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  • gsgs
    replied
    Re: list of reassortment studies

    [from another thread, I want it here too]

    June 12th, 2014, 06:06 AM
    gsgs
    Registered User Join Date: Feb 2006
    Location: germany
    Posts: 10,542

    Re: Genes found in nature yield 1918-like virus with pandemic potential

    --------------------------------------------------------------------------------


    Found it here:
    http://www.cell.com/cell-host-microb...2814%2900163-2


    7 amino acid in polymerases and HA conferred
    droplet transmission in ferrets,

    Interestingly, for most viral proteins
    (except for hemagglutinin [HA], neuraminidase [NA], and PB1-
    F2), we found avian influenza virus proteins that differed from
    their 1918 counterparts by only a limited number of amino acids
    (Table S1).

    [that's because of the bird-index (they didn't know that ?!) 1918 flu has
    46=8+7+10+9+7+5 amino-acid differences from the index in the 6 inner segments.]


    PB2 of A/blue-winged teal/Ohio/926/2002 (H3N8), 00,01,04,03,03,01
    PB1 of A/blue-winged teal/Alberta(ALB)/286/77(H3N6), 02,03,--,--,01,--
    PA of A/pintail duck/ALB/219/77 (H1N1), 05,00,03,02,00,05
    HA of A/pintail duck/ALB/238/79 (H1N1), 03,01,06,03,00,02*
    NP of A/blue-winged teal/Ohio/908/2002 (H1N1), 03,04,08,02,02,02
    NA of A/mallard/duck/ALB/46/77 (H1N1), 05,02,01,02,01,00
    M of A/duck/Germany/113/95(H9N2), 02,--,03,05,01,03
    NS of A/canvasback duck/Alberta/102/76 (H3N6), 04,03,--,--,01,03

    -------------------
    -
    N375S,E383D,R584H
    R269K,S400L,K716R,

    V105M,L136M

    T121A
    D209N,R224G,S291N
    -------------------------


    8 , 6, 9 , 7 , 6, 4 amino acids from the 1918 virus.

    [so it has already 0+1+1+2+1+1=6 of the 46 mutations from 1918 in the inner segments (13%),
    which presumably almost randomly appeared in some birds, but not systematically, not together,
    I wished they had taken the pure bird-index-virus plus HA,NA]

    [I remember, Taubenberger et.al. did something similar some years ago,
    they called the bird-index "avian consensus", was it mentioned ? The word
    "consensus" doesn't appear in the Kawaoka paper (57 pages .pdf, 14+suppl.)]

    ================================================== =====

    the Taubenberger paper : (2012)
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416129/


    http://www.flutrackers.com/forum/showpost.php?p=489304

    > segments 1,2,3,5,6,7,8, from A/Green Wing Teal/Ohio/175/1986 (H2N1)
    > segment 4 from A/mallard/Ohio/265/1987 (H1N9)
    > similar to the avian influenza virus consensus (see below),


    3,0,7,-,3,-,0,1 A/Green Wing Teal/Ohio/175/1986 (H2N1) [9004]
    4,3,5,-,2,-,0,1 A/mallard/Ohio/265/1987 (H1N9) [10751]
    8,7,10,-,9,-,7,5 A/Brevig Mission/1/1918(H1N1) [3]
    0,0,0,0,0,0,0,0 A/bird-index-a/2000(H3N8) {1]


    ==============================================


    A/blue-winged teal/Ohio/926/2002 (H3N8)
    A/blue-winged teal/Alberta(ALB)/286/77(H3N6)
    A/pintail duck/ALB/219/77 (H1N1)
    ====
    A/blue-winged teal/Ohio/908/2002 (H1N1)
    ====
    A/duck/Germany/113/95(H9N2)
    A/canvasback duck/Alberta/102/76 (H3N6)

    A/pintail duck/ALB/238/79 (H1N1)
    A/mallard/duck/ALB/46/77 (H1N1)
    8,6,9,33,7,31,6,4
    20 (PB1-F2),

    0,1,4,3,3,1
    2,3,-,-,1,-
    5,0,3,2,0,5
    3,1,6,3,0,2
    3,4,8,2,2,2
    5,2,1,2,1,0
    2,1,3,5,1,3
    4,3,-,-,1,3

    -------------------reading the whole paper ...-----------------------
    Interestingly, for most viral proteins
    (except for hemagglutinin [HA], neuraminidase [NA], and PB1-
    F2), we found avian influenza virus proteins that differed from
    their 1918 counterparts by only a limited number of amino acids
    (Table S1).

    grew well in Madin-Darby canine kidney (MDCK) cells and
    embryonated chicken eggs
    growth was comparable to that of the 1918 virus

    intermediate pathogenicity in mice, mild in ferrets

    The 1918 PB2 and HA Genes Contribute to Enhanced
    Pathogenicity and Transmissibility in Ferrets

    We were unable to generate 1918-like avian
    PB2-627K:HA-190D/225D virus in embryonated chicken eggs,

    PB2-627K:HA-89ED/190D/225D virus group and found three
    additional mutations, HA-S113N, PB2-A684D, and PA-V253M


    (E627K and A684D in PB2;
    E89D, S113N, I187T, E190D, G225D, and D265V in HA; V253M
    in PA; and T232I in NP) may be associated with efficient 1918-
    like avian virus transmission in ferrets.


    --------------------------------------------------
    46,>A/Brevig Mission/1/1918(H1N1)
    T108A(1),V114I(1),A199S(1),L475M(1),I539V(1),D567N (1),E627K(1),K702R(1)
    K54R(2),N375S(2),E383D(2),V473L(2),L576I(2),V645M( 2),S654N(2)
    P28L(3),D55N(3),V100A(3),C241Y(3),K312R(3),I322V(3 ),E382D(3),S400L(3),T552S(3),K716R(3)

    G16D(5),V33I(5),R100I(5),V105M(5),L136M(5),L283P(5 ),F313Y(5),Q357K(5),N473S(5)

    T121A(7),L234I(7),G267E(7),E269G(7),K271R(7),S273N (7),Q331K(7)
    E70K(8),I178V(8),D209N(8),E227K(8),S291N(8)
    -----------------------------------------------------------

    ===============================================
    http://www.cidrap.umn.edu/news-persp...irborne-spread
    E627K(1),H99Y(2),H103Y(4),T156A(4),Q222L(4) or G224S(4)
    ================================================== =
    Attached Files

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.ncbi.nlm.nih.gov/pubmed/24429367
    888m8mm8 is transmissable in guinea pigs like m (while 88888888 doesn't transmit at all)
    8:PR8,m=******

    ------m- allows filamentous viruses, enhances NA-activity

    ------------------------------------------------------------------------------

    http://www.ncbi.nlm.nih.gov/pubmed/24224823
    888888e8 is filamentous

    8:PR8
    e:equine H3N8
    ------------------------------------------------------------------------------------

    ==========edit================
    from post 1:
    Attached Files rspmf3.txt (1.23 MB, 7730 views)

    7730 views is unusual, and it's a big file of 1.23 MB
    I wonder whether it has to do with the DOS-attacks ?!

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    no reassortment (yet) , just 10 passagings in ferrets:
    http://jvi.asm.org/content/early/201...65-13.abstract

    hp H7N1(Italy,2013) + T81I(PB2),V284M(NP),R95K(M1),Q211K(M1),K313R(HA)

    is droplet transmissible in ferrets and virulent


    1978sweu had A284V(5) , bird-index has TARQ , H7N9 has 313V

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.ncbi.nlm.nih.gov/pubmed/24510170
    February 2014 , Differences in transmissibility and pathogenicity of reassortants
    between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

    a=A/Swine/Jiangsu/48/2010(pH1N1) [JS48]
    b=A/Swine/Jiangsu/285/2010(pH1N1) [JS285]
    9=A/Swine/Taizhou/5/08(H9N2-9.4.2.5) (TZ5)
    1=A/California/04/2009(pH1N1) [CA04]

    999a9999 ++tgp +pm
    999b9999 ++tgp +pm
    99919999 ++tgp +pm
    999a9a99 +tgp +++pm
    999b9b99 +tgp ++pm
    99919199 +tgp ++pm

    tgp:transmissibility in guinea pigs
    pm:pathogenicity in mice
    +:increased
    ------------------------------------------------------------
    Related Content

    Novel genetic reassortants in H9N2 and their diverse pathogenicity to mice November 2011
    Attenuation of a human H9N2 in mammalian host by reassortment with an avian flu July 2004
    Experimental infection of non-human primates with avian H9N2 October 2013
    Variability of NS1 proteins among H9N2 isolated in Israel during 2000–2009 December 2010
    An NA-deficient pH1N1 mutant can efficiently replicate in cultured cells
    ------------------------------------------------------------------------------

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.ncbi.nlm.nih.gov/pubmed/24131710
    novel transfection based inoculation system to select reassortant viruses under in vivo selective pressure
    risk of reassortment between H9N2 and pH1N1
    ----------------------------------------------------------------

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory
    tract of ferrets , Kawaoka et.al. 2008
    Substitution of single genes from the 1918 virus in the genetic background of
    (A/Kawasaki/173/2001; K173) virus did not markedly alter the pattern of infection.
    That is, the reassortants grew well in nasal turbinates, but only sporadically (if
    at all) in the trachea and lungs. One exception was the 1918PB1/K173 reassortant,
    which replicated efficiently in lung tissues as well as the upper respiratory tract.
    A reassortant virus expressing the 1918 viral RNA polymerase complex (PA, PB1, and PB2)
    and nucleoprotein showed virulence properties in the upper and lower
    respiratory tracts of ferrets that closely resembled those of wild-
    type 1918 virus. Our findings strongly implicate the viral RNA
    polymerase complex as a major determinant of the pathogenicity
    of the 1918 pandemic virus. This new insight may aid in identifying
    virulence factors in future pandemic viruses that could be targeted
    with antiviral compounds.
    pathogenesis ? pandemic influenza


    BKKKKKKK
    KBKKKKKK lung
    KKBKKKKK
    KKKBKKKK
    KKKKBKKK
    KKKKKBKK
    KKKKKKBK
    KKKKKKKB
    BBBKBKKK virulent in ferrets

    --------------------------------------------------------------------------------------------------

    Cited By ...

    Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory tract of ferrets
    Tokiko Watanabe, Shinji Watanabe, Kyoko Shinya, Jin Hyun Kim, Masato Hatta, Yoshihiro Kawaoka
    Proc Natl Acad Sci U S A. 2009 January 13; 106(2): 588–592. Published online 2008 December 29. doi: 10.1073/pnas.0806959106
    PMCID:
    PMC2626747
    Article PubReader PDF–877K Supplementary Material
    Is Cited by the Following 24 Articles in this Archive:


    Code:
    A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico
    Reconstruction of the 1918 Influenza Virus: Unexpected Rewards from the Past
    Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice
    Engineering H5N1 avian influenza viruses to study human adaptation
    Integrated Clinical, Pathologic, Virologic, and Transcriptomic Analysis of H5N1 Influenza Virus-Induced Viral Pneumonia in the Rhesus Macaque
    Restored PB1-F2 in the 2009 Pandemic H1N1 Influenza Virus Has Minimal Effects in Swine
    PB1-F2 Modulates Early Host Responses but Does Not Affect the Pathogenesis of H1N1 Seasonal Influenza Virus
    Mutations in Polymerase Genes Enhanced the Virulence of 2009 Pandemic H1N1 Influenza Virus in Mice
    Decoding the Distribution of Glycan Receptors for Human-Adapted Influenza A Viruses in Ferret Respiratory Tract
    Autopsy series of 68 cases dying before and during the 1918 influenza pandemic peak
    Insights on influenza pathogenesis from the grave
    The pathogenesis of influenza virus infections: the contributions of virus and host factors
    Pathogenesis of the 1918 Pandemic Influenza Virus
    The contribution of animals models to the understanding of the host-range and virulence of influenza A viruses
    PB2 Residue 158 Is a Pathogenic Determinant of Pandemic H1N1 and H5 Influenza A Viruses in Mice 
    The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets
    The PB2-E627K Mutation Attenuates Viruses Containing the 2009 H1N1 Influenza Pandemic Polymerase
    MicroRNA Expression and Virulence in Pandemic Influenza Virus-Infected Mice 
    Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells
    Attenuated Strains of Influenza A Viruses Do Not Induce Degradation of RNA Polymerase II 
    Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System
    Emergence and pandemic potential of swine-origin H1N1 influenza virus
    Nuclear Factor 90 Negatively Regulates Influenza Virus Replication by Interacting with Viral Nucleoprotein 
    Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://mbio.asm.org/content/1/1/e00067-10.full

    VN1203=V=A/Vietnam/1203/2004(H5N1)
    CH58=C=A/Ck/Vietnam/C58/2004(H5N1)
    CA09=M=A/California/04/2009(pH1N1)
    NY312=N=A/New York/312/2001(H1N1)
    S09=O=A/green winged teal/Ohio/175/1986(H2N1)
    1918=8=A/Brevig Mission/1/1918(H1N1)

    6=1918-K627E=68888888
    E=NY-K627E=ENNNNNNN
    K=S09-E627K=KOOOOOOO
    CA09-E627K=XMMMMMMM
    CA09-D701N=YMMMMMMM

    rNY312=NY312=NNNNNNNN
    rNY312-K27E=ENNNNNNN
    1918RNP=888N8NNNN
    1918RNP-K627E=688N8NNN
    VN1203RNP=VVVNVNNN
    S09RNP=OOONONNN
    S09RNP-E627K=KOONONNN
    CA09RNP=MMMNMNNN
    CA09RNP-E627K=XMMNMNNN
    CA09RNP-D701N=YMMNMNNN
    =VVVCCCCC
    =CCCVVVVV


    TABLE 1
    Characteristics of viruses evaluated in this study
    Name,Genotype,Nadir weight-change (% of baseline),Lung titer (PFU/g) at 3dpi,at 5dpi, Histopathology
    -----------------------------------------------------------------------------------------------------------------------------------------------
    rNY312,NY312,−0.62%,2.6e3,2.7e3,fif+vrva
    rNY312-K627E,NY312, PB2-K627E,−0.57%,4.1e3,3.5e3,fif+vrva
    1918RNP,1918RNP:NY312,−15.9%,7.7e5,6.5e5,mmnb+abva
    1918RNP-K627E,1918RNP;PB2-K627E;NY312,+0.54%,4.4e4,2.6e5,fif+mbva
    VN1203RNP,VN1203RNP:NY312,−15.7%,4.2e5,2.6e3,mmnb+ abva
    S09RNP,S09RNP:NY312,−4.93%,5.2e4,1.4e2,fa+pava
    S09RNP-E627K,S09RNP;PB2-E627K:NY312,−8.06%,9.9e5,2.3e5,mb+obva
    CA09RNP,CA09RNP:NY312,−4.18%,2.2e5,4.1e4,mfnb+llob va
    CA09RNP-E627K,CA09RNP;PB2-E627K:NY312,−0.83%,1.1e3,4.3e2,fif+vrva
    CA09RNP-D701N,CA09RNP;PB2-D701N:NY312,−1.71%,1.2e4,2.5e3,fif,vrva

    fif:Few inflammatory foci
    vrva:very rare viral antigen
    mmnb:Moderate-marked necrotizing bronchiolitis
    abva:abundant bronchiolar viral antigen
    mbva:minimal bronchiolar viral antigen
    fa:Focal alveolitis
    pava:predominantly alveolar viral antigen
    mb:Mild bronchiolitis
    obva:moderate bronchiolar viral antigen
    mfnb:Mild, focal necrotizing bronchiolitis
    llobva:low levels of bronchiolar viral antigen



    ------------add others to this table, add columns for MDCK,alveolar cells,ferret pathology,ferret transmission,humans

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393695/ , July 2012
    summary:
    virulent mouse-adapted H9N2 (147L,627K) , 8 lung passages-->M147L(1),V250G,E627K,L226Q(4),R210K(7)
    in A/chicken/Shandong/16/05(H9N2) [that virus is not at genbank,
    Shandong however is in NE-China, so presumably quite different from the H9N2
    that led to H7N9 (as are other H9N2 from Shandong)]
    -----------------------------------------------------
    more details and keywords:
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393695/

    Increased virulence was detectable after 8 sequential lung passages in mice.
    Five amino acid substitutions were found in the genome of SD16-MA compared
    with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K).
    [none of the avian viruses at genbank have L at 147 in PB2 : I=7410,M=82,T=966,V=236]

    Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1
    genome segments increased virus replication; however, only the mouse-adapted PB2
    significantly increased virulence. Although the PB2 E627K amino acid substitution
    enhanced viral polymerase activity and replication, none of the single mutations of
    mouse adapted PB2 could confer increased virulence on the SD16 backbone.
    The combination of M147L and E627K significantly enhanced viral replication ability
    and virulence in mice. Thus, our results show that the combination of PB2 amino
    acids at position 147 and 627 is critical for the increased pathogenicity of H9N2
    influenza virus in mammalian host.

    A/Chicken/Hebei/4/2008 virus caused acute respiratory distress syndrome (ARDS) in mice,

    A mouse-adapted H9N2 virus, generated by serial lung-to-lung passage, gained improved
    growth characteristics on mammalian cells, extended tissue tropism in mice, and was lethal for mice [17].

    Isoleucine residue at position 97 in PA protein plays a key role in enhanced virulence in mice and is
    implicated in the adaptation of avian influenza viruses to mammalian hosts [27].
    -------------------------------------------------------------------------------

    http://jvi.asm.org/content/early/201...88-12.full.pdf
    http://jvi.asm.org/content/early/201...88-12.full.pdf
    H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a
    shorter neuraminidase (NA) stalk have become predominant in China. The A316S
    was shown to increase HA cleavage efficiency when combining with short stalk NA,
    and the short stalk NA improved NA enzyme activity and release of virus from
    erythrocytes. Single or combination of these mutations strengthened the virulence of
    H9N2 virus in chickens and mice.
    -----------------------------------------------------------------------------------------
    BJ/94-like lineage
    1994–2004, 79.8–95.4% of Chinese H9N2 influenza viruses
    possessed the PARSSR/GL amino acid sequence motif in the HA cleavage site



    ------------------
    20. Sun, Y., J. Pu, Z. Jiang, T. Guan, Y. Xia, Q. Xu, L. Liu, B. Ma, F. Tian, E.
    G. Brown, and J. Liu. 2010. Genotypic evolution and antigenic drift of H9N2
    influenza viruses in China from 1994 to 2008. Vet Microbiol 146:215-225.
    five series (BJ/94-, G1-, BG-, F/98- and Aq-series
    BJ/94,F/98 in N and S China, others in S-China
    BJ/94 <2000,F/98 since 2004
    --------------
    26. Xu, K. M., G. J. Smith, J. Bahl, L. Duan, H. Tai, D. Vijaykrishna, J. Wang,
    J. X. Zhang, K. S. Li, X. H. Fan, R. G. Webster, H. Chen, J. S. Peiris, and
    Y. Guan. 2007. The genesis and evolution of H9N2 influenza viruses in
    poultry from southern China, 2000 to 2005. J Virol 81:10389-10401.
    http://jvi.asm.org/content/81/19/10389.short
    Chicken/Beijing/1/94 (Ck/Bei-like) and Quail/Hong Kong/G1/97 (G1-like)
    Duck/Hong Kong/Y439/97 (Y439-like or Korean-like)
    Qa/HK/G1/97, Dk/HK/Y280/97, and Ck/HK/G9/97,
    A total of 89 H9N2 viruses were isolated from 49,150 duck samples
    ----------------------------------------------------------------------

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.sciencemag.org/content/ea...cience.1240532
    The viruses isolated from birds were nonpathogenic in chickens, ducks, and mice;
    however, the viruses isolated from humans caused up to 30&#37; body weight loss in mice.
    Most importantly, one virus isolated from humans was highly transmissible in ferrets by
    respiratory droplets.

    Three human viruses (SH/1, SH/2, and AH/1)

    and even though their HA and NA proteins were genetically quite similar, at least one H7N9
    isolate transmitted readily via respiratory droplets among ferrets.

    four of the five viruses tested could be transmitted between ferrets in direct contact with
    each other, and one transmitted with high efficiency via respiratory droplets.

    H7N9 in one ferret exposed to those infected with one of the bird strains and two human
    strains isolated from some of the first patients in Shanghai

    virus in all three ferrets exposed to animals with AH/1

    mouse study: SH/1, SH/2, AH/1, CK/S1053, PG/S1069, PG/S1421

    ferret transmission study: SH/1, SH/2, AH/1 ,CK/S1053 , PG/S1421

    For the respiratory droplet transmission studies, groups of three ferrets were
    inoculated i.n. with 106 EID50

    -------------------------------------------------
    amino-acid differences from the index:
    SH/1:4,3,0,9,5,2,1,1
    SH/2:2,0,0,0,0,1,0,0
    AH/1:1,0,0,0,0,0,0,0
    CK/S1053:0,0,0,0,0,6,1,0
    PG/S1421:0,0,0,0,0,0,0,0

    nucleotide-differences from the index:
    SH/1:8,13,3,12,35,8,1,2
    SH/2:3,1,0,1,0,1,0,0
    AH/1:1,1,1,0,0,1,0,0
    CK/S1053:2,1,0,3,0,8,1,0
    PG/S1421:1,1,0,1,2,3,1,0

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    Kash, Taubenberger,2012
    http://jvi.asm.org/content/early/201...87-12.full.pdf
    http://jvi.asm.org/content/86/17/9211.full.pdf

    x=1918flu, a="avian consensus A/green-winged teal/Ohio/175/1986(H2N1) plus HA from Ohio/265
    [3,0,7,-,3,-,0,1]


    xaxxxxxx,xxaxxxxx,xxxaxxxx,xxxxaxxx,xxxxxaxx,xxxxx xax,xxxxxxxa
    inintranasally infected mice. replicated and caused disease equivalent to p1918.
    axxxxxxx also, when E627K(1) was added

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    2011: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143117/

    e=A/swine/Fujian/204/2007 (H1N1)
    t=A/swine/Guangdong/1222/2006 (H1N2)
    rH1N1=ttttteet

    rH1N1 in mice had higher replicability and pathogenicity than sH1N1,eH1N1
    but similar to the pH1N1 and tH1N2
    in guinea pigs rH1N1 was not transmissible, but p2009 was
    HA and NS contributed to the transmission of pH1N1.
    HA+NA of pH1N1 gave good contact transmission among guinea pigs.
    -----------------------------------------------------------------------
    [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within
    10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets
    the TRS viruses were moderately pathogenic in ferrets and grew efficiently in both the upper and
    lower respiratory tracts. All North American TRS viruses studied were transmitted between ferrets
    via direct contact. However, their transmissibility by respiratory droplets was related to their HA
    and NA lineages: TRS viruses with human-like HA and NA were transmitted most efficiently, those
    with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like
    HA and human-like NA (N2) showed intermediate transmissibility.
    ------------------------------------------------------------------
    Abstract
    We previously reported that A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory droplet'transmissible virus (CT-Sw/1204) had acquired simultaneous HAD225G and NAS315N mutations. Incorporating these mutations into the non-pathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or remarkable disease. We, therefore, exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did individual expression of polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA225G and NA315N modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet'contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.


    CT-Sw/1204 (=A/Sw/Korea/1204/2009(H1N2)+D225G(4)+S315N(6)
    is virulent and transmissible in ferrets
    segments 1,2,3 are responsible for enhanced replication and pathogenicity in mice
    1,2,3 from Sw/1204 and 4,5,6,7,8 from A/Sw/Korea/1130/2009(H1N2) and D225G(4) and S315N(6)
    was transmissible in ferrets
    1130 and 1204 are triple reass.
    -------------------------------------------------------------------------------------------
    http://jvi.asm.org/content/83/16/8131.full.pdf
    2005-2009 : H5N1(THA05+H3N2(WY05) , ferrets attenuated, not transmissible

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    Josh P. • 4 hours ago
    −+

    ..I agree with your Vincent, this is quality science and much more relevant than the gain of function
    experiments done last year in ferrets. I will tell you this, the PA segment of H1N1 is a nasty ******.
    I find this segment increases the replication fitness within seven segments of H3N2 swine over the
    parent strain in both human and swine cells when I make reassortments. The more alarming scenario
    that isn't mentioned is that multiple genome segments of pH1N1 are circulating in the swine population
    right now..including pH1N1 PA. However there is a little surveillance data on internal influenza genes
    as mostly just sequencing is done with NA, HA and sometimes M. I hypothesize based on my co-infection
    reassortment studies in swine (Hopefully some upcoming animal data) and data in the influenza online
    database (although limited) that the PA gene segment of pH1N1 will be an issue for years to come.
    I bet if you took H7N9 and replaced the PA gene with pH1N1 you would get a very nasty virus...this
    could very well happen in pigs. 50 million pigs in the H7N9-infected region, knowing how reassortments
    behave is VERY important.

    Leave a comment:


  • gsgs
    replied
    Re: reassortment studies

    http://www.ncbi.nlm.nih.gov/pubmed/23639426
    > Conversely, HA and each of the three polymerase segments, alone or in combination,
    > of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone.

    I'm not sure, what that means. 55551111 reassorts or 71771111 or 11191111 ?

    > Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or
    > H11N6 avian viruses or carried different combination of avian origin polymerase segments,
    > exerted a higher replication effectiveness than that of the parental viruses.

    111s1111 , 111e1111 , 55511111 , 71711111 all replicate better than 11111111

    > These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic
    > avian influenza viruses should not be misjudged in the prediction of the next pandemic.

    nothing should be misjudged in any prediction

    ----------------------------------------------------------------------------
    http://www.ncbi.nlm.nih.gov/pubmed/23639426
    well working reassortments in A549 cells are:

    111s1111 (?)
    x11x1111
    1x1x1111
    11xx1111
    xxxx1111

    111s1111
    111e1111
    a1111111,1a111111,11a11111,aa111111,1aa11111,a1a11 111,aaa11111

    1:H1N1pdm,a:avian,e:H11N6,s:old seasonal H1N1,x:{H1N1,H3N2,H11,H10,H9,H7,H1av}

    -------------------------------------------------------------------------------

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.sciencemag.org/content/ea...cience.1229455

    both PA and NS of H1N1 made H5N1
    transmissible by droplet in guinea pigs, without death


    http://www.nature.com/news/scientist...rborne-1.12925

    could spread through the air between guinea pigs in adjacent cages, as long as they carried
    either or both of two genes from H1N1 called PA and NS


    but in 293T cells 6,7,8 were selected from H1N1p , 67 from H3N2, 6,7 from H1N1s
    in MDCK cells few differences , NA alone was worse
    in wdNHBE cells wt was best
    in ferrets 6,7(1) were selected from the mix
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3604002/
    Reassortment between Avian H5N1 and Human Influenza Viruses
    Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments
    H1N1, H3N2 and pandemic H1N1 , post 4 above

    Leave a comment:


  • gsgs
    replied
    Re: list of reassortment studies

    http://www.plosone.org/article/info%...l.pone.0059550
    Although the Kd' ~300 pM for WF10 HA binding to human receptor is 5 fold higher
    than that of 2009 H1N1 HA [7], a reassorted virus with HA and NA from WF10 and
    other internal genes from a human-adapted H3N2 virus did not show respiratory droplet
    transmission in ferrets [15].

    Repeated passaging of this reassorted virus in ferrets led to a strain (RCP10) that had
    additional mutations in HA and NA and transmitted via respiratory droplets in ferrets.
    One of the mutations Thr-189→Ala is in the RBS of H9 HA while the other mutation
    is in HA2 close to the transmembrane region (unlikely to impact RBS features and
    hence receptor binding). It was demonstrated that both these mutations are needed
    for conferring respiratory droplet transmission.
    Thr-189→Ala mutation is needed
    H9 HA a single mutation Q226L might be sufficient
    Leu-226 in context of His-156 and Lys-137 in the Qa88 RBS provides a more optimal
    environment than Leu-226 in the context of Gln-156 and Arg-137 in WF10 RBS for
    achieving a higher quantitative human receptor affinity
    While H9N2 subtype is yet to adapt to the human host, reassorted strains with H9 HA
    and NA have acquired as few as 2 amino acid changes in HA and a single Ile-28→Val
    change in NA to confer respiratory droplet transmission in ferrets
    Such an outcome has not been possible with HAs from other avian subtypes

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