China - H7N9 Human Isolates on Deposit at GISAID

[sharon sanders]

hat tip Mixin


A/Anhui/1/2013
EPI_ISL_138739
Submitted by WHO Chinese National Influenza Center

A/Shanghai/2/2013
EPI_ISL_138738
Submitted by WHO Chinese National Influenza Center

A/Shanghai/1/2013
EPI_ISL_138737
Submitted by WHO Chinese National Influenza Center


We would like to remind our posters that GISAID does not allow any public posting of any information about data from their database.

http://platform.gisaid.org/epi3/frontend#5f9a08

FluTrackers News and Data thread on this subject:

China - Three confirmed cases of human infection with H7N9 avian influenza - Two fatalities

[mixin]

Re: China - H7N9 Human Isolates on Deposit at GISAID

First I need to say that I have no experience with reading H7N9 sequences and a disclaimer that I am not a sequencer, virologist, etc..

When I aligned the HA amino acids, Anhui and Shanghai/2 are identical but Shanghai/1 has a number of differences. These sequences surprise me often; I would have thought the 2 from Shanghai would have been identical.

I then decided to align the humans with some of the more recent birds and here is a list of the ones that have HAs:

A/wild bird/Korea/A14/11 2011/02/01 EPI_ISL_120868 | AH7N9 | HA
A/spot-billed duck/Korea/447/11 2011/04/01 EPI_ISL_120871 | AH7N9 | HA
A/wild bird/Korea/A3/11 2011/02/01 EPI_ISL_120869 | AH7N9 | HA
A/wild bird/Korea/A9/11 2011/02/01 EPI_ISL_120881 | AH7N9 | HA
A/goose/Nebraska/17097-4/2011 2011/04/05 EPI_ISL_130360 | AH7N9 | HA
A/guinea fowl/Nebraska/17096-1/2011 2011/04/05 EPI_ISL_130362 | AH7N9 | HA
A/northern shoverl/Mississippi/11OS145/2011 2011/01/08 EPI_ISL_135036 | AH7N9 | HA
A/Shanghai/1/2013 2013/01/01 EPI_ISL_138737 | AH7N9 | HA
A/Shanghai/2/2013 2013/01/01 EPI_ISL_138738 | AH7N9 | HA
A/Anhui/1/2013 2013/01/01 EPI_ISL_138739 | AH7N9 | H

In the HA nucleotides, we clearly have 3 separate groups; the Asian birds, the US birds and the humans.

I see the humans have S31N in the amino acid M2 gene.

I'm sorry I'm lacking the ability to see any reassortment. Maybe JJackson will come along and take a look.

[sharon sanders]

Re: China - H7N9 Human Isolates on Deposit at GISAID

hat tip NS1 at Genewurx

Initial GeneWurx Impressions

This event is not unprecedented as to H7 classification. A higher interest will occur if the epidemiology demonstrates relationships among the human victims.

Recall that H7 is the only other primarily Avian hosted HA classification outside of H5 that has human fatality confirmed?

H7N7 Netherlands from Veterinarian Post-Mortem Lung Tissue

. . . . NetherlandsTeeffelen219_57M_2003_04_17_f (
. . . . . . . . GISAID HA EPI234814
. . . . . . . . GISAID Isolate EPI_ISL_67238


Also recall that human H7 infection is on record (Netherlands and UK) and that large groups of human poultry workers have fallen ill from H7N7, including during the initial Scherpenzeel outbreak. Eye infection is generally the presenting symptom.

Avian H7N7 and H7N3 are widespread. H7N7, in particular, hosts significant and increasing homology to current pH1N1 genetic acquisition. We, at GeneWurx, consider H7N7 to be an important candidate as one of several zoonotic sub-segment donators to pH1N1.

The H7N9 Anhui HA is listed as most similar to avian H7N3 (95%, 1617/1685nu) and H7N7 (95%, 1601/1682nu) according to current deposits / software at GISAID, an initial signal of moderate-to-low potential for reassortment. With no exact match, that potential reassortment obviously does not occur from any sample on record. The H7N9 Anhui NA GISAID BLAST-based evaluation shows homology to most HxN9 serotypes, again with no exact matches.

Due to the inherent limitations of the software design, BLAST is generally inconclusive on these types of 'Ultimate Origin' matters unless an exact match is found in the database.

[sharon sanders]

Re: China - H7N9 Human Isolates on Deposit at GISAID

hat tip NS1 at Genewurx

These H7N9 HA segments are unrelated genetically to the previous human H7N7 fatality:

. . . . NetherlandsTeeffelen219_57M_2003_04_17_f (
. . . . . . . . GISAID HA EPI234814
. . . . . . . . GISAID Isolate EPI_ISL_67238

The H7N9 human sequences under investigation do NOT show the H7N7 fatality polybasic extended cleavage area, 'aagaggaggaggaga'/'KRRRR'. The H7N9 human sequences demonstrate a typical H7N9 cleavage area.

Nor do any of the H7N9 human sequences carry any of the HA amino polymorphisms found on the H7N7 Fatality (I13S, A143T, K416R). They each are unremarkable at those 3 amino acid positions.

However, a significant quantity and positioning of HA variation from existing H7N9 avian sequences, including antigenic areas, do exist and will be reported after numbering pattern resolution.

Administration Note:

Until sample dates are received, we are logically imputing sample dates from the female as the onset date (known previous male deaths promote swabbing) and from the males as the date of death (post-mortem tissue). We are also imputing, until further data is released, that the Shanghai1 sequence represents the first death, 87M; ergo, Shanghai2 arises from the 27 year old male.

? AnhuiChuzhouCity1_E1_35F _2013_03_15_s onset 2013-03-15
? Shanghai2_E1_27M_2013_03_03_10_f onset 2013-02-19
? Shanghai1_E1_87M_2013_03_04_f onset 2013-02-19

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Please review the attached preliminary GeneWurx overview of the H7N9 Emergent Human sequences filed Sunday at GISAID for details concerning Rare and Novel antigenic Hemagglutinin polymorphisms and the relationships suggested to the currently circulating human pH1N1.

[Giuseppe]

Re: China - H7N9 Human Isolates on Deposit at GISAID

From China CDC:

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

2012 article on China H9N2 pathogenicity with implications for humans:

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038233

.

[sharon sanders]

Re: China - H7N9 Human Isolates on Deposit at GISAID

2012 article on China H9N2 pathogenicity with implications for humans:

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038233

.

This study is Open Access.


Research Article
A Single E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus Increases Virulence by Inducing Higher Glucocorticoids (GCs) Level

• Jin Tian equal contributor,
  
  
• Wenbao Qi equal contributor,
  
  
• Xiaokang Li,
  
• Jun He,
  
• Peirong Jiao,
  
• Changhui Zhang,
  
• Guo-Qian Liu,
  
• Ming Liao mail
  
  

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• Abstract
• Introduction
• Materials and Methods
• Results
• Discussion
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Abstract

While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB₂ residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that V_K627 and rTs_E627K infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rV_K627E and Ts_E627.

Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by V_K627 and rTs_E627K infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung.

These data demonstrated that V_K627 and rTs_E627K infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV.

Citation: Tian J, Qi W, Li X, He J, Jiao P, et al. (2012) A Single E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus Increases Virulence by Inducing Higher Glucocorticoids (GCs) Level. PLoS ONE 7(6): e38233. doi:10.1371/journal.pone.0038233
Editor: Nupur Gangopadhyay, University of Pittsburgh, United States of America
Received: January 11, 2012; Accepted: May 1, 2012; Published: June 13, 2012
Copyright: ? 2012 Tian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (No. 81001375). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.

Introduction

H9N2 subtype avian influenza virus was first isolated in turkeys in the U.S. in 1966 [1]. Since 1998, H9N2 viruses have been isolated in pigs and humans in Hong Kong and Mainland China, and the infected displayed an influenza-like illness [2]. These findings indicate the H9N2 avian influenza virus takes on rapid evolution [2]. At the same time, the pressure of vaccine and natural immunity may contribute to substantial virus evolution, which also leads to virus reassorting with other influenza viruses [3]. Multiple studies show some gene segments from newly isolated H9N2 viruses in southeastern and Eastern China possess H5N1 internal genomes [4], [5].
Rapid evolution also leads to enhanced pathogenicity for the virus in mammals and poultry. Evidence shows H9N2 avian-human reassortant virus has enhanced replication and efficient transmission in ferrets [6]. Following adaptation in the ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a human H3N2 backbone could transmit efficiently via respiratory droplets, creating a clinical infection similar to human influenza infections [7]. In 2010, Hye-Ryoung Kim?s research results showed that three H9N2 reassortant viruses generated from the H5N2 viruses of domestic ducks without pre-adaptation were recovered at high titers in chickens [8].
The evidence on human cases of avian influenza infection in Hong Kong and mainland China leads to more attention to the role of H9N2 avian influenza viruses in human disease [9]. Albeit causing a mild disease in H9N2 virus-infected humans, H9N2 viruses have repeatedly infected humans [10]. Furthermore, some of the H9N2 influenza viruses currently circulating in southern China have molecular features that allow them to preferentially bind to α-2,6-NeuAcGal receptors [11]. Previous studies by Rui Wu et al. indicated that mouse-adapted H9N2 influenza viruses could replicate efficiently and be transmitted among mice through both contact and respiratory droplet routes [12]. Moreover, more evidence shows that H9N2 AIVs causing severe disease in experimentally infected mice without prior adaptation are increasing [13]?[14]. However, the mechanism regarding enhanced pathogenicity to mice for H9N2 virus is less known.
More evidence has demonstrated that PB₂ residue 627 is a key host range and virulent determinant of influenza A viruses [15] and PB₂ E627K mutation can directly elevate the enzyme kinetics of influenza polymerase that facilitates virus replication in mammalian cells [16]. In H5N1 virus, a single-amino-acid substitution in PB₂ residue 627 is associated with systemic infection and impaired T-cell activation in mice [17]. H9N2 AIV prior to adaptation in mice shows multiple amino acid substitutions that include PB₂ E627K are involved [18]. So far, there are no reports on the impact of a single residue substitution in PB₂ residue 627 of H9N2 virus on host defense and immune responses. Moreover, H5N1 virus could infect thymus, spleen, and lymphonode and destroy the immune response against virus [19], but H9N2 virus used in our study could not be isolated in these tissues, which suggests the different mechanism of enhanced virulence from H5N1 virus.
Glucocorticoids (GCs) display potent immunomodulatory activities, including the ability to induce T lymphocytes apoptosis and inhibit inflammatory response [20], but high GCs level may be detrimental for host immune response [21]. In our study, we demonstrated that the enhanced virulence for H9N2 AIV correlated with a higher GCs level. Higher GCs titer in plasma of mice induced apoptosis increase in thymus cortex, which impaired the T cells development and led to T cells depletion in lymphoid and lung tissues. Moreover, higher GCs also suppressed the pro-inflammatory cytokines and chemokines level in lungs of mice, which led to the reduction of inflammatory cells infiltration. Finally, the inhibition of host immune defense response contributed to susceptibility to virus infection. GCs were required to protect hosts from lethal immunopathology [21], but the GCs level beyond physiological concentration would destroy the immune response against virus infection, which may be rendered as one of the mechanisms of immunosuppression induced by influenza virus.

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Figure 1. Comparison of weight loss and lung virus titers.
Mice (n = 20/group) were inoculated i.n with 10⁴ PFU for V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). The weight from eight mice per group was monitored daily (A). Lungs from three mice per group per time point were harvested for virus titration at indicated days p.i. (B). Virus titers were given in units of log₁₀PFU per ml. The data shown represents mean ? standard deviation (SD) for three independent experiments. *p<0.05 between V_K627 and rV_K627E; ?p<0.05 between Ts_E627 and rTs_E627K.
doi:10.1371/journal.pone.0038233.g001

Materials and Methods

Viruses

The viruses used in study were H9N2 AIV A/chicken/Guangdong/Ts/2004 (Ts_627E) and A/chicken/Guangdong/V/2008 (V_K627). Recombinant viruses (rV_K627E and rTs_E627K) were produced by eight-plasmid reverse genetics systems introduced below. Virus stocks propagated in the allantoic cavity of 9- to 11-day-old embryonated specific-pathogen-free hen?s eggs at 37?C. The allantoic fluids were harvested at 48h post-inoculation. The viral titer was determined by plaque assay on MDCK cells (ATCC) in duplicate. In brief, confluent monolayer of MDCK cells were prepared in six-well plates, infected with 10-fold dilutions of virus at 37?C for 2 h. The inoculum was removed, and washed and then overlaid with MEM containing 1% agarose and 2 ?g/ml of TPCK-treated trypsin. After plaques had formed at 48?72 h post-infection, the agarose was removed and cells were stained with 0.5% crystal violet in 10% formaldehyde solution. The plaques were visualized and manually counted.
Construction of Plasmids

A bidirectional transcription vector (pDL) was used to establish eight-plasmid reverse genetic systems. The pDL contains human RNA pol I promoter and murine terminator sequences, which are flanked by the RNA polymerase II promoter of human cytomegalovirus and SV40 late polyadenylation signal. Two BsmB I restriction sites were utilized to clone viral full-length cDNA between RNA pol I promoter and terminator. The viral cDNAs were amplified by RT-PCR with primers containing BsmB I sites (primers are available upon request), and then digested with BsmBI and cloned into the BsmBI sites of the pDL vector. The resulting plasmids (pDL-V-PB2, -PB1, -PA, -HA, -NP, -NA, -M and ?NS; pDL-TS-PB2, -PB1, -PA, -HA,-NP, -NA, -M and ?NS) were confirmed by sequencing (primers are available upon request). Mutations were introduced into the PB2 gene by site-directed mutagenesis kit (Invitrogen). The resulting plasmids are pDL-V-PB2-627E and pDL-TS-PB2-627K, which were confirmed by sequencing. The plasmids for transfection were prepared by using the Perfectprep Plasmid mini kit (Eppendorf, Hamburg, Germany).
Generation of Recombinant Viruses

A monolayer of 293T cells (ATCC)with approximately 90% confluence in six-well plates was transfected with 5 ?g of the eight plasmids (0.6 ?g/each plasmid) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer?s instructions. Briefly, 5 ?g of plasmids and 10 ?L of lipofectamine 2000 were mixed, incubated at room temperature for 30 min, and then added to the cells. After 6 hours incubation at 37?C, the mixture was replaced with DMEM containing 2% fetal bovine serum and 0.2 ?g/mL TPCK-treated trypsin (Sigma-Aldrich). The supernatant was harvested after 2 days incubation and 100 ?L of supernatant was injected into an embryonated egg for virus propagation. The inoculated eggs were incubated for 3 days and the allantoic supernatant was collected and tested by hemagglutination assay. The rescued viruses were confirmed by sequencing of the whole viral genome.
Mice Infections

Four-week-old female BALB/c mice (Experimental Animal Centre of Guangdong Province, P.R. China) were anesthetized with dry ice and intranasally (i.n.) inoculated with 10⁴PFU of influenza virus (sublethal inoculum for a prolonged disease course) diluted in 50 ?L of sterile, endotoxin-free PBS or 50 ?L of sterile PBS (mock group). Mice (n = 8/group) were weighed before infection, and then monitored daily for weight loss as a measure of morbidity. All animal research was conducted under the guidance of CDC?s Institutional Animal Care and Use Committee and in an Association for Assessment and Accreditation of Laboratory Animal Care International- accredited facility. Our animal research in our study had been approved by Guangdong Province Animal Disease Control Center.
Mifepristone Treatment

The mice (n = 14/group) were treated with 0.1 mg/g (initial weight) of mifepristone (RU486) (Sigma-Aldrich) (suspended in 100 ?L of 2% ethyl alcohol) via intraperitoneal administration beginning on day 1 before infection and continued daily until the end of the experiments. The control mice were injected with 100 ?L of 2% ethyl alcohol. Both groups were challenged with 10⁴PFU of V_K627 and rTs_E627K. Weight loss (n = 8/group) was monitored daily as a measure of morbidity. On day 5 post-infection, mice (n = 6/group) from the treatment and control groups were euthanized. The lungs from three mice per group were collected for analysis of virus titer and cytokines. The thymus and lungs from another three mice per group were harvested. Apoptosis and CD4⁺CD8⁺ cells in thymus and T cells and inflammatory in lungs were analyzed by flow cytometry.

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Figure 2. Lung cell characterization following infection.
Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). Lungs from three mice per virus group per time point were harvested and single cell suspensions were prepared. The percentages of T cells and inflammatory cells were determined by appropriate gating on labeled cells. The numbers of CD4⁺ T cells (A), CD8⁺ T cells (B), macrophages (C), neutrophils (D), and dentritic cells (E) were calculated by multiplying the percentage of each cell type by the total number of viable lung cells. Baseline cell numbers from PBS inoculated mice (n = 5) are shown as a dashed lines in each graph. The data shown represents mean ? SD for three independent experiments. *p<0.05 between V_K627 and rV_K627E; ?p<0.05 between Ts_E627 and rTs_E627K.
doi:10.1371/journal.pone.0038233.g002
Flow Cytometric Analysis

Mice (n = 12/group) were infected with 10⁴PFU of V_K627, rV_K627E, Ts_E627, rTs_E627K. The peripheral blood, lung and thymus from three mice per group per time point were collected. Lung was washed with cold PBS and homogenized individually in 2 ml of collagenase B (Sigma-Aldrich) at a concentration of 2 mg/ml in RPMI 1640 (Gibco BRL, Grand Island, N.Y.) and incubated for 30 min in a 37?C water bath. Subsequently, the enzyme-digested lung tissues were filtered through a 200-micron nylon mesh to obtain a single cell suspension. The erythrocytes were lysed by treatment with 0.83% of NH₄Cl-Tris buffer, and the remaining cells were washed and resuspended in PBS. Thymus was gently passed through a 200-micron nylon mesh, lysed with NH₄Cl-Tris buffer, and single cell suspensions were washed and resuspended in PBS. Next, 0.1 ml of blood or single cell suspensions containing 10⁶ cells was incubated on ice for 10 min with anti-Fc block (anti-CD16/32). Specific cell populations were stained with anti-CD4, anti-CD8 and anti-CD3 for T cells analysis and anti-CD11b, anti-CD11c, anti-Ly6G/6, and anti-MHCII for inflammatory cells analysis. All the mAbs were purchased from eBioscience. After being stained for 30 min at 4?C, the erythrocytes in blood samples were lysed with Optiman C (Beckman), and then the samples were added to 1 ml PBS and analyzed on FACSCalibur flow cytometer (BD Bioscience). Other samples, following staining for 30 min at 4?C, were washed twice, resuspended in 1 ml of 2% paraformaldehyde, and analyzed on FACSCalibur flow cytometer. Inflammatory cells were differentiated by expression of cell-specific markers as indicated in reference [22]. A total of 10,000 gated events were performed in three independent experiments. The number of viable cells per sample was determined by using a Coulter counter (Beckman), and individual cell subsets were calculated by multiplying the percentage of each cell type (as determined by FACS) by the total number of viable cells per tissue.
Histopathology Analysis

Mice (n = 3/goup) were infected with V_K627, rV_K627E, Ts_E627, rTs_E627K (10⁴PFU). At day 5 p.i., three thymuses from each group were fixed in 4% formalin, routinely processed, and embedded in paraffin. Routine hematoxylin-and-eosin-stained sections were examined as previously described [19].
Analysis of Apoptosis in Thymus

Mice (n = 9/goup) were infected with V_K627, rV_K627E, Ts_E627, rTs_E627K (10⁴PFU). At day 3 and 5 p.i., three thymuses from each group were collected. The single cell suspensions from thymus were prepared and 0.1 ml of cell suspensions containing 10⁶ cells was stained with annexin-V and PI (Invitrogen) according to the manufacturer?s instructions. Following staining, the cells were immediately analyzed on a FACSCalibur flow cytometer. The characterization of early apoptotic cells was distinguished as described [23]. A total of 10,000 gated events were performed in three independent experiments. Late apoptotic cells with DNA strand breaks were identified in histological paraffin sections using the in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit (Sigma-Aldrich). A total of six paraffin sections from three mice per group, sacrificed at day 5 p.i., were prepared according to the manufacturer?s instructions. The brown cells were apoptotic cells.
Lungs Virus Titrations, Cytokines, and Glucocorticoids Analysis

Mice (n = 12/group) were infected with 10⁴PFU of V_K627, rV_K627E, Ts_E627, rTs_E627K. The lungs from three mice per group per time point were removed at the indicated time point and stored at −70?C for cytokines and virus titer analysis. At the same time, peripheral blood of mice from the orbital plexus of anesthetized mice was collected and centrifuged to prepare for the plasma. The three lungs from each group per time point were homogenized in 1 ml of cold PBS. The homogenate was pelleted by centrifugation and the virus titer was determined by plaque assay on MDCK cells in duplicate. The titers are reported as plaque forming units per ml PBS (PFU/ml). With the use of ELISA kits (R&D Systems), the clarified lung homogenates were assayed for IL-6, IFN-γ, IL-1β, TNF-α, MIP-1α, MIP-2 and MCP-1 following the manufacturer?s instructions. The levels of cortisol in plasma from three mice per group per time point were measured according to the manufacturer?s instructions by ELISA (Enzo).
Quantitative Real-time PCR

Mice (n = 12/group) were inoculated i.n. with 10⁴PFU of V_K627 and rTs_E627K. The three thymuses per group per time point were harvested from day 1 to 7 p.i. Total RNA was isolated from the homogenate using TRIzol reagent (Invitrogen). The allantoic fluids from eggs inoculated with virus and lungs from infected mice were worked as the positive control. The cDNA was achieved with reverse-transcibed kit (Promega). Quantitative real-time PCR was used to determine the expression of beta-actin and influenza virus M gene with SYBR Green PCR kit (TAKARA). The relative expression values of M gene were normalized to the expression value of the β-actin gene. The qPCR programs and primer sequences could be supplied if needed.
Statistical Analysis

Statistical significance of differences between experimental groups was determined through the use of the paired, nonparametric Student?s t test. P<0.05 was thought significant difference.

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Figure 3. Lung cytokines response.
Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). Lungs from three mice per virus group per time point were harvested and homogenized in 1 ml of PBS. Cytokine levels were measured individually and in duplicate. Baseline cytokine levels from PBS inoculated mice (n = 5) are shown as a dashed line in each cytokine graph. The data shown represents mean ? SD for three independent experiments. *p<0.05 between V_K627 and rV_K627E; ?p<0.05 between Ts_E627 and rTs_E627K.
doi:10.1371/journal.pone.0038233.g003

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Figure 4. Histopathology and CD4⁺CD8⁺ thymocytes in the thymus.
Mice (n = 6/group) were infected i.n. with 10⁴ PFU of V_K627, rV_K627E, Ts_E627, rTs_E627K, and PBS (mock mice). At 5 day p.i. the thymuses from each group were removed. (A) Three thymuses per group were processed for hematoxylin and eosin. Five sections per tissue were analyzed. Magnification, ?50. (B) Another three thymuses per group were prepared for single cell suspension. Following staining, the percentages of CD4⁺CD8⁺ thymocytes were examined by flow cytometry. Three independent experiments yielded consistent results.
doi:10.1371/journal.pone.0038233.g004

Results

A Single-amino-acid Change in the PB2 Protein Affects the Replicative Capacity and Pathogenicity of H9N2 Viruses in Mice

To perform a comparison of mortality of each virus, mice (n = 8/group) were inoculated i.n. with each virus (10⁴PFU). The mice infected with V_K627 or rTs_E627K showed the greatest signs of illness, such as ruffled fur and severe morbidity (24.1% and 23.6% weight loss at day 7) (Fig.1A). However, the mice infected with rV_K627E or Ts_E627 showed lighter signs of illness without weight loss (Fig.1A). To determine viral replication in the lungs, lungs from three mice infected with 10⁴ PFU of each virus per group were collected on day 1, 3, 5 and 7 p.i. and viral load in supernatant of lungs homogenizer was quantified in MDCK cells by plaque assay (Fig. 1B). The virus titers in V_K627-infected lungs were significantly higher (*p = 0.031) than titers observed in rV_K627E-infected lungs over the course of infection. The mutation, E627K in PB₂, remarkably increased the replicative capacity of rTs_E627K in lungs. Compared with that in Ts_E627-infected mice, lung virus titer in rTs_E627K-infected mice was increased by approximately tenfold (?p = 0.029) at day 1 p.i., and the significantly higher virus titers from rTs_E627K-infected lungs were also observed on days 3 and 7 p.i. All the data showed that the virulence and replicative capacity of the H9N2 virus could be affected by the PB₂ residue 627.

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Table 1. Analysis of apoptosis in thymus.
doi:10.1371/journal.pone.0038233.t001
V_K627 and rTs_E627K Infections Decrease the Numbers of T Cells and Inflammatory Cells Infiltrating into Lung

To explore the factors for the enhanced morbidity of H9N2 AIV, the numbers of T cells and inflammatory cells in lungs were quantified (Fig. 2). Following infection with 10⁴ PFU of each virus, lungs from three mice per group were collected at day 1, 3, 5 and 7 p.i. and single cell suspensions were prepared. The percentages of lung immune cells were determined by flow cytometry. After V_K627 and rTs_E627K infection, the numbers of CD4⁺ and CD8⁺ T cells displayed an identical or lower level compared with mock group from day 1 to 5 p.i., but exhibited a progressive increase in rV_K627E and Ts_E627 groups (Figs. 2A and 2B). V_K627 and rTs_E627K infection significantly reduced the numbers of CD4⁺ and CD8⁺ T cells at day 3 p.i. compared with the numbers detected in rV_K627E and Ts_E627 groups. On day 5 p.i., a reduction of at least thrice as few CD4⁺ and CD8⁺ T cells (*p = 0.005, ?p = 0.012) was observed in V_K627 and rTs_E627K groups. The inflammatory cells examined in V_K627 and rTs_E627K groups rapidly mounted to the peak from day 1 to 3 p.i., but fell off drastically from day 5 to 7 p.i. (Figs. 2C?2E). In contrast, the numbers in rV_K627E and Ts_E627 groups exhibited a progressive increase (Figs. 2C?2E). These data indicated that V_K627 and rTs_E627K infection resulted in depletion of T cells and inflammatory cells in lung.

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Figure 5. TUNEL assay.
Following infection with 10⁴ PFU of V_K627, rV_K627E, Ts_E627, rTs_E627K, and PBS (mock mice), mice (n = 3/group) were euthanized on day 3 p.i. and thymuses were removed and fixed in 4% formalin. Apoptotic cells were identified in histological sections using the TUNEL assay. Three sections per tissue were analyzed Magnification, ?400. Three independent experiments yielded consistent results.
doi:10.1371/journal.pone.0038233.g005
V_K627 and rTs_E627K Infections Result in Diminished Proinflammatory Cytokines and Chemokines Production

Many cytokines in the lung are believed to contribute to the recruitment of inflammatory cells. Since the previous experiments established that V_K627 and rTs_E627K infection resulted in a depletion of inflammatory cells, we next determined whether critical cytokine and chemokine responses might also be limited in the lung following infection. After infection with each virus (10⁴ PFU), lungs from three mice per group were collected at day 1, 3, 5 and 7 p.i. and the levels of cytokines (IL-6, IL-1β, TNF-α, IFN-γ) and chemokines (MIP-2, MIP-1α, MCP-1) in lungs were analyzed by ELISA. All cytokines and chemokines were produced well above the constitutive levels 1 day after infection with each virus (Figs. 3A?3G). However, the production of IL-6, IL-1β, IFN-γ and MIP-1α was strikingly reduced in V_K627 and rTs_E627K groups on day 5 to 7 p.i., compared with those in rV_K627E and Ts_E627 groups (Figs. 3A, 3B, 3D, 3G). The levels of TNF-α (Fig. 3C) in V_K627 group were remarkably lower than the levels in rV_K627E group from day 3 to 7 p.i., but the similar decrease was detected only in rTs_E627K group at day 5 and 7 p.i. Moreover, the protein levels of MIP-2 were reduced to <25% in the rTs_E627K group compared with Ts_E627 group at day 5 and 7 p.i. (Fig. 3E). These data revealed V_K627 and rTs_E627K infection led to the diminished production of critical cytokines and chemokines. To examine whether the reduced levels of cytokines and chemokines were caused by elevated IL-10 production, the titer of IL-10 was analyzed (Fig. 3H). Although all the viruses? infection decreased the production of IL-10, a lower level was observed in V_K627 and rTs_E627K groups than that in rV_K627E and Ts_E627 groups. So the reduced production of cytokines and chemokines were not caused by higher IL-10 titer.

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Figure 6. GCs level in plasma and RU486 treatment.
(A) Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). The plasma from three mice per virus group per time point was prepared and GCs level in plasma was measured by ELISA. Baseline GCs levels from PBS inoculated mice (n = 5) are shown as a dashed line in graph. *p<0.05 between V_K627 and rV_K627E; ^Λp<0.05 between Ts_E627 and rTs_E627K; (B?D) Following RU486 treatment, mice (n = 6/group) were infected i.n. with 10⁴ PFU of V_K627 (V_K627-RU) and rTs_E627K (rTs_E627K-RU). The control groups were named with V_K627-C and rTs_E627K-C. All the mice were sacrificed at day 5 p.i. Lungs and thymuses from three mice of each group were removed and single cell suspensions were prepared. The percentages of apoptosis (B) and CD4⁺CD8⁺ thymocytes (C) in thymus and T cells and inflammatory cells in lung were measured by flow cytometry. The numbers of T cells and inflammatory cells (D) were calculated by multiplying the percentage of each cell type by the total number of viable lung cells. Lungs from another three mice in each group were removed and homogenized in 1 ml of PBS. Cytokines levels (D) were measured individually and in duplicate. The data shown in B and C represents three independent experiments and the data shown in A and D represents mean ? SD for three independent experiments. *p<0.05 between V_K627-RU and V_K627-C; ?p<0.05 between rTs_E627K-RU and rTs_E627K-C.
doi:10.1371/journal.pone.0038233.g006

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Figure 7. Weight change and viral burden following RU486 treatment.
Following RU486 treatment, mice (n = 11/group) were infected i.n. with 10⁴ PFU of V_K627 (V_K627-RU) and rTs_E627K (rTs_E627K-RU). (A) Mice (n = 8/group) were weighed daily from 0 day p.i. to 7 day p.i. (B) At 5 day p.i., the lungs from three mice per group were removed and homogenized in 1 ml of PBS, and virus titer was determined by plaque assay. The data shown in A and B represents mean ? SD for three independent experiments. *p<0.05 between V_K627-RU and V_K627-C; ?p<0.05 between rTs_E627K-RU and rTs_E627K-C.
doi:10.1371/journal.pone.0038233.g007
V_K627 and rTs_E627K Infections Interfere with T Cells Development

The depletion of T cells in V_K627 and rTs_E627K groups were not only observed in lung, but also detected in peripheral blood (Fig. S1A). Whether both virus infections impaired the T cells development in thymus was examined. After infection with each virus (10⁴ PFU), the thymuses from six mice per group were removed at day 5 p.i., and three thymuses were used for analysis of histopathology and another three for analysis of CD4⁺CD8⁺ thymocytes by flow cytometry. Visual atrophy of thymus (Fig. S1B) was observed in V_K627 and rTs_E627K groups. The examination of histopathology (Fig.4A) showed that the normal corticomedullary differentiation was lost following V_K627 and rTs_E627K infection, and the cortex was almost absent. The analysis of lymphocyte populations from thymus showed the percents (4.5?1.5%) of CD4⁺CD8⁺ thymocytes in V_K627- and rTs_E627K-infected mice were far lower than the percents (82.3?4.3%) observed in rV_K627E and Ts_E627 infection groups at day 5 p.i. (Fig. 4B). Moreover, the mature CD4⁺ and CD8⁺ T cells in V_K627 and rTs_E627K groups took on remarkably lower numbers than the numbers in rV_K627E and Ts_E627 groups from day 3 to 7 p.i. (Fig. S1C). These data revealed V_K627 and rTs_E627K infection interfered with T cells development and caused T cells lymphopenia in thymus.
Increased Apoptosis in Cortex of Thymus may be Responsible for the Reduced Percent of CD4⁺CD8⁺ Thymocytes

To check whether the depletion of thymus was induced by increased apoptosis, both flow cytometry and TUNEL assay were performed. After being challenged with each virus (10⁴ PFU), thymuses from three mice per group were collected at day 3 and 5 p.i. and single cell suspensions were prepared. Early apoptosis was analyzed by flow cytometry. The results showed the early apoptosis in V_K627 and rTs_E627K groups had a significant increase compared with that in rV_K627E and Ts_E627 groups at day 3 and 5 p.i. (Table 1). Situ detection of late apoptotic cells in paraffin-embedded thymus sections from two mice per group was achieved by TUNEL assay at day 5 p.i. (Fig. 5). Following infection, many apoptotic cells were pronounced in cortex from V_K627- and rTs_E627K-infected mice, but not in medulla. In the rV_K627E- or Ts_E627-infected mice, no apoptotic cells could be detected in the cortex and medulla. Cortex of thymus is the location of negative selection for CD4⁺CD8⁺ thymocytes. So, the reduced percent of CD4⁺CD8⁺ thymocytes may result from the increased apoptosis in cortex of thymus.
The Higher GC Levels in the Plasma of V_K627- and rTs_E627K- Infected Mice are an Important Factor for the Increased Apoptosis in Thymus and the Decreased Infiltration of T Cells and Inflammatory Cells in Lung

Apoptosis is regarded as a host defense mechanism against virus infections that works by removing foreign nucleic acids from an infected host [24]. Following infection with 10⁴PFU of V_K627 and rTs_E627K, the levels of virus nucleic acids in thymuses from three mice per group were analyzed from day 1 to 7 p.i. by qPCR. But the virus nucleic acids could not be detected during the whole course of infection (data not shown).
We subsequently found the mice infected with each virus showed a sustained increase level of GCs in the plasma compared with the mock group (Fig. 6A). But the levels of GCs in V_K627 and rTs_E627K groups were significantly higher than rV_K627E and Ts_E627 groups from day 1 to 7 p.i. So we hypothesized the increased apoptosis in thymus was caused by higher GCs levels. Next, whether blocking the glucocorticoid receptors (GR) could decrease thymocytes apoptosis after V_K627 or rTs_E627K infection was examined. The RU48-treated mice were infected i.n. with 10⁴PFU of V_K627 (V_K627-RU) and rTs_E627K (rTs_E627K-RU). At day 5 p.i., the lungs and thymuses from three mice per group were harvested and single cell suspensions were prepared. The early and late apoptosis in thymus of V_K627-RU or rTs_E627K-RU groups was strongly decreased at day 5 p.i. compared with mock groups (Fig. 6B). Importantly, the percentages of CD4⁺CD8⁺ thymocytes in both RU486-treated groups recovered to 75.2?5.5%, which was significantly higher than mock groups (11.5?2.1%) (Fig. 6C). RU486 treatment also significantly increased the infiltration of T cells in the lungs (Fig. 6D). Endogenous and pharmacologic GCs limit inflammatory cascades by modulating a wide range of inflammatory molecules, including many cytokines [21]. Indeed, blockade of GR resulted in the dramatic increase in production of inflammatory cytokines (IL-6, IL-1β, TNF-α) and chemokines (MIP-1α, MIP-2) (Fig. 6D). Moreover, the dentritic cells in RU486-treated groups had a remarkable increase (>3 folds) (*p = 0.0002, ?p = 0.012). But the significant increase of macrophage was observed only in V_K627-RU group, and the similar increase of neutrophils was only observed in rTs_E627K-RU group (Fig. 6D). So, the higher level of GCs played an important role in the decreased T cells, and inflammatory cells in V_K627- and rTs_E627K-infected lungs.
Blockade of Glucocorticoid Receptors Results in Protection to V_K627 and rTs_E627K Challenge in Mice

To examine the effect of blockade of GR on virus infection, RU486-treated mice (n = 11/group) were challenged with 10⁴PFU of V_K627 and rTs_E627K. The weight loss (n = 8/group) was monitored daily. At day 5 p.i., virus loads of lungs from three mice per group were determined by plaque assay. We noted that the V_K627-RU- and rTs_E627K-RU-infected mice experienced less weight loss compared with mock mice from day 4 to 7 p.i. (Fig. 7A). The virus loads of lungs in both RU486-treated groups were decreased at day 5 p.i. but significant differences (?p = 0.013) were observed only between rTs_E627K-RU and rTs_E627K-C (Fig. 7B).

Discussion

The PB₂ residue 627 has been identified as an important determinant of host range restriction [25] and virulence and replicative efficiency in animal models [15], [17], [26]. In the in vivo infection experiment, the replicative efficiency and virulence of rV_K627E was obviously lower than V_627K and the replicative efficiency and virulence of rTs_E627K was significantly enhanced compared with Ts_E627, which suggested that PB₂ residue 627 substitutions affected the replicative efficiency and virulence in vivo. However, it is more important to understand how the presence of lysine at position 627 of the PB2 protein affects virus-host interactions and is sufficient to allow the virus to replicate quickly. In our study, we found PB₂ residue 627 substitution affected host immune defense response and contributed to susceptibility to virus infection. However, whether there were other amino acids in the genomes of H9N2 AIV that could contribute to same effect needs to be investigated further.
The proinflammatory cytokine response is responsible for recruiting immune effector cells to clear the virus infection [27]. Nevertheless, this response, with inappropriate activation or inefficient regulation, may contribute to severe lung viral pneumonia and serious complications of infection [28], [29]. It has been proposed that the increased pathogenicity for 1918 and H5N1 influenza virus infection is related to excessive early cytokine response, immune cell recruitment, and poor outcome [30]. But highly lethal H5N1 influenza (HK483) could reduce inflammatory cells recruitment, which is considered as the cause of virus not being cleared from tissues [19]. So, the enhanced morbidity for V_K627 and rTs_E627K infections was associated with the reduction of inflammatory cells in lungs.
Recently several studies have established a role for CD8⁺ T cells during the innate immune response against bacterial and parasite infection [31]. A lack of CD8⁺ T cells led to increased viral replication and morbidity in mice infected with A/Puerto Rico/8/34 (PR8). Moreover, CD4⁺ T cells can direct CD8⁺ T cells response by secreting TypeI panel cytokines (IFN-γ, IL-2, TNF-α), modulating the magnitude and duration of CD8⁺ T cells response and driving B cells production of antibody to neutralize viral particles [32]. The insufficient infiltration of CD4⁺ and CD8⁺ T cells in lungs may impair the host immune response. So, suppressive T cells response also contributed to the enhanced morbidity for V_K627 and rTs_E627K infections.
Macrophages and neutrophils can secrete chemokines and cytokines that can act in an autocrine fashion, which in turn can promote the migration of those cells and other leukocytes into lung tissue [28]. Contrary to the widely recognized theory, the expression of cytokines is mainly regulated by pulmonary endothelium [33]. We found the levels of proinflammatory cytokines and chemokines examined in the lung were significantly inhibited at day 5 and 7 p.i. So, fewer cells migrating into lung may be associated with the suppressive expression of cytokines in lung of V_K627 or rTs_E627K-infected mice. The mice deficient in either IL-1β, TNF-α, IFN-γ increased their mortality due to influenza virus infection compared with wild-type control mice [34]. The reduced level for IL-6, IL-1β, TNF-α, and IFN-γ in lung may be a reason for the enhanced morbidity in V_K627 and rTs_E627K infections.
Leukopenia has been demonstrated following infection with many of viruses, and a transient leukopenia could occur following infection with human influenza subtypes in humans [19], [35]. However, the mechanism of lymphopenia remains less known. To investigate a possible mechanism for T cells lymphopenia in peripheral and lymphoid tissue after V_K627 and rTs_E627K infection, we focused on the thymus. In addition to the thymus suffering from severe atrophy, the histopathology examination of thymus revealed that V_K627 or rTs_E627K infection induced the cortex of thymus to be reduced. More importantly, the percent of CD4⁺CD8⁺ thymocytes in V_K627- or rTs_E627K-infected mice underwent a processive reduction. So, inability of the thymus to reproduce the peripheral T lymphocyte compartment may represent one mechanism of T cells reduction in peripheral tissues [35].
Influenza virus-induced apoptosis was primarily thought to be a host defense mechanism to limit virus replication and clear viruses from the host [36]; however, the virus has abilities not only to overcome but to utilize apoptosis for its efficient replication [37]. Because H5N1 virus could be detected in these lymphoid organs, it is difficult to judge increased apoptosis induced by virus directly or indirectly [19]. In our research, the data demonstrated that V_627K and rTs_627K did not directly participate in increasing apoptosis in thymus because no virus nucleic acids were detected in thymus. GCs are important for T cell selection development and AICD [38], [39]. GCs have long been known to induce cell death (apoptosis) in the thymus (40). The CD4⁺CD8⁺TCR^low subset, although expressing less than half the GR density of CD4^?CD8^?TCR^? cells, is the most sensitive subset to GCs-induced apoptosis [41], with which the results in our study were consistent. Moreover, after RU486 treatment, the apoptosis in the thymus of mice infected with V_K627 or rTs_E627K was decreased, and the CD4⁺CD8⁺ thymocytes and lung T cells were increased at day 5 p.i. So the GCs play an important role in increased apoptosis indirectly induced by influenza virus infection.
In the infection with herpes simplex virus-1 (HSV-1), the exposure to stress or corticosterone in the earliest stages of infection is sufficient to suppress the subsequent antiviral immune response in a glucocorticoid receptor-dependent manner [42]. The ability of popliteal lymph nodes-derived dendritic cells to prime HSV-1?specific CD8+ T cells is functionally impaired and the administration of the GR antagonist completely prevented stress from reducing the numbers of activated, functional CD8+ T cells [42]. Recent study proves early GCs exposure would increase the risk of developing critical disease in humans infected by pandemic influenza A (H1N1) virus infection [43]. Influenza virus infection could trigger a stress response leading to a sustained increase in serum GCs, which compromised innate immune response, but virus-induced GCs production is necessary to prevent lethal immunopathology during coinfection [21]. So, we hypothesized the physiological level of GCs may be beneficial to regulating host immune response by modulating the production of cytokines, but higher levels may be detrimental to host defense against virus infection. After RU486 treatment, the titers of cytokines were increased, which resulted in enhancement of inflammatory cells infiltration. More importantly, the virus load in lungs had a reduced trend and the weight loss of mice was decreased following treatment with RU486. But the two cell types other than dentritic cells did not fully recover the same level as in rV_K627E- or Ts_E627-infected mice. So, the higher GCs level triggered by the V_K627 or rTs_E627K infection may not be the sole factor causing increased virulence for H9N2 virus expressing PB₂ 627K, but it played an important role in enhancing pathogenicity in mice.
The interaction between the neuroendocrine and immune systems is now well demonstrated. More evidence has placed hormones and neuropeptides among potent immunomodulators, participating in various aspects of immune system function in both health and disease [44]. Hypothalamic- pituitary-adrenal (HPA) axis can be stimulated following infection of many viruses, resulting in the release of adrenal GCs [45]. The production of IL-6 and IL-1β after virus infection can stimulate HPA axis, which leads to the release of GCs [46]. However, another study revealed that the high level of serum GCs induced by influenza virus infection was in part independent of systemic inflammatory cytokines [21]. So, the exact mechanism linking infection-induced tissue damage to the HPA axis activation is currently unknown.

Supporting Information

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Analysis of T cells in blood and thymus of infected mice. Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). Blood and thymuses from three mice per group per time point were collected. The percents of CD4⁺ and CD8⁺ T cells in blood (A) were analyzed by flow cytometry. The morphology of thymuses (B) was photoed and single cell suspension was prepared. The percentages of T cells (as determined by appropriate gating on labeled cells) were examined and the numbers of T cells (C) in thymus were calculated by multiplying the percentage of each cell type by the total number of viable thymus cells. Baseline from PBS inoculated mice is shown as a dashed line in each graph. The data shown in A and B represents mean ? SD for three independent experiments. *p<0.05 between V_K627 and rV_K627E; ?p<0.05 between Ts_E627 and rTs_E627K.





Figure S1.
Analysis of T cells in blood and thymus of infected mice. Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). Blood and thymuses from three mice per group per time point were collected. The percents of CD4⁺ and CD8⁺ T cells in blood (A) were analyzed by flow cytometry. The morphology of thymuses (B) was photoed and single cell suspension was prepared. The percentages of T cells (as determined by appropriate gating on labeled cells) were examined and the numbers of T cells (C) in thymus were calculated by multiplying the percentage of each cell type by the total number of viable thymus cells. Baseline from PBS inoculated mice is shown as a dashed line in each graph. The data shown in A and B represents mean ? SD for three independent experiments. *p<0.05 between V_K627 and rV_K627E; ?p<0.05 between Ts_E627 and rTs_E627K.
doi:10.1371/journal.pone.0038233.s001
(TIF)

Author Contributions

Conceived and designed the experiments: JT WQ ML. Performed the experiments: JT CZ. Analyzed the data: JT XL JH. Contributed reagents/materials/analysis tools: GQL PJ. Wrote the paper: JT.

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35. 35. Vogel AB, Haasbach E, Reiling SJ, Droebner K, Klingel K, et al. (2010) Highly pathogenic influenza virus infection of the thymus interferes with T lymphocyte development. J Immunol 185: 4824?34. doi: 10.4049/jimmunol.0903631. Find this article online
36. 36. Teodoro JG, Branton PE (1997) Regulation of apoptosis by viral gene products. J Virol 71: 1739?46. Find this article online
37. 37. Takizawa T, Nakanishi Y (2010) Role and Pathological Significance of Apoptosis Induced by Influenza Virus Infection. The Open Antimicrobial Agents Journal 2: 22?5. doi: 10.2174/1876518101002020022. Find this article online
38. 38. Brewer JA, Kanagawa O, Sleckman BP, Muglia LJ (2002) Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo. J Immunol 169: 1837?43. Find this article online
39. 39. Hughes FM Jr, Cidlowski JA (1998) Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA degradation. J Steroid Biochem Mol Biol 65: 207?17. doi: 10.1016/S0960-0760(97)00188-X. Find this article online
40. 40. Wilckens T, De Rijk R (1997) Glucocorticoids and immune function: unknown dimensions and new frontiers. Immunol Today 18: 418?24. doi: 10.1016/S0167-5699(97)01111-0. Find this article online
41. 41. Wiegers GJ, Knoflach M, Bock G, Niederegger H, Dietrich H, et al. (2001) CD4(+)CD8(+)TCR(low) thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid-induced apoptosis. Eur J Immunol 31: 2293?301. doi: 10.1002/1521-4141(200108)31:8<2293::AID-IMMU2293>3.0.CO;2-I. Find this article online
42. 42. Elftman MD, Hunzeker JT, Mellinger JC, Bonneau RH, Norbury CC, et al. (2010) Stress-induced glucocorticoids at the earliest stages of herpes simplex virus-1 infection suppress subsequent antiviral immunity, implicating impaired dendritic cell function. J Immunol 184: 1867?75. doi: 10.4049/jimmunol.0902469. Find this article online
43. 43. Han K, Ma H, An X, Su Y, Chen J, et al. (2011) Early use of glucocorticoids was a risk factor for critical disease and death from pH1N1 infection. Clin Infect Dis 53: 326?33. doi: 10.1093/cid/cir398. Find this article online
44. 44. Herold MJ, McPherson KG, Reichardt HM (2006) Glucocorticoids in T cell apoptosis and function. Cell Mol Life Sci 63: 60?72. doi: 10.1007/s00018-005-5390-y. Find this article online
45. 45. Silverman MN, Pearce BD, Biron CA, Miller AH (2005) Immune modulation of the hypothalamic-pituitary-adrenal (HPA) axis during viral infection. Viral Immunol 18: 41?78. doi: 10.1089/vim.2005.18.41. Find this article online
46. 46. Chrousos GP (2000) The stress response and immune function: clinical implications. The 1999 Novera H. Spector Lecture. Ann N Y Acad Sci 917: 38?67. doi: 10.1111/j.1749-6632.2000.tb05371.x. Find this article online

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

GISAID Citations

A table demonstrating lateral sub-segment data transfer potential is found in the earlier preliminary overview.

H7N9 HA aa197 equates to 188T wildtype, the defining polymorphism for the dominant pH1N1 subclade. The 3 H7N9 fatality sequences are wildtype at that amino acid position and have also established an adjacent pH1N1 like revision to 189A (198A H7 numbering), the wildtype for aa189 on Pandemic H1N1. Add 226L (2 sequences) and transmission benefits.

Side by Side Matches
Human pH1N1 and emergent zoonotic H7N9
188T
189A

Numbering on the polymorphism lists is absolute (no offset), but the antigenic center of the HA can be managed to H3 numbering with a -9, e.g. 235L becomes 226L. Synonymous changes have been removed from the lists pending verification.

HA Polymorphisms
GISAID

Hospitalised

No sequences available from GISAID for current Hospitalised cases

Fatal

. . . . ChinaHangzhou1_C1_38M_2013_03_24_f (
. . . . . . . . GISAID HA EPI440095
. . . . . . . . GISAID Isolate EPI_ISL_138977
. . . . . . . . Clinical: 2013-03-07 Symptom Onset
. . . . . . . . Clinical: 2013-03-18 InPatient
. . . . . . . . Clinical: 2013-03-27 Fatality
. . . . . . . . 105 Polymorphisms (17 Amino and 88 Silent)
. . . . . . . . 11I,
. . . . . . . . 130A,
. . . . . . . . 183S,
. . . . . . . . 188V,
. . . . . . . . 195V,
. . . . . . . . 198A,
. . . . . . . . 211V,
. . . . . . . . 217N,
. . . . . . . . 235I [226I Human H3N2 Current wildtype],
. . . . . . . . . . . . [H3N2 commercial poultry
. . . . . . . . . . . . . . . . with HA 155, 156, 158, 159 VxX revisions
. . . . . . . . . . . . . . . . . . and G228S, known virulence marker],
. . . . . . . . 285N,
. . . . . . . . 307D,
. . . . . . . . 321R,
. . . . . . . . 410N,
. . . . . . . . 427I,
. . . . . . . . 455D,
. . . . . . . . 462K,
. . . . . . . . 541V)

. . . . ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f (
. . . . . . . . GISAID HA EPI439507
. . . . . . . . GISAID Isolate EPI_ISL_138739
. . . . . . . . Clinical: 2013-03-09 Symptom Onset
. . . . . . . . Clinical: 2013-03-14 Fever 39&#176; C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-18 Fever 40&#176; C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-19 InPatient, Pneumonia
. . . . . . . . Clinical: 2013-03-20 Transfer Critical Care, Severe Pneumonia,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Septic Shock, Heart Inflammation,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver Dysfunction, Acute Kidney Damage,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain Inflammation
. . . . . . . . Clinical: 2013-04-02 Remains in Critical Care
. . . . . . . . Clinical: 2013-04-09 Fatality
. . . . . . . . Clinical Tx: ECMO, Continuous Renal Replacement Therapy
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 104 Polymorphisms (17 Amino and 87 Silent)
. . . . . . . . 11I,
. . . . . . . . 130A,
. . . . . . . . 183S,
. . . . . . . . 188V,
. . . . . . . . 195V,
. . . . . . . . 198A,
. . . . . . . . 211V,
. . . . . . . . 217N,
. . . . . . . . 235L,
. . . . . . . . 285N,
. . . . . . . . 307D,
. . . . . . . . 321R,
. . . . . . . . 410N,
. . . . . . . . 427I,
. . . . . . . . 455D,
. . . . . . . . 462K,
. . . . . . . . 541V)

. . . . ChinaShanghai2_E1_27M_2013_03_05_f (
. . . . . . . . GISAID HA EPI439502
. . . . . . . . GISAID Isolate EPI_ISL_138738
. . . . . . . . Clinical: 2013-02-27 Symptom Onset
. . . . . . . . Clinical: 2013-03-04 InPatient
. . . . . . . . Clinical: 2013-03-06 Transfer Critical Care, Severe Pneumonia
. . . . . . . . Clinical: 2013-03-10 Fatality
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 105 Polymorphisms (17 Amino and 88 Silent)
. . . . . . . . 11I,
. . . . . . . . 130A,
. . . . . . . . 183S,
. . . . . . . . 188V,
. . . . . . . . 195V,
. . . . . . . . 198A,
. . . . . . . . 211V,
. . . . . . . . 217N,
. . . . . . . . 235L,
. . . . . . . . 285N,
. . . . . . . . 307D,
. . . . . . . . 321R,
. . . . . . . . 410N,
. . . . . . . . 427I,
. . . . . . . . 455D,
. . . . . . . . 462K,
. . . . . . . . 541V)

. . . . ChinaShanghai1_E1_87M_2013_02_26_f (
. . . . . . . . GISAID HA EPI439486
. . . . . . . . GISAID Isolate EPI_ISL_138737
. . . . . . . . Clinical: 2013-02-18 Symptom Onset
. . . . . . . . Clinical: 2013-02-25 InPatient
. . . . . . . . Clinical: 2013-03-04 Fatality with Brain Inflammation
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 103 Polymorphisms (15 Amino and 88 Silent)
. . . . . . . . 11I,
. . . . . . . . 130A,
. . . . . . . . 146S,
. . . . . . . . 188V,
. . . . . . . . 198A,
. . . . . . . . 211V,
. . . . . . . . 217N,
. . . . . . . . 230T,
. . . . . . . . 285D,
. . . . . . . . 292Y,
. . . . . . . . 307D,
. . . . . . . . 321R,
. . . . . . . . 427I,
. . . . . . . . 455D,
. . . . . . . . 462K)


NA Polymorphisms

Hospitalised

No sequences available from GISAID for current Hospitalised cases

Fatal

. . . . ChinaHangzhou1_C1_38M_2013_03_24_f (
. . . . . . . . GISAID NA EPI440096
. . . . . . . . GISAID Isolate EPI_ISL_138977
. . . . . . . . Clinical: 2013-03-07 Symptom Onset
. . . . . . . . Clinical: 2013-03-18 InPatient
. . . . . . . . Clinical: 2013-03-27 Fatality
. . . . . . . . 33 Polymorphisms (10 Amino and 23 Silent)
. . . . . . . . 16I [H5N1 Human Fatality China 2011],
. . . . . . . . . . . [H5N1 Human Cambodia 2005],
. . . . . . . . . . . [H5N1 Avian Rare (58) Scotland, Middle East and Asia]
. . . . . . . . . . . [pH1N1 Rare (35) Worldwide including Scotland and 1 US Low Reactor],
. . . . . . . . . . . [avH1N1farm],
. . . . . . . . 19A,
. . . . . . . . 40G,
. . . . . . . . 53T,
. . . . . . . . 81T,
. . . . . . . . 84N,
. . . . . . . . 112S,
. . . . . . . . syn142R (cGA) [syn141R (cGA)],
. . . . . . . . 335I,
. . . . . . . . 359A,
. . . . . . . . 401A)

. . . . ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f (
. . . . . . . . GISAID NA EPI439509
. . . . . . . . GISAID Isolate EPI_ISL_138739
. . . . . . . . Clinical: 2013-03-09 Symptom Onset
. . . . . . . . Clinical: 2013-03-14 Fever 39&#176; C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-18 Fever 40&#176; C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-19 InPatient, Pneumonia
. . . . . . . . Clinical: 2013-03-20 Transfer Critical Care, Severe Pneumonia,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Septic Shock, Heart Inflammation,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver Dysfunction, Acute Kidney Damage,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain Inflammation
. . . . . . . . Clinical: 2013-04-02 Remains in Critical Care
. . . . . . . . Clinical: 2013-04-09 Fatality
. . . . . . . . Clinical Tx: ECMO, Continuous Renal Replacement Therapy
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 33 Polymorphisms (10 Amino and 23 Silent)
. . . . . . . . 16I [H5N1 Human Fatality China 2011],
. . . . . . . . . . . [H5N1 Human Cambodia 2005],
. . . . . . . . . . . [H5N1 Avian Rare (58) Scotland, Middle East and Asia]
. . . . . . . . . . . [pH1N1 Rare (35) Worldwide including Scotland and 1 US Low Reactor],
. . . . . . . . . . . [avH1N1farm],
. . . . . . . . 19A,
. . . . . . . . 40G,
. . . . . . . . 53T,
. . . . . . . . 81T,
. . . . . . . . 84N,
. . . . . . . . 112S,
. . . . . . . . 335I,
. . . . . . . . 359A,
. . . . . . . . 401A,
. . . . . . . . syn435E (GAa) [syn419E (GAa)])

. . . . ChinaShanghai2_E1_27M_2013_03_05_f (
. . . . . . . . GISAID NA EPI439500
. . . . . . . . GISAID Isolate EPI_ISL_138738
. . . . . . . . Clinical: 2013-02-27 Symptom Onset
. . . . . . . . Clinical: 2013-03-04 InPatient
. . . . . . . . Clinical: 2013-03-06 Transfer Critical Care, Severe Pneumonia
. . . . . . . . Clinical: 2013-03-10 Fatality
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 33 Polymorphisms (11 Amino and 22 Silent)
. . . . . . . . 16I [H5N1 Human Fatality China 2011],
. . . . . . . . . . . [H5N1 Human Cambodia 2005],
. . . . . . . . . . . [H5N1 Avian Rare (58) Scotland, Middle East and Asia]
. . . . . . . . . . . [pH1N1 Rare (35) Worldwide including Scotland and 1 US Low Reactor],
. . . . . . . . . . . [avH1N1farm],
. . . . . . . . 19A,
. . . . . . . . 26M,
. . . . . . . . 40G,
. . . . . . . . 53T,
. . . . . . . . 81T,
. . . . . . . . 84N,
. . . . . . . . 112S,
. . . . . . . . 335I,
. . . . . . . . 359A,
. . . . . . . . 401A)

. . . . ChinaShanghai1_E1_87M_2013_02_26_f (
. . . . . . . . GISAID NA EPI439487
. . . . . . . . GISAID Isolate EPI_ISL_138737
. . . . . . . . Clinical: 2013-02-18 Symptom Onset
. . . . . . . . Clinical: 2013-02-25 InPatient
. . . . . . . . Clinical: 2013-03-04 Fatality with Brain Inflammation
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 32 Polymorphisms (10 Amino and 22 Silent)
. . . . . . . . 16I [H5N1 Human Fatality China 2011],
. . . . . . . . . . . [H5N1 Human Cambodia 2005],
. . . . . . . . . . . [H5N1 Avian Rare (58) Scotland, Middle East and Asia]
. . . . . . . . . . . [pH1N1 Rare (35) Worldwide including Scotland and 1 US Low Reactor],
. . . . . . . . . . . [avH1N1farm],
. . . . . . . . 19A,
. . . . . . . . 53T,
. . . . . . . . 81T,
. . . . . . . . 84N,
. . . . . . . . 112S,
. . . . . . . . 294K [293K],
. . . . . . . . . . . .[~292K N2 Numbering TamiFlu Resistance Marker (Aoki, et al, Antiviral Therapy)],
. . . . . . . . 335I,
. . . . . . . . 359A,
. . . . . . . . 401A)

We acknowledge the authors of the 2013-04-11 New England Journal of Medicine Original Article, "Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus" (Rongbao Gao, M.D., Bin Cao, M.D., Yunwen Hu, M.D., Zijian Feng, M.D., M.P.H.,Dayan Wang, M.D., Wanfu Hu, M.D., Jian Chen, M.D., Zhijun Jie, M.D.,Haibo Qiu, M.D., Ph.D., Ke Xu, M.D., Xuewei Xu, M.D., Hongzhou Lu, M.D., Ph.D.,Wenfei Zhu, M.D., Zhancheng Gao, M.D., Nijuan Xiang, M.D., Yinzhong Shen, M.D.,Zebao He, M.D., Yong Gu, M.D., Zhiyong Zhang, M.D., Yi Yang, M.D., Ph.D.,Xiang Zhao, M.D., Lei Zhou, M.D., Xiaodan Li, M.D., Shumei Zou, M.D.,Ye Zhang, M.D., Xiyan Li, M.D., Lei Yang, M.D., Junfeng Guo, M.D., Jie Dong, M.D.,Qun Li, M.D., Libo Dong, M.D., Yun Zhu, M.D., Tian Bai, M.D., Shiwen Wang, M.D.,Pei Hao, M.D., Weizhong Yang, M.D., Yanping Zhang, M.D., Jun Han, M.D.,Hongjie Yu, M.D., Dexin Li, M.D., George F. Gao, Ph.D., Guizhen Wu, M.D.,Yu Wang, M.D., Zhenghong Yuan, Ph.D., and Yuelong Shu, Ph.D.) on which several Clinical Progression and Treatment Modality specifics within this genetic analysis were updated.

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-04-01-22_17_09_666340 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Though H9N2 is regaled as the nearest relative on file for these internal gene segments of the H7N9 emerging zoonosis, note the highlighted areas including a fatal H5N1 human, pH1N1 in swine (with human homology) and sH3N2. These H7N9 sequences have developed from pedigrees not entirely disjunctive from human infection. The BLAST listings are abbreviated to save trees.

. . . . ChinaShanghai1_E1_87M_2013_02_26_f
. . . . . . . . GISAID Isolate EPI_ISL_138737

Details are found in attached PDF.
Citations are found in post #5.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Version 2 of Preliminary H7N9 Fatalities Overview Available

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Cross Serotype Homology
Human emergent zoonotic H7N9

Synonymous changes have been removed from the lists pending verification.

HA Polymorphisms
GISAID

Hospitalised

No sequences available from GISAID for current Hospitalised cases

Fatal

. . . . ChinaHangzhou1_C1_38M_2013_03_24_f (
. . . . . . . . GISAID HA EPI440095
. . . . . . . . GISAID Isolate EPI_ISL_138977
. . . . . . . . Clinical: 2013-03-07 Symptom Onset
. . . . . . . . Clinical: 2013-03-18 InPatient
. . . . . . . . Clinical: 2013-03-27 Fatality
. . . . . . . . 105 Polymorphisms (17 Amino and 88 Silent)
. . . . . . . . 11I [#4I H3N2, H7N7, H10N7],
. . . . . . . . 130A [118A],
. . . . . . . . . . . . [H7N7 Rare (2): Korea 2007 Passerine & commercial poultry species],
. . . . . . . . . . . . [H7N3 Rare (5): China Zhejiang 2011 (3) Wet Market Surveillance,
. . . . . . . . . . . . . . . . . . . . . . . Japan Shimane 2006 (2) Migratory Duck],
. . . . . . . . . . . . [avH1N1 with 453K],
. . . . . . . . 183S [174S Novel to H7],
. . . . . . . . . . . . [avH3N2 with 226L],
. . . . . . . . 188V [179V avH3N2],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 195V [186V H7N7 Rare],
. . . . . . . . . . . . [sH3N2 Vax Component (Vict361X217A)],
. . . . . . . . . . . . [H13N9 Avian wildtype],
. . . . . . . . . . . . [H13N2 Mammal wildtype],
. . . . . . . . . . . . [H5N1 Gain of Function Residue aa182:
. . . . . . . . . . . . . . . PubMed PMC2903244, 17108965],
. . . . . . . . 198A [189A H5N1 & pH1N1 wildtype],
. . . . . . . . 211V [202V],
. . . . . . . . . . . . [H5N1 Human Rare, 13 Cases (Asia-12, Egypt-1)],
. . . . . . . . . . . . [H5N1 China Fatalities February 2013 (2)
. . . . . . . . . . . . . . . . 21F & 31M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality January 2012
. . . . . . . . . . . . . . . . 39M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality December 2011
. . . . . . . . . . . . . . . . 39M with host species transition signals],
. . . . . . . . . . . . [H5N1 China Human 2009
. . . . . . . . . . . . . . . . 23F & 29M Recovered, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Fatality December 2008
. . . . . . . . . . . . . . . . 19F, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Pigeon 2010],
. . . . . . . . . . . . [H5N1 China Wet Market Surveillance January 2009
. . . . . . . . . . . . . . . . Hebei & Guizhou from water, feces and environment],
. . . . . . . . . . . . [H5N1 Egypt Human 2011],
. . . . . . . . . . . . [H5N1 Vietnam Fatality January 2004 (1)],
. . . . . . . . . . . . [H5N1 Vietnam Human 2004 (4)],
. . . . . . . . . . . . [H5N1 Vietnam Civet 2005],
. . . . . . . . . . . . [H5N1 Vietnam Quail 2005],
. . . . . . . . . . . . [H5N2 Asia Avian],
. . . . . . . . . . . . [H5N2 Russia, Sweden, Switzerland, Portugal & Italy Avian],
. . . . . . . . . . . . [H5N3 Asia Avian],
. . . . . . . . . . . . [H5N3 Australia, Portugal & Italy Avian],
. . . . . . . . . . . . [H6N1],
. . . . . . . . . . . . [avH3N2 & pH1N1 wildtype],
. . . . . . . . . . . . [avH1N1farm: pH1N1 Ultimate Origin reservoir],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 217N [208N H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . 235I [226I Human H3N2 Current wildtype],
. . . . . . . . . . . . [H3N2 commercial poultry
. . . . . . . . . . . . . . . . with HA 155, 156, 158, 159 VxX revisions
. . . . . . . . . . . . . . . . . . and G228S, known virulence marker],
. . . . . . . . 285N [277N avH3N2 with 158E],
. . . . . . . . . . . . [avH1N1 wildtype, H5N1, H9N2],
. . . . . . . . 307D [299D Canine H3N2],
. . . . . . . . . . . . [Human H3N2 Singular (US 2012)],
. . . . . . . . . . . . [Avian H3N8],
. . . . . . . . 321R [313R],
. . . . . . . . . . . . [H5N1 LAd with 137S, 160A, 178V, 189A, 202V, 277N, 401N, 533V],
. . . . . . . . . . . . [H9N2],
. . . . . . . . syn405I (ATc) [syn396I (ATc)],
. . . . . . . . . . . . . . . . . . .[avH3N2 with HA 226I & 277N],
. . . . . . . . 410N [401N H5N1 & pH1N1 wildtype],
. . . . . . . . . . . . [H7N7 Mammal - Equine and Seal],
. . . . . . . . . . . . [H7N7 Avian],
. . . . . . . . 427I [418I H9N2 wildtype],
. . . . . . . . 455D [446D H3N2 Human Rare (9), 2013 US with 128A & 300T mix wt, 2012-2, 2000-4, 1993-1, 1990-1],
. . . . . . . . . . . . [H7N3 Avian Commercial Poultry species],
. . . . . . . . . . . . [H7N2, H7N6 Avian Commercial Poultry species],
. . . . . . . . 462K [453K pH1N1 Clade1.Upsilon HighCFR],
. . . . . . . . . . . . [avH1N1, H3N2, H6N1, H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 541V [533V H5N1 wildtype],
. . . . . . . . . . . . [sH3N2 South Africa 2012])

. . . . ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f (
. . . . . . . . GISAID HA EPI439507
. . . . . . . . GISAID Isolate EPI_ISL_138739
. . . . . . . . Clinical: 2013-03-09 Symptom Onset
. . . . . . . . Clinical: 2013-03-14 Fever 39? C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-18 Fever 40? C, OutPatient AntiViral
. . . . . . . . Clinical: 2013-03-19 InPatient, Pneumonia
. . . . . . . . Clinical: 2013-03-20 Transfer Critical Care, Severe Pneumonia,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Septic Shock, Heart Inflammation,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver Dysfunction, Acute Kidney Damage,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain Inflammation
. . . . . . . . Clinical: 2013-04-02 Remains in Critical Care
. . . . . . . . Clinical: 2013-04-09 Fatality
. . . . . . . . Clinical Tx: ECMO, Continuous Renal Replacement Therapy
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 104 Polymorphisms (17 Amino and 87 Silent)
. . . . . . . . 11I [#4I H3N2, H7N7, H10N7],
. . . . . . . . 130A [118A],
. . . . . . . . . . . . [H7N7 Rare (2): Korea 2007 Passerine & commercial poultry species],
. . . . . . . . . . . . [H7N3 Rare (5): China Zhejiang 2011 (3) Wet Market Surveillance,
. . . . . . . . . . . . . . . . . . . . . . . Japan Shimane 2006 (2) Migratory Duck],
. . . . . . . . 183S [174S Novel to H7],
. . . . . . . . . . . . [avH3N2 with 226L],
. . . . . . . . 188V [179V avH3N2],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 195V [186V H7N7 Rare],
. . . . . . . . . . . . [sH3N2 Vax Component (Vict361X217A)],
. . . . . . . . . . . . [H13N9 Avian wildtype],
. . . . . . . . . . . . [H13N2 Mammal wildtype],
. . . . . . . . . . . . [H5N1 Gain of Function Residue aa182:
. . . . . . . . . . . . . . . PubMed PMC2903244, 17108965],
. . . . . . . . 198A [189A H5N1 & pH1N1 wildtype],
. . . . . . . . 211V [202V],
. . . . . . . . . . . . [H5N1 Human Rare, 13 Cases (Asia-12, Egypt-1)],
. . . . . . . . . . . . [H5N1 China Fatalities February 2013 (2)
. . . . . . . . . . . . . . . . 21F & 31M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality January 2012
. . . . . . . . . . . . . . . . 39M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality December 2011
. . . . . . . . . . . . . . . . 39M with host species transition signals],
. . . . . . . . . . . . [H5N1 China Human 2009
. . . . . . . . . . . . . . . . 23F & 29M Recovered, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Fatality December 2008
. . . . . . . . . . . . . . . . 19F, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Pigeon 2010],
. . . . . . . . . . . . [H5N1 China Wet Market Surveillance January 2009
. . . . . . . . . . . . . . . . Hebei & Guizhou from water, feces and environment],
. . . . . . . . . . . . [H5N1 Egypt Human 2011],
. . . . . . . . . . . . [H5N1 Vietnam Fatality January 2004 (1)],
. . . . . . . . . . . . [H5N1 Vietnam Human 2004 (4)],
. . . . . . . . . . . . [H5N1 Vietnam Civet 2005],
. . . . . . . . . . . . [H5N1 Vietnam Quail 2005],
. . . . . . . . . . . . [H5N2 Asia Avian],
. . . . . . . . . . . . [H5N2 Russia, Sweden, Switzerland, Portugal & Italy Avian],
. . . . . . . . . . . . [H5N3 Asia Avian],
. . . . . . . . . . . . [H5N3 Australia, Portugal & Italy Avian],
. . . . . . . . . . . . [H6N1],
. . . . . . . . . . . . [avH3N2 & pH1N1 wildtype],
. . . . . . . . . . . . [avH1N1farm: pH1N1 Ultimate Origin reservoir],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 217N [208N H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . 235L [226L],
. . . . . . . . . . . . [avH3N2],
. . . . . . . . . . . . [pH1N1 sw],
. . . . . . . . . . . . [H9N2 with 208N],
. . . . . . . . 285N [277N avH3N2 with 158E],
. . . . . . . . . . . . [avH1N1 wildtype, H5N1, H9N2],
. . . . . . . . 307D [299D Canine H3N2],
. . . . . . . . . . . . [Human H3N2 Singular (US 2012)],
. . . . . . . . . . . . [Avian H3N8],
. . . . . . . . 321R [313R],
. . . . . . . . . . . . [H5N1 LAd with 137S, 160A, 178V, 189A, 202V, 277N, 401N, 533V],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 410N [401N H5N1 & pH1N1 wildtype],
. . . . . . . . . . . . [H7N7 Mammal - Equine and Seal],
. . . . . . . . . . . . [H7N7 Avian],
. . . . . . . . 427I [418I H9N2 wildtype],
. . . . . . . . 455D [446D H3N2 Human Rare (9), 2013 US with 128A & 300T mix wt, 2012-2, 2000-4, 1993-1, 1990-1],
. . . . . . . . . . . . [H7N3 Avian Commercial Poultry species],
. . . . . . . . . . . . [H7N2, H7N6 Avian Commercial Poultry species],
. . . . . . . . 462K [453K pH1N1 Clade1.Upsilon HighCFR],
. . . . . . . . . . . . [avH1N1, H3N2, H6N1, H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 541V [533V H5N1 wildtype],
. . . . . . . . . . . . [sH3N2 South Africa 2012])

. . . . ChinaShanghai2_E1_27M_2013_03_05_f (
. . . . . . . . GISAID HA EPI439502
. . . . . . . . GISAID Isolate EPI_ISL_138738
. . . . . . . . Clinical: 2013-02-27 Symptom Onset
. . . . . . . . Clinical: 2013-03-04 InPatient
. . . . . . . . Clinical: 2013-03-06 Transfer Critical Care, Severe Pneumonia
. . . . . . . . Clinical: 2013-03-10 Fatality
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 105 Polymorphisms (17 Amino and 88 Silent)
. . . . . . . . 11I [#4I H3N2, H7N7, H10N7],
. . . . . . . . 130A [118A],
. . . . . . . . . . . . [H7N7 Rare (2): Korea 2007 Passerine & commercial poultry species],
. . . . . . . . . . . . [H7N3 Rare (5): China Zhejiang 2011 (3) Wet Market Surveillance,
. . . . . . . . . . . . . . . . . . . . . . . Japan Shimane 2006 (2) Migratory Duck],
. . . . . . . . 183S [174S Novel to H7],
. . . . . . . . . . . . [avH3N2 with 226L],
. . . . . . . . 188V [179V avH3N2],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 195V [186V H7N7 Rare],
. . . . . . . . . . . . [sH3N2 Vax Component (Vict361X217A)],
. . . . . . . . . . . . [H13N9 Avian wildtype],
. . . . . . . . . . . . [H13N2 Mammal wildtype],
. . . . . . . . . . . . [H5N1 Gain of Function Residue aa182:
. . . . . . . . . . . . . . . PubMed PMC2903244, 17108965],
. . . . . . . . 198A [189A H5N1 & pH1N1 wildtype],
. . . . . . . . 211V [202V],
. . . . . . . . . . . . [H5N1 Human Rare, 13 Cases (Asia-12, Egypt-1)],
. . . . . . . . . . . . [H5N1 China Fatalities February 2013 (2)
. . . . . . . . . . . . . . . . 21F & 31M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality January 2012
. . . . . . . . . . . . . . . . 39M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality December 2011
. . . . . . . . . . . . . . . . 39M with host species transition signals],
. . . . . . . . . . . . [H5N1 China Human 2009
. . . . . . . . . . . . . . . . 23F & 29M Recovered, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Fatality December 2008
. . . . . . . . . . . . . . . . 19F, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Pigeon 2010],
. . . . . . . . . . . . [H5N1 China Wet Market Surveillance January 2009
. . . . . . . . . . . . . . . . Hebei & Guizhou from water, feces and environment],
. . . . . . . . . . . . [H5N1 Egypt Human 2011],
. . . . . . . . . . . . [H5N1 Vietnam Fatality January 2004 (1)],
. . . . . . . . . . . . [H5N1 Vietnam Human 2004 (4)],
. . . . . . . . . . . . [H5N1 Vietnam Civet 2005],
. . . . . . . . . . . . [H5N1 Vietnam Quail 2005],
. . . . . . . . . . . . [H5N2 Asia Avian],
. . . . . . . . . . . . [H5N2 Russia, Sweden, Switzerland, Portugal & Italy Avian],
. . . . . . . . . . . . [H5N3 Asia Avian],
. . . . . . . . . . . . [H5N3 Australia, Portugal & Italy Avian],
. . . . . . . . . . . . [H6N1],
. . . . . . . . . . . . [avH3N2 & pH1N1 wildtype],
. . . . . . . . . . . . [avH1N1farm: pH1N1 Ultimate Origin reservoir],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 217N [208N H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . 235L [226L],
. . . . . . . . . . . . [avH3N2],
. . . . . . . . . . . . [pH1N1 sw],
. . . . . . . . . . . . [H9N2 with 208N],
. . . . . . . . 285N [277N avH3N2 with 158E],
. . . . . . . . . . . . [avH1N1 wildtype, H5N1, H9N2],
. . . . . . . . 307D [299D Canine H3N2],
. . . . . . . . . . . . [Human H3N2 Singular (US 2012)],
. . . . . . . . . . . . [Avian H3N8],
. . . . . . . . 321R [313R],
. . . . . . . . . . . . [H5N1 LAd with 137S, 160A, 178V, 189A, 202V, 277N, 401N, 533V],
. . . . . . . . . . . . [H9N2],
. . . . . . . . syn337K (AAa) [syn329K (AAa)],
. . . . . . . . 410N [401N H5N1 & pH1N1 wildtype],
. . . . . . . . . . . . [H7N7 Mammal - Equine and Seal],
. . . . . . . . . . . . [H7N7 Avian],
. . . . . . . . 427I [418I H9N2 wildtype],
. . . . . . . . 455D [446D H3N2 Human Rare (9), 2013 US with 128A & 300T mix wt, 2012-2, 2000-4, 1993-1, 1990-1],
. . . . . . . . . . . . [H7N3 Avian Commercial Poultry species],
. . . . . . . . . . . . [H7N2, H7N6 Avian Commercial Poultry species],
. . . . . . . . 462K [453K pH1N1 Clade1.Upsilon HighCFR],
. . . . . . . . . . . . [avH1N1, H3N2, H6N1, H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 541V [533V H5N1 wildtype],
. . . . . . . . . . . . [sH3N2 South Africa 2012])

. . . . ChinaShanghai1_E1_87M_2013_02_26_f (
. . . . . . . . GISAID HA EPI439486
. . . . . . . . GISAID Isolate EPI_ISL_138737
. . . . . . . . Clinical: 2013-02-18 Symptom Onset
. . . . . . . . Clinical: 2013-02-25 InPatient
. . . . . . . . Clinical: 2013-03-04 Fatality with Brain Inflammation
. . . . . . . . Clinical Rx: Oseltamivir, Steroid, IV Immunoglobulin, Antibiotic Package
. . . . . . . . 103 Polymorphisms (15 Amino and 88 Silent)
. . . . . . . . 11I [#4I H3N2, H7N7, H10N7],
. . . . . . . . 130A [118A],
. . . . . . . . . . . . [H7N7 Rare (2): Korea 2007 Passerine & commercial poultry species],
. . . . . . . . . . . . [H7N3 Rare (5): China Zhejiang 2011 (3) Wet Market Surveillance,
. . . . . . . . . . . . . . . . . . . . . . . Japan Shimane 2006 (2) Migratory Duck],
. . . . . . . . 146S [138S],
. . . . . . . . . . . . [H9N2 Human Asia 2009 (2)],
. . . . . . . . . . . . [H9N2 China anser 2011],
. . . . . . . . . . . . [H9N2 China quail, et al Wet Market Surveillance, 2000-2005],
. . . . . . . . . . . . [H9N2 Middle East commercial poultry, 2003-2012],
. . . . . . . . . . . . [H7N7 Near-Passerine, Companion Species Singular & Distant-to-Relation],
. . . . . . . . . . . . [H7N7 Amino Residue 138T Singular equine, Distant-to-Relation, SeroType Transition with H3N8 internal involvement],
. . . . . . . . . . . . [H5N1 Human Fatality],
. . . . . . . . . . . . [H5N9 Commercial Poultry species],
. . . . . . . . . . . . [H3N2 Human Rare (95):
. . . . . . . . . . . . . . . . Re-Emergent in Coastal US:
. . . . . . . . . . . . . . . . 2013 (3):
. . . . . . . . . . . . . . . . . . . EPI442834 North Carolina05_2013_03_07,
. . . . . . . . . . . . . . . . . . . EPI441082 Oregon01_E3_19F_2013_01_11,
. . . . . . . . . . . . . . . . . . . EPI418249 Texas01_2013_01_08,
. . . . . . . . . . . . . . . . 2012 (8),
. . . . . . . . . . . . . . . . 2011 (19),
. . . . . . . . . . . . . . . . 2010 (9), 2009 (20), 2008 (20) , 2007 (15), 2006 (1)],
. . . . . . . . . . . . [avH3N2 SouthDakota],
. . . . . . . . . . . . [pH1N1 Human Rare (6)],
. . . . . . . . . . . . [zH1N1 Mammal (Mink 2012)],
. . . . . . . . . . . . [avH1N1farm: pH1N1 Ultimate Origin reservoir],
. . . . . . . . . . . . [H6N1, H10N7],
. . . . . . . . 188V [179V avH3N2],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 198A [189A H5N1 & pH1N1 wildtype],
. . . . . . . . 211V [202V],
. . . . . . . . . . . . [H5N1 Human Rare, 13 Cases (Asia-12, Egypt-1)],
. . . . . . . . . . . . [H5N1 China Fatalities February 2013 (2)
. . . . . . . . . . . . . . . . 21F & 31M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality January 2012
. . . . . . . . . . . . . . . . 39M with host species transition signals,
. . . . . . . . . . . . . . . . . . . including 185S matching H7N9 wildtype],
. . . . . . . . . . . . [H5N1 China Fatality December 2011
. . . . . . . . . . . . . . . . 39M with host species transition signals],
. . . . . . . . . . . . [H5N1 China Human 2009
. . . . . . . . . . . . . . . . 23F & 29M Recovered, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Fatality December 2008
. . . . . . . . . . . . . . . . 19F, associated with Wet Market],
. . . . . . . . . . . . [H5N1 China Pigeon 2010],
. . . . . . . . . . . . [H5N1 China Wet Market Surveillance January 2009
. . . . . . . . . . . . . . . . Hebei & Guizhou from water, feces and environment],
. . . . . . . . . . . . [H5N1 Egypt Human 2011],
. . . . . . . . . . . . [H5N1 Vietnam Fatality January 2004 (1)],
. . . . . . . . . . . . [H5N1 Vietnam Human 2004 (4)],
. . . . . . . . . . . . [H5N1 Vietnam Civet 2005],
. . . . . . . . . . . . [H5N1 Vietnam Quail 2005],
. . . . . . . . . . . . [H5N2 Asia Avian],
. . . . . . . . . . . . [H5N2 Russia, Sweden, Switzerland, Portugal & Italy Avian],
. . . . . . . . . . . . [H5N3 Asia Avian],
. . . . . . . . . . . . [H5N3 Australia, Portugal & Italy Avian],
. . . . . . . . . . . . [H6N1],
. . . . . . . . . . . . [avH3N2 & pH1N1 wildtype],
. . . . . . . . . . . . [avH1N1farm: pH1N1 Ultimate Origin reservoir],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918]
. . . . . . . . 217N [208N H9N2],
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . 230T [221T pH1N1 Rare (2), US 2009-06 & Cuba 2009],
. . . . . . . . 285D [277D pH1N1 wildtype],
. . . . . . . . 292Y [284Y H7N7 Avian Rare (8, During Netherlands human outbreak)],
. . . . . . . . 307D [299D Canine H3N2],
. . . . . . . . . . . . [Human H3N2 Singular (US 2012)],
. . . . . . . . . . . . [Avian H3N8],
. . . . . . . . 321R [313R],
. . . . . . . . . . . . [H5N1 LAd with 137S, 160A, 178V, 189A, 202V, 277N, 401N, 533V],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 427I [418I H9N2 wildtype],
. . . . . . . . 455D [446D H3N2 Human Rare (9), 2013 US with 128A & 300T mix wt, 2012-2, 2000-4, 1993-1, 1990-1],
. . . . . . . . . . . . [H7N3 Avian Commercial Poultry species],
. . . . . . . . . . . . [H7N2, H7N6 Avian Commercial Poultry species],
. . . . . . . . 462K [453K pH1N1 Clade1.Upsilon HighCFR],
. . . . . . . . . . . . [avH1N1, H3N2, H6N1, H9N2]
. . . . . . . . . . . . [WSN_1933, WSZ_1933],
. . . . . . . . . . . . [1918])


GISAID Citations

We acknowledge the authors, originating and submitting laboratories of the sequences from GISAID?s EpiFlu? Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

We acknowledge the authors of the 2013-04-11 New England Journal of Medicine Original Article, "Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus" (Rongbao Gao, M.D., Bin Cao, M.D., Yunwen Hu, M.D., Zijian Feng, M.D., M.P.H.,Dayan Wang, M.D., Wanfu Hu, M.D., Jian Chen, M.D., Zhijun Jie, M.D.,Haibo Qiu, M.D., Ph.D., Ke Xu, M.D., Xuewei Xu, M.D., Hongzhou Lu, M.D., Ph.D.,Wenfei Zhu, M.D., Zhancheng Gao, M.D., Nijuan Xiang, M.D., Yinzhong Shen, M.D.,Zebao He, M.D., Yong Gu, M.D., Zhiyong Zhang, M.D., Yi Yang, M.D., Ph.D.,Xiang Zhao, M.D., Lei Zhou, M.D., Xiaodan Li, M.D., Shumei Zou, M.D.,Ye Zhang, M.D., Xiyan Li, M.D., Lei Yang, M.D., Junfeng Guo, M.D., Jie Dong, M.D.,Qun Li, M.D., Libo Dong, M.D., Yun Zhu, M.D., Tian Bai, M.D., Shiwen Wang, M.D.,Pei Hao, M.D., Weizhong Yang, M.D., Yanping Zhang, M.D., Jun Han, M.D.,Hongjie Yu, M.D., Dexin Li, M.D., George F. Gao, Ph.D., Guizhen Wu, M.D.,Yu Wang, M.D., Zhenghong Yuan, Ph.D., and Yuelong Shu, Ph.D.) on which several Clinical Progression and Treatment Modality specifics within this genetic analysis were updated.
__________________

[mixin]

Re: China - H7N9 Human Isolates on Deposit at GISAID

NS1,
Can you share with us what sequence(s) you are using for a consensus? It looks like A/brambling/Beijing/16/2012 (A/H9N2) would be a good one for some of the segments.

[Giuseppe]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Could we scan into antigenic site of the H7N9?

Is it possible to extrapolate conserved epitopes of H5/H9 viruses?

This may help to explain the lack of paediatric patients so far.

Children, playing outdoor, may have some kind of previous immunity after H5/H9 infection...

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

NS1,
Can you share with us what sequence(s) you are using for a consensus? It looks like A/brambling/Beijing/16/2012 (A/H9N2) would be a good one for some of the segments.

The HA and NA segments are not reassortments from H9. We constructed reference sequences based on the H7N9 avian sub-clade most resembling the human sequences.

The HA and NA of the human sequences are quite divergent from the typical H7N9 (if a typical idea can be drawn from a population of 27), but 2 very distinct sub-clades emerge on inspection. An item sampled 2011_04 is the most recent isolate from the sub-clade that is loosely similar to the human H7N9 set, but no individual item will be an obvious selection for a reference point.