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Dynamics of hantavirus infections in humans and animals in Wuhan city, Hubei, China
Infection, Genetics and Evolution 12 (2012) 1614–1621
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Dynamics of hantavirus infections in humans and animals in Wuhan city, Hubei, China Yan-Jun Kang a,1, Dun-Jin Zhou b,1, Jun-Hua Tian b,1, Bin Yu b, Wen-Ping Guo a, Wen Wang a, Ming-Hui Li a, Tai-Ping Wu b, Jin-Song Peng b, Alexander Plyusnin a,c, Yong-Zhen Zhang a,⇑ a
State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute for Communicable Disease Control and Prevention, Changping Liuzi 5, 102206 Beijing, China Wuhan Center for Disease Control and Prevention, 430022 Wuhan, Hubei Province, China c Department of Virology, Research Programs Unit, Infection Biology Research Program, Haartman Institute, University of Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 12 April 2012 Received in revised form 24 July 2012 Accepted 29 July 2012 Available online 12 August 2012 Keywords: Hemorrhagic fever with renal syndrome Hantaan virus Seoul virus Epidemiology Dynamics
a b s t r a c t Hemorrhagic fever with renal syndrome (HFRS) has been a significant public problem since the first cases were reported in 1961 in Wuhan city (capital of Hubei province of China). Epidemiological surveys were carried out to better understand the dynamics of hantavirus infection in humans and animals in Wuhan. During 1961–2011, a total of 21,820 HFRS cases were registered in Wuhan. The two large epidemics had occurred during 1970–1991. They reached peaks in 1973 and 1983, respectively. There have been <10 cases since 2005. The disease occurred in the whole region including the downtown areas, but mainly in two districts. Although in 1980s and 1990s HFRS cases mainly recorded in August and winter, since 2000 the disease has mainly occurred in spring and summer. In this study, hantaviruses were identified in Apodemus mice, Rattus rats, and Mus mice by indirect immunofluorescent-assay and RT-PCR. Serological and genetic analyses showed that Hantaan virus (HTNV) and Seoul virus (SEOV) co-circulated in rodents. Phylogenetic analysis of hantaviral genome sequences revealed a novel genetic lineage of HTNV circulating in rodents in Wuhan. Another lineage of HTNV was closely related to the lineages from the provinces located in the origin and delta of Yangtze River. Remarkably, SEOV variants identified in Wuhan were more closely related to the variants found outside China. Results of the present study showed that HFRS cases in Wuhan are caused by HTNV and SEOV. Phylogenetic analysis of the hantavirus sequences revealed that a novel genetic lineage of HTNV is present in rodents in Wuhan. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses is one of important (re)emerging zoonotic diseases (Jonsson et al., 2010). This disease is a major public health problem in Europe and Asia (Kariwa et al., 2007; Vapalahti et al., 2003), especially in China (Zhang et al., 2010b). Although the incidence of HFRS has dramatically decreased after the enforcement of comprehensive preventive measures over the past decades, the annual cases and the annual fatality reported in China still remain the highest in the world (Zhang et al., 2010b). Hantaviruses are negative, single stranded RNA viruses in the family Bunyaviridae. Unlike other members of the family, ⇑ Corresponding author. Address: State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute of Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping Liuzi 5, Beijing 102206, China. Tel.: +86 10 58900782; fax: +86 10 58900700. E-mail address: [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this work and share first authorship. 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.07.017
hantaviruses are carried and transmitted by the small animals (rodents and insectivores) (Jonsson et al., 2010; Plyusnin et al., 2011). The hantaviral genome consists of three segments: small (S), medium (M), and large (L) which encode the nucleocapsid protein, two envelope glycoproteins and RNA polymerase, respectively (Plyusnin et al., 1996). At least 23 approved species and 30 tentative hantavirus species have been identified worldwide (Plyusnin et al., 2011). In China, eleven species (or tentative species) of hantaviruses have been identified (Guo et al., 2011; Wang et al., 2000; Zhang et al., 2010b, 2011). These viruses include Amur/ Soochong virus (ASV), Da bie shan virus (DBSV), Gou virus (GOUV), Hantaan virus (HTNV), Hokkaido virus (HOKV), Khabarovsk virus (KHAV), Luxi virus (LXV), Seoul virus (SEOV), Thottapalayam virus (TPMV), Vladivostok virus (VLAV), and Yuanjiang virus (YUJV). So far, HFRS caused by ASV, HTNV, and SEOV have been recognized in China (Wang et al., 2000; Zhang et al., 2010b, 2011). Whether other viruses are pathogenic to humans remains unknown. Wuhan, the capital of Hubei province, is one of the largest cities in China. It includes both urban and rural areas, with more than 7.8 million of population. It is situated in the intersection of the central
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Fig. 1. Map of Wuhan city with location of sampling sites (N) and its location in China. Wuhan city and Hubei province are highlighted in red and black in the map of China, respectively. The geographic distribution of human HFRS cases reported during the period of 1990–2011 in Wuhan. The incidence of human HFRS cases is shown in red color level, and the Yangtze River is shown in blue (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
reaches of the Yangtze River and Hanshui River, and is an important transport center in central China. The water transportation was especially important in the past due to the Yangtze River which crosses ten provinces and is the largest river in China and even in the whole Asia (Fig. 1). The climate in the area is a subtropical humid monsoon, with sufficient rainfall, which supports plenty of vegetables and crops for a high density of rodents. Wuhan has always been one of the most seriously HFRS-affected areas in China since the first cases reported in 1961 (Tian et al., 2008). Particularly, more than 2000 HFRS cases were reported in 1983. Previous epidemiological investigations in Wuhan revealed the presence of hantaviral antibodies or antigens in several rodent species including the striped field mice (Apodemus agrarius), house mice (Mus musculus), and Norway rats (Rattus norvegicus) (Tian et al., 2008; Zhao and Lin, 2002). Although the incidence of HFRS has been relatively low in recent years, the disease is still reported, even in Wuhan downtown. So far, no molecular epidemiological investigations have been carried out in Wuhan. Thus, epidemiological surveys of hantavirus infection and its dynamics in humans and animals would be instrumental to reveal the dynamics of HFRS in humans and hantavirus infection in rodents in Wuhan, and may help in the prevention of the disease in Wuhan and other large cities both in- and outside China. In this study, the hantavirus infection and its dynamics in humans and rodents in Wuhan are described. 2. Materials and methods 2.1. HFRS cases data collection The records of HFRS cases were obtained from Wuhan Center of Disease Control and Prevention. HFRS cases had been defined
according to a national standard of clinical criteria before 1982, and were further confirmed by detecting hantavirus specific antibodies in serum samples from 1982. 2.2. Trapping of rodents Rodents were trapped with snap-traps in the urban and rural areas (Fig. 1) during spring and August according to the protocols described previously (Mills et al., 1995). Trapped animals were identified according to the previous criteria (Chen, 1987). Lung and kidney tissue samples collected from trapped animals were stored immediately in liquid nitrogen, and then transported to the laboratory in Beijing for further processing. 2.3. Detection of hantaviral antigen Hantaviral antigens in the lung or kidney tissue (frozen sections) of rodents were detected by using indirect immunofluorescent-assay (IFA) according to the method described previously (Zhang et al., 2009a), with rabbit anti-SEOV/L99 and HTNV/76118 hantavirus antibodies prepared by our laboratory and FITC-labeled goat anti-rabbit IgG antibodies (Sigma, St. Louis, MO, USA). 2.4. Serologic assays IFA was used to test immunoglobulin IgG and IgM antibodies against HTNV or SEOV in human serum samples by the method as described previously (Zhang et al., 2009a). Briefly, IgG and IgM IFAs were carried out with HTNV (strain 76–118)- and SEOV (strain L99)-infected Vero E6 cells on slides. FITC-labeled rabbit anti-human IgG or IgM antibodies (Sigma, St. Louis, MO, USA) were used.
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Fig. 2. Annual numbers of HFRS cases in Wuhan, 1961–2011. Incidence rates are cases/100,000 population shown in blue. Mortality rates are shown in red (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.5. Reverse transcription-PCR (RT-PCR) and sequencing In order to amplify partial S and M segment sequences of hantaviruses, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from lung or kidney tissues and human serum samples according to the manufacturer’s instruction. cDNAs for the M or S segments were synthesized with AMV reverse transcriptase (Promega, Beijing, China) in the presence of primer P14 based on the terminus of hantaviral genome (Schmaljohn et al., 1986). Partial M or partial S segment sequences of HTNV or SEOV were amplified as described by (Zhang et al., 2009b). Complete M and S segment sequences were obtained as described previously (Zou et al., 2008b). QIAquick gel extraction kits (Qiagen, Beijing, China) were employed to purify PCR products according to the manufacturer’s instructions. Purified DNA fragments were directly subjected to sequencing by using the ABI-PRISM dye termination sequencing kit and an ABI 373-A genetic analyzer. 2.6. Phylogenetic analysis Hantaiviral M and S segment sequences were aligned by ClustalW algorithm (integrated in Mega5 package), and edited manually under Mega5 (Tamura et al., 2011). The nucleotide and amino acid sequence identities were calculated by using Lasergene7 software (Burland, 2000). Phylogenetic relationships of hantaviruses were estimated using Bayesian Markov Chain Monte Carlo (MCMC) method in MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001), with a gamma-distribution model determined by jModelTest version 0.1 (Posada, 2008). For comparison, hantaviral sequences were also retrieved from GenBank. The sequences retrieved from GenBank or obtained in this study are listed in Supplementary Tables 1 and 2. 3. Results 3.1. Hantavirus infection in humans in Wuhan Since 1961, in which the first case was reported, HFRS cases have occurred in Wuhan each year. From 1961 through 2011, there were four major epidemics with a total of 21,820 cases (an average incidence rate of 7.76/100,000 population) in Wuhan. The first epidemic occurred during 1961–1969, with a total of 1486 HFRS cases and 99 deaths. Subsequently, two large epidemics had appeared during 1970–1991. They reached peaks in 1973 (1980 cases,
Fig. 3. The proportion of HFRS cases in Jiangxia district, Xinzhou district, urban area, and other areas during the period of 1985–2011 in Wuhan.
38.92 cases/100,000 population) and 1983 (2239 cases, 37.67 cases/100,000 population), respectively. After the two epidemics, the small epidemic occurred during 1992–2004, with a total of 1142 cases. Since 2005, the annual number of HFRS cases has been less than 10 cases each year. During 1961–2011, a total of 633 deaths caused by HFRS were recorded in Wuhan, with median fatality rate 2.9%. The highest fatality rates occurred when HFRS cases appeared in 1960s, with up to 30.77%, indicating both the severity of HFRS caused by HTNV and a poor knowledge of how to treat it. Remarkably, the high death rates still occurred during 1988–1994, and were as high as 13.70% in 1991. Although Wuhan as a provincial capital offered somewhat better medical care, two patients died of HFRS in 2004 and 2011 respectively. These deaths might be due to not receiving timely treatment because of the low economic status in rural area. 3.2. Geographic distribution and dynamic of HFRS cases over the past three decades Over the past 26 years, HFRS cases were reported in all districts of Wuhan, but mainly occurred in districts Jiangxia and Xinzhou
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Fig. 4. The monthly proportion of HFRS cases during 1985–2011 in Wuhan (a) and its districts: the urban districts (b), Jianxia district (c), and Xinzhou district (d).
3.3. Seasonal distribution
Table 1 Detection of hantaviral antibodies and RNA genome by IFA and PCR in the serum samples from HFRS patients in Wuhan, 2011. Patient No.
IgG assay HTNV
09 10 12 16 27 29
320 40 640 640 320 640
*
IgM assay
For the whole region, there was one major peak in winter during 1985–1990 (Fig. 4a). Obviously, in 1990s, the peak in April–July was observed, whereas the HFRS incidence was the highest during November through February. Notably, the peaks in spring and summer became higher than in August and winter in 2000s. The seasonal distribution was also analyzed location-wise. During 1985–1989, the HFRS cases occurred mainly in winter in both urban and rural areas (Fig. 4b–d). However, the peak in April–July appeared in both the urban areas and Xinzhou in 1990s, whereas there was only one major peak in winter in Jiangxia. In 2000s, the disease still occurred mainly in August and winter in Jiangxia (Fig. 4c). However, the incidence rate became higher in spring than in winter in both urban areas and Xinzhou (Fig. 4b and d). In addition, the small peak in winter still appeared in the urban areas (Fig. 4b).
PCR detection
SEOV
HTNV
SEOV
640 160 160 320 160 160
10 40 20 10
40 20 40 10 -
+ + +
*
Numbers represent the endpoint titers of antibodies against hantaviruses (HTNV or SEOV) antibodies in the human serum samples. HTNV or SEOV was determined by analyses of recovered genome sequence.
(Figs. 1 and 3). Notably, most of HFRS cases occurred in Jiangxia, accounting for 48.03% of the total cases during 1985–1989 and 44.56% during 1990–1994, and still 30% at present, respectively. The incidences in the urban areas were also higher, with respectively 8.45% and 8.86% of the total cases during same periods. In addition, the HFRS cases in Xinzhou increased gradually, with 31.75% of the total cases during 1995–1999, and became the highest at present, with 36.36% of all HFRS cases during 2000–2011. More importantly, sporadic cases had been reported even in the downtown areas over the past ten years, even with 2 cases in 2011 occurred in Wuchang and Hongshan districts.
3.4. Serologic and genetic investigation of patient serum samples Serum samples were collected from 6 of 8 patients at day 1 of admission (2–9 days post the onset of fever). Specific IgG and IgM antibodies against HTNV or SEOV were detected by IFA (Table 1). In 4 of 6 sera, the IgG titers against HTNV were 2- or 4-fold higher than those against SEOV, while the remaining two serum samples showed higher titers against SEOV. In addition, the viral
Table 2 The prevalence of hantaviruses in different rodent species in various regions of Wuhan.
*
Species
Urban region
Caidian
Huangpi
Jiangxia
Xinzhou
Total
A. agrarius R. flavipectus R. norvegicus M. musculus N. niviventer Total
1/0/0* 168/8/8 180/13/9 84/1/1 0/0/0 433/22/18
30/0/0 3/0/0 0/0/0 0/0/0 0/0/0 33/0/0
1/0/0 18/0/0 0/0/0 18/0/0 1/0/0 38/0/0
67/1/1 16/0/0 3/0/0 10/0/0 1/0/0 97/1/1
15/0/0 9/0/0 0/0/0 87/3/2 0/0/0 111/3/2
114/1/1 214/8/8 183/13/9 199/4/3 2/0/0 712/26/21
Rodents trapped/positive for hantavirus antigen/RNA.
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Fig. 5. Phylogenetic relationships between HTNV variants described in this study and found previously in and outside China. Bayesian trees were based on the S (a) and M (b) segment sequences. Dobrava virus (DOBV) was used as the outgroup. Numbers (>0.5) above or below nodes indicate posterior node probabilities. The sequences recovered in this study are shown in red. The accession numbers of the sequences retrieved from GenBank are listed in Supplementary Table 1.The accession numbers of the sequences obtained in this study are listed in Supplementary Table 2.
genome sequences were recovered from 3 patient serum samples using HTNV-specific primers, but not from the serum samples with higher titers of antibodies against SEOV. 3.5. Hantaviruses infection in rodents To gain more insight on hantaviruses circulating in rodents in Wuhan, rodents were trapped in the fields and residential areas during the period of 2007–2011 (Fig. 1). A total of 712 rodents were captured (Table 2). These rodents included 114 A. agrarius, 214 Rattus flavipectus, 183 R. Norvegicus, 199 M. Musculus and 2 Niviventer niviventer trapped at eight locations. Clearly, A. agrarius is the dominant species in Jiangxia and Caidian, while M. musculus is the dominant species in Xinzhou. Both R. norvegicus and R. flavipectus are highly prevalent in urban areas and both R. flavipectus and M. musculus are the major hosts in Huangpi. Hantaviral antigens were identified in a total of 21 (2.95%) lung or kidney samples collected from these rodents. 3.6. Phylogenetic analysis Partial M (nt 1970–2329) and partial S (nt 475–1011) segment sequences were recovered from three human serum samples. The hantaviral sequences obtained in this study were named respectively as WuhanAa-, WuhanRn-, WuhanRf-, WuhanMm-, WuhanHu-, and one isolate HubeiHu02 (obtained from HFRS patient serum samples collected from Wuhan in 2002 by our laboratory) (Figs. 5 and 6). Probably because some samples were not fresh from captured rodents with snap-traps or were not collected from patients during the early phase respectively, our attempts to amplify hantaviral genome sequences were not successful from these rodent or human samples. Genetic analyses of these sequences indicated that the sequences recovered from one A. agrarius sample
(WuhanAaJ10), two human serum samples (WuhanHu12, WuhanHu29), and the isolate (HubeiHu02) were very closely related to each other, with 96.4–98.1% nucleotide identity for the partial M segment sequences and 95.9–98% nucleotide identity for the partial S segment sequences. These sequences showed higher identity to the known HTNV variants (<10.3% nucleotide differences for the M segment sequences and <8.6% nucleotide differences for the S segment sequences) than other hantavirus species (>25.1% and >24.8%). They clustered together and formed a new distinct genetic lineage (Fig. 5), and showed a close evolutionary relationship with the sequences recovered from A. agrarius mice and humans from Guizhou and Tianjin within the lineage #2 (Zou et al., 2008a). However, the strain WuhanHu16 had more than 10% nucleotide sequence differences from the strains WuhanAaJ10, WuhanHu12, WuhanHu29, and HubeiHu02. The strain clustered together with the isolate HV114 from Hubei province (Xiao et al., 1993) and strains from Guizhou, Zhejiang and Jiangsu provinces, which belong to the lineage #3 (Zou et al., 2008a). Remarkably, Guizhou, Hubei, Jiangsu and Zhejiang provinces are located, respectively, in upstream, middle reaches, and downstream of the Yangtze River. Sequences recovered from R. norvegicus, R. flavipectus, and M. musculus, were also very closely related to each other, with less than 3.9% nucleotide sequence differences for the partial M segment and 2.7% nucleotide sequence differences for the partial S segment. These sequences shared higher identity with known SEOV variants including those recovered previously from Wuhan and other parts of Hubei province (Lin et al., 2012) than with HTNV sequences recovered from A. agrarius or corresponding sequences of other hantavirus species (<73.9% nucleotide identity for the M segment sequences and <76.4% nucleotide identity for the partial S segment sequences). On the phylogenetic trees based on either M or S segment sequences (Fig. 6), all viruses from Rattus rats, and Mus mice
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Fig. 6. Phylogenetic relationships between SEOV variants described in this study and found previously in and outside China. Bayesian trees were based on the S (a) and M (b) segment sequences. Dobrava virus (DOBV) was used as the outgroup. Numbers (>0.5) above or below nodes indicate posterior node probabilities. The sequences recovered in this study are shown in red. The accession numbers of the sequences retrieved from GenBank are listed in (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
identified in this study belong to the phylogroup A of SEOV (Lin et al., 2012). On the M-tree, most of sequences obtained in this study clustered together within the phylogroup A of SEOV (Lin et al., 2012). In contrast, WuhanMm24 was closely related to the stain HubeiRn55 and strains from Japan, South Korea, and Singapore (Lin et al., 2012). These viruses formed another small cluster within the phylogroup A. On the S-tree, WuhanMm24 clustered together with the majority of strains identified in Wuhan in this study, while the clustering pattern of all other sequences was similar to that on the M-tree suggesting that genetic reassortment within the phylogroup A might have occurred. In addition, the isolate Hubeihuman1 clustered together with those outside of China. Finally, all known sequences from Wuhan and Hubei fell into four small clusters.
4. Discussion Like the whole country (Zhang et al., 2010b), Wuhan was seriously affected by the disease in 1970s and 1980s (Fig. 2). Remarkably, the annual numbers have decreased significantly from more
than 2000 in 1983 to less than 10 since 2005, indicating the disease is controlled effectively. As hantaviruses are still seriously prevalent in rodents in both rural and urban areas, more efforts are needed to further prevent human infection from rodents. The occurrence and epidemics of HFRS is influenced by both natural (e.g., ecological) and social (e.g., economic, occupational) factors (Bi et al., 2002; Chen and Qiu, 1994; Schmaljohn and Hjelle, 1997; Zhang et al., 2009b, 2010a). For example, most HFRS cases (>70%) occurred in rural areas, especially in those which belong to the humid and semi-humid climate zones in China (Bi et al., 2002; Zhang et al., 2010b). High rodent density and poor housing conditions in residential areas are responsible for most HFRS epidemics (Zhang et al., 2010b). Previous epidemiologic surveys revealed a linkage between human HFRS epidemics and high density of rodents, and hence their hantaviruses, in residential areas and fields in Wuhan (Chen et al., 1992; Fang et al., 1995). Especially in Jiangxia, which is the major agricultural areas of Wuhan, the higher capturing rate of A. agrarius mice in fields (especially in fields of rice) than in other regions has caused the higher incidence of HFRS. When compared with 1980s (Chen et al., 1992), the density of rodents in both fields and residential
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areas in Wuhan began to decrease significantly in 1990s (Chen et al., 2001; Fang et al., 1995). In accord with the recent surveys of rodent density (Wu et al., 2007), our surveys also revealed lower capturing rates of rodents in both fields and residential areas when compared with 1990s (Chen et al., 2001; Fang et al., 1995) and the beginning of 2000s (Tian et al., 2008). In addition, like other parts of China, in 1970s and 1980s the rural areas in Wuhan had been of the low socioeconomic status. Thus, the relatively low density of rodents and the significant improvement of housing conditions in recent years might have mainly contributed to the decrease tendency of HFRS incidence over the last decade in Wuhan. The peak of HFRS associated with SEOV carried by Rattus rats occurred in the spring, whereas HFRS associated with HTNV carried by Apodemus mice occurred mainly in the winter (Chen et al., 1986). The two peaks were related to the high densities of R. norvegicus rats in residential areas in spring and A. agrarius mice in fields in August and winter respectively (Chen et al., 1992, 2001) There are at least 20 species of rodents and insectivores in Wuhan (Liu and Yang, 2006). Previous surveys revealed that the dominant species of hantavirus carriers in Wuhan was A. agrarius 91.59% in fields and R. norvegicus 49.65% in residential areas, respectively, while hantavirus antigens were also detected in other species such as R. flavipectus and M. musculus (Chen et al., 1992, 2001). As most HFRS cases occurred in winter in 1990s, it means that HFRS cases were mainly transmitted by A. agrarius, even in urban areas. In the present study, hantavirus antigens were detected in these four species. Obviously, Rattus rats and Mus mice have become the major carriers of hantaviruses in Wuhan, except Caidian and Jianxia districts. Most cases occurred in spring and summer suggesting that they were associated with high density of Rattus rats in residential areas in 2000s. Like in other parts of China (Zhang et al., 2010a,b), SEOV and its carriers Rattus rats have become the primary source of human infection. In addition, Mus mice, are highly prevalent in urban and Xinzhou areas, and carry SEOV, suggesting that they also play an important role in the transmission of hantaviruses to human, particularly in urban areas. Serological and genetic analyses indicated that the HFRS cases occurred in urban areas were caused by HTNV. Further studies are needed to know whether these cases have been transmitted from the local rodents. There are at least nine genetic lineages of HTNV circulating in Apodemus mice in Eastern Asia including China; generally they show geographical clustering (Zou et al., 2008a). Phylogenetic analyses of the sequences recovered in this study showed that there are two lineages of HTNV variants circulating in rodents in Wuhan. Of these variants, WuhanHu12, WuhanHu29, HubeiHu02, and WuhanAaJ10 formed a new cluster on both M- and S-trees. These data suggest that at least some of human HFRS cases have been caused by the local lineage of HTNV variants. However, the strain WuhanHu16 clustered together with those isolated in other parts of Hubei province or in Guizhou, Jiangsu, and Zhejiang provinces, suggesting the spread of HTNV which might follow the migration of Apodemus mice along the Yangtze River. Although SEOV generally shows high genetic diversity and also displays geographic clustering pattern like other hantaviruses, the first SEOV phylogroup includes the majority of the known Chinese variants and also all known non-Chinese variants and displays a worldwide distribution resulted from the migration of Norway rats (Lin et al., 2012). Remarkably, all known SEOV variants identified in this study or previously in Wuhan/Hubei fell into four small clusters with the phylogroup A. More importantly, some of these viruses are closely related to those found outside of China (Lin et al., 2012; Wang et al., 2000). These data suggest that the convenient transportation (especially on water) in Wuhan may accelerate the translocation of Rattus rats and consequently the spread of SEOV between Wuhan and other parts of China or even outside China. Thus, these data strongly support our recent hypothesis that
the present worldwide distribution of SEOV is resulted from the migration of Norway rats (Lin et al., 2012). 5. Conclusion Both HTNV and SEOV are co-circulating in rodents in Wuhan. Although most of HFRS cases had caused by HTNV in both 1980s and 1990s, SEOV has become the dominant species to cause human HFRS today. A novel genetic lineage of HTNV is circulating in rodents in Wuhan. HTNV strains found in Wuhan are closely related to those from the provinces located in upstream, middle reaches, and downstream of the Yangtze River, and SEOV variants identified in Wuhan are more closely to those found outside China. These data suggest that the convenient transportation (especially on water) might have accelerated the spread of HTNV and SEOV between Wuhan and other parts of China and even outside China. Acknowledgments This study was supported by Grants 2003BA712A08-02 from the Chinese Ministry of Science and Technology and by the State Key Laboratory for Infectious Disease Prevention and Control (2011SKLID101). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2012. 07.017. References Bi, P., Donald, K., Tong, S., Ni, J., Parton, K., 2002. Climatic, reservoir and occupational variables and the transmission of haemorrhagic fever with renal syndrome in China. Int. J. Epidemiol. 31, 189–193. Burland, T.G., 2000. DNASTAR’s Lasergene sequence analysis software. Methods Mol. Biol. 132, 71–91. Chen, D.L., Yu, Q.F., Wang, S.Y., Fu, T.X., Wu, Y.X., Rao, K.M., Li, H., Tian, W.D., 2001. Surveillance of hemorrhagic fever with renal syndrome in Jiangxia district of Wuhan. In: Chen, H.X., Luo, C.W. (Eds.), Hemorrhagic fever with renal syndrome: Studies and the surveillance and application of vaccine, Hong Kong Medical Publisher, pp. 307–311 (in Chinese). Chen, D.L., Zhang, L., Luo, W.P., Zhang, L.Q., Ma, Z.J., Zhang, M.W., Huang, J.S., Fu, T.X., Rao, H.X., Li, H., 1992. Surveillance of epidemiological fever during 1984–1990 in Wuchang county. In: Chen, H.X., Wang, Z., Tang, S.Z. (Eds.), Surveillance on the epidemic hemorrhagic fever in China. Beijing Science & Technology Press, pp. 282–289 (in Chinese). Chen, H.X., 1987. Classification and Identification of Medical Animals. The Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing (in Chinese). Chen, H.X., Qiu, F.X., 1994. Studies on the environment structure of natural nidi and epidemic areas of hemorrhagic fever with renal syndrome in China. Chin. Med. J. 107, 107–112. Chen, H.X., Qiu, F.X., Dong, B.J., Ji, S.Z., Li, Y.T., Wang, Y., Wang, H.M., Zuo, G.F., Tao, X.X., Gao, S.Y., 1986. Epidemiological studies on hemorrhagic fever with renal syndrome in China. J. Infect. Dis. 154, 394–398. Guo, W.P., Lin, X.D., Wang, W., Zhang, X.H., Chen, Y., Cao, J.H., Ni, Q.X., Li, W.C., Li, M.H., Plyusnin, A., Zhang, Y.Z., 2011. A new subtype of Thottapalayam virus carried by the Asian house shrew (Suncus murinus) in China. Infect. Genet. Evol. 11, 1862–1867. Fang, Y., Huang, X.H., Zhou, Q., Hu, E.P., Wu, H.X., 1995. The trend of epidemic situation changes of hemorrhagic fever with renal syndrome (HFRS) in early 1990s in Hubei province. Chin. J. Vector Biol. Control 6, 111–113 (in Chinese). Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Jonsson, C.B., Figueiredo, L.T., Vapalahti, O., 2010. A global perspective on hantavirus ecology, epidemiology, and disease. Clin. Microbiol. Rev. 23, 412–441. Kariwa, H., Yoshimatsu, K., Arikawa, J., 2007. Hantavirus infection in East Asia. Comp. Immunol. Microbiol. Infect. Dis. 30, 341–356. Lin, X.D., Guo, W.P., Wang, W., Zou, Y., Hao, Z.Y., Zhou, D.J., Dong, X., Qu, Y.G., Li, M.H., Tian, H.F., Wen, J.F., Plyusnin, A., Xu, J., Zhang, Y.Z., 2012. Migration of norway rats resulted in the worldwide distribution of seoul hantavirus today. J. Virol. 86, 972–981.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Dynamics of hantavirus infections in humans and animals in Wuhan city, Hubei, China Yan-Jun Kang a,1, Dun-Jin Zhou b,1, Jun-Hua Tian b,1, Bin Yu b, Wen-Ping Guo a, Wen Wang a, Ming-Hui Li a, Tai-Ping Wu b, Jin-Song Peng b, Alexander Plyusnin a,c, Yong-Zhen Zhang a,⇑ a
State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute for Communicable Disease Control and Prevention, Changping Liuzi 5, 102206 Beijing, China Wuhan Center for Disease Control and Prevention, 430022 Wuhan, Hubei Province, China c Department of Virology, Research Programs Unit, Infection Biology Research Program, Haartman Institute, University of Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 12 April 2012 Received in revised form 24 July 2012 Accepted 29 July 2012 Available online 12 August 2012 Keywords: Hemorrhagic fever with renal syndrome Hantaan virus Seoul virus Epidemiology Dynamics
a b s t r a c t Hemorrhagic fever with renal syndrome (HFRS) has been a significant public problem since the first cases were reported in 1961 in Wuhan city (capital of Hubei province of China). Epidemiological surveys were carried out to better understand the dynamics of hantavirus infection in humans and animals in Wuhan. During 1961–2011, a total of 21,820 HFRS cases were registered in Wuhan. The two large epidemics had occurred during 1970–1991. They reached peaks in 1973 and 1983, respectively. There have been <10 cases since 2005. The disease occurred in the whole region including the downtown areas, but mainly in two districts. Although in 1980s and 1990s HFRS cases mainly recorded in August and winter, since 2000 the disease has mainly occurred in spring and summer. In this study, hantaviruses were identified in Apodemus mice, Rattus rats, and Mus mice by indirect immunofluorescent-assay and RT-PCR. Serological and genetic analyses showed that Hantaan virus (HTNV) and Seoul virus (SEOV) co-circulated in rodents. Phylogenetic analysis of hantaviral genome sequences revealed a novel genetic lineage of HTNV circulating in rodents in Wuhan. Another lineage of HTNV was closely related to the lineages from the provinces located in the origin and delta of Yangtze River. Remarkably, SEOV variants identified in Wuhan were more closely related to the variants found outside China. Results of the present study showed that HFRS cases in Wuhan are caused by HTNV and SEOV. Phylogenetic analysis of the hantavirus sequences revealed that a novel genetic lineage of HTNV is present in rodents in Wuhan. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses is one of important (re)emerging zoonotic diseases (Jonsson et al., 2010). This disease is a major public health problem in Europe and Asia (Kariwa et al., 2007; Vapalahti et al., 2003), especially in China (Zhang et al., 2010b). Although the incidence of HFRS has dramatically decreased after the enforcement of comprehensive preventive measures over the past decades, the annual cases and the annual fatality reported in China still remain the highest in the world (Zhang et al., 2010b). Hantaviruses are negative, single stranded RNA viruses in the family Bunyaviridae. Unlike other members of the family, ⇑ Corresponding author. Address: State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute of Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping Liuzi 5, Beijing 102206, China. Tel.: +86 10 58900782; fax: +86 10 58900700. E-mail address: [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this work and share first authorship. 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.07.017
hantaviruses are carried and transmitted by the small animals (rodents and insectivores) (Jonsson et al., 2010; Plyusnin et al., 2011). The hantaviral genome consists of three segments: small (S), medium (M), and large (L) which encode the nucleocapsid protein, two envelope glycoproteins and RNA polymerase, respectively (Plyusnin et al., 1996). At least 23 approved species and 30 tentative hantavirus species have been identified worldwide (Plyusnin et al., 2011). In China, eleven species (or tentative species) of hantaviruses have been identified (Guo et al., 2011; Wang et al., 2000; Zhang et al., 2010b, 2011). These viruses include Amur/ Soochong virus (ASV), Da bie shan virus (DBSV), Gou virus (GOUV), Hantaan virus (HTNV), Hokkaido virus (HOKV), Khabarovsk virus (KHAV), Luxi virus (LXV), Seoul virus (SEOV), Thottapalayam virus (TPMV), Vladivostok virus (VLAV), and Yuanjiang virus (YUJV). So far, HFRS caused by ASV, HTNV, and SEOV have been recognized in China (Wang et al., 2000; Zhang et al., 2010b, 2011). Whether other viruses are pathogenic to humans remains unknown. Wuhan, the capital of Hubei province, is one of the largest cities in China. It includes both urban and rural areas, with more than 7.8 million of population. It is situated in the intersection of the central
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Fig. 1. Map of Wuhan city with location of sampling sites (N) and its location in China. Wuhan city and Hubei province are highlighted in red and black in the map of China, respectively. The geographic distribution of human HFRS cases reported during the period of 1990–2011 in Wuhan. The incidence of human HFRS cases is shown in red color level, and the Yangtze River is shown in blue (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
reaches of the Yangtze River and Hanshui River, and is an important transport center in central China. The water transportation was especially important in the past due to the Yangtze River which crosses ten provinces and is the largest river in China and even in the whole Asia (Fig. 1). The climate in the area is a subtropical humid monsoon, with sufficient rainfall, which supports plenty of vegetables and crops for a high density of rodents. Wuhan has always been one of the most seriously HFRS-affected areas in China since the first cases reported in 1961 (Tian et al., 2008). Particularly, more than 2000 HFRS cases were reported in 1983. Previous epidemiological investigations in Wuhan revealed the presence of hantaviral antibodies or antigens in several rodent species including the striped field mice (Apodemus agrarius), house mice (Mus musculus), and Norway rats (Rattus norvegicus) (Tian et al., 2008; Zhao and Lin, 2002). Although the incidence of HFRS has been relatively low in recent years, the disease is still reported, even in Wuhan downtown. So far, no molecular epidemiological investigations have been carried out in Wuhan. Thus, epidemiological surveys of hantavirus infection and its dynamics in humans and animals would be instrumental to reveal the dynamics of HFRS in humans and hantavirus infection in rodents in Wuhan, and may help in the prevention of the disease in Wuhan and other large cities both in- and outside China. In this study, the hantavirus infection and its dynamics in humans and rodents in Wuhan are described. 2. Materials and methods 2.1. HFRS cases data collection The records of HFRS cases were obtained from Wuhan Center of Disease Control and Prevention. HFRS cases had been defined
according to a national standard of clinical criteria before 1982, and were further confirmed by detecting hantavirus specific antibodies in serum samples from 1982. 2.2. Trapping of rodents Rodents were trapped with snap-traps in the urban and rural areas (Fig. 1) during spring and August according to the protocols described previously (Mills et al., 1995). Trapped animals were identified according to the previous criteria (Chen, 1987). Lung and kidney tissue samples collected from trapped animals were stored immediately in liquid nitrogen, and then transported to the laboratory in Beijing for further processing. 2.3. Detection of hantaviral antigen Hantaviral antigens in the lung or kidney tissue (frozen sections) of rodents were detected by using indirect immunofluorescent-assay (IFA) according to the method described previously (Zhang et al., 2009a), with rabbit anti-SEOV/L99 and HTNV/76118 hantavirus antibodies prepared by our laboratory and FITC-labeled goat anti-rabbit IgG antibodies (Sigma, St. Louis, MO, USA). 2.4. Serologic assays IFA was used to test immunoglobulin IgG and IgM antibodies against HTNV or SEOV in human serum samples by the method as described previously (Zhang et al., 2009a). Briefly, IgG and IgM IFAs were carried out with HTNV (strain 76–118)- and SEOV (strain L99)-infected Vero E6 cells on slides. FITC-labeled rabbit anti-human IgG or IgM antibodies (Sigma, St. Louis, MO, USA) were used.
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Fig. 2. Annual numbers of HFRS cases in Wuhan, 1961–2011. Incidence rates are cases/100,000 population shown in blue. Mortality rates are shown in red (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.5. Reverse transcription-PCR (RT-PCR) and sequencing In order to amplify partial S and M segment sequences of hantaviruses, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from lung or kidney tissues and human serum samples according to the manufacturer’s instruction. cDNAs for the M or S segments were synthesized with AMV reverse transcriptase (Promega, Beijing, China) in the presence of primer P14 based on the terminus of hantaviral genome (Schmaljohn et al., 1986). Partial M or partial S segment sequences of HTNV or SEOV were amplified as described by (Zhang et al., 2009b). Complete M and S segment sequences were obtained as described previously (Zou et al., 2008b). QIAquick gel extraction kits (Qiagen, Beijing, China) were employed to purify PCR products according to the manufacturer’s instructions. Purified DNA fragments were directly subjected to sequencing by using the ABI-PRISM dye termination sequencing kit and an ABI 373-A genetic analyzer. 2.6. Phylogenetic analysis Hantaiviral M and S segment sequences were aligned by ClustalW algorithm (integrated in Mega5 package), and edited manually under Mega5 (Tamura et al., 2011). The nucleotide and amino acid sequence identities were calculated by using Lasergene7 software (Burland, 2000). Phylogenetic relationships of hantaviruses were estimated using Bayesian Markov Chain Monte Carlo (MCMC) method in MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001), with a gamma-distribution model determined by jModelTest version 0.1 (Posada, 2008). For comparison, hantaviral sequences were also retrieved from GenBank. The sequences retrieved from GenBank or obtained in this study are listed in Supplementary Tables 1 and 2. 3. Results 3.1. Hantavirus infection in humans in Wuhan Since 1961, in which the first case was reported, HFRS cases have occurred in Wuhan each year. From 1961 through 2011, there were four major epidemics with a total of 21,820 cases (an average incidence rate of 7.76/100,000 population) in Wuhan. The first epidemic occurred during 1961–1969, with a total of 1486 HFRS cases and 99 deaths. Subsequently, two large epidemics had appeared during 1970–1991. They reached peaks in 1973 (1980 cases,
Fig. 3. The proportion of HFRS cases in Jiangxia district, Xinzhou district, urban area, and other areas during the period of 1985–2011 in Wuhan.
38.92 cases/100,000 population) and 1983 (2239 cases, 37.67 cases/100,000 population), respectively. After the two epidemics, the small epidemic occurred during 1992–2004, with a total of 1142 cases. Since 2005, the annual number of HFRS cases has been less than 10 cases each year. During 1961–2011, a total of 633 deaths caused by HFRS were recorded in Wuhan, with median fatality rate 2.9%. The highest fatality rates occurred when HFRS cases appeared in 1960s, with up to 30.77%, indicating both the severity of HFRS caused by HTNV and a poor knowledge of how to treat it. Remarkably, the high death rates still occurred during 1988–1994, and were as high as 13.70% in 1991. Although Wuhan as a provincial capital offered somewhat better medical care, two patients died of HFRS in 2004 and 2011 respectively. These deaths might be due to not receiving timely treatment because of the low economic status in rural area. 3.2. Geographic distribution and dynamic of HFRS cases over the past three decades Over the past 26 years, HFRS cases were reported in all districts of Wuhan, but mainly occurred in districts Jiangxia and Xinzhou
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Fig. 4. The monthly proportion of HFRS cases during 1985–2011 in Wuhan (a) and its districts: the urban districts (b), Jianxia district (c), and Xinzhou district (d).
3.3. Seasonal distribution
Table 1 Detection of hantaviral antibodies and RNA genome by IFA and PCR in the serum samples from HFRS patients in Wuhan, 2011. Patient No.
IgG assay HTNV
09 10 12 16 27 29
320 40 640 640 320 640
*
IgM assay
For the whole region, there was one major peak in winter during 1985–1990 (Fig. 4a). Obviously, in 1990s, the peak in April–July was observed, whereas the HFRS incidence was the highest during November through February. Notably, the peaks in spring and summer became higher than in August and winter in 2000s. The seasonal distribution was also analyzed location-wise. During 1985–1989, the HFRS cases occurred mainly in winter in both urban and rural areas (Fig. 4b–d). However, the peak in April–July appeared in both the urban areas and Xinzhou in 1990s, whereas there was only one major peak in winter in Jiangxia. In 2000s, the disease still occurred mainly in August and winter in Jiangxia (Fig. 4c). However, the incidence rate became higher in spring than in winter in both urban areas and Xinzhou (Fig. 4b and d). In addition, the small peak in winter still appeared in the urban areas (Fig. 4b).
PCR detection
SEOV
HTNV
SEOV
640 160 160 320 160 160
10 40 20 10
40 20 40 10 -
+ + +
*
Numbers represent the endpoint titers of antibodies against hantaviruses (HTNV or SEOV) antibodies in the human serum samples. HTNV or SEOV was determined by analyses of recovered genome sequence.
(Figs. 1 and 3). Notably, most of HFRS cases occurred in Jiangxia, accounting for 48.03% of the total cases during 1985–1989 and 44.56% during 1990–1994, and still 30% at present, respectively. The incidences in the urban areas were also higher, with respectively 8.45% and 8.86% of the total cases during same periods. In addition, the HFRS cases in Xinzhou increased gradually, with 31.75% of the total cases during 1995–1999, and became the highest at present, with 36.36% of all HFRS cases during 2000–2011. More importantly, sporadic cases had been reported even in the downtown areas over the past ten years, even with 2 cases in 2011 occurred in Wuchang and Hongshan districts.
3.4. Serologic and genetic investigation of patient serum samples Serum samples were collected from 6 of 8 patients at day 1 of admission (2–9 days post the onset of fever). Specific IgG and IgM antibodies against HTNV or SEOV were detected by IFA (Table 1). In 4 of 6 sera, the IgG titers against HTNV were 2- or 4-fold higher than those against SEOV, while the remaining two serum samples showed higher titers against SEOV. In addition, the viral
Table 2 The prevalence of hantaviruses in different rodent species in various regions of Wuhan.
*
Species
Urban region
Caidian
Huangpi
Jiangxia
Xinzhou
Total
A. agrarius R. flavipectus R. norvegicus M. musculus N. niviventer Total
1/0/0* 168/8/8 180/13/9 84/1/1 0/0/0 433/22/18
30/0/0 3/0/0 0/0/0 0/0/0 0/0/0 33/0/0
1/0/0 18/0/0 0/0/0 18/0/0 1/0/0 38/0/0
67/1/1 16/0/0 3/0/0 10/0/0 1/0/0 97/1/1
15/0/0 9/0/0 0/0/0 87/3/2 0/0/0 111/3/2
114/1/1 214/8/8 183/13/9 199/4/3 2/0/0 712/26/21
Rodents trapped/positive for hantavirus antigen/RNA.
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Fig. 5. Phylogenetic relationships between HTNV variants described in this study and found previously in and outside China. Bayesian trees were based on the S (a) and M (b) segment sequences. Dobrava virus (DOBV) was used as the outgroup. Numbers (>0.5) above or below nodes indicate posterior node probabilities. The sequences recovered in this study are shown in red. The accession numbers of the sequences retrieved from GenBank are listed in Supplementary Table 1.The accession numbers of the sequences obtained in this study are listed in Supplementary Table 2.
genome sequences were recovered from 3 patient serum samples using HTNV-specific primers, but not from the serum samples with higher titers of antibodies against SEOV. 3.5. Hantaviruses infection in rodents To gain more insight on hantaviruses circulating in rodents in Wuhan, rodents were trapped in the fields and residential areas during the period of 2007–2011 (Fig. 1). A total of 712 rodents were captured (Table 2). These rodents included 114 A. agrarius, 214 Rattus flavipectus, 183 R. Norvegicus, 199 M. Musculus and 2 Niviventer niviventer trapped at eight locations. Clearly, A. agrarius is the dominant species in Jiangxia and Caidian, while M. musculus is the dominant species in Xinzhou. Both R. norvegicus and R. flavipectus are highly prevalent in urban areas and both R. flavipectus and M. musculus are the major hosts in Huangpi. Hantaviral antigens were identified in a total of 21 (2.95%) lung or kidney samples collected from these rodents. 3.6. Phylogenetic analysis Partial M (nt 1970–2329) and partial S (nt 475–1011) segment sequences were recovered from three human serum samples. The hantaviral sequences obtained in this study were named respectively as WuhanAa-, WuhanRn-, WuhanRf-, WuhanMm-, WuhanHu-, and one isolate HubeiHu02 (obtained from HFRS patient serum samples collected from Wuhan in 2002 by our laboratory) (Figs. 5 and 6). Probably because some samples were not fresh from captured rodents with snap-traps or were not collected from patients during the early phase respectively, our attempts to amplify hantaviral genome sequences were not successful from these rodent or human samples. Genetic analyses of these sequences indicated that the sequences recovered from one A. agrarius sample
(WuhanAaJ10), two human serum samples (WuhanHu12, WuhanHu29), and the isolate (HubeiHu02) were very closely related to each other, with 96.4–98.1% nucleotide identity for the partial M segment sequences and 95.9–98% nucleotide identity for the partial S segment sequences. These sequences showed higher identity to the known HTNV variants (<10.3% nucleotide differences for the M segment sequences and <8.6% nucleotide differences for the S segment sequences) than other hantavirus species (>25.1% and >24.8%). They clustered together and formed a new distinct genetic lineage (Fig. 5), and showed a close evolutionary relationship with the sequences recovered from A. agrarius mice and humans from Guizhou and Tianjin within the lineage #2 (Zou et al., 2008a). However, the strain WuhanHu16 had more than 10% nucleotide sequence differences from the strains WuhanAaJ10, WuhanHu12, WuhanHu29, and HubeiHu02. The strain clustered together with the isolate HV114 from Hubei province (Xiao et al., 1993) and strains from Guizhou, Zhejiang and Jiangsu provinces, which belong to the lineage #3 (Zou et al., 2008a). Remarkably, Guizhou, Hubei, Jiangsu and Zhejiang provinces are located, respectively, in upstream, middle reaches, and downstream of the Yangtze River. Sequences recovered from R. norvegicus, R. flavipectus, and M. musculus, were also very closely related to each other, with less than 3.9% nucleotide sequence differences for the partial M segment and 2.7% nucleotide sequence differences for the partial S segment. These sequences shared higher identity with known SEOV variants including those recovered previously from Wuhan and other parts of Hubei province (Lin et al., 2012) than with HTNV sequences recovered from A. agrarius or corresponding sequences of other hantavirus species (<73.9% nucleotide identity for the M segment sequences and <76.4% nucleotide identity for the partial S segment sequences). On the phylogenetic trees based on either M or S segment sequences (Fig. 6), all viruses from Rattus rats, and Mus mice
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Fig. 6. Phylogenetic relationships between SEOV variants described in this study and found previously in and outside China. Bayesian trees were based on the S (a) and M (b) segment sequences. Dobrava virus (DOBV) was used as the outgroup. Numbers (>0.5) above or below nodes indicate posterior node probabilities. The sequences recovered in this study are shown in red. The accession numbers of the sequences retrieved from GenBank are listed in (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
identified in this study belong to the phylogroup A of SEOV (Lin et al., 2012). On the M-tree, most of sequences obtained in this study clustered together within the phylogroup A of SEOV (Lin et al., 2012). In contrast, WuhanMm24 was closely related to the stain HubeiRn55 and strains from Japan, South Korea, and Singapore (Lin et al., 2012). These viruses formed another small cluster within the phylogroup A. On the S-tree, WuhanMm24 clustered together with the majority of strains identified in Wuhan in this study, while the clustering pattern of all other sequences was similar to that on the M-tree suggesting that genetic reassortment within the phylogroup A might have occurred. In addition, the isolate Hubeihuman1 clustered together with those outside of China. Finally, all known sequences from Wuhan and Hubei fell into four small clusters.
4. Discussion Like the whole country (Zhang et al., 2010b), Wuhan was seriously affected by the disease in 1970s and 1980s (Fig. 2). Remarkably, the annual numbers have decreased significantly from more
than 2000 in 1983 to less than 10 since 2005, indicating the disease is controlled effectively. As hantaviruses are still seriously prevalent in rodents in both rural and urban areas, more efforts are needed to further prevent human infection from rodents. The occurrence and epidemics of HFRS is influenced by both natural (e.g., ecological) and social (e.g., economic, occupational) factors (Bi et al., 2002; Chen and Qiu, 1994; Schmaljohn and Hjelle, 1997; Zhang et al., 2009b, 2010a). For example, most HFRS cases (>70%) occurred in rural areas, especially in those which belong to the humid and semi-humid climate zones in China (Bi et al., 2002; Zhang et al., 2010b). High rodent density and poor housing conditions in residential areas are responsible for most HFRS epidemics (Zhang et al., 2010b). Previous epidemiologic surveys revealed a linkage between human HFRS epidemics and high density of rodents, and hence their hantaviruses, in residential areas and fields in Wuhan (Chen et al., 1992; Fang et al., 1995). Especially in Jiangxia, which is the major agricultural areas of Wuhan, the higher capturing rate of A. agrarius mice in fields (especially in fields of rice) than in other regions has caused the higher incidence of HFRS. When compared with 1980s (Chen et al., 1992), the density of rodents in both fields and residential
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areas in Wuhan began to decrease significantly in 1990s (Chen et al., 2001; Fang et al., 1995). In accord with the recent surveys of rodent density (Wu et al., 2007), our surveys also revealed lower capturing rates of rodents in both fields and residential areas when compared with 1990s (Chen et al., 2001; Fang et al., 1995) and the beginning of 2000s (Tian et al., 2008). In addition, like other parts of China, in 1970s and 1980s the rural areas in Wuhan had been of the low socioeconomic status. Thus, the relatively low density of rodents and the significant improvement of housing conditions in recent years might have mainly contributed to the decrease tendency of HFRS incidence over the last decade in Wuhan. The peak of HFRS associated with SEOV carried by Rattus rats occurred in the spring, whereas HFRS associated with HTNV carried by Apodemus mice occurred mainly in the winter (Chen et al., 1986). The two peaks were related to the high densities of R. norvegicus rats in residential areas in spring and A. agrarius mice in fields in August and winter respectively (Chen et al., 1992, 2001) There are at least 20 species of rodents and insectivores in Wuhan (Liu and Yang, 2006). Previous surveys revealed that the dominant species of hantavirus carriers in Wuhan was A. agrarius 91.59% in fields and R. norvegicus 49.65% in residential areas, respectively, while hantavirus antigens were also detected in other species such as R. flavipectus and M. musculus (Chen et al., 1992, 2001). As most HFRS cases occurred in winter in 1990s, it means that HFRS cases were mainly transmitted by A. agrarius, even in urban areas. In the present study, hantavirus antigens were detected in these four species. Obviously, Rattus rats and Mus mice have become the major carriers of hantaviruses in Wuhan, except Caidian and Jianxia districts. Most cases occurred in spring and summer suggesting that they were associated with high density of Rattus rats in residential areas in 2000s. Like in other parts of China (Zhang et al., 2010a,b), SEOV and its carriers Rattus rats have become the primary source of human infection. In addition, Mus mice, are highly prevalent in urban and Xinzhou areas, and carry SEOV, suggesting that they also play an important role in the transmission of hantaviruses to human, particularly in urban areas. Serological and genetic analyses indicated that the HFRS cases occurred in urban areas were caused by HTNV. Further studies are needed to know whether these cases have been transmitted from the local rodents. There are at least nine genetic lineages of HTNV circulating in Apodemus mice in Eastern Asia including China; generally they show geographical clustering (Zou et al., 2008a). Phylogenetic analyses of the sequences recovered in this study showed that there are two lineages of HTNV variants circulating in rodents in Wuhan. Of these variants, WuhanHu12, WuhanHu29, HubeiHu02, and WuhanAaJ10 formed a new cluster on both M- and S-trees. These data suggest that at least some of human HFRS cases have been caused by the local lineage of HTNV variants. However, the strain WuhanHu16 clustered together with those isolated in other parts of Hubei province or in Guizhou, Jiangsu, and Zhejiang provinces, suggesting the spread of HTNV which might follow the migration of Apodemus mice along the Yangtze River. Although SEOV generally shows high genetic diversity and also displays geographic clustering pattern like other hantaviruses, the first SEOV phylogroup includes the majority of the known Chinese variants and also all known non-Chinese variants and displays a worldwide distribution resulted from the migration of Norway rats (Lin et al., 2012). Remarkably, all known SEOV variants identified in this study or previously in Wuhan/Hubei fell into four small clusters with the phylogroup A. More importantly, some of these viruses are closely related to those found outside of China (Lin et al., 2012; Wang et al., 2000). These data suggest that the convenient transportation (especially on water) in Wuhan may accelerate the translocation of Rattus rats and consequently the spread of SEOV between Wuhan and other parts of China or even outside China. Thus, these data strongly support our recent hypothesis that
the present worldwide distribution of SEOV is resulted from the migration of Norway rats (Lin et al., 2012). 5. Conclusion Both HTNV and SEOV are co-circulating in rodents in Wuhan. Although most of HFRS cases had caused by HTNV in both 1980s and 1990s, SEOV has become the dominant species to cause human HFRS today. A novel genetic lineage of HTNV is circulating in rodents in Wuhan. HTNV strains found in Wuhan are closely related to those from the provinces located in upstream, middle reaches, and downstream of the Yangtze River, and SEOV variants identified in Wuhan are more closely to those found outside China. These data suggest that the convenient transportation (especially on water) might have accelerated the spread of HTNV and SEOV between Wuhan and other parts of China and even outside China. Acknowledgments This study was supported by Grants 2003BA712A08-02 from the Chinese Ministry of Science and Technology and by the State Key Laboratory for Infectious Disease Prevention and Control (2011SKLID101). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2012. 07.017. References Bi, P., Donald, K., Tong, S., Ni, J., Parton, K., 2002. Climatic, reservoir and occupational variables and the transmission of haemorrhagic fever with renal syndrome in China. Int. J. Epidemiol. 31, 189–193. Burland, T.G., 2000. DNASTAR’s Lasergene sequence analysis software. Methods Mol. Biol. 132, 71–91. Chen, D.L., Yu, Q.F., Wang, S.Y., Fu, T.X., Wu, Y.X., Rao, K.M., Li, H., Tian, W.D., 2001. Surveillance of hemorrhagic fever with renal syndrome in Jiangxia district of Wuhan. In: Chen, H.X., Luo, C.W. (Eds.), Hemorrhagic fever with renal syndrome: Studies and the surveillance and application of vaccine, Hong Kong Medical Publisher, pp. 307–311 (in Chinese). Chen, D.L., Zhang, L., Luo, W.P., Zhang, L.Q., Ma, Z.J., Zhang, M.W., Huang, J.S., Fu, T.X., Rao, H.X., Li, H., 1992. Surveillance of epidemiological fever during 1984–1990 in Wuchang county. In: Chen, H.X., Wang, Z., Tang, S.Z. (Eds.), Surveillance on the epidemic hemorrhagic fever in China. Beijing Science & Technology Press, pp. 282–289 (in Chinese). Chen, H.X., 1987. Classification and Identification of Medical Animals. The Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing (in Chinese). Chen, H.X., Qiu, F.X., 1994. Studies on the environment structure of natural nidi and epidemic areas of hemorrhagic fever with renal syndrome in China. Chin. Med. J. 107, 107–112. Chen, H.X., Qiu, F.X., Dong, B.J., Ji, S.Z., Li, Y.T., Wang, Y., Wang, H.M., Zuo, G.F., Tao, X.X., Gao, S.Y., 1986. Epidemiological studies on hemorrhagic fever with renal syndrome in China. J. Infect. Dis. 154, 394–398. Guo, W.P., Lin, X.D., Wang, W., Zhang, X.H., Chen, Y., Cao, J.H., Ni, Q.X., Li, W.C., Li, M.H., Plyusnin, A., Zhang, Y.Z., 2011. A new subtype of Thottapalayam virus carried by the Asian house shrew (Suncus murinus) in China. Infect. Genet. Evol. 11, 1862–1867. Fang, Y., Huang, X.H., Zhou, Q., Hu, E.P., Wu, H.X., 1995. The trend of epidemic situation changes of hemorrhagic fever with renal syndrome (HFRS) in early 1990s in Hubei province. Chin. J. Vector Biol. Control 6, 111–113 (in Chinese). Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Jonsson, C.B., Figueiredo, L.T., Vapalahti, O., 2010. A global perspective on hantavirus ecology, epidemiology, and disease. Clin. Microbiol. Rev. 23, 412–441. Kariwa, H., Yoshimatsu, K., Arikawa, J., 2007. Hantavirus infection in East Asia. Comp. Immunol. Microbiol. Infect. Dis. 30, 341–356. Lin, X.D., Guo, W.P., Wang, W., Zou, Y., Hao, Z.Y., Zhou, D.J., Dong, X., Qu, Y.G., Li, M.H., Tian, H.F., Wen, J.F., Plyusnin, A., Xu, J., Zhang, Y.Z., 2012. Migration of norway rats resulted in the worldwide distribution of seoul hantavirus today. J. Virol. 86, 972–981.
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