China - H7N9 Human Isolates on Deposit at GISAID

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Fatal H5N1 Homology
to
Emergent H7N9
from
Shanghai in March

Fudan University released emergent H7N9 segments at GenBank for a case sampled on March 5, 2013 in Shanghai. No metadata or clinical outcome was provided. The case follows course having HA 226L, but is novel due to HA 16A (1A) in combination to the 291H (283H) previously reported on the fatal ChinaShanghai3_E1_73M_2013_02_27_f. ChinaShanghai4664T_2013_03_05 is the first human emergent H7N9 case without the synonymous changes at HA 365H (356H) and 366Q (357Q) and appears to be an recombinant of the emergent human H7N9 with contributions from non-emergent avian H7N9 sequences.

The Neuraminidase shows 5' variance by remaining wildtype at aa16 and aa19, two positions that have changed consistently to the same values on 100% of emergent H7N9, human, avian and environmental. The NA does revise in that area to 13M with a two base revision and to 26M that is found on the fatal ChinaShanghai2_E1_27M_2013_03_10_f. Additionally, NA 285K (284K) is on display.

The PB2 continues the 100% human trend of mammalian adaptation at either aa627 or aa701 with Lysine at 627. Furthermore, the PB2 distinguishes this segment with the novel 354V, a polymorphism found on a 2009 pH1N1 case in China.

The PB1 carries 4 "silent" polymorphisms off ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f, marking the genetic maturation process: syn208K (AAg) [neH7N9], syn222L (CTt) [ChinaShanghai2_E1_27M_2013_03_05_f, neH7N9 Czech Avian (2)], syn544A (GCc) [all emergent H7N9 except the divergent PB1 of pgnChinaShanghaiS1069_2013_04_02, many neH7N9] and syn662T (ACt) [neH7N9, emergent H7N9 pgnChinaShanghaiS1069_2013_04_02].

Fatal H5N1 Homology to ChinaShanghai4664T_2013_03_05 PA

The PA shows a compelling list of 19 polymorphisms off ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f, including 86V, 206V and 621S with syn58G (GGc) and syn681F (TTc), the first of which is implicated in H5N1 fatality in 2008 and in 2009 each in young Asian adults with one from a Chinese Wet Market exposure, the last of which appears on 2 additional emergent H7N9 sequences: ChinaZhejiangDTIDZJU01_xM_2013_04_03 & ChinaZhejiang2_64M_2013_04_03.

• CY098729.1 ChinaXinjiang1_E2_31F_2009_01_10_f [Novel, Distance-to-Relation Top 10 = 8 to 12nu]
• HM114608.1 VietnamNinhBinhUT31413II_M2_27M_2008_02_13_f [Novel, Distance-to-Relation Top 10 = 3 to 31nu]

Between the two fatal H5N1 sequences, additional homology with the H7N9 PA polymorphic superset occurs extensively with non-emergent H7N9 avian isolates and definitively for emergent H7N9, including syn582R (AGg), syn588S (TCc) syn589L (CTt), syn597E (GAg), syn598A (GCc), syn599E (GAg), syn600S (TCt), syn681F (TTc), syn715L (CTg) & 716K for this sequence under investigation.

Therefore, the item of highest interest concerning the Polymerase Acidic segment is the suggestion of multiple revision, sub-segment genetic transfer from fatal H5N1 human pathogens in unsurveilled circulation throughout Asia. Ten polymorphisms dispersed into 5 distinct areas of the Polymerase Acidic segment bring into question any application of the convenient terms, "random" and "mutation", used so often by the patrons of the two pillars philosophy.

The issue is compounded by the fact that at least one section of the PA from these fatal H5N1 cases (aa582 to aa600) is in wide and current circulation informing genetic maturation of H5N1, H10N7 and various other serotypes including H6Nx. Homology to current H5N1 human fatalities includes ChinaGuizhou2_E1E1_31M_2013_02_09_f and ChinaGuizhou1_E1E1_21F_2013_02_08_f with HA 202V. These two human fatalities from three months ago in Guizhou also carry HA 185S similar to H7N9, but rare to H5N1; all but one of the 185S H5N1 cases are documented as fatal.

Now we may begin to recognise the footfalls of a disease that approaches quietly but leaves these faint impressions in the dust. Hemagglutinin 202V and groups of Polymerase Acidic changes from H5N1 cases are documented with human fatality. Must two pillars science continue to dissemble that segment-level BLAST results demonstrate the genetic maturation process.

PA Potential Recombination (Grouped)

• syn582R (AGg), 3rd base, A1746G
• syn583R (CGt), 3rd base, C1749T

• syn586L (CTc), 3rd base, T1758C

• syn588S (TCc), 3rd base, T1764C
• syn589L (CTt) , 3rd base, C1767T
• syn590Q (CAg), 3rd base, A1770G

• syn597E (GAg), 3rd base, A1791G
• syn598A (GCc), 3rd base, T1794C
• syn599E (GAg), 3rd base, A1797G
• syn600S (TCt), 3rd base, C1800T

• syn608T (ACa), 3rd base, C1824A
• syn609K (AAg), 3rd base, A1827G

• syn715L (CTg), 3rd base, A2145G
• 716K (AaA), 2nd base, G2147A

HA Polymorphisms

. . . . ChinaShanghai4664T_2013_03_05 (
. . . . . . . . GenBank HA KC853228
. . . . . . . . 104 Polymorphisms (19 Amino and 85 Silent)
. . . . . . . . 11I [#4I],
. . . . . . . . 16A [1A],
. . . . . . . . . . . [pH1N1 wildtype],
. . . . . . . . . . . [H7N9 Avian and Environment],
. . . . . . . . . . . [H7N7 Avian & Mammal Rare (20):
. . . . . . . . . . . . . . . . . America anas 2011 (2),
. . . . . . . . . . . . . . . . . America anas 2010 (3),
. . . . . . . . . . . . . . . . . America anas 2009 (2),
. . . . . . . . . . . . . . . . . America anas 2009 (2),
. . . . . . . . . . . . . . . . . America anas 2007 (2),
. . . . . . . . . . . . . . . . . America anas 2006 (3),
. . . . . . . . . . . . . . . . . America environment 2007,
. . . . . . . . . . . . . . . . . Alaska anser 2006,
. . . . . . . . . . . . . . . . . Egypt anas 2004 with HA 243I,
. . . . . . . . . . . . . . . . . America anas 1989,
. . . . . . . . . . . . . . . . . America anas 1988,
. . . . . . . . . . . . . . . . . America seal 1980],
. . . . . . . . . . . [H7N6, N5, N4 Avian Rare],
. . . . . . . . . . . [H7N3 Human & Avian:
. . . . . . . . . . . . . . . . . Mexico Human 2012,
. . . . . . . . . . . . . . . . . Mexico gal 2012 (2),
. . . . . . . . . . . . . . . . . Worldwide extensive 1971-2011],
. . . . . . . . . . . [H7N2 Avian, extensive including emu, gal, quail],
. . . . . . . . . . . [H7N1 Avian Rare: rhea (3) & anas (2)],
. . . . . . . . . . . [H5N1 Avian and Environment Rare (13):
. . . . . . . . . . . . . . . . . Egypt anas 2011,
. . . . . . . . . . . . . . . . . China Xinjiang Wet Market env 2009 (2),
. . . . . . . . . . . . . . . . . Bangladesh gal 2008,
. . . . . . . . . . . . . . . . . Egypt gal 2008,
. . . . . . . . . . . . . . . . . Vietnam anas 2007 (3),
. . . . . . . . . . . . . . . . . Benin gal 2007 (2),
. . . . . . . . . . . . . . . . . Romania gal 2005,
. . . . . . . . . . . . . . . . . China Anhui Shitai anas 2003,
. . . . . . . . . . . . . . . . . China Guangdong Shantou anas 2003],
. . . . . . . . . . . [H5N2 Avian Rare (5):
. . . . . . . . . . . . . . . . . Germany anas 1986 (2),
. . . . . . . . . . . . . . . . . Hong Kong anas 1978 (3)],
. . . . . . . . . . . [H5N8 Avian Rare (2):
. . . . . . . . . . . . . . . . . Ireland gal, anas 1983],
. . . . . . . . 130A [118A],
. . . . . . . . 183S [174S],
. . . . . . . . 188V [179V],
. . . . . . . . 195V [186V],
. . . . . . . . 198A [189A],
. . . . . . . . 211V [202V],
. . . . . . . . 217N [208N],
. . . . . . . . 235L [226L],
. . . . . . . . 285N [277N],
. . . . . . . . 291H [283H],
. . . . . . . . . . . . [Emergent H7N9 ChinaShanghai3_E1_73M_2013_02_27_f],
. . . . . . . . 307D [299D],
. . . . . . . . 321R [313R],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . 541V [533V])

NA Polymorphisms

. . . . ChinaShanghai4664T_2013_03_05 (
. . . . . . . . GISAID NA KC853231
. . . . . . . . 33 Polymorphisms (11 Amino and 22 Silent)
. . . . . . . . 13M [13M],
. . . . . . . . . . . [pH1N1 wildtype LAd2],
. . . . . . . . 26M [26M],
. . . . . . . . . . . [Emergent H7N9 ChinaShanghai2_E1_27M_2013_03_10_f],
. . . . . . . . . . . [H1N2 swine England 2009-09-28, NA distance to relative=37nu],
. . . . . . . . . . . [sH3N2 Human wildtype LAu2],
. . . . . . . . . . . [avH3N2 LAu2 Vietnam 2011, Quebec 2006],
. . . . . . . . . . . [H9N2 LAu2],
. . . . . . . . . . . [H11N2 LAu2],
. . . . . . . . . . . [H6N2 LAu2 Vietnam 2012,
. . . . . . . . . . . . . . . . . . . China Guizhou, Jiangxi, Shantou 2007],
. . . . . . . . . . . [H2N2 LAu2 America 1995],
. . . . . . . . . . . [H1N2 LAu2 Delaware 1994],
. . . . . . . . 40G [40G],
. . . . . . . . 53T [53T],
. . . . . . . . 81T [80T],
. . . . . . . . 84N [83N],
. . . . . . . . 112S [111S],
. . . . . . . . 285K [284K],
. . . . . . . . . . . .[pH1N1.Upsilon LAd3],
. . . . . . . . . . . .[H5N1 LAd3],
. . . . . . . . . . . .[avH1N1 LAd3],
. . . . . . . . 335I [332I],
. . . . . . . . 359A [355A],
. . . . . . . . 401A [397A])

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-05-04-01_30_56_379780 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Fatal H5N1 Karo Cluster Homology
to
Emergent H7N9
from
First Fujian Case

April 24, 2013

The Fujian Center for Disease Control released emergent H7N9 segments at GenBank for the first case in their province. The 65 year old male was sampled on April 24, 2013. No metadata or clinical outcome was provided by the depositors, although the FluTrackers 2013 Human Case List [#116] indicates an onset of April 18 and hospitalisation at April 23. The patient was discharged on May 13.

The case follows course having HA 226L, but is made novel by the NA 148E (147E). The Neuraminidase polymorphism is found on the Indonesian H5N1 human Karo cluster from May 2006, including the fatalities. Due to the absence of Polymerase segments, determination must wait concerning the mammalian adaptations of either PB2 627K / 701N that have been found on 100% of the previously deposited human emergent H7N9 cases .

HA Polymorphisms

. . . . ChinaFujianLongyanCity1_65M_2013_04_24_s (
. . . . . . . . GenBank HA KC994453
. . . . . . . . 104 Polymorphisms (17 Amino and 87 Silent)
. . . . . . . . 11I [#4I],
. . . . . . . . 130A [118A],
. . . . . . . . 183S [174S],
. . . . . . . . 188V [179V],
. . . . . . . . 195V [186V],
. . . . . . . . 198A [189A],
. . . . . . . . 211V [202V],
. . . . . . . . 217N [208N],
. . . . . . . . 235L [226L],
. . . . . . . . 285N [277N],
. . . . . . . . 307D [299D],
. . . . . . . . 321R [313R],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . 541V [533V])

NA Polymorphisms

. . . . ChinaFujianLongyanCity1_65M_2013_04_24_s (
. . . . . . . . GenBank NA KC994454
. . . . . . . . 33 Polymorphisms (11 Amino and 22 Silent)
. . . . . . . . 16I [16I],
. . . . . . . . 19A [19A],
. . . . . . . . 40G [40G],
. . . . . . . . 53T [53T],
. . . . . . . . 81T [80T],
. . . . . . . . 84N [83N],
. . . . . . . . 112S [111S],
. . . . . . . . 148E [147E],
. . . . . . . . . . . .[H5N1 Indonesia Karo Cluster Fatalities May 2006
. . . . . . . . . . . . . . . . with dispersed ΣH7N9 NA homology],
. . . . . . . . . . . .[H5N1 Indonesia gal, anas 2005, 2006],
. . . . . . . . 335I [332I],
. . . . . . . . 359A [355A],
. . . . . . . . 401A [397A])

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-05-09-01_39_38_988890 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Receptor Binding Site Novelty
on
Emergent H7N9
from
First Taiwan Case

April 24, 2013

The Taiwan Center for Disease Control released emergent H7N9 segments at GISAID on 2013-05-09 from the first case in the Republic of China, that appears to have been imported from the mainland. The 53 year old male was sampled on April 24, 2013. Age, gender, location and isolate passage metadata were provided by the depositors, but no patient status was made available. The FluTrackers 2013 Human Case List [#110] indicates an onset of April 12th and hospitalisation at April 16th for the man who had been working in Suzhou, Jiangsu.

The case demonstrates novelty in 6 of 8 segments: HA, NA, MP, PB1, PB2 and PA. Although conclusions may not be drawn because the H7N9 reservoir continues to be dramatically under-surveilled, the aggressive rate of diversification within the superset of emergent human H7N9 suggests a viral reservoir that is transitioning to a new host and promiscuously seeking genetic acquisition. This quite "confused" organism does not appear to be willing to sit idle and, ergo, may remain in confusion for some time as branches continue to be created with little to no foliage.

Polymerase Basic 2 shows typical emergent H7N9 mammalian adaptation with the more common 627K continuing the trend of all human emergent H7N9 cases carrying either 627K or 701N. One synonymous polymorphism locates at syn179S (TCc) and is novel to H7N9, but is previously found on avian H2N2 and H12N3 upon segments otherwise dis-similar to the item under review. A second synonymous change, syn321S (TCg), is also novel to H7N9 and is previously found on human H1N1 Vietnam (distance-to-relation=5) and avian H3N2 Korea (multiple species). The aa321 change is most frequently found in H6Nx avian species throughout China.

Polymerase Basic 1 shows 4 changes off an Anhui1 reference, 3 synonymous, 2 previously found in H7N9:

• syn221A (GCg)
• syn364L (TTg) [neH7N9]
• syn544A (GCc) [emergent H7N9]
• 632A

Polymerase Acidic is also polymorphic with 3 changes off an Anhui1 reference, 2 synonymous, 2 previously found in H7N9:

• syn58G (GGc) [emergent H7N9]
• syn93S (AGc) [novel to emergent H7N9, common to neH7N9]
• 268F [novel to emergent H7N9]

The Hemagglutinin carries 226P and 291N while the Neuraminidase is made novel by a 68N [67N]. Homology to a circulating seasonal human H3N2 (HA 291N) that is displaying 6 times higher mortality than last year (graphs for US state of Minnesota in attached PDF) may demonstrate another move by this emergent H7N9 reservoir toward a convergence / ReAssortment event.

?H7N9 HA aa226 shows a diverse set of 4 distinct values with most sequences related to H3N2 Avian 226L or H3N2 Human 226I and all motifs having fatal cases except the novel 226P from the sequence under profile here:

• HA 226P
  • TaiwanTaipei1_E1_53M_2013_04_24_s
• HA 226I
  • ChinaJiangsu01_E1_45F_2013_03_30_s
  • ChinaZhejiang1_37M_2013_03_24_f
  • ChinaHangzhou1_C1_38M_2013_03_24_f
• HA 226L
  • ChinaFujianLongyanCity1_65M_2013_04_24_s
  • envChinaHangzhou34_2013_04_04
  • ChinaZhejiang2_64M_2013_04_03
  • ChinaZhejiangDTIDZJU01_2013_04_03
  • envChinaShanghaiS1088_2013_04_03
  • ckChinaShanghaiS1053_2013_04_03
  • ChinaHangzhou3_79M_2013_04_02_s
  • pgnChinaShanghaiS1069_2013_04_02
  • ckChinaJiangsuK89_2013_04_01
  • gsChinaJiangsuK27_2013_04_01
  • ChinaHangzhou2_67M_2013_03_25_f
  • ChinaAnhuiChuzhouCity1_E1_35F_2013_03_15
  • ChinaShanghai2_E1_27M_2013_03_10_f
  • ChinaShanghai4_E1_63M_2013_03_09
  • ChinaShanghai4664T_2013_03_05
  • ChinaShanghai3_E1_73M_2013_02_27_f
• HA wildtype Q226
  • ckChinaZhejiangDTIDZJU01_2013_04
  • ChinaShanghai1_E1_87M_2013_02_26_f

Risk factors increase when human seasonal H3N2 wildtype values like the HA 291N occur on emergent H7N9 human sequences. This HA carries 3 notable synonymous changes at aa113 [101], aa294 [286] and aa471 [462]. That final syn462E (GAg) is the H5N1 wildtype value. The Neuraminidase polymorphism is potentially sourced from pH1N1 NA wildtype 68N, a downstream LookAside.

HA Polymorphisms

. . . . TaiwanTaipei1_E1_53M_2013_04_24_s (
. . . . . . . . GISAID HA EPI445912
. . . . . . . . GISAID Isolate EPI_ISL_140356
. . . . . . . . 108 Polymorphisms (18 Amino and 90 Silent)
. . . . . . . . 11I [#4I],
. . . . . . . . syn113E (GAg) [syn101E (GAg)],
. . . . . . . . . . . . . . . . . . . [Emergent H7N9:
. . . . . . . . . . . . . . . . . . . . . . . . . ChinaHangzhou3_79M_2013_04_02_s,
. . . . . . . . . . . . . . . . . . . . . . . . . gs/ckChinaJiangsuK27_2013_04_01],
. . . . . . . . . . . . . . . . . . . [Non-Emergent H7N9],
. . . . . . . . 130A [118A],
. . . . . . . . 183S [174S],
. . . . . . . . 188V [179V],
. . . . . . . . 195V [186V],
. . . . . . . . 198A [189A],
. . . . . . . . 211V [202V],
. . . . . . . . 217N [208N],
. . . . . . . . 235P [226P],
. . . . . . . . 285N [277N],
. . . . . . . . syn294G (GGg) [syn286G (GGg)],
. . . . . . . . . . . . . . . . . . . [Non-Emergent H7N9],
. . . . . . . . 299N [291N],
. . . . . . . . . . . .[sH3N2 wildtype],
. . . . . . . . 307D [299D],
. . . . . . . . 321R [313R],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . syn471E (GAg) [syn462E (GAg)],
. . . . . . . . . . . . . . . . . . . [Non-Emergent H7N9],
. . . . . . . . . . . . . . . . . . . [H5N1 Human and Avian wildtype],
. . . . . . . . 541V [533V])


NA Polymorphisms

. . . . TaiwanTaipei1_E1_53M_2013_04_24_s (
. . . . . . . . GISAID NA EPI445914
. . . . . . . . GISAID Isolate EPI_ISL_140356
. . . . . . . . 33 Polymorphisms (11 Amino and 22 Silent)
. . . . . . . . 16I [16I],
. . . . . . . . 19A [19A],
. . . . . . . . 40G [40G],
. . . . . . . . 53T [53T],
. . . . . . . . 68N [67N],
. . . . . . . . . . .[pH1N1 LAd],
. . . . . . . . . . .[H5N1 LAd2],
. . . . . . . . . . .[H9N2 LAd3],
. . . . . . . . . . .[H6N1 LAd3],
. . . . . . . . 81T [80T],
. . . . . . . . 84N [83N],
. . . . . . . . 112S [111S],
. . . . . . . . 335I [332I],
. . . . . . . . 359A [355A],
. . . . . . . . 401A [397A])

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-05-09-08_38_46_966420 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Additional mammalian adaptation indicated in extensive PB2 and HA Genetic Tracking updates for Human Emergent H7N9 from Zhejiang Province

• ChinaHangzhou3_79M_2013_04_02_s
• ChinaHangzhou2_67M_2013_03_25_f

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Fatal pH1N1 Homology
to
Emergent H7N9
Early Shanghai Divergences

Fudan University released emergent H7N9 segments at GenBank on May 13, 2013 for early divergent cases from Shanghai. No metadata or clinical outcome was provided for the 3 Hemagglutinin and 2 Polymerase Basic 2 fragments.

Please tell us that you have remaining original sample to implement this perfect use case to supersede Sanger for NextGen.

The human emergent H7N9 sequences exhibit disturbing homology of contiguous sub-segment genetics to mammalian-adapted serotypes. From aa355 to aa364 (aa346-aa355) on the Hemagglutinin segments, these deposits bear a striking resemblance to well-adapted human motifs that presently circulate widely in swine, humans and birds. At four positions (355, 356, 357 & 361) identical polymorphisms occur across all three Shanghai sequences, none of which have been previously seen in the H7N9 reservoir.

The 3 HA segments are also missing the syn359G (GGt) [syn350G (GGt)] that is carried by all other emergent H7N9 (human, avian, environment) and is wildtype on the currently circulating human seasonal H3N2. One 2010 sH3N2 strain from Africa matches these emergent H7N9 for 28 of 29 of the divergent nucleotides, excepting the 3rd base of aa350 that remains as wildtype sH3N2 and matches 100% of the previous emergent H7N9 deposits at that position. A divergent human H1N1 from 2005 (host species transitional) that has previously been under investigation for pH1N1 contributions also demonstrates homology for 28 of 29 of the H7N9 divergent nucleotides, excepting the final residue.

Homology to the first H7N9 human fatality occurs at 3 notable positions on 2 of these HA segments:

ChinaShanghai4665T_2013_03_05
ChinaShanghai4655T_2013_02_26
~ ChinaShanghai1_E1_87M_2013_02_26_f
. . . . . . . . aa410 wildtype, syn490S (AGc), aa541 wildtype

Key mammalian adaptation markers (627K, 701N) cannot be evaluated due to PB2 fragments only covering aa272 and aa345, aa348 respectively. However, potential contiguous sub-segment genetic acquisition of previously human-adapted genetics exists at the beginning of these 2 PB2 segments. The first 21 nucleotides of these PB2 sequences bear 5 synonymous polymorphisms in common that have each been individually found on non-emergent H7N9, but never on emergent H7N9. The string of 21 nucleotides has, however, travelled as a unit on H5N2 swine, H3N2 swine as recently as 2012, H1N2 swine, H1N1 swine and on 20 human pH1N1 sequences, including at least 2 fatalities, surfacing on 2009-06-01 in England and being last documented in China at the end of 2009.

pH1N1 geography sharing PB2 homology with the current emergent H7N9 human Shanghai sequences:

• England (5) with 1 HA 225E and 1 Fatality
• America (4)
• Scotland (2)
• Netherlands (3) with 1 HA 225N
• Denmark
• Russia with HA 225G Fatality
• Australia
• Peru
• Canada
• China

PB2 Links

. . . . ChinaShanghai4665T_2013_03_05 (
. . . . . . . . GenBank PB2 KF006371
. . . . . . . . PB2 Truncated before aa272,
. . . . . . . . PB2 Truncated after aa345)

. . . . ChinaShanghai4659T_2013_02_27 (
. . . . . . . . GenBank PB2 KF006370
. . . . . . . . PB2 Truncated before aa272,
. . . . . . . . PB2 Truncated after aa348)

HA Polymorphisms

. . . . ChinaShanghai4665T_2013_03_05 (
. . . . . . . . GenBank HA KF006369
. . . . . . . . 33 Polymorphisms (4 Amino and 29 Silent)
. . . . . . . . HA Truncated before aa355 [HA Truncated before aa346],
. . . . . . . . syn355G (GGa) [syn346G (GGa)],
. . . . . . . . . . . . [pH1N1.Upsilon High-CFR],
. . . . . . . . . . . . [sH3N2 wildtype],
. . . . . . . . . . . . [H5N1 wildtype],
. . . . . . . . 356M [347M],
. . . . . . . . . . . . [pH1N1 wildtype],
. . . . . . . . . . . . [sH3N2 wildtype],
. . . . . . . . . . . . [H5N1 wildtype],
. . . . . . . . syn357I (ATa) [syn348I (ATa)],
. . . . . . . . . . . . . . . . . . . [pH1N1 2009, 2010, 2011 & 2012,
. . . . . . . . . . . . . . . . . . . . . . . . including American DoD and severe cases in China],
. . . . . . . . . . . . . . . . . . . [vH1N1 Human America from swine 2011
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Canada from swine 2012],
. . . . . . . . . . . . . . . . . . . [H1N2 Human India 2009],
. . . . . . . . . . . . . . . . . . . [sH3N2 Africa 2010],
. . . . . . . . . . . . . . . . . . . [H3N2 Avian America 2011, 2010 extensive],
. . . . . . . . . . . . . . . . . . . [H3N8 extensive],
. . . . . . . . . . . . . . . . . . . [avH1N1farm],
. . . . . . . . . . . . . . . . . . . [swH1N1 extensive],
. . . . . . . . syn361Y (TAc) [syn352Y (TAc)],
. . . . . . . . . . . . . . . . . . . [pH1N1 wildtype],
. . . . . . . . . . . . . . . . . . . [sH3N2 wildtype],
. . . . . . . . . . . . . . . . . . . [H5N1 Human Indonesia 2012 (2),
. . . . . . . . . . . . . . . . . . . . . . . . . . . . Indonesia 2011 Fatality,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . Egypt 2008],
. . . . . . . . . . . . . . . . . . . [H5N1 Avian Extensive
. . . . . . . . . . . . . . . . . . . . . . . . . . . . China Guizhou gal 2008,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . Egypt gal 2008, 2009,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . Nigeria gal 2007],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . syn490S (AGc) [syn481S (AGc)],
. . . . . . . . . . . . . . . . . . . [H7N7],
. . . . . . . . . . . . . . . . . . . [H9N2],
. . . . . . . . . . . . . . . . . . . [H5N1],
. . . . . . . . . . . . . . . . . . . [avH1N1farm])

. . . . ChinaShanghai4659T_2013_02_27 (
. . . . . . . . GenBank HA KF006368
. . . . . . . . 34 Polymorphisms (6 Amino and 28 Silent)
. . . . . . . . HA Truncated before aa355 [HA Truncated before aa346],
. . . . . . . . syn355G (GGa) [syn346G (GGa)],
. . . . . . . . 356M [347M],
. . . . . . . . syn357I (ATa) [syn348I (ATa)],
. . . . . . . . syn361Y (TAc) [syn352Y (TAc)],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . 541V [533V])

. . . . ChinaShanghai4655T_2013_02_26 (
. . . . . . . . GenBank HA KF006367
. . . . . . . . 33 Polymorphisms (4 Amino and 29 Silent)
. . . . . . . . HA Truncated before aa355 [HA Truncated before aa346],
. . . . . . . . syn355G (GGa) [syn346G (GGa)],
. . . . . . . . 356M [347M],
. . . . . . . . syn357I (ATa) [syn348I (ATa)],
. . . . . . . . syn361Y (TAc) [syn352Y (TAc)],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . syn490S (AGc) [syn481S (AGc)])

H7N9 Influenza Hemagglutinin Segments elucidated at 2013-05-16-09_54_36_316350 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Two Months
is
Too Long

These GeneWurx comments were originally written on an earlier thread concerning a joint production of various CDC units of China that was web published on 2013-05-22 in the New England Journal of Medicine Correspondence entitled Live-Animal Markets and Influenza A (H7N9) Virus Infection:

Summary

A finding of 228S on the emergent human H7N9 Hemagglutinin, an experimental potentiator of human transmission, apparently de-motivated the authors to publish immediately. Intentionally withholding this data, from a hospitalisation on 2013-03-23 and an admitted tracheal sample taken on 2013-03-28, until now is clearly faulty thinking if, in fact, the reported genetic results are validated. The paper appears to have been written some time ago because the analyses, including the phylogenetic trees, are missing current sequences for comparative purpose.

Aged Phylogenetic Trees

Figure 1. Phylogenetic Tree of Full-Length Matrix Protein (M) Gene of Novel H7N9 Virus. (clickable from original paper) caption (emphasis ours):

The two isolated viral strains of H7N9 — Env/Nanjing and Nanjing/1 — had a remarkable degree of separation from the three other available human strains (Shanghai 1, Shanghai 2, and Anhui 1). Phylogenetic analysis showed that the M genes in the novel virus were derived from avian H9N2 viruses, among others.

The authors did create the illusion of currency by updating the first paragraph with case count and fatality count statistics prior to publication, but the phylogenetic tree caption in Figure 1 and the trees for all segments discussed in the supplement indicate a very early date of production. The comparative set of 2 early Shanghai and the single Anhui human sequences represented the full, public human set for only 5 days, from 2013-03-31 until 2013-04-04. On April 5, 2013, ChinaHangzhou1_C1_38M_2013_03_24_f was deposited at GISAID, a sequence sampled only four days before the Nanjing1 sequence that is now being profiled today for the first time (New England Journal of Medicine).

Furthermore, we would estimate that the authors of this current paper had earlier access than the public to all sequences produced by the CDC units of China. At any rate, the comparatives found on their trees (if restricted to public data) are suggestive of an analytic run prior to April 5, 2013, more than 6 weeks prior to the publication.

Viral researchers and epidemiologists are well aware of what can happen in 30 days during a pandemic emergence. Perhaps we should take caution prior to our next standing ovation for the ostensible transparency and cooperation of these organisations that withhold critical data at inflection points of human history. Who, in the public health leadership, would possibly advise a group of bench scientists to withhold such data? And who in that group of bench scientists would ever allow such information to be withheld?

The narrative indicates HA 228S, but the actual chart of polymorphisms per segment found in the supplement shows that the two sequences under discussion do NOT have the 228S, but remain at 228G wildtype? The Env/Nanjing and Nanjing/1 columns next to HA G228S are notated as 'G', the same as all other H7N9 sequences.

Narrative

From Page 1, Paragraph 4, Sentence 1 of the original paper (emphasis ours):

Two viral strains, one from the patient (Nanjing/1) and one from poultry-cage specimens obtained in the neighboring stall (Env/Nanjing), were successfully isolated (GenBank numbers, KC896771-KC896778 for the case specimen and KC896763-KC896770 for the environmental specimen).

From Page 1, Paragraph 4, Sentence 3 of the original paper (emphasis ours):

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A, Q226L, and G228S (H3 numbering) in the receptor-binding site of hemagglutinin.

Data in Supplement

From Page 12 of the supplement document (labeled 'Page 11'), we have excerpted the lines of interest (emphasis ours, dots employed for web spacing):

Table S1. Analysis of the Key Amino Acid Positions in Different Viral Proteins Related with Interspecies Transmission or Drug Resistance.

. . . . . . . . . . . . . . . . . . . . . . . . . . . Env/Nanjing Nanjing/1 Shanghai/1 Shanghai/2 Anhui/1
HA Receptor binding site (RBS) G228S . . . . . G . . . . . . . G . . . . . . G . . . . . . G . . . . . G . . .

Is this disjunction between the narrative and the actual data yet another publication error in a peer-reviewed journal concerning this potential H7N9 pandemic emergence? Does the peer-review process, at some point, involve a reading of the paper?

The sequences are not yet available at GenBank for verification.

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

The two referenced Nanjing sequences have been made available at GenBank now and do NOT show HA G228S.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

New England Journal of Medicine
Retraction of HA G228S Claim
without
Erratum

GeneWurx comments were originally written on an earlier thread and in earlier posts on this thread concerning a joint production of various CDC units of China that was web published on 2013-05-22 in the New England Journal of Medicine Correspondence entitled Live-Animal Markets and Influenza A (H7N9) Virus Infection:

While we are grateful and relieved to see the Chinese CDC (joint team) remove the claim of HA G228S from the peer-reviewed New England Journal of Medicine discussion, we are also dismayed that no erratum is notated nor any versioning indicated so that those who were mis-led by the error may be suitably relieved of their first impression concerns and may properly cascade their retractions. The small print at NEJM does, however, indicate that an update took place on May 23, but does not document the subject matter or attribution of the revision.

This letter was published on May 22, 2013, and updated on May 23, 2013, at NEJM.org

Narrative

From Page 1, Paragraph 4, Sentence 3 of the original paper (emphasis ours):

Original Publication

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A, Q226L, and G228S (H3 numbering) in the receptor-binding site of hemagglutinin.

Current Web Image

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A and Q226L (H3 numbering) in the receptor-binding site of hemagglutinin.

BP2 versus PB2

If interest remains toward accuracy, the authors may wish to involve their scribes in reading the next sentence after today's correction. Making another update there will add to readability of the segment name that carries the very important mammalian determinant at Polymerase Basic 2 aa627. BP2 should, perhaps be restated as the more common abbreviation, "PB2"? Secondarily, it's never too late for the editorial team at NEJM to have a reading of the full text.

From Page 1, Paragraph 4, Sentence 4 of the updated paper (emphasis ours):

The two isolates showed E rather than K at position 627 of BP2.

PB2 627E has been and remains the standard amino for non-mammal-hosted emergent H7N9, so finding that value in a poultry cage should be expected and is not remarkable. On the other hand, their finding of a human sequence without PB2 627K or PB2 701N was NOVEL and is most certainly a matter of international interest though the specimen was taken from an upper respiratory location. Broncho-alveolar lavage, the gold standard for emergent H7N9, may have produced a more representative Influenza organism with PB2 mammalian adaptations.

No explanation by any of the joint authors from the various Chinese CDC organisations has been added to the discussion concerning the extensive delay in publication of these sequences.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Mammalian Adaptation
of
Emergent H7N9
Neuraminidase

A human sequence, ChinaNanjing1_2013_03_28 [KC896776], was placed on deposit at GenBank by the Jiangsu CDC on 2013-05-23. Additionally, on 2013-05-26 the Zhejiang University School of Medicine deposited a human sequence, ChinaZhejiangHZ1_2013_04 [KF055467], also at GenBank with similar Neuraminidase findings. We estimate that the Zhejiang sequence is from Hangzhou due to name encoding (HZ) and sequence similarity to an earlier Hangzhou sequence.

As of these deposits, the Neuraminidase of emergent ΣH7N9 now has 5 instances of 305V, an amino value found as wildtype in human pH1N1 and in avian H1N1. Furthermore, the amino value is wildtype in H5N1, H6N1 and H9N2, including human cases.

. . . . . . . . 305V [304V],
. . . . . . . . . . . . [Emergent H7N9 ChinaZhejiangHZ1_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . ChinaHangzhou3_79M_2013_04_02_s [Analysis],
. . . . . . . . . . . . . . . . . . . . . . . . ChinaNanjing1_2013_03_28,
. . . . . . . . . . . . . . . . . . . . . . . . envChinaShanghaiS1088_2013_04_03,
. . . . . . . . . . . . . . . . . . . . . . . . ckChinaShanghaiS1053_2013_04_03],
. . . . . . . . . . . . [pH1N1 WildType
. . . . . . . . . . . . . . . . . . . . . . . . . with codon GTt [107] Re-Emergence North America:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2012 Toddler & Adult TamiFlu Resistance],
. . . . . . . . . . . . [avH1N1 WildType],
. . . . . . . . . . . . [H5N1 WildType],
. . . . . . . . . . . . [H6N1 WildType],
. . . . . . . . . . . . [H9N2 WildType]

. . . . . . . . 345I [341I],
. . . . . . . . . . . . [Emergent H7N9 ChinaZhejiangHZ1_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . ChinaHangzhou3_79M_2013_04_02_s [Analysis],
. . . . . . . . . . . . . . . . . . . . . . . . ChinaNanjing1_2013_03_28],
. . . . . . . . . . . . [pH1N1 Australia 2009-11],
. . . . . . . . . . . . [H5N1 LAu China ostrich 2003,
. . . . . . . . . . . . . . . . . . . . China anas 2001]

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

TamiFlu Resistance
via
NA 292K
in
Taiwan and Shanghai

For the sake of clarity, these findings were also cited in the recent WHO/China Joint Mission assessment paper: http://www.who.int/influenza/human_a...eport2013u.pdf


(...)

The first three isolates contained a number of genetic signatures previously associated in other subtypes with low pathogenicity in poultry, enhanced capacity for mammalian infection, resistance to the adamantane class of antiviral drugs and sensitivity to the neuraminidase inhibitors oseltamivir and zanamivir. These signatures include the following:

• A single arginine at the HA cleavage site, consistent with low pathogenicity in poultry;
• Deletion of five amino acids in the NA stalk, associated with adaptation to poultry;
• Q226L substitution in the HA, associated with enhanced binding to the α-2,6-linked sialylated receptors found in the mammalian respiratory tract;
• E627K in the PB2 protein, associated with viral replication at the lower temperature of the mammalian respiratory tract;
• S31N in the M2 protein, conferring resistance to adamantanes;
• Absence of the H275Y substitution in the NA, associated with resistance to the oseltamivir in H1N1 viruses;
• R292K in one virus (A/Shanghai/1/2013), associated with markedly reduced sensitivity to oseltamivir and modestly reduced sensitivity to zanamivir.

In vitro analyses confirmed that all three viruses bound both α -2,3- and α -2,6 linked sialic acids, suggesting an ability to bind to both avian and mammalian cells.

This analysis was published first at 2013-05-27-08:43 on an earlier thread entitled Drug-resistant H7N9 strains to change treatment: researcher as an up-to-the-minute TamiFlu Resistance summary of emergent ΣH7N9 in reply to Giuseppe's excellent synoptic of the press release. Sequences found only at GenBank, only at GISAID and at both were aggregated into a superset, de-duplicated, transformed and evaluated for this analysis:

The original deposit of the Taiwan E1 passage does not show the TamiFlu Resistance marker, but 2 isolates of ostensibly (no metadata) alternate tissue samples from a subsequent Taiwan deposit show the same change as the emergent H7N9 index case from Shanghai and two other May samplings from the Pasteur Shanghai release.

At this time, 5 of the emergent H7N9 human sequences indicate genotypic TamiFlu Resistance.

Another researcher today published suspiciously parallel, but un-attributed, material approximately 2 hours later at 10:41 AM. This pattern of a 15 minute to 2 hour reproduction cycle has drawn our focus and is observed weekly. The re-publication this morning unfortunately misrepresents the molecular mechanism. When a researcher cannot be troubled to conduct original research, then a direct quote with attribution is in order to prevent misrepresentation.

That individual appears to be interchanging this particular NA R292K (N2 numbering) genotypic marker with the amino acid position 294 (N2 numbering) polymorphism, NA N294S, that is also associated with TamiFlu Resistance on other backgrounds. Aoki, Boivin and Rogers discussed the molecular mechanism for 292K in Antiviral Therapy 12:603-616, 2007, Influenza virus susceptibility and resistance to oseltamivir [pdf] as defined by Kati, et al in Antimicrobial Agents and Chemotherapy, 2002, In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication [pdf].

We would like to clarify specifically that the emergent H7N9 TamiFlu Resistance is being discussed due to the mechanism at amino acid position 292 (N2 numbering) as defined in the Aoki, et al review, though the polymorphism is found in absolute ordination on the H7N9 at aa294 of the Neuraminidase sequences. In order to support Cross Serotype Analysis, the polymorphism is also carried alternately as NA 293K in GeneWurx's present modeling instantiation.

Additionally, we formally declare that GeneWurx RnR Analytic reporting on emergent H7N9 stands separate from those who re-publish our findings without attribution and with insertional errors.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Fatalities
Associated With
TamiFlu Resistance
via
NA 292K

Of the 5 sequences representing 4 human cases that carry the NA 292K polymorphism, 2 fatalities have occurred, both in Shanghai. At least 3 of the 4 cases required treatment involving in extremis measures such as ECMO.

Today's publication in The Lancet from Dr. Malik Peiris and an extensive multi-disciplinary team of Chinese organisations defines clinical progressions, treatments, outcomes and experimental findings including discussions on two TamiFlu Resistance markers, NA 292K and NA 152K. Their initial study reveals that the single 292K polymorphism increases oseltamivir resistance 100 fold.

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

We have shown that A/Shanghai/1/2013 (H7N9) virus isolate contains a mixed population of Arg/Lys at position 292 of the NA gene, and by purifying virus plaques that carry NA Arg292 and Lys292, we have noted that this mutation increases resistance to oseltamivir by 100-fold and zanamivir by 30-fold in a fluorescence-based NA inhibition assay (unpublished data).

In patient 6 (from the mechanical ventilation group), we found emergence of Arg152Lys mutation in the NA gene. This mutation was first reported from an
immunocompromised patient infected with influenza B after zanamivir treatment.13 Using the baculovirus expressed N9 NA protein, it had been shown to exhibit mild resistance to both zanamivir and oseltamivir in vitro.14

We have been concerned that the few detailed clinical progressions may not have fully reported sepsis. This paper provides insight confirming one type of clinical similarity to other zoonotics including many pH1N1 cases and H5N1 human infections:

We detected viral RNA in the serum obtained at some time during the clinical illness of 12 (86%) of the 14 patients (appendix; all three patients in the ECMO group, all four patients in the mechanical ventilation group, and five of seven patients in the pneumonia group).

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

From Tamiflu-Resistance Gene in H7N9 Bird Flu Spurs Drug Tests thread:

Almost all patients responded to oseltamivir treatment.

In some ECMO patients, and / or under corticosteroids treatment, R292K mutation in the NA happened, despite initial viral suppression.

During the course of treatment, a viral load 'rebound' was observed and it was suggested that either the immune status of patient or the concurrent use of steroids made the emersion of R292K much easy than in other patients.

Mixed population of susceptible and resistant to oseltamivir H7N9 viruses in certain cases has been noted from ALL the WHO updates so far.

Since the old age of the patients and the severity of the disease in these cases, this represents an expected finding as happened for H5N1 cases in the past.

Functional antivirals tests performed on R292K isolates demonstrated they are susceptible to oseltamivir and zanamivir albeit with some degree of variability.

It should be used a combination therapy in order to safeguard the limited arsenal of antivirals currently available for treatment and for pandemic preparedness stockpile.

An expedited clinical testing of such combinations should be performed as soon as possible in addition to an enhance surveillance for resistant strain in human and animals.

GM

Almost all Influenza cases self-resolve without treatment of any kind (~99.1 to 99.4%).

We can hardly nominate this situation as a clinical success when a substance designed to prevent the release of viral RNA from human cells preceded a finding of viral RNA in the blood from 86% (12 of 14) of professionally-treated patients. As you may recall, virus in the blood is a rarity in human influenza infections and is a direct indicator of increased morbidity. The entire cohort of 14 patients was treated aggressively with oseltamivir.

A 100 fold reduction in inhibition due to the NA 292K is no minor matter when 5-fold increase in IC50 is a traditional boundary for gathering statistics. Drug resistance was recorded as early as 5 days after treatment initiation.

Table 1: Demographic details, therapy, and outcome of patients with A/H7N9 infection appears to indicate that multi-drug antiviral therapy contributed at this time to only 1 successful discharge (Patient 8 at 22 days) with 1 remaining under standard treatment (Patient 5 at 37 days) and with 1 remaining on ECMO (Patient 3 at 46 days) at the time of data gathering. One death has also occurred after multi-drug treatment (Patient 2 at 19 days).

Ergo, at the time of data gathering, the "Released versus Deceased" counts are equal for this cohort (1/1) . . . demonstrating something much less than a stellar summary.

Despite the continual applause at these self-declared victories, the actual data predicts a future catastrophic clinical failing (even where logistics allow heroic measures) should emergent H7N9 transmission increase . . .

We would like to see an emergence of transparency in China by their reporting all of the current cases before the world is faced with a simultaneous emergence of H7N9 pathogen on all populated continents.

Their publication concerning the Beijing case is a start. Now let's put all the data on the table so that strategies may be developed that show potential for success.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

TamiFlu Resistance
via
NA 292K

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

Four of the fourteen Sanger Neuraminidase sequences deposited at GenBank by Fudan University today and associated with the 2013-05-28 AntiViral Resistance paper (Malik Peiris and associates, The Lancet) show NA 292K. Two patients were indicated in the paper as developing antiviral resistance at NA 292K.

We are grateful to Dr. Peiris for coordinating production of the cross-reference information with the authoring organisations concerning serialisation and patient assignment for the samplings.

NA 292K

Our preliminary groupings for the four genotypic AntiViral Resistant sequences are estimated until we receive the master cross-reference. These groups represent either patient 2 or patient 3 from the tables in the paper. ECMO was required for each. Patient 2 expired at 19 days post-onset; patient 3 remained under treatment at 46 days (as of May 18, 2013). Group 1 is very likely Patient 2 who died during ECMO treatment. At least one fatality is represented in these 2 groupings.

We have arranged Group 1 due to Hemagglutinin homology:

Group 1 with 3 Sequences
Probable as Patient 2 Fatality
Drug Resistance by Treatment Day 4

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028381]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028380]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028379]

Group 2 with 1 Sequence
Probable as Patient 3

• ChinaShanghai4842T_56M_2013_04_13_TmX_s [KF028383]

NA Polymorphism Summary

ChinaShanghai4798T_62M_2013_04_12_TmX_s [KF028387] shows 152K and is likely Patient 6 who was mechanically ventilated and successfully discharged 35 days from onset. The polymorphism developed after day 3 of oseltamivir treatment and by the sampling date on day 5 of treatment.

ChinaShanghai4821T_2013_04_12_s [KF028392] shows NA 285K [284K] with first and third base polymorphisms. Only the emergent H7N9 ChinaShanghai4664T_2013_03_05 [KC853231] and a single instance from H4N9 avian show this polymorphism in the data record.

HA Polymorphism Summary

Three HA sequences carry a synonymous change at the third base of aa471 producing syn471E (GAg) [syn462E (GAg)]:

Probable as Patient 2 Fatality

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028376]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028375]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028374]

All three are also TamiFlu Resistant and are from a patient that required ECMO.

This syn462E (GAg) polymorphism has been seen in non-emergent H7N9 and in 5 emergent Human H7N9 sequences, including 3 from the single Taiwan case and 2 from the Pasteur Shanghai GISAID deposit of preliminary sequences on 2013-05-23.

The revision is also found in Pandemic H1N1 2009 (pH1N1), H5N1 (wildtype), H7N3 (extensive), H7N7 (rare, passerine), H9N2 (extensive, ostrich) and H9N9. Four of the nine instances within pH1N1 (all host transition from 2009) are from China and three others are from neighboring Asian countries. Argentina shows 2 sequences from subjects aged 2 years and younger. Seven of the eight pH1N1 sequences with this polymorphism and age metadata are pediatric cases and one of those Chinese sequences was published as part of a drug resistance investigation.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

GISAID Citations

Zhejiang Fatal H7N9
with
Divergent NA, PB2 701N and Aberrant PA

We would like to thank the researchers at Zhejiang University for their GISAID deposit on 2013-04-17 that included this divergent passaging of one emergent H7N9 fatal human sequence. The Hemagglutinin and Polymerase Basic 1 are stock emergent H7N9, ordinary. However, the Polymerase Acidic segment carries 36 polymorphisms with striking Human H5N1 sub-segment homology to:

• China Wet Market 2009 ~23 polymorphisms
• Vietnam Fatality 2008 . ~21 polymorphisms

The Polymerase complex segments (PB2, PB1, PA) were referenced against their respective ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f segments, the Hemagglutinin and Neuraminidase against a pseudo-consensus.

PB2 Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PB2 EPI441801
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 3 Polymorphisms (3 Amino and 0 Silent)
. . . . . . . . 395S [ChinaShanghai1_E1_87M_2013_03_04_f],
. . . . . . . . 627E [ChinaZhejiang2_64M_2013_04_03_f,
. . . . . . . . . . . . . . Nanjing1_45F_2013_03_28,
. . . . . . . . . . . . . . TaiwanTaipeiT02081_53M_2013_04_22_s],
. . . . . . . . . . . . . . [H7N9 Avian],
. . . . . . . . 701N [ChinaZhejiang2_64M_2013_04_03_f])

PB1 Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PB1 EPI441800
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 1 Polymorphisms (0 Amino and 1 Silent)
. . . . . . . . syn544A (GCc))

PA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PA EPI441795
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 36 Polymorphisms (5 Amino and 31 Silent)
. . . . . . . . . . . . . . ~ H5N1 Human China Wet Market 2009 (23 polymorphisms)
. . . . . . . . . . . . . . ~ H5N1 Human Vietnam Fatality 2008 (21 polymorphisms)
. . . . . . . . syn7Q (CAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn58G (GGc) [H5N1 Human China, Vietnam],
. . . . . . . . syn62I (ATt) [H5N1 Human China, Vietnam],
. . . . . . . . 70V,
. . . . . . . . syn98T (ACg),
. . . . . . . . 100V [H5N1 Human China, Vietnam],
. . . . . . . . syn116R (CGa) [H5N1 Human China, Vietnam],
. . . . . . . . syn136N (AAc) [H5N1 Human China],
. . . . . . . . syn146H (CAc) [H5N1 Human China, Vietnam],
. . . . . . . . syn148F (TTc) [H5N1 Human China, Vietnam],
. . . . . . . . syn203E (GAg),
. . . . . . . . syn248Q (CAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn257I (ATc),
. . . . . . . . syn262R (AGg) [H5N1 Human China, Vietnam (3rd Base)],
. . . . . . . . 272D [H5N1 Human China, Vietnam],
. . . . . . . . syn318K (AAg),
. . . . . . . . syn390L (cTG) [ChinaShanghai1_E1_87M_2013_03_04_f],
. . . . . . . . . . . . . . . . . . [H5N1 Human China, Vietnam],
. . . . . . . . 394D [H5N1 Human China, Vietnam],
. . . . . . . . 409S [H5N1 Human China, Vietnam (2nd Base)],
. . . . . . . . syn414A (GCa) [H5N1 Human China, Vietnam],
. . . . . . . . syn417L (tTG) [H5N1 Human China, Vietnam],
. . . . . . . . syn419D (GAt) [H5N1 Human China, Vietnam],
. . . . . . . . syn433A (GCt) [H5N1 Human China, Vietnam],
. . . . . . . . syn446F (TTc) [H5N1 Human China, Vietnam],
. . . . . . . . syn483I (ATc),
. . . . . . . . syn489C (TGc) [H5N1 Human China, Vietnam],
. . . . . . . . syn500L (CTa),
. . . . . . . . syn508R (AGg),
. . . . . . . . syn510H (CAc) [H5N1 Human China],
. . . . . . . . syn558S (TCt),
. . . . . . . . syn609K (AAg) [ChinaShanghai4664T_2013_03_05],
. . . . . . . . syn616S (TCt),
. . . . . . . . syn623E (GAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn625P (CCc),
. . . . . . . . syn648S (AGt),
. . . . . . . . syn681F (TTc) [H5N1 Human China, Vietnam])

HA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID HA EPI441794
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 104 Polymorphisms (17 Amino and 87 Silent)
. . . . . . . . 11I [#4I],
. . . . . . . . 130A [118A],
. . . . . . . . 183S [174S],
. . . . . . . . 188V [179V],
. . . . . . . . 195V [186V],
. . . . . . . . 198A [189A],
. . . . . . . . 211V [202V],
. . . . . . . . 217N [208N],
. . . . . . . . 235L [226L],
. . . . . . . . 285N [277N],
. . . . . . . . 307D [299D],
. . . . . . . . 321R [313R],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . 541V [533V])

NA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID NA EPI441797
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 35 Polymorphisms (13 Amino and 22 Silent)
. . . . . . . . 16I [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 [19A],
. . . . . . . . . . . [avH1N1, H9N2],
. . . . . . . . 40G [40G],
. . . . . . . . . . . [H9N2],
. . . . . . . . 53T [53T],
. . . . . . . . 81T [80T],
. . . . . . . . . . . [avH1N1],
. . . . . . . . 84N [83N],
. . . . . . . . 112S [111S],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 335I [332I],
. . . . . . . . . . . .[H6N1],
. . . . . . . . 359A [355A],
. . . . . . . . 401A [397A]
. . . . . . . . . . . . [H6N1],
. . . . . . . . 439L [437L],
. . . . . . . . 442G [439G],
. . . . . . . . . . . . . . [Emergent H7N9 ckZhejiangDTIDZJU01_2013_04],
. . . . . . . . 443D [440D])

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-04-23-00_30_18_809650 by GeneWurx see.PolyDetector v0, Copyright 2007-2013
H7N9 Influenza PolymeraseBasic2, PolymeraseBasic1 and PolymeraseAcidic Segments elucidated at 2013-06-02-02_04_58_819480 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

GISAID Citations

Novel PB2, Divergent PB1 & Novel PA
from
First Fujian Case

We would like to thank the researchers at the Fujian Center for Disease Control and Prevention for their Emergent H7N9 supplementary GISAID deposit on 2013-06-04 dated 2013-05-27. The Polymerase and NP segments were deposited under a separate isolate identifier than the earlier HA, NA, MP and NS deposit leaving questions concerning passage history for the first GISAID isolate identifier (140881). No metadata or clinical outcome was provided by the depositors, although the FluTrackers 2013 Human Case List [#116] indicates an onset of April 18 and hospitalisation at April 23. The patient was discharged on May 13. This 65 year old male subject was sampled on April 24, 2013 and passage was annotated as 'E1'.

The case follows course having PB2 627K and a stock emergent H7N9 Hemagglutinin segment, but was originally made novel by the NA 148E (147E). The Neuraminidase polymorphism is found on the Indonesian H5N1 human Karo cluster from May 2006. The Fujian emergent H7N9 HA and NA were discussed on May 9, 2013 at FluTrackers post, Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case, [#107] within this thread.

This divergent Polymerase Basic 1 segment carries 7 polymorphisms, 5 that are not previously found on emergent H7N9. However, the bracketing polymorphisms, PB1 56A and syn544A (GCc), are previously found within ΣeH7N9.

The Polymerase complex segments (PB2, PB1, PA) were referenced against their respective ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f segments.

PB2 Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PB2 EPI453827
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 2 Polymorphisms (1 Amino and 1 Silent)
. . . . . . . . syn244Y (TAt) [eH7N9 ckChinaZhejiangDTIDZJU01_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . envChinaHangzhou34_2013_04_04],
. . . . . . . . 420L [H7N9 Novel])


PB1 Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PB1 EPI453828
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 7 Polymorphisms (5 Amino and 2 Silent)
. . . . . . . . PB1 Truncated before aa2,
. . . . . . . . 56A [eH7N9 pgnChinaShanghaiS1069_2013_04_02],
. . . . . . . . syn64P (CCt) [H5N1 China Human Fatality Cluster February 2013 with HA 202V:
. . . . . . . . . . . . . . . . . . . . . . . [EPI420390] ChinaGuizhou1_E1_21F_2013_02_08_f,
. . . . . . . . . . . . . . . . . . . . . . . [EPI420767] ChinaGuizhou2_E1E1_31M_2013_02_09_f],
. . . . . . . . . . . . . . . . . . [H5N1 China Hunan environment Dongting Lake 2008],
. . . . . . . . . . . . . . . . . . [H5N1 Bangladesh Avian gal 2012],
. . . . . . . . . . . . . . . . . . [H5N1 America Avian anas 2010],
. . . . . . . . . . . . . . . . . . [H5N1 India Avian gal 2010],
. . . . . . . . . . . . . . . . . . [H5N1 India Avian pas 2008],
. . . . . . . . . . . . . . . . . . [H9N2 China Avian gal 2011],
. . . . . . . . . . . . . . . . . . [neH7N9 Avian Alaska & Korea],
. . . . . . . . . . . . . . . . . . [H7N7 America Avian anas 2010],
. . . . . . . . . . . . . . . . . . [H7N7 Korea Avian anas 2009],
. . . . . . . . . . . . . . . . . . [H7N7 Korea Avian pas 2007],
. . . . . . . . . . . . . . . . . . [H7N7 Australia Avian pas 1985],
. . . . . . . . . . . . . . . . . . [H7N6 Japan Avian quail 2009],
. . . . . . . . . . . . . . . . . . [H3N2 China Avian anas 2011],
. . . . . . . . . . . . . . . . . . [H11N9 China Jiangxi Avian environment 2009],
. . . . . . . . . . . . . . . . . . [H11N9 America Avian anas 2009],
. . . . . . . . 338G [H7N9 Novel],
. . . . . . . . 373P [H7N9 Novel],
. . . . . . . . 376V [H7N9 Novel],
. . . . . . . . 377G [H7N9 Novel],
. . . . . . . . syn544A (GCc) [H7N9 Widespread (18)],
. . . . . . . . . . . . . . . . . . . [H5N1 China Human Fatality Cluster February 2013 with HA 202V:
. . . . . . . . . . . . . . . . . . . . . . . . [EPI420390] ChinaGuizhou1_E1_21F_2013_02_08_f,
. . . . . . . . . . . . . . . . . . . . . . . . [EPI420767] ChinaGuizhou2_E1E1_31M_2013_02_09_f])

PA Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PA EPI453837
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 3 Polymorphisms (2 Amino and 1 Silent)
. . . . . . . . syn58G (GGc),
. . . . . . . . 201T [eH7N9 Novel],
. . . . . . . . . . . . [neH7N9 dk_w_spotbillKorea447_2011_04],
. . . . . . . . 403V [H7N9 Novel])

H7N9 Influenza PolymeraseBasic2, PolymeraseBasic1 and PolymeraseAcidic Segments elucidated at 2013-06-04-03_11_36_299990 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & 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

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion