Journal of Clinical Virology 70 (2015) 16–22

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Journal of Clinical Virology

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Short communication

Living poultry markets in rural area: Human infection with H7N9virus re-emerges in Zhejiang Province, China, in winter 2014

Yi Suna,1, Henghui Wangb,1, Xiuyu Loua,1, Haiyan Maoa, Yong Yanb, Enfu Chena,Xinying Wanga, Zhen Lia, Jian Gaoa, Xiaofei Fub, Zhongwen Chenb, Youjun Fengc,Zhiping Chena, Yanjun Zhanga,c,∗

a Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang, Chinab Jiaxing Center for Disease Control and Prevention, Dongyang, Zhejiang, Chinac Center for Infection & Immunity, Department of Medical Microbiology & Parasitology, School of Basic Medical Sciences, Zhejiang University, Hangzhou,Zhejiang 310058, China

a r t i c l e i n f o

Article history:Received 7 January 2015Received in revised form 4 June 2015Accepted 18 June 2015

Keywords:Avian influenzaH7N9Live poultry marketRural area

a b s t r a c t

Background: Avian influenza A H7N9 virus, previously undetected in humans, has caused infections inmany areas in China since February 2013. Here we report the re-emergence of a case of H7N9 in ruralJiaxing city, Zhejiang Province, in the winter of 2014.Objectives: To understand (1) the clinical syndrome, epidemiological and virological characteristics of thiscase; (2) the importance of controlling live poultry markets (LPMs) in rural areas.Study design: There is one patient and 16 contacts, including 4 family members living in the same house-hold, and 12 medical personnel. Pharyngeal swabs and serum samples were collected from the patientand her contacts. Environment samples were also obtained from the local LPMs. We conducted detailedclinical and epidemiological investigations and laboratory work, including viral RNA extraction, RT-PCRdetection and sequencing. Characteristic and phylogenetic analyses were performed using the obtainedsequences.Results: H7N9s were detected in environmental samples collected in LPMs in Jiaxing, Zhejiang. Unknownmutations were discovered in amino acids in the sample from the patient. The strain from the patientwas in a clade different from isolates obtained in 2013 in phylogenetic trees of HA, NA and PB2.Conclusions: A severe case of H7N9 was identified in early winter, 2014. Epidemiological and clinical testswere consistent with patterns reported previously, while laboratory findings showed the virus to bedifferent. Live poultry markets in rural Zhejiang Province are in need of closer supervision and enhancedmanagement.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Background

Human infections with avian influenza virus A (H7N9) haveposed serious public health concerns globally. The largest numberof confirmed cases of human H7N9 infections has been in ZhejiangProvince, China, since the first case was diagnosed in spring, 2013.Given that live poultry markets (LPM) play a critical role in thedevelopment of H7N9 [1], the Zhejiang government permanentlyshut down all LPMs in urban areas, but not in rural areas, in July

∗ Corresponding author at: Zhejiang Provincial Center for Disease Con-trol and Prevention, 3399 Binsheng Road, Hangzhou, Zhejiang, China, 310051.Fax: +86 0571 87115198.

E-mail address: yjzhang@cdc.zj.cn (Y. Zhang).1 These authors contributes equally.

2014. The management and control of rural/suburban markets isdifficult because of the numerous, often small, LPMs in such places.Here we report on a random human case of H7N9 infection in Jiax-ing in the winter of 2014. To the best of our knowledge, it was thefirst case of the flu season after closure of the LPMs in Zhejiang.

2. Objectives

The objectives were to understand: (1) the clinical syndrome,epidemiological and virological characteristics of this case; (2) theimportance of controlling live poultry markets (LPMs) in rural areas.

3. Study design

Definitions for probable, confirmed, severe and surveillancecases are based on ‘Diagnosis and treatment program for human

http://dx.doi.org/10.1016/j.jcv.2015.06.0921386-6532/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Y. Sun et al. / Journal of Clinical Virology 70 (2015) 16–22 17

Table 1Environmental surveillance of avian influenza viruses in Jiaxing rural area in November 2014. Hx in the table means influenza subtypes except H5, H7 and H9.

Location No. of environment samples collected No. of positive samples

Hx H5 H7 H9 Total

LPMs in Xinten village 7 4 0 1 0 5Other LPMs in Jiaxing 37 2 4 2 8 13Total 44 6 4 3 8 18

infection with H7N9 avian influenza (2014 edition)’ [2]. With per-mission of the patient and her contacts, including 4 family members(husband, son, son’s wife and grandson) in the same household and12 medical personnel, pharyngeal swabs and serum samples werecollected. With permission from the local government, 44 envi-ronmental samples, comprising live poultry stools, pluckings andcontainers, were collected from markets in rural Jiaxing. The sam-ples were stored in 3–5 ml of Hanks solution, containing 100 U/mlpenicillin and 100 �g/ml streptomycin, at −70 ◦C until analysis.

4. Laboratory testing

Viral RNA was extracted using the RNeasy Mini kit (Qiagen, CA,USA) according to the manufacturer’s instructions. Procedures forRT-PCR reactions followed WHO’s document on ‘Real-time RT-PCRprotocol for the detection of avian influenza A (H7N9) virus’ (http://www.who.int/influenza/gisrs laboratory/en/). We sequenced thesample based on the Ion torrent PGM platform [3]. We performedmultiple alignments with data matrixes from H7N9 sequencesobtained in Zhejiang in 2013 (Zhejiang/1/2013, Zhejiang/22/2013)and the WHO recommended H7N9 vaccine virus (Anhui/1/2013)[3–5] to determine the sequences and to elucidate variations.

Phylogenetic trees were generated using Mega 6.0.5 with otherselected H7N9 virus strains from GISAID [6]. The viral sequencesfrom this patient (A/Zhejiang/17/2014) were deposited in GISAID(EPI552396-EPI552401, EPI587894-EPI587895).

5. Results

The medical record of the patient, a 59-year old femalewith chronic bronchitis and hypertension, suggested a history ofnephroliths, Nelson’s syndrome, gastrohelcosis and penicillin ana-phylaxis. The patient was recorded as exhibiting a fever of 41 ◦Cand a paroxysmal cough on November 17. She was hospitalized onNovember 20, admitted to the ICU on November 24 and confirmedto be H7N9-positive on November 25. She was suffering from ARDSand received mechanical ventilation, a tracheotomy, and was fittedwith a tracheal cannula. A combination of ceftazidime, imipenemand cilasatin sodium, moxifloxacin and methylprednisolonesodium succinate was administered. Antiviral therapy (oseltamivir)was initiated 7 days after onset of illness. Her white cell count wasmeasured at 2.8 ∼ 3.9 × 109/L, and neutrophilic granulocyte per-centage was 85–89%. Computed tomography (CT) chest scan indi-cated the syndrome of bilateral pneumonia (Supplemental Fig. 1).

Table 2Substitutions in segments in each novel H7N9 of avian origin. Substitutions consistent with Chinese CDC isolations as previously describe are not presented below [4].

Genes Site A/Zhejiang/17/2014 A/Zhejiang/22/2013 A/Zhejiang/1/2013 A/Anhui/1/2013

HA(H3 Numbering) 32 A G G G127 N S S S134 V A A A177 I L L L226 L L L I388 A E E E493 R S S S

NA 21 T I I I99 T M M M247 P S S S327 S T N N334 S N N N370 T A A A

PB2 191 E K K K339 E K K K473 V M M M511 I V V V535 L M M M570 I M M M627 E E K K647 V I I I

PB1 54 R K K KPA 57 Q R R R

100 V A A A138 K I I I182 L M M M296 N S S S394 D N N N556 R Q Q Q

NP 217 V I I I239 V M M M

18 Y. Sun et al. / Journal of Clinical Virology 70 (2015) 16–22

Fig. 1. Phylogenetic trees for H7, N9 and PB2 segments of A/Zhejiang/17/2014 (H7N9) obtained in this study. Sequences with black triangle were isolated in Zhejiang during2013–2014. Sequence with red circle (A/Zhejiang/17/2014) was strain obtained in our study. A: H7 tree; B: N9 tree; C: PB2.

Y. Sun et al. / Journal of Clinical Virology 70 (2015) 16–22 19

Fig. 1. (Continued)

20 Y. Sun et al. / Journal of Clinical Virology 70 (2015) 16–22

Fig. 1. (Continued)

Y. Sun et al. / Journal of Clinical Virology 70 (2015) 16–22 21

6. Epidemiological investigation

Further investigation revealed that the patient visited the livepoultry market on November 5 and purchased two live chickens,which were kept on her balcony. Of particular note, the patientfed the chickens once a day and cleaned the area around themone or two times per day without protection. Her exposure con-tinued until the chickens were slaughtered. The demographic andepidemiologic characteristics are described in Supplemental Table1.

The LPM visited by the patient is in the center of Xinten village,a rural area where the live poultry market operates daily, 10 kilo-meters from the main city of Jiaxing. Results from environmentalsurveillance determined the total positive rate for H7 was 6.82%(3/44), with a total of 40.91 (18/44) (Table 1). Some environmen-tal samples were detected to be a mixture of H7 and H9 viruses.It is strongly suspected that LPMs are a significant cause of H7N9infections in humans. The LPM in Xinten thus provides a seriouspotential for transmitting the H7N9 virus.

7. Laboratory analysis

Real-time PCR analyses of throat swabs suggested that thepatient was positive for avian H7N9 nucleic acid, with CTvalues of 26; all 16 contacts were negative. This sample,A/Zhejiang/17/2014(H7N9) resulted in a total of 4,189,123 readswith a mean length of 153 bp, of which more than 3,000,000 readspassed quality filtering. Average coverage for this sample was 4021.The genomic sequence analyses of the H7N9 virus isolate identi-fied with 94.9% ∼ 98.7% of known H7N9 viruses at the nucleotidelevel. Given that the substitutions plus variations were summa-rized by the China CDC (Table 2) [4], we found the amino acids ofZhejiang/17/2014 virus to be basically similar to other H7N9 in Zhe-jiang, especially at the mammalian adaptive receptor-binding siteQ226L and G228S in HA, and the antiviral-resistance site R294Kin NA. Zhejiang/17/2014 received glutamic acid instead of lysineat position 627 in PB2, indicating that this strain may not have theability for enhanced viral replication in culture. We also anticipatedthat virulence of Zhejiang/17/2014 was attenuated in mammals inthat a mutation of A100 V in PA was present [3,7].

Extensive phylogenetic analyses further demonstrated that Zhe-jiang/17/2014 shared high homology with other strains, includingenvironmental samples isolated in 2014, as they clustered into oneclade of the trees not only for the HA and NA segments, but alsofor such internal genes as PB2 (Fig. 1). The structure of the phyloge-netic tree showed that Zhejiang/17/2014 had diverged from strainsisolated in Zhejiang in 2013. This observation was generally consis-tent with prior conclusions that H7N9 has evolved genetic diversitylocally with reassortment endemic H9N2 [8].

8. Discussion

We are the first to report the re-emergence of human H7N9infection in the winter of 2014, several months after the last casewas confirmed in China in the spring of 2014. The source of theinfection is believed to be the LPM in rural Jiaxing city, ZhejiangProvince, since the patient had a history of exposure to poultry andpositive results were obtained from tests of environmental sam-ples taken in the Xinten market of Jiaxing. Clinical features of thisre-emergent case were similar to cases reported previously, butwith a different genotype from strains isolated in Zhejiang in 2013.The several new genetic substitutions require further functionalresearch. It appears possible that endemic H7N9 may initiate a newwave of infections during the influenza season, even though LPMs

in urban areas were permanently closed by the government in July,2014.

Several sporadic cases of H7N9 were identified and reported bythe government in Yongkang and Shaoxing, Zhejiang, this winter.From the findings of an epidemiological investigation, all cases areconsidered to be related to local LPMs in rural areas or exposure tolive poultry. LPMs in rural areas require immediate enhanced man-agement. Long term surveillance remains essential for providingearly warning to the public of the potential for viral transmission.

Conflict of interest

I approved the final manuscript.

Funding

This work was supported by the Zhejiang Provincial Program forthe Cultivation of High-level Innovative Health Talents, Program forZhejiang Leading Team of Science and Technology (2011R50021),Monitor Technology Platform of Infectious Diseases of the StateMajor Science and Technology Special Projects during China’s 12thfive-year plan (2012ZX10004-210) and Jiaxing Key Laboratory ofPathogenic Microorganisms. Dr. Youjun Feng is a recipient of the“Young 1000 Talents” Award.

Competing interests

The authors declare that they have no competing interests.

Ethics approval

This study was approved by the ethics committee of the Zhe-jiang Provincial Center for Disease Control and Prevention (ZJCDC),China.

Acknowledgements

The authors acknowledge that the analyses were made possibleusing publicly accessible GISAID virological and genetic moleculardata provided by many labs. We thank Jianyong Luo, Jiaxing Cen-ter for Disease Control and Prevention, for providing samples andemergency response. David Boufford is gratefully acknowledgedfor editing the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcv.2015.06.092

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