nosocomial transmission of avian influenza a (h7n9) virus in … · 05-05-2015 · yi, huai-ming;...

Confidential: For Review O

nly

Nosocomial Transmission of Avian Influenza A (H7N9) Virus

in China: Epidemiological Investigation

Journal: BMJ

Manuscript ID: BMJ.2015.026821

Article Type: Research

BMJ Journal: BMJ

Date Submitted by the Author: 05-May-2015

Complete List of Authors: Ma, Mai-Juan; Beijing Institute of Microbiology and Epidemiology, Fang, Chun-Fu; Quzhou Center for Disease Control and Prevention, Zhan, Bing-Dong; Quzhou Center for Disease Control and Prevention, Lai, Shi-Ming; Quzhou Center for Disease Control and Prevention, Hu, Yi; Beijing Institute of Microbiology and Epidemiology,

Yang, Xiao-Xian; Beijing Institute of Microbiology and Epidemiology, Li, Jing; Beijing Institute of Microbiology and Epidemiology, Cao, Guo-Ping; Beijing Institute of Microbiology and Epidemiology, Zhou, Jing-Jing; Beijing Institute of Microbiology and Epidemiology, Zhang, Jian-Min; Quzhou Center for Disease Control and Prevention, Wang, Shuang-Qing; Quzhou Center for Disease Control and Prevention, Hu, Xiao-Long; Quzhou Center for Disease Control and Prevention, Li, Yin-Jun; Beijing Institute of Microbiology and Epidemiology, Yao, Hongwu; State Key Laboratory of Pathogen and Biosecurity, Li, Xin-Lou; Beijing Institute of Microbiology and Epidemiology, Chen, Enfu; Zhejiang Provincial Center for Disease Control and Prevention, Yi, Huai-Ming; Changshan County Center for Disease Control and

Prevention, Xu, Wei-Dong; Kecheng District People’s Hospital, Jiang, Jiafu; Beijing Institute of Microbiology and Epidemiology, ; Gray, Gregory; Duke University, Fang, Li-Qun; Beijing Institute of Microbiology and Epidemiology, Cao, Wu-Chun; Beijing Institute of Microbiology and Epidemiology,

Keywords: Avian influenza A (H7N9) virus, Nosocomial, Human-to-human transmission, Close contacts, China

https://mc.manuscriptcentral.com/bmj

BMJ


nly

1

5/5/2015

Nosocomial Transmission of Avian Influenza A (H7N9) Virus in China:

An Epidemiological Investigation

Chun-Fu Fang director1,a

, Mai-Juan Ma epidemiologist2,a

, Bing-Dong Zhan epidemiologist1,a

,

Shi-Ming Lai deputy director1, Yi Hu virologist

2, Xiao-Xian Yang molecular biologist

2, Jing Li

virologist 2, Guo-Ping Cao epidemiologist

1, Jing-Jing Zhou molecular biologist

2, Jian-Min

Zhang public health officer1, Shuang-Qing Wang public health officer

1, Xiao-Long Hu public

health officer , Yin-Jun Li public health officer

2, Hong-Wu Yao molecular biologist

2, Xin-Lou Li

molecular biologist2, En-Fu Chen epidemiologist

3, Huai-Ming Yi epidemiologist

4, Wei-Dong

Xu doctor5, Jia-Fu Jiang epidemiologist

2, Gregory C. Gray professor

6,*, Li-Qun Fang

epidemiologist2,*

, Wu-Chun Cao professor and director2,*

1Quzhou Center for Disease Control and Prevention, Quzhou 324000, China;

2State Key

Laboratory of Pathogen and Security, Beijing Institute of Microbiology and Epidemiology,

Beijing 100071, China; 3Zhejiang Provincial Center for Disease Control and Prevention,

Hangzhou 310051, China; 4Changshan County Center for Disease Control and Prevention,

Changshan 324200, China; 5Kecheng District People’s Hospital, Quzhou 324000, China;

6Division of Infectious Diseases, Global Health Institute, & Nicholas School of the Environment,

Duke University, Duke University Medical Center, Durham, NC, 27710, USA

a These authors contributed equally to this manuscript.

*Correspondence to: G C Gray [email protected], L Q Fang [email protected], and W

C Cao [email protected].

Running title: H7N9 Nosocomial Transmission

Page 1 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

2

Keywords: Avian influenza A (H7N9) virus; Nosocomial; Human-to-human transmission; close

contacts; China;

Word counts: 3092

Page 2 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

3

Abstract

OBJECTIVE To investigate a cluster of two hospitalized H7N9-infected patients in February

2015.

DESIGN A cross-sectional epidemiological investigation.

SETTING Quzhou, Zhejiang province, China

PARTICIPANT Two patients, their close contacts, live bird market workers, and relevant

environments were studied. Samples collected from the two patients, their close contacts, their

home environments and live bird markets were examined using rRT-PCR and viral culture, a

hemagglutination inhibition (HI) assay and validated with a microneutralization (MN) assay. The

viral genome of H7N9 viruses were studied with full genome sequence and phylogenetic

analyses. Solid-phase binding assays were used to identify viral receptor-binding properties.

MAIN OUTCOMES MEASURES Clinical data, results of serological assays, viral

phylogenetic tree and receptor-binding properties.

RESULTS The index patient was an otherwise healthy 49-yr old male who developed symptoms

five to six days after his exposure to poultry. A 57-yr old male, with a history of chronic

obstructive pulmonary disease, also developed symptomatic H7N9 infection 5 to 6 days after he

shared a hospital room with the index case. Of 38 close contacts, one doctor developed mild

respiratory symptoms and one nurse without respiratory symptoms developed transiently HI

elevated antibodies against H7N9 virus but were both negative for H7N9 by rRT-PCR studies of

throat swabs and MN assays. The H7N9 viruses from both the index and secondary case were

nearly identical to H7N9 isolates collected from the live poultry market. Through solid-phase

binding assays the H7N9 isolates were found to have the capability to bind both the human and

avian receptors.

Page 3 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

4

CONCLUSIONS Our investigative data strongly support the premise that nosocomial, human-

to-human H7N9 transmission occurred in this setting. These findings undergird the need for

aggressive hospital infection control practices in settings where human H7N9 infections may

occur.

WHAT IS ALREADY KNOWN ON THIS TOPIC

Since the first human infections with novel avian influenza A (H7N9) virus were detected in

February 2013, China has document threes waves of infection. Extensive human-to-human

transmission remains seriously concerning. While evidence for human-to-human transmission

has been sparse, the potential for such transmission competence is real and of the worldwide

concern.

WHAT THIS PAPER ADDS

Our investigation provides some of the first evidence of human-to-human H7N9 virus

transmission.

Page 4 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

5

Introduction

Since the first recognized human infections with novel avian influenza A (H7N9) virus were

detected in February 2013, mainland China has document threes waves of infection, and 620

laboratory-confirmed human cases with 235 deaths (as of February 28th

, 2015).1 The epidemic is

clearly, clandestinely spreading among healthy chickens in increasing numbers of China’s

provinces and cities.2 Unfortunately, as H7N9 surveillance in poor among China’s chickens,

humans are now serving as sentinels for this very disconcerting spread.3 4

Thus far evidence for human-to-human H7N9 transmission has been limited to ~16 family

clusters. Some argue that these household clusters reflect a genetic familial predisposition for

infection.5-14

Were occupational or nosocomial H7N9 clusters recognized to have occurred this

might reflect an alarming increased transmission capability for the viruses. Due to the genetic

diversity and persistent expansion15

of the virus, the possibility of such viral genetic changes

seems quite possible. Here we report our investigation in a possible cluster of nosocomial,

human-to-human H7N9 transmission which occurred in Quzhou City of Zhejiang Province in

China.

Methods

Epidemiology investigation and samples collection

The Zhejiang Provincial Centers for Disease Control and Prevention (ZPCDC) was notified

of the first case in this human H7N9 cluster on February 24th

, 2015. Epidemiologists from the

ZPCDC, Quzhou City CDC (QCDC), and Changshan County CDC (CCDC) conducted an

outbreak investigation. The investigation included interviews of hospital staff, patients, patients’

family members, close contacts of patients, poultry market workers, and a review of medical

records. A questionnaire was developed and employed to ascertain the cases’ demographic

Page 5 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

6

information, recent animal exposures, and contact history with febrile or suspected H7N9

infected patients.

Throat swab specimens were collected from two human cases who were diagnosed with

H7N9 virus infection. For the index case, environmental specimens including fecal specimens

and smear swab were collected from 2 chickens he recently bought at a live bird market A, and

from chickens in the live bird market where the index case had purchased two chickens before he

developed symptoms. For the secondary case, swab samples were obtained from trash containers,

from chicken feces at his neighbor’s home (200m away) as well as from a nearby farm (1km

away). Paired serum samples (separated by at least three weeks) from close contacts of the two

H7N9-infected patients (and throat swabs if the close contacts with signs or symptoms of

influenza) were collected to evaluate asymptomatic or subclinical H7N9 infections.

Real time RT-PCR and virus isolation

The viral RNA of each swab sample was extracted using QIAamp MinElute Virus Spin Kit

(Cat.No.57704, Qiagen) following the manufacturer’s directions. RNA was then screened with a

rRT-PCR assay targeting the influenza matrix gene.16

Specimens which were influenza A virus-

positive were next tested for the H7N9 virus using a rRT-PCR assay targeting the HA and NA

genes.17

Next, H7N9-positive specimens was inoculated into allantoic cavities of 9 to 11-day-old

specific-pathogen-free (SPF) embryonated chicken eggs. Allantoic fluid was harvested after

incubation at 72 h at 37°C.

Genome sequencing and phylogenetic analysis

Whole viral genomes of the viruses were amplified using universal primers for influenza A

virus.18

PCR products were then sequenced employing Ion Torrent PGM technology with a 318

chip (Life Technologies, Grand Island, NY, USA) as previously described.19

Full genome

Page 6 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

7

sequences of the viruses were deposited in the GISAID (accession number: KR351265-

KR351272 for index case—A/Quzhou/1/2015(H7N9), KR351273- KR351280 for secondary

case A/Quzhou/2/2015(H7N9) and KR351260- KR351264 for one H7N9 positive environmental

specimen—A/chicken/Quzhou/1/2015(H7N9)).

The Maximum likelihood (ML) phylogenetic trees model was used to generate the

phylogenetic trees of each gene segment of influenza virus. We used the General Time

Reversible Nucleotide substitution model and Gamma distributed with Invariant site (G+I) rates

among sites with a bootstrapping resampling process (1000 replications) implemented in MEGA

version 6.0.6 (http://www.megasoftware.net/).

Serological and receptor binding assays

Sera from close contacts were screened with a horse RBC, hemagglutination inhibition (HI)

assay against H7N9 virus.20

If HI titers were ≥1:20, a microneutralization assay adapted from

Rowe21

was used to confirm results. A viral isolate from the index patient (A/Quzhou/1/2015

[H7N9]) was used in the HI and MN assays.

Receptor binding specificity of A/Quzhou/1/2015/H7N9 was analyzed by a solid-phase direct

binding assay biotinylated sialylglycopolymers: 3’-sialyllactose-PAA-biotin (3’SL-PAA, 3’

Neu5Aca2-3Galb1-4Glc) and 6’-sialyllactosamine-PAA-biotin (6’SLN-PAA, 6’ Neu5Aca2-

6Galb1-4Glcb) (Cat. No. 01-038, 01-039, Glycotech, Gaithersburg, MD) as our previously

described.22

The A/California/07/2009 (H1N1) and A/Chicken/Jiangsu/927/2014 (H5N1) were

used as controls.

Results

Page 7 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

8

After examining medical records, interviews, and questionnaire data, a timeline (Fig. 1) was

developed documenting the sequence of events. A table (Table 1) was also created to capture the

H7N9 patients’ epidemiological and clinical characteristics.

Patients and possible infection source

The index case was a 49 year-old male, pneumatic drill operator (Table 1) who worked in a

local mine and lived with his wife, two daughters and one son. On February 9, 2015 (Fig. 1), he

and his two daughters purchased vegetables and two live chickens from stalls (Figure S1 in

appendix) of live poultry market A. They kept the chickens in their family’s yard and fed the

chickens several times each day prior to the index case’s illness. Until he brought home the two

live chickens they had not raised chickens and other animals before. He had no known other

market exposures during the seven days prior to his illness. On February 16th

, he developed a

fever, cough, and sore throat and sought medical care with a temperature of 37.5oC. Diagnosed

with a cold, he received two days of oral antibiotic therapy at home for recovery. On February

18th

he present same symptoms with a temperature of 37.8°C. Later on February 18th

, he visited

district hospital (hospital A), where his chest radiograph demonstrated pneumonia (right lung).

He was admitted to an internal medicine respiratory disease ward with a diagnosis of bacterial

pneumonia. He was treated with amoxicillin and levofloxacin. On February 19th

the patient was

noted to still have fever (40°C) and a cough. A computed tomography revealed right lung

pneumonia and his medications changed to dexamethasone, indomethacin, amoxicillin,

cefuroxime sodium, levofloxacin, and supplemental oxygen for 2 hours/day via nasal cannula. A

2nd

computed tomography on February 22nd

documented right and left lung infiltrates. On

February 23rd

, he was transferred to hospital B and a molecular assay for H7N9 was positive on

Page 8 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

9

February 24th

. The patient was next transferred to reference hospital C on February 25th

,

intubated put on a ventilator, and treated with oseltamivir. The patient died on April 20.

The second case was a 57 year-old male farmer who lived only with his wife. His home was

located in a village >8km from the index case. During the six days before the onset of his illness

(February 18th

-23rd

), he shared a ward room (Figure S2 in appendix) in hospital A with the index

patient. He had no know history of exposure to live poultry during the two weeks prior to his

illness. He had a history of chronic obstructive pulmonary disease (COPD) having several recent

hospitalizations for this condition. On February 15th

, he was admitted to his district hospital’s

(hospital A) internal medicine ward with a diagnoses of aggravated COPD and bronchitis.

Routine blood testing identified no abnormality, except for an increased C reactive protein (CRP)

of 47.1 mg/L). The patient was prescribed oxygen by nasal cannula and cefoperazone-sulbactam,

and his symptoms improved and his examination results were considered as normal next day. He

was discharged to home on February 23rd

as recovered from his illness. During hospitalization,

he had no fever, runny nose, sore throat of influenza-like illness symptoms. On February 24th

, the

patient developed a fever (40°C) and cough. His village clinician visited his home on February

25th

, when the patient was noted to have a fever (38.4°C), sore throat, body aches, paroxysmal

cough, expectoration, malaise and chest pain and prescribed anti-inflammatory medication and

rehydration therapy. As he was a close contact of the index case, he was transported to a negative

pressure room of county hospital (hospital D) for treatment. Upon auscultation his breath sounds

were reported to be rough with a few moist rales. His white blood cell count was elevated

(17.8x109/L) and he had a left shift (neutrophils 97.8%) with a CRP of 233mg/L. On February

25th

, the CCDC collected a throat swab sample which was found to be positive for H7N9 by the

QCDC. The ZCDC’s laboratory confirmed that the patient was infected with H7N9 virus On

Page 9 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

10

February 26th

and was treated with oseltamivir. The patient died on March 2. A third patient with

diabetes mellitus also shared the room with the two patients of interest but he never developed

signs or symptoms of influenza.

Suspected infection of doctor and nurse

Twenty-eight close contacts were identified for index case (Figure S2 in appendix) and ten

close contacts were identified for the secondary case. A male doctor who cared for the index

patient developed a light dry cough on February 23rd

and later a fever (37.7°C) on February 25th

which persisted to February 27th

. On February 27th

he began a seven day course of oseltamivir

and recovered from his symptoms. On February 26th

, three throat swabs were collected from the

doctor and they were negative for H7N9 virus. However, the doctor was found to have an

elevated HI titer against H7N9 on February 23rd

(HI titer 1:40 but MN titer <1:10). However, a

follow-up sera collected on March 28th

demonstrated no sustained elevation (HI and MN titer

<1:10). A nurse close contact of the index case also had an elevated HI titer (1:40 but MN titer

<1:10) on her February 23rd

sera, however, she denied ever having any signs or symptoms of

infection. No other close contacts were found to have clinical or serological evidence of H7N9

infection.

Identity of viruses from patients and their environments

Two of the four home chicken fecal specimens and five of the eleven live poultry market

environmental swabs were positive for H7N9 viral genes. All environmental samples for the

second case were negative for molecular evidence of H7N9 virus. One viral strain from the index

case was successfully cultured and studied with full genome sequencing. No other viral swabs

were culture positive. However, one throat swab from the secondary case was successfully

studied with full genome sequence and one environmental swab taken at live poultry market A

Page 10 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

11

yielded sequence data for all gene segments save for PB1, PB2, and PA. These three viruses had

high degrees of similarity when considering both nucleotide (99.8%-100%) and amino acid

(99.6%-100%) coding. The remaining RT-PCR positive samples failed to yield interpretable

sequence data likely due to low viral loads.

Phylogenetic analysis using 100 most highly similarity sequences showed that HA and NA

genes of the three H7N9 viruses belonged to the same clade and were genetically very similar to

sequences isolated from chickens in Jiangxi province in 2014 but different from the H7N9

viruses identified in China in 2013 (Figure 2). Similar to the HA and NA genes, all internal

genes of the viruses belonged to the same clade and clustered together with H7N9 and H9N2

viruses isolated from China (Figure S3 in appendix). Our phylogenetic analyses suggested

different origins for other genes. The NS gene which was closely related to

A/chicken/shanghai/015/2014 (H9N2). The MP and PA internal genes were very similar to

H7N9 viruses isolated from Jiangxi in 2014 and Zhejiang Provinces

(A/chicken/Huzhou/3765/2013(H7N9)). The NP, PB1, and PB2 internal genes were most closely

related to H7N9 viruses isolated in Jiangxi Province and Shanghai in 2014.

The three H7N9 viruses were examined for key mutations associated with virulence and

mammalian adaption (Table 2). The HA protein of each of the viruses had a single basic amino

acid (PEIPKGR↓G) at the cleavage site, indicating low pathogenic effects in poultry.

Substitution at S138A, L186V, and Q226L (H3 numbering) in the HA protein were observed for

three viruses suggesting a possible increased affinity for binding human α2,6-linked sialic acid

receptors. Regarding human-like and mammalian-adapting signatures, substitutions at L89V in

PB2, I368V in PB1, P42S in NS1, and N30D and T215A in M1 were observed in all viruses.

Substitution at E627K in PB2 that involved in mammalian adaptation were also identified. The

Page 11 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

12

69-73 amino acid deletion was observed in the stalk region of NA residue, and no substitution of

G292K amino acid was observed in the NA gene, indicating that the viruses should still be

sensitive to oseltamivir, zanamivir, and peramivir. However, S31N substitution in M2 protein

(associated with adamantane resistance) was noted in all three viruses.

Increased receptor binding specificity

To characterize the receptor-binding properties of A/Quzhou/1/2015/H7N9 (QZ1-H7N9) at

the virus level, we analyzed their receptor-binding properties through solid-phase binding assays

using the 2009 pandemic influenza virus isolate [CA07-H1N1, A/California/07/2009 (H1N1)]

and avian H5N1 influenza virus isolate in our laboratory [JS927-H5N1,

A/Chicken/Jiangsu/927/2014 (H5N1)] as control viruses that have typical human or avian

receptor specificity, respectively. QZ1-H7N9 binds both the human and avian receptor (Fig. 3A).

In contrast, CA07-H1N1 specifically binds the human receptor (Fig. 3B), and JS927-H5N1

specifically binds the avian receptor (Fig. 3C).

Discussion

Our findings strongly suggest that the H7N9 virus live poultry market was the most possible

source of influenza H7N9 virus infection for the index case as no other animals were kept at his

home and he had no exposure to poultry before he bought two chickens from live poultry market.

While we cannot completely rule out an unidentified environmental exposure that might explain

the H7N9 infection in the 2nd

patient, study data seem compelling that transmission occurred in

the hospital setting. Previous reports of family clusters of H7N9 virus infection imply either a

common familial genetic susceptibility to the virus or common environmental exposures.7 9 12

Our data are unique in that the index case and secondary case are quite unrelated.

Page 12 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

13

Our epidemiological findings further suggest that the live chickens from market A were the

original source of infections for the index case. This is based upon the positive H7N9 swabs

from the index case’s live chickens and finding evidence for a H7N9 virus very similar to the

two patients at the live animal market where these chickens were first purchased. This

implication of live bird markets as a H7N9 amplifying source is consistent with previous H7N9

reports.23

Exposure to live bird markets are recognized as a risk factor for previous human H5N1

infections in mainland China24

and Hong Kong25

, and as well as for human H7N9 infections in

other parts of mainland China.26-28

In fact, shutting down live poultry markets has been strongly

correlated with marked decline in human H7N9 infections.29 30

Our epidemiological investigations also strongly support the index case as the origin of

secondary case’s H7N9 infection. First, he developed symptoms five days after 5 days of contact

with the index case at hospital A. Second, he had no history of visiting a live market or a poultry

farm for two weeks prior to his illness. Third, poultry and environmental samples collected from

his home and village were negative for H7N9 virus. Hence, it seem most likely that the H7N9

virus was transmitted from the index case to the secondary case.

Based on genetic analysis, our results showed that the H7N9 viruses we isolated were quite

similar again suggesting this was nosocomial cluster of avian influenza H7N9 virus infection.

Phylogenetic analysis revealed that our viral HA and NA genes were most closely to H7N9

viruses isolated in the Jiangxi Province of China in second wave of human infection with H7N9

virus in 2014. However, the internal genes of the three viruses had different evolutionary origins,

suggesting H7N9 virus continues to undergo reassortment.15

Our studies of the H7N9 viruses’

key molecular characteristics also indicated that the viruses shared common characteristics,

specifically changes in receptor binding sites S138A, G186V, and G226L in the HA protein, and

Page 13 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

14

at E627K in the PB2 protein. Receptor binding assays also revealed that the H7N9 viruses

isolated from the patients had the ability to bind both the human and avian receptors. These

results suggested that the virus from the live poultry market had gained the ability to be

transmitted from chickens to humans. This might be consistent with the increased number of

human H7N9 cases in the most recent H7N9 wave.

Important strengths and differences in relation to other studies

Our study data suggested that the secondary cases most likely acquired the H7N9 virus from

the index case as he shared the same room with the index case. It is uncertain how the index case

transmitted his virus to the secondary case. Was it through direct contact or through aerosol? We

can only speculate but the fact that the index case frequently coughed and used a spittoon is

suggestive of bioaerosol transmission.

Implications of the study

Soon after their caring for the two H7N9 infected patients, one doctor and one nurse

developed transiently elevated HI antibodies (1:40) against H7N9 virus. The doctor also had

upper respiratory tract symptoms, was RT-PCR negative for virus, but was treated with

oseltamivir, the nurse had no symptoms. While we cannot be certain if these HI elevations were

due to true H7N9 infections in the medical workers, their rapid decline in titers seems to argue

against it. Kinetic studies of the serological response in mildly symptomatic H5N1 infected

patients have documented declines over time 31

but not as rapid as those observed in these two

medical workers. Hence, it seems most likely that the elevated titers we observed here were due

to some other cause which might include cross-reacting antibody from other infecting viruses or

from vaccines or laboratory variation. It is interesting that the diabetes patient shared the same

ward room for 5 days as did the index and secondary case and apparently was not infected H7N9

Page 14 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

15

virus. It is possible that the closer proximity the secondary case had with the index case and his

COPD, put him at increased risk of H7N9 infections compared to the diabetes patient.

Weaknesses of the study

This study had a number of limitations. While we had a number of RT-PCR H7N9 positive

swabs none yielded virus by culture and only one provided sequence data, so we could not fully

implicate the index case’s chickens as the original H7N9 source. Due to delays in diagnosis and

frequent movement of the patients, we did not receive serum samples from the two patients and

thus we could not examine their serological response to H7N9 viruses.

Unanswered questions and future research

With the continuing, unchecked geographical spread of H7N9 in mainland China and the

likely continuing genetic changes occurring in the virus, this investigations evidence of human-

to-human transmission portends a rather ominous outlook for future human morbidity and

mortality due to these H7N9 viruses.

We thank the doctors, the nurses, and other medical staff who helped us in this work. We also

thanks the live animal market workers and patients’ family members who also guided us.

Contributors: MJM, GCG, LQF, and WCC designed the study, and drafted the manuscript. FCF,

BDZ, SML, GPC, JMZ, SQW, XLH, EFC, YJL, JFJ, HMY, and WDX conducted the

epidemiological investigation and collected samples. MJM, YH, XXY, JL, JJZ, HWY, and XLL

performed laboratory assays and data interpretations. All authors contributed to the development

of the manuscript and approved the final draft. LQF, GCG, and WCC are guarantors.

Funding: WCC was partly supported by the Program of International Science and Technology

Cooperation (2013DFA30800) of the Ministry of Science and Technology of China and the

Page 15 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

16

Basic Work on Special Program for Science and Technology Research (2013FY114600). MJM

was partly supported by the National Natural Science Foundation of China (No.81402730). LQF

is partly supported by the Special Program for Prevention and Control of Infectious Diseases in

China (No. 2013ZX10004218). GCG was partly supported by US NIH Grant 1R01-AI108993.

The funding bodies had no role in study design, data collection and analysis, preparation of the

manuscript, or the decision to publish.

Declaration of interests: All authors declare that no support from any organization for the

submitted work; no financial relationships with any organizations that might have an interest in

the submitted work in the previous three years; no other relationships or activities that could

appear to have influenced the submitted work.

Ethical approval: An ethics waiver was granted and authorized under National Emergent Public

Health Events Act. According to this Act, collection of data related to H7N9 cases was an

important part in epidemic analyses and subsequent control measures. Therefore, the

investigation was exempt from institutional board assessment.

Transparency declaration: MJM, GCG, LQF, and WCC affirm that the manuscript is an honest,

accurate, and transparent account of the study being reported; that no important aspects of the

study have been omitted; and that any discrepancies from the study as planned (and, if relevant,

registered) have been explained.

Data sharing: Requests for de-identified study data will be reviewed and considered by the

corresponding authors.

Page 16 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

17

References

1. FluTrackers. FluTrackers 2013-15 Human Case List of Provincial/Ministry of

Health/Government Confirmed Influenza A(H7N9) Cases, 2015.

2. Liu Y, Paquette SG, Zhang L, Leon AJ, Liu W, Xiuming W, et al. The Third Wave: H7N9

Endemic Reassortant Viruses and Patient Clusters. J Infect Dev Ctries 2015;9(2):122-7.

3. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-

origin influenza A (H7N9) virus. N Engl J Med 2013;368(20):1888-97.

4. Wu A, Su C, Wang D, Peng Y, Liu M, Hua S, et al. Sequential reassortments underlie diverse

influenza H7N9 genotypes in China. Cell Host Microbe 2013;14(4):446-52.

5. Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of human infections with

avian influenza A(H7N9) virus in China. N Engl J Med 2014;370(6):520-32.

6. Liu T, Bi Z, Wang X, Li Z, Ding S, Wang L, et al. One family cluster of avian influenza

A(H7N9) virus infection in Shandong, China. BMC Infect Dis 2014;14:98.

7. Ding H, Chen Y, Yu Z, Horby PW, Wang F, Hu J, et al. A family cluster of three confirmed

cases infected with avian influenza A (H7N9) virus in Zhejiang Province of China. BMC

Infect Dis 2014;14(1):3846.

8. Xiao XC, Li KB, Chen ZQ, Di B, Yang ZC, Yuan J, et al. Transmission of avian influenza

A(H7N9) virus from father to child: a report of limited person-to-person transmission,

Guangzhou, China, January 2014. Euro Surveill 2014;19(25).

9. Hu J, Zhu Y, Zhao B, Li J, Liu L, Gu K, et al. Limited human-to-human transmission of avian

influenza A(H7N9) virus, Shanghai, China, March to April 2013. Euro Surveill

2014;19(25).

Page 17 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

18

10. Qi X, Qian YH, Bao CJ, Guo XL, Cui LB, Tang FY, et al. Probable person to person

transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013:

epidemiological investigation. BMJ 2013;347:f4752.

11. Yi L, Guan D, Kang M, Wu J, Zeng X, Lu J, et al. Family clusters of avian influenza A

H7N9 virus infection in Guangdong Province, China. J Clin Microbiol 2015;53(1):22-8.

12. Gao HN, Yao HP, Liang WF, Wu XX, Wu HB, Wu NP, et al. Viral genome and antiviral

drug sensitivity analysis of two patients from a family cluster caused by the influenza

A(H7N9) virus in Zhejiang, China, 2013. Int J Infect Dis 2014;29:254-8.

13. Mao H, Guo B, Wang F, Sun Y, Lou X, Chen Y, et al. A study of family clustering in two

young girls with novel avian influenza A (H7N9) in Dongyang, Zhejiang Province, in

2014. J Clin Virol 2015;63:18-24.

14. Chen Z, Liu H, Lu J, Luo L, Li K, Liu Y, et al. Asymptomatic, mild, and severe influenza

A(H7N9) virus infection in humans, Guangzhou, China. Emerg Infect Dis

2014;20(9):1535-40.

15. Lam TT, Zhou B, Wang J, Chai Y, Shen Y, Chen X, et al. Dissemination, divergence and

establishment of H7N9 influenza viruses in China. Nature 2015.

16. WH. O. Real-time RT-PCR Protocol for the Detection of Avian Influenza A(H7N9) Virus.

2013.

17. Organization WH. Real-time RT-PCR protocol for the detection of avian influenza A(H7N9)

virus, 2014.

18. Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, et al. Single-

reaction genomic amplification accelerates sequencing and vaccine production for classical

and Swine origin human influenza a viruses. J Virol 2009;83(19):10309-13.

Page 18 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

19

19. Liu W, Fan H, Raghwani J, Lam TT, Li J, Pybus OG, et al. Occurrence and reassortment of

avian influenza A (H7N9) viruses derived from coinfected birds in China. J Virol

2014;88(22):13344-51.

20. Organization WH. Serological detection of avian influenza A(H7N9) virus infections by

modified horse red blood cells haemagglutination-inhibition assay, 2013.

21. Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim W, et al. Detection of

antibody to avian influenza A (H5N1) virus in human serum by using a combination of

serologic assays. J Clin Microbiol 1999;37(4):937-43.

22. Ma MJ, Yang XX, Qian YH, Zhao SY, Hua S, Wang TC, et al. Characterization of a novel

reassortant influenza A virus (H2N2) from a domestic duck in Eastern China. Sci Rep

2014;4:7588.

23. Webster RG. Wet markets--a continuing source of severe acute respiratory syndrome and

influenza? Lancet 2004;363(9404):234-6.

24. Yu H, Feng Z, Zhang X, Xiang N, Huai Y, Zhou L, et al. Human influenza A (H5N1) cases,

urban areas of People's Republic of China, 2005-2006. Emerg Infect Dis 2007;13(7):1061-

4.

25. Mounts AW, Kwong H, Izurieta HS, Ho Y, Au T, Lee M, et al. Case-control study of risk

factors for avian influenza A (H5N1) disease, Hong Kong, 1997. J Infect Dis

1999;180(2):505-8.

26. Wang L, Cowling BJ, Wu P, Yu J, Li F, Zeng L, et al. Human exposure to live poultry and

psychological and behavioral responses to influenza A(H7N9), China. Emerg Infect Dis

2014;20(8):1296-305.

Page 19 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

20

27. Liu B, Havers F, Chen E, Yuan Z, Yuan H, Ou J, et al. Risk factors for influenza A(H7N9)

disease--China, 2013. Clin Infect Dis 2014;59(6):787-94.

28. Bao CJ, Cui LB, Zhou MH, Hong L, Gao GF, Wang H. Live-animal markets and influenza A

(H7N9) virus infection. N Engl J Med 2013;368(24):2337-9.

29. Wu P, Jiang H, Wu JT, Chen E, He J, Zhou H, et al. Poultry market closures and human

infection with influenza A(H7N9) virus, China, 2013-14. Emerg Infect Dis

2014;20(11):1891-4.

30. He Y, Liu P, Tang S, Chen Y, Pei E, Zhao B, et al. Live poultry market closure and control

of avian influenza A(H7N9), Shanghai, China. Emerg Infect Dis 2014;20(9):1565-6.

31. Buchy P, Vong S, Chu S, Garcia JM, Hien TT, Hien VM, et al. Kinetics of neutralizing

antibodies in patients naturally infected by H5N1 virus. PLoS One 2010;5(5):e10864.

Page 20 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Confidential: For Review Only

21

Table 1. Epidemiological and clinical features of the two confirmed H7N9 cases upon first admission

Characteristic Index case Second case

Age (yrs) 49 57

Sex Male Male

Occupation Pneumatic drill operator Farmer

Type of exposure Visited free market and bought two chickens Shared the same room with the index case

Underlying medical disorders Chronic obstructive pulmonary disease No

Smoking (yrs) 30 No

Relation between two patients Wardmate Wardmate

Onset of illness (mo-day-year) 2-16-2015 2-24-2015

Admission to hospital (mo-day-year) 2-18-2015 2-25-2015

Sign(s) of illness Fever, cough, and sore throat Fever and cough

Temperature (°C) 38.8 40.0

White blood count (×109/liter) 6.4 6.7

Neutrophils (×109/liter) 1.68 5.4

Lymphocytes (×109/liter) 0.35 1.02

Platelets (×109/liter) 146 193

C-reactive protein (mg/L) 29.5 47.1

PaCO2 (mm Hg) N/A 46

PaO2 (mm Hg) N/A 58

Saturation of peripheral oxygen (%) 95% 68%

Chest radiography Pneumonia NA

Page 21 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


22

Mechanical ventilation Yes Yes

Oseltamivir treatment Yes Yes

Oxygen treatment Yes Yes

Outcome Died Died

NA = not applicable.

Page 22 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


23

Table 2. Key molecular characteristics of four H7N9 viruses identified in this study.

Gene Position A/Quzhou/1/2015 A/Quzhou/2/2015 A/Chicken/Quzhou/1/2015 Comments

HA

Cleavage PEIPKGR↓G EIPKGR↓G PEIPKGR↓G Pathogenic to poultry

S138A A A A

RBS position, altered receptor

specificity

G186V V V V

Q226L L L L

G228S G G G

NA 63–73 Yes Yes Yes Increased virulence in mice

R292K R R R Osteltamivir and zanamivir resistance

PB2 L89V V V NA Mammalian host adaption and increased

virulence in mice E627K K K NA

PB1 H99Y H H NA

H5 virus transmissible among ferrets I368V V V NA

M1 N30D D D D Increased virulence in mice

T215A A A A

M2 S31N N N N Antiviral resistance (amantadine)

NS1 P42S S S S Increased virulence in mice

NA = not applicable.

Page 23 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

24

Legend for Figures

Figure 1. Timeline of events associated with the human H7N9 infections, Quzhou City of

Zhejiang Province, China, 2015. (rRT-PCR = real-time reverse transcriptase-polymerase chain

reaction; COPD = chronic obstructive pulmonary disease; HI=hemagglutination inhibition).

Figure 2. Phylogenetic tree of the HA and NA genes of the H7N9 viruses isolated or sequenced

from patients or their environments in Zhejiang Province, China. Supporting bootstrap values

greater than 75 are shown. Scale bars indicates nucleotide substitutions per site. H7N9 viruses

isolated or sequenced were market with solid red circles.

Figure 3. Characterization of receptor-binding properties at virus level. Binding of virus to a2,3-

lingked (3’SL-PAA) or a2,6-lingked (6’SL-PAA) sialylgycan receptors was determined by solid-

phase binding assays. (A) QZ1-H7N9 (A/Quzhou/1/2015/H7N9) virus; (B) CA07-H1N1,

(A/California/07/2009) virus; (C) JS927-H5N1 (A/Chicken/Jiangsu/927/2014) virus.

Page 24 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

Timeline of events associated with the human H7N9 infections, Quzhou City of Zhejiang Province, China, 2015. (rRT-PCR = real-time reverse transcriptase-polymerase chain reaction; COPD = chronic obstructive

pulmonary disease; HI=hemagglutination inhibition) 558x189mm (300 x 300 DPI)

Page 25 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

hylogenetic tree of the HA and NA genes of the H7N9 viruses isolated or sequenced from patients or their environments in Zhejiang Province, China. Supporting bootstrap values greater than 75 are shown. Scale

bars indicates nucleotide substitutions per site. H7N9 viruses isolated or sequenced were market with solid red circles.

81x33mm (300 x 300 DPI)

Page 26 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

Characterization of receptor-binding properties at virus level. Binding of virus to a2,3-lingked (3’SL-PAA) or a2,6-lingked (6’SL-PAA) sialylgycan receptors was determined by solid-phase binding assays. (A) QZ1-H7N9

(A/Quzhou/1/2015/H7N9) virus; (B) CA07-H1N1, (A/California/07/2009) virus; (C) JS927-H5N1

(A/Chicken/Jiangsu/927/2014) virus. 120x266mm (300 x 300 DPI)

Page 27 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

1

May 5, 2015

Appendix 1: Supplementary figures

Figure S1. Location of two H7N9 infected patients’ home, and the live poultry stall at

live bird market A where the index case bought his two chickens, Quzhou City of

Zhejiang Province, China, February 2014.

Page 28 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

2

Figure S2. Diagram of the patients’ shared hospital ward.

Page 29 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

3

Figure S3. Phylogenetic tree of the internal genes of the H7N9 viruses isolated or

sequenced from patients or their environments in Zhejiang Province, China.

Supporting bootstrap values greater than 75 are shown. Scale bars indicates nucleotide

substitutions per site. H7N9 viruses isolated or sequenced were market with solid red

circles.

Page 30 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

4

Page 31 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960


nly

5

Page 32 of 32


BMJ

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

nosocomial transmission of avian influenza a (h7n9) virus in … · 05-05-2015 · yi, huai-ming;...

Documents