Clinical Microbiology and Infection 24 (2018) 321e323
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Clinical Microbiology and Infection
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Letter to the Editor
Human infection with H9N2 avian influenza in northern China
Dear Sir,
The first case of a human infected with avian influenza A(H9N2)
virus was reported in 1998, China [1]. Since then, H9N2 has become
a global, public health concern. According to a World Health Orga-
nization report, at least 37 laboratory-confirmed cases of human
infection with avian influenza H9N2 had been reported globally
by the end of June 2017. Until now, most cases were found in south-
ern China and South Eastern Asian countries, whereas no cases
were reported in the northern area. Here we reporte the epidemi-
ological and virological characteristics of the first and another hu-
man infection with H9N2 influenza in northern China.
The first case (case 1) was identified by virological surveillance
of influenza-like illness in Beijing on 12 December 2016. A 3-
year-old boy developed initial symptoms of fever on 11 December
2016. At 1 day after illness onset, he was admitted to a paediatric
hospital, identified as influenza A RNA positive and then confirmed
as having H9N2 infection using real-time RT-PCR using commercial
kits (Shuoshi, Jiangsu, China). The patient received symptomatic
treatment and was recovered 5 days after onset without antiviral
treatment and hospitalization. Another mild H9N2-infected case
(case 2) was a 32-year-old male worker. The patient suffered
from headache and fever on 28 April 2017 and was confirmed as
having H9N2 infection by real-time RT-PCR 2 days later during
his initial medical attention. Oseltamivir was used after the diag-
nosis, and nucleic acid testing for influenza A showed a negative
result 11 days after illness onset. No abnormalities were observed
among close contacts of the two cases at the time of reporting. It
is worth noting that case 1 has close contact with a live poultry
market about 7 days before onset, while case 2 showed no such
contact. Moreover, no H9N2 virus was detected in case 1 and case
2-related environment.
Although only one of these two cases had a clear record of
recent contact with avians and an avian-related environment, pre-
vious studies showed that poultry as well as a few wild birds were
the most common source of H9N2 infections [3]. During the same
period, we performed environmental surveillance to investigate
H9N2 circulating. In all, 2970 samples were collected from the envi-
ronment of poultry farms, street poultry vendors and wild birds in
six suburban areas of Beijing in 2016. A total of 15 H9N2 RNA-
positive samples (0.51%) were detected. Among them, most viruses
were found in street poultry vendors (14/15, 93.33%) and in
September (6/15, 40%) (see Supplementary material, Table S1).
Then viral RNA was extracted from two H9N2 viruses in patients
and five H9N2 environmental isolates using QIAmp Viral Mini Kit
(Qiagen, Hilden, Germany), and sequenced using an Ion Torrent
PGM system (Applied Biosystems, Foster City, CA, USA) (NCBI
https://doi.org/10.1016/j.cmi.2017.10.026
1198-743X/© 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
accession no. MF440688eMF440743). Phylogenetic inference
showed that all seven H9N2 viruses belonged to clade 4.2.5
(BJ94/Y280-like) low pathogenic avian influenza (Fig. 1) [2]. More-
over, HA-228G and HA-226L substitution (H3 numbering) in the HA
sequences of all viruses indicated dual receptor-binding with avian
a-2,3-glycan receptors and human a-2,6-glycan receptors. There
was no amino acid deletion at the stalk region of the NA protein,
which was related to the virus adaptation and increase in virulence.
Adaptive mutations of internal genes in A/Beijing/1/2016 and A/
Beijing/1/2017 were also investigated. Although human adaptive
sites of PB2-627V, PB1-P13 and PA-409N were found in PB2, PB1
and PA gene, respectively, no mutation was found at PB2-591Q
and 701N in two cases viruses, retaining the avian characteristics.
In addition, all viruses isolated from cases and the environment car-
ried adamantane-resistant mutation of S31N in the M2 gene but
neither of them showed an NAI-resistant mutation in NA gene,
such as H274Y, R292K and N294S. In addition, high similarity and
significant homology were observed in all six internal genes be-
tween tested viruses and human infecting H7N9 viruses.
In this study, the first case, and one following H9N2 laboratory-
confirmed case, in northern China are reported. Compared with the
southern area, the risk of H9N2 infection in northern China is rela-
tively low. A recent study suggested that seroprevalences of the H9
G9 and G1 lineage in northern China were 2.34% and 0.81%, respec-
tively, which were significantly lower than in the southern area
(3.42% and 1.37%, respectively) [3]. Even under these circum-
stances, two H9N2-infected cases were identified by influenza-
like illness surveillance, indicating that the true infection number
of H9N2 in northern China and even in other low epidemic areas
may be underestimated, because most infected individuals only
show mild clinical symptoms so it is difficult to find using regular
surveillance systems [1]. We also noticed that the H9N2 viruses
derived from two patients and those isolated from the environment
distributed in different clusters in the HA phylogenetic tree, which
might suggest genetic diversity and a wide spread of H9N2 in the
Beijing area.
Another important issue is the high-risk populations in H9N2
infection. Previous serological investigations indicated that the
highest seroprevalences of H9N2 could be found in occupations
with high levels of exposure to live poultry [3,4]. Our data also sug-
gest that almost all H9N2 viruses found in the environment were
associated with poultry vendors (93.33%). Next, more importantly,
it has been proved that the internal genes of H7N9 virus were
donated by H9N2 in the Beijing area (A/brambling/Beijing/16/
2012) [5]. We also show that the H9N2 viruses found in patients
and the environment shared high homology with currently circu-
lating H7N9 virus, indicating its key role in the origin and evolution
322 Letter to the Editor / Clinical Microbiology and Infection 24 (2018) 321e323
Fig. 1. Phylogenetic tree of H9 gene (a) and PB2, PB1, PA, NP, NA, MP and NS gene (b) of avian influenza A (H9N2) viruses in Beijing. The phylogenetic trees were constructed using
MEGA (v6.0.4) program and employing the neighbour-joining method with a bootstrap of 1000.
of H7N9. Hence, a vigilant virological and serological surveillance
for H9 in humans, poultry and wild birds is essential, not only for
H9N2, but also for HxNy virus in the future.
Transparency declaration
The authors report no potential conflicts of interest.
Funding
This work was supported by The Capital Health Research and
Development of Special (2014-1-1011), Beijing Young Top-notch
Talent Project (2014000021223ZK36), and Beijing Natural Science
Foundation (7152075).
Acknowledgements
We thank staff members of the Capital Institute of Paediatrics,
Chaoyang District Centres for Disease Control and Prevention and
Fangshan District Centres for Disease Control and Prevention for
their help with field investigation, administration and data
collection.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.cmi.2017.10.026.
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Janetanakit T, et al. Influenza A(H9N2) virus, Myanmar, 2014-2015. Emerg Infect
Dis 2017;23:1041e3.
[3] Li X, Tian B, Jianfang Z, Yongkun C, Xiaodan L, Wenfei Z, et al. A comprehensive
retrospective study of the seroprevalence of H9N2 avian influenza viruses in
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[4] Yu Q, Liu L, Pu J, Zhao J, Sun Y, Shen G, et al. Risk perceptions for avian influenza
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 Letter to the Editor / Clinical Microbiology and Infection 24 (2018) 321e323
Y. Pan
Institute for Infectious Disease and Endemic Disease Control, Beijing
Centre for Disease Prevention and Control (CDC), Beijing, China
Research Centre for Preventive Medicine of Beijing, Beijing, China
Capital Medical University School of Public Health, Beijing, China
S. Cui, Y. Sun, X. Zhang, C. Ma, W. Shi, X. Peng, G. Lu, D. Zhang,
Y. Liu, S. Wu
Institute for Infectious Disease and Endemic Disease Control,
Beijing Centre for Disease Prevention and Control (CDC), Beijing,
China
Research Centre for Preventive Medicine of Beijing, Beijing, China
P. Yang**
Institute for Infectious Disease and Endemic Disease Control, Beijing
Centre for Disease Prevention and Control (CDC), Beijing, China
Research Centre for Preventive Medicine of Beijing, Beijing, China
Capital Medical University School of Public Health, Beijing, China
323
Q. Wang*
Institute for Infectious Disease and Endemic Disease Control, Beijing
Centre for Disease Prevention and Control (CDC), Beijing, China
Research Centre for Preventive Medicine of Beijing, Beijing, China
*
Corresponding author. Q. Wang, Institute for Infectious Disease
and Endemic Disease Control, Beijing Centre for Disease
Prevention and Control (CDC), Beijing, China.
E-mail address: bjcdcxm@126.com (Q. Wang).
**
Corresponding author. P. Yang, Institute for Infectious Disease
and Endemic Disease Control, Beijing Centre for Disease
Prevention and Control (CDC), Beijing, China
E-mail address: yangpengcdc@163.com (P. Yang).
10 September 2017
Available online 26 November 2017
Editor: L Leibovici