Announcement

**NS1** · April 11, 2013, 01:16 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Potential H5N1 Involvement
in H7N9 Hemagglutinin Emergence

The 211 amino residue is highly conserved in H7 Hemagglutinin as Isoleucine, but the position has seen previous variance to Valine in isolates from 7 horses almost 40 years ago and from 4 avian isolates prior to 1933. The Valine revision located on all emergent H7N9, including the Wet Market avian and environmental samples, is also recently seen as a polymorphism on Fatal Human H5N1. More importantly, this 211V (202V) that is Rare to H7 is conspicuously enough also Rare to H5. Although the Valine is found less than 150 times (147 Cases) in the GISAID database of 5,195 H5N1 HA segments, human fatalities are represented in 5 of the calendar years between 2004 and today, with 4 of those 6 deaths in a 14 month window from December 28, 2011 to February 9, 2013.

The polymorphism is known to be associated with Wet Markets, as 6 environmental samples from 2009 in Hebei and Guizhou traced to dominant Valine at this position within the H5N1 serotype. The 3 human H5N1 cases in the 2008/2009 period, including a fatality, are also associated with Wet Markets. One civet, one quail and one pigeon feature this polymorphism in H5N1. Additional avian host types numbering 18 and inclusive of commercial poultry species have carried the polymorphism in H5N1, H5N2 and H5N3 between 1993 and 2012. The change is not reported in avian H5N1 after 2011, but is reported in duck H5N2 and H5N3 during 2012.

Perhaps we are looking at the work of a potential host species transitioner for this H7 background . . . not quite mammalian, not quite avian . . . a buffer of some sort?

. . . . . . . . 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]

Ongoing Sequence Analysis - Open-Access, Full-Text
GISAID Citation

**AlaskaDenise** · April 11, 2013, 02:50 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

The paper discusses mutations between 2 patients (H2H), where the second patient had only ONE mutation different (presumed related to antiviral use). That may address JJackson's question - the lack of variation. As GSGS posted, it changed during treatment of one patient:

Emergence of Oseltamivir-Resistant Pandemic H1N1 Virus during Prophylaxis

(snipped)

The 2009 H1N1 viral isolate collected from the index patient before oseltamivir therapy was susceptible to oseltamivir (50% inhibitory concentration, 0.27 nM) and zanamivir (50% inhibitory concentration, 0.18 nM), whereas the father's 2009 H1N1 viral isolate was resistant to oseltamivir (50% inhibitory concentration, >400 nM) but susceptible to zanamivir (50% inhibitory concentration, 0.12 nM). Complete 2009 H1N1 virus genomes of the father's virus (GenBank accession number, FN434454) differed from the index patient's virus (GenBank accession number, FN434445) by only one substitution (H275Y) in the neuraminidase protein.

Another case supporting GSGS's comment about resistance developing during treatment is here:

Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection

Influenza A (H5N1) virus with an amino acid substitution in neuraminidase conferring high-level resistance to oseltamivir was isolated from two of eight Vietnamese patients during oseltamivir treatment. Both patients died of influenza A (H5N1) virus infection, despite early initiation of treatment in one patient. Surviving patients had rapid declines in the viral load to undetectable levels during treatment. These observations suggest that resistance can emerge during the currently recommended regimen of oseltamivir therapy and may be associated with clinical deterioration and that the strategy for the treatment of influenza A (H5N1) virus infection should include additional antiviral agents......

.

**NS1** · April 11, 2013, 04:05 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Camster,

What is the probability that US agencies will announce emergent H7 cases along the coasts before the end of the month?

**Giuseppe** · April 12, 2013, 03:56 AM

China - H7N9 Human Isolates on Deposit at GISAID

[Source: Eurosurveillance, full text: (LINK). Abstract, edited.]

Eurosurveillance, Volume 18, Issue 15, 11 April 2013

Rapid communications

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013

T Kageyama<SUP>1</SUP><SUP>,2</SUP>, S Fujisaki<SUP>1</SUP><SUP>,2</SUP>, E Takashita<SUP>1</SUP>, H Xu<SUP>1</SUP>, S Yamada<SUP>3</SUP>, Y Uchida<SUP>4</SUP>, G Neumann<SUP>5</SUP>, T Saito<SUP>4</SUP><SUP>,6</SUP>, Y Kawaoka<SUP>3</SUP><SUP>,5</SUP><SUP>,7</SUP><SUP>,8</SUP>, M Tashiro ()<SUP>1</SUP>

Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan
These authors contributed equally to this work
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization, Ibaraki, Japan
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States
The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan
ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan
Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan

<HR>
Citation style for this article: Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M. Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013. Euro Surveill. 2013;18(15):pii=20453. Available online: http://www.eurosurveillance.org/View...rticleId=20453

Date of submission: 08 April 2013 <HR>
Novel influenza viruses of the H7N9 subtype have infected 33 and killed nine people in China as of 10 April 2013. Their haemagglutinin (HA) and neuraminidase genes probably originated from Eurasian avian influenza viruses; the remaining genes are closely related to avian H9N2 influenza viruses. Several characteristic amino acid changes in HA and the PB2 RNA polymerase subunit probably facilitate binding to human-type receptors and efficient replication in mammals, respectively, highlighting the pandemic potential of the novel viruses.

<HR>

Humans are rarely infected with avian influenza viruses, with the exception of highly pathogenic avian influenza A(H5N1) viruses, which have caused 634 infections and 371 deaths as of 12 March 2013 [1]. A few isolated cases of human infection with viruses of the H7N2, H7N3, and H7N5 subtypes have been reported, but none were fatal [2-11]. In 2003, in the Netherlands, 89 people were infected with an influenza virus of the H7N7 subtype that caused conjunctivitis and one fatality [5,7].

On 19 February 2013, an 87 year-old man in Shanghai developed a respiratory infection and died on 4 March, and on 27 February 2013, a 27 year-old pork seller in a Shanghai market became ill and died on 10 March. A 35 year-old woman in Chuzhou City in Anhui province (west of Shanghai), who had contact with poultry, became ill on 15 March 2013, and remains hospitalised in critical condition. There is no known epidemiological relationship among these three cases. A 38 year-old man in Hangzhou (Zhejiang province, south of Shanghai) became ill on 7 March 2013 and died on 27 March.

All four cases presented with respiratory infections that progressed to severe pneumonia and breathing difficulties.

On 31 March 2013, the Chinese Centre for Disease Control and Prevention announced the isolation in embryonated eggs of avian influenza viruses of the H7N9 subtype (designated A/Shanghai/1/2013, A/Shanghai/2/2013, and A/Anhui/1/2013) from the first three cases. The sequences of the coding regions of all eight viral genes were deposited in the influenza sequence database of the Global Initiative on Sharing All Influenza Data (GISAID) on 31 March (Table 1). On 5 April 2013, the Hangzhou Center for Disease Control and Prevention deposited the haemagglutinin (HA), neuraminidase (NA), and matrix (M) gene sequences of A/Hongzhou/1/2013 virus (Table 1), which was isolated in cell culture from samples obtained from the 38 year-old man.

Table 1. Origin of influenza A(H7N9) isolates included in the phylogenetic analysis, China, February?April 2013 (n=7)

__________________

All four human influenza A(H7N9) viruses are similar at the nucleotide and amino acid levels, suggesting a common ancestor. The HA gene of the novel viruses belongs to the Eurasian lineage of avian influenza viruses and shares ca. 95% identity with the HA genes of low pathogenic avian influenza A(H7N3) viruses isolated in 2011 in Zhejiang province (south of Shanghai) (Figure 1, Table 2). The NA gene of the novel viruses is ca. 96% identical to the low pathogenic avian influenza A(H11N9) viruses isolated in 2010 in the Czech Republic (Figure 1, Table 2).

Figure 1. Phylogenetic analysis of the haemagglutinin (A) and neuraminidase (B) genes of the novel influenza A(H7N9) viruses, China, February - April 2013 (n=7)

__________________

Table 2. Nucleotide identity of novel influenza A(H7N9) virus genes and their closest relative, China, February - April 2013

__________________

The sequences of the remaining viral genes are closely related (>97% identity) to avian influenza A(H9N2) viruses, which recently circulated in poultry in Shanghai, Zhejiang, Jiangsu, and neighbouring provinces of Shanghai (Table 2, Figure 2).

These findings strongly suggest that the novel influenza A(H7N9) viruses are reassortants that acquired their H7 HA and N9 NA genes from avian influenza viruses, and their remaining genes from recent influenza A(H9N2) poultry viruses (Figure 1, Figure 3, Table 2).

Figure 2. Phylogenetic analysis of the six remaining genes of the novel influenza A(H7N9) viruses, China, February ? April, 2013 (n=7)

__________________

Figure3. Schematic diagram of novel influenza A(H7N9) virus generation

__________________

At the nucleotide level, A/Shanghai/2/2013, A/Anhui/1/2013, and A/Hangzhou/1/2013 share more than 99% identity and differ by no more than three nucleotides per gene, even though they were isolated in different cities several hundred kilometres apart. On 7 April 2013, the Harbin Veterinary Research Institute deposited the full genome sequences of isolates from a pigeon (A/pigeon/Shanghai/S1069/2013), a chicken (A/chicken/Shanghai/S1053/2013), and an environmental sample (A/environment/Shanghai/S1088/2013) that were collected on 2 and 3 April from a Shanghai market (Table 1). All eight genes of these three isolates are similar to those of A/Shanghai/2/2013 and A/Anhui/1/2013 at the nucleotide level, except for the PB1 gene of A/pigeon/Shanghai/S1069/2013, which belongs to a different lineage than the PB1 of the other H7N9 isolates (Figures 1 and 2).

Interestingly, A/Shanghai/1/2013 and A/Shanghai/2/2013 differ by 52 nucleotides (for example, there are 13 nucleotide and nine amino acid differences in their HA sequences) even though these two cases were identified in the same city and at around the same time. These findings suggest that A/Shanghai/2/2013, A/Anhui/1/2013, A/Hangzhou/1/2013, as well as the viruses from the chicken and the environment, share a closely related source of infection, whereas A/Shanghai/1/2013 and A/pigeon/Shanghai/S1069/2013 are likely to have originated from other sources.

Highly pathogenic avian influenza viruses are characterised by a series of basic amino acids at the HA cleavage site that enable systemic virus spread. The HA cleavage sequence of the novel influenza A(H7N9) viruses possesses a single basic amino acid (EIPKGR*GL; *indicates the cleavage site), suggesting that these viruses are of low pathogenicity in avian species.

The amino acid sequence of the receptor-binding site (RBS) of HA determines preference for human- or avian-type receptors. At this site, A/Shanghai1/2013 encodes an S138A mutation (H3 numbering; Figure 4, Table 3), whereas A/Shanghai/2/2013, A/Anhui/1/2013, the two avian isolates, and the virus from the environmental sample encode G186V and Q226L mutations; any of these three mutations could increase the binding of avian H5 and H7 viruses to human-type receptors [12-14]. The finding of mammalian-adapting mutations in the RBS of these novel viruses is cause for concern. The A/Hangzhou/1/2013 isolate encodes isoleucine at position 226, which is found in seasonal influenza A(H3N2) viruses.

Figure 4. Amino acid changes in the novel influenza A(H7N9) viruses that may affect their receptor-binding properties, China, February - April 2013 (n=7)

__________________

Table 3. Selected characteristic amino acids of the novel influenza A(H7N9) viruses, China, February - April 2013 (n=7)

__________________

In addition, all seven influenza A(H7N9) viruses possess a T160A substitution (H3 numbering; Table 3) in HA, which is found in recently circulating H7 viruses; this mutation leads to the loss of an N-glycosylation site at position 158 (H3 numbering; position 149 in H7 numbering), which results in increased virus binding to human-type receptors [15].

Lysine at position 627 of the polymerase PB2 protein is essential for the efficient replication of avian influenza viruses in mammals [16] and has been detected in highly pathogenic avian influenza A(H5N1) viruses and in the influenza A(H7N7) virus isolated from the fatal case in the Netherlands in 2003 [17]. PB2-627K is rare among avian H9N2 PB2 proteins (i.e. it has been found in only five of 827 isolates). In keeping with this finding, the avian and environmental influenza A(H7N9) viruses analysed here encode PB2-627E. By contrast, all four human H7N9 viruses analysed here encode PB2-627K (Table 3).

Antiviral compounds are the first line of defense against novel influenza viruses until vaccines become available. All seven novel influenza A(H7N9) viruses sequenced to date encode the S31N substitution in the viral ion channel M2 (encoded by the M segment) (Table 3), which confers resistance to ion channel inhibitors [18,19]. Based on the sequences of their NA proteins, all H7N9 viruses analysed here, with the exception of A/Shanghai/1/2013, should be sensitive to neuraminidase inhibitors (Table 3). However, the R294K mutation in the NA protein of A/Shanghai/1/2013 is known to confer resistance to NA inhibitors in N2 and N9 subtype viruses [20], and is therefore of great concern.

All H7N9 viruses encode a deletion at positions 69?73 of the NA stalk region (Table 3), which is reported to occur upon virus adaptation to terrestrial birds. This finding suggests that the novel H7N9 viruses (or their ancestor) may have circulated in terrestrial birds before infecting humans.

Moreover, this deletion is associated with increased virulence in mammals [21].

The influenza A virus PB1-F2 protein (encoded by the PB1 segment) is also associated with virulence. The available sequences indicate that the H7N9 PB1 genes of all of the human viruses encode a full-length PB1-F2 of 90 amino acids, but lack the N66S mutation that is associated with the increased pathogenicity of the 1918 pandemic virus and the highly pathogenic avian influenza A(H5N1) viruses [22].

Interestingly, the pigeon isolate encodes a truncated PB1-F2 of only 25 amino acids; the significance of this truncation is unknown.

The NS1 protein (encoded by the NS segment) is an interferon antagonist with several functions in the viral life cycle. All available H7N9 NS1 sequences lack the C-terminal PDZ domain-binding motif; the lack of the PDZ domain-binding motif may attenuate these viruses in mammals [23].

Other amino acids in the NS1 and matrix (M1; encoded by the M segment) proteins of the novel viruses are also associated with increased virulence (Table 3) [24.25]. However, these amino acids are found in many avian influenza viruses, and therefore, their significance for the biological properties of the novel influenza A(H7N9) viruses is currently unclear.

In conclusion, we here present a biological evaluation of the sequences of the avian influenza A(H7N9) viruses that caused fatal human infections in China. These viruses possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans and raise concerns regarding their pandemic potential.
__________________

Acknowledgements

We are grateful to Dr. Shu Yuelong, Chinese National Influenza Center, Chinese Center for Disease Control and Prevention, Beijing, China, for his rapid publication of the entire gene sequence data of A(H7N9) viruses isolated from human cases in China, and also for his information sharing and advice to this study. We also thank Susan Watson for scientific editing. This work was supported by Grants-in-Aid for Pandemic Influenza Research (TK, SF, HX, and MT) and Grant-in-Aid for Specially Promoted Research (MT) from the Ministry of Health, Labour and Welfare, Japan, by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C)(YK), by a Grant-in-Aid for Specially Promoted Research, by the Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (YK), and by ERATO, Japan (YK).

Authors contributions

Designed the analyses: TK, SF, ET, SY, GN, YK, MT. Analysed and interpreted data: TK, SF, ET, HX, SY, YU, GN, YK, MT. Drafted the article: TK, SF. Revised the article: ET, GN, TS, YK, MT.

Conflict of interest

None declared. <HR>This manuscript was accepted on 11 March 2013 and published online on 12 March 2013. <HR>
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**NS1** · April 12, 2013, 08:31 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Eurosurveillance, Volume 18, Issue 15, 11 April 2013
Rapid communications
Genetic analysis of novel avian A(H7N9) influenza viruses isolated from Patients in China, February to April 2013
Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M.
Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.
Euro Surveill. 2013;18(15):pii=20453.

We have contacted the authors of this paper requesting clarification on two matters that are potentially scribe errors:

The notation for the ChinaShanghai1_E1_87M_2013_02_26_f Receptor Binding Site polymorphism HA A138S in the text of the paper (prior to the monomers diagram) appears to be reversed in the statement, 'encodes an S138A mutation'. The diagram in Figure 4 is properly annotated with A138S / A128S.
On Table 3 Amino Characteristics, ?No Deletion? is stated under ?Avian Influenza Viruses? for the NA segment though the reference is to a paper detailing the H5N1 deletion (short stalk v long stalk). The H7N9 deletion pattern is certainly shorter and primarily in a downstream location with only a small overlap to the classic H5N1 NA deletion, but other avian influenza serotypes that are ?of concern? also show the larger upstream deletion.

Their paper pointedly illuminates the fact of cross-segment combinations of polymorphisms that each have previous experience with mammals or laboratory-confirmed mammalian-adaptation correlates. Rapid progression from onset to fatality occurs during host species transitions. A virus that is "confused" or carrying genetics for two different host classes conducts operations using methods and in places that are aberrant.

We continue to be perplexed that authors are referencing the H7N9 wildtype HA 160A as a T160A 'mutation'. While we agree that the value Alanine at amino acid position 160 is demonstrated to have a negating effect on motif glycosylation and, as such, is of very high interest, these emergent H7N9 sequences have not mutated / revised / polymorphed from Threonine to Alanine according to the data record, but have pre-existed as Alanine at aa160.

At the moment, details concerning the genetics of the offending pathogen, including the Novel H7N9 HA Q226I adaptation arising at a critical Receptor Binding Site potentially from human H3N2, are available in the GeneWurx Cross Serotype Homology Analysis (Open-Access, Full-Text version). The genetics comparatives are under comprehensive discussion on the wider thread, entitled "China - H7N9 Human Isolates on Deposit at GISAID".

GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

**NS1** · April 12, 2013, 08:32 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Originally posted by NS1 View Post

Eurosurveillance, Volume 18, Issue 15, 11 April 2013
Rapid communications
Genetic analysis of novel avian A(H7N9) influenza viruses isolated from Patients in China, February to April 2013
Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M.
Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.
Euro Surveill. 2013;18(15):pii=20453.

We have contacted the authors of this paper requesting clarification on two matters that are potentially scribe errors:

The notation for the ChinaShanghai1_E1_87M_2013_02_26_f Receptor Binding Site polymorphism HA A138S in the text of the paper (prior to the monomers diagram) appears to be reversed in the statement, 'encodes an S138A mutation'. The diagram in Figure 4 is properly annotated with A138S / A128S.
On Table 3 Amino Characteristics, ?No Deletion? is stated under ?Avian Influenza Viruses? for the NA segment though the reference is to a paper detailing the H5N1 deletion (short stalk v long stalk). The H7N9 deletion pattern is certainly shorter and primarily in a downstream location with only a small overlap to the classic H5N1 NA deletion, but other avian influenza serotypes that are ?of concern? also show the larger upstream deletion.

Their paper pointedly illuminates the fact of cross-segment combinations of polymorphisms that each have previous experience with mammals or laboratory-confirmed mammalian-adaptation correlates. Rapid progression from onset to fatality occurs during host species transitions. A virus that is "confused" or carrying genetics for two different host classes conducts operations using methods and in places that are aberrant.

We continue to be perplexed that authors are referencing the H7N9 wildtype HA 160A as a T160A 'mutation'. While we agree that the value Alanine at amino acid position 160 is demonstrated to have a negating effect on motif glycosylation and, as such, is of very high interest, these emergent H7N9 sequences have not mutated / revised / polymorphed from Threonine to Alanine according to the data record, but have pre-existed as Alanine at aa160.

At the moment, details concerning the genetics of the offending pathogen, including the Novel H7N9 HA Q226I adaptation arising at a critical Receptor Binding Site potentially from human H3N2, are available in the GeneWurx Cross Serotype Homology Analysis (Open-Access, Full-Text version). The genetics comparatives are under comprehensive discussion on the wider thread, entitled "China - H7N9 Human Isolates on Deposit at GISAID".

GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

Even at the weekend, Japan's NIID lead author responded immediately and kindly initiated a review of the material.

**NS1** · April 14, 2013, 02:15 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Next Potential H7N9 Emergence
Guizhou and Hunan

Guizhou and Hunan are probable points of early emergence based on the limited genetic data from 7 sequences (4 human, 2 avian, 1 environment).

Guizhou holds a higher probability of emergence, but obviously a lower probability of timely reporting.

Guizhou also demonstrates a higher potential for additional genetic revision concerning pathogenicity upon emergence in that region.

H7N9 Human Isolates on Deposit at GISAID carries a cumulative discussion and up-to-date analysis of emergent H7N9 genetic features related to human and avian pathogenicity of this outbreak.

Ongoing Sequence Analysis - Open-Access, Full-Text
GISAID Citation

**NS1** · April 14, 2013, 05:55 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Potential Passerine Involvement
as Emergent H7N9
Reservoir

Current emergent H7N9 Hemagglutinin genetic evidence suggests that a wider species list of Passerines should be sampled and sequenced for Influenza A H5 and H7 serotypes in Shanghai, Guizhou, Hong Kong, Japan & Korea, including the following list in order of descending importance:

Pica pica and other Pica genus species
Cissa chinensis and other Cissa genus species
Garrulus lidthi
Hirundo rustica
Delichon urbicum

One or more of these Passerine species may presently carry genetics related to the HA emergence.

Detailed surveillance of these species in three areas of Guangdong province may prove beneficial:

Shenzhen
Guangzhou
Huadong

**NS1** · April 15, 2013, 04:52 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

H5N1 Wet Market sub-segment relationships Updated

Sequence Analysis - Open-Access, Full-Text
GISAID Citation

**Gert van der Hoek** · April 15, 2013, 06:10 AM

China - H7N9 Human Isolates on Deposit at GISAID

ANALYSIS-Gene swapping makes new China bird flu a moving target

Mon, 15 Apr 2013 09:34 GMT

* Deadly new H7N9 flu is mix of three avian flu strains

* Virus previously unknown in humans has now killed 13

* Genetic data available so far suggests H7N9 is unstable

By Kate Kelland, Health and Science Correspondent

LONDON, April 15 (Reuters) - A new bird flu virus that has killed 13 people in China is still evolving, making it hard for scientists to predict how dangerous it might become.

Influenza experts say the H7N9 strain is probably still swapping genes with other strains, seeking to select ones that might make it fitter.

If it succeeds, the world could be facing the threat of a deadly flu pandemic. But it may also fail and just fizzle out.

More: Alertnet

**sharon sanders** · April 15, 2013, 11:12 AM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Originally posted by Gert van der Hoek View Post

ANALYSIS-Gene swapping makes new China bird flu a moving target...More: Alertnet

The experts' opinions are reflected in the original analysis in this thread:

1) "Even with just the three (gene) sequences we have available, there's some evidence that one doesn't quite fit with the other two. So we might think this virus is still fishing around for a genetic constellation that its happy with," said Wendy Barclay, a flu virologist at Imperial College London. "Maybe there are other viruses out there that it is still exchanging genes with until it gets to a stable constellation."

2) Yoshihiro Kawaoka of the University of Wisconsin and Masato Tashiro at the National Institute of Infectious Diseases in Tokyo, said the H7N9 sequences "possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans". These features, the scientists wrote, "raise concerns regarding their pandemic potential".

3) "We know H7 viruses can spill over into humans ... and for me the most important thing to find out now is from which species do we think this H7N9 is spilling over," said Ab Osterhaus, head of viroscience of the Erasmus Medical Centre in the Netherlands. "Is it one species? Are there different species? At this stage we are still lacking a lot of data."

Thanks everyone! A great contribution by all.

**NS1** · April 15, 2013, 11:22 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> Originally Posted by sharon sanders
The experts' opinions are reflected in the original analysis in this thread:

1) "Even with just the three (gene) sequences we have available, there's some evidence that one doesn't quite fit with the other two. So we might think this virus is still fishing around for a genetic constellation that its happy with," said Wendy Barclay, a flu virologist at Imperial College London. "Maybe there are other viruses out there that it is still exchanging genes with until it gets to a stable constellation."

2) Yoshihiro Kawaoka of the University of Wisconsin and Masato Tashiro at the National Institute of Infectious Diseases in Tokyo, said the H7N9 sequences "possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans". These features, the scientists wrote, "raise concerns regarding their pandemic potential".

3) "We know H7 viruses can spill over into humans ... and for me the most important thing to find out now is from which species do we think this H7N9 is spilling over," said Ab Osterhaus, head of viroscience of the Erasmus Medical Centre in the Netherlands. "Is it one species? Are there different species? At this stage we are still lacking a lot of data."

Thanks everyone! A great contribution by all.
</td></tr></tbody></table>
Expert#1 on 2013-04-15:

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> 1) "Even with just the three (gene) sequences we have available, there's some evidence that one doesn't quite fit with the other two. So we might think this virus is still fishing around for a genetic constellation that its happy with," said Wendy Barclay, a flu virologist at Imperial College London. "Maybe there are other viruses out there that it is still exchanging genes with until it gets to a stable constellation." </td></tr></tbody></table>
As previously discussed on April 10, 2013 in FluTracker's-hosted, GeneWurx original research based on precise RnR genetic evaluation of emergent H7N9 and the Cross Serotype Relationships to active human and avian influenza:

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> If this competent HA becomes less interested in the dead-ending, a potential exists for a reassortment to a more capable and compatible NA. The founding genetics of this current emanation of emergent H7N9 demonstrates multiple signal pathways toward N2 affinity with nominal secondary potential for mammal N8. The Gain of Function scalability ranges from the potentially lower risk H9N2 to the significantly higher risk donation from the circulating Vaccine-Escape, High-CFR H3N2.

That circulating human H3N2 has overseen mortality rates in the United States between 5 and 17 times last season's totals and between 200% and 300% of the previous state records in the states of Indiana, Minnesota and Pennsylvania. Though H3N2 is a long "understood" serotype with a multi-decade history of vaccine production, in the state of Indiana this year, 39% of the fatalities had confirmed vaccinations and another 34% conveniently were annotated as an "unknown" status. Only 27% of the fatalities were confirmed unvaccinated. Our reports from 2013 Week 13 (cumulative) are attached to this post.

The emergent H7N9 cases, while not 80% over 60 years old, demonstrate a weighting toward older citizens. Does this emergent H7N9 have something in common with the circulating human H3N2 that describes this affinity for older hosts or is the age skewing a small population anomaly that will self-correct as the sample size increases? If the emergent H7N9 does, in fact, prove to have interchange behaviour with sH3N2, will a convergent H3N2 NA reassortment be an attractive option to this competent HA and the currently stable 6 internal genes of the emergent H7N9? </td></tr></tbody></table>
Paper#2 on 2013-04-11:

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> 2) Yoshihiro Kawaoka of the University of Wisconsin and Masato Tashiro at the National Institute of Infectious Diseases in Tokyo, said the H7N9 sequences "possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans". These features, the scientists wrote, "raise concerns regarding their pandemic potential". </td></tr></tbody></table>
2013-03-31 GeneWurx Open Access at FluTrackers, Rare and Novel H7N9 Hemagglutinin Polymorphisms and their relationship to human pH1N1.

2013-04-02 GeneWurx Open Access at FluTrackers, Re-Assortment Validation and assertation of human homology.

2013-04-03 GeneWurx Open Access at FluTrackers, Cross Serotype Homology evaluation (First published 2013-04-03, Updated twice daily).

2013-04-04 GeneWurx Open Access at FluTrackers, Mammalian Gain of Function evaluation

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> We are grateful to the samplers, the depositing labs and the GISAID consortium for making the small batch of evaluation material available on 2013-03-31 during this zoonotic epidemic emergence of H7N9 that has the very unfortunate combination of a High Case Fatality Rate and Low clinical intervention effectiveness [GISAID Citations].

The H7N9 human cases show clinically and experimentally-confirmed Gain of Function traits on a minimum of 3 gene segments:

Hemagglutinin 226L
Neuraminidase aa69-73 stalk deletion (15 base)
Polymerase Basic 2 627K

The H7N9 sequences are related at the polymorphism level to widely circulating human virus patterns including pH1N1, sH3N2 and to fatal human H5N1. Each component of the H7N9 human cases carries sub-segment genetics that have seen mammalian adaptation, including the external proteins, Hemagglutinin and Neuraminidase.

An up-to-date and comprehensive follow-up evaluation for each HA polymorphism found on the 3 human H7N9 sequences was provided Wednesday, 2013-04-03, demonstrating the Cross Serotype Homology details. The preliminary H7N9 Novelty Evaluation and Relationship Potential to pH1N1 with detailed Hemagglutinin amino-level polymorphism surfacing was made available for preview on 2013-03-31, formalised at midnight and is now being versioned.

Genetic specifics concerning animal origins and human / mammal adaption are under analysis. The full text of those intermediate reports are currently in pre-publication viewing status and are available at the following links. These compilations will be updated as labs release new sequences.

GeneWurx Human Emergent H7N9 Cross Serotype Homology (External Genes)
GeneWurx Human Emergent H7N9 Rare and Novel Hemagglutinin Antigens (External Genes)
GeneWurx Human Emergent H7N9 ReAssortment Assessment (Internal Genes)

Additional sequences from early emergence fatal and recovered cases are critical to understanding the immune dynamics required for establishing a bulkhead toward the protection of public health. </td></tr></tbody></table>
2013-04-05 GeneWurx Open Access at FluTrackers, Human Adaptation

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> While multiple laboratories have investigated Avian Influenza models at amino acid position 186 (H3 numbering) and have found that variation toward Lysine and Asparagine on certain backgrounds acts as a co-effector toward increased infectivity of cell types in the human upper respiratory tract, the genetic record extends that potential with amino acid variations perhaps yet uncharacterised. The 2 most recent human H7N9 sequences carry a revision at aa186, HA 186V.

ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f
ChinaShanghai2_E1_27M_2013_03_05_f

The Valine at this position is novel to H7N9, but is found within the wider H7 reservoir under 40 times and essentially in species considered as commercial poultry. The HA 186V is also found as wildtype in the H13 reservoir among serotypes often considered "dead-end" for avian host progression outside of gulls. Oddly enough, H13N2 has been documented in sea mammals and does carry the Valine in the instance under review. Additionally, the H13N2 positions 186, 187 and 188 correspond to these current H7N9 human sequences.

Perhaps H13 is not "dead-end" at all, but is some type of intermediate platform for preservation or movement between species groups. That speculation will not likely be answered in the near future, but we do have now HA 186V and two trailing amino values that correspond to this new H7N9 motif from an H13N2 sea mammal collection and the H7N9 novel motif of 188T wildtype + 189A revision matching the most commonly circulating pH1N1 subclade at 188T + 189A wildtype.

We may come to find that HA 186V on this background is an intermediate or a full co-effector of alpha 2,6 sialic binding. The combination of 186V / 226L on these H7N9 sequences may be found to parallel the 186K / 226L mutants evaluated on the H5N1 Gain of Function studies.

. . . . . . . . 195V [186V H7N7 Rare],
. . . . . . . . . . . . [H13N9 Avian wildtype],
. . . . . . . . . . . . [H13N2 Mammal wildtype],
. . . . . . . . . . . . [H5N1 Gain of Function Residue aa182:
. . . . . . . . . . . . . . . PubMed PMC2903244, 17108965] </td></tr></tbody></table>

2013-04-05 GeneWurx Open Access at FluTrackers, Receptor Binding Site Transition to Novel Value Q226I

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> . . . . . . . . 235I [226I Human H3N2 Current wildtype],
. . . . . . . . . . . . [H3N2 commercial poultry
. . . . . . . . . . . . . . . . with HA 155, 156, 158, 159 VxX revisions
. . . . . . . . . . . . . . . . . . and G228S, known virulence marker],

HA 237S (H3 228S) and 255T (H3 246T) may be closer than we'd like to consider.

This HA 226I is novel to H7N9, but is found in avian H3N2 samples from commercial poultry species and in the most widely circulating human Influenza, H3N2, as the wildtype. Those HA sequences from the poultry bear sub-segment genomic homology to these human H7N9 fatalities, as well as homology to currently circulating human H3N2. The H7N9 HA varies at syn405I (ATc) from the current human cases.

At the NA segment, a synonymous revision occurs from the current set of human H7N9 cases, syn142R (cGA).

Though migratory birds have proven capable of carrying the HA 226I / 228S, the combination is known also in swine as recently as 2010 in North America and 2007 in Asia. avH3N2 is also on record with 226L / 228S in North America early in the last decade. The migratory birds sampled in South Dakota six years ago with 226I / 238S share downstream elements from aa228 up to 51 bases with currently circulating human H3N2 (Oceania, United States & Europe). HA 277N (H7N9 285N) is found in correlation on avian H3N2 segments that have variation at 226, including all 226I bearing segments. HA 138S (H7N9 146S) like our current index case, ChinaShanghai1_E1_87M_2013_02_26_f, is also located on the avian H3N2 segments bearing 226I.

Potential for step-wise interchange exists. </td></tr></tbody></table>
2013-04-12 GeneWurx Review of Eurosurveillance, Volume 18, Issue 15, 11 April 2013, with error communication to authors.

Expert#3 on 2013-04-15:

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset"> 3) "We know H7 viruses can spill over into humans ... and for me the most important thing to find out now is from which species do we think this H7N9 is spilling over," said Ab Osterhaus, head of viroscience of the Erasmus Medical Centre in the Netherlands. "Is it one species? Are there different species? At this stage we are still lacking a lot of data." </td></tr></tbody></table>
2013-04-14 GeneWurx Open Access at FluTrackers, Potential Passerine Involvement as Reservoir with sample requests for specific geographies and species.

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"><tbody><tr><td class="alt2" style="border:1px inset">
Current emergent H7N9 Hemagglutinin genetic evidence suggests that a wider species list of Passerines should be sampled and sequenced for Influenza A H5 and H7 serotypes in Shanghai, Guizhou, Hong Kong, Japan & Korea, including the following list in order of descending importance:

Pica pica and other Pica genus species
Cissa chinensis and other Cissa genus species
Garrulus lidthi
Hirundo rustica
Delichon urbicum

One or more of these Passerine species may presently carry genetics related to the HA emergence.

Detailed surveillance of these species in three areas of Guangdong province may prove beneficial:

Shenzhen
Guangzhou
Huadong

</td></tr></tbody></table>

**Laidback Al** · April 16, 2013, 05:49 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

China ? Map of Potential Emergence of H7N9

<!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w

oNotPromoteQF/> <w:LidThemeOther>EN-US</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables/> <w:SnapToGridInCell/> <w:WrapTextWithPunct/> <w:UseAsianBreakRules/> <w

ontGrowAutofit/> <w:SplitPgBreakAndParaMark/> <w:EnableOpenTypeKerning/> <w

ontFlipMirrorIndents/> <w:OverrideTableStyleHps/> </w:Compatibility> <m:mathPr> <m:mathFont m:val="Cambria Math"/> <m:brkBin m:val="before"/> <m:brkBinSub m:val="--"/> <m:smallFrac m:val="off"/> <m:dispDef/> <m:lMargin m:val="0"/> <m:rMargin m:val="0"/> <m:defJc m:val="centerGroup"/> <m:wrapIndent m:val="1440"/> <m:intLim m:val="subSup"/> <m:naryLim m:val="undOvr"/> </m:mathPr></w:WordDocument> </xml><![endif]-->Based on the GeneWurx post by NS1, Guizhou and Hunan hold the potential for H7N9 emergence.

Link: http://www.flutrackers.com/forum/sho...0&postcount=68

**NS1** · April 16, 2013, 06:14 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

Tremendous, Al!

That's exactly the visual we'd have built if we had your talent. Now readers have a truly communicative idea of the data projections.

Let's stand on the hope that the strong transmission of data (your map) will lead the public and the public health leaders to actions that weaken the disease transmission and reduce morbidity in these potential areas of emergence colored in yellow.

**NS1** · April 17, 2013, 05:48 PM

Re: China - H7N9 Human Isolates on Deposit at GISAID

A Tale
of
Two Sequences

"It was the best of times, it was the worst of times . . ." 1

The good news:

Item A
The 69 year old recovered son of the 87M (Fatal 2013-03-04) has now been retrospectively confirmed via serology as having suffered from H7N9. This familial aggregation creates the possibility of H2H, but is not determinant of H2H. Isolation and sequencing of samples taken from the 69M would rule in or rule out homology of disease. The emergent H7N9 in the father carried markers that distinguish a potential separate introduction from the later cases. As such, a sequence from the son may rule in clustering so that further epidemiology studies will be encouraged.

Item B
Today, a report arose from Nanjing, Jiangsu province dated 2013-04-16 concerning the first wild bird found infected with emergent H7N9. A passerine bird in the wild has been sampled, tested positive and sequenced with confirmation of emergent H7N9.

On April 14, 2013, we requested additional samples from passerines to verify the trend of data showing that one or more passerine species is involved in the Hemagglutinin genetic progression chain, pooling sub-segment polymorphisms into a highly probable reservoir.

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"> <tbody><tr> <td class="alt2" style="border:1px inset">

H7N9 bird flu virus found in Wild Pigeon
video in English.

. . . the genetic sequence of the virus is very similar to that discovered in pigeons sold at a Shanghai market.

</td> </tr> </tbody></table>
The description, "very similar," does not communicate 100% homology, nor does it exclude 100% homology to pgnChinaShanghaiS1069_2013_04_02. With involvement at critical HA aminos from H5N1 samples (including a pigeon and 3 mammal species) that match emergent H7N9 Wet Market sequences, these passerine sequences become essential to understanding the genetic development pace and multiple lineages.

Quote:
<table border="0" cellpadding="6" cellspacing="0" width="100%"> <tbody><tr> <td class="alt2" style="border:1px inset">

Shanghai Wet Market Surveillance
2013-04-07

GISAID Citations

HA Polymorphisms

. . . . pgnChinaShanghaiS1069_2013_04_02 (
. . . . . . . . GISAID HA EPI440701
. . . . . . . . GISAID Isolate EPI_ISL_138985
. . . . . . . . 105 Polymorphisms (18 Amino and 87 Silent)
. . . . . . . . 11I,
. . . . . . . . 130A,
. . . . . . . . 183S,
. . . . . . . . 188V,
. . . . . . . . 195V,
. . . . . . . . 198A,
. . . . . . . . 211V,
. . . . . . . . 217N,
. . . . . . . . 235L,
. . . . . . . . 252I [243I],
. . . . . . . . . . . .[H3N2 Human Rare (4):
. . . . . . . . . . . . . . . . . 2012-12-27 USNavy with UltraAmbiguity,
. . . . . . . . . . . . . . . . . 2009 Hong Kong,
. . . . . . . . . . . . . . . . . 2008 Florida,
. . . . . . . . . . . . . . . . . 2005 USNavy Distant-to-Relative],
. . . . . . . . . . . .[H7N7 Avian Rare (13), 2004-2006, 2008, 2009],
. . . . . . . . . . . .[H7N1 Avian Rare (4), 2000, 2004, 2008, 2009 including
. . . . . . . . . . . . . . . . . non-gallinaceous commercial avian,
. . . . . . . . . . . . . . . . . Ostrich 2000 Pneumo- & Neuro-Tropic to Mice with PB2 627K],
. . . . . . . . . . . .[H7N8 Avian Singular],
. . . . . . . . . . . .[H7N3 Avian Rare (6), 2006, 2007],
. . . . . . . . . . . .[H7N2 Avian Rare (2), 2006, 2007],
. . . . . . . . . . . .[H5N1 Mammals (Human, Tiger, Raccoon Dog)],
. . . . . . . . . . . .[H5N1 Avian (7 Species)],
. . . . . . . . . . . .[H5N2 Mexico commercial avian (gallinaceous), 1994, 1995],
. . . . . . . . . . . .[H5N3 Hong Kong commercial avian (anser), 1976],
. . . . . . . . 285N,
. . . . . . . . 307D,
. . . . . . . . 321R,
. . . . . . . . 410N,
. . . . . . . . 427I,
. . . . . . . . 455D,
. . . . . . . . 462K,
. . . . . . . . 541V)

NA Polymorphisms

. . . . pgnChinaShanghaiS1069_2013_04_02 (
. . . . . . . . GISAID NA EPI440700
. . . . . . . . GISAID Isolate EPI_ISL_138985
. . . . . . . . 33 Polymorphisms (10 Amino and 23 Silent)
. . . . . . . . 16I,
. . . . . . . . 19A,
. . . . . . . . 40G,
. . . . . . . . 53T,
. . . . . . . . 81T,
. . . . . . . . 84N,
. . . . . . . . 112S,
. . . . . . . . 335I,
. . . . . . . . 359A,
. . . . . . . . 401A) </td> </tr> </tbody></table>
The other news:

Sequences from these two cases (Shanghai 69M and pgnChinaNanjing) are not available at this time.

Footnotes
1. Charles Dickens, A Tale of Two Cities (London: All the Year Round, 1859), 1.

Announcement

China - H7N9 Human Isolates on Deposit at GISAID

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