Development and evaluation of a new real‐time RT‐PCR assay for detecting the latest H9N2 influenza viruses capable of causing human infection

Abstract

The H9N2 subtype of avian influenza A viruses (AIVs) has spread among domestic poultry and wild bird worldwide. H9N2 AIV is sporadically transmitted to humans from avian species. A total of 42 laboratory‐confirmed cases of non‐fatal human infection with the Eurasian Y280 and G1 lineages have been reported in China, Hong Kong, Bangladesh, and Egypt since 1997. H9N2 AIV infections in poultry have become endemic in Asia and the Middle East and are a major source of viral internal genes for other AIV subtypes, such that continuous monitoring of H9N2 AIV is recommended. In this study, a new, one‐step, real‐time RT‐PCR assay was developed to detect two major Eurasian H9 lineages of AIVs capable of causing human infection. The sensitivity of this assay was determined using in vitro‐transcribed RNA, and the detection limit was approximately 3 copies/reaction. In this assay, no cross‐reactivity was observed against RNAs from H1‐15 subtypes of influenza A viruses, influenza B viruses, and other viral respiratory pathogens. In addition, this assay could detect the H9 HA gene from artificially reconstituted clinical samples spiked with H9N2 virus without any non‐specific reactions. Therefore, this assay is highly sensitive and specific for H9 HA detection. The assay is useful both for diagnostic purposes in cases of suspected human infection with influenza H9N2 viruses and for the surveillance of both avian and human influenza viruses.

Full text

Received: 11 October 2018
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Revised: 20 December 2018
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Accepted: 24 December 2018
DOI: 10.1111/1348-0421.12666
ORIGINAL ARTICLE
Virology
Development and evaluation of a new real-time RT-PCR assay
for detecting the latest H9N2 influenza viruses capable of
causing human infection
Shinji Saito
1
|
Ikuyo Takayama
1
|
Mina Nakauchi
1
|
Shiho Nagata
1
|
Kunihiro Oba
2
|
Takato Odagiri
1
|
Tsutomu Kageyama
1
1
Influenza Virus Research Center, National
Institute of Infectious Diseases, 4-7-1
Gakuen, Musashimurayama, Tokyo 208-
0011, Japan
2
Department of Pediatrics, Showa General
Hospital, 8-1-1 Hanakoganei, Kodaira,
Tokyo 187-8510, Japan
Correspondence
Tsutomu Kageyama, Influenza Virus
Research Center, National Institute of
Infectious Diseases, 4-7-1 Gakuen,
Musashimurayama, Tokyo 208-0011, Japan.
Email: tkage@nih.go.jp
Funding information
AMED, Grant number: JP18fk0108030
Abstract
The H9N2 subtype of avian influenza A viruses (AIV) has spread among domestic
poultry and wild birds worldwide. H9N2 AIV is sporadically transmitted to humans
from avian species. A total of 42 laboratory-confirmed cases of non-fatal human
infection with the Eurasian Y280 and G1 lineages have been reported in China, Hong
Kong, Bangladesh and Egypt since 1997. H9N2 AIV infections in poultry have
become endemic in Asia and the Middle East and are a major source of viral internal
genes for other AIV subtypes, such that continuous monitoring of H9N2 AIV is
recommended. In this study, a new, one-step, real-time RT-PCR assay was developed
to detect two major Eurasian H9 lineages of AIV capable of causing human infection.
The sensitivity of this assay was determined using in vitro-transcribed RNA, and the
detection limit was approximately 3 copies/reaction. In this assay, no cross-reactivity
was observed against RNA from H1–15 subtypes of influenza A viruses, influenza B
viruses and other viral respiratory pathogens. In addition, this assay could detect the
H9 hemagglutinin (HA) gene from artificially reconstituted clinical samples spiked
with H9N2 virus without any non-specific reactions. Therefore, this assay is highly
sensitive and specific for H9 HA detection. The assay is useful both for diagnostic
purposes in cases of suspected human infection with influenza H9N2 viruses and for
the surveillance of both avian and human influenza viruses.
KEYWORDS
avian influenza, diagnosis, H9N2, influenza, real-time RT-PCR
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd
Abbreviations: AIV, avian influenza A virus; Cp, crossing point; Ct, threshold cycle; GISAID, Global Initiative on Sharing All Influenza Data; HA,
hemagglutinin; NA, neuraminidase; rRT-PCR, real-time RT-PCR.
Microbiol Immunol. 2019;63:21–31. wileyonlinelibrary.com/journal/mim
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1
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INTRODUCTION
Influenza A viruses are single-stranded, negative-sense RNA
viruses belonging to the Orthomyxoviridae family. The natural
host of influenza A viruses are wild aquatic birds, with 16
hemagglutinin (HA) and nine neuraminidase (NA) subtypes
identified in avian species [1]. Avian influenza viruses (AIV)
of the H9N2 subtype circulate primarily among wild birds and
domestic poultry, but the viruses can infect swine and humans
as well. A total of 42 cases of non-fatal human infection were
reported in Asia and the Middle East as of March 2018 (http://
www.who.int/influenza/human_animal_interface/
HAI_Risk_Assessment/en/) [2–9].
H9N2 viruses have been widely and consistently isolated
worldwide since their first isolation from turkeys in Wisconsin,
USA, in 1966 [10]. H9N2 viruses are divided into a North
American lineage and a Eurasian lineage [11]. The North
American lineage H9N2 viruses are typically detected in
shorebirds and wild ducks, and no cases of human infection
have been reported to date [12]. The Eurasian lineage of H9N2
AIV circulating in Asia, the Middle East and Europe have been
classified into two major lineages, Y280 and G1, and one minor
Korean lineage. Since 1997, sporadic laboratory-confirmed cases
of avian-to-human transmission of Y280-lineage viruses in China
and G1-lineage viruses in China, Hong Kong, Bangladesh and
Egypt have been reported (http://www.who.int/influenza/
human_animal_interface/HAI_Risk_Assessment/en/) [2–9].
However, the results of serologic studies in Asia and the Middle
East suggest that the number of humans infected by H9N2 AIV is
much greater than the number of laboratory-confirmed cases [13–
18]. It is thus important to monitor H9N2 AIV in wild birds and
poultry in order to assess the risk for human infection.
Molecular diagnostic techniques such as the PCR method
can be used as diagnostic tools for virus identification and
assessing viral infection. In particular, real-time RT-PCR (rRT-
PCR) is one of the most widely used methods for detecting viral
genes, and rRT-PCR assays for detecting H9 viruses have been
reported [19–22]. However, the sequences of probes and primers
used in rRT-PCR in previous studies were designed for detecting
viruses of the North American lineage or past circulated G1-
lineage H9 AIV. These methods did not use minor groove binder
(MGB) probes, resulting in different conditions for these assays
compared with the assay used in Japan for detecting other
influenza viruses. Therefore, our newly developed, one-step
rRT-PCR assay was designed to detect both recent Y280- and
G1-lineage H9 AIV, including those causing human infection.
2
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MATERIALS AND METHODS
2.1
|
Primer and probe design
The nucleotide sequences of the HA genes of H9 subtype AIV
were aligned using ClustalX 2.1 software with the sequences
of all human viruses registered to date and avian viruses in
Asia and the Middle East registered after 2014 in the
GenBank/EMBL/DDBJ (https://www.ncbi.nlm.nih.gov/
genbank/) and Global Initiative on Sharing All Influenza
Data (GISAID) (http://www.gisaid.org) databases [23,24].
On the basis of highly conserved sequences in the HA1
region, primers were designed to detect as many human and
avian viruses as possible by rRT-PCR using the MGB
TaqMan
®
probe (Thermo Fisher Scientific, Waltham, MA,
USA). The sequences and positions of the primers and probes
are listed in Table 1.
2.2
|
One-step rRT-PCR assay
The reaction was performed using AgPath-ID™one-step RT-
PCR reagents (Thermo Fisher Scientific) in a 25 μL reaction
mixture containing 12.5 μL of 2× RT-PCR buffer, 1 μLof
25× RT-PCR enzyme mix, 0.1 μL (20 U) of RNase inhibitor
(Thermo Fisher Scientific), 600 nM each forward and reverse
primer, 100 nM TaqMan MGB probe and 5 μL of RNA
template. The rRT-PCR assays were carried out using a
LightCycler
®
480 II (Roche, Basel, Switzerland) under the
following conditions: 50°C for 10 min, 95°C for 10 min, and
45 cycles of 15 s at 95°C, 30 s at 56°C and 15 s at 72°C.
Amplification data were collected at 56°C (annealing step)
and analyzed according to the second derivative maximum
method in the LightCycler
®
480 SW1.5 software.
2.3
|
Viruses and viral RNA extraction
H1–15 subtypes of influenza A viruses, influenza B viruses
and 19 viral respiratory pathogens stored in our laboratory
were used in this study (Tables 2–4). Viral RNA was
extracted from 140 μL cultures of each virus propagated in
embryonated chicken eggs to 60 μL of AVE (elution buffer
supplied with the kit) using a QIAamp
®
viral RNA mini kit
(Qiagen, Hilden, Germany) according to the manufacturer's
instructions. In this study, the copy number of the M gene of
the 18 H9 viruses was determined quantitatively as previously
described [25] using an influenza A (type A) rRT-PCR assay
targeted to the universal M gene of all influenza A
viruses [26]. The threshold cycle (Ct) values of viral RNA
extracted from 19 viral respiratory pathogens were deter-
mined using the multiplex real-time PCR assay described
previously, with minor modifications [27].
2.4
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Clinical specimens and identification of
seasonal influenza viruses
Nasal swabs or aspirate samples collected from patients with
influenza-like illness were collected at Showa General
Hospital, Japan, between 2014 and 2016 and suspended
using a UTM 360 C kit (Copan, Brescia, Italy). The study
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SAITO ET AL.

protocol was approved by the ethics committees of both the
National Institute of Infectious Diseases and Showa General
Hospital, and the study was performed in compliance with the
Declaration of Helsinki. Informed consent was obtained from
all patients. Total RNA was extracted using the QIAamp viral
RNA mini kit (Qiagen) according to the manufacturer's
instructions. The type and subtype of influenza viruses in each
clinical sample were determined using rRT-PCR as previ-
ously described [26,28].
2.5
|
Phylogenetic analysis of the H9 HA gene
Phylogenetic analysis of the H9 HA gene was performed
using Molecular Evolutionary Genetics Analysis software
(MEGA, version 7.0) [29]. Evolutionary history was inferred
using the neighbor-joining method [30]. Evolutionary
distances were computed using Kimura's two-parameter
method [31]. Bootstrap values of the HA genes were
calculated from 1000 replicates [32].
2.6
|
Preparation of in vitro-transcribed RNA
To evaluate the sensitivity of the H9 rRT-PCR assay, three
in vitro-transcribed full-length H9 HA gene RNA were
used. RNA transcripts of the full-length H9 HA gene were
synthesized from artificial DNA (Eurofins Genomics,
Tokyo, Japan) of A/Hunan/44558/2015 (H9N2) (GISAID
accession no. EPI680526), A/chicken/Bangladesh/28182/
2016 (H9N2) (WSS1378750) and A/chicken/Egypt/
F12173D/2016 (H9N2) (EPI953355) using the following
procedure. The H9 HA artificial DNA were amplified by
PCR using Phusion high-fidelity DNA polymerase (New
England BioLabs, Ipswich, MA, USA) with the paired
primers T7 + Stop-R (5′-TAATACGACTCACTA-
TAGGGTTA-3′) and Hunan-F (5′-ATGGAGACAGTAT-
CACTAATAACTA-3′), Bangladesh-F (5′-ATGGAAACA
GTAACACTGTTGAC-3′)orEgypt-F(5′-ATGGAAA-
TAATACCACTGATG-3′) (underline indicates the T7
promoter sequence) according to the manufacturer's
instructions. RNA were transcribedusingtheT7Ribo-
MAX™Express large-scale RNA production system
(Promega, Madison, WI, USA) and treated with TURBO
®
DNase (Thermo Fisher Scientific) to degrade the template
DNA according to the manufacturer's instructions. dNTP
and NTP were removed using MicroSpin G-25 columns
(GE Healthcare, Piscataway, NJ, USA) according to the
manufacturer's instructions. Transcribed RNA were quan-
tified using a NanoDrop™spectrometer (Thermo Fisher
Scientific), and the copy number was then calculated. The
integrity of transcribed RNA was assessed using a 2100
BioAnalyzer (Agilent Technologies, Santa Clara, CA,
USA). Transcript dilutions were performed in nuclease-
free water containing 10 ng/μL of carrier RNA (Qiagen).
2.7
|
Validation and evaluation of the H9 rRT-
PCR assay
The analytical sensitivity of the assay was assessed by
testing serial dilutions of two quantified in vitro-tran-
scribed RNA from A/Hunan/44558/2015 (H9N2) of the
Y280 lineage and A/chicken/Egypt/F12173D/2016 (H9N2)
of the G1 lineage using six replicates of each concentration.
The limit of detection was calculated using StatPlus
®
Professional Version 2009 for Windows (Build 5.8.4.3) by
probit regression analysis [33] with a 95% probability end-
point.
For evaluation of the H9 rRT-PCR assay, extracted RNA
were prepared at 1, 10 and 100 copies/μL based on the number
of M genes determined using type A rRT-PCR for 10 Y280
viruses, three G1 viruses, four Korean viruses and one North
American virus, and three synthetic RNA were also prepared
at 1, 10 and 100 copies/μL. The H9 rRT-PCR assay was
performed in triplicate for each dilution. Results were
considered to be positive when the crossing point (Cp) value
was given by the second derivative maximum method in the
Light Cycler
®
480 SW1.5 software. For positive samples with
Cp > 40 (flagged as “late Cp call”[last five cycles] with high
uncertainty by the LightCycler 480 software), the Light
Cycler 480 software applied 40.00 to the sample. The number
of positive results per test number and Cp values are shown in
Table 2.
The specificity of the H9 rRT-PCR assay was validated
using RNA extracted from 24 representative subtype
viruses except for the H9 subtype of influenza A and
three influenza B viruses, and 19 viral respiratory
pathogens (Tables 3 and 4).
TABLE 1 Primers and probe used in the H9 real-time RT-PCR assay
Name Sequence (5–3′)†Position
‡
Product size (bp)
NIID-H9 TMPrimer-F1 AATGTYCCTGTGACACATGCCAAAGA 121–146
NIID-H9 TMPrimer-R1 AGRTCACAAGAAGGRTTGCCATA 238–260 140
NIID-H9 Probe1 (FAM)CATYCCATTRTGCTCTGTGTGGAG(MGB) 151–174
†Probe was labeled with FAM at the 5′-end and minor groove binder at the 3′-end.
‡
Nucleotide numbering is based on the HA gene CDS of A/Hong Kong/308/2014 (H9N2).
SAITO ET AL.
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23

TABLE 2 Detection limit of the new H9 rRT-PCR assay in comparison with the type A rRT-PCR assay
Type A rRT-PCR assay H9 rRT-PCR assay
Lineage Virus strain
M gene copy
number
†
No. of positive
results
Cp value
‡
(average ± SD)
No. of positive
results
Cp value
‡
(average ± SD)
Y280 A/chicken/Hong Kong/G9/
1997 (H9N2)
5 3/3 37.54 ± 0.58 3/3 36.33 ± 0.44
A/Hong Kong/308/2014
(H9N2)
5 2/3 38.6 3/3 37.88 ± 1.01
A/swine/Hong Kong/9/98
(H9N2)
5 3/3 37.82 ± 0.54 3/3 37.82 ± 0.54
A/Hunan/44558/2015 (H9N2)
§
5
¶
Not available 3/3 38.77 ± 1.19
A/duck/Japan/AQ-HE5/2015
(H9N2)
5 3/3 37.56 ± 0.55 3/3 37.51 ± 0.49
A/chicken/Japan/AQ-HE14/
2015 (H9N2)
5 3/3 37.23 ± 0.54 3/3 39.12 ± 0.76
A/duck/Japan/AQ-HE28/2015
(H9N2)
5 3/3 38.04 ± 0.82 3/3 38.49 ± 1.45
A/chicken/Japan/AQ-HE61/
2015 (H9N2)
5 3/3 38.83 ± 1.05 3/3 37.37 ± 0.39
A/chicken/Japan/AQ-HE28-
28/2016 (H9N2)
5 3/3 38.19 ± 1.03 3/3 37.86 ± 0.11
A/chicken/Japan/AQ-HE28-
50/2016 (H9N2)
5 3/3 38.59 ± 1.23 3/3 37.45 ± 0.14
A/chicken/Japan/AQ-HE28-
57/2016 (H9N2)
5 3/3 38.25 ± 0.23 3/3 38.13 ± 0.54
G1 A/Hong Kong/1073/99 (H9N2) 5 3/3 38.72 ± 1.13 3/3 38.30 ± 0.83
A/chicken/Bangladesh/28182/
2016 (H9N2)
§
5
¶
Not available 2/3 38.88
A/chicken/Egypt/F12173D/
2016 (H9N2)
§
5
¶
Not available 3/3 36.94 ± 0.19
A/parakeet/Chiba/1/97 (H9N2) 5 3/3 38.45 ± 0.42 3/3 36.10 ± 0.39
A/parakeet/Narita/92a/98
(H9N2)
5 3/3 38.09 ± 1.24 3/3 36.40 ± 0.60
Korean A/duck/Hong Kong/448/78
(H9N2)
5 3/3 38.21 ± 0.06 3/3 37.50 ± 0.56
A/duck/Hong Kong/702/79
(H9N5)
5 3/3 37.83 ± 1.09 3/3 37.94 ± 1.79
A/duck/Hokkaido/31/97
(H9N2)
500
50
5
3/3
3/3
3/3
31.16 ± 0.04
34.46 ± 0.53
38.54 ± 1.41
2/3
0/3
0/3
40.00
-
-
A/duck/Fukui/3/2005 (H9N1) 5 3/3 38.61 ± 1.21 2/3 40.00
North
American
A/turkey/Wisconsin/1/66
(H9N2)
500
50
5
3/3
3/3
2/3
31.07 ± 0.09
34.45 ± 0.22
38.47
3/3
1/3
0/3
38.39 ± 0.47
40.00
-
†Copy number of the M gene corresponding to the detection limit of the H9 HA gene (copies/reaction).
‡
Crossing point (Cp) values were analyzed according to the second derivative maximum method in the Light Cycler
®
480 SW1.5 software. The Cp value of 40.00 was
detectable.
§In vitro-transcribed RNA was used for the HA gene of each isolate.
¶
Copy number of the H9 HA gene (copies/reaction).
rRT-PCR, real-time RT-PCR
24
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SAITO ET AL.

2.8
|
Evaluation of the H9 rRT-PCR assay
using artificially reconstituted clinical samples
Artificially reconstituted clinical samples spiked with H9N2
virus (130 μLofH1N1pdm09,H3N2ortypeBpositive
clinical specimens, or influenza A and B viruses negative
clinical specimens + 10 μL of A/Hong Kong/308/2014
[H9N2]), those not spiked with H9N2 virus (130 μLof
H1N1pdm09, H3N2 or type B positive clinical specimens,
or influenza A and B viruses negative clinical speci-
mens + 10 μL of PBS) and diluted H9N2 virus (130 μLof
PBS + 10 μL of A/Hong Kong/308/2014 [H9N2]) were
prepared. Total RNA was extracted using the QIAamp viral
RNA mini kit (Qiagen) according to the manufacturer's
instructions. Simultaneous with the H9 rRT-PCR assay,
type A, B/NS, H1pdm and H3 rRT-PCR assays were
performed using primer and probe sets previously described
under the same conditions used for the H9 rRT-PCR
assay [26,28].
3
|
RESULTS
The phylogenetic tree based on the H9 HA gene, including the
21 H9 AIV examined in this study and 44 representative H9
AIV from the GenBank/EMBL/DDBJ and GISAID data-
bases, is shown in Figure 1. The detection limit of the H9 rRT-
PCR assay was determined by testing six replicates each of
10-fold serial dilutions of 5.0 × 10
7
copies/reaction of in
vitro-transcribed full-length H9 HA RNA derived from
isolate A/Hunan/44558/2015 (H9N2) of the Y280 lineage and
isolate A/chicken/Egypt/F12173D/2016 (H9N2) of the G1
lineage (Figure 1). An amplification plot for A/Hunan/44558/
2015 (H9N2) obtained as raw data is shown in Figure S1.
TABLE 3 Panel of non-H9 influenza viruses used in the H9 rRT-PCR assay
Sample Strain or sample name Type or subtype Type A or B/NS rRT-PCR assays†H9 rRT-PCR assay
†
Virus isolate A/duck/Alberta/35/76 H1N1 18.84 N.D.
A/Brisbane/59/2007 H1N1 25.45 N.D.
A/Narita/1/2009 H1N1pdm09 23.91 N.D.
A/duck/Germany/1215/73 H2N3 19.82 N.D.
A/duck/Ukraine/1/63 H3N8 18.11 N.D.
A/Uruguay/716/2007 H3N2 25.20 N.D.
A/Indiana/12/2012 H3N2v 21.75 N.D.
A/duck/Czechoslovakia/56 H4N6 19.60 N.D.
A/duck/Hyogo/1/2010 H4N6 23.64 N.D.
A/blow fly/Kyoto/93/2004 H5N1 26.24 N.D.
A/chicken/Ibaraki/1/2005 H5N2 18.17 N.D.
A/white swan/Hokkaido/4/2011 H5N1 18.66 N.D.
A/turkey/Massachusetts/3740/65 H6N2 23.29 N.D.
A/duck/Hong Kong/301/78 H7N1 21.24 N.D.
A/duck/Fukui/1/2004 H7N7 24.65 N.D.
A/Anhui/1/2013 H7N9 24.62 N.D.
A/turkey/Ontario/6118/68 H8N4 22.57 N.D.
A/duck/Shizuoka/45/2011 H8N4 21.84 N.D.
A/chicken/Germany/N/49 H10N7 17.86 N.D.
A/duck/England/56 H11N6 19.00 N.D.
A/duck/Alberta/60/76 H12N5 19.39 N.D.
A/gull/Maryland/704/77 H13N6 19.60 N.D.
A/mallard/Gurjev/263/82 H14N5 18.92 N.D.
A/duck/Australia/341/83 H15N8 19.20 N.D.
B/Florida/04/2006 Type B 25.40 N.D.
B/Brisbane/60/2008 Type B 29.61 N.D.
B/Massachusetts/2/2012 Type B 30.07 N.D.
†Crossing point values were determined using the second derivative maximum method in Light Cycler
®
480 SW1.5 software.
N.D., not detected; rRT-PCR, real-time RT-PCR.
SAITO ET AL.
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25

Probit regression analysis of the data from the two viruses
tested showed detection limits of 3.1 and 1.8 copies/reaction,
respectively. The efficiency of the assay for the two viruses
tested was 96.3% and 93.8%, respectively; the R
2
value was
0.99 for each virus, and the slope of the standard curve was
−3.41 and −3.48 in the range between 5.0 and 5.0 × 10
7
copies/reaction, respectively (Figure 2).
The H9 AIV examined in this study were classified into
four lineages (Figure 1). The AIV of 11 belonged to Y280,
five AIV belonged to G1, four AIV belonged to the Korean
lineage and one AIV belonged to the North American lineage;
all were detected by the H9 rRT-PCR assay (Table 2).
Phylogenetically, H9N2 viruses isolated from smuggled
meats illegally imported into Japan, including A/duck/Japan/
AQ-HE5/2015 (H9N2), A/chicken/Japan/AQ-HE14/2015
(H9N2), A/duck/Japan/AQ-HE28/2015 (H9N2), A/chicken/
Japan/AQ-HE61/2015 (H9N2), A/chicken/Japan/AQ-HE28-
28/2016 (H9N2), A/chicken/Japan/AQ-HE28-50/2016
(H9N2) and A/chicken/Japan/AQ-HE28-57/2016 (H9N2),
were classified into the Y280 lineage along with closely
related AIV recently isolated from poultry in China (Figure 1).
All RNA extracted from the AIV (except for A/duck/
Hokkaido/31/97 [H9N2], belonging to the Korean lineage,
and A/turkey/Wisconsin/1/66 [H9N2], belonging to the North
American lineage) and both G1- and one of the Y280-lineage
virus in vitro-transcribed H9 HA RNA could be detected at
concentrations corresponding to 5, 50 and 500 copies of the M
gene RNA per reaction and 5, 50 and 500 copies of H9 HA
gene per reaction using the newly established H9 rRT-PCR
assay (Table 2 and data not shown). For these AIV, the H9
rRT-PCR assay exhibited amplification plots similar to the
type A rRT-PCR assay (Figure S2a). A/duck/Hokkaido/31/97
(Korean lineage) and A/turkey/Wisconsin/1/66 (North
American lineage), which were isolated over 20 years ago,
could also be detected using the H9 rRT-PCR assay at RNA
concentrations corresponding to a minimum of 500 and 50
copies/reaction of M gene RNA in viral extracted RNA,
respectively (Table 2). However, both viruses could be
detected at 5, 50 and 500 copies of M gene RNA per reaction
using the type A rRT-PCR assay, suggesting that the H9 rRT-
PCR assay is less sensitive against these past prevalent viruses
(Figure S2b).
The cross-reactivity of the H9 rRT-PCR assay was
evaluated against other HA subtypes, H1 through H15 (except
for H9) influenza A viruses, influenza B viruses and 19 other
respiratory viruses (Tables 3 and 4). No cross-reactivity was
observed against RNA derived from these isolates, and no
non-specific reactions were observed (Tables 3 and 4).
To demonstrate the robustness of the H9 rRT-PCR
assay, we evaluated artificially reconstituted clinical
TABLE 4 Panel of non-influenza respiratory pathogens used in the H9 rRT-PCR assay
Respiratory pathogen Other PCR assay†H9 rRT-PCR assay
‡
Respiratory syncytial virus A 24.1 N.D.
Respiratory syncytial virus B 26.0 N.D.
Human parainfluenza virus type 1 (strain C35) 17.5 N.D.
Human parainfluenza virus type 2 (strain GREER) 18.5 N.D.
Human parainfluenza virus type 3 (strain Washington/1957 C243) 18.5 N.D.
Human parainfluenza virus type 4a (strain M-25) 22.1 N.D.
Human parainfluenza virus type 4b (strain CH19503) 20.0 N.D.
Human rhinovirus type A 30.9 N.D.
Human rhinovirus type B 28.6 N.D.
Human metapneumovirus type A1 26.1 N.D.
Human metapneumovirus type B2 25.7 N.D.
Human coronavirus OC43 26.8 N.D.
Human coronavirus 229E 25.9 N.D.
Human coronavirus NL63 27.0 N.D.
Human coronavirus HKU1 25.0 N.D.
Human bocavirus 24.2 N.D.
Human enterovirus 28.9 N.D.
Human adenovirus 2 27.0 N.D.
Human adenovirus 4 30.0 N.D.
†These results were obtained by multiplex real-time PCR assay as described in the main text. Threshold cycle values were determined using 7500 software, version 2.3.
‡
Crossing point values were determined using the second derivative maximum method in Light Cycler
®
480 SW1.5 software.
N.D., not detected; rRT-PCR, real-time RT-PCR.
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specimens with or without seasonal influenza viruses and
spiked or not spiked with the H9N2 virus. The H9 rRT-PCR
assays could detect the HA gene of the H9N2 virus from all
12 artificially reconstituted samples spiked with the H9N2
virus, and no non-specific reactions were observed in the 12
clinical samples not spiked with the H9N2 virus (Table 5).
The Cp values for all 12 artificially reconstituted samples in
the H9 rRT-PCR assay were almost the same as the Cp
value for PBS spiked with the same amount of the H9N2
virus.
FIGURE 1 Phylogenetic tree for the H9 HA genes. The tree was constructed using the neighbor-joining method with MEGA7 software.
Evolutionary distances were computed using the Kimura two-parameter method. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) and values of more than 50% are shown next to the branches. The viruses used in this
study are shown in bold. *Viruses for which in vitro-transcribed RNA was used for rRT-PCR
SAITO ET AL.
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4
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DISCUSSION
Among H9N2 viruses, those of the Y280 lineage are
spreading primarily in poultry in China, and this has resulted
in occasional cases of human infection. H9N2 viruses of the
G1 lineage are more prevalent in poultry in Asia and the
Middle East, with recent reports of sporadic transmission to
humans in China, Hong Kong, Bangladesh and Egypt. We
therefore developed a new H9 rRT-PCR assay that is highly
sensitive and specific for viruses of the Y280 and G1 lineages.
Our H9 rRT-PCR assay also exhibited good linearity
(R
2
= 0.99) and high sensitivity for detecting in vitro-
transcribed HA gene RNA from the Y280-lineage virus A/
Hunan/44558/2015 (H9N2) and the G1-lineage virus A/
chicken/Egypt/F12173D/2016 (H9N2) (Figure 2). The H9
HA gene RNA was detected for all isolates of both lineages
examined in this study at the equivalent of a minimum of 5
copies of the M gene per reaction (Table 2). Therefore, the
sensitivity of the H9 rRT-PCR assay is comparable to that of
our previously developed rRT-PCR assay for the universal
detection of M genes of all influenza A viruses [26]. The
sensitivity of the assay was at least 10- and 100-fold lower for
A/duck/Hokkaido/31/97 (Korean lineage) and A/turkey/
Wisconsin/1/66 (North American lineage), respectively,
compared with other Y280- or G1-lineage viruses. The A/
duck/Hokkaido/31/97 virus had one mismatch in the forward
primer region and four mismatches in the reverse primer
region, whereas the A/turkey/Wisconsin/1/66 virus had four
mismatches in the forward primer region, three mismatches in
the reverse primer region and one mismatch in the probe
region. As these viruses had many more mismatches in the
primer/probe regions compared with the other viruses
examined, the decrease in sensitivity was attributed to these
mismatches. However, the mismatches in the primer and
probe sequences are not conserved in recently described
viruses of the Y280 and G1 lineages (data not shown).
Moreover, there are no reports to date of human infections
caused by viruses of the Korean and North American lineages
derived from poultry. Reports of Y280- and G1-lineage
viruses with an HA-Q226L substitution are increas-
ing [7,8,34]. The adaptation of Y280 and G1 lineages to
humans may be dependent on HA-L226, which prefers α-2,6-
linked sialic acids [35]. These results suggest that the H9 rRT-
PCR assay described here is highly sensitive for current
epidemic strains of the Y280 and G1 lineages in poultry that
are transmissible to humans.
The viral load of clinical specimens from patients infected
with AIV is often very low compared with specimens from
seasonal influenza cases [36]. Therefore, a highly sensitive
detection system is needed to diagnose cases of human
infection with AIV. An evaluation of the H9 rRT-PCR assay
using artificially reconstituted clinical samples showed that
the assay could detect a small viral load (Cp > 30) of H9N2
when the specimen contained a viral load of seasonal
influenza A virus higher (Cp < 23.25) than the amount of
spiked H9N2 virus (Table 5). In all artificially reconstituted
clinical specimens containing influenza B virus and non-
influenza viruses, the Cp values for the H9 and type A rRT-
PCR assays were almost the same as that for diluted H9N2
virus in PBS. These results suggested that the sensitivity of
the H9 rRT-PCR assay is sufficient and that there are no non-
specific reactions or interference in analyses of clinical
specimens with and without seasonal influenza virus.
Even though H9N2 avian influenza viruses circulate
worldwide, only 42 cases of human infection were confirmed
between 1997 and May 2018. However, the results of
serologic studies in Asia and the Middle East suggest that the
actual number of humans infected with H9N2 AIV is much
greater than the number of confirmed cases [13–18]. For
example, the seroprevalence among avian-exposed humans in
Egypt is reportedly between 5.6% and 7.5% [16]. Indeed, H9
HA was shown to have a human influenza virus-like binding
FIGURE 2 Dynamic range of the H9 rRT-PCR assay. Standard curve (crossing point [Cp] value vs log
10
concentration) for serial dilutions
of in vitro-transcribed RNA of the HA gene of (a) A/Hunan/44558/2015 (H9N2), Y280 lineage and (b) A/chicken/Egypt/F12173D/2016 (H9N2),
G1 lineage. The standard curve was generated using the average Cp values obtained from six replicates. The correlation coefficient (R
2
) and
slope of the standard curve are shown in each graph
28
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SAITO ET AL.

property [37]. In addition, H9N2 viruses have been identified
as a major source of six internal genes in H5N1 [38],
H7N9 [39] and H10N8 [40] viruses. Therefore, given the
pandemic potential of H9N2 viruses, continuous monitoring
and surveillance of these viruses using the highly sensitive
rRT-PCR assay is needed.
At present, the risk of H9N2 exposure may be limited to
countries in which the viruses are endemic in poultry, such
as China, Egypt and Bangladesh. However, the virus can be
transferred to other countries through AIV-infected migra-
tory birds, illegal importation of raw poultry products from
birds infected with AIV [41], or travel by persons infected
with an AIV, such as the case of an H7N9 HP AIV human
infection in Taiwan in 2017 (http://www.who.int/csr/don/
22-february-2017-ah7n9-china/en/). These dangers high-
light the need for a highly sensitive and specific system for
detecting H9N2 viruses even in countries with a low risk of
infection.
In our previous study, we developed rRT-PCR assays to
detect types of influenza A and B viruses, determine
subtypes of H1pdm09, former H1 (Russian flu), H3, H5 and
H7 influenza A viruses, and discriminate between the
Victoria and Yamagata lineages of influenza B viruses.
These assays can be performed under the same conditions as
the H9 assay described in the present report (http://www.
who.int/influenza/gisrs_laboratory/molecular_diagnosis/en/
) [25,26,28]. Hence, by combining these methods, influenza
viruses can be easily and simultaneously identified with
respect to type and subtype or lineage with high sensitivity
for diagnostic and monitoring purposes.
TABLE 5 Detection of the H9 HA gene from artificially reconstituted clinical samples spiked with H9N2 virus
Sample
name
Type or
subtype
Virus-
spike
Type A rRT-PCR
assay†
H9 rRT-PCR
assay
†
Other type/subtype rRT-PCR
assays
†‡
F16-9 H1N1pdm09 + 21.05 31.56 20.59
−21.06 N.D. 20.58
F16-26 H1N1pdm09 + 20.62 31.62 20.74
−20.64 N.D. 20.78
F16-61 H1N1pdm09 + 23.88 31.21 23.25
−23.01 N.D. 23.06
F14-53 H3N2 + 19.13 31.68 18.54
−19.06 N.D. 18.48
F15-7 H3N2 + 18.30 31.84 17.84
−19.31 N.D. 17.99
F16-17 H3N2 + 16.73 31.72 16.31
−17.45 N.D. 16.48
F15-15 Type B + 32.61 32.42 23.12
−N.D. N.D. 22.77
F16-44 Type B + 32.31 32.02 21.52
−N.D. N.D. 21.16
F16-56 Type B + 33.32 31.35 18.75
−N.D. N.D. 18.90
F16-52 −+ 32.35 31.57 N.T.
−N.D. N.D. N.T.
F16-68 −+ 32.64 31.70 N.T.
−N.D. N.D. N.T.
F16-76 −+ 32.12 31.67 N.T.
−N.D. N.D. N.T.
PBS −+ 31.51 31.43 N.T.
−N.D. N.D. N.T.
†Crossing point values were determined using the second derivative maximum method in Light Cycler
®
480 SW1.5 software.
‡
Other assays were as follows: H1pdm rRT-PCR assay for F16-9, F16-26, and F16-61; H3 rRT-PCR assay for F14-53, F15-7, and F16-17; B/NS rRT-PCR assay for F15-
15, F16-44, and F16-56.
N.D., not detected; N.T., not tested; rRT-PCR, real-time RT-PCR.
SAITO ET AL.
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29

In summary, our newly developed rRT-PCR assay is
capable of detecting two major Eurasian H9 lineages of AIV
known to cause human infection. This assay can serve as a
useful tool for not only highly sensitive and specific
laboratory diagnostic testing for H9 infections in humans
but also surveillance and monitoring of the spread of H9 AIV,
including those that circulate among avian species and infect
humans.
ACKNOWLEDGMENTS
The authors thank the St Jude Children's Research Hospital,
USA, for providing the A/Hong Kong/308/2014 (H9N2) isolate.
The authors also thank the National Institute for Biological
Standards and Control, a Centre of the Medicines and Healthcare
Products Regulatory Agency, UK, for providing the A/chicken/
Hong Kong/G9/1997 (H9N2) and A/Hong Kong/1073/99
(H9N2) isolates; Animal Quarantine Service, Ministry of
Agriculture, Forestry and Fisheries, Japan, for providing the
A/duck/Japan/AQ-HE5/2015 (H9N2), A/chicken/Japan/AQ-
HE14/2015 (H9N2), A/duck/Japan/AQ-HE28/2015 (H9N2),
A/chicken/Japan/AQ-HE61/2015 (H9N2), A/chicken/Japan/
AQ-HE28-28/2016 (H9N2), A/chicken/Japan/AQ-HE28-50/
2016 (H9N2) and A/chicken/Japan/AQ-HE28-57/2016
(H9N2) isolates; the authors, originating and submitting
laboratories of the sequences from GISAID's EpiFlu™
Database, on which this research is based; and Drs Hideyuki
Kubo and Atsushi Kaida, Osaka City Institute of Public Health
and Environmental Sciences, for providing DNA/RNA of viral
respiratory pathogens. This research was supported by AMED
under grant number JP18fk0108030.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest
regarding this manuscript.
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in
the supporting information tab for this article.
How to cite this article: Saito S, Takayama I,
Nakauchi M, et al. Development and evaluation of a
new real-time RT-PCR assay for detecting the latest
H9N2 influenza viruses capable of causing human
infection. Microbiol Immunol. 2019;63:21–31.
https://doi.org/10.1111/1348-0421.12666
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