recombination in H5-flu-viruses

gsgs · Mar 22, 2006

I finished examining the H5-viruses for recombination-candidates.
11 very clear recombinants, the candidate which has most possible
partners gets a "*".\\\\

Code:

PB2:Tree sparrow/Henan/2/2004(H5N1)  *Tree sparrow/Henan/4/2004(H5N1)
PB2:Ck/Korea/ES/03(H5N1)             *SCk/HK/YU100/2002(H5N1)
PB2:goose/Guangxi/914/2004(H5N1)     *Dk/Guangxi/668/2004(H5N1)
PB2:*WildDk/Guangdong/314/2004(H5N1)  Ck/Henan/13/2004(H5N1)
PB1:Ck/Yamaguchi/7/2004(H5N1)        *Ck/Hebei/718/2001(H5N1)
PB1:Gf/HK/38/2002(H5N1)              *Ck/HK/37.4/2002(H5N1)
PA: Ck/HK/31.4/02(H5N1)              *Ck/HK/37.4/2002(H5N1)
PA: *SCk/HK/YU100/2002(H5N1)          Ck/HK/31.2/2002(H5N1)
NP: Gf/HK/38/2002(H5N1)              *Ck/HK/37.4/2002(H5N1)
NA: Ck/Hong Kong/258/97(H5N1)        *Ck/Hebei/718/2001(H5N1)
NA: *tree sparrow/Henan/3/2004(H5N1)  tree sparrow/Henan/4/2004(H5N1)

gsgs · Mar 23, 2006

Re: recombination in H5-flu-viruses

it seems that recombination prefers other genes than HA in H5-viruses.
I didn't find any clear example of recombination in H5-HA. OK, I mean
"this" sort of recombination, Niman would identify other sorts of
recombination which even result in single nucleotide-changes.

But, as the two recent papers about binding of H5N1 to human
respiratory-tract-cells show, recombination of H5N1 might not be
required and a series of single-nucleotide-changes could do the trick.

I measured the "clustering" of mutations in the genes
(this might indicate recombination):

Code:

PB2 223:496, 7497:15,353:620,,120:599,  979: 7, 637:1131
PB1  75:428,  483:17,266:590,, 81:492,  385: 5, 620: 996
PA  192:383,  820:17,340:651,, 77:484,  448: 5,1119:1387
HA  385:526,  859:47,489:660,,156:499,  360:15,1072:1224
NP  310:413,  584:15,260:663,,163:298,  281: 4, 876:1253
NA  169:527,10414:24,244:673,,176:369, 2341: 2, 830:1242
M   357:473, 2104: 5,224:755,,106,127,15054: 1, 735:1793
NS  204:510,42870:16, 39:764,, 27:343,34789: 3, 602:1966

the exact meaning of the numbers is hard to explain,
but the 2nd value after ":" is always from a randomized run.
The 2nd part after ",," considers only 3rd position mutations (in codon),
these often don't change the properties of the virus.
The NS gene shows high tendency for clustering the mutations but
without showing the clear identifiable candiates for recombination as
in the 11 examples above, so there could be other reasons for the
clustering - the head-part could be more susceptible to mutations
than the tail-part or vice versa.
Maybe I should restrict to mutations which don't change an
amino-acid ? These should occur more "randomly".

gsgs · Mar 26, 2006

Re: recombination in H5-flu-viruses

here are H1 and H5 recombination candidates. First entry has most
partners, second entry is best partner for first. (but there are probably
many others)

Code:

H1:244,272,240,796,351,383,299,238 sequences for the 8 segments 
PB2,PB1,PA,HA,NP,NA,M,NS examined
 
[FONT=Arial]PB2:Swine/Illinois/100085A/01(H1N2)     Swine/Ontario/53518/03(H1N1)
PB1:Swine/Ontario/57561/03(H1N1)        Mallard/Alberta/130/2003(H1N1)
PA :WSN/33(H1N1)                        Swine/Italy/1513-1/98(H1N1)     
PA :Swine/Ontario/57561/03(H1N1)        Human/Denver/57(H1N1)     
PA :Swine/Tennessee/26/77(H1N1)         Swine/Ontario/53518/03(H1N1)
NA :Human/USA/1995(H1N1)                Human/Slovakia/2000(H1N1)    
HA :Swine/Japan/1992(H1N1)              Human/USA/1991(H1N1)
NS :Swine/Korea/S10/2004(H1N1)          Swine/Korea/S175/2004(H1N1)
 
NP :0005250  Swine/Canada/2003(H1N1)       Swine/USA/2001(H1N2)   
NA :0016467  Swine/Canada/2003(H1N1)       Human/USA/1994(H1N1)
PA :0406947  Swine/Alberta/56626/03(H1N1)  Swine/USA/1931(H1N1)     
PB2:2895191  Swine/Tennessee/24/77(H1N1)   Swine/Ontario/55383/04(H1N2) 
 
---------------------------------------------------------------------------
 
H5:384,382,374,734,402,594,477,518 sequences for the 8 segments examined
 
PB2:Tree sparrow/Henan/4/2004(H5N1)  Tree sparrow/Henan/2/2004(H5N1) 
PB2:SCk/HK/YU100/2002(H5N1)          Ck/Korea/ES/03(H5N1)            
PB2:Dk/Guangxi/668/2004(H5N1)        goose/Guangxi/914/2004(H5N1)     
PB2:WildDk/Guangdong/314/2004(H5N1)  Ck/Henan/13/2004(H5N1)
PB1:Ck/Hebei/718/2001(H5N1)          Ck/Yamaguchi/7/2004(H5N1)   
PB1:Ck/HK/37.4/2002(H5N1)            Gf/HK/38/2002(H5N1)              
PA :Ck/HK/37.4/2002(H5N1)            Ck/HK/31.4/02(H5N1)              
PA :SCk/HK/YU100/2002(H5N1)          Ck/HK/31.2/2002(H5N1)
NP :Ck/HK/37.4/2002(H5N1)            Gf/HK/38/2002(H5N1)      
NA :Ck/Hebei/718/2001(H5N1)          Ck/Hong Kong/258/97(H5N1)       
NA :tree sparrow/Henan/3/2004(H5N1)  tree sparrow/Henan/4/2004(H5N1)
 
PA :0256204  Ck/Hebei/108/02(H5N1)   Dk/Shanghai/35/2002(H5N1) 
 
----------------------------------------------------------------------
[/FONT]

gsgs · Mar 31, 2006

Re: recombination in H5-flu-viruses

I measured the clustering of mutations of the genes
(this might indicate recombination):

Code:

H5:384,382,374,402,594,477,518 sequences examined

     l5 (p=) l5 (#)  (p=)      same 3 
--------------------------------------------------------
PB2 223:496, 7497:15,353:620,,120:599,  979: 7, 637:1131
PB1  75:428,  483:17,266:590,, 81:492,  385: 5, 620: 996
PA  192:383,  820:17,340:651,, 77:484,  448: 5,1119:1387
HA  385:526,  859:47,489:660,,156:499,  360:15,1072:1224
NP  310:413,  584:15,260:663,,163:298,  281: 4, 876:1253
NA  169:527,10414:24,244:673,,176:369, 2341: 2, 830:1242
M   357:473, 2104: 5,224:755,,106:127,15054: 1, 735:1793
NS  204:510,42870:16, 39:764,, 27:343,34789: 3, 602:1966

H1:244,272,240,796,351,383,299,238 sequences examined
--------------------------------------------------------
PB2 334:442,  652: 5,550:648,,294:596,   37: 4, 781: 888
PB1  38:510,  212: 4,686:754,, 29:459,  128: 5, 855:1110
PA   93:327, 1002: 7,268:657,,149:405,  192: 2, 642:1063
HA  253:554,10977:62,352:720,,232:528, 1909:10,1507:1405
NP  267:514, 3728: 4,485:781,,288:605,   33: 2, 892:1168
NA   55:397,  974:16,527:788,,379:557,  925: 4, 922:1131
M   259:479, 4087: 4,122:763,,391:---, 1164: 0, 621:1728
NS  166:632, 2109: 2,322:789,, 94:---, 5135: 0,1280:1939

The 12 numbers in
PB2:223:496, 7497:15,353:620,,120:599, 979: 7, 637:1131
are obtained by:

recom pb2_h5 l5 b100 i020 (average p) recom pb2_h5 l5 b100 i020 r66 (average p)
recom pb2_h5 l5 b100 i020 (number of pairs) recom pb2_h5 l5 b100 i020 r66 (number of pairs)
recom pb2_h5 b100 i020 (average p) recom pb2_h5 b100 i020 r66 (average p)
recom pb2_h5 l5 b100 i020 3 (average p) recom pb2_h5 l5 b100 i020 r66 3 (average p)
recom pb2_h5 l5 b100 i020 3 (number of pairs) recom pb2_h5 l5 b100 i020 r66 3 (number of pairs)
recom pb2_h5 b100 i020 3 (average p) recom pb2_h5 b100 i020 r66 3 (average p)

HenryN · Mar 31, 2006

Re: recombination in H5-flu-viruses

gsgs said:

here are H1 and H5 recombination candidates. First entry has most
partners, second entry is best partner for first. (but there are probably
many others)

Code:

H1:244,272,240,796,351,383,299,238 sequences for the 8 segments 
PB2,PB1,PA,HA,NP,NA,M,NS examined
 
[FONT=Arial]PB2:Swine/Illinois/100085A/01(H1N2)     Swine/Ontario/53518/03(H1N1)
PB1:Swine/Ontario/57561/03(H1N1)        Mallard/Alberta/130/2003(H1N1)
PA :WSN/33(H1N1)                        Swine/Italy/1513-1/98(H1N1)     
PA :Swine/Ontario/57561/03(H1N1)        Human/Denver/57(H1N1)     
PA :Swine/Tennessee/26/77(H1N1)         Swine/Ontario/53518/03(H1N1)
NA :Human/USA/1995(H1N1)                Human/Slovakia/2000(H1N1)    
HA :Swine/Japan/1992(H1N1)              Human/USA/1991(H1N1)
NS :Swine/Korea/S10/2004(H1N1)          Swine/Korea/S175/2004(H1N1)
 
NP :0005250  Swine/Canada/2003(H1N1)       Swine/USA/2001(H1N2)   
NA :0016467  Swine/Canada/2003(H1N1)       Human/USA/1994(H1N1)
PA :0406947  Swine/Alberta/56626/03(H1N1)  Swine/USA/1931(H1N1)     
PB2:2895191  Swine/Tennessee/24/77(H1N1)   Swine/Ontario/55383/04(H1N2) 
 
---------------------------------------------------------------------------
 
H5:384,382,374,734,402,594,477,518 sequences for the 8 segments examined
 
PB2:Tree sparrow/Henan/4/2004(H5N1)  Tree sparrow/Henan/2/2004(H5N1) 
PB2:SCk/HK/YU100/2002(H5N1)          Ck/Korea/ES/03(H5N1)            
PB2:Dk/Guangxi/668/2004(H5N1)        goose/Guangxi/914/2004(H5N1)     
PB2:WildDk/Guangdong/314/2004(H5N1)  Ck/Henan/13/2004(H5N1)
PB1:Ck/Hebei/718/2001(H5N1)          Ck/Yamaguchi/7/2004(H5N1)   
PB1:Ck/HK/37.4/2002(H5N1)            Gf/HK/38/2002(H5N1)              
PA :Ck/HK/37.4/2002(H5N1)            Ck/HK/31.4/02(H5N1)              
PA :SCk/HK/YU100/2002(H5N1)          Ck/HK/31.2/2002(H5N1)
NP :Ck/HK/37.4/2002(H5N1)            Gf/HK/38/2002(H5N1)      
NA :Ck/Hebei/718/2001(H5N1)          Ck/Hong Kong/258/97(H5N1)       
NA :tree sparrow/Henan/3/2004(H5N1)  tree sparrow/Henan/4/2004(H5N1)
 
PA :0256204  Ck/Hebei/108/02(H5N1)   Dk/Shanghai/35/2002(H5N1) 
 
----------------------------------------------------------------------
[/FONT]

Yes, there are many more. In the examples above for PA you have Tennessee/26 paring up with Ontario 53518. However, it also pairs up with 5 other isoaltes from that series (11112, 55383, 23866, 48235, 57561). The location of the pairs vary from isolate to isolate, but the matches are EXACT and the EXACT match generall is for over 1000 BP for each isolate.

Similary Tennessee/24 pairs up with 5 of these isolates in PB2 (23866, 57561, 48235, 55383, 56626). Other than 57561/56626 which pair up with each other on either side of the Tennessee homology, the sequences then are quite divergent outside of the Tennessee homology (55383 and 53518 match each other for the first 550BP and then 53518, which has no Tennesssee sequence is an exact match with the Korean sequence.

Recombination is very clear in all 7 isolates in both of these genes (and there are more examples for the other 6 gene segments).

gsgs · Mar 31, 2006

Re: recombination in H5-flu-viruses

this is probably since I only considered recombinations
AB+CD--> AD or CB , so that's the lower half in page 43
of your patent-pdf.

BTW. the other sort, as in the upper half ABC+DEF-->AEC or DBF
can we assume
it's from two consecutive lower half recombinations ?

HenryN · Mar 31, 2006

Re: recombination in H5-flu-viruses

gsgs said:
this is probably since I only considered recombinations
AB+CD--> AD or CB , so that's the lower half in page 43
of your patent-pdf.

BTW. the other sort, as in the upper half ABC+DEF-->AEC or DBF
can we assume
it's from two consecutive lower half recombinations ?

In some instances the second parent is not known. Thus, when two sequences are exact matchs for 1/3 of the gene and then suddenl;y diverge, one or both are recombinants, even if the divergent sequence(s) are not in the database. This can be easily seen in the swine sequences, because most of then are paired. Thus, they exactly match each other in several genes and then suddenly diverge in different directions in the paired genes.

gsgs · Mar 31, 2006

Re: recombination in H5-flu-viruses

I'll try this ABC+DEF-->AEC thing too, but it's much slower.

In the above lists, each isolate only occurs once and then with
the best possible partner. The other pairings with one of the
already used partners are omitted - else the list would be too long.

I should also replace the "3" option by considering only A-G,C-T
transformations in the 3rd places. These don't change the amino acid
and thus not the properties of the virus.

I would have expected that these mutations were completely random
then, but I found some papers indicating that they probably aren't,
not sure. The statistics of this sort of mutations might be useful
to estimate the frequency of recombinations vs. random
point-mutations.

gsgs · Nov 7, 2006

Re: recombination in H5-flu-viruses

many new H5N1-sequences had been published since that analysis in March, so here is an update with the actual H5N1-sequences from genbank. My best candidates for recombination are:

[the smaller the first number, the more likely it's indeed recombination]

see http://magictour.free.fr/reco.l8
for pictures of the sequence-differences

Code:

[FONT=Fixedsys]pb2
0000000000000  151 SCk/HK/YU100/2002            86 CK/Nakorn-Patom/THA/CU-K2/04
0000000000000  205 WildDk/Guangdong/314/2004   518 CK/Jilin/hg/2002
0000000000000  265 Dk/Guangxi/668/2004         266 Gs/Guangxi/914/2004
0000000000000  511 CK/Henan/wu/2004            512 CK/Hubei/wj/1997
0000000000000  704 CK/Guiyang/1655/2006        583 Dk/Guiyang/2231/2005  
0000000000000  695 Dk/Guiyang/1588/2006        697 Dk/Guiyang/1722/2006
0000000000001  532 mallard/Guangxi/wt/2004     521 CK/Jilin/ha/2003  
0000000000033  238 Tree sparrow/Henan/4/2004   236 Tree sparrow/Henan/2/2004 
0001489804835  519 CK/Jilin/hj/2003            441 dog/THA-Suphanburi/KU-08/04 
0003222184991  506 CK/Hubei/wi/1997            237 Tree sparrow/Henan/3/2004 
0003380760408  523 CK/Jilin/hf/2002            517 CK/Jilin/he/2002  
0004758580771  503 CK/Hubei/wf/2002            329 CK/THA/Nontaburi/CK-162/05 
0007612835529  558 CK/Jilin/hl/2004            190 CK/Korea/ES/03 
 
pb1
0000000000000  331 CK/Hebei/718/2001        55 Ck/HongKong/YU822.2/01 
0000000000013  528 swine/Guangxi/wz/2004   561 CK/Jilin/hn/2003
0000000001586  519 CK/Jilin/xw/2003        186 Dk/Hong Kong/821/02 
0000000003119  149 Ck/HK/37.4/2002         147 Gf/HK/38/2002 
0000003360058  513 CK/Jilin/hg/2002        562 Dk/Hubei/wq/2003
0000004613246  564 CK/Jilin/xv/2002        428 Vietnam/CL01/2004  
0000005730406  512 CK/Jilin/he/2002         80 Goose/Hong Kong/ww100/01 
0000349988645  525 Gs/Guangdong/xb/2001    330 CK/Hebei/108/02
0002070938847  563 CK/Jilin/hp/2003        530 cygnus cygnus/Iran/754/2006 
0030102891771  507 CK/Henan/wu/2004        511 CK/Hunan/fg/2004
0103143415485  502 CK/Hubei/wf/2002        118 Ck/HK/409.1/02   
0160346378962  176 CK/Jilin/9/2004         510 CK/Jiangsu/cz1/2002
0322974187812  518 CK/Jilin/hd/2002        524 Gs/Fujian/bb/2003
0336153924881  201 WildDk/Guangdong/314/04 556 cygnus olor/Italy/742/2006
0355783065525  517 CK/Jilin/ha/2003        332 CK/Hebei/326/2005   
 
pa
0000000000000  150 Ck/HK/37.4/2002              94 Dk/Guangdong/22/2002 
0000000000000  517 CK/Jilin/hg/2002            125 Ck/Indonesia/2A/2003 
0000000000000  151 SCk/HK/YU100/2002           149 Ck/HK/31.2/2002 
0000000035927  513 CK/Hubei/wm/1997            179 swine/Shandong/2/03  
0000000066064  321 CK/Hebei/108/02             103 Dk/Shanghai/35/2002
0000000196838  522 CK/Jilin/xw/2003             87 Dk/Fujian/01/2002  
0000000874887  516 CK/Jilin/he/2002            514 CK/Jiangsu/cz1/2002  
0000013650073  222 Dk/Fujian/1734/05           569 Ck/Thailand/NP-172/06
0000018479557  512 CK/Hubei/wl/1997            180 swine/Fujian/1/2003  
0000045022087  520 CK/Jilin/ha/2003            177 CK/Jilin/9/2004 
0000128813714  524 Dk/Hubei/wp/2003            563 Dk/Hubei/wq/2003
0000161592619  526 Dk/Zhejiang/bj/2002          44 Dk/Hong Kong/2986.1/2000
0000187384186  504 CK/Hubei/wi/1997            515 CK/Hunan/fg/2004
0000530602832  426 CK/Vietnam/134/2004         242 Dk/Viet Nam/TV-V2/2004 
0061751925411  518 CK/Jilin/hj/2003            191 egret/Hong Kong/757.2/03  
0088737223542  565 CK/Jilin/xv/2002            193 Dk/Korea/ESD1/03 
 
ha
0000584340341  764 Dk/Zhejiang/bj/2002         743 Dk/Hubei/wg/2002  
0892659101122  512 Dk/Vietnam/8/05              26 Hong Kong/532/97  
 
np
0000000000000  170 Ck/HK/37.4/2002      106 Ck/HK/31.4/02  
0000000612300  449 Dk/Guangxi/13/2004   584 CK/Jilin/he/2002
0004165278925  570 CK/Hubei/wl/1997     307 Tree sparrow/Henan/3/2004 
0005657958799  578 CK/Henan/wu/2004     249 Indonesia/CDC582/2006 
0440168002743  589 CK/Jilin/hl/2004     114 Dk/Fujian/01/2002 
0974755968792  591 CK/Jilin/hn/2003     344 Viet Nam/HG-178/2004  
 
na
0000000000000  711 CK/Jilin/xw/2003          99 CK/Vietnam/CM/2004  
0000000051534  262 tree sparrow/Henan/4/04  762 swine/Anhui/ca/2004
0000092268723  508 CK/Hebei/718/2001         47 Gs/Hong Kong/3014.8/2000  
0000546945844  703 CK/Jiangsu/cz1/2002      707 CK/Hubei/wo/2003
0089600166367   63 Ck/HongKong/YU562/01     710 CK/Jilin/hf/2002
0920683591659  691 Dk/Hubei/wg/2002         717 goose/Guangdong/xb/2001
 
m
0001055937111  549 Dk/Minnesota/1525/1981   203 swan/Guangxi/307/2004   
0002468596632  575 CK/Jilin/hf/2002         566 CK/Jiangsu/cz1/2002  
0004583784749  568 CK/Jilin/he/2002         492 human/Zhejiang/16/2006   
 
ns
0000021543505  618 CK/Jilin/hg/2002        211 swine/Fujian/F1/2001 
0948505644666  286 Dk/Minnesota/1525/1981  205 swine/Shandong/2/03 
[/FONT]

recombination in H5-flu-viruses

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