Journal of General Virology (2008), 89, 1987–1997
DOI 10.1099/vir.0.2008/000497-0
Molecular diversity and phylogeny of Hantaan virus in Guizhou, China: evidence for Guizhou as a radiation center of the present Hantaan virus Yang Zou,1,2 Jing Hu,3 Zhao-Xiao Wang,3 Ding-Ming Wang,3 Ming-Hui Li,1 Guo-Dong Ren,1 Zheng-Xiu Duan,1 Zhen F. Fu,4 Alexander Plyusnin5 and Yong-Zhen Zhang1 Correspondence Yong-Zhen Zhang
[email protected]
1
Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping Liuzi 5, 102206 Beijing, PR China
2
Beijing Friendship Hospital, Affiliate of Capital Medical University, Beijing, PR China
3
Guizhou Center for Disease Control and Prevention, Guiyang, Guizhou Province, PR China
4
Department of Pathology, University of Georgia, Athens, GA 30602, USA
5
Department of Virology, Haartman Institute, University of Helsinki, Finland
Received 18 January 2008 Accepted 10 April 2008
To gain further insight into the molecular epidemiology of Hantaan virus (HTNV) in Guizhou, China, rodents were captured in this region in 2004 and 2005. In addition, serum samples were collected from four patients. Ten hantaviruses were isolated successfully in cell culture from four humans, two Apodemus agrarius, three Rattus norvegicus and one Rattus nitidus. The nucleotide sequences for their small (S), medium (M) and partial large (L) segments were determined. Phylogenetic analysis of the S and M segment sequences revealed that all of these isolates belong to the species HTNV, suggesting a spillover of HTNV from A. agrarius to Rattus rats. All available isolates from Guizhou were divided into four distinct groups either in the S segment tree or in the M segment tree. The clustering pattern of these isolates in the S segment tree was not in agreement with that in the M or L segment tree, showing that genetic reassortment between HTNV had occurred naturally. Analysis of the S segment sequences from available HTNV strains indicated that they formed three clades. The first clade, which comprised only viruses from Guizhou, was the outgroup of clades II and III. The viruses in the second clade were found in Guizhou and mainly in the far-east Asian region, including China. However, the viruses in the third clade were found in most areas of China, including Guizhou, in which haemorrhagic fever with renal syndrome (HFRS) is endemic. Our results reveal that the highest genetic diversity of HTNV is in a limited geographical region of Guizhou, and suggest that Guizhou might be a radiation centre of the present form of HTNV.
INTRODUCTION Haemorrhagic fever with renal syndrome (HFRS) is an important human disease; 60 000 to 100 000 cases requiring hospitalization are reported annually worldwide, with the majority occurring in China (Johnson, 1999; Zhang et al., 2004). The disease is caused by hantaviruses, members of the genus Hantavirus in the family Bunyaviridae (Nichol et al., 2005). The virus genome consists of three separate segments of negative-stranded RNA referred to as small (S), medium (M), and large (L) segments, which encode GenBank accession numbers of the S, M and L segment sequences determined in this study are given in Table 1. Two supplementary tables and a supplementary figure are available with the online version of this paper.
2008/000497 G 2008 SGM Printed in Great Britain
nucleocapsid protein (NP), two envelope glycoproteins (Gn and Gc) and viral RNA-dependent RNA polymerase (RdRP), respectively (Plyusnin et al., 1996). In contrast to other genera of the Bunyaviridae, hantaviruses are not transmitted by arthropods (Schmaljohn & Hjelle, 1997). They establish a chronic infection that causes no apparent harm to rodents of the Muridae family, which are their natural hosts (Childs et al., 1989; Hutchinson et al., 2000; Kurata et al., 1983; Li et al., 1995; Netski et al., 1999). Currently, at least 22 distinct hantavirus species have been identified worldwide; all of these hantaviruses except Thottapalayam virus (TPMV) are carried by rodents (Nichol et al., 2005). Each rodent-borne hantavirus species appears to be associated primarily with one (or a few closely related) rodent species (Nichol et al., 2005; Plyusnin 1987
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& Morzunov, 2001). Most of the current data support the notion that hantaviruses have co-evolved with their respective rodent hosts during the long-term evolution of this group of viruses (Morzunov et al., 1998; Nichol et al., 2005; Plyusnin et al., 1996; Plyusnin & Morzunov, 2001). Guizhou province is located in the south-western part of China. The province has always been one of the most seriously affected areas in China since the first hantavirus outbreak was reported in 1962 (Wang et al., 1989, 2003; Zhang et al., 2004). More than 5000 HFRS patients were reported in 1985 alone (Wang et al., 1989). Forty-five species of rodents have been found in Guizhou (Li et al., 1999) and during the past two decades, hantavirus-reactive antibodies and/or antigens have been identified in at least 12 rodent species in Guizhou (Chen et al., 1999; Wang et al., 1989, 2003). However, the mouse Apodemus agrarius has been identified as the major reservoir of hantavirus. Phylogenetic analysis of partial M and S segment sequences indicates that Hantaan virus (HTNV) displays high genetic diversity in Guizhou (Wang et al., 2000) and at least three distinct phylogroups of HTNV and one new variant of Seoul virus (SEOV) have been found to circulate in Guizhou (Zou et al., 2008). Spillover of HTNV from A. agrarius to Rattus norvegicus has been reported in Guizhou. Circumstantial evidence suggests that genetic reassortment between HTNV and SEOV has also occurred naturally during or after the spillover. In addition, Guizhou is the main constituent of the Yunnan–Guizhou plateau, which may be a significant factor in lineage isolation of Apodemus spp. (Xia, 1984; Suzuki et al., 2003). Thus, further studies of hantaviruses in Guizhou would be helpful to clarify the phylogeny of HTNV and to shed light on the prevention and control of the diseases it causes.
Sampling and screening, and virus propagation. Rodents were captured in HFRS endemic areas in Guizhou with snap-traps, which were set at five metre intervals and baited with peanuts (Fig. 2), between the spring of 2004 and the autumn of 2005. Lung tissue was obtained from trapped animals. Serum samples were obtained from two patients with acute HFRS, which was diagnosed by using clinical criteria and the presence of IgM antibodies to HTNV. Hantavirusspecific antigens in rat lungs were detected by indirect immunofluorescence assay (IFA), as described by Lee et al. (1978). Antigenpositive lung tissues were homogenized. The supernatants and serum samples from HFRS patients were used to inoculate Vero E6 cell monolayers as described by Lee (1999). After 2 h adsorption, the tissue suspension and the sera were removed, and maintenance medium [Dulbecco’s modified Eagle medium supplemented with 2 % heat-inactivated fetal calf serum (FCS), 100 mg penicillin ml21 and 100 mg streptomycin ml21] was added to the cells. Cells were incubated at 37 uC with 5 % CO2 in an incubator. On day 21 postinoculation (p.i.), cells were suspended by trypsin treatment, and part of the suspended cells was cultured with fresh Vero E6 cells. A sample of the cells was fixed onto glass slides for detection of hantavirus antigen by IFA. The four viruses that were isolated in 1986 (above) were propagated in Vero E6 cells. RT-PCR. Primer P14 (Schmaljohn et al., 1986) was used for reverse transcription of the S, M, and L segments from total RNA by using avian myeloblastosis virus reverse transcriptase (Promega). The S segment was amplified by using the primers S1 and S2 (Puthavathana et al., 1992). The M segment was amplified as two overlapping fragments using two initial primer pairs, M1 and HV-MFO and HVMRO and M4 (Shi et al., 1998; Sun et al., 2005), for the first round of amplification. In the second round of amplification, the primer pairs M1 and HMF, and HMR and M4 (Shi et al., 1998; Sun et al., 2005) were used to amplify the two fragments for HTNV. Partial L segment [nucleotides (nt) 2750–3200] sequences were amplified by using primer pair HTN-L-F1 and HTN-L-R1 for initial PCR and primer pair HTN-L-F2 and HTN-L-R2 for the second round of amplification (Klempa et al., 2006).
In the present study, we recovered the complete S and M sequences and also partial L segment sequences from the Guizhou HTNV variants isolated both in this study and previously, and compared these sequences with those of HTNV isolates reported previously in China, far-eastern Russia and South Korea. Our data indicated that there are at least four distinct phylogroups of HTNV in Guizhou. Further analysis of the S segment sequences showed that the HTNV isolates from China, far-eastern Russia and South Korea form six distinct phylogroups, which cluster into three clades. The viruses in clade I have only been found in Guizhou; these became the outgroup of clades II and III. The viruses in the second clade are found in Guizhou and mainly in far-eastern Asia (China, Russia and South Korea). The viruses in the third clade are distributed in most HFRS areas of China, including Guizhou.
PCR products were separated by electrophoresis and purified from gel slices by using the agarose gel DNA purification kit (TaKaRa Biotechnology) according to the manufacturer’s instructions. Purified DNA fragments were cloned into the pMD18-T vector (TaKaRa Biotechnology). The ligated products were transformed into Escherichia coli JM109 competent cells. DNA sequencing was performed with the ABI-PRISM Dye Terminator Sequencing kit and an ABI 373A Genetic Analyzer. The nucleotide sequences of at least two clones from each isolate were determined.
METHODS
RESULTS
Phylogenetic analysis. The
PHYLIP program package (version 3.65) was used to construct phylogenetic trees by using the maximumlikehood (ML) and the maximum-parsimony (MP) methods with 1000 bootstrap replicates. Alignments were prepared with CLUSTAL W (version 1.83) by first translating nucleotide sequences into amino acid sequences with DNASTAR (version 5.01). Nucleotide or amino acid identities were also calculated by using DNASTAR. The hantavirus sequences available in GenBank that are listed in Table 1 were also retrieved for analysis.
Virus. The four hantaviruses (CGAa4MP9, CGAa4P15, CGAa1011
and CGAa1015) used in this study were isolated from A. agrarius in Guizhou in 1986. These viruses were stored in the Department of Hemorrhagic Fever of the Institute of Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, China. 1988
Screening of rodents and isolation of hantaviruses from the rodent lung tissues A total of 301 rodents (106 A. agrarius, 157 R. norvegicus and 38 Rattus nitidus) were captured between 2004 and Journal of General Virology 89
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Fig. 1. Phylogenetic trees of hantaviruses based on S (a), M (b) and partial L (c) segment sequences. The trees were constructed by using the maximum-likehood (ML) method. Dobrava virus (DOBV) was an outgroup in the trees. Numbers above branches are percentage bootstrap support values for 1000 replicates; only values greater than 50 % are shown. Sequences obtained from Guizhou are shown in bold.
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Table 1. Hantavirus strains used for analysis in this study Strain
Source
Location
GenBank accession no. S
HTNV AA1719 AA2499 AA1028 Bao14 HTN261 CJAp93 CGHu1 S85-46 LR1 CUMC-B11 76-118 Lee CFC94-2 Maaji-2 CGRn2616 CGHu2 CGRni1 CGAa2 CGRn2618 CGRn45 CGAa31P9 CGRn15 CGHu5 CGHu4 A16 TJJ16 CGAa75 CGHu3 CGAa31MP7 Q32 CGRn9415 CGRn8316 CGRn5310 CGRn93P8 CGRn93MP8 CGRn53 A9 Hu Z10 Z5 CGAa1015 CGAa1011 CGAa4MP9 CGAa4P15 84FLi Chen4 SN7 RG9 E142 AP1371 AP1168 AP708 Solovey-AP61-1999 Solovey-AP63-1999
1990
A. agrarius A. agrarius A. agrarius A. agrarius A. agrarius A. peninsulae Human A. agrarius Vaccine Not known A. agrarius Human Human Human R. norvegicus Human R. nitidus A. agrarius R. norvegicus R. norvegicus A. agrarius R. norvegicus Human Human A. agrarius N. confucianus A. agrarius Human A. agrarius A. agrarius R. norvegicus R. norvegicus R. norvegicus R. norvegicus R. norvegicus R. norvegicus A. agrarius Human Human Not known A. agrarius A. agrarius A. agrarius A. agrarius Human Human N. confucianus Vaccine E. eleusis A. peninsulae A. peninsulae A. peninsulae A. peninsulae A. peninsulae
Khabarovsk, Russia Khabarovsk, Russia Khabarovsk, Russia Heilongjiang, China Hainan, China Jilin, China Guizhou, China Sichuan, China China Korea South Korea South Korea Korea Korea Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Shaanxi, China Tianjin, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Jiangsu, China Hubei, China Zhejiang, China Zhejiang, China Guizhou, China Guizhou, China Guizhou, China Guizhou, China Shaanxi, China Anhui, China Sichuan, China Guangzhou, China Yunnan, China Khabarovsk, Russia Khabarovsk, Russia Khabarovsk, Russia Solovey, Russia Solovey, Russia
AF427319 AF427320 AF427318 AB127998 AF252259 EF208929 EU092218 AF288659 AF288294 U37768 M14626 2 X95077 AF321095 EU363811 EU363813 EU363812 EU092219 EU363808 EU092221 EF990910 EU363810 EU990908 EU990909 AF288646 AY839871 EU092220 EU363809 EF990911 AB027097 EF990902 EF990903 EF990906 EF990904 EF990905 EF990907 AF329390 AB027111 AF184987 EF103195 EF990912 EF990913 EF990915 EF990914 AY017064 AB027101 AF288657 AF288296 AF288644 AF427324 AF427323 AF427322 AB071183 AB071184
M 2 2 2 AB127995 2 EF208930 EU092222 AF288658 AF288293 U38177 M14627 D00377 2 2 EU363816 EU363819 EU363815 EU092223 EU363817 EU092225 EF990924 EU363814 EU990922 EU990923 AF288645 EU074672 EU092224 EU363818 EF990925 DQ371905 EF990916 EF990917 EF990920 EF990918 EF990919 EF990921 AF035831 2 AF276987 EU074224 EF990926 EF990927 EF990929 EF990928 AF345636 2 AF288656 2 2 2 2 2 2 2
L 2 2 2 2 2 EU092230 EU092226 2 2 2 X55901 2 2 2 2 2 2 EU092227 2 EU092229 EF990938 2 EF990935 EF990937 2 2 EU092228 2 EF990939 DQ371906 EF990930 EF990931 EF990934 EF990932 EF990933 EF990935 AF293665 2 AF189155 2 EF990940 EF990941 EF990943 EF990942 AF336826 2 2 2 2 2 2 2 2 2
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Table 1. cont. Strain
Source
Location
GenBank accession no. S
H5 B78 Liu JilinAp06 Hojo HV-114 H8205 SC-1 SC-2 SEOV L99 DOBV Dobrava-Belgrade Poroia
L
Human Human Human A. peninsulae Human Human Human A. peninsulae A. peninsulae
Heilongjiang, China Shandong, China Shandong, China Jilin, China South Korea China China South Korea South Korea
AB127996 AB127997 AF288649 EF121324 2 2 2 AY675349 2
AB127993 AB127994 AF288648 EF371454 D00376 L08753 AB030232 AY675353 DQ056293
2 2 2 2 2 2 2 DQ056292 AY675354
R. loseda
Jiangxi, China
AF288299
AF288298
AF288297
A. flavicollis A. flavicollis
Slovenia Greece
L41916 2
L33685 2
– AJ410617
2005 in Guizhou. The hantavirus antigen was detected in tissue samples from two A. agrarius, three R. norvegicus and one R. nitidus. These hantavirus-positive lung tissues were homogenized. The supernatants from the lung homogenates were inoculated onto Vero E6 cells. At 55–63 days p.i., hantavirus antigen-positive cells were found by IFA in cells inoculated with each of the samples. These isolates from A. agrarius (two), R. norvegicus (three) and R. nitidus (one) were designated CGAa31MP7, CGAa31P9, CGRn15, CGRn2616, CGRn2618 and CGRni1, respectively. Serum samples collected from four patients with acute HFRS were treated similarly to the samples above. Four isolates from humans were identified and designated CGHu2, CGHu3, CGHu4, CGHu5. Molecular diversity and phylogenetic relationship of HTNV strains from Guizhou Complete S and M sequences and partial L segment sequences were determined for all ten isolates, as well as for four viruses isolated previously (CGAa4MP9, CGAa4P15, CGAa1011 and CGAa1015). Phylogenetic ML trees were constructed by using the S and M segment sequences from Guizhou that were recovered in the present study and in previous studies (Table 1; available in GenBank) (Fig 1a, strains in bold type were recovered in this study). This tree indicates that all known isolates from Guizhou, regardless of their source, belong to HTNV and could be divided into four distinct groups (S1, S2, S3 and S4). These data also suggest that the spillover of HTNV occurred naturally from A. agrarius to R. norvegicus or R. nitidus. The first group contained only one human isolate . The Guizhou viruses in the second group were divided into two distinct lineages. The Guizhou viruses in the third group could be also divided into two lineages. Phylogroup S4 comprised only viruses from Guizhou, and these viruses were further http://vir.sgmjournals.org
M
divided into two distinct lineages, suggesting a high degree of molecular diversity of HTNV in Guizhou. In the phylogenetic tree generated from the M segment sequences (Fig. 1b), all the Guizhou isolates belonged to HTNV except the reassortants CGRn8316 and CGRn9415 (Zou et al., 2008). Analysis of the M segment sequences also divided the Guizhou HTNV variants into four groups (M1, M2, M3 and M4). However, the relationship among these variants was different from that in the phylogenetic tree based on S sequences. The S4 variants, which are distinct from the other three groups in the tree based on the S segment, clustered together with the viruses (CGAa2, CGAa75, CGRn15, CGRn45, CGRn2616, CGRn2618 and CGHu3) from S2, and comprise the group M4. These data suggest that the genetic reassortment within HTNV might have occurred naturally. In order to gain more insights into hantavirus reassortment, partial L segment sequences (nt 2750–3200) were recovered and analysed. Similar to the phylogenetic trees based on the S and M segment sequences, all Guizhou HTNV strains were divided into four groups (L1, L2, L3 and L4) (Fig. 1c). The clustering of the Guizhou viruses in the L tree was congruent with their clustering in the M tree, but different from the clustering in the S tree. These data supported our hypothesis that genetic reassortment within HTNV occurs in nature. Comparison of the S sequences revealed that the nucleotide divergence among all Guizhou HTNV isolates ranged from 0.1 to 16 % (see Supplementary Table S1, available in JGV Online). The amino acid divergence varied from 0.2 to 4 %. There was no significant amino acid difference among groups except between group S1 and other groups. The M segment nucleotide divergence among all Guizhou HTNV isolates varied from 0.1 to 17 %, similar to the S segment divergence, but the amino acid difference varied from 0.4 1991
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to 7 %, and was higher than that for the S segment (see Supplementary Table S2). Furthermore, the divergence among groups ranged from 10 to 17 % at the nucleotide level and from 5 to 7 % at the amino acid level, and the difference among lineage within group varied from 5 to 7 % at the nucleotide level and from 1 to 2 % at the amino acid level, respectively. These data suggest that more nonsynonymous substitutions occurred in the M segment than in the S segment. The geographical distribution of HTNV in Guizhou Overall, HTNV strains clustered geographically in Guizhou (Fig. 2). The first group (S1) consisted of only one isolate, CGHu1, which was derived from a human and was found in Zunyi; the third group (S3) had six isolates, which were detected in Anshun and Rongjiang; and viruses of the fourth group (S4) were detected in Changshun. In contrast, the group S2 viruses were distributed widely, and were found in most of the HFRS endemic areas of the province (Cengong, Kaiyang, Shiqian, Xingyi and Zunyi). Furthermore, the viruses isolated between 2001 and 2005 also belong to group S2. These data suggest that this group has been predominant in Guizhou in recent times. Phylogenetic relationship of HTNV from Guizhou with those from other parts of China To determine the phylogenetic relationship between the Guizhou isolates and those from other parts of China and far-eastern Asia, we also analysed all available S and M segment sequences of HTNV strains from Guizhou and those distributed in other parts of China, far-eastern Russia and South Korea (Table 1). In the phylogenetic tree based on the S segment sequences, all HTNV isolates formed
three clades (Fig. 1a). The first clade consisted of viruses (group S4) derived only from A. agrarius in Guizhou. The second clade comprised two groups (S1 and S6), derived mainly from humans, A. agrarius and A. peninsulae in north-eastern China (Sun et al., 2001; Wang et al., 2000; Zhang et al., 2007), as well as from far-eastern Russia (Lokugamage et al., 2004) and South Korea (Baek et al., 2006; Lee et al., 1978), both of which share borders with China. The third clade included three groups (S2, S3 and S5), derived mainly from A. agrarius in HFRS endemic areas of China including Guizhou. The viruses from S2 grouped together with isolates A16 from A. agrarius in Shaanxi (Yao et al., 2001a) and TJJ16 from Niviventer confucianus in Tianjin (Li et al., 2005). Interestingly, the Guizhou viruses from S3 formed a group with the isolates Z5 derived from A. agrarius (sequence can be found under GenBank accession number EF103195), Z10 derived from a human in Zhejiang (Yao et al., 2001b), A9 from A. agrarius in Jiangsu (Shi et al., 1998), which shares borders with Zhejiang, and Hu isolated from a human in Hubei (Wang et al., 2000). The group S5 in the third clade contains strains 84FLi from a human in Shaanxi, Chen4 from a human in Anhui (Liang et al., 1994), SN7 from N. confucianus in Sichuan (Yao et al., 2002) and E142 from Eothenomys eleusis in Yunnan (this sequence can be found under GenBank accession number AF288644). The tree constructed by using the MP method had the same topology as that constructed by the ML method, supported by higher bootstrap values (Supplementary Fig. S1). In contrast to the topology of the S tree, all HTNV isolates formed seven distinct phylogroups and showed monophyletic ancestry in the tree constructed from the M segment sequences (Fig. 1b). Furthermore, strains Z5 (EF103195) and Z10 (Yao et al., 2001b) formed group M7, which was different from the other S3 viruses, although there was only weak bootstrap support for the M7 group. The tree constructed by using the MP method had a similar topology to that constructed by using the ML method (Supplementary Fig. S1). However, the viruses in group M7 clustered together with the viruses in group M3 in the MP tree. Multiple alignments of the deduced amino acid sequences of the M and S coding region
Fig. 2. Map of Guizhou, showing capture sites of A. agrarius, R. norvegicus and R. nitidus and the geographical distribution of HTNV S, M and L phylogroups. 1992
It has previously been reported that all genetic lineages of Puumala virus (PUUV) possess specific amino acid ‘signatures’ in the NP sequences (Sironen et al., 2001). Although a high degree of nucleotide variation of the S segment is present among different groups, only a few nonsynonymous substitutions were observed for the NP sequence. Furthermore, unlike PUUV, groups S3 and S5 do not possess specific amino acid ‘signatures’. The other groups possess specific amino acid ‘signatures’ such as group S1, aa 52 changes from AAV; group S2, aa 295 IAV; group S4, aa 241 SAG; and group S6, aa 9 RAK, aa 28 AAR, aa 215 IAV and aa 416 VAL. In addition, the Journal of General Virology 89
Genetic characterization of hantavirus from Guizhou
following non-synonymous substitutions are shared by several groups: substitution at aa 43 (TAA) is shared by group S3 strains except Z5 and Z10, nearly half of the viruses in group S2 and most of the viruses in group S6; at aa 124 (IAV) by groups S1 and S6; at aa 256 (HAL) by groups S1 and S4; at aa 290 (SAA) by groups S1 and S3 except the strains Z5 and Z10; and at aa 290 (SAT) by groups S4 and S6, and strains Z5 and Z10 from group S3. Comparing GnGc protein sequences (encoded by the M segment) may provide clues to antigenic, as well as genetic, diversity (Schmaljohn & Hjelle, 1997). All genetic groups possess specific amino acid ‘signatures’ in the deduced GnGc protein sequence, which are displayed in bold in Fig. 3. Interestingly, groups M3 and M7 shared a total of 14 identical non-synonymous substitutions, and groups M5 and M7 also share eight identical nonsynonymous substitutions, suggesting that these groups may be closely related.
DISCUSSION To gain a better understanding of the molecular diversity of HTNV isolates from Guizhou and of their evolutionary relationship with HTNV from other parts of China and fareastern Asia, S, M and partial L segment sequences were determined for a total of 14 isolates obtained from patients and from rodents (A. agrarius, R. norvegicus and R. nitidus). Phylogenetic analysis of these sequences revealed that at least four distinct groups of HTNV are cocirculating in Guizhou. Further analysis of the S segment sequences indicated that the available HTNV isolates could be divided into three clades. In particular, the first clade, which has been found exclusively in Guizhou, formed an outgroup for the other two clades. Genetic analysis of S and M segments from 14 isolates showed a high degree of genetic diversity of HTNV in Guizhou. The previous phylogenetic analysis of partial M segment sequences of the isolates showed that four distinct
Fig. 3. Multiple alignments of amino acid sequences of the M segment. * some strains in the group do not contain the signatures. Bold type, specific amino acid ‘signatures’ in the deduced GnGc protein sequence. http://vir.sgmjournals.org
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lineages of HTNV are present in A. agrarius in Guizhou (Wang et al., 2000). Recently, we found three distinct phylogroups of HTNV in Guizhou (Zou et al., 2008). In the present study, phylogenetic analysis of S and M segment sequences indicated that HTNV isolates could be divided into four distinct phylogroups and seven distinct lineages (Fig. 1). These viruses show up to 17 % divergence at the nucleotide level and up to 7 % at the amino acid level for the M segment. Furthermore, there is evidence that genetic reassortment has occurred for group S4 viruses and viruses from group S2 (CGHu3, CGAa2, CGAa75, CGRn15, CGRn45, CGRn2616, and CGRn2618, see below for explanation). Therefore, there may be two unrecognized groups of HTNV co-circulating in the rodent population in Guizhou. In addition, the S segment sequences from group S4 viruses, which have not been found in other parts of China or far-east Asia, are distinct from those of all other known HTNV. These data may suggest that a new variant of HTNV may circulate in rodents in Guizhou. In addition, group S5 virus has been detected recently in Guizhou (data not shown). Thus, a total of six phylogroups of HTNV could be distinguished by using S and M segment sequences, which indicates a remarkably high genetic diversity of HTNV within a small geographical region (Fig. 2). Several studies have shown that genetic reassortment can occur naturally within hantaviruses (Henderson et al., 1995; Li et al., 1995) or experimentally between hantaviruses (Razzauti et al., 2008; Rizvanov et al., 2004; Rodriguez et al., 1998). This genetic reassortment occurs more frequently between closely related strains than genetically distant hantaviruses (Henderson et al., 1995; Li et al., 1995; Khaiboullina et al., 2005; Razzauti et al., 2008). Recently, we provided evidence that genetic reassortment between HTNV and SEOV has occurred naturally in Guizhou (Zou et al., 2008). In the present study, the clustering pattern of the viruses isolated from Guizhou in the tree based on S segment sequences was in disagreement with that in the trees based on either M or partial L segment sequences. It was obvious that the viruses in group S2 could be divided into two distinct groups in the trees constructed with the M or partial L segment (Fig. 1). Particularly, all viruses in group S4 were also grouped into group M2 in the tree based on the M segment sequence, and into group L4 in the tree based on partial L segment sequences. For these viruses, their respective amino acid ‘signatures’ on the NP and the GnGc protein sequences also suggested that genetic reassortment had occurred between groups within HTNV. These data indicate that genetic reassortment has occurred within HTNV in Guizhou. In addition, phylogenetic analysis of these sequences also revealed that virus A16 isolated from A. agrarius in Shaanxi province was a reassortant (Fig. 1). Together, our results suggest that genetic reassortment of hantaviruses may be more common than expected when closely related rodent species are sympatric (Zou et al., 2008) and that it is one of the mechanisms that generate HTNV genetic diversity. 1994
Similar to other members of the family Bunyaviridae, the hantaviral NP plays an important role in virus replication (Blakqori et al., 2003; Flick et al., 2003; Flick & Pettersson, 2001; Kaukinen et al., 2005). Glycoproteins are known to mediate cell attachment and fusion (Tsai et al., 1984; Arikawa et al., 1985; Ogino et al., 2004; Okuno et al., 1986; Tischler et al., 2005) and are presumed to be the major element involved in induction of neutralizing antibodies during hantavirus infection (Khaiboullina et al., 2005). Although the nucleotide sequences of the M and S segments of any two HTNV have approximately the same degree of divergence (Supplementary Tables S1 and S2), the amino acid sequences of the S segment are less variable than those of the M segment (Supplementary Table S2). This means that more synonymous substitutions occur for the S segment and that more non-synonymous substitutions occur for the M segment. Unlike in PUUV (Sironen et al., 2001), the small number of non-synonymous substitutions also leads to a lack of specific amino acid ‘signatures’ in the NP of each phylogenetic group of HTNV. On the other hand, phylogenetic analysis of the S segment sequences of HTNVs indicates that there are three clades with five groups (Fig. 1a), while these viruses form seven groups in the tree based on M segment sequences (Fig. 1b). Hence, comparison of deduced N protein sequences among distinct groups of hantaviruses could not provide clues to localized genetic features of each group of HTNV. However, comparision of deduced GnGc protein sequences may provide clues to the antigenic as well as genetic diversity among hantaviruses (Schmaljohn & Hjelle, 1997). Analysis of the GnGc protein sequence showed that all groups of HTNV have specific amino acid ‘signatures’ (Fig. 3). Viral glycoproteins are often associated with high levels of non-synonymous diversity and provide some of the best examples in nature of positive selection (Yang & Bielawski, 2000; Valarcher et al., 2000; Holmes et al., 2002). It has been suggested that changes in the hypervariable region might represent adaptation to host-specific characteristics of the immune response (Hughes & Friedman, 2000). Earlier studies also found that the S, M and L segments of Amur–Soochong viruses isolated from humans in China (Liang et al., 1994) and carried by Apodemus peninsulae (Liang et al., 1994; Lokugamage et al., 2004; Baek et al., 2006) diverged from hantaviruses isolated from A. agrarius by 15, 22 and 21 % at the nucleotide level and 3, 9 and 4 % at the amino acid level, respectively. Considering the co-evolution of hantaviruses with their hosts (Plyusnin & Morzunov, 2001), these data suggest that hantaviral S and M segments may face different selection pressures from their host. Therefore, analysis of the S segment may provide more information about the earliest original relationship of HTNV, but comparison of the M segment may yield more information about the adaptation of HTNV to its local host. The current distribution of hantaviruses is the result of rodent migration and the history of virus–host cospeciation events. Different characteristics of hantaviruses Journal of General Virology 89
Genetic characterization of hantavirus from Guizhou
have emerged as adaptations to the distinct genetic environment of their rodent hosts (Plyusnin & Morzunov, 2001). Molecular data have provided strong evidence that Apodemus mice diverged about 8–10 million years ago (Serizawa et al., 2000). The Hengduan Mountains region was hypothesized to have played an important role in the evolutionary history of Apodemus since the Pleistocene era (Liu et al., 2004). It has been proposed that the Hengduan Mountains region might be a radiation centre of the present Apodemus species (Xia, 1984; Musser et al., 1996). The Yunnan–Guizhou plateau, which connects and overlaps with the Hengduan Mountains region, is also thought to play an important role in lineage isolation in Apodemus (Suzuki et al., 2003). The second radiation of Apodemus involved the divergence of A. agrarius, A. peninsulae, Apodemus semotus and Apodemus speciosus, and these four Asian species can be treated as a monophyletic group (Serizawa et al., 2000). Furthermore, these species could be integrated into the A. agrarius group within Apodemus (Liu et al., 2004; Serizawa et al., 2000). It is suggested that the ancestral HTNV might first have migrated into Guizhou (Wang et al., 2000). In the present study, phylogenetic analysis of the S segment sequence revealed that all HTNV isolates could be divided into three clades with six distinct genetic groups. In particular, the first clade, which has been found only in Guizhou to date, was the nearest to the ancestral node separating HTNV from other hantaviruses in the tree based on S segment sequences (Fig. 1a). Furthermore, this clade became the outgroup for both viruses in clades II and III, supported by
high bootstrap values (100 %). Hence, the prototype viruses of the group S4 reassortant viruses might have diversified earlier than the other two clades. These data also suggest that viruses in clades II and III have a common origin, although the bootstrap support for clade II and also for the monophyly of clades II and III was low. Taken together, a hypothesis for the evolutionary history of HTNV is illustrated in Fig. 4 and described here. An ancestor of hantaviruses carried by Apodemus mice in the Hengduan Mountains region or the Yunnan–Guizhou plateau diversified into two groups. The first group resulted in the divergence of clade I and the second group resulted in the divergence of most HTNV carried by A. agrarius, which diversified into two clades. Clade II evolved into group S1, associated with A. agrarius, and group S6, associated with A. peninsulae. Following radiation of A. peninsulae, this latter group diversified into strain A16 in Shaanxi (Yao et al., 2001a), B78 in Shandong, H5 in Heilongjiang (Liang et al., 1994) and Amur–Soochong viruses in far-eastern Russia (Lokugamage et al., 2004) and South Korea (Baek et al., 2006). As A. agrarius and A. peninsulae are closely related (Liu et al., 2004; Serizawa et al., 2000) and often inhabit the same forest, HTNV is closely associated with both species (Zhang et al., 2007). The ancestor of clade III diversified into groups S2, S3 and S5, and then spread and further diversified following rodent migration to the north and the east. However, knowledge of the genetic diversity and geographical distribution of hantaviurses carried by Apodemus is limited
Fig. 4. Geographical distribution of HTNV in China, far-eastern Russia and Korea. Immigration routes were deduced from the tree based on S segment sequences and are indicated by arrows. In addition, some immigration routes from Guizhou to Hainandao, Inner Mongolia, Liaoning and other provinces were deduced from partial S segment sequences (data not shown). http://vir.sgmjournals.org
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and thus further studies of HTNV in Guizhou and neighbouring areas will help to clarify the phylogeny of HTNV. In conclusion, our results reveal that HTNV displays extremely high genetic diversity in Guizhou. In general, the strains are clustered geographically, but group S2 viruses are predominant. Furthermore, Guizhou might be a radiation centre of the present HTNV and might play an important role in the evolution of HTNV.
pp. 1–6. Edited by H. W. Lee, C. Calisher & C. Schmaljohn. Seoul: Seoul Asan Institute for Life Sciences. Kaukinen, P., Vaheri, A. & Plyusnin, A. (2005). Hantavirus
nucleocapsid protein: a multifunctional molecule with both housekeeping and ambassadorial duties. Arch Virol 150, 1693–1713. Khaiboullina, S. F., Morzunov, S. P. & St Jeor, S. C. (2005).
Hantaviruses: molecular biology, evolution and pathogenesis. Curr Mol Med 5, 773–790. Klempa, B., Fichet-Calvet, E., Lecompte, E., Auste, B., Aniskin, V., Meisel, H., Denys, C., Koivogui, L., ter Meulen, J. & Kruger, D. H. (2006). Hantavirus in African wood mouse, Guinea. Emerg Infect Dis
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ACKNOWLEDGEMENTS
Kurata, T., Tsai, T. F., Bauer, S. P. & McCormick, J. B. (1983).
This study was supported by the Chinese Ministry of Science and Technology (grants no. 2001DIA40037, 2002DIB40095 and 2003BA712A08-02).
Lee, H. W. (1999). Virus isolation. In Manual of Hemorrhagic Fever
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