1 2 3 the h9 subtype of avian influenza viruses (h9aivs ... · 2 23 abstract: an...
TRANSCRIPT
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Title Page: 1
Development of an immunochromatographic strip for rapid detection of 2
the H9 subtype of avian influenza viruses (H9AIVs) 3
Fuhu Peng a
, Zheng Wang a
, Shuhui Zhang a, Renwei Wu
a, Sishun Hu
a, Zili Li
a, Xiliang 4
Wang a
, Dingren Bi a * 5
6
a Laboratory of Animal Microbiology and Immunology, State Key Laboratory of Agricultural 7
Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 8
People΄s Republic of China 9
10
Running title: 11
Detection of the H9 subtype of avian influenza viruses 12
13
14
15
*Corresponding author. 16
Name: DingRen Bi, 17
Address: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 18
430070, P.R. China 19
Tel: +86-27-87280208 Fax: +86-27-87280408 20
Email: bidingren@mail. hzau. edu. cn 21
22
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00273-07 CVI Accepts, published online ahead of print on 16 January 2008
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ABSTRACT: An immunochromatographic strip was developed for the detection of the H9 23
subtype of avian influenza viruses (H9AIVs) in poultry using two McAbs, 4C4 for H9AIV 24
hemagglutinin and 4D4 for nucleoprotein (NP), respectively. The 4C4 was labeled with colloidal 25
gold as a detection reagent, and the 4D4 was blotted on the test line while a goat anti-mouse 26
antibody was used on the control line of the nitrocellulose membrane. In comparison with the 27
hemagglutination (HA) and hemagglutination inhibition (HI) test, the strip was specific for 28
detecting H9AIV with the sensitivity at 0.25 HA units within 10 min. Storage of the strips at 29
room temperature for six months or at 4 °C for 12 months did not change their sensitivity and 30
specificity. Evaluation of the strip on experimental tracheal and cloacal swab samples collected 31
from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 32
post-inoculation, earlier than the appearance of clinical symptoms. Application of the strip for 33
analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry 34
farms showed that four farms were infected by H9AIV. Further characterization of 10 positive 35
and 30 negative samples randomly selected showed that no single sample was false positive or 36
negative, as determined by the standard virus isolation and HI assays. Therefore, the 37
immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity and 38
stability. This, together with the advantages of rapid detection and easy operation without 39
requirement for special skills and equipments, makes it suitable for the on-site detection and 40
differentiation of H9AIVs from other viruses in poultry. 41
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Key words: H9 subtype of avian influenza viruses; immunochromatographic strip; McAb 43
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INTRODUCTION 46
Influenza viruses (family Orthomyxoviridae) are classified into three types, A, B and C, 47
based on the antigenicity of viral nucleoproteins and matrix proteins (18). Influenza viruses are 48
further classified into different subtypes, according to the antigenicities of the hemagglutinin 49
(HA) and neuraminidase (NA) surface glycoproteins, Belonging to type A virus, avian influenza 50
virus (AIV) has 16 different HA and 9 NA subtypes (5, 31). AIV is categorized into two groups 51
based on its virulence. Infections of poultry with highly pathogenic AIV (HPAIV) usually result 52
in a very high mortality, up to 100%, while infection with low-pathogenicity AIV (LPAIV) can 53
be asymptomatic or often induce mild respiratory symptoms. Interestingly, infection with LPAIV 54
can rapidly lead to a reduction in egg production in poultry. Importantly, LPAIVs can serve as 55
progenitors to HPAIVs. Therefore, early detection and control of LPAIV infection will be 56
economically and healthfully significant. 57
Since the H9N2 subtype of AIV was discovered from turkeys in Wisconsin in 1966 (11), the 58
H9N2AIV-mediated outbreaks have been reported in many countries in the world (1, 7, 16, 20, 59
24, 25). In mainland China, H9N2 AIV was first isolated from chickens in the Guangdong 60
province in 1994 (4), and a huge outbreak occurred in 1998, which subsequently spread to 61
several provinces in China. During the outbreak and spreading, most chickens infected with 62
H9N2 AIV showed clinical symptoms such as mild respiratory signs, edema around the eyes, 63
diarrhea, and the laying of soft-shelled eggs, with a severe drop in egg production and a 5-15% 64
mortality rate, leading to severe economic losses in the poultry industries (19). In addition, 65
surveillance in the poultry markets in Hong Kong in 1997 revealed that the H9AIVs were 66
co-circulating with the HPAI H5N1 viruses, raising the concern of genetic recombination 67
between these viruses (37). Importantly, accumulated evidence has demonstrated that H9N2 AIV 68
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can infect mammals, including humans (8, 21). H9AIVs have also been considered to be one of 69
the candidates for the next potential pandemic. Hence, the rapid identification of the virus has 70
important clinical, economic, and epidemiological implications. 71
Various laboratory methods are currently available for the detection and surveillance of 72
H9AIVs, and include virus isolation, hemagglutination (HA) assay, hemagglutination inhibition 73
(HI) test (14) and RT-PCR (3). However, these assays are laborious and time consuming, 74
difficult to incorporate into automated procedure, and require laboratory operation, skilled 75
technicians, and special equipment/facilities. Therefore, development of a sensitive, specific, and 76
easily performed assay is crucial for the rapid detection and surveillance of H9AIV infection and 77
spreading. 78
Immunochromatographic assay is a new immunochromatographic technique in which a 79
cellulose membrane is used as the carrier, and a colloidal gold-labeled antigen or antibody is 80
used as the tracer. The method has been widely used for the diagnosis of many contagious 81
human diseases and the detection of bioactive molecules, hormones, haptens, and others (9, 30). 82
Recently, it has been efficiently applied to detect bovine viral diarrhea and white spot syndrome 83
viruses (15, 27). We have successfully developed immunochromatographic strips for detecting 84
antibodies against AIV (22) and sulfadiazine (28, 29). To extend these studies, we have recently 85
developed an immunochromatographic strip, which can specifically, sensitively, and rapidly 86
detect H9AIV. Here, we report the development and validation of this assay system and discuss 87
its implication in the surveillance of H9AIV. 88
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MATERIALS AND METHODS 90
Equipment 91
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The transmission electron microscopy (TEM) images were recorded with a Hitachi H600 92
transmission electron microscope (Hitachi Instrument Co., Tokyo, Japan). ZX1000 Dispensing 93
Platform and CM4000 Guillotine Cutter (BioDot, Irvine, USA) were used for the preparation of 94
test strips. 95
Chemicals and special reagents 96
Gold chloride (HAuCl4·3H2O), sodium citrate (C6H5Na3O7·2H2O), bovine serum 97
albumin (BSA), polyvinylpyrrolidone K30 and tween-20 were purchased from Sigma (St. Louis, 98
MO, USA). The recombinant nucleocapsid protein (rNP) was generated and purified in our 99
laboratory as previously described (12). In brief, the cDNA for the H9 AIV nucleoprotein (NP) 100
gene (1497bp) was obtained from H9N2 virus by reverse transcription polymerase chain reaction 101
(RT-PCR), and cloned into an expression vector pGEX-KG in E.coli to generate the recombinant 102
plasmid pKG-NP. Following transformation, the expression of fusion protein GST-NP was 103
induced using 1mmol/L isopropylthio-β-D-galactoside (IPTG) in E. coli. Subsequently, the 104
recombinant GST-NP was purified with the GSTrapTM HP column (Amersham Biosciences, 105
USA), according to the manufacture΄s instructions. The eluted NP proteins were analyzed by 106
SDS–PAGE. The goat anti-mouse antibody was obtained from Sino-American Biotechnology 107
Co. (Luoyang, China). Nitrocellulose membranes, glass fibers and absorbent paper were 108
purchased from Millipore Corporation (Bedford, MA, USA). 109
Viruses and antigens 110
Influenza A/chicken/China/HSS/1999 (H9N2) was isolated from an AIV-infected chicken in 111
the Hubei province in 1998 (19). Reference antigens of each subtype of H5, H7, or H9 AIV and 112
specific sera against each subtype of AIV were obtained from Harbin Veterinary Research 113
Institute (Harbin, China). Other reference viruses, including Newcastle disease (ND), infectious 114
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bronchitis (IB), infectious bursal disease (IBD), infectious laryngotracheitis (ILT), and fowl 115
adenovirus (FA), and reference sera specific for NDV were obtained from China Institute of 116
Veterinary Drug Control (Beijing, China). 117
Chickens, infection and sample preparations 118
Influenza A/chicken/China/HSS/1999 (H9N2) was propagated in the allantoic cavity of 119
10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (Spafas Poultry Co., Jinan, 120
China). Following incubation at 37 ˚C for 48 h, the allantoic fluids were harvested (26) and their 121
viral contents were determined using HA assay by mixing 25µl allantoic fluids with an equal 122
volume of 0.5% [v/v] chicken red blood cells in PBS in V-bottomed microtitre plates (10). 123
A total of 25 white leghorn bio-clean chickens at 6-weeks of age were housed in animal 124
experimental facilities of Huazhong Agricultural University, China, and experimentally infected 125
intra-nasally with 0.2 ml of H9N2-infected allantoic fluid (128 HA units of H9N2). A group of 126
White Leghorn bio-clean chickens inoculated with allantoic fluid from unmanipulated eggs was 127
used as the control. Their cloacal and tracheal swab samples were collected one day before 128
inoculation and every other day after inoculation, up to 11 days post-inoculation (p.i.). On day 3 129
post-inoculation, four infected and two control chickens were sacrificed, and their tracheas, lungs, 130
hearts, livers, spleens, kidneys, and muscles were collected. The collected cloacal and tracheal 131
samples were pre-treated by dipping the swabs into 1 ml distilled water in 1.5 ml centrifuge 132
tubes with gently stirring and extruding. Following settling for a couple of minutes, their 133
supernatants were collected for the strip tests. Individual organ sample, including trachea, lung, 134
heart, liver, spleen, kidney, and muscle collected from a chicken, was homogenized in 10 135
volumes of distilled water. After settling for 15 min, their supernatants were collected as samples 136
for the strip tests. 137
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Preparation of anti-AIV monoclonal antibodies (McAbs) 138
The McAbs against the NP or H9 subtype hemagglutinin of AIV were produced as 139
previously described (33, 36). Briefly, BALB/c mice at 6-8 weeks of age were immunized 140
subcutaneouly with 0.1ml of H9N2 virus (512 units of HA) emulsified in 50% complete Freund’s 141
adjuvant. The mice were boosted with the same amount of antigen in 50% incomplete Freund’s 142
adjuvant every 15 days for 4 times, followed by intraperitoneal injection with 0.2ml of H9N2 143
virus (1024 units of HA). Three days later, their splenic mononuclear cells were isolated and 144
fused with murine myeloma cells (SP2/0) using 50% PEG. The hybridomas were generated 145
through the selection of HAT medium. The supernatants of hybridoma cultures from each well 146
were screened using the rNP-based ELISA and H9 subtype hemagglutinin-specific HI assays. 147
The positive hybridoma cells were cloned by a limiting dilution and the stable hybridoma clones 148
were injected into BALB/c nude mice. 149
The IgG1 McAbs, 4C4 recognizing the H9 subtype hemagglutinin or 4D4 for NP, were 150
purified from mouse ascetic fluids using sequential precipitation with caprylic acid and 151
ammonium sulfate (23) and dialyzed against the phosphate buffer (pH 7.4) at 4°C. Their purities 152
were characterized by SDS-PAGE, and their specificities and affinities were demonstrated by 153
ELISA (13). 154
Preparation of colloidal gold and colloidal gold-McAb conjugate 155
Colloidal gold was prepared as previously reported (6) with minor modifications. Briefly, 156
200 ml of 0.01% (w/v) HAuCl4 in doubly distilled water in a 500-ml round-bottom flask with 157
rapid stirring was heated up to boiling, and then 3.6 ml of 1% trisodium citrate was added to the 158
solution. After boiling for an additional 15 min, the colloid gold solution was continually stirred 159
and gradually cooled down. Its pH was adjusted to 8.2 using 1% potassium carbonate (w/v) 160
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followed by storage at 4 °C in a dark-colored glass bottle. 161
McAb 4C4 (300µl, 1mg/ml) was mixed with 20 ml of colloidal gold solution. The mixture 162
was stirred vigorously for 30 min, and added with 2.5ml of 10% (w/v) BSA to block excess 163
reactivity of the gold colloid, followed by stirring the mixture for an additional 30 min. After 164
centrifugation at 6,000×g at 4°C for 45 min, the resulting conjugate pellet was re-suspended and 165
washed twice with 2-mM borax buffer (pH 9.0) containing 0.1% (w/v) polyethylene glycol 166
(MW:20000), followed by re-suspending them in 1ml of the same buffer. The size and shape of 167
unconjugated colloidal gold and that of colloidal gold conjugated to antibodies were 168
characterized using TEM measurements according to a standard procedure (32). 169
Preparation of the immunochromatographic strip 170
The immunochromatographic strip was comprised of four components: sample pad, 171
conjugate pad, nitrocellulose membrane, and absorbent pad as illustrated in Fig. 1. The sample 172
pads (cellulose fiber, Millipore, CAT: CFSP223000) and the conjugate pads (glass–fiber 173
membrane, Millipore, CAT: GFCP203000) were treated with 20mM phosphate buffer containing 174
2% BSA, 2.5% sucrose, 1% tween-20, 0.3% polyvinylpyrrolidone K30 and 0.02% sodium azide, 175
pH 7.4, and dried at 37 °C. The McAb 4D4 (1mg/ml) or the goat anti-mouse antibody (1mg/ml) 176
in PBS were dispensed at the test or control lines on the nitrocellulose membrane (Millipore, 177
CAT: SHF01200225) using the BioDot XYZ Platform at a rate of 0.9µl/cm and a speed of 4 178
cm/s and then dried at 37 °C. The McAb 4C4-colloidal gold conjugate was applied to the treated 179
conjugate pad at a rate of 10µl/cm (about 1.5µg/cm) and then lyophilized completely. The 180
absorption pad, nitrocellulose membrane, pre-treated conjugate pad, and sample pad were 181
assembled as a strip and attached to a plastic scale board with a 1-2 mm overlap sequentially. 182
The assembled plate was cut into pieces 3 mm wide using a CM 4000 Cutter (Bio-Dot). The 183
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generated strip products were packaged in a plastic bag with desiccant, and stored at 4°C or the 184
indicated condition. 185
Principle of immunochromatographic assay and procedure for test 186
During the assay process, the liquid sample is applied to the sample pad and it rapidly 187
diffuses into the conjugate pad. If the sample contains H9AIV antigen, the sample will react with 188
the colloidal gold-4C4 conjugate to form antigen-colloidal gold-4C4 complex. The complex will 189
move along on the nitrocellulose membrane chromatographically due to capillary action. 190
Eventually, the complex will react with immobilized anti-NP McAb 4D4 on the test line to form 191
a color band. The excess conjugate, or free conjugate if the sample does not contain H9AIV 192
antigen, will migrate along the membrane to the control line where it will interact with 193
immobilized goat anti-mouse antibody to form a color band. Therefore, a positive sample will 194
display two bands at the test and control lines while a negative sample will show only one band 195
at the control line within 10 min. The composition of the immunochromatographic strip test is 196
illustrated in Fig. 2. 197
Specificity, sensitivity and stability of the immunochromatographic strip 198
Allantoic fluid harvested from H9N2-infected eggs at 256 HA unit, allantoic fluids from 199
unmanipulated eggs, standard H5, H7 or H9 subtypes of AIV antigens (5 mg/ml) and other 200
viruses commonly infected in poultry, including NDV (256 HA units/ml), IBV (105.0 EID50/ml), 201
IBDV (105.0 ELD50/ml), ILTV (107.2 EID50/ml), and FAV (EDS76V, 1024 HA units/ml), were 202
simultaneously tested by the immunochromatographic strips. Allantoic fluids with different units 203
of HA were used to evaluate the sensitivity of the immunochromatographic strip. To determine 204
the stability, immunochromatographic strips were stored at room temperature (RT) or 4°C, and 205
used for testing positive (16 HA units) and negative samples every fifteen or thirty days. 206
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Diagnosis of H9AIV infection in field 207 The immunochromatographic strips were applied in diagnosing H9AIV infection. A total of 208
157 tracheal or cloacal swabs were collected from chickens with respiratory symptoms consistent 209
with AIV infection on five chicken farms in Hubei province. Potential H9AIV infection was 210
assessed in duplicate using the immunochromatographic strips. Ten positive and thirty negative 211
samples tested by the immunochromatographic strips were randomly selected and re-tested using 212
the standard virus isolation and HI assays (10, 34). Briefly, sample supernatants were pre-treated 213
with antibiotic-antimycotic solution. Approximate 200 µl supernatant of individual sample was 214
inoculated into 9-to-10-day-old SPF embryonated chicken eggs. On day 5 p.i., the allantoic 215
fluids were harvested and tested for the presence of H9AIV using the HA and HI test and the 216
H9N2-specific sera (Harbin Veterinary Research Institute, Harbin, China). 217
218
RESULTS 219
Specificity and sensitivity of the immunochromatographic strip 220
To determine the specificity of the immunochromatographic strip, allantoic fluids harvested 221
from H9N2 infected eggs at 64 units of HA, allantoic fluids from unmanipulated eggs, together 222
with standard antigens of H5, H7, or H9AIV and other viruses, were characterized using the 223
immunochromatographic strips simultaneously (Fig. 3A). Clearly, while all of the samples tested 224
showed one strong band on the control line, only allantoic fluid harvested from H9N2 infected 225
eggs and the standard H9 AIV antigen displayed an additional band on the test line on the 226
immunochromatographic strips. This suggests that the immunochromatographic strip can 227
specifically detect H9AIV, but not other tested AIVs, NDV, IBV, IBDV, ILTV, and FAV, 228
common infectious viruses in poultry. This high specificity of the immunochromatographic strip 229
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allows discrimination of H9AIV from other common virus-mediated infections in poultry. 230
The allantoic fluids containing H9N2 AIV at 64 units/ml of HA were serially diluted and 231
evaluated for the sensitivity of the immunochromatographic strip (Fig. 3B). When the allantoic 232
fluids were diluted from 1:2 to 1:256 (from 32 to 0.25 HA units), two clear bands at the test and 233
control lines were observed. Therefore, the sensitivity of the immunochromatographic strip 234
reached a level of 0.25 units of HA antigens. Notably, the densities of the bands for varying 235
concentrations of HA antigens on the test lines gradually declined from the strongest band at 32 236
units of HA to 0.25 units of HA, suggesting that the efficacy of detecting H9AIVs by this strip is 237
dose-dependent. Furthermore, similar results were observed by repeated testing of these samples 238
for 10 times (data not shown), indicating the high reproducibility of this experimental system. 239
Therefore, the immunochromatographic strip for the detection of H9AIVs is both highly specific 240
and sensitive and is reproducible. 241
Stability of the immunochromatographic strip 242
To determine the stability, the immunochromatographic strips were randomly sampled. They 243
were stored at RT or 4 °C up to 12 months, and their specificity and sensitivity for the detection 244
of H9AIVs were tested every 15-30 days (Table 1). The immunochromatographic strips stored at 245
4 °C for 12 months showed persistent sensitivity and could detect 0.25 HA units of H9AIVs, a 246
sensitivity as same as a strip produced freshly. In contrast, although the strips kept at RT for 6 247
months did not alter their sensitivity for detecting H9AIVs, continual storage of them at RT for 9 248
months reduced their sensitivity by 50% and extension of storage time to 12 months further 249
decreased the sensitivity of the strips. Importantly, the specificity of the 250
immunochromatographic strip for the detection of H9AIVs did not change, as evidenced by no 251
single negative sample becoming a false positive regardless of storage conditions tested. 252
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Apparently, the immunochromatographic strip can be stored at 4 °C for at least 12 months and at 253
RT for 6 months without loss in sensitivity and specificity for the detection of H9AIVs. 254
255
Evaluation of the immunochromatographic strip for testing samples from experimental 256
infected chickens 257
To evaluate whether the immunochromatographic strip could detect clinical samples, groups 258
of White Leghorn chickens were inoculated intra-nasally with allantoic fluids containing H9N2 259
viruses or not. Following infection, their tracheal and cloacal swab samples were longitudinally 260
collected and tested using the immunochromatographic strips in Table 2. Control chickens 261
inoculated with healthy allantoic fluid showed no single positive for the H9AIV infection 262
throughout the observation period. In contrast, 25 out of 25 tracheal and 23 out of 25 cloacal 263
swab samples from H9N2 infected chickens were identified as positive for H9AIVs by the strips 264
on day 3 p.i., when no clinical sign was observed in all experimental chickens. Analysis of their 265
sera revealed that no anti-H9AIV antibody was detected before infection and the titers of 266
anti-H9AIV antibodies reached about 1:16 in most infected chickens on day 5 p.i. as determined 267
by the HI assays (data not shown). Hence, the immunochromatographic strip can detect early 268
H9AIV infection in chickens. Notably, the positive rates of the detection of H9AIVs in this 269
experimental model gradually declined with time. On day 11 or 9 p.i., there was no detectable 270
H9AIVs in tracheal or cloacal swab samples, respectively, which may reflect specific 271
antibody-mediated viral clearance. 272
Given that all the chickens experimentally infected showed no clinical sign on day 3 p.i., 4 273
infected and 2 control chickens were randomly selected and sacrificed. Their trachea, lung, heart, 274
liver, spleen, kidney, and muscle samples were analyzed using the strips. All the tracheal 275
samples from the infected chickens were positive for H9AIV infection, however, the results from 276
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other organ samples were negative except that some organ samples from one infected chicken 277
were questionably positive. These results are consistent with the notion that the H9AIVs mainly 278
spread via a respiratory route. Our data suggest that the tracheal or cloacal swab samples are the 279
most appropriate for the immunochromatographic strip test. 280
Clinical application of the immunochromatographic strip 281
To further validate the application of the immunochromatographic strip at the clinic, a total 282
of 157 cloacal and tracheal swab samples were collected from chickens on 5 poultry farms where 283
the housed chickens were suspected to have viral infection. Characterization of these samples 284
revealed that 26 out of 157 cloacal and tracheal swab samples were positive for H9AIV infection 285
(Table 3). Notably, 10 positive and 30 negative samples were randomly selected from the pools 286
of strip-positive or negative samples and inoculated into SPF embryonated chicken eggs. Five 287
days later, their allantoic fluids were harvested and tested using the HA and HI assays. Allantoic 288
fluids of the 10 positive samples showed varying titers (4-64 units) of HA and were all positive 289
for H9AIV as determined by the HI assay (Table 4). Interestingly, allantoic fluids of the 30 290
negative samples tested by the strip tests were still negative for H9AIVs in the HI assay, 291
although 14 of these were positive by HA and allantoic fluids of 7 had evidence of NDV by HI 292
assay. Thus, the results from the immunochromatographic strip tests were consistent with the 293
data from the highly sensitive and specific virus isolation test. These findings suggest that the 294
immunochromatographic strip may be used for the detection and differentiation of H9AIV in 295
clinical diagnosis. 296
297
DISCUSSION 298
The epidemic of AI has resulted in a tremendous economic loss and has potentially affected 299
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human health. Since the 1990s, specific vaccination has been employed in many countries. 300
However, epidemiological studies have indicated that the LPAIV infections, especially caused 301
by the H9 subtype of LPAIVs, have become more ubiquitous and endemic in poultry in many 302
countries (2). Furthermore, infection with H9AIVs usually is asymptomatic or induces mild 303
respiratory symptoms, but causes a rapid drop in egg production in poultry, which is similar to 304
that of other OIE listed viruses, such as NDVs, IBVs or ILTV in vaccinated poultry. Therefore, 305
the development of an easy operating assay for the on-site detection of H9AIVs will be 306
significant in the surveillance of H9AIV infection and spreading. 307
We have successfully developed the immunochromatographic strip for the detection of 308
H9AIVs using the colloidal gold-conjugated 4C4, a McAb specific for the hemagglutinin of 309
H9AIV, as the detection antibody, and the 4D4, an anti-NP McAb, as the precipitation reagent 310
on the membrane. Analysis of the specificity showed that the strip was specific for the detection 311
of H9AIVs and H9AIV antigens and reacted with neither the allantoic fluid from unmanipulated 312
eggs, H5 or H7AIV antigens, nor other tested viruses, NDV, IBV, IBDV, ILTV and FAV. 313
Characterization of the sensitivity revealed that the strip could detect H9AIVs at 0.25 HA units 314
within 10 min, which was similar to that of the HI assay. The sensitivity and specificity of the 315
strips did not decrease after they were stored at RT for six months or at 4 °C for 12 months, a 316
demonstration of the high stability. Furthermore, evaluation of the strip on experimental tracheal 317
and cloacal swab samples collected from H9N2-infected or control chickens revealed that the 318
strip could be used for detecting H9N2 viruses on day 3 p.i., which was earlier than the 319
appearance of clinical symptoms following H9AIV infection. Conceivably, this strip can be used 320
for the early detection of H9AIV infection. In addition, application of the strip in the analysis of 321
157 tracheal or cloacal samples from potentially infected chickens on 5 poultry farms showed 322
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that 4 farms were infected by H9AIV. Further characterization of 10 positive and 30 negative 323
samples randomly selected using the standard virus isolation, HA, and HI assays showed that no 324
single sample was false positive or negative. Therefore, the immunochromatographic strip for 325
detection of H9AIVs has high specificity, sensitivity and stability. 326
Currently, there are several assays available for the detection of H9AIVs. The HA and HI 327
assays, the gold standard tests, are labor-intensive and time-consuming, requiring several 328
controls freshly prepared for standardization, which makes them unsuitable for the rapid and 329
on-site characterization of AIV infection. Similarly, RT-PCR can detect sub-type of virus with 330
high sensitivity (3), however, it usually requires special primers, laboratory operation, skilled 331
technicians and special equipment, which makes it difficult for the rapid and on-site detection of 332
viruses in the fields. In comparison with these assays, the strip we developed can be used for the 333
rapid detection of H9AIVs as the results can be read directly by the naked eye within 10 min. 334
This assay is easily operated and can be performed by farmers. Collectively, the 335
immunochromatographic strip we have developed has many advantages and can be potentially 336
used for the on-site detection of H9AIVs. 337
The specificity and sensitivity of the immunochromatographic strip are largely dependent on 338
the following factors. First, the quality of the McAbs used in the strip test is crucial for the 339
specificity and sensitivity of the strip. Both McAbs we used specifically recognize H9AIVs and 340
have a high affinity for their antigen epitopes. Second, pre-treatment of the sample pad and the 341
conjugate pad is important for enhancing the release speed of the conjugate and for reducing 342
non-specificity. Furthermore, careful choice of a membrane is critical for high specificity, 343
sensitivity and rapid detection as the wicking rate and speed of liquid diffusion on the membrane 344
are key characters for the suitability of membranes (35). Membranes with a larger pore-size give 345
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a lower wicking rate and higher diffusion rate, which benefits the rapid detection test. However, 346
this kind of membrane usually has a lower capacity for protein binding, potential leading to a 347
low sensitivity, as the pore sizes in membranes are inversely correlated to the protein binding 348
capacity of the membrane. We found that the 120 s/4cm nitrocellulose membrane was optimal in 349
our experimental system. 350
In summary, we have successfully developed a highly specific and sensitive 351
immunochromatographic strip for the detection of H9AIVs. Application of this strip at the clinic 352
demonstrated that this strip could be used for the rapid, on-site and early detection of H9AIVs. 353
The development of this strip provides a screening tool for the differential diagnosis of the 354
H9AIV infection from other subtypes of AIV and virus-mediated diseases in poultry. Further 355
extension of this first generation of immunochromatographic strip to a multiple 356
immunochromatographic assay for the detection of other AIVs and viruses may help 357
differentially diagnose common poultry diseases, such as Newcastle disease, avian infectious 358
bronchitis, avian infectious laryngotracheitis, and others. Thus, our findings provide a basis for 359
the design of new test strips for the surveillance of virus-mediated diseases in poultry. 360
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TABLE 1. Specificity and sensitivity of the immunochromatographic strip at various
storage conditions
Storage time Positive sample Negative sample
0 month
RT 1:64 - 4°C 1:64 -
3 month RT 1:64 - 4°C 1:64 -
6 month RT 1:64 - 4°C 1:64 -
9 month RT 1:32 - 4°C 1:64 -
12 month RT 1:8 - 4°C 1:64 -
A total of 20 positive samples and 10 negative samples were simultaneously analyzed and
repeated for ten times. Data are presented as mean titer of each sample group. (-) negative results
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TABLE 2. Evaluation of the immunochromatographic strip in detecting H9N2 in tracheal
and cloacal swab samples from experimentally infected chickens
Sample Group No. of positive samples over total samples
collected on different days post inoculation
Positive/
total
3 5 7 9 11
Tracheal Infected 25/25 14/21 5/21 2/21 0/21 46/109
Control 0/10 0/7 0/7 0/7 0/7 0/38
Cloacal Infected 23/25 10/21 2/21 0/21 0/21 35/109
Control 0/10 0/7 0/7 0/7 0/7 0/38
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TABLE 3. Diagnosis of H9AIV infection in field Farm
No. of tracheal or cloacal
swabs of chickens
No. of
positive
Positive
rate (%)
1 37 15 40.5
2 16 1 6.3
3 24 0 0
4 57 3 5.3
5 23 7 30.4
Total 157 26 16.6
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TABLE 4. Comparative analysis of samples randomly selected
No of sample
Strip test HA titers HI tests to detect specific viruses
H9AIV H5AIV H7AIV NDV
1 ﹢ 4 ﹢ ﹣ ﹣ ﹣
2 ﹢ 8 ﹢ ﹣ ﹣ ﹣
3 ﹢ 32 ﹢ ﹣ ﹣ ﹣
4 ﹢ 4 ﹢ ﹣ ﹣ ﹣
5 ﹢ 8 ﹢ ﹣ ﹣ ﹣
6 ﹢ 16 ﹢ ﹣ ﹣ ﹣
7 ﹢ 16 ﹢ ﹣ ﹣ ﹣
8 ﹢ 64 ﹢ ﹣ ﹣ ﹣
9 ﹢ 8 ﹢ ﹣ ﹣ ﹣
10 ﹢ 16 ﹢ ﹣ ﹣ ﹣
11-18 ﹣ 10 a ﹣ ﹣ ﹣ ﹣
18-24 ﹣ 22 a ﹣ ﹣ ﹣ ﹢
24-40 ﹣ 0 ﹣ ﹣ ﹣ ﹣
Allantoic fluids from 10 and 30 strip positive and negative samples, respectively, were
inoculated into embryonated eggs. Allantoic fluids harvested at day 5 p.i., were retested by the
strip test and HA. Samples 1-10 were from initially positive samples while samples 11-40 were
initially strip negative samples.
a the average HA titer
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Fig. 1. Schematic diagram of the immunochromatographic strip
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Fig. 2. Sample results of the immunochromatographic strip test ACCEPTED
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Fig. 3. Specificity and sensitivity of the immunochromatographic strip. Negative allantoic fluids
(AF) of healthy SPF embryonated chicken egg, Positive allantoic fluids from H9N2 AIV-infected
chicken eggs, reference standard antigens of H9 , H5 or H7 subtypes of AIV, and other
viruses including NDV, IBV, IBDV, ILTV and FAV were simultaneously characterized by the
strips (A). Similar patterns of results were observed from 20 times of repeated experiments. The
64 HA units of H9N2 AIV was serially diluted and tested with the strips for determining the
sensitivity of the strip (B).
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