 615–1141 (527 bp) for HTNV (numbering as for strain Q32, AB027097), respectively, were obtained. The corresponding locations of amino acids (aa) were 183–368 (SEOV), and 194–368 (HTNV), which fell into the variable region (aa 155–429) of N protein including some serotype-specific epitopes (Yoshimatsu et al., 2003). For these samples the partial M segment sequences covered nt 53–490 (438 bp) for SEOV (numbering as for strain Z37, AF1190119) and nt 61–496 (436 bp) for HTNV (numbering as for strain Q32, DQ371905), respectively, were amplified and sequenced. The corresponding locations of amino acids were 3–148 (SEOV), and 8–152 (HTNV), which covered parts of the signal peptide and the N-terminal of Gn protein. 3.3. mtDNA sequencing and host phylogeny Sequencing of mtDNA D-loop regions from hantavirus-positive lung tissues confirmed the morphological species determination. Phylogenetic reconstructions of rodent host relationships with the ML and NJ methods placed A. agrarius and R. norvegicus in two different and highly supported clades, and formed two sub-clades among Brown Norway rats from different sampling sites in Hubei (Fig. 2A). All Brown Norway rats from Nanzhang (central hills) were placed in lineage Rn-I, showing geographical clustering. Fifteen Brown Norway rats from Jiangxia, Qichun and Xinzhou formed lineage Rn-II. In addition, three rats (MA28, MQ2-20 and MX38), which were from Jiangxia, Qichun, and Xinzhou, respectively, did not cluster with other sequences. Five striped field mice, including one from Jiangxia and four from Xinzhou, clustered with a sample from Shandong province of China. 3.4. Phylogenetic analyses of hantavirus sequences To assess phylogenetic relationships of the hantaviruses in Hubei, we used partial S- and M-segment sequences of 30 hantavirus variants from rodent samples. Both of ML, NJ methods showed similar tree topology. Phylogenetic trees demonstrated
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Fig. 2. Phylogenetic trees (ML trees) based on mtDNA D-loop sequences, partial S- and M-segment sequences of hantaviruses. (A) Mitochondrial D-loop phylogenetic tree of hantavirus-positive A. agrarius and R. norvegicus with other sequences from GenBank (899 nt). (B) S-segment phylogenetic tree of HTNVs and SEOVs (560 nt). (C) M-segment phylogenetic tree of HTNVs and SEOVs (438 nt). Taxon names are color-coded according to their geographic origins. Rodent samples are labeled by the geographic origin and year of collection. Hantaviruses are labeled with rodent host, geographic origin and year of collection. Star symbols () are attached to virus samples suggesting reassortants. Numbers at nodes indicate bootstrap values for ML method (before slash) and NJ method (after slash). Only support values of >50% for main branches are displayed. Branch lengths are proportional to the number of nucleotide substitutions. Abbreviations: Aa, A. agrarius; Rn, R. norvegicus; JX, Jiangxia; XZ, Xinzhou; NZ, Nanzhang; QC, Qinchun. The accession numbers of D-loop sequences used for comparison are: Shangdong/China (DQ359172), USA (DQ673916), Czech (AY58820), WKY (DQ673907), and Sweden (FJ919765). The sequences of Microtus kikuchii (NC 003041) and Peromyscus maniculatus pallescens (EU140794) were used as the outgroup. The accession numbers of hantavirus sequences used for comparison are list in Supplementary Table. The sequences of Sin Nombre virus strain NM-H10 (NC 005216, NC 005215) were used as the outgroup.
J. Liu et al. / Virus Research 163 (2012) 439–447
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Table 2 Descriptive statistics of genetic variation of hantavirus partial S- and M-segment sequences in Hubei, China. Type
Segment
Lineage
Na
Nhb
Sc
Hdd mean ± SD)
SEO SEO SEO SEO SEO SEO SEO HTN HTN
S S S M M M M S M
#3 #7 #8 #3 #7 #8 #9 #10 #10
7 7 9 7 7 8 3 4 4
3 3 3 2 2 3 3 2 4
5 3 5 2 1 5 11 18 12
0.52 0.52 0.64 0.29 0.57 0.68 1.00 0.50 1.00
a b c d e
± ± ± ± ± ± ± ± ±
0.21 0.21 0.13 0.20 0.12 0.12 0.27 0.27 0.18
nt diversity (%) (mean ± SD)
aa diversity (%) (mean ± SD)
0.27 ± 0.11 0.15 ± 0.08 0.30 ± 0.09 0.13 ± 0.09 0.13 ± 0.03 0.37 ± 0.17 NAe NA NA
0.33 ± 0.23 0.15 ± 0.16 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.57 + 0.42 NA NA NA
N, number of small mammals. Nh, number of alleles. S, number of polymorphic (segregating) sites. Hd, allele diversity. NA, unapplicable, because the number of samples is less than seven.
that several previously undefined lineages of SEOV and one novel lineage of HTNV were distributed in Hubei in a geographic clustering pattern. No evidence of recombination was found for either of the partial S- and M-segment sequences by using multiple recombination-detection methods.
3.4.1. Phylogenetic analyses of SEOV sequences Previous phylogenetic analyses of SEOV based on partial Sand M-segment sequences had revealed six genetic lineages (Shi et al., 2003; Wang et al., 2000; Zhang et al., 2010c). For the viruses belonging to lineage #2, the available partial sequences are not overlapping with the sequences obtained in this study, so they are not included in our phylogenetic analysis. Herein, the partial Ssegment sequences of SEOV variants identified in Hubei fell into three lineages, SEO-S-#3 (both of ML and NJ bootstraps showed moderate supports), #7 (ML bootstrap showed moderate support, and NJ bootstrap showed high support), and #8 (both of ML and NJ bootstraps showed high supports) (Fig. 2B). Among these lineages, the latter two were previously unidentified. In addition, three variants (MA35, MQ2-17 and MQ2-20) did not cluster with other sequences. Seven variants from Jiangxia (central Jianghan plain), with hosts belonging to lineage Rn-II, fell into lineage SEO-S#3, clustering with some SEOV variants from Shangdong (JUN5-14), Beijing (BjHD01) and Zhejiang (Z37 and ZT10) of China. Lineage SEO-S-#7 consisted of seven samples from Nanzhang (central hills), and showed a geographical clustering in accordance with that of the hosts (lineage Rn-I). Lineage SEO-S-#8 was composed of four samples from Xinzhou (Jianghan plain) and five from Qichun (southeast low hills), with most hosts belonging to lineage Rn-II. It is noteworthy that one virus, MX81, which was from A. agrarius in Jianghan plain, were grouped in lineage SEO-S-#8, suggesting spillover infection of SEOV from R. norvegicus to A. agrarius. In the phylogenetic tree based on partial M-segment sequences of SEOVs (Fig. 2C), the Hubei variants fell into four lineages (SEO-M#3, #7, #8, and #9), and one sample (MQ2-20) did not cluster with other sequences. Comparing with the clustering patterns of lineages based on partial S-segment sequences, those of the lineages SEO-M-#3 and #7 were similar to the former, although those of the lineages SEO-M-#8 and #9 showed differences. Sample MQ2-17, of which the partial S-segment sequence did not cluster with other sequences, clustered into lineage SEO-M-#8. Three samples (MA35, MX81 and MQ2) formed lineage SEO-M-#9 (both of ML and NJ bootstraps showed moderate supports), which was not formed based on partial S-segment sequences. The differences in the clustering patterns of lineages of SEOV based on partial S- and M-segments indicated that inter-lineage reassortment may occur among viruses from Jiangxia (Jianghan plain), Xinzhou (Jianghan plain) and Qichun (southeast low hills).
3.4.2. Phylogenetic analyses of HTNV sequences Previous phylogenetic analysis of both S and M segments revealed nine genetic lineages of HTNV (Wang et al., 2000). Da Bie Shan virus (DBSV) and Amur-Soochong virus (ASV), which were assigned to lineages HTN-#1 and HTN-#2 previously, had been classified as new hantaviruses (Jiang et al., 2007; Zhang et al., 2010c). So the lineages of HTNV were numbered from #3 in Fig. 2B and C. Herein, four HTNV variants from Xinzhou and Jiangxia (Jianghan plain) formed a highly supported novel lineage, HTN-S-#10 (Fig. 2B). The partial M-segment sequences of these variants also clustered in the same pattern (Fig. 2C), and this lineage shared a common ancestor with viruses from Guizhou, Yunnan and Tianjin of China (Zhang et al., 2010a; Zou et al., 2008a). Taken together, three/four lineages of SEOV and one lineage of HTNV, based on partial S- and M-segment sequences, were identified from rodents in Hubei, China.
3.5. Molecular analysis of hantavirus sequences A pairwise comparison of partial SEOV S- and M-segment sequences from Hubei revealed a low level of divergence within lineages. Levels of the average nucleotide (amino acid) divergences of partial S-segment sequences in lineages SEO-S-#3, #7, and #8 ranged from 0.15% to 0.30% (0.00–0.33%), and those of partial Msegment sequences in lineages SEO-M-#3, #7, and #8 ranged from 0.13% to 0.37% (0.00–0.57%) (Table 2). Between the SEOV lineages from Hubei, the average nucleotide (amino acid) diversities of the partial S-segment ranged from 1.36% to 3.55% (0.00–1.10%), and those of the partial M-segment were higher, ranging from 2.55% to 5.48% (0.00–3.50%) (Tables 3 and 4). It is noteworthy that the partial M-segment sequence of MQ220 showed higher nucleotide (amino acid) divergence from other Hubei SEOV variants, ranging from 4.53% to 5.48% (2.10–3.50%) (Table 4). Comparing with the representative SEOV sequences in GenBank, the partial S- and M-segment nucleotide sequences of Hubei SEO variants were most closely related with strain Z37 from Zhejiang province, except for the partial M-segment of MQ2-20, which was more similar to strain L99 from Jiangxi province (Tables 3 and 4). The partial N protein amino acid sequences of SEOV variants in lineages SEO-S-#8, and samples MA35, MQ2-17 and MQ2-20 were identical with strain 80-39 from Japan. The partial Gn protein amino acid sequences of variants in lineage SEO-M-#7 and #9 were identical with that of strain Z37. Comparing with other representative HTNV strains, HTN-#10 showed nucleotide (amino acid) divergences ranging from 10.79% to 13.85% (0.69–3.43%), and 10.14% to 16.43% (1.40–9.79%) for partial S- and M-segments, respectively (Tables 3 and 4).
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Table 3 Nucleotide and amino acid percent divergence among lineages of analyzed partial S segments of hantaviruses and representative sequences from GenBank. SEO-S-#3
SEO-S-#7 3.21
1.10 0.92 0.92 0.92 0.92 0.37 0.92 1.47 3.11 0.37 2.01 24.68 23.44 23.99 24.54 25.64 25.09
0.18 0.18 0.18 0.18 0.73 0.18 0.73 2.38 0.73 1.28 24.86 23.63 24.18 24.73 25.82 25.27
SEO-S-#8 2.81 2.01 0.00 0.00 0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
MA35 3.32 1.83 1.97
MQ2-17 3.55 2.22 2.58 2.38
0.00 0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
MQ2-20
Z37
3.32 1.36 1.97 1.46 2.19 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
2.45 2.40 2.76 2.56 2.74 2.56 0.55 1.10 2.75 0.00 1.65 24.31 23.08 23.63 24.18 25.27 24.73
80-39
SR-11
IR461
4.27 2.56 3.24 2.74 3.29 2.74 3.47
4.83 3.00 3.98 3.66 3.29 3.29 4.02 2.74
4.63 3.47 3.52 2.74 3.47 3.11 3.84 3.66 4.20
0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
2.75 1.10 1.65 24.86 23.63 24.18 24.73 25.82 25.27
2.75 3.30 25.41 24.18 24.73 25.27 26.37 25.82
L99 4.51 3.29 3.98 3.66 3.66 3.29 3.66 3.66 3.84 4.20 1.65 24.31 23.08 23.63 24.18 25.27 24.73
Gou3
HTN-S-#10
76-118
Q32
84FLi
A9
Z10
13.89 12.43 13.21 12.80 13.16 12.61 13.89 12.98 12.98 13.35 12.61
28.32 28.02 28.02 28.47 27.97 28.43 28.38 28.66 28.66 28.56 28.47 30.30
29.31 28.52 28.88 29.25 28.52 28.15 28.70 29.25 30.16 29.07 28.15 30.16 13.85
29.54 29.43 29.58 29.80 28.88 29.80 29.62 29.98 29.25 30.16 28.52 29.62 10.79 16.27
31.20 30.71 31.03 31.44 31.26 31.26 30.90 31.44 31.08 30.90 30.35 31.26 11.06 14.99 9.14
28.82 28.70 28.70 29.25 28.15 28.88 28.70 29.62 28.34 29.07 28.70 29.25 11.84 17.00 11.70 12.98
28.05 27.79 27.97 28.34 27.61 27.61 28.15 28.34 27.79 28.88 27.97 30.16 11.79 15.36 10.97 10.97 11.33
25.41 24.18 24.73 25.27 26.37 25.82
2.88 1.24 0.69 3.43 1.79
2.75 2.20 3.85 2.75
0.55 3.30 1.65
2.75 1.10
3.30
Nucleotide divergence, above diagonal; amino acid divergence, below the diagonal. All results are based on the pairwise analysis of 41 sequences, with 524-nt positions corresponding to 174 aa in the final data set.
Table 4 Nucleotide and amino acid percent divergence among lineages of analyzed partial M segments of hantaviruses and representative sequences from GenBank. SEO-M-#3 SEO-M-#3 SEO-M-#7 SEO-M-#8 SEO-M-#9 MQ2-20 Z37 80-39 SR-11 IR461 L99 Gou3 HTN-M-#10 76-118 Q32 84FLi A9 Z10
SEO-M-#7 3.13
1.40 1.84 1.40 3.50 1.40 3.50 2.80 3.50 2.80 5.59 34.97 36.36 35.66 35.66 37.76 37.06
0.44 0.00 2.10 0.00 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
SEO-M-#8 3.67 2.55 0.44 2.53 0.44 2.53 1.84 2.53 1.84 4.63 34.70 36.10 35.40 35.40 37.50 37.50
SEO-M-#9 3.81 3.61 4.43 2.10 0.00 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
MQ2-20 4.66 4.53 5.48 4.82 2.10 4.20 3.50 4.20 3.50 4.20 34.27 34.97 34.97 34.27 36.36 36.36
Z37 2.16 2.90 3.85 3.03 4.20 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
80-39
SR-11
IR461
4.20 4.53 5.48 4.97 5.59 3.96
3.73 3.36 4.55 3.81 4.43 3.50 4.43
5.36 5.23 6.06 5.05 6.29 4.90 5.83 5.59
3.50 4.20 3.50 6.29 34.97 36.36 35.66 35.66 37.76 37.76
3.50 2.80 5.59 35.66 37.06 36.36 36.36 38.46 38.46
3.50 6.29 34.97 34.97 35.66 34.27 36.36 36.36
L99 3.26 3.13 4.08 3.57 3.73 3.03 4.66 3.50 4.90 5.59 34.97 35.66 35.66 34.97 37.06 37.06
Gou3
HTN-M-#10
76-118
Q32
84FLi
A9
Z10
14.22 14.92 14.71 14.69 15.15 13.99 14.45 14.22 15.62 13.99
30.05 31.49 31.50 31.27 28.90 30.07 30.42 31.12 30.19 30.07 31.53
31.47 32.30 32.81 32.40 31.00 31.93 31.00 32.17 32.40 31.70 29.60 14.92
32.80 33.80 32.90 33.72 32.87 32.40 32.17 33.57 32.63 33.33 31.93 10.14 16.78
31.70 32.17 31.96 32.40 31.70 31.47 31.47 31.93 31.70 31.70 33.57 15.50 17.02 14.92
33.50 34.27 34.82 34.50 32.87 32.87 33.80 34.03 34.03 33.80 34.27 16.43 16.55 17.72 12.12
30.70 31.14 31.09 31.24 31.47 30.54 30.54 31.70 30.54 30.77 31.70 16.26 16.32 16.08 13.99 13.29
33.57 33.57 34.27 33.57 35.66 35.66
5.59 1.40 6.99 9.79 9.79
6.99 7.69 9.79 11.89
5.59 9.09 10.49
7.69 9.09
8.39
Nucleotide divergence, above diagonal; amino acid divergence, below the diagonal. All results are based on the pairwise analysis of 41 sequences, with 436-nt positions corresponding to 145 aa in the final data.
J. Liu et al. / Virus Research 163 (2012) 439–447
SEO-S-#3 SEO-S-#7 SEO-S-#8 MA35 MQ2-17 MQ2-20 Z37 80-39 SR-11 IR461 L99 Gou3 HTN-S-#10 76-118 Q32 84FLi A9 Z10
J. Liu et al. / Virus Research 163 (2012) 439–447
3.6. Comparison of the amino acid sequences and identification of lineage-specific amino acid residues A comparison of the deduced partial N protein sequences spanning amino acids 183–368 (numbering in the N protein of strain Z37) of the novel SEOV variants with strain Z37 revealed three conservative amino acid exchanges: F225L in MA2, D231E in MA28 (both samples belonging to lineage SEO-S-#3), and T233A in MB1-7 (lineage SEO-S-#7). A comparison of the deduced signal peptide and N terminus of Gn protein covering amino acid positions 3–148 (numbering in glycoprotein precursor of strain Z37) with strain Z37 revealed more exchanges than for the partial N protein (Supplementary Fig. S1). In lineage SEO-M-#3, all the samples from Jiangxia (Jianghan plain) had conservative amino acid exchanges L6F and V11I. In the novel lineage SEO-M-#8, four samples had amino acid exchanges E58D, and one sample (MQ2-17) had an A8T amino acid substitution. The sample MQ2-20 had three unique amino acid substitutions, G14D, F15V, and R24K. Comparing with closely related strain Q32, the deduced partial N protein sequences spanning amino acids 194–368 (numbering as for strain Q32) of variants in lineage HTN-S-#10 showed a V296I substitution, which falls in the epitope region for monoclonal antibody C24B4 and may affect the antigenicity of N protein (Yoshimatsu et al., 1996). Another substitution, V307I, was observed for members in lineage HTN-S-#10 except for HV004. The deduced partial Gn protein sequences of HTNV variants showed conservative amino acid exchanges as T55A and I116V in all members in lineage HTN-M-#10.
4. Discussion In this paper, we describe a comprehensive study on the presence of HTNV and SEOV in four geographically diverse HFRS epidemic areas of Hubei, central-south China from 2000 to 2009. In the past decade, although the geographic area of epidemics was expanding, the incidence and mortality of HFRS had declined in China (Chen and Qiu, 1993; Zhang et al., 2010c). Genetic analyses of the endemic hantaviruses and their hosts will be helpful for understanding the changing epidemiology of HFRS. In this study, the majority of collected rodents in Hubei were Brown Norway rats, and the corresponding SEOV was predominant hantavirus. Similar patterns had been observed in other regions of China (Dai et al., 2007; Zhang et al., 2010b; Zhou et al., 2009) in recent years. In addition, HFRS infections caused by SEOV have emerged in northern China (Inner Mongolia and Beijing) where it had not been reported before (Jiang et al., 2008; Zhang et al., 2009b), suggesting the expansion of SEOV epidemic areas. In the past two decades, China experienced fast socioeconomic development and urbanization progress, which might increase the migration frequencies of rats and give them more chances to contact with humans. So ratcarried SEOV may have more opportunities to be transmitted to humans than field mouse-carried HTNV. It is known that human SEOV infections cause a milder form of HFRS, and the percentage of inapparent infection is higher than for HTNV (Chen et al., 1986). We presume that besides comprehensive preventive measures, the increase in the prevalence of SEOV is part of the reasons for the declined incidence and mortality of HFRS in China. Our data revealed that multiple phylogenetic lineages (#3, #7, #8, and #9) of SEOV co-circulate in rodents in Hubei, which is remarkable for the epidemiology of SEOV. SEOVs belonging to lineages #1 and #4 had been identified from HFRS patient and rodents in Hubei before (Liang et al., 1994; Xiong et al., 2010). Taken together, there are at least six phylogenetic lineages of SEOVs in Hubei, including three already known (#1, #3 and #4) and three
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novel lineages (#7–#9). It suggested that Hubei province is an important epidemic focus of SEOV. Unlike other hantaviruses, SEOV has a worldwide distribution, both in the Old and New Worlds. Most of the related HFRS cases are found in China, but little is known about the origin of this virus. By locating the geographic distribution of phylogenetic lineages of SEOV, we found that multiple lineages, namely #1 (Shi et al., 2003), #3 (Zhang et al., 2010b), #4 (Wang et al., 2000), #5 (Wang et al., 2000), and #7–#9, circulate in middle and lower reaches of the Yangtze River (involving six provinces: Hubei, Hunan, Jiangxi, Anhui, Zhejiang, and Jiangsu). However, only two or three lineages of SEOV are distributed in other parts of China, such as #1, #3 and #4 in the northeast (Zhang et al., 2009a,b); #1–#3 in the North China Plain (Sun et al., 2005; Zuo et al., 2008); and #1 and #2 in South China (Wang et al., 2000; Yang et al., 2006). In addition, the variants in SEO-#5 which are from Zhejing province, show higher genetic diversity from the majority of SEOVs (Wang et al., 2000) and occupy a basal position in the phylogenetic tree, indicating an early evolutionary divergence. In the other countries of the world, only lineages #4 and #6 were found (Heyman et al., 2004; Shi et al., 2003; Xiao et al., 1994). The abundance of the phylogenetic lineages of SEOV in the middle and lower reaches of the Yangtze River demonstrates that these regions are important epidemic nidi for SEOV. Our molecular analysis also supports this presumption. Most of the known SEOV variants (from China as well as those from Japan, South Korea, the U.S. and United Kingdom) are genetically homogeneous, irrespective of geographic distribution, suggesting these viruses may share a recent common ancestor and distribute following the expansion of R. norvegicus. Studies in France (Heyman et al., 2004) and the United States (Mantooth et al., 2001) indicated that SEOVs were not indigenous and were introduced from Asia into the new endemic areas in associated with their hosts. The current distribution of hantaviruses is the result of migration and the natural history of their rodent hosts. The discovery of fossils demonstrate that R. norvegicus originate in lower reaches of Yangtze River and then expanded to other parts of China in late Pleistocene (Wu et al., 2008). The middle and lower reaches of Yangtze River are mainly consisted of alluvial plains and hills. The climate is north subtropical, with plenty of rainfall. These areas had been culture centers of China in the history over 2500 years ago. These ecological conditions are favorable for the thriving of rats. The prosperous commercial trading and well developed waterway transportation may assist in the global distribution of R. norvegicus in the Holocene. Taken together, we propose that the middle and lower reaches of the Yangtze River are radiation centers for most known phylogenetic lineages of SEOVs. The worldwide dispersion of Brown Norway rats may spread ancestors of the current SEOVs around the world. Phylogenetic analyses based on partial S- and M-segment sequences indicated 4 probable inter-lineage reassortants of SEOV. The frequency (13.3%, 4/30) of the reassortment is not significantly different when compared with two other observations: 10.0% (2/20, Fisher’s Exact Test P = 1.00) was reported previously in SNV from North America (Black et al., 2009) and 32% (8/25, Fisher’s Exact Test P = 0.11) in PUUV in northern Finland (Razzauti et al., 2009). It suggests that the corresponding hosts from Jiangxia, Xinzhou (both in Jianghan plain) and Qichun (southeast low hills) had close interactions and viruses were transmitted among them horizontally. Since hantaviruses possess segmented genomes, the possibility of genetic reassortment between different hantavirus variants to achieve high infectivity and adapt to new hosts exists, as already known for influenza viruses. L segment sequences and longer sequences for S- and M-segments are needed to confirm the reassortment among the viruses. And further surveillance should be carried out to clarify
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whether these reassortant variants will disappear or exist persistently, or even cause severe epidemiologic consequences. Herein, we found for the first time that SEOV existed simultaneously in A. agrarius and R. norvegicus, suggesting that a spillover event of SEOV might occur naturally in Hubei. The spillover sample MX81 was a reassortant with the S-#8–M-#9 type. Our data suggested an inter-species interaction between A. agrarius and SEOV-infected R. norvegicus. Previous ecological surveillance in the middle and lower reaches of the Yangtze River showed that striped field mice live in fields, while Brown Norway rats live both in the field and houses, and migrate between the two habitats seasonally (Li et al., 2005a; Zhang et al., 2003). Hantavirus could spill over into sympatric and syntopic species, particularly during irruption in rodent reservoir population (Kang et al., 2009). However, more evidences are needed to confirm the spillover infection of SEOV to A. agrarius. In addition to SEOVs, HTNVs were also identified from rodents in this study. HTNV strains belonging to lineages #7 and #9 (Wang et al., 2000) had been detected from HFRS patients in Hubei previously (Ding et al., 2003; Xu et al., 2004). Herein, a new distinct genetic lineage HTN-#10 consisted of variants from Jianghan plain was formed, which is closely related with strains from the North China plain and the Yunnan-Guizhou plateau in the southwest. However, HTNV belonging to this novel lineage had not been detected in HFRS patients yet. Further studies are warranted to illuminate the pathogenic role of this new HTNV. In conclusion, this study demonstrates that SEOV and HTNV were co-circulating in Hubei. There were at least three phylogenetic lineages of SEOVs circulating in Hubei. A novel genetic lineage of HTNV was recovered in rodents. Based on phylogenetic analysis of SEOVs and rodent fossils discovered before, we propose that the middle and lower reaches of Yangtze River are radiation centers for most known phylogenetic lineages of SEOVs. Probable inter-lineage reassortment and spillover infection of SEOV were observed. Further studies are warranted to clarify the biological characteristics of the new found hantavirus variants and the epidemiology impact of them.
Acknowledgements This work was supported by the National High Technology Research and Development 863 Program of China (No. 2007AA02Z465), a grant from the National Natural Science Foundation of China (NSFC project No. 30770096), National Natural Science Foundation of Jiangsu province (BK2010537), and the Open Grant of State Key Laboratory of Virology (2010006), Scientific Research Foundation of the State Education Ministry for Returned Chinese Scholars (30115), and the Fundamental Research Funds for the Central Universities (3082003).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.11.006.
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Genetic analysis of hantaviruses and their rodent hosts in central-south China Jing Liu a,1, Dong-Ying Liu a,b,1, Wen Chen a,1, Jin-Lin Li a, Fan Luo a, Qing Li c, Jia-Xin Ling a, Yuan-Yuan Liu a, Hai-Rong Xiong a , Xiao-Hua Ding a , Wei Hou a , Yun Zhang d , Shi-Yue Li e , Jie Wang d , Zhan-Qiu Yang a,∗ a
State Key Laboratory of Virology, Institute of Medical Virology, School of Medicine, Wuhan University, Wuhan 430071, PR China Department of Microbiology, School of Medicine, Wuhan University, Wuhan 430071, PR China Department of Basic Medicine, Xiamen University Medical College, Xiamen 361005, PR China d Institute of Military Medical Sciences, Nanjing Command, Nanjing 210002, PR China e School of Public Health, Wuhan University, Wuhan 430071, PR China b c
a r t i c l e
i n f o
Article history: Received 26 August 2011 Received in revised form 2 November 2011 Accepted 4 November 2011 Available online 17 November 2011 Keywords: Hantavirus Phylogeny Rodents
a b s t r a c t Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped viruses with a negative-sense RNA genome organized into three segments: L (large), M (medium) and S (small) (Plyusnin, 2002). The L segment encodes an RNA-dependent RNA polymerase and shows the highest homology among sequences (Khaiboullina et al., 2005). The M segment encodes two envelope glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid (N) protein (Plyusnin et al., 1996). Variations in the M and S segments may alter the antigenicity and virulence of hantaviruses (Zhang et al., 2007). Two human diseases are associated with hantavirus infections: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas (Jonsson et al., 2010). Unlike the rest of the family Bunyaviridae, hantaviruses are not arthropod-borne but rather are transmitted by rodents and insectivores (Plyusnin et al., 1996). The first hantavirus described,
∗ Corresponding author at: State Key Laboratory of Virology, Institute of Medical Virology, School of Medicine, Wuhan University, 185 Donghu Road, Wuhan 430071, PR China. Tel.: +86 27 68759136; fax: +86 27 68758766. E-mail addresses: [email protected], [email protected] (Z.-Q. Yang). 1 These authors contributed equally to this article.
Hantaan virus (HTNV), was isolated from striped field mouse (Apodemus agrarius) in 1976 (Lee and Lee., 1976). This landmark study launched the discovery of many different sero/genotypes of hantaviruses, such as Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV) and Puumala virus (PUUV) in Eurasia; Sin Nombre virus (SNV), Andes virus (ANDV), and related viruses in the American continents (Schmaljohn and Hjelle, 1997). Each rodentborne hantavirus species appears to be primarily associated with one (or a few closely related) specific rodent species. The substantial similarity in the phylogenetic relationships of hantaviruses and their reservoir hosts have been observed and explained by a long-standing history of hantavirus-host co-evolution and codivergence (Morzunov et al., 1998; Plyusnin and Morzunov, 2001). However, the applicability of the co-divergence theory has recently been questioned and preferential host switching and local adaptation has been suggested as an alternative explanation for the convergent phylogenies (Ramsden et al., 2009). The ecological and phylogenetic relationships between some hantaviruses (PUUV, Dekonenko et al., 2003; Nemirov et al., 2010; SAAV, Nemirov et al., 2002; Tula virus, Schmidt-Chanasit et al., 2010; SNV-like viruses, Morzunov et al., 1998; and ANDV Torres-Perez et al., 2010) and their rodent hosts have been studied, suggesting that the distribution and natural history of the reservoir hosts play important roles in the geographic distribution and epidemiology of hantavirusrelated diseases.
0168-1702/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.11.006
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HTNV and SEOV, mainly carried by striped field mice (A. agrarius) and Brown Norway rats (Rattus norvegicus), respectively, are two major agents of HFRS (Chen and Qiu, 1993; Wang et al., 2000; Zou et al., 2008c). HFRS is highly endemic in China accounting for 90% of the total cases in the world (Fang et al., 2010). Hubei province, which is located along the Yangtze River in the centralsouth China, had been one of the most severe epidemic areas of HFRS, with the highest annual incidence 49.68 cases/100,000 individuals in 1983 (Cohen et al., 1981; Wang et al., 1990; Xiao et al., 1993). A SEOV strain (Hubei-1) highly identical with strain 80-39, and a HTNV strain (HV114) closely related with strain A9 were isolated from HFRS patients in 1970s and 1980s, respectively (Lee et al., 1980; Liang et al., 1994; Xiao et al., 1993; Xu et al., 2004, 2007). Our previous study on variability of Gn gene of hantaviruses in HFRS patients revealed four types of restriction fragment length polymorphism (RFLP) pattern of HTNV and one RFLP pattern of SEOV (Li et al., 2005b), suggesting the presence of uncharacterized genetic variants of hantaviruses in Hubei. However, the evolution and prevalence of hantaviruses in their natural reservoirs in Hubei have not been fully assessed. In the present study, we sought to investigate the phylogenetic relationships, genetic diversity and geographic distribution of HTNVs and SEOVs in rodent reservoirs in Hubei. The existence of novel genetic variants of HTNV and/or SEOV was expected. The study is valuable for a better understanding of the evolution of hantaviruses, and control of hemorrhagic fever with renal syndrome. In this study, rodents were trapped and screened for the presence of hantaviruses in the period from 2000 to 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene of rodents were identified from lung tissues. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analyses revealed several novel genetic lineages of SEOV and one of HTNV in Hubei, which showed geographic clustering. Probable inter-lineage reassortment and spillover infection of SEOV were observed. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs. 2. Materials and methods 2.1. Rodent samples From 2000 to 2009, rodents were captured with snap-traps, which were generally set at five meters and baited with peanuts in both residential areas and fields. Trapping was conducted at five location in four geographically diverse HFRS epidemic areas in Hubei province, China: including Dangyang (southwest mountains), Nanzhang (central hills), Xinzhou and Jiangxia (Jianghan plain), and Qichun (southeast low hills) (Fig. 1). Trapped animals were identified as described previously (Chen et al., 1986; Zhang et al., 2010b). All the animal research was conducted in accordance with the internationally accepted principles and guidelines for Care and Use of Laboratory Animals of Wuhan University. Lung tissues were collected and stored at −80 ◦ C until use. 2.2. Detection of hantavirus antigens Hantavirus-specific antigens in rodent lungs were detected by indirect immunofluorescence assay (IFA), as described previously (Zou et al., 2008b). Scattered, granular fluorescence in the cytoplasm was considered a positive reaction. 2.3. RT-PCR and sequencing Total RNA was extracted from rodent lung tissues using the RNAprep Tissue Kit (Qiagen, Germany). First-strand cDNA
Fig. 1. Map showing the trapping sites for the rodents in Hubei province, China.
was generated from total RNA using random primers and moloney murine leukemia virus reverse transcriptase (Promega, USA). The primers for S- and M-segments were designed (Supplementary Table 1) according to HTNV and SEOV sequences obtained through GenBank. Partial S segments (nt 588–1147 for SEOV, nt 615–1141 for HTNV) were amplified using three pairs of primers: HTN-S558F/HTN-S830R, HTN-S774F/S1007R, and S927F/S1168R for HTNVs; SEO-S567F/SEO-S870R, SEOS802F/S1007R, and S927F/S1168R for SEOVs. Partial M segments (nt 53–490 for SEOV, nt 61–496 for HTNV) were amplified using two pairs of primers: HTN-M40F/HTN-M282R and HTN-M255F/HTN-M517R for HTNVs; SEO-M32F/SEO-M343R and SEO-M238F/SEO-M511R for SEOVs. The total volume of each PCR reaction was 50 l and contained 5 l of cDNA, 0.2 M of the forward and reverse primers, 200 M of dNTPs, 1 unit Taq DNA polymerase (Tiangen, China), 10 mM Tris/HCl (pH 8.3), 50 mM KCl and 1.5 mM MgCl2 . The PCR reaction was run under the following conditions: 5 min at 94 ◦ C; 30 cycles, each cycle consisting of 45 s at 94 ◦ C, 45 s at 55 ◦ C, and 45 s at 72 ◦ C; followed by a final extension step of 5 min at 72 ◦ C. RT-PCR products were gel-purified, and sequenced with an ABI 3730 automatic sequencer. 2.4. PCR and sequencing of mitochondrial (mt) DNA D-loop sequences Total DNA was extracted from hantavirus-positive lung tissues using the DNAprep Tissue Kit (Qiagen, Germany). The entire 899nucleotide region of the D-loop of mtDNA was amplified by PCR, using primers DF (5 -GTCAACTCCCAAAGCTGAAATTC-3 ) and DR
J. Liu et al. / Virus Research 163 (2012) 439–447
(5 -TCTCGAGATTTTCAGTGTCTTGCTTT-3 ). PCR was performed in 50 l reaction mixtures, containing 0.5 g of DNA template, 0.1 M of each primer, 200 M of dNTPs, and 2 units Taq DNA polymerase. Cycling conditions consisted of an initial denaturation of 3 min at 94 ◦ C; 35 cycles, each cycle consisting of 1 min at 94 ◦ C, 1 min at 50 ◦ C, and 1 min at 72 ◦ C; followed by a final extension step at 72 ◦ C for 10 min. Amplified DNA was gel-purified and then sequenced with an ABI 3730 automatic sequencer. 2.5. Phylogenetic analyses Multiple sequence alignment was carried out using ClustalX 2.0 (Larkin et al., 2007) with default parameters and revised with BioEdit 7.0 (Hall, 1999) manually. The occurrence of recombination events among hantaviruses sequences were evaluated using multiple recombination-detection methods, including RDP, GENECONV, MaxChi, Chimaero and 3seq implemented in the RDP3 Beta 4.9 program (Martin et al., 2005). The best substitution models were estimated as the GTR + I + G, GTR + G and HKY + G for partial S-, M-segments, and mtDNA D-loop sequences, respectively, as selected by using jModelTest version 0.1 (Posada, 2008). Phylogenetic trees were constructed using maximum-likelihood (ML) and neighbor-joining (NJ) analyses in PAUP* 4.0 (Swofford, 2002). Node support was evaluated by bootstrap analysis of 1000 iterations, using ML and NJ trees in PAUP* 4.0. We considered clades with bootstrap values ≥95% as highly supported, and 70–95% as moderately supported (Alfaro et al., 2003; Hillis and Bull, 1993). For the hantaviruses sequences, Sin Nombre virus strain NM-H10 was used as the outgroup. For the mtDNA D-loop sequences, Microtus kikuchii and Peromyscus maniculatus pallescens belonging to the family Cricetidae were used as the outgroup to root the phylogenetic tree. 2.6. Molecular diversity analysis For the partial S- and M-segment sequences of hantavirus variants, estimates of genetic divergence were obtained by pairwise analysis using MEGA 5.0 (Tamura et al., 2011). Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated from the dataset. The number of alleles, allele diversity, and nucleotide diversity were calculated with DnaSP 5.00 (Librado and Rozas, 2009). 2.7. Nucleotide sequence accession numbers The partial S- and and mtDNA D-loop have been deposited HQ655891–HQ655992. used in the study were Table 2).
M-segment sequences of hantaviruses, sequences described in this study in GenBank with accession numbers Other previously published sequences obtained from GenBank (Supplementary
3. Results 3.1. Hantavirus infection in rodents Between 2000 and 2009, a total of 687 rodents including 148 (21.54%) A. agrarius, 507 (73.80%) R. norvegicus and 32 (4.66%) Rattus tanezumi were captured from five trapping sites in Hubei province (Fig. 1). Lung tissues were screened using IFA to detect hantavirus antigens, and 46 (6.70%) samples, including 11/148 (7.43%) A. agrarius, 35/507 (6.90%) R. norvegicus and no R. tanezumi samples were identified positive. The numbers of trapped and hantavirus-antigen positive animals at each location are summarized in Table 1. The total percentages of positive rodents were similar among all the
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Table 1 Detection of hantavirus antigen in rodents trapped at different sites in Hubei, China. Site
No. of hantavirus antigen positive rodents/no. of trapped rodents (%) A. agrarius
R. norvegicus
R. tanezumi
Total
Nanzhang Dangyang Jiangxia Qichun Xinzhou
0/10 2/28 (7.14) 1/23 (4.35) 0/6 8/81 (9.88)
8/77 (10.39) 5/73 (6.85) 8/125 (6.40) 7/75 (9.21) 7/157 (4.46)
0/18 0 0 0/14 0
8/105 (7.62) 7/101 (6.93) 9/148 (6.08) 7/95 (7.37) 15/238 (6.30)
Total
11/148 (7.43)
35/507 (6.90)
0/32
46/876 (6.70)
locations, ranging from 6.30% to 7.62%, although, the positive percentage rates of A. agrarius and R. norvegicus varied among different sampling sites. In summary, hantavirus antigens were detected in A. agrarius and R. norvegicus, with the latter as the predominant rodent species. 3.2. RT-PCR and sequence analysis Partial S-segment sequence analysis indicated that among the 46 hantavirus-positive samples, 15.22% (7/46) were HTNV, and 84.78% (39/46) were SEOV variants. For 16 samples (13 SEOVs, 3 HTNV), only amplifications with primers S927F/S1168R (product length 242 bp) were successful, and these sequences were not included in further analysis because of their short lengths. For 30 samples, including 26 SEOV and 4 HTNV variants, partial S-segment covered nucleotide (nt) 588–1147 (560 bp) for SEOV (numbering as for strain Z37, AF187082) and nt 615–1141 (527 bp) for HTNV (numbering as for strain Q32, AB027097), respectively, were obtained. The corresponding locations of amino acids (aa) were 183–368 (SEOV), and 194–368 (HTNV), which fell into the variable region (aa 155–429) of N protein including some serotype-specific epitopes (Yoshimatsu et al., 2003). For these samples the partial M segment sequences covered nt 53–490 (438 bp) for SEOV (numbering as for strain Z37, AF1190119) and nt 61–496 (436 bp) for HTNV (numbering as for strain Q32, DQ371905), respectively, were amplified and sequenced. The corresponding locations of amino acids were 3–148 (SEOV), and 8–152 (HTNV), which covered parts of the signal peptide and the N-terminal of Gn protein. 3.3. mtDNA sequencing and host phylogeny Sequencing of mtDNA D-loop regions from hantavirus-positive lung tissues confirmed the morphological species determination. Phylogenetic reconstructions of rodent host relationships with the ML and NJ methods placed A. agrarius and R. norvegicus in two different and highly supported clades, and formed two sub-clades among Brown Norway rats from different sampling sites in Hubei (Fig. 2A). All Brown Norway rats from Nanzhang (central hills) were placed in lineage Rn-I, showing geographical clustering. Fifteen Brown Norway rats from Jiangxia, Qichun and Xinzhou formed lineage Rn-II. In addition, three rats (MA28, MQ2-20 and MX38), which were from Jiangxia, Qichun, and Xinzhou, respectively, did not cluster with other sequences. Five striped field mice, including one from Jiangxia and four from Xinzhou, clustered with a sample from Shandong province of China. 3.4. Phylogenetic analyses of hantavirus sequences To assess phylogenetic relationships of the hantaviruses in Hubei, we used partial S- and M-segment sequences of 30 hantavirus variants from rodent samples. Both of ML, NJ methods showed similar tree topology. Phylogenetic trees demonstrated
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Fig. 2. Phylogenetic trees (ML trees) based on mtDNA D-loop sequences, partial S- and M-segment sequences of hantaviruses. (A) Mitochondrial D-loop phylogenetic tree of hantavirus-positive A. agrarius and R. norvegicus with other sequences from GenBank (899 nt). (B) S-segment phylogenetic tree of HTNVs and SEOVs (560 nt). (C) M-segment phylogenetic tree of HTNVs and SEOVs (438 nt). Taxon names are color-coded according to their geographic origins. Rodent samples are labeled by the geographic origin and year of collection. Hantaviruses are labeled with rodent host, geographic origin and year of collection. Star symbols () are attached to virus samples suggesting reassortants. Numbers at nodes indicate bootstrap values for ML method (before slash) and NJ method (after slash). Only support values of >50% for main branches are displayed. Branch lengths are proportional to the number of nucleotide substitutions. Abbreviations: Aa, A. agrarius; Rn, R. norvegicus; JX, Jiangxia; XZ, Xinzhou; NZ, Nanzhang; QC, Qinchun. The accession numbers of D-loop sequences used for comparison are: Shangdong/China (DQ359172), USA (DQ673916), Czech (AY58820), WKY (DQ673907), and Sweden (FJ919765). The sequences of Microtus kikuchii (NC 003041) and Peromyscus maniculatus pallescens (EU140794) were used as the outgroup. The accession numbers of hantavirus sequences used for comparison are list in Supplementary Table. The sequences of Sin Nombre virus strain NM-H10 (NC 005216, NC 005215) were used as the outgroup.
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Table 2 Descriptive statistics of genetic variation of hantavirus partial S- and M-segment sequences in Hubei, China. Type
Segment
Lineage
Na
Nhb
Sc
Hdd mean ± SD)
SEO SEO SEO SEO SEO SEO SEO HTN HTN
S S S M M M M S M
#3 #7 #8 #3 #7 #8 #9 #10 #10
7 7 9 7 7 8 3 4 4
3 3 3 2 2 3 3 2 4
5 3 5 2 1 5 11 18 12
0.52 0.52 0.64 0.29 0.57 0.68 1.00 0.50 1.00
a b c d e
± ± ± ± ± ± ± ± ±
0.21 0.21 0.13 0.20 0.12 0.12 0.27 0.27 0.18
nt diversity (%) (mean ± SD)
aa diversity (%) (mean ± SD)
0.27 ± 0.11 0.15 ± 0.08 0.30 ± 0.09 0.13 ± 0.09 0.13 ± 0.03 0.37 ± 0.17 NAe NA NA
0.33 ± 0.23 0.15 ± 0.16 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.57 + 0.42 NA NA NA
N, number of small mammals. Nh, number of alleles. S, number of polymorphic (segregating) sites. Hd, allele diversity. NA, unapplicable, because the number of samples is less than seven.
that several previously undefined lineages of SEOV and one novel lineage of HTNV were distributed in Hubei in a geographic clustering pattern. No evidence of recombination was found for either of the partial S- and M-segment sequences by using multiple recombination-detection methods.
3.4.1. Phylogenetic analyses of SEOV sequences Previous phylogenetic analyses of SEOV based on partial Sand M-segment sequences had revealed six genetic lineages (Shi et al., 2003; Wang et al., 2000; Zhang et al., 2010c). For the viruses belonging to lineage #2, the available partial sequences are not overlapping with the sequences obtained in this study, so they are not included in our phylogenetic analysis. Herein, the partial Ssegment sequences of SEOV variants identified in Hubei fell into three lineages, SEO-S-#3 (both of ML and NJ bootstraps showed moderate supports), #7 (ML bootstrap showed moderate support, and NJ bootstrap showed high support), and #8 (both of ML and NJ bootstraps showed high supports) (Fig. 2B). Among these lineages, the latter two were previously unidentified. In addition, three variants (MA35, MQ2-17 and MQ2-20) did not cluster with other sequences. Seven variants from Jiangxia (central Jianghan plain), with hosts belonging to lineage Rn-II, fell into lineage SEO-S#3, clustering with some SEOV variants from Shangdong (JUN5-14), Beijing (BjHD01) and Zhejiang (Z37 and ZT10) of China. Lineage SEO-S-#7 consisted of seven samples from Nanzhang (central hills), and showed a geographical clustering in accordance with that of the hosts (lineage Rn-I). Lineage SEO-S-#8 was composed of four samples from Xinzhou (Jianghan plain) and five from Qichun (southeast low hills), with most hosts belonging to lineage Rn-II. It is noteworthy that one virus, MX81, which was from A. agrarius in Jianghan plain, were grouped in lineage SEO-S-#8, suggesting spillover infection of SEOV from R. norvegicus to A. agrarius. In the phylogenetic tree based on partial M-segment sequences of SEOVs (Fig. 2C), the Hubei variants fell into four lineages (SEO-M#3, #7, #8, and #9), and one sample (MQ2-20) did not cluster with other sequences. Comparing with the clustering patterns of lineages based on partial S-segment sequences, those of the lineages SEO-M-#3 and #7 were similar to the former, although those of the lineages SEO-M-#8 and #9 showed differences. Sample MQ2-17, of which the partial S-segment sequence did not cluster with other sequences, clustered into lineage SEO-M-#8. Three samples (MA35, MX81 and MQ2) formed lineage SEO-M-#9 (both of ML and NJ bootstraps showed moderate supports), which was not formed based on partial S-segment sequences. The differences in the clustering patterns of lineages of SEOV based on partial S- and M-segments indicated that inter-lineage reassortment may occur among viruses from Jiangxia (Jianghan plain), Xinzhou (Jianghan plain) and Qichun (southeast low hills).
3.4.2. Phylogenetic analyses of HTNV sequences Previous phylogenetic analysis of both S and M segments revealed nine genetic lineages of HTNV (Wang et al., 2000). Da Bie Shan virus (DBSV) and Amur-Soochong virus (ASV), which were assigned to lineages HTN-#1 and HTN-#2 previously, had been classified as new hantaviruses (Jiang et al., 2007; Zhang et al., 2010c). So the lineages of HTNV were numbered from #3 in Fig. 2B and C. Herein, four HTNV variants from Xinzhou and Jiangxia (Jianghan plain) formed a highly supported novel lineage, HTN-S-#10 (Fig. 2B). The partial M-segment sequences of these variants also clustered in the same pattern (Fig. 2C), and this lineage shared a common ancestor with viruses from Guizhou, Yunnan and Tianjin of China (Zhang et al., 2010a; Zou et al., 2008a). Taken together, three/four lineages of SEOV and one lineage of HTNV, based on partial S- and M-segment sequences, were identified from rodents in Hubei, China.
3.5. Molecular analysis of hantavirus sequences A pairwise comparison of partial SEOV S- and M-segment sequences from Hubei revealed a low level of divergence within lineages. Levels of the average nucleotide (amino acid) divergences of partial S-segment sequences in lineages SEO-S-#3, #7, and #8 ranged from 0.15% to 0.30% (0.00–0.33%), and those of partial Msegment sequences in lineages SEO-M-#3, #7, and #8 ranged from 0.13% to 0.37% (0.00–0.57%) (Table 2). Between the SEOV lineages from Hubei, the average nucleotide (amino acid) diversities of the partial S-segment ranged from 1.36% to 3.55% (0.00–1.10%), and those of the partial M-segment were higher, ranging from 2.55% to 5.48% (0.00–3.50%) (Tables 3 and 4). It is noteworthy that the partial M-segment sequence of MQ220 showed higher nucleotide (amino acid) divergence from other Hubei SEOV variants, ranging from 4.53% to 5.48% (2.10–3.50%) (Table 4). Comparing with the representative SEOV sequences in GenBank, the partial S- and M-segment nucleotide sequences of Hubei SEO variants were most closely related with strain Z37 from Zhejiang province, except for the partial M-segment of MQ2-20, which was more similar to strain L99 from Jiangxi province (Tables 3 and 4). The partial N protein amino acid sequences of SEOV variants in lineages SEO-S-#8, and samples MA35, MQ2-17 and MQ2-20 were identical with strain 80-39 from Japan. The partial Gn protein amino acid sequences of variants in lineage SEO-M-#7 and #9 were identical with that of strain Z37. Comparing with other representative HTNV strains, HTN-#10 showed nucleotide (amino acid) divergences ranging from 10.79% to 13.85% (0.69–3.43%), and 10.14% to 16.43% (1.40–9.79%) for partial S- and M-segments, respectively (Tables 3 and 4).
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Table 3 Nucleotide and amino acid percent divergence among lineages of analyzed partial S segments of hantaviruses and representative sequences from GenBank. SEO-S-#3
SEO-S-#7 3.21
1.10 0.92 0.92 0.92 0.92 0.37 0.92 1.47 3.11 0.37 2.01 24.68 23.44 23.99 24.54 25.64 25.09
0.18 0.18 0.18 0.18 0.73 0.18 0.73 2.38 0.73 1.28 24.86 23.63 24.18 24.73 25.82 25.27
SEO-S-#8 2.81 2.01 0.00 0.00 0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
MA35 3.32 1.83 1.97
MQ2-17 3.55 2.22 2.58 2.38
0.00 0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
0.00 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
MQ2-20
Z37
3.32 1.36 1.97 1.46 2.19 0.55 0.00 0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
2.45 2.40 2.76 2.56 2.74 2.56 0.55 1.10 2.75 0.00 1.65 24.31 23.08 23.63 24.18 25.27 24.73
80-39
SR-11
IR461
4.27 2.56 3.24 2.74 3.29 2.74 3.47
4.83 3.00 3.98 3.66 3.29 3.29 4.02 2.74
4.63 3.47 3.52 2.74 3.47 3.11 3.84 3.66 4.20
0.55 2.20 0.55 1.10 24.86 23.63 24.18 24.73 25.82 25.27
2.75 1.10 1.65 24.86 23.63 24.18 24.73 25.82 25.27
2.75 3.30 25.41 24.18 24.73 25.27 26.37 25.82
L99 4.51 3.29 3.98 3.66 3.66 3.29 3.66 3.66 3.84 4.20 1.65 24.31 23.08 23.63 24.18 25.27 24.73
Gou3
HTN-S-#10
76-118
Q32
84FLi
A9
Z10
13.89 12.43 13.21 12.80 13.16 12.61 13.89 12.98 12.98 13.35 12.61
28.32 28.02 28.02 28.47 27.97 28.43 28.38 28.66 28.66 28.56 28.47 30.30
29.31 28.52 28.88 29.25 28.52 28.15 28.70 29.25 30.16 29.07 28.15 30.16 13.85
29.54 29.43 29.58 29.80 28.88 29.80 29.62 29.98 29.25 30.16 28.52 29.62 10.79 16.27
31.20 30.71 31.03 31.44 31.26 31.26 30.90 31.44 31.08 30.90 30.35 31.26 11.06 14.99 9.14
28.82 28.70 28.70 29.25 28.15 28.88 28.70 29.62 28.34 29.07 28.70 29.25 11.84 17.00 11.70 12.98
28.05 27.79 27.97 28.34 27.61 27.61 28.15 28.34 27.79 28.88 27.97 30.16 11.79 15.36 10.97 10.97 11.33
25.41 24.18 24.73 25.27 26.37 25.82
2.88 1.24 0.69 3.43 1.79
2.75 2.20 3.85 2.75
0.55 3.30 1.65
2.75 1.10
3.30
Nucleotide divergence, above diagonal; amino acid divergence, below the diagonal. All results are based on the pairwise analysis of 41 sequences, with 524-nt positions corresponding to 174 aa in the final data set.
Table 4 Nucleotide and amino acid percent divergence among lineages of analyzed partial M segments of hantaviruses and representative sequences from GenBank. SEO-M-#3 SEO-M-#3 SEO-M-#7 SEO-M-#8 SEO-M-#9 MQ2-20 Z37 80-39 SR-11 IR461 L99 Gou3 HTN-M-#10 76-118 Q32 84FLi A9 Z10
SEO-M-#7 3.13
1.40 1.84 1.40 3.50 1.40 3.50 2.80 3.50 2.80 5.59 34.97 36.36 35.66 35.66 37.76 37.06
0.44 0.00 2.10 0.00 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
SEO-M-#8 3.67 2.55 0.44 2.53 0.44 2.53 1.84 2.53 1.84 4.63 34.70 36.10 35.40 35.40 37.50 37.50
SEO-M-#9 3.81 3.61 4.43 2.10 0.00 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
MQ2-20 4.66 4.53 5.48 4.82 2.10 4.20 3.50 4.20 3.50 4.20 34.27 34.97 34.97 34.27 36.36 36.36
Z37 2.16 2.90 3.85 3.03 4.20 2.10 1.40 2.10 1.40 4.20 34.27 35.66 34.97 34.97 37.06 37.06
80-39
SR-11
IR461
4.20 4.53 5.48 4.97 5.59 3.96
3.73 3.36 4.55 3.81 4.43 3.50 4.43
5.36 5.23 6.06 5.05 6.29 4.90 5.83 5.59
3.50 4.20 3.50 6.29 34.97 36.36 35.66 35.66 37.76 37.76
3.50 2.80 5.59 35.66 37.06 36.36 36.36 38.46 38.46
3.50 6.29 34.97 34.97 35.66 34.27 36.36 36.36
L99 3.26 3.13 4.08 3.57 3.73 3.03 4.66 3.50 4.90 5.59 34.97 35.66 35.66 34.97 37.06 37.06
Gou3
HTN-M-#10
76-118
Q32
84FLi
A9
Z10
14.22 14.92 14.71 14.69 15.15 13.99 14.45 14.22 15.62 13.99
30.05 31.49 31.50 31.27 28.90 30.07 30.42 31.12 30.19 30.07 31.53
31.47 32.30 32.81 32.40 31.00 31.93 31.00 32.17 32.40 31.70 29.60 14.92
32.80 33.80 32.90 33.72 32.87 32.40 32.17 33.57 32.63 33.33 31.93 10.14 16.78
31.70 32.17 31.96 32.40 31.70 31.47 31.47 31.93 31.70 31.70 33.57 15.50 17.02 14.92
33.50 34.27 34.82 34.50 32.87 32.87 33.80 34.03 34.03 33.80 34.27 16.43 16.55 17.72 12.12
30.70 31.14 31.09 31.24 31.47 30.54 30.54 31.70 30.54 30.77 31.70 16.26 16.32 16.08 13.99 13.29
33.57 33.57 34.27 33.57 35.66 35.66
5.59 1.40 6.99 9.79 9.79
6.99 7.69 9.79 11.89
5.59 9.09 10.49
7.69 9.09
8.39
Nucleotide divergence, above diagonal; amino acid divergence, below the diagonal. All results are based on the pairwise analysis of 41 sequences, with 436-nt positions corresponding to 145 aa in the final data.
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SEO-S-#3 SEO-S-#7 SEO-S-#8 MA35 MQ2-17 MQ2-20 Z37 80-39 SR-11 IR461 L99 Gou3 HTN-S-#10 76-118 Q32 84FLi A9 Z10
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3.6. Comparison of the amino acid sequences and identification of lineage-specific amino acid residues A comparison of the deduced partial N protein sequences spanning amino acids 183–368 (numbering in the N protein of strain Z37) of the novel SEOV variants with strain Z37 revealed three conservative amino acid exchanges: F225L in MA2, D231E in MA28 (both samples belonging to lineage SEO-S-#3), and T233A in MB1-7 (lineage SEO-S-#7). A comparison of the deduced signal peptide and N terminus of Gn protein covering amino acid positions 3–148 (numbering in glycoprotein precursor of strain Z37) with strain Z37 revealed more exchanges than for the partial N protein (Supplementary Fig. S1). In lineage SEO-M-#3, all the samples from Jiangxia (Jianghan plain) had conservative amino acid exchanges L6F and V11I. In the novel lineage SEO-M-#8, four samples had amino acid exchanges E58D, and one sample (MQ2-17) had an A8T amino acid substitution. The sample MQ2-20 had three unique amino acid substitutions, G14D, F15V, and R24K. Comparing with closely related strain Q32, the deduced partial N protein sequences spanning amino acids 194–368 (numbering as for strain Q32) of variants in lineage HTN-S-#10 showed a V296I substitution, which falls in the epitope region for monoclonal antibody C24B4 and may affect the antigenicity of N protein (Yoshimatsu et al., 1996). Another substitution, V307I, was observed for members in lineage HTN-S-#10 except for HV004. The deduced partial Gn protein sequences of HTNV variants showed conservative amino acid exchanges as T55A and I116V in all members in lineage HTN-M-#10.
4. Discussion In this paper, we describe a comprehensive study on the presence of HTNV and SEOV in four geographically diverse HFRS epidemic areas of Hubei, central-south China from 2000 to 2009. In the past decade, although the geographic area of epidemics was expanding, the incidence and mortality of HFRS had declined in China (Chen and Qiu, 1993; Zhang et al., 2010c). Genetic analyses of the endemic hantaviruses and their hosts will be helpful for understanding the changing epidemiology of HFRS. In this study, the majority of collected rodents in Hubei were Brown Norway rats, and the corresponding SEOV was predominant hantavirus. Similar patterns had been observed in other regions of China (Dai et al., 2007; Zhang et al., 2010b; Zhou et al., 2009) in recent years. In addition, HFRS infections caused by SEOV have emerged in northern China (Inner Mongolia and Beijing) where it had not been reported before (Jiang et al., 2008; Zhang et al., 2009b), suggesting the expansion of SEOV epidemic areas. In the past two decades, China experienced fast socioeconomic development and urbanization progress, which might increase the migration frequencies of rats and give them more chances to contact with humans. So ratcarried SEOV may have more opportunities to be transmitted to humans than field mouse-carried HTNV. It is known that human SEOV infections cause a milder form of HFRS, and the percentage of inapparent infection is higher than for HTNV (Chen et al., 1986). We presume that besides comprehensive preventive measures, the increase in the prevalence of SEOV is part of the reasons for the declined incidence and mortality of HFRS in China. Our data revealed that multiple phylogenetic lineages (#3, #7, #8, and #9) of SEOV co-circulate in rodents in Hubei, which is remarkable for the epidemiology of SEOV. SEOVs belonging to lineages #1 and #4 had been identified from HFRS patient and rodents in Hubei before (Liang et al., 1994; Xiong et al., 2010). Taken together, there are at least six phylogenetic lineages of SEOVs in Hubei, including three already known (#1, #3 and #4) and three
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novel lineages (#7–#9). It suggested that Hubei province is an important epidemic focus of SEOV. Unlike other hantaviruses, SEOV has a worldwide distribution, both in the Old and New Worlds. Most of the related HFRS cases are found in China, but little is known about the origin of this virus. By locating the geographic distribution of phylogenetic lineages of SEOV, we found that multiple lineages, namely #1 (Shi et al., 2003), #3 (Zhang et al., 2010b), #4 (Wang et al., 2000), #5 (Wang et al., 2000), and #7–#9, circulate in middle and lower reaches of the Yangtze River (involving six provinces: Hubei, Hunan, Jiangxi, Anhui, Zhejiang, and Jiangsu). However, only two or three lineages of SEOV are distributed in other parts of China, such as #1, #3 and #4 in the northeast (Zhang et al., 2009a,b); #1–#3 in the North China Plain (Sun et al., 2005; Zuo et al., 2008); and #1 and #2 in South China (Wang et al., 2000; Yang et al., 2006). In addition, the variants in SEO-#5 which are from Zhejing province, show higher genetic diversity from the majority of SEOVs (Wang et al., 2000) and occupy a basal position in the phylogenetic tree, indicating an early evolutionary divergence. In the other countries of the world, only lineages #4 and #6 were found (Heyman et al., 2004; Shi et al., 2003; Xiao et al., 1994). The abundance of the phylogenetic lineages of SEOV in the middle and lower reaches of the Yangtze River demonstrates that these regions are important epidemic nidi for SEOV. Our molecular analysis also supports this presumption. Most of the known SEOV variants (from China as well as those from Japan, South Korea, the U.S. and United Kingdom) are genetically homogeneous, irrespective of geographic distribution, suggesting these viruses may share a recent common ancestor and distribute following the expansion of R. norvegicus. Studies in France (Heyman et al., 2004) and the United States (Mantooth et al., 2001) indicated that SEOVs were not indigenous and were introduced from Asia into the new endemic areas in associated with their hosts. The current distribution of hantaviruses is the result of migration and the natural history of their rodent hosts. The discovery of fossils demonstrate that R. norvegicus originate in lower reaches of Yangtze River and then expanded to other parts of China in late Pleistocene (Wu et al., 2008). The middle and lower reaches of Yangtze River are mainly consisted of alluvial plains and hills. The climate is north subtropical, with plenty of rainfall. These areas had been culture centers of China in the history over 2500 years ago. These ecological conditions are favorable for the thriving of rats. The prosperous commercial trading and well developed waterway transportation may assist in the global distribution of R. norvegicus in the Holocene. Taken together, we propose that the middle and lower reaches of the Yangtze River are radiation centers for most known phylogenetic lineages of SEOVs. The worldwide dispersion of Brown Norway rats may spread ancestors of the current SEOVs around the world. Phylogenetic analyses based on partial S- and M-segment sequences indicated 4 probable inter-lineage reassortants of SEOV. The frequency (13.3%, 4/30) of the reassortment is not significantly different when compared with two other observations: 10.0% (2/20, Fisher’s Exact Test P = 1.00) was reported previously in SNV from North America (Black et al., 2009) and 32% (8/25, Fisher’s Exact Test P = 0.11) in PUUV in northern Finland (Razzauti et al., 2009). It suggests that the corresponding hosts from Jiangxia, Xinzhou (both in Jianghan plain) and Qichun (southeast low hills) had close interactions and viruses were transmitted among them horizontally. Since hantaviruses possess segmented genomes, the possibility of genetic reassortment between different hantavirus variants to achieve high infectivity and adapt to new hosts exists, as already known for influenza viruses. L segment sequences and longer sequences for S- and M-segments are needed to confirm the reassortment among the viruses. And further surveillance should be carried out to clarify
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whether these reassortant variants will disappear or exist persistently, or even cause severe epidemiologic consequences. Herein, we found for the first time that SEOV existed simultaneously in A. agrarius and R. norvegicus, suggesting that a spillover event of SEOV might occur naturally in Hubei. The spillover sample MX81 was a reassortant with the S-#8–M-#9 type. Our data suggested an inter-species interaction between A. agrarius and SEOV-infected R. norvegicus. Previous ecological surveillance in the middle and lower reaches of the Yangtze River showed that striped field mice live in fields, while Brown Norway rats live both in the field and houses, and migrate between the two habitats seasonally (Li et al., 2005a; Zhang et al., 2003). Hantavirus could spill over into sympatric and syntopic species, particularly during irruption in rodent reservoir population (Kang et al., 2009). However, more evidences are needed to confirm the spillover infection of SEOV to A. agrarius. In addition to SEOVs, HTNVs were also identified from rodents in this study. HTNV strains belonging to lineages #7 and #9 (Wang et al., 2000) had been detected from HFRS patients in Hubei previously (Ding et al., 2003; Xu et al., 2004). Herein, a new distinct genetic lineage HTN-#10 consisted of variants from Jianghan plain was formed, which is closely related with strains from the North China plain and the Yunnan-Guizhou plateau in the southwest. However, HTNV belonging to this novel lineage had not been detected in HFRS patients yet. Further studies are warranted to illuminate the pathogenic role of this new HTNV. In conclusion, this study demonstrates that SEOV and HTNV were co-circulating in Hubei. There were at least three phylogenetic lineages of SEOVs circulating in Hubei. A novel genetic lineage of HTNV was recovered in rodents. Based on phylogenetic analysis of SEOVs and rodent fossils discovered before, we propose that the middle and lower reaches of Yangtze River are radiation centers for most known phylogenetic lineages of SEOVs. Probable inter-lineage reassortment and spillover infection of SEOV were observed. Further studies are warranted to clarify the biological characteristics of the new found hantavirus variants and the epidemiology impact of them.
Acknowledgements This work was supported by the National High Technology Research and Development 863 Program of China (No. 2007AA02Z465), a grant from the National Natural Science Foundation of China (NSFC project No. 30770096), National Natural Science Foundation of Jiangsu province (BK2010537), and the Open Grant of State Key Laboratory of Virology (2010006), Scientific Research Foundation of the State Education Ministry for Returned Chinese Scholars (30115), and the Fundamental Research Funds for the Central Universities (3082003).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.11.006.
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