JVI Accepted Manuscript Posted Online 24 February 2016 J. Virol. doi:10.1128/JVI.00013-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Manuscript
2 3
Neuraminidase Activity and The Resistance of 2009 Pandemic H1N1 Influenza Virus to
4
Antiviral Activity in Bronchoalveolar Fluid
5 6
Kanyarat Ruangrung1, Ornpreya Suptawiwat1, Kittipong Maneechotesuwan2,
7
Chompunuch Boonarkart1, Warunya Chakritbudsabong6, Jirawatna Assawabhumi2,
8
Parvapan Bhattarakosol5, Mongkol Uiprasertkul3, Pilaipan Puthavathana1, 8,
9
Witthawat Wiriyarat6, Anan Jongkaewwattana7, Prasert Auewarakul1, 4*
10 11
1
12
Thailand
13
2
14
Medicine Siriraj Hospital, Mahidol University, Thailand
15
3
16
4
17
5
18
6
19
7
20
Biotechnology, Thailand
21
8
22
Thailand
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Division of Respiratory Diseases and Tuberculosis, Department of Medicine, Faculty of
Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand Institute of Molecular Biosciences, Mahidol University, Thailand; Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Thailand; Faculty of Veterinary Science, Mahidol University, Thailand and Virology and Cell Technology Laboratory, National Center for Genetic Engineering and
Center of Research and Innovation, Faculty of Medical Technology, Mahidol University,
23 24 25
*Corresponding author: Prasert Auewarakul, MD, Dr med
26
Mailing address: Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol
27
University, Bangkok 10700, Thailand
28
Tel: +662 4197059
29
Fax: +662 4182663
30
E-mail: [email protected]
31 1
32
KEYWORDS: neuraminidase/ resistance / oseltamivir/ pandemic H1N1 2009/ influenza A
33
virus/ antiviral/ bronchoalveolar fluid
34 35
RUNNING TITLE: The Resistance of 2009 pdmH1N1 Influenza Virus to BALF
36 37
ABSTRACT
38
Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be
39
a frontline innate defense against the virus. Several anti-viral factors, including surfactant protein
40
D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza was previously
41
shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza
42
strains have different sensitivity to the overall anti-influenza activity of human bronchoalveolar
43
fluid was not known. We compared the sensitivity of 2009 pandemic H1N1, seasonal H1N1, and
44
seasonal H3N2 influenza strains to an inhibition by human bronchoalveolar lavage (BAL). The
45
pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL than the H3N2
46
strains. The BAL anti-influenza activity could be enhanced by Oseltamivir, indicating that the
47
viral neuraminidase (NA) activity could provide a resistance to the anti-viral defense. In
48
accordance with this finding, the BAL anti-influenza activity was found to be sensitive to
49
sialidase. The Oseltamivir-resistant mutation, H275Y, rendered the pandemic H1N1 virus but not
50
the seasonal H1N1 virus more sensitive to BAL. Since only the seasonal H1N1 but not the
51
pandemic H1N1 had compensatory mutations that allowed Oseltamivir-resistant strains to
52
maintain NA enzymatic activity and transmission fitness, the resistance to BAL of the drug
53
resistant seasonal H1N1 virus might play a role in the viral fitness.
54 55
2
56
IMPORTANCE
57
Human airway secretion contains anti-influenza activity. Different influenza strains may
58
vary in their susceptibility to this antiviral activity. Here we show that the 2009 pandemic and
59
seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage than
60
H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the
61
pandemic virus was determined by its NA gene and that the NA inhibitor resistant mutation,
62
H275Y, abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which
63
had compensatory mutations that maintained the fitness of drug resistant strains. Therefore, the
64
innate respiratory tract defense may be a barrier against NA inhibitor resistant mutants and
65
evasion of this defense may play a role in emergence and spreading of drug resistant strains.
66
3
67
INTRODUCTION
68
Despite being regarded as a mild pandemic, the H1N1 influenza virus can cause
69
unusually severe diseases, which can rapidly progress to respiratory failure and death (1).
70
Comparative studies in the same outbreak season showed that patients infected with the
71
pandemic H1N1 virus are more likely to develop severe disease and consequently die than those
72
infected with seasonal influenza virus (2). In addition, animal experiments in mice, ferrets and
73
macaques have shown that the H1N1 2009 pandemic influenza virus is more virulent and
74
replicates more efficiently in lungs than seasonal influenza virus (3, 4). These suggest that the
75
pandemic virus may invade lungs more effectively.
76
Innate defense plays important roles in susceptibility to viral infection and pathogenesis.
77
Soluble anti-viral factors are important components of innate defense in the respiratory tract.
78
Several soluble factors with anti-influenza activity have been identified in BAL, including
79
surfactant protein (SP)-D, SP-A, scavenger receptor gp340, long pentraxin PTX3, L-ficolin, H-
80
ficolin, serum amyloid P (5-12). These factors are present in serum and respiratory secretions.
81
Serum influenza inhibitors can be classified as α-, β-, and γ-inhibitors according to their physical
82
properties and mechanisms of inhibition (13-15). Because α- and γ-inhibitors are sialylated
83
glycoproteins or receptor analogues that compete with sialic acid in the binding of influenza HA,
84
they are heat-resistant and Ca++-independent, but can be destroyed by sialidase or receptor
85
destroying enzyme (RDE). In contrast, β-inhibitors, such as SP-D, are heat labile, require Ca++
86
and can be inhibited by some monosaccharides, but are resistant to RDE. They are lectins that
87
bind influenza HA via carbohydrate moiety on glycosylation sites of HA, and inhibit HA
88
function probably through steric hindrance. In addition, exosome from human bronchial
89
epithelial cells has been shown to exhibit anti-influenza activity (16).
90
Among these known anti-influenza factors, SP-D is believed to be the main contributor to
91
the BAL anti-influenza activity (6). It was shown that the 2009 H1N1 pandemic virus is resistant
92
to SP-D because it had less N-linked glycosylation site on the globular head of hemagglutinin
93
(HA) (17, 18). However, whether susceptibility to other innate anti-viral factors, especially α-
94
and γ-inhibitors, contributes to the virulence of the 2009 pandemic influenza is not known. The
95
antiviral activity of α- and γ-inhibitors can be detected without interference from β-inhibitor by
4
96
performing the assay in a condition without calcium ion. We therefore assessed the sensitivity of
97
2009 pandemic influenza strains in comparison with seasonal influenza strains, to human BAL.
98 99
MATERIALS AND METHODS
100
Ethics statement. The human study was approved by the Ethics Committee of Faculty of
101
Medicine Siriraj Hospital (Siriraj Institutional Review Board), which is in full compliance with
102
the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines and the International
103
Conference on Harmonization in Good Clinical Practice (ICH-GCP). The study was performed
104
under protocol COA No.SI572/2009 ‘‘Sensitivity of Influenza 2009 H1N1 to antiviral factors in
105
bronchoalveolar lavage’’. All human subjects provided written informed consent.
106 107
BAL samples. BAL samples were collected from 52 patients aged 30-96 years (mean + SD = 64
108
+ 12) with a male-to-female ratio of 1:1.17, who underwent routine bronchoscopy and
109
bronchoalveolar lavage for investigation of suspected lung cancer. None of the patients were
110
being treated with any anti-viral treatment. Bronchoalveolar lavage with 0.9% normal saline was
111
performed at a non-lesional lung segment. The retrieved fluid was collected and immediately
112
carried on ice to the laboratory. The BAL samples were centrifuged at 1,500g, 4°C for 5 minutes.
113
The supernatant was stored frozen in aliquots at -80ºC.
114 115
Viral strains. The 2009 pandemic H1N1 (pdmH1N1) influenza virus strains were isolated from
116
patients with influenza-liked illness in Thailand during 2009 – 2011 (A/Nonthaburi/102/2009,
117
A/Thailand/104/2009,
118
Seasonal influenza strains used in this study are A/Thailand/Siriraj-03/2006 (sH1N1) (A/New
119
Caledonia/20/99-like virus), A/Thailand/Siriraj-12/2006 (sH1N1) (A/New Caledonia/20/99-like
120
virus), A/Thailand/SirirajICRC_SEA-3479/2008 (sH1N1) (A/Brisbane/59/2007-like virus),
121
A/Thailand/Siriraj-03/2004 (H3N2) (A/Fujian/411/02-like virus) and A/Thailand/Siriraj-04/2003
122
(H3N2). All the viruses were isolated and propagated for less than 10 passages in Madin-Darby
123
Canine Kidney (MDCK) cells with 1 mML-1-tosylamido-2-phenylethyl chloromethyl ketone
124
(TPCK)-treated trypsin (SIGMA-ALDRICH®) in Minimum Essential Medium (MEM)
125
(GIBCO®).
A/Thailand/MVCU-013/2009
5
and
A/Thailand/ICRC_NSN_1/2011).
126 127
Antiviral activity. The antiviral activity in BAL samples was assessed using hemagglutination
128
inhibition (HI) and neutralization (NT) assays as previously described for serum antibody assay
129
(19), except that the samples were not treated by sialidase prior to the assay. Briefly, for HI
130
assay, BAL was pre-absorbed with 0.5% goose red blood cell (GRBC) suspension to eliminate
131
non-specific agglutinins, serially diluted in phosphate buffered saline (PBS) with and without 5
132
mM CaCl2, mixed with 4 hemagglutination unit of virus and 0.5% GRBC in a 96-well U-shaped
133
microtiter plate, incubated at 4ºC for 30 minutes. HI titers were read as reciprocal of the highest
134
dilution with complete hemagglutination. For NT assay, BAL was serially diluted in MEM plus
135
1 mM TPCK-treated trypsin (with 1.8 mM CaCl2) and mixed with 25 TCID50 (50% tissue
136
culture infectious dose) of virus, incubated at 37°C for an hour and inoculated onto MDCK cells
137
in a 96-well tissue culture plate. Briefly, 3x104 cells in 200 μl MEM plus 10% heat-inactivated
138
fetal bovine serum (FBS) (GIBCO®) were seeded to each well and incubated at 37°C overnight).
139
After an overnight incubation at 37°C, the tissue culture plate was washed and fixed with 80%
140
cold acetone in 1X PBS, the level of viral infection was measured using an antiviral
141
nucleoprotein monoclonal antibody and a peroxidase-conjugated secondary antibody. Optical
142
densities were measured and NT titers with 50% inhibition of specific signals were calculated.
143
BAL sample was treated with Receptor Destroying Enzyme (RDE II DENKA SEIKEN) (1:2)
144
37°C overnight. RDE was inactivated at 56ºC for 30 minutes before the HI testing. Protease
145
treatment was done by adding 30U of Proteinase K (SIGMA-ALDRICH) to 1 ml of BAL
146
sample, incubated at 37°C overnight and inactivated at 92ºC for 10 minutes.
147 148
Reverse genetics viruses. The HA and NA genes of selected pdmH1N1 strains were amplified
149
from RNA extracted from infected MDCK cells, cloned into the reverse genetic plasmid
150
pHw2000 and sequenced. Specific mutations were introduced into the NA gene by PCR site-
151
directed mutagenesis with Dpn I digestion. Reverse genetic viruses were produced in HEK293T
152
cells by co-transfecting pHw2000 plasmids carrying the cloned HA and NA genes of the 2009
153
pandemic virus and the other 6 genes from the A/Puerto Rico/8/1934 (H1N1)(PR8) strain as
154
previously described (20).
155
6
156
IgA and IgG depletion. IgA and IgG in BAL sample were precipitated using a lectin (Jacalin)
157
from Artocarpus integrifolia cross-linked with 4% beaded agarose (SIGMA-ALDRICH) (21)
158
and Pierce™ protein G agarose (Thermo scientific), respectively. Two hundred and fifty
159
microlitre of BAL sample was added with 100 μl of settle agarose bead and mixed thoroughly.
160
The mixture was rotated and incubated at 4°C overnight. The agarose beads were precipitated
161
from sample by centrifugation at 1,000 rpm for 30 seconds. The supernatant was collected. To
162
precipitate total protein in sample, 10 μl of supernatant was added with 250 μl of absolute
163
ethanol, mixed thoroughly and centrifuged at 13,000 rpm 4°C for an hour. The supernatant was
164
discarded and dried. Twenty microliter of reducing loading dye was added, mixed thoroughly
165
and incubated at 70ºC for 10 minutes. The sample was run in sodium dodecyl sulfate-
166
polyacrylamide gel electrophoresis (SDS-PAGE) and performed Western blotting. IgA and IgG
167
were detected using 1: 2000 of Goat anti-human IgA HRP-labeled antibody (SIGMA-
168
ALDRICH) and 1: 2000 of Goat anti-human IgG HRP-labeled antibody (Invitrogen) in 2%
169
bovine serum albumin (SIGMA-ALDRICH), respectively. Both IgA and IgG detected antibody
170
were developed with DAB (3, 3'-diaminobenzidine) HRP substrate (SIGMA-ALDRICH). One
171
hundred microliters of IgA and IgG depleted sample were further measured for HI titer.
172 173
Animal experiment. Ferrets aged 6-18 months were screened for influenza HI antibody with a
174
2009 pandemic H1N1 strain. Three animals with no HI titer were randomly assigned into each
175
experimental group. The animals were intra-tracheally inoculated with either MVCU-013/2009
176
or 104/2009 at the dosage of 106 TCID50 in 1.2 ml volume. The animals were kept separately in
177
animal isolators in a BSL-3 laboratory. The animals were observed clinically every day, and at
178
day 10 post-infection, they were euthanized and lung tissue was collected and processed for
179
pathological study and RNA extraction for viral load measurement. The tissue was cut in small
180
pieces in TriZol solution, and RNA was extracted using the manufacture’s protocol. Viral RNA
181
was measured by a real time RT-PCR using a primer pair for Influenza A M gene and TaqMan®
182
probes (5’6-FAM and 3’BHQ1) (CDC protocol of real time RT-PCR for influenza A (H1N1)
183
(22, 23).
184 185
Statistical analysis. HI/NT titers and NT %Inhibition were shown in geometric mean with 95%
186
Confidence Interval (CI) and arithmetic mean+SEM, respectively. The comparisons of HI and 7
187
NT titers and NT %Inhibition were tested by paired/un-paired t-test (GraphPad Prism version 5.1
188
Software).
189 190
RESULTS
191
H1N1 influenza viruses were less sensitive to human BAL
192
BAL samples from 24 subjects were tested for their NT activity against two H1N1
193
pandemic
(A/Nonthaburi/102/2009
and
A/Thailand/104/2009),
two
seasonal
H3N2
194
(A/Thailand/Siriraj03/2004 and A/Thailand/Siriraj04/2003), and two seasonal H1N1 strains
195
(A/Thailand/Siriraj03/2006 and A/Thailand/Siriraj12/2006). Together as a group, pandemic and
196
seasonal H1N1 viruses had lower BAL NT titers as compared to the H3N2 virus (t-test, p
1
Manuscript
2 3
Neuraminidase Activity and The Resistance of 2009 Pandemic H1N1 Influenza Virus to
4
Antiviral Activity in Bronchoalveolar Fluid
5 6
Kanyarat Ruangrung1, Ornpreya Suptawiwat1, Kittipong Maneechotesuwan2,
7
Chompunuch Boonarkart1, Warunya Chakritbudsabong6, Jirawatna Assawabhumi2,
8
Parvapan Bhattarakosol5, Mongkol Uiprasertkul3, Pilaipan Puthavathana1, 8,
9
Witthawat Wiriyarat6, Anan Jongkaewwattana7, Prasert Auewarakul1, 4*
10 11
1
12
Thailand
13
2
14
Medicine Siriraj Hospital, Mahidol University, Thailand
15
3
16
4
17
5
18
6
19
7
20
Biotechnology, Thailand
21
8
22
Thailand
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Division of Respiratory Diseases and Tuberculosis, Department of Medicine, Faculty of
Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand Institute of Molecular Biosciences, Mahidol University, Thailand; Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Thailand; Faculty of Veterinary Science, Mahidol University, Thailand and Virology and Cell Technology Laboratory, National Center for Genetic Engineering and
Center of Research and Innovation, Faculty of Medical Technology, Mahidol University,
23 24 25
*Corresponding author: Prasert Auewarakul, MD, Dr med
26
Mailing address: Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol
27
University, Bangkok 10700, Thailand
28
Tel: +662 4197059
29
Fax: +662 4182663
30
E-mail: [email protected]
31 1
32
KEYWORDS: neuraminidase/ resistance / oseltamivir/ pandemic H1N1 2009/ influenza A
33
virus/ antiviral/ bronchoalveolar fluid
34 35
RUNNING TITLE: The Resistance of 2009 pdmH1N1 Influenza Virus to BALF
36 37
ABSTRACT
38
Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be
39
a frontline innate defense against the virus. Several anti-viral factors, including surfactant protein
40
D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza was previously
41
shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza
42
strains have different sensitivity to the overall anti-influenza activity of human bronchoalveolar
43
fluid was not known. We compared the sensitivity of 2009 pandemic H1N1, seasonal H1N1, and
44
seasonal H3N2 influenza strains to an inhibition by human bronchoalveolar lavage (BAL). The
45
pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL than the H3N2
46
strains. The BAL anti-influenza activity could be enhanced by Oseltamivir, indicating that the
47
viral neuraminidase (NA) activity could provide a resistance to the anti-viral defense. In
48
accordance with this finding, the BAL anti-influenza activity was found to be sensitive to
49
sialidase. The Oseltamivir-resistant mutation, H275Y, rendered the pandemic H1N1 virus but not
50
the seasonal H1N1 virus more sensitive to BAL. Since only the seasonal H1N1 but not the
51
pandemic H1N1 had compensatory mutations that allowed Oseltamivir-resistant strains to
52
maintain NA enzymatic activity and transmission fitness, the resistance to BAL of the drug
53
resistant seasonal H1N1 virus might play a role in the viral fitness.
54 55
2
56
IMPORTANCE
57
Human airway secretion contains anti-influenza activity. Different influenza strains may
58
vary in their susceptibility to this antiviral activity. Here we show that the 2009 pandemic and
59
seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage than
60
H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the
61
pandemic virus was determined by its NA gene and that the NA inhibitor resistant mutation,
62
H275Y, abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which
63
had compensatory mutations that maintained the fitness of drug resistant strains. Therefore, the
64
innate respiratory tract defense may be a barrier against NA inhibitor resistant mutants and
65
evasion of this defense may play a role in emergence and spreading of drug resistant strains.
66
3
67
INTRODUCTION
68
Despite being regarded as a mild pandemic, the H1N1 influenza virus can cause
69
unusually severe diseases, which can rapidly progress to respiratory failure and death (1).
70
Comparative studies in the same outbreak season showed that patients infected with the
71
pandemic H1N1 virus are more likely to develop severe disease and consequently die than those
72
infected with seasonal influenza virus (2). In addition, animal experiments in mice, ferrets and
73
macaques have shown that the H1N1 2009 pandemic influenza virus is more virulent and
74
replicates more efficiently in lungs than seasonal influenza virus (3, 4). These suggest that the
75
pandemic virus may invade lungs more effectively.
76
Innate defense plays important roles in susceptibility to viral infection and pathogenesis.
77
Soluble anti-viral factors are important components of innate defense in the respiratory tract.
78
Several soluble factors with anti-influenza activity have been identified in BAL, including
79
surfactant protein (SP)-D, SP-A, scavenger receptor gp340, long pentraxin PTX3, L-ficolin, H-
80
ficolin, serum amyloid P (5-12). These factors are present in serum and respiratory secretions.
81
Serum influenza inhibitors can be classified as α-, β-, and γ-inhibitors according to their physical
82
properties and mechanisms of inhibition (13-15). Because α- and γ-inhibitors are sialylated
83
glycoproteins or receptor analogues that compete with sialic acid in the binding of influenza HA,
84
they are heat-resistant and Ca++-independent, but can be destroyed by sialidase or receptor
85
destroying enzyme (RDE). In contrast, β-inhibitors, such as SP-D, are heat labile, require Ca++
86
and can be inhibited by some monosaccharides, but are resistant to RDE. They are lectins that
87
bind influenza HA via carbohydrate moiety on glycosylation sites of HA, and inhibit HA
88
function probably through steric hindrance. In addition, exosome from human bronchial
89
epithelial cells has been shown to exhibit anti-influenza activity (16).
90
Among these known anti-influenza factors, SP-D is believed to be the main contributor to
91
the BAL anti-influenza activity (6). It was shown that the 2009 H1N1 pandemic virus is resistant
92
to SP-D because it had less N-linked glycosylation site on the globular head of hemagglutinin
93
(HA) (17, 18). However, whether susceptibility to other innate anti-viral factors, especially α-
94
and γ-inhibitors, contributes to the virulence of the 2009 pandemic influenza is not known. The
95
antiviral activity of α- and γ-inhibitors can be detected without interference from β-inhibitor by
4
96
performing the assay in a condition without calcium ion. We therefore assessed the sensitivity of
97
2009 pandemic influenza strains in comparison with seasonal influenza strains, to human BAL.
98 99
MATERIALS AND METHODS
100
Ethics statement. The human study was approved by the Ethics Committee of Faculty of
101
Medicine Siriraj Hospital (Siriraj Institutional Review Board), which is in full compliance with
102
the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines and the International
103
Conference on Harmonization in Good Clinical Practice (ICH-GCP). The study was performed
104
under protocol COA No.SI572/2009 ‘‘Sensitivity of Influenza 2009 H1N1 to antiviral factors in
105
bronchoalveolar lavage’’. All human subjects provided written informed consent.
106 107
BAL samples. BAL samples were collected from 52 patients aged 30-96 years (mean + SD = 64
108
+ 12) with a male-to-female ratio of 1:1.17, who underwent routine bronchoscopy and
109
bronchoalveolar lavage for investigation of suspected lung cancer. None of the patients were
110
being treated with any anti-viral treatment. Bronchoalveolar lavage with 0.9% normal saline was
111
performed at a non-lesional lung segment. The retrieved fluid was collected and immediately
112
carried on ice to the laboratory. The BAL samples were centrifuged at 1,500g, 4°C for 5 minutes.
113
The supernatant was stored frozen in aliquots at -80ºC.
114 115
Viral strains. The 2009 pandemic H1N1 (pdmH1N1) influenza virus strains were isolated from
116
patients with influenza-liked illness in Thailand during 2009 – 2011 (A/Nonthaburi/102/2009,
117
A/Thailand/104/2009,
118
Seasonal influenza strains used in this study are A/Thailand/Siriraj-03/2006 (sH1N1) (A/New
119
Caledonia/20/99-like virus), A/Thailand/Siriraj-12/2006 (sH1N1) (A/New Caledonia/20/99-like
120
virus), A/Thailand/SirirajICRC_SEA-3479/2008 (sH1N1) (A/Brisbane/59/2007-like virus),
121
A/Thailand/Siriraj-03/2004 (H3N2) (A/Fujian/411/02-like virus) and A/Thailand/Siriraj-04/2003
122
(H3N2). All the viruses were isolated and propagated for less than 10 passages in Madin-Darby
123
Canine Kidney (MDCK) cells with 1 mML-1-tosylamido-2-phenylethyl chloromethyl ketone
124
(TPCK)-treated trypsin (SIGMA-ALDRICH®) in Minimum Essential Medium (MEM)
125
(GIBCO®).
A/Thailand/MVCU-013/2009
5
and
A/Thailand/ICRC_NSN_1/2011).
126 127
Antiviral activity. The antiviral activity in BAL samples was assessed using hemagglutination
128
inhibition (HI) and neutralization (NT) assays as previously described for serum antibody assay
129
(19), except that the samples were not treated by sialidase prior to the assay. Briefly, for HI
130
assay, BAL was pre-absorbed with 0.5% goose red blood cell (GRBC) suspension to eliminate
131
non-specific agglutinins, serially diluted in phosphate buffered saline (PBS) with and without 5
132
mM CaCl2, mixed with 4 hemagglutination unit of virus and 0.5% GRBC in a 96-well U-shaped
133
microtiter plate, incubated at 4ºC for 30 minutes. HI titers were read as reciprocal of the highest
134
dilution with complete hemagglutination. For NT assay, BAL was serially diluted in MEM plus
135
1 mM TPCK-treated trypsin (with 1.8 mM CaCl2) and mixed with 25 TCID50 (50% tissue
136
culture infectious dose) of virus, incubated at 37°C for an hour and inoculated onto MDCK cells
137
in a 96-well tissue culture plate. Briefly, 3x104 cells in 200 μl MEM plus 10% heat-inactivated
138
fetal bovine serum (FBS) (GIBCO®) were seeded to each well and incubated at 37°C overnight).
139
After an overnight incubation at 37°C, the tissue culture plate was washed and fixed with 80%
140
cold acetone in 1X PBS, the level of viral infection was measured using an antiviral
141
nucleoprotein monoclonal antibody and a peroxidase-conjugated secondary antibody. Optical
142
densities were measured and NT titers with 50% inhibition of specific signals were calculated.
143
BAL sample was treated with Receptor Destroying Enzyme (RDE II DENKA SEIKEN) (1:2)
144
37°C overnight. RDE was inactivated at 56ºC for 30 minutes before the HI testing. Protease
145
treatment was done by adding 30U of Proteinase K (SIGMA-ALDRICH) to 1 ml of BAL
146
sample, incubated at 37°C overnight and inactivated at 92ºC for 10 minutes.
147 148
Reverse genetics viruses. The HA and NA genes of selected pdmH1N1 strains were amplified
149
from RNA extracted from infected MDCK cells, cloned into the reverse genetic plasmid
150
pHw2000 and sequenced. Specific mutations were introduced into the NA gene by PCR site-
151
directed mutagenesis with Dpn I digestion. Reverse genetic viruses were produced in HEK293T
152
cells by co-transfecting pHw2000 plasmids carrying the cloned HA and NA genes of the 2009
153
pandemic virus and the other 6 genes from the A/Puerto Rico/8/1934 (H1N1)(PR8) strain as
154
previously described (20).
155
6
156
IgA and IgG depletion. IgA and IgG in BAL sample were precipitated using a lectin (Jacalin)
157
from Artocarpus integrifolia cross-linked with 4% beaded agarose (SIGMA-ALDRICH) (21)
158
and Pierce™ protein G agarose (Thermo scientific), respectively. Two hundred and fifty
159
microlitre of BAL sample was added with 100 μl of settle agarose bead and mixed thoroughly.
160
The mixture was rotated and incubated at 4°C overnight. The agarose beads were precipitated
161
from sample by centrifugation at 1,000 rpm for 30 seconds. The supernatant was collected. To
162
precipitate total protein in sample, 10 μl of supernatant was added with 250 μl of absolute
163
ethanol, mixed thoroughly and centrifuged at 13,000 rpm 4°C for an hour. The supernatant was
164
discarded and dried. Twenty microliter of reducing loading dye was added, mixed thoroughly
165
and incubated at 70ºC for 10 minutes. The sample was run in sodium dodecyl sulfate-
166
polyacrylamide gel electrophoresis (SDS-PAGE) and performed Western blotting. IgA and IgG
167
were detected using 1: 2000 of Goat anti-human IgA HRP-labeled antibody (SIGMA-
168
ALDRICH) and 1: 2000 of Goat anti-human IgG HRP-labeled antibody (Invitrogen) in 2%
169
bovine serum albumin (SIGMA-ALDRICH), respectively. Both IgA and IgG detected antibody
170
were developed with DAB (3, 3'-diaminobenzidine) HRP substrate (SIGMA-ALDRICH). One
171
hundred microliters of IgA and IgG depleted sample were further measured for HI titer.
172 173
Animal experiment. Ferrets aged 6-18 months were screened for influenza HI antibody with a
174
2009 pandemic H1N1 strain. Three animals with no HI titer were randomly assigned into each
175
experimental group. The animals were intra-tracheally inoculated with either MVCU-013/2009
176
or 104/2009 at the dosage of 106 TCID50 in 1.2 ml volume. The animals were kept separately in
177
animal isolators in a BSL-3 laboratory. The animals were observed clinically every day, and at
178
day 10 post-infection, they were euthanized and lung tissue was collected and processed for
179
pathological study and RNA extraction for viral load measurement. The tissue was cut in small
180
pieces in TriZol solution, and RNA was extracted using the manufacture’s protocol. Viral RNA
181
was measured by a real time RT-PCR using a primer pair for Influenza A M gene and TaqMan®
182
probes (5’6-FAM and 3’BHQ1) (CDC protocol of real time RT-PCR for influenza A (H1N1)
183
(22, 23).
184 185
Statistical analysis. HI/NT titers and NT %Inhibition were shown in geometric mean with 95%
186
Confidence Interval (CI) and arithmetic mean+SEM, respectively. The comparisons of HI and 7
187
NT titers and NT %Inhibition were tested by paired/un-paired t-test (GraphPad Prism version 5.1
188
Software).
189 190
RESULTS
191
H1N1 influenza viruses were less sensitive to human BAL
192
BAL samples from 24 subjects were tested for their NT activity against two H1N1
193
pandemic
(A/Nonthaburi/102/2009
and
A/Thailand/104/2009),
two
seasonal
H3N2
194
(A/Thailand/Siriraj03/2004 and A/Thailand/Siriraj04/2003), and two seasonal H1N1 strains
195
(A/Thailand/Siriraj03/2006 and A/Thailand/Siriraj12/2006). Together as a group, pandemic and
196
seasonal H1N1 viruses had lower BAL NT titers as compared to the H3N2 virus (t-test, p
