Phylogenetic analysis based on mitochondrial DNA sequences of wild rats, and the relationship with Seoul virus infection in Hubei, China

  • Dong-Ying Liu,

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China,
    Department of Microbiology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

  • Jing Liu,

    Affiliation School of Health Sciences, Wuhan University, Wuhan 430071, China

  • Bing-Yu Liu,

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

  • Yuan-Yuan Liu,

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

  • Hai-Rong Xiong,

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

  • Wei Hou,

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

  • Zhan-Qiu Yang

    zqyang@whu.edu.cn

    Affiliation State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

    http://orcid.org/0000-0003-3839-938X

Phylogenetic analysis based on mitochondrial DNA sequences of wild rats, and the relationship with Seoul virus infection in Hubei, China

  • Dong-Ying Liu, 
  • Jing Liu, 
  • Bing-Yu Liu, 
  • Yuan-Yuan Liu, 
  • Hai-Rong Xiong, 
  • Wei Hou, 
  • Zhan-Qiu Yang
x

Abstract

Seoul virus (SEOV), which is predominantly carried by Rattus norvegicus, is one of the major causes of hemorrhagic fever with renal syndrome (HFRS) in China. Hubei province, located in the central south of China, has experienced some of the most severe epidemics of HFRS. To investigate the mitochondrial DNA (mtDNA)-based phylogenetics of wild rats in Hubei, and the relationship with SEOV infection, 664 wild rats were captured from five trapping sites in Hubei from 2000–2009 and 2014–2015. Using reverse-transcription (RT)-PCR, 41 (6.17%) rats were found to be positive for SEOV infection. The SEOV-positive percentage in Yichang was significantly lower than that in other areas. The mtDNA D-loop and cytochrome b (cyt-b) genes of 103 rats were sequenced. Among these animals, 37 were SEOV-positive. The reconstruction of the phylogenetic relationship (based on the complete D-loop and cyt-b sequences) allowed the rats to be categorized into two lineages, R. norvegicus and Rattus nitidus, with the former including the majority of the rats. For both the D-loop and cyt-b genes, 18 haplotypes were identified. The geographic distributions of the different haplotypes were significantly different. There were no significant differences in the SEOV-positive percentages between different haplotypes. There were three sub-lineages for the D-loop, and two for cyt-b. The SEOV-positive percentages for each of the sub-lineages did not significantly differ. This indicates that the SEOV-positive percentage is not related to the mtDNA D-loop or cyt-b haplotype or the sub-lineage of rats from Hubei..

INTRODUCTION

Seoul virus (SEOV) (genus Hantavirus, family Bunyaviridae), which is mainly carried by Rattus norvegicus (Norway rat), is a major cause of hemorrhagic fever with renal syndrome (HFRS) in China. Currently, the Hantavirus genus includes 23 distinct species and 30 provisional species (Plyusnin et al., 2012). Hantaviruses are hosted and transmitted by rodents, insectivores, and bats (Zhang, 2014). In most cases, there is a “one hantavirus, one host” relationship. Both co-speciation (Plyusnin and Sironen, 2014) and host-switching (Ramsden et al., 2009) have had an important impact on hantavirus evolution (Guo et al., 2013).

Two human diseases are caused by hantaviruses: HFRS in Eurasia, and hantavirus pulmonary syndrome (HPS) in the Americas (Watson et al., 2014). Studies on the ecological and phylogenetic relationships between hantaviruses and their hosts have suggested that the distribution and evolution of the reservoir hosts play important roles in the geographic distribution and epidemiology of hantavirus-related diseases (Schlegel et al., 2012; Bennett et al., 2014; Yanagihara et al., 2014; Schmidt et al., 2016).

HFRS is highly endemic in China. Hantaan virus (HTNV), which is mainly carried by Apodemus agrarius (Striped Field Mouse), and SEOV, which is mainly carried by R. norvegicus, are the major causes of HFRS (Chen et al., 1986a; Song, 1999; Zhang et al., 2014a; Cao et al., 2016). Hubei province, which is located in the central south of China, has experienced some of the most severe epidemics of HFRS (Zhang, 1990; Zhang et al., 2014b). SEOV and HTNV co-circulate in Hubei (Kang et al., 2012). Phylogenetic analysis (based on partial S- and M-segment sequences) revealed that there are several lineages of SEOV and a novel genetic lineage of HTNV in Jiangxia, Xinzhou, Qichun and Nanzhang of Hubui (Li et al., 2012; Liu et al., 2012). Analysis of epidemiological data from Hubei has indicated that the HFRS cases that occurred in the 1980s and 1990s were mainly caused by HTNV, whereas the proportion of HFRS cases caused by SEOV increased greatly in the 2000s (Zhang et al., 2014b).

Mitochondria are involved in virus-host interaction processes, such as apoptosis (Neumann et al., 2015; Zan et al., 2016) and auto/mitophagy (Ruggieri et al., 2014). Mitochondrial DNA (mtDNA) activates several innate immune pathways involving Toll-like receptor 9 (TLR9), the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, and stimulator of interferon genes (STING) signaling. In addition to regulating antiviral signaling, mtDNA contributes to inflammatory diseases after cellular damage and stress (Fang et al., 2016).

Genetic analysis based on mtDNA sequences has been carried out to identify the animal species that act as hantavirus reservoirs and to clarify the evolutionary relationships among the reservoir hosts (Morzunov et al., 1998; Torres-Perez et al., 2010; Lin et al., 2012; Liu et al., 2012; Hugot et al., 2014; Schmidt et al., 2016). However, the association between SEOV infection and the mtDNA characteristics of the reservoir hosts in Hubei, China, has not been fully demonstrated. This information would provide a better understanding of the mtDNA genetic background of wild rats in Hubei, shed light on the relationship between SEOV infection and mtDNA genetic diversity, and could be valuable for the control of HFRS. To fill this gap, wild rats were captured in Hubei during 2000–2009 and 2014–2015. The lungs of the rats were screened for SEOV using specific reverse-transcription (RT)-PCR. The mitochondrial D-loop and cytochrome b (cyt-b) gene sequences were then identified from the lung tissues and subjected to phylogenetic analysis.

MATERIALS AND METHODS

Rodent samples

During 2000–2009 and 2014–2015, wild rats were captured with snap-traps, which were generally set at 5 m apart and baited with peanuts in both residential areas and fields. Trapping was conducted at five locations that are known to be HFRS epidemic areas in Hubei: Nanzhang (NZ), Yichang (YC), Xinzhou (XZ), Jiangxia (JX), and Qichun (QC) (Figure 1). All the animal research was conducted in accordance with internationally accepted principles and the Wuhan University Guidelines on the Care and Use of Laboratory Animals. The trapped animals were identified as described previously (Chen et al., 1986b). Lung tissues were collected and stored at –80 °C until use.

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Fig 1. Map showing the trapping sites (▲) for wild rats in Hubei province, China.

RT-PCR of the SEOV partial S-segment sequence

Total RNA was extracted from the rodent lung tissues using an RNAprep Tissue Kit (Tiangen, Beijing, China). First-strand cDNA was generated from the total RNA using random primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Beijing, China). The SEOV partial S-segment sequences were amplified with primers S927F (5′-GATTGAAGATAT TGAGTCACC-3′) and S1168R (5′-GTTGTATCCCCATT GATTGTG-3′). The total volume of each PCR reaction was 50 µL, comprising 5 µL cDNA, 0.2 µmol/L of the forward and reverse primers, 200 µmol/L deoxynucleotide triphosphates (dNTPs), 1 unit Taq DNA polymerase (Tiangen, Beijing, China), 10 mmol/L Tris/HCl, (pH 8.3), 50 mmol/L KCl, and 1.5 mmol/L MgCl2. The PCR cycling conditions were as follows: 5 min at 94 °C; 30 cycles consisting of 45 s at 94 °C, 45 s at 55 °C, and 20 s at 72 °C; and a final extension step of 5 min at 72 °C. The PCR products were detected by running them on an agarose gel and staining them with ethidium bromide, and they were then view under ultraviolet light.

PCR and sequencing of the mtDNA D-loop and cyt-b genes

To verify the species carried by the hantavirus-infected rodents and to study their phylogenetic relationships, genomic DNA was extracted from the lung tissues using a DNAprep Tissue Kit (Tiangen, Beijing, China). The entire 899-nucleotide D-loop region of the mtDNA was amplified by PCR, using primers DF (5′-GTCAACT CCCAAAGCTGAAATTC-3′) and DR (5′-TCTCGAGATTTTCAGTGTCTTGCTTT-3′). The entire 1143-nucleotide cyt-b region of the mtDNA was amplified by PCR, using primers BF (5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′) and BR (5′-AACTGCAGTC ATCTCCGGTTTACAAGAC-3′). The total volume of each PCR reaction was 50 µL, comprising 0.5 µg of DNA template, 0.1 µmol/L of each primer, 200 μmol/L of dNTPs, and 2 units of Taq DNA polymerase (Tiangen, Beijing, China). The cycling conditions consisted of an initial denaturation of 3 min at 94 °C; 35 cycles consisting of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C; and a final extension step at 72 °C for 10 min. The PCR products were gel-purified, and sequenced using an ABI 3730 automatic sequencer (Torrance, CA, USA).

Molecular diversity analysis

For the mtDNA D-loop and cyt-b sequences, several population parameters, i.e., haplotype number (h), haplotype diversity (h.d.), and nucleotide diversity (π), were calculated using DnaSP v5.0 (Librado and Rozas, 2009). All positions containing gaps and missing data were eliminated from the dataset.

Phylogenetic analyses

Multiple sequence alignment was carried out using ClustalW (Larkin et al., 2007) with default parameters, and the analysis was revised using BioEdit 7.2 (Hall, 1999). D-loop or cyt-b sequences of rats from other parts of China and other countries were also included in the phylogenetic analysis. For each haplotype, one sequence was included in the phylogenetic analysis. The phylogenetic relationships for first the D-loop and then the cyt-b sequences were reconstructed using Bayesian Markov Chain Monte Carlo (MCMC) runs over 500,000 generations, as implemented in MrBayes 3.1 (Ronquist and Huelsenbeck, 2003). For both analyses, a majority rule (50%) consensus tree was constructed after burn-in of an initial 1250 trees. For both the mtDNA D-loop and cyt-b sequences, Microtus kikuchii (a species of vole that belongs to the family Cricetidea), was used as the outgroup to root the phylogenetic tree.

Statistical analyses

Using the likelihood ratio chi-square test, the SEOV-positive percentage among different geographic locations, different mtDNA haplotypes, and different mtDNA phylogenetic sub-lineages were evaluated, along with the geographic distribution of the mtDNA haplotypes.

Nucleotide sequence accession numbers

The new mtDNA D-loop and cyt-b sequences of the rodents described in this study have been deposited in GenBank under accession numbers KY356101-KY356194, and MF062462-MF062465 (Supplementary Table S1). Other previously published sequences used in the study were obtained from GenBank (Supplementary Table S2).

RESULTS

Hantavirus infection in rodents

During 2000–2009 and 2014–2015, 664 wild rats were captured from five trapping sites in Hubei. Using RT-PCR, 41 (6.17%) wild rats were found to be positive of SEOV infection (Table 1). The SEOV-positive percentage in Yichang was significantly lower than that in other areas (P < 0.01).

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Table 1. Detection of SEOV (using RT-PCR) in wild rats from Hubei, China
Site RT-PCRpositive Total Percentage
Nanzhang 16 160 10.00
Yichang 1 126 0.79**
Xinzhou 10 178 5.62
Jiangxia 7 125 5.60
Qichun 8 75 10.67
Total 41 664 6.17
Note: **P < 0.01 (chi-square test)

mtDNA sequencing, phylogeny, and relationship with SEOV infection

In this study, the D-loop and cyt-b genes of 103 rats were sequenced. Among these animals, 37 were SEOV positive. For both the D-loop and cyt-b genes, 18 haplotypes were identified (Tables 2, 3).

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Table 2. Number of rats with each D-loop haplotype (D1–18) by geographic location
Haplotype Nanzhang Yichang Xinzhou Jiangxia Qichun All
D1 4/14 (28.57) 1/7 (14.29) 5/21 (23.81)
D2 4/19 (21.05) 4/19 (21.05)
D3 6/9 (66.67) 0/9 (0) 6/18 (33.33)
D4 5/7 (71.43) 5/7 (71.43)
D5 4/6 (66.67) 1/1 (100) 5/7 (71.43)
D6 3/6 (50.00) 3/6 (50.00)
D7 2/5 (40.00) 2/5 (40.00)
D8 0/4 (0) 0/4 (0)
D9 2/3 (66.67) 2/3 (66.67)
D10 1/2 (50.00) 1/2 (50.00)
D11 0/2 (0) 0/2 (0)
D12 1/2 (50.00) 1/2 (50.00)
D13 1/1 (100.00) 1/1 (100.00)
D14 0/1 (0) 0/1 (0)
D15 1/1 (100.00) 1/1 (100.00)
D16 1/1 (100) 1/1 (100.00)
D17 0/1 (0) 0/1 (0)
D18 0/2 (0) 0/2 (0)
All 15/31 (48.39) 1/16 (6.25) 9/32 (28.13) 5/7 (71.43) 7/17 (41.18) 37/103 (35.92)
Note: For each cell, the figures are the no. of SEOV-positive rats/no. of sequenced rats (%). The chi-square test of the difference in the SEOV-positive percentage among the different haplotypes indicated no significant differences (P > 0.05).The chi-square test of the difference in the geographic distribution among the different haplotypes indicated a significant difference (P < 0.01).

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Table 3. Number of rats with each cyt-b haplotype (B1–18) by geographic location
Haplotype Nanzhang Yichang Xinzhou Jiangxia Qichun All
B1 5/14 (35.71) 1/7 (14.29) 1/2 (50.00) 7/23 (30.43)
B2 6/9 (66.67) 0/9 (0) 1/1 (100.00) 7/19 (36.84)
B3 5/19 (26.32) 5/19 (26.32)
B4 1/6 (14.28) 1/6 (16.67)
B5 4/6 (66.66) 4/6 (66.66)
B6 4/6 (66.66) 4/6 (66.66)
B7 3/6 (50.00) 3/6 (50.00)
B8 2/4 (50.00) 2/4 (50.00)
B9 0/3 (0) 0/3 (0)
B10 0/2 (0) 0/2 (0)
B11 0/1 (0) 0/1 (0)
B12 1/1 (100.00) 1/1 (100.00)
B13 1/1 (100.00) 1/1 (100.00)
B14 1/1 (100.00) 1/1 (100.00)
B15 1/1 (100.00) 1/1 (100.00)
B16 0/1 (0) 0/1 (0)
B17 0/1 (0) 0/1 (0)
B18 0/2 (0) 0/2 (0)
All 16/31 (51.61) 1/16 (6.25) 8/32 (25.00) 5/7 (71.43) 7/17 (38.89) 37/103 (35.92)
Note: For each cell, the figures are the no. of SEOV-positive rats/no. of sequenced rats (%). The chi-square test of the difference in the SEOV-positive percentage among the different haplotypes indicated no significant differences (P > 0.05).The chi-square test of the difference in the geographic distribution among the different haplotypes indicated a significant difference (P < 0.01).

Using Bayesian methods to reconstruct the phylogenetic relationships based on complete D-loop or cyt-b sequences allowed the rats to be categorized into two lineages, R. norvegicus and Rattus nitidus, both with high support values (Figure 2). Lineage R. norvegicus contained the majority of the rats, i.e., 101 rats, with 37 (36.63%) being SEOV positive. Lineage R. nitidus included two rats (both from Qichun in Hubei), both of which were SEOV negative.

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Fig 2. Bayesian reconstruction of (A) D-loop and (B) cyt-b phylogenetic trees. The haplotypes of mtDNA D-loop and cyt-b identified in this study are presented in boldface. The number of SEOV-positive rats/mumber of sequenced rats (percentage) and geographic locations are noted after each haplotype. The haplotype numbers of reference sequences (obtained from GenBank) are marked according to previous descriptions (Song et al.,2014). The branches are labeled with the Bayesian posterior possibilities (cut-off > 50%). The scale bar shows number of substitutions per mucleotide.

mtDNA D-loop

The lineage R. norvegicus consisted of 17 haplotypes, with a haplotype diversity of 1.00 ± 0.02 and a nucleotide diversity of 0.74 ± 0.08% (Table 4). Some of the rats from Hubei formed three sub-lineages, D-I to D-III, while the others could not be classified into clusters (Figure 2A). Sub-lineage D-I included 40 rats, with six haplotypes (D2, D5–D7, D11, and D17). Sub-lineage D-II included six rats, with two haplotypes. Sub-lineage D-III included 19 rats, with two haplotypes. There were 36 rats, with seven haplotypes (D-others), that were not classified into sub-lineages (Table 4). The SEOV-positive percentages for the sub-lineages were 35% (D-I), 16.67% (D-II), 36.84% (D-III), and 41.67% (D-others). There were no significant differences in the SEOV-positive percentages between the sub-lineages when evaluated with a chi-square test (P > 0.05).

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Table 4. Descriptive statistics of genetic variation of D-loop and cyt-b sequences of R. norvegicus and R. nitidus in Hubei, China
Gene Lineage Na Nhb Sc Hdd (mean ± SD) πe (%) (mean ± SD)
D-loop D-I 40 6 12 1.00 ± 0.10 0.47 ± 0.09
D-loop D-II 6 2 1 1.00 ± 0.50 0.11 ± 0.06
D-loop D-III 19 2 2 1.00 ± 0.50 0.22 ± 0.11
D-loop D-others 36 7 18 1.00 ± 0.76 0.65 ± 0.16
D-loop R. norvegicus 101 17 35 1.00 ± 0.02 0.74 ± 0.08
D-loop R. nitidus 2 1 0 0 NA
D-loop All 103 18 88 1.00 ± 0.02 1.48 ± 0.64
cyt-b B-I 7 2 1 1.00 ± 0.50 0.09 ± 0.04
cyt-b B-II 4 2 1 1.00 ± 0.50 0.09 ± 0.04
cyt-b B-others 90 13 19 1.00 ± 0.03 0.33 ± 0.04
cyt-b R. norvegicus 101 17 31 1.00 ± 0.02 0.50 ± 0.10
cyt-b R. nitidus 2 1 0 0 NA
cyt-b All 103 18 84 1.00 ± 0.02 1.04 ± 0.48
Note: aN, number of rats; bNh, number of haplotypes; cS, number of polymorphic (segregating) sites; dHd, haplotype diversity; eπ, nucleotide diversity; fNA, non-applicable, because the number of samples was less than seven.

Haplotype D12 clustered with a rat from Hainan, China. Haplotype D15 clustered with a rat from Denmark. Other rats from Japan, France, and Germany did not cluster with any of the rats from Hubei in our study.

For each D-loop haplotype, the SEOV-positive percentage varied, but there were no significant differences when evaluated with a chi-square test (P > 0.05) (Table 2). Most of the rats in this study were in haplotypes D1–D3, and they were distributed in Nanzhang, Yichang, and Xinzhou. The numbers of D-loop haplotypes in Qichun (seven haplotypes) and Xinzhou (six haplotypes) were larger than that in any other area (Table 2, Figure 3A). The geographic distributions of the different D-loop haplotypes were significantly different when evaluated with a chi-square test (P < 0.01).

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Fig 3. Geographic distribution of haplotypes of mtDNA (A) D-loop or (B) cyt-b. The geographic distributions among the different D-loop and cyt-b haplotypes were evaluated with chi-square tests, and there was a significant difference for both (P < 0.01).

cyt-b gene

The lineage R. norvegicus comprised 17 haplotypes, with a haplotype diversity of 1.00 ± 0.02 and a nucleotide diversity of 0.50 ± 0.10% (Table 4). Two sub-lineages, B-I and B-II, were formed. Sub-lineage B-I included six rats, with two haplotypes. Sub-lineage B-II involved four rats, with two haplotypes. There were 90 rats, with 13 haplotypes (B-others), that were not classified into sub-lineages (Figure 2B, Table 4). The SEOV-positive percentage for the sub-lineages were 71.42% (B-I), 0% (B-II), and 32.56% (B-others). There were no significant differences in the SEOV-positive percentages between the sub-lineages when evaluated with a chi-square test (P > 0.05).

Lineage B-I was closely related to a rat from Guangdong, China. Haplotype B13 clustered with a rat from Wuhan, Hubei, China (Lin et al., 2012), and two from Europe. Haplotype B11 clustered with a rat from Fujian, China (Lin et al., 2012). Other previously described rat cyt-b sequences did not cluster with any of the sequences in this study.

For each cyt-b haplotype, the SEOV-positive percentage varied, but there were no significant differences when evaluated with a chi-square test (P > 0.05) (Table 3). The haplotypes B1-B3 included most of the rats in this study, and they were distributed in Nanzhang, Yichang, Xinzhou, and Jiangxia. The numbers of cyt-b haplotypes in Xinzhou (seven haplotypes) and Qichun (six haplotypes) were larger than that in any of the other areas (Table 3, Figure 3B). The geographic distributions of different cyt-b haplotypes were significantly different when evaluated with a chi-square test (P < 0.01).

DISCUSSION

Norway rats are reservoir hosts for several zoonotic pathogens that infect humans, such as hantaviruses (particularly SEOV), hepatitis E virus, Leptospira interrogans, and Toxoplasma gondii (Meerburg et al., 2009). As the main reservoir of SEOV, the distribution of Norway rats determines the spread of SEOV (Lin et al., 2012). SEOV has been found across the world, from Asia (Kariwa et al., 1994; Ibrahim et al., 1996; Guo et al., 2016) to Africa (Avsic-Zupanc et al., 2015), Europe (Heyman et al., 2004; Heyman et al., 2009; Plyusnina et al., 2012; Dupinay et al., 2014), and the Americas (Childs et al., 1987; Cueto et al., 2008; Costa et al., 2014).

In the past, it was thought that Norway rats originated in the area bordering northern China and Mongolia (Hedrich, 2000; Lin et al., 2012). However, recent fossil analyses indicate that they may have originated in Southwestern China around 1.2–1.6 million years ago (Mya) (Jin et al., 2008; Wu and Wang, 2012). In addition, a phylogeographic analysis based on mtDNA cyt-b and D-loop sequences indicated that the Norway rat originated in southern China about 1.3 Mya (Song et al., 2014). The analysis of rat fossils collected in the Choukoutien Cave in northern China further indicated that the species arrived there about 0.14 Mya and was widely distributed across most of China and adjacent Asian countries about 0.01–0.13 Mya (Wu and Wang, 2012).

During the 15th century, Norway rats began their spread across the globe (Aplin et al., 2003). The Norway rats migrated to Europe in the 18th century (Barnett, 2002). They reached North America on the ships of the new settlers by the middle of the 18th century (Grzimek, 1968). The worldwide spread of Norway rats can be directly attributed to their relationship with humans (Robinson, 1965). The current worldwide distribution of the Norway rats followed the distribution of human activities, and it might have taken place within the last few centuries (Lin et al., 2012).

In China, HFRS is still serious public health problem even though comprehensive prevention measures have been implemented. HTNV and SEOV are the main causes of HFRS in China. SEOV-positive rats have been found in all the provinces of China except for Qinghai (Zhang et al., 2010; Hu et al., 2015). The proportion of SEOV-related HFRS cases increased greatly in the 2000s in Hubei (Zhang et al., 2014b).

In this study, using RT-PCR, SEOV was detected in rats captured in five areas in Hubei. The SEOV-positive percentage was 6.17%. The SEOV-positive percentage in Yichang was significantly lower than that in all the other areas (P < 0.01). It has previously been reported that rodent density and incidences of HFRS decreased and were maintained at low levels in the Three Gorges reservoir region, which includes Yichang (Chang et al., 2016). Further continuous surveillance will be needed to monitor hantavirus infections in rodent populations.

Mitochondrial genes are associated with the course of disease caused by several viruses. Hepatitis C virus persistence is strongly associated with mitochondrial dysfunction, with liver mtDNA genetic diversity being linked to disease progression (Campo et al., 2016). The Amerindian mtDNA haplogroup B2 enhances risk of cervical cancer caused by human papillomavirus, and deregulation of mitochondrial genes may be involved (Guardado-Estrada et al., 2012). The frequency of mtDNA D-loop mutations in non-neoplastic tissue was found to be higher in HBV-infected patients with hepatocellular carcinoma than in HBV non-infected patients with hepatocellular carcinoma (Gwak et al., 2011). Mitochondrial cyt-b is a mediator of FAS-induced apoptosis (Komarov et al., 2008). Forced expression of cyt-b mutations induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells (Dasgupta et al., 2009). It has been reported that, according to phylogenetic trees based on mtDNA D-loop or cyt-b gene sequences of Apodemus agrarius, hantavirus-positive and hantavirus-negative mice belonged to the same cluster (Yang et al., 2016).

In this study, the reconstruction of the phylogenetic relationship between rats based on complete D-loop or cyt-b sequences allowed the rats to be categorized into two lineages, R. norvegicus and R. nitidus, with the former group containing the majority of the rats. These two species are sister species in the genus Rattus, and cannot be easily distinguished from one another. R. norvegicus lives with humans in and around residential areas, while R. nitidus lives only on farmland in hilly areas. In the mountainous areas of Guizhou, Yunan, Zhejiang, and Hunan provinces of China, hantaviruses were isolated from R. nitidus (Zou et al., 2008; Zhou et al., 2009; Lin et al., 2012). In this study, both of the rats that were categorized as R. nitidus were SEOV negative. More samples are therefore needed to clarify the level of hantavirus infection in R. nitidus in Hubei province.

The geographic distribution of the different mtDNA D-loop or cyt-b haplotypes were significantly different. This indicates that the mtDNA genes of rats vary in different areas of Hubei. For R. norvegicus, 17 haplotypes of mtDNA D-loop or cyt-b genes were identified in Hubei. There were no significant differences among the SEOV-positive percentages for the different haplotypes. There were three sub-lineages based on the D-loop sequences, and two based on the cyt-b sequences. The SEOV-positive percentages for the sub-lineages did not significantly differ. Further studies with immunity-related genes, such as major histocompatibility complex (MHC) class I, MHC class II, C4A component of the complement system, tumor necrosis factor (TNF), and interleukin-1 receptor antagonist (IL-1RA) (Charbonnel et al., 2014) may shed more light on the relationship between hantavirus infections and host genetic background.

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China (81402728, 81371865).

COMPLIANCE WITH ETHICS GUIDELINES

The authors declared that they have no conflict of interests. The study was approved by the Ethics Committees of School of Basic Medical Sciences, Wuhan University. All institutional and national guidelines for the care and use of laboratory animals were followed.

AUTHOR CONTRIBUTIONS

ZQY and DYL designed the experiments. JL, BYL, YYL, HRX and WH carried out the experiments. DYL analyzed the data. DYL and ZQY wrote the manuscript. All authors read and approved the final manuscript. Supplementary tables are available on the websites of Virologica Sinica: www.virosin.org; link.springer.com/journal/12250.

SUPPLEMENTARY MATERIAL

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Table S1. GenBank number for each haplotype of mtDNA D-loop or cyt-b
Haplotype GenBank number
D1 MF062462
D2 MF062463
D3 MF062464
D4 KY356160
D5 KY356153
D6 KY356169
D7 KY356186
D8 KY356167
D9 KY356190
D10 KY356179
D11 KY356181
D12 KY356185
D13 HQ655894
D14 KY356156
D15 KY356174
D16 HQ655911
D17 KY356184
D18 KY356172
B1 KY356138
B2 KY356105
B3 MF062465
B4 KY356126
B5 KY356102
B6 KY356113
B7 KY356116
B8 KY356139
B9 KY356144
B10 KY356135
B11 KY356108
B12 KY356115
B13 KY356124
B14 KY356129
B15 KY356130
B16 KY356136
B17 KY356142
B18 KY356122

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Table S2. The geographic origin, haplotype and length of reference sequences from GenBank
Species Continent Country Sample location (if known) Strain or ID Gene Haplotype Sequence length (bp) GenBank No.
Rattus
norvegicus
Europe France 83 D-loop D1 499 JX887169
Rattus
norvegicus
Wild/Mcwi D-loop D1 898 DQ673916
Rattus
norvegicus
Europe France 17 D-loop D2 664 JX887165
Rattus
norvegicus
Europe Germany Ludwigshafen 3999 D-loop D3 664 JX887170
Rattus
norvegicus
T2DN/Mcwi D-loop D3 899 DQ673915
Rattus
norvegicus
Europe Germany Drensteinfurt 3748 D-loop D4 664 JX887166
Rattus
norvegicus
Asia Japan Japan/Tku D-loop D5 664 DQ673917
Rattus
norvegicus
Europe Germany Velen 3273 D-loop D6 664 JX887167
Rattus
norvegicus
Europe Germany Magdeburg 3234 D-loop D7 654 JX887171
Rattus
norvegicus
Europe Germany Dorsten 3599 D-loop D8 664 JX887173
Rattus
norvegicus
Europe Germany Drensteinfurt 3493 D-loop D9 664 JX887174
Rattus
norvegicus
Europe France 111 D-loop D10 499 JX887172
Rattus
norvegicus
Europe Denmark Copenhagen D-loop D12 664 AJ428514
Rattus
norvegicus
Asia Vietnam specimen 947 D-loop D13 300 U13748
Rattus
norvegicus
Asia China Hainan DS05 D-loop D14 469 HM031630
Rattus
norvegicus
GH/OmrMcwi D-loop D23 664 DQ673911
Rattus
norvegicus
GK/Far D-loop D24 664 DQ673912
Rattus
norvegicus
WKY/NCrl D-loop D25 664 DQ673907
Rattus
norvegicus
BN/SsNHsdMCW D-loop 898 NC_001665
Rattus
norvegicus
Sprague/Dawley D-loop 897 X04734
Rattus
norvegicus
Wistar D-loop 899 X52757
Rattus
nitidus
Asia China Sichuan D-loop 897 KX058347
Rattus
Rattus
RNZRrTit01 D-loop 898 EU273707
Rattus
Rattus
M.D. D-loop 898 X04735
Rattus
tanezumi
RJPNAna02 D-loop 901 EU273712
Microtus
Kikuchii
D-loop 922 NC003041
Rattus
norvegicus
Europe France 17 cyt-b C1 549 JX887163
Rattus
norvegicus
Europe Germany Ludwigshafen 3999 cyt-b C2 549 JX887164
Rattus
norvegicus
Europe Germany Olfen 3862 cyt-b C3 549 JX887161
Rattus
norvegicus
Europe France 91 cyt-b C4 549 JX887160
Rattus
norvegicus
Europe France 83 cyt-b C5 549 JX887162
Rattus
norvegicus
Europe Denmark Denmark cyt-b C6 549 AJ428514
Rattus
norvegicus
Asia Japan cyt-b C7 549 DQ673917
Rattus
norvegicus
Asia China Hainan cyt-b C8 549 HM031679
Rattus
norvegicus
Asia China Hainan cyt-b C9 549 HM031682
Rattus
norvegicus
Asia China Inner Mongolia cyt-b C10 549 GU592954
Rattus
norvegicus
Asia China Hebei cyt-b C11 549 GU592956
Rattus
norvegicus
Asia China Guangdong cyt-b C12 549 GU592960
Rattus
norvegicus
Asia China Guangdong cyt-b C13 549 GU592961
Rattus
norvegicus
Asia China Hebei cyt-b C14 549 GU592963
Rattus
norvegicus
Asia China Heilongjiang cyt-b C15 549 GU592964
Rattus
norvegicus
Asia China Henan cyt-b C16 549 GU592966
Rattus
norvegicus
Asia China Hebei cyt-b C17 549 GU592962
Rattus
norvegicus
Asia China Liaoning cyt-b C18 549 GU592970
Rattus
norvegicus
Asia China Hunan cyt-b C19 549 GU592974
Rattus
norvegicus
Asia China Jiangsu cyt-b C20 549 GU592975
Rattus
norvegicus
Asia China Jilin cyt-b C21 549 GU592979
Rattus
norvegicus
Asia China Jilin cyt-b C22 549 GU592980
Rattus
norvegicus
Asia China Jilin cyt-b C23 549 GU592981
Rattus
norvegicus
Asia China Shandong cyt-b C24 549 GU592982
Rattus
norvegicus
Asia China Fujian cyt-b C25 549 GU592983
Rattus
norvegicus
Asia China Liaoning cyt-b C26 549 GU592988
Rattus
norvegicus
Asia China Hubei cyt-b C27 549 GU592991
Rattus
norvegicus
Asia China Inner Mongolia cyt-b C28 549 GU592993
Rattus
norvegicus
Asia China Inner Mongolia cyt-b C29 549 GU592994
Rattus
norvegicus
Asia China Yunnan cyt-b C30 549 GU592997
Rattus
norvegicus
Asia Vietnam cyt-b C31 549 AB355903
Rattus
norvegicus
Asia Vietnam cyt-b C32 549 FJ842277
Rattus
norvegicus
Asia Vietnam cyt-b C33 549 FJ842278
Rattus
norvegicus
Asia Vietnam cyt-b C34 549 FR775887
Rattus
norvegicus
Asia Thailand cyt-b C35 549 HM217429
Rattus
norvegicus
Asia Indonesia cyt-b C36 549 FJ842279
Rattus
norvegicus
Africa South Africa cyt-b C37 549 FJ842274
Rattus
norvegicus
Africa South Africa cyt-b C38 549 DQ439839
Rattus
norvegicus
WKY/NCrl cyt-b C39 549 DQ673907
Rattus
nitidus
Asia China Zhejiang YongjiaRn40 cyt-b 1140 GU592990
Rattus
nitidus
Asia China Zhejiang YongjiaRn14 cyt-b 1140 GU592995
Rattus
nitidus
Asia China Zhejiang YongjiaRn56 cyt-b 1140 GU592965
Rattus
nitidus
Asia China Hunan NYA039 cyt-b 1140 GU592985
Rattus
nitidus
Asia China Tibet R120516 cyt-b 1143 KC735129
Rattus
nitidus
Asia China cyt-b 1140 KX058347
Rattus
nitidus
Asia India Mao CAUII344 cyt-b 1140 AB973108
Rattus
nitidus
Asia India Ukhrul CAUII2012 cyt-b 1140 AB973109
Rattus
nitidus
Asia Viet Nam Dak Lak pr. Eawy D29 cyt-b 1140 FR775883
Rattus
nitidus
Asia Viet Nam Gia Lai pr, Pleiku Z40 cyt-b 1140 FR775884
Rattus
tanezumi
RJPNAna02 9Oct06 cyt-b 1140 EU273712
Rattus
Rattus
Africa South Africa ARC101 cyt-b 1140 DQ439830
Rattus
Rattus
RNZRrTit01 cyt-b 1140 EU273707
Microtuskukuchii cyt-b 1143 AF348082

References

  1. 1. Aplin KP, Chesser T, Ten Have J. 2003. Evolution biology of the genus Rattus: profile of an archetypal rodent pest. In: Rats, mice and people: rodent biology and management, Singleton GR, Hinds LA, Krebs CJ, et al. (eds). Canberra: Australian Centre for International Agriculture Research. pp 487–498.
  2. 2. Avsic-Zupanc T, Saksida A, Korva M. 2015. Hantavirus infections. Clin Microbiol Infect.
  3. 3. Barnett SA. 2002. The story of rats: their impact on us, and our impact on them. Australia: Allen and Uwin, Crows Nest. pp. 17–18.
  4. 4. Bennett SN, Gu SH, Kang HJ, Arai S, Yanagihara R. 2014. Reconstructing the evolutionary origins and phylogeography of hantaviruses. Trends Microbiol, 22: 473–482.
  5. 5. Campo DS, Roh HJ, Pearlman BL, Fierer DS, Ramachandran S, Vaughan G, Hinds A, Dimitrova Z, Skums P, Khudyakov Y. 2016. Increased Mitochondrial Genetic Diversity in Persons Infected With Hepatitis C Virus. Cell Mol Gastroenterol Hepatol, 2: 676–684.
  6. 6. Cao S, Ma J, Cheng C, Ju W, Wang Y. 2016. Genetic characterization of hantaviruses isolated from rodents in the port cities of Heilongjiang, China, in 2014. BMC Vet Res, 12: 69.
  7. 7. Chang ZR, Lu L, Mao DQ, Pan HM, Feng LG, Yang XB, Liu FF, He YY, Zhang J, Yang WZ. 2016. Dynamics of Rodent and Rodent-borne Disease during Construction of the Three Gorges Reservoir from 1997 to 2012. Biomed Environ Sci, 29: 197–204.
  8. 8. Charbonnel N, Pages M, Sironen T, Henttonen H, Vapalahti O, Mustonen J, Vaheri A. 2014. Immunogenetic factors affecting susceptibility of humans and rodents to hantaviruses and the clinical course of hantaviral disease in humans. Viruses, 6: 2214–2241.
  9. 9. Chen HX, Qiu FX, Dong BJ, Ji SZ, Li YT, Wang Y, Wang HM, Zuo GF, Tao XX, Gao SY. 1986a. Epidemiological studies on hemorrhagic fever with renal syndrome in China. J Infect Dis, 154: 394–398.
  10. 10. Chen HX, Qiu FX, Dong BJ, Ji SZ, Li YT, Wang Y, Wang HM, Zuo GF, Tao XX, Gao SY. 1986b. Epidemiological studies on hemorrhagic fever with renal syndrome in China. J Infect Dis, 154: 394–398.
  11. 11. Childs JE, Korch GW, Glass GE, LeDuc JW, Shah KV. 1987. Epizootiology of Hantavirus infections in Baltimore: isolation of a virus from Norway rats, and characteristics of infected rat populations. Am J Epidemiol, 126: 55–68.
  12. 12. Costa F, Porter FH, Rodrigues G, Farias H, de Faria MT, Wunder EA, Osikowicz LM, Kosoy MY, Reis MG, Ko AI, Childs JE. 2014. Infections by Leptospira interrogans, Seoul virus, and Bartonella spp. among Norway rats (Rattus norvegicus) from the urban slum environment in Brazil. Vector Borne Zoonotic Dis, 14: 33–40.
  13. 13. Cueto GR, Cavia R, Bellomo C, Padula PJ, Suarez OV. 2008. Prevalence of hantavirus infection in wild Rattus norvegicus and R. rattus populations of Buenos Aires City, Argentina. Trop Med Int Health, 13: 46–51.
  14. 14. Dasgupta S, Hoque MO, Upadhyay S, Sidransky D. 2009. Forced cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells. Int J Cancer, 125: 2829–2835.
  15. 15. Dupinay T, Pounder KC, Ayral F, Laaberki MH, Marston DA, Lacote S, Rey C, Barbet F, Voller K, Nazaret N, Artois M, Marianneau P, Lachuer J, Fooks AR, Pepin M, Legras-Lachuer C, McElhinney LM. 2014. Detection and genetic characterization of Seoul virus from commensal brown rats in France. Virol J, 11: 32.
  16. 16. Fang C, Wei X, Wei Y. 2016. Mitochondrial DNA in the regulation of innate immune responses. Protein Cell, 7: 11–16.
  17. 17. Grzimek B. 1968. Animal life encyclopedia. New York, NY: Van Nostrand Reinhold, pp. 579.
  18. 18. Guardado-Estrada M, Medina-Martinez I, Juarez-Torres E, Roman-Bassaure E, Macias L, Alfaro A, Alcantara-Vazquez A, Alonso P, Gomez G, Cruz-Talonia F, Serna L, Munoz-Cortez S, Borges-Ibanez M, Espinosa A, Kofman S, Berumen J. 2012. The Amerindian mtDNA haplogroup B2 enhances the risk of HPV for cervical cancer: de-regulation of mitochondrial genes may be involved. J Hum Genet, 57: 269–276.
  19. 19. Guo G, Sheng J, Wu X, Wang Y, Guo L, Zhang X, Yao H. 2016. Seoul virus in the Brown Rat (Rattus norvegicus) from Urumqi, Xinjiang, Northwest of China. J Wildl Dis, 52: 705–708.
  20. 20. Guo WP, Lin XD, Wang W, Tian JH, Cong ML, Zhang HL, Wang MR, Zhou RH, Wang JB, Li MH, Xu J, Holmes EC, Zhang YZ. 2013. Phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents. PLoS Pathog, 9: e1003159.
  21. 21. Gwak GY, Lee DH, Moon TG, Choi MS, Lee JH, Koh KC, Paik SW, Joh JW, Yoo BC. 2011. The correlation of hepatitis B virus pre-S mutation with mitochondrial D-loop mutations and common deletions in hepatocellular carcinoma. Hepatogastroenterology, 58: 522–528.
  22. 22. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser., 41: 95–98.
  23. 23. Hedrich H. 2000. History, Strains and Models. In: The laboratory Rat (Handbook of Experimental Animals), Krinke GJ and Buton T (eds). London: Academic Press. pp. 1871–1895.
  24. 24. Heyman P, Baert K, Plyusnina A, Cochez C, Lundkvist A, Esbroeck MV, Goossens E, Vandenvelde C, Plyusnin A, Stuyck J. 2009. Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium. Scand J Infect Dis, 41: 51–56.
  25. 25. Heyman P, Plyusnina A, Berny P, Cochez C, Artois M, Zizi M, Pirnay JP, Plyusnin A. 2004. Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France. Eur J Clin Microbiol Infect Dis, 23: 711–717.
  26. 26. Hu T, Fan Q, Hu X, Deng B, Chen G, Gu L, Li M, Zheng Y, Yuan G, Qiu W, Jiang X, Zhang F. 2015. Molecular and serological evidence for Seoul virus in rats (Rattus norvegicus) in Zhangmu, Tibet, China. Arch Virol, 160: 1353–1357.
  27. 27. Hugot JP, Gu SH, Feliu C, Ventur J, Ribas A, Dormion J, Yanagihara R, Nicolas V. 2014. Genetic Diversity of Talpa Europaea and Nova Hanta Virus (NVAV) in France. Bull Acad Vet Fr, 167.
  28. 28. Ibrahim IN, Sudomo M, Morita C, Uemura S, Muramatsu Y, Ueno H, Kitamura T. 1996. Seroepidemiological survey of wild rats for Seoul virus in Indonesia. Jpn J Med Sci Biol, 49: 69–74.
  29. 29. Jin C, Qin D, Pan W, Wang Y, Zhang Y, Deng C, Zheng J. 2008. Micromammals of the Giantpithecus fauna from Sanhe Cave, Chongzuo, Guangxi. Quaternary Sciences, 28: 1129–1137.
  30. 30. Kang YJ, Zhou DJ, Tian JH, Yu B, Guo WP, Wang W, Li MH, Wu TP, Peng JS, Plyusnin A, Zhang YZ. 2012. Dynamics of hantavirus infections in humans and animals in Wuhan city, Hubei, China. Infect Genet Evol, 12: 1614–1621.
  31. 31. Kariwa H, Isegawa Y, Arikawa J, Takashima I, Ueda S, Yamanishi K, Hashimoto N. 1994. Comparison of nucleotide sequences of M genome segments among Seoul virus strains isolated from eastern Asia. Virus Res, 33: 27–38.
  32. 32. Komarov AP, Rokhlin OW, Yu CA, Gudkov AV. 2008. Functional genetic screening reveals the role of mitochondrial cytochrome b as a mediator of FAS-induced apoptosis. Proc Natl Acad Sci U S A, 105: 14453–14458.
  33. 33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics, 23: 2947–2948.
  34. 34. Li JL, Ling JX, Liu DY, Liu J, Liu YY, Wei F, Luo F, Chen W, Zhang YH, Xiong HR, Hou W, Yang ZQ. 2012. Genetic characterization of a new subtype of Hantaan virus isolated from a hemorrhagic fever with renal syndrome (HFRS) epidemic area in Hubei Province, China. Arch Virol, 157: 1981–1987.
  35. 35. Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25: 1451–1452.
  36. 36. Lin XD, Guo WP, Wang W, Zou Y, Hao ZY, Zhou DJ, Dong X, Qu YG, Li MH, Tian HF, Wen JF, Plyusnin A, Xu J, Zhang YZ. 2012. Migration of Norway rats resulted in the worldwide distribution of Seoul hantavirus today. J Virol, 86: 972–981.
  37. 37. Liu J, Liu DY, Chen W, Li JL, Luo F, Li Q, Ling JX, Liu YY, Xiong HR, Ding XH, Hou W, Zhang Y, Li SY, Wang J, Yang ZQ. 2012. Genetic analysis of hantaviruses and their rodent hosts in central-south China. Virus Res, 163: 439–447.
  38. 38. Meerburg BG, Singleton GR, Kijlstra A. 2009. Rodent-borne diseases and their risks for public health. Crit Rev Microbiol, 35: 221–270.
  39. 39. Morzunov SP, Rowe JE, Ksiazek TG, Peters CJ, St Jeor SC, Nichol ST. 1998. Genetic analysis of the diversity and origin of hantaviruses in Peromyscus leucopus mice in North America. J Virol, 72: 57–64.
  40. 40. Neumann S, El Maadidi S, Faletti L, Haun F, Labib S, Schejtman A, Maurer U, Borner C. 2015. How do viruses control mitochondria-mediated apoptosis?. Virus Res, 209: 45–55.
  41. 41. Plyusnin A, Beaty Bj, Elliott RM, Goldbach R, Kormelink R, Lundkvist A, Schmaljohn CS, Tesh RB. 2012. Bunyaviridae. In: Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses, King AMQ, Lefkowitz EJ, Adams MJ, et al. (eds). San Diego: Elsevier. pp. 725–741.
  42. 42. Plyusnin A, Sironen T. 2014. Evolution of hantaviruses: co-speciation with reservoir hosts for more than 100 MYR. Virus Res, 187: 22–26.
  43. 43. Plyusnina A, Heyman P, Baert K, Stuyck J, Cochez C, Plyusnin A. 2012. Genetic characterization of seoul hantavirus originated from norway rats (Rattus norvegicus) captured in Belgium. J Med Virol, 84: 1298–1303.
  44. 44. Ramsden C, Holmes EC, Charleston MA. 2009. Hantavirus evolution in relation to its rodent and insectivore hosts: no evidence for codivergence. Mol Biol Evol, 26: 143–153.
  45. 45. Robinson R. 1965. Genetics of the Norway rats. Oxford, United Kingdom: Pergamon.
  46. 46. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19: 1572–1574.
  47. 47. Ruggieri V, Mazzoccoli C, Pazienza V, Andriulli A, Capitanio N, Piccoli C. 2014. Hepatitis C virus, mitochondria and auto/mitophagy: exploiting a host defense mechanism. World J Gastroenterol, 20: 2624–2633.
  48. 48. Schlegel M, Radosa L, Rosenfeld UM, Schmidt S, Triebenbacher C, Lohr PW, Fuchs D, Heroldova M, Janova E, Stanko M, Mosansky L, Fricova J, Pejcoch M, Suchomel J, Purchart L, Groschup MH, Kruger DH, Klempa B, Ulrich RG. 2012. Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe. Virus Genes, 45: 48–55.
  49. 49. Schmidt S, Saxenhofer M, Drewes S, Schlegel M, Wanka KM, Frank R, Klimpel S, von Blanckenhagen F, Maaz D, Herden C, Freise J, Wolf R, Stubbe M, Borkenhagen P, Ansorge H, Eccard JA, Lang J, Jourdain E, Jacob J, Marianneau P, Heckel G, Ulrich RG. 2016. High genetic structuring of Tula hantavirus. Arch Virol, 161: 1135–1149.
  50. 50. Song G. 1999. Epidemiological progresses of hemorrhagic fever with renal syndrome in China. Chin Med J (Engl), 112: 472–477.
  51. 51. Song Y, Lan Z, Kohn MH. 2014. Mitochondrial DNA phylogeography of the Norway rat. PLoS One, 9: e88425.
  52. 52. Torres-Perez F, Palma RE, Hjelle B, Ferres M, Cook JA. 2010. Andes virus infections in the rodent reservoir and in humans vary across contrasting landscapes in Chile. Infect Genet Evol, 10: 820–825.
  53. 53. Watson DC, Sargianou M, Papa A, Chra P, Starakis I, Panos G. 2014. Epidemiology of Hantavirus infections in humans: a comprehensive, global overview. Crit Rev Microbiol, 40: 261–272.
  54. 54. Wu X, Wang Y. 2012. Fossil materials and migrations of Mus musculus and Rattus norvegicus. Research of China's Frontier Archaeology, 11: 344–353.
  55. 55. Yanagihara R, Gu SH, Arai S, Kang HJ, Song JW. 2014. Hantaviruses: rediscovery and new beginnings. Virus Res, 187: 6–14.
  56. 56. Yang Z, Yao P, Zhu H, Xu F, Yue M, Xie R, Sun Y, Xu Z, Wang C, Zhang Y. 2016. Co-divergence of hantavirus with its hosts' mithochondrial D-loop and cyt-b sequences. Chin J Public Health, 32: 205–207.
  57. 57. Zan J, Liu J, Zhou JW, Wang HL, Mo KK, Yan Y, Xu YB, Liao M, Su S, Hu RL, Zhou JY. 2016. Rabies virus matrix protein induces apoptosis by targeting mitochondria. Exp Cell Res, 347: 83–94.
  58. 58. Zhang S, Wang S, Yin W, Liang M, Li J, Zhang Q, Feng Z, Li D. 2014a. Epidemic characteristics of hemorrhagic fever with renal syndrome in China, 2006-2012. BMC Infect Dis, 14: 384.
  59. 59. Zhang XF. 1990. Epidemiological survey of epidemic haemorrhagic fever in Xianning County. Chin J Prev Med, 24: 351–353.
  60. 60. Zhang YH, Ge L, Liu L, Huo XX, Xiong HR, Liu YY, Liu DY, Luo F, Li JL, Ling JX, Chen W, Liu J, Hou W, Zhang Y, Fan H, Yang ZQ. 2014b. The epidemic characteristics and changing trend of hemorrhagic fever with renal syndrome in Hubei Province, China. PLoS One, 9: e92700.
  61. 61. Zhang YZ. 2014. Discovery of hantaviruses in bats and insectivores and the evolution of the genus Hantavirus. Virus Res, 187: 15–21.
  62. 62. Zhang YZ, Zou Y, Fu ZF, Plyusnin A. 2010. Hantavirus infections in humans and animals, China. Emerg Infect Dis, 16: 1195–1203.
  63. 63. Zhou JH, Zhang HL, Wang JL, Yang WH, Mi ZQ, Zhang YZ, Song XY, Hu QL, Dong YK, Pu WH, Hu HM, Gao LF, Yuan QH, Ya HX, Feng Y. 2009. Survey on host animal and molecular epidemiology of hantavirus in Chuxiong prefecture, Yunnan province. Chin J Epidemiol, 30: 239–242.
  64. 64. Zou Y, Hu J, Wang ZX, Wang DM, Li MH, Ren GD, Duan ZX, Fu ZF, Plyusnin A, Zhang YZ. 2008. Molecular diversity and phylogeny of Hantaan virus in Guizhou, China: evidence for Guizhou as a radiation center of the present Hantaan virus. J Gen Virol, 89: 1987–1997.