Rhiza Labs FluTracker Forum

Two more HA sequences from A/Nagano/RC1/2009 have been released at GISAID. The initial sequence was released at NIID in December and the sequence had D225G. The August infection (33M) was fatal. The two new sequences were from autopsy lung. One sequence had a mixture of G158E with wild type as well as D225G with D225N. The second sequence had G158E and D225N, supporting selection in the fatal lung infection as well as the presence of two or more distinct psuedospecies at position 225.

The recent HA sequences as well as the other seven gene segments will be available at Genbank shortly.

The appearance and modulation of these markers in autopsy lung raises concerns about the true level of D225G, D225N, and G158E in patients represented in teh sequence database.

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Influenza A virus (A/Nagano/RC1/2009(H1N1)) HA gene for hemagglutinin (HA-Major), complete cds, segment: 4
FeaturesSequenceLOCUS AB538389 1742 bp cRNA linear VRL 20-APR-2010
DEFINITION Influenza A virus (A/Nagano/RC1/2009(H1N1)) HA gene for
hemagglutinin (HA-Major), complete cds, segment: 4.
ACCESSION AB538389
VERSION AB538389.1 GI:294862209
DBLINK Project:37813
KEYWORDS .
SOURCE Influenza A virus (A/Nagano/RC1/2009(H1N1))
ORGANISM Influenza A virus (A/Nagano/RC1/2009(H1N1))
Viruses; ssRNA negative-strand viruses; Orthomyxoviridae;
Influenzavirus A.
REFERENCE 1
AUTHORS Kuroda,M., Katano,H., Nakajima,N., Tobiume,M., Ainai,A.,
Sekizuka,T., Hasegawa,H., Tashiro,M., Sasaki,Y., Arakawa,Y.,
Hata,S., Watanabe,M. and Sata,T.
TITLE Characterization of Quasispecies of Pandemic 2009 Influenza A Virus
(A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA
Sequence
JOURNAL PLoS ONE (2010) In press
REFERENCE 2
AUTHORS Nakajima,N., Hata,S., Sato,Y., Tobiume,M., Katano,H., Kaneko,K.,
Nagata,N., Kataoka,M., Ainai,A., Hasegawa,H., Tashiro,M.,
Kuroda,M., Odai,T., Urasawa,N., Ogino,T., Hanaoka,H., Watanabe,M.
and Sata,T.
TITLE The first autopsy case of pandemic influenza (A/H1N1pdm) virus
infection in Japan: detection of a high copy number of the virus in
type II alveolar epithelial cells by pathological and virological
examination
JOURNAL Jpn. J. Infect. Dis. 63 (1), 67-71 (2010)
PUBMED 20093768
REFERENCE 3 (bases 1 to 1742)
AUTHORS Kuroda,M. and Sekizuka,T.
TITLE Direct Submission
JOURNAL Submitted (16-DEC-2009) Contact:Makoto Kuroda National Institute of
Infectious Diseases, Pathogen Genomics Center; 1-23-1,
Shinjyuku-ku, Tokyo 162-8640, Japan
FEATURES Location/Qualifiers
source 1..1742
/organism="Influenza A virus (A/Nagano/RC1/2009(H1N1))"
/mol_type="viral cRNA"
/strain="A/Nagano/RC1/2009(H1N1)"
/serotype="H1N1"
/isolation_source="gender:M; age:33; Autopsy lung"
/host="Homo sapiens"
/db_xref="taxon:702698"
/segment="4"
/tissue_type="lung"
/country="Japan: Nagano"
/collection_date="27-Aug-2009"
/note="Consensus sequence obtained by 40 mer read mapping.
Sequencing by Illumina Genome Analyzer II.;
lineage=swl"
gene 12..1712
/gene="HA"
CDS 12..1712
/gene="HA"
/note="HA-Major (detected at 75%)"
/codon_start=1
/product="hemagglutinin"
/protein_id="BAJ05802.1"
/db_xref="GI:294862210"
/translation="MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVT
HSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETTSS
DNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKS
FYKNLIWLVKKXNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFV
GTSRYSKKFKPEIAIRPKVRXQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMER
NAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATG
LRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEI
TNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENER
TLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSE
EAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI"
ORIGIN
1 aagcaacaaa aatgaaggca atactagtag ttctgctata tacatttgca accgcaaatg
61 cagacacatt atgtataggt tatcatgcga acaattcaac agacactgta gacacagtac
121 tagaaaagaa tgtaacggta acacactctg ttaaccttct agaagacaag cataacggga
181 aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac attgctggct
241 ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg tcctacattg
301 tggaaacaac tagttcagac aatggaacgt gttacccagg agatttcatc gattatgagg
361 agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga
421 caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcatgctg
481 gagcaaaaag cttctacaaa aatttaatat ggctagttaa aaaagraaat tcatacccaa
541 agctcagcaa atcctacatt aatgataaag ggaaagaagt cctcgtgcta tggggcattc
601 accatccatc tactagtgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt
661 ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaata agacccaaag
721 tgaggrrtca agaagggaga atgaactatt actggacact agtagagccg ggagacaaaa
781 taacattcga agcaactgga aatctagtgg taccgagata tgcattcgca atggaaagaa
841 atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc
901 agacacccaa gggtgctata aacaccagcc tcccatttca gaatatacat ccgatcacaa
961 ttggaaaatg tccaaaatat gtaaaaagca caaaattgag actggccaca ggattgagga
1021 atgtcccttc tattcaatct agaggcctat ttggggccat tgccggtttc attgaagggg
1081 ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaaatgag caggggtcag
1141 gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact aacaaagtaa
1201 attctgttat tgaaaagatg aatacacagt tcacagcagt aggtaaagag ttcaaccacc
1261 tggaaaaaag aatagagaat ttaaataaaa aagttgatga tggtttcctg gacatttgga
1321 cttacaatgc cgaactgttg gttctattgg aaaatgaaag aactttggac taccacgatt
1381 caaatgtgaa gaacttatat gaaaaggtaa gaagccagtt aaaaaacaat gccaaggaaa
1441 ttggaaacgg ctgctttgaa ttttaccaca aatgcgataa cacgtgcatg gaaagtgtca
1501 aaaatgggac ttatgactac ccaaaatact cagaggaagc aaaattaaac agagaagaaa
1561 tagatggggt aaagctggaa tcaacaagga tttaccagat tttggcgatc tattcaactg
1621 tcgccagttc attggtactg gtagtctccc tgggggcaat cagtttctgg atgtgctcta
1681 atgggtctct acagtgtaga atatgtattt aacattagga tttcagaagc atgagaaaaa
1741 ac

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LOCUS AB538394 1742 bp cRNA linear VRL 20-APR-2010
DEFINITION Influenza A virus (A/Nagano/RC1/2009(H1N1)) HA gene for
hemagglutinin (HA-minor), complete cds, segment: 4.
ACCESSION AB538394
VERSION AB538394.1 GI:294862221
DBLINK Project:37813
KEYWORDS .
SOURCE Influenza A virus (A/Nagano/RC1/2009(H1N1))
ORGANISM Influenza A virus (A/Nagano/RC1/2009(H1N1))
Viruses; ssRNA negative-strand viruses; Orthomyxoviridae;
Influenzavirus A.
REFERENCE 1
AUTHORS Kuroda,M., Katano,H., Nakajima,N., Tobiume,M., Ainai,A.,
Sekizuka,T., Hasegawa,H., Tashiro,M., Sasaki,Y., Arakawa,Y.,
Hata,S., Watanabe,M. and Sata,T.
TITLE Characterization of Quasispecies of Pandemic 2009 Influenza A Virus
(A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA
Sequence
JOURNAL PLoS ONE (2010) In press
REFERENCE 2
AUTHORS Nakajima,N., Hata,S., Sato,Y., Tobiume,M., Katano,H., Kaneko,K.,
Nagata,N., Kataoka,M., Ainai,A., Hasegawa,H., Tashiro,M.,
Kuroda,M., Odai,T., Urasawa,N., Ogino,T., Hanaoka,H., Watanabe,M.
and Sata,T.
TITLE The first autopsy case of pandemic influenza (A/H1N1pdm) virus
infection in Japan: detection of a high copy number of the virus in
type II alveolar epithelial cells by pathological and virological
examination
JOURNAL Jpn. J. Infect. Dis. 63 (1), 67-71 (2010)
PUBMED 20093768
REFERENCE 3 (bases 1 to 1742)
AUTHORS Kuroda,M. and Sekizuka,T.
TITLE Direct Submission
JOURNAL Submitted (16-DEC-2009) Contact:Makoto Kuroda National Institute of
Infectious Diseases, Pathogen Genomics Center; 1-23-1,
Shinjyuku-ku, Tokyo 162-8640, Japan
FEATURES Location/Qualifiers
source 1..1742
/organism="Influenza A virus (A/Nagano/RC1/2009(H1N1))"
/mol_type="viral cRNA"
/strain="A/Nagano/RC1/2009(H1N1)"
/serotype="H1N1"
/isolation_source="gender:M; age:33; Autopsy lung"
/host="Homo sapiens"
/db_xref="taxon:702698"
/segment="4"
/tissue_type="lung"
/country="Japan: Nagano"
/collection_date="27-Aug-2009"
/note="Consensus sequence obtained by 40 mer read mapping.
Sequencing by Illumina Genome Analyzer II.;
lineage=swl"
gene 12..1712
/gene="HA"
CDS 12..1712
/gene="HA"
/note="HA-minor (detected at 25%)"
/codon_start=1
/product="hemagglutinin"
/protein_id="BAJ05809.1"
/db_xref="GI:294862222"
/translation="MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVT
HSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETTSS
DNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKS
FYKNLIWLVKKENSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFV
GTSRYSKKFKPEIAIRPKVRNQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMER
NAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATG
LRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEI
TNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENER
TLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSE
EAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI"
ORIGIN
1 aagcaacaaa aatgaaggca atactagtag ttctgctata tacatttgca accgcaaatg
61 cagacacatt atgtataggt tatcatgcga acaattcaac agacactgta gacacagtac
121 tagaaaagaa tgtaacggta acacactctg ttaaccttct agaagacaag cataacggga
181 aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac attgctggct
241 ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg tcctacattg
301 tggaaacaac tagttcagac aatggaacgt gttacccagg agatttcatc gattatgagg
361 agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga
421 caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcatgctg
481 gagcaaaaag cttctacaaa aatttaatat ggctagttaa aaaagaaaat tcatacccaa
541 agctcagcaa atcctacatt aatgataaag ggaaagaagt cctcgtgcta tggggcattc
601 accatccatc tactagtgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt
661 ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaata agacccaaag
721 tgaggaatca agaagggaga atgaactatt actggacact agtagagccg ggagacaaaa
781 taacattcga agcaactgga aatctagtgg taccgagata tgcattcgca atggaaagaa
841 atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc
901 agacacccaa gggtgctata aacaccagcc tcccatttca gaatatacat ccgatcacaa
961 ttggaaaatg tccaaaatat gtaaaaagca caaaattgag actggccaca ggattgagga
1021 atgtcccttc tattcaatct agaggcctat ttggggccat tgccggtttc attgaagggg
1081 ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaaatgag caggggtcag
1141 gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact aacaaagtaa
1201 attctgttat tgaaaagatg aatacacagt tcacagcagt aggtaaagag ttcaaccacc
1261 tggaaaaaag aatagagaat ttaaataaaa aagttgatga tggtttcctg gacatttgga
1321 cttacaatgc cgaactgttg gttctattgg aaaatgaaag aactttggac taccacgatt
1381 caaatgtgaa gaacttatat gaaaaggtaa gaagccagtt aaaaaacaat gccaaggaaa
1441 ttggaaacgg ctgctttgaa ttttaccaca aatgcgataa cacgtgcatg gaaagtgtca
1501 aaaatgggac ttatgactac ccaaaatact cagaggaagc aaaattaaac agagaagaaa
1561 tagatggggt aaagctggaa tcaacaagga tttaccagat tttggcgatc tattcaactg
1621 tcgccagttc attggtactg gtagtctccc tgggggcaat cagtttctgg atgtgctcta
1681 atgggtctct acagtgtaga atatgtattt aacattagga tttcagaagc atgagaaaaa
1741 ac

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The above characterization sheet indicate G158E and D225N were a minor sepcies (25%), while the mixed signal (G158E with wild type, as well as D225G with D225N) sequence was at 75%.

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The First Autopsy Case of Pandemic Influenza (A/H1N1pdm) Virus Infection in Japan: Detection of a High Copy Number of the Virus in Type II Alveolar Epithelial Cells by Pathological and Virological Examination

Noriko Nakajima1, Satoru Hata2, Yuko Sato1, Minoru Tobiume1, Harutaka Katano1, Keiko Kaneko1, Noriyo Nagata1, Michiyo Kataoka1, Akira Ainai3, Hideki Hasegawa1,3, Masato Tashiro3, Makoto Kuroda4, Tamami Odai5, Nobuyuki Urasawa5, Tomoyoshi Ogino6, Hiroaki Hanaoka6, Masahide Watanabe6, and Tetsutaro Sata1*

1Department of Pathology, 3Influenza Virus Research Center, and 4Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo 162-8640; and 2Department of Clinical Laboratory, 5Department of Cardiology, and 6Department of Pathology, Nagano Red Cross Hospital, Nagano 380-8582, Japan

(Received December 15, 2009. Accepted January 5, 2010)



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*Corresponding author: Mailing address: Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-5285-1111, Fax: +81-3-5285-1189, E-mail: tsata@nih.go.jp



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SUMMARY: We report the pathological and virological findings of the first autopsy case of the 2009 pandemic influenza (A/H1N1pdm) virus infection in Japan. A man aged 33 years with chronic heart failure due to dilated cardiomyopathy, mild diabetes mellitus, atopic dermatitis, bronchial asthma, and obesity died of respiratory failure and multiple organ dysfunction syndrome. Macroscopic examination showed　severe　plumonary　 edema　and　microscopically the lung sections showed very　early exudative-stage　diffuse　alveolar　damage (DAD). Immunohistochemistry revealed proliferation of the influenza (A/H1N1pdm) virus in alveolar epithelial cells, some of which expressed SAα2-3Gal on the cell surface. Influenza (A/H1N1pdm) virus genomic RNA and mRNA were also detected in alveolar epithelial cells. Real-time PCR revealed 723 copies/cell in the left lower lung section from which the influenza (A/H1N1pdm) virus was isolated. Electron microscopic analysis revealed filamentous viral particles in the lung tissue. The concentrations of various cytokines/chemokines in the serum and the autopsied lung tissue were measured. IL-2R, IL-6, IL-8, IL-10, IFN-α, MCP-1, and MIG levels were elevated in both. These findings indicated a case of viral pneumonia caused by influenza (A/H1N1pdm) virus infection, showing characteristic pathological findings of the early stage of DAD.

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Patient tested negative on rapid test. No oseltamivir given. H1N1 positive at autopsy.

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These data raise serious concerns over how reprentative sequences are of the pH1N1 infections. There are three different HA sequences from the same patient. One has D225G, one has D225G with D225N, and one has D225N. Similarly, there were three different results for position 158 (wild type, wild type with G158E, and G158E).

The presence of D225G and D225N in the same fatally infected patient has been reported in Ukraine, Moldova, Mexico, and the US. In addition the Duke cluster, which involved three fatalities, had D225G in the index case (in two seperate collections), D225N in another, and wild type in a third. WHO and CDC have yet to acknowledge transmission of D225G or D225N in this cluster or any other cluster.

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Commentary

http://www.recombinomics.com/News/04201 ... seudo.html

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