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    Epidemics 5 (2013) 1 10

    Contents lists available at SciVerse ScienceDirect


    jo u rn al hom epa g e: www.elsev ier .com/ locate /ep idemics

    volutionary and molecular analysis of the emergent severe fever withhrombocytopenia syndrome virus

    ommy Tsan-Yuk Lama,1, Wei Liub,1, Thomas A. Bowdenc,1, Ning Cuid, Lu Zhuangb, Kun Liub,ao-Yun Zhangb, Wu-Chun Caob, Oliver G. Pybusa,

    Department of Zoology, University of Oxford, Oxford, United KingdomState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaDivision of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United KingdomThe 154 Hospital, People Liberation Army, Xinyang 464000, Henan, PR China

    r t i c l e i n f o

    rticle history:eceived 4 July 2012ccepted 13 September 2012vailable online xxx

    eywords:hlebovirusevere fever with thrombocytopeniayndromeick-borne diseasehylogeneticvolutionucleoprotein structure

    a b s t r a c t

    In 2009, a novel Bunyavirus, called severe fever with thrombocytopenia syndrome virus (SFTSV) was iden-tified in the vicinity of Huaiyangshan, China. Clinical symptoms of this zoonotic virus included severefever, thrombocytopenia, and leukocytopenia, with a mortality rate of 10%. By the end of 2011 thedisease associated with this pathogen had been reported from eleven Chinese provinces and human-to-human transmission suspected. However, current understanding of the evolution and molecularepidemiology of SFTSV before and after its identification is limited. To address this we undertake phylo-genetic, evolutionary and structural analyses of all available SFTSV genetic sequences, including a newSFTSV complete genome isolated from a patient from Henan in 2011. Our discovery of a mosaic L seg-ment sequence, which is descended from two major circulating lineages of SFTSV in China, represents thefirst evidence that homologous recombination plays a role in SFTSV evolution. Selection analyses indicatethat negative selection is predominant in SFTSV genes, yet differences in selective forces among genes areconsistent between Phlebovirus species. Further analysis reveals structural conservation between SFTSVand Rift Valley fever virus in the residues of their nucleocapsids that are responsible for oligomerisation

    and RNA-binding, suggesting the viruses share similar modes of higher-order assembly. We reconstructthe epidemic history of SFTSV using molecular clock and coalescent-based methods, revealing that theextant SFTSV lineages originated 50150 years ago, and that the viral population experienced a recentgrowth phase that concurs with and extends the earliest serological reports of SFTSV infection. Takentogether, our combined structural and phylogenetic analyses shed light into the evolutionary behaviour

    f othe

    of SFTSV in the context o


    Reports of severe fever with thrombocytopenia syndromeSFTS) emerged between March and July, 2009, in rural areasf central China. The syndrome was characterized by fever, gas-rointestinal symptoms, thrombocytopenia and leukocytopenia inndividuals bitten by ticks (Yu et al., 2011). Differential diagnosisnitially included human anaplasmosis, leptospirosis and hemor-hagic fever with renal syndrome; however, the causative pathogen

    ould not be ascertained. A few months later, a novel Phlebovirusfamily Bunyaviridae) was reported as the cause of SFTS, through

    olecular characterization and epidemiologic investigations of the

    Corresponding author at: Department of Zoology, South Parks Road, Oxford OX1PS, United Kingdom. Tel.: +44 01865 271274; fax: +44 01865 310447.

    E-mail address: [email protected] (O.G. Pybus).1 These authors contributed equally.

    755-4365/$ see front matter 2012 Elsevier B.V. All rights reserved.ttp://

    r, better-known, pathogenic Phleboviruses. 2012 Elsevier B.V. All rights reserved.

    virus isolated from patients blood (Yu et al., 2011). From June 2009to September 2010, SFTSV RNA or specific antibodies were detectedin the serum of 171 of 241 hospitalized patients who met the casedefinition for SFTS (in Henan, Hubei, Shandong, Liaoning, Anhuiand Jiangsu provinces) (Yu et al., 2011). Two names are used inthe literature to refer to the virus: severe fever with thrombocy-topenia syndrome virus (Yu et al., 2011; SFTSV; used hereafter)or Huaiyangshan virus (Zhang et al., 2012b). Like many othermembers of the Phlebovirus genus, SFTSV uses ticks as a putativehost vector and is likely to be transmitted to human populationsfrom an animal reservoir via blood feeding (Elliott, 2008). Previ-ous studies have detected SFTSV in Haemaphysalis longicornis andRhipicephalus microplus ticks collected from a number of domes-tic animals, including cattle, buffalo, goats, cats and dogs (Zhang

    et al., 2011, 2012b). Although transmission of SFTSV is currentlypoorly understood, viral genome sequences obtained from infectedticks on animals are highly similar to those obtained from humanisolates, suggesting zoonotic transmission (Zhang et al., 2012b).[email protected]/10.1016/j.epidem.2012.09.002

  • 2 T.T.-Y. Lam et al. / Epid

    Fig. 1. Map showing the provinces in mainland China that have officially reportedSFTSV in 201011. The red triangle indicates the first SFTS case (in Dingyuan County,Clt






    huzhou City, Anhui, in September, 2006) that was retrospectively confirmed byaboratory testing (Liu et al., 2012). (For interpretation of the references to colour inhis figure legend, the reader is referred to the web version of the article.)

    owever, a number of human infection clusters have also beendentified, suggesting the possibility of human-to-human transmis-ion (Bao et al., 2011; Liu et al., 2012; Xu et al., 2011).

    Starting from October 2010, active surveillance for human SFTSVnfections has been undertaken by the Chinese Centre for Diseaseontrol and Prevention (CCDC) (Ministry of Health, China, 2010).y the end of 2011, SFTS had been reported in eleven provinces

    ncluding Henan, Hubei, Anhui, Shandong, Jiangsu, Zhejiang, Liao-ing, Yunnan, Guangxi, Jiangxi and Shannxi (Fig. 1). The mortalityf SFTSV infections is between 8% and 16%, according to differenttudies (Table 1).

    The genome of SFTSV, like other Phleboviruses, comprises threeegments: L (large), M (medium), and S (small). Nucleocapsid (N)nd non-structural (NS) proteins are encoded in the two non-verlapping ORFs of the S segment, whereas the L and M segmentsncode the RNA-dependent RNA polymerase (RdRp) and Gn-Gcnvelope glycoproteins (GP), respectively (Yu et al., 2011). Whole-enome sequencing and analysis confirmed that SFTSV is a novelunyavirus and is most closely related to Uukuniemi virus (pairwiseucleotide similarities for the L, M and S segments are 34%, 24% and9%, respectively; Zhang et al., 2012b). All SFTSV sequences cur-ently available in GenBank are similar regardless of their samplingocations (>90% sequence similarity; Zhang et al., 2012b), suggest-ng a relatively recent common ancestor.

    The broad geographic distribution throughout China, relativelyigh case fatality rate, and potential for human-to-human trans-

    ission render SFTSV a notable threat to public health, both

    egionally and internationally. Despite this, very little is knownbout the molecular epidemiology and structure of this zoonoticathogen. In order to address this paucity of information, we have

    able 1aboratory-confirmed and fatal cases of SFTSV infection in China.

    Study Laboratory-confirmedcases

    Fatal cases Mortality rate(%)

    Yu et al. (2011) 171 21 12.28Liu et al. (2012) 14 2 14.29Zhang et al. (2012a) 49 8 16.33Liu and Liu (2011) 17 2 11.76Zhang et al. (2011) 33 5 15.15Yu et al. (2012) 61 5 8.2

    emics 5 (2013) 1 10

    undertaken comprehensive phylogenetic, evolutionary and struc-tural analyses of SFTSV genetic sequences, and report the completegenome sequence of a new SFTSV isolate, obtained in 2011 from apatient from Henan province. We estimate SFTSVs date of origin,rate of evolution and epidemic history, and test for the presence ofviral recombination and reassortment. Through comparative anal-yses of the genome sequences of SFTSV and related Phleboviruses(such as Rift Valley fever virus) we infer structural properties of theSFTSV nucleoprotein and glycoproteins, and explore the effects ofnatural selection on SFTSV protein evolution.

    Materials and methods

    Sample collection, virus isolation and sequencing

    Patients who met criteria for probable SFTS cases accordingto the national guidelines (Ministry of Health, China, 2010) wererecruited at an army hospital in the Xinyang administrative districtof Henan Province. One patient was confirmed to be infected withSFTSV in June 2010. Briefly, sera samples from this patient wereobtained three days following the onset of illness. Serum was inoc-ulated on DH82 cells which were cultured at 37 C in a 5% carbondioxide atmosphere with twice-weekly media changes. One isolate,generated by primer-walking strategies as previously described(Zhang et al., 2011) (named WCH-2011/HN/China/isolate97), wasused to derive the whole genome sequence. The resulting negative-sense RNA genome was 11,491 n