Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

I have to go through this paper in more detail, but it appears to have set up a number of hoops that have been raised high, and ignores the jumping through lower hoops.

The paper really focuses on "obvious" recombination and then throws out examples at the ends of the genes and is left with two examples, which have identity with earlier isolates. These are considered to be lab artifacts because the sequences are conserved over a long time period.

The paper acknowledges the possibility of short regions of recombination, which would be missed by the analysis method used (and therefore doesn't address obvious recombination between H1N1 and H3N2 internal genes).

The paper also does not address multiple recombination events over time, which would split recombined regions, as was demonstrated in the swine Nature Precedings paper, which the authors deem as "controversial".

The paper does address the obvious examples of recombination in the swine sequences, which would be difficult to explain by lab error, because it would require multiple contaminants (two 1977 Tennessee swine, one 1997 North Carolina Swine, one 2002 Korean swine, and 1 1931 Iowa swine), just to explain the recombination in PB2 and PA.

There are some obvious examples of recombination in a series of human south Korean HA sequences, which are also hard to explain by lab contamination. I am not sure why these sequences were not addressed, although the paper seems to focused more on explaining away data it doesn't like, or excluding clear examples for various reasons (I suspect exclusion is because the sequences are partial or full gene segments were not released).

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

For the two candidate recombinants A/New York/11/2003 (PB2) and
188 A/Christchurch/14/2004 (NP), it is also puzzling that the parental sequences were sampled 25
189 and 31 years apart, respectively. Hence, for one of these recombination events to have
190 occurred, a lineage of viruses closely related to an ?archaic? virus (either A/Hong Kong/14/1974
191 or A/Beijing/1/1968) must have circulated until at least 1999 and recombined with A/New
192 York/424/1999 or A/New York/153/1999. Given the rapid rate of influenza A virus mutation
193 through frequent RNA polymerase error, as well as the rapid lineage turnover driven by positive
194 selection on the major antigenic proteins (6, 11, 12, 23), this scenario seems extremely unlikely.
195 Thus, laboratory error, such as template switching during amplification in a mixed or contaminated sample, is a likely explanation of these apparent homologous
196 recombination
197 events.

I'm not sure if there are errors in the paper, but I don't see anything that remotely looks like recombination in A/Christchurch/14/2004 (NP).

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

I have this in my NP-file :

NP:
p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+ 95 A/Christchurch/14/2004(H3N2) A/quail/Shantou/1218/2003(H6N1)

but it involves H6N1.
I'll check later.

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

p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+  95    >A/Christchurch/14/04(H3N2)     >A/Qa/Shantou/1218/03(H6N1)
p=0.0000089305815657764 1445  345 l:1496 cut: 300 dif:  51=  24+  27    >A/Christchurch/14/04(H3N2)     >A/HK/498/97(H3N2)
p=0.0000245520022496002 1445  481 l:1001 cut: 750 dif: 205= 130+  75    >A/Christchurch/14/04(H3N2)     >A/Dk/Shantou/2088/01(H9N2)
p=0.0000422254888616796 1445  334 l:1496 cut: 750 dif: 275= 108+ 167    >A/Christchurch/14/04(H3N2)     >A/Sw/Nebraska/209/98(H3N2)


checking for 2 cuts :

p=0.0000000000000000000 1445 5480 l:1064  973-1053 dif: 246= 186+  60    >A/Christchurch/14/04(H3N2)     >A/Qa/Shantou/1218/03(H6N1)
p=0.0000000000005338106 1445 5674 l: 287  153- 273 dif:  99=  29+  70    >A/Christchurch/14/04(H3N2)     >A/PR/8/34(H1N1)

p=0.0000000055769896136 1445 4944 l:1474 1433-1473 dif: 303= 278+  25    >A/Christchurch/14/04(H3N2)     >A/Sw/Hangzhou/1/06(H9N2)
p=0.0000000203857481050 1445  323 l:1405   33-1353 dif: 293=  40+ 253    >A/Christchurch/14/04(H3N2)     >A/China) segment 5 nucleoprotein(NP) gene, partial cds
p=0.0000000324854822614 1445  334 l:1496  753-1333 dif: 275= 128+ 147    >A/Christchurch/14/04(H3N2)     >A/Sw/Nebraska/209/98(H3N2)
p=0.0000000758549573162 1445 4784 l:1496  773-1333 dif: 268= 129+ 139    >A/Christchurch/14/04(H3N2)     >A/Sw/British Columbia/28103/05(H3N2)
p=0.0000001155391775063 1445 4785 l:1496  773-1333 dif: 267= 129+ 138    >A/Christchurch/14/04(H3N2)     >A/Sw/Manitoba/12707/05(H3N2)
p=0.0000001402953788517 1445 4718 l:1496 1113-1173 dif: 303= 273+  30    >A/Christchurch/14/04(H3N2)     >A/Dk/Guangxi/13/04(H5N1)
p=0.0000001751326206486 1445 4782 l:1496  773-1333 dif: 266= 129+ 137    >A/Christchurch/14/04(H3N2)     >A/Ontario/RV1273/05(H3N2)

but that PR/34 sequence is a mess, so it's probably not relevant:

------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------.......oo...o.o...
o.o......o.o..............o...........o..o.....o..........o.
...........o...o................o....o.........o..oo.o..o...
.............o...o....oo.o.ooooooo..ooooooooo..-------------
------------------------------------------------------------
-----.o.o.oo.oo..ooooooo.ooo...oooo.o.o.oooo...o..o..oo.oo..
o.----------------------------------------------------------
------------------------------------------------------------
---------------------.oo.o....o...oooo.oo.oo...ooooo.o.oo..o
..oo--------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
--------------------------------------------------------

the Shantou-quail looks irrelevant too (sequencing/alignment error)

se7a5.10
>A/Christchurch/14/04(H3N2)
>A/Qa/Shantou/1218/03(H6N1)
........o..............o........o.............o...o..o......
.....o........o..o.....o..o..o.oo...o...o.........o..o..o..o
..o..o..............o...........oo........o.oo.o..o.........
o.o..........o......o........o...........o.....o.o..........
.....o.....o...........o..o...........o..o..o..o..oo.o..ooo.
..o..o..o.....o..o..o..oo.o..o...........o..o..o..oo....o...
..o.....o........o..o.....o...........o........o............
.....oo.o........o...........o...........o.....o.....o.....o
.......................o..o..o.....o..o..o..o..o........o...
..o.....o..o...o.......o..............oo.o..o...o.o.....o...
.....o.....o.................o.....o..o.o......o.o..........
..............o..o.....o..............o...............o.....
..o..oo.......oo.o..o..o..o........o..o........o..o...o.....
o.o..o...o.o..oo.......o...o.o..o..o..o..o.....o.....o....o.
..o....o...o...o.......o..o........o.o...o..o.....o..o..o...
.....o......ooo..o..o.....o.....oo.o.o...o...........o..o...
........----------------------------------------------------
--------------------------------.oooooooo..oooo.ooooo.oooo.o
o.ooooooooo.ooooo.oo.o..oo..ooo.oooo.o.o.oooooooo.o..oooo.oo
o.ooo.------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
------------------------------------------------------------
--------------------------------------------------------

[Oracle]

Look at the lead authors CV.

It's a kid, a postdoc less than 2 years out of his PhD.

Don't get your panties in a wad, Henry. They arbitrarily set the limit at 100 nucleotide chunks, while admitting to finding many shorter sequences that may well have supported your point of increasing viral utilization of homologous recombination. These authors are looking for large sequence exchanges, but I don't think that's how this type of adaptation works. It's fine tuning of a normally benign virus-turned-pathogen to a susceptible host and local environment, amplification of pre-existing *very low* probability phenotypes that are carried within a larger pool in circulation.

Don't worry, boyo. You'll be vindicated shortly.

The key is to understand that recombination is ordinarily a rare event (over the course of decades to as long as a hundred years or more). You're catching evidence that it's a syncopated process and presently very much in evidence.

[HenryN]

Re: Look at the lead authors CV.

It's a kid, a postdoc less than 2 years out of his PhD.

Don't get your panties in a wad, Henry. They arbitrarily set the limit at 100 nucleotide chunks, while admitting to finding many shorter sequences that may well have supported your point of increasing viral utilization of homologous recombination. These authors are looking for large sequence exchanges, but I don't think that's how this type of adaptation works. It's fine tuning of a normally benign virus-turned-pathogen to a susceptible host and local environment, amplification of pre-existing *very low* probability phenotypes that are carried within a larger pool in circulation.

Don't worry, boyo. You'll be vindicated shortly.

The key is to understand that recombination is ordinarily a rare event (over the course of decades to as long as a hundred years or more). You're catching evidence that it's a syncopated process and presently very much in evidence.

This paper is a good vehicle for addressing this issue. The senoir authors (Holmes and Taughtenberger) have been avoiding recombination for some time. Holmes called the recombination in HA of 1918 "differential evolution" (even though the recombination is actually in all 8 gene segments), while Taughtenberger is looking for the 1918 precursor, when it is actually a recombinant between H1N1 human and swine (WSN/33 and swine/Iowa/15/31).

This paper presents a good opportunity to attack this head on. There are some OBVIOUS examples in HA sequences from South Korea on the human side, and the paper is indirectly stating that the OBVIOUS examples in the Canadian swine are lab artifacts, involving MULTIPLE prior isolates.

The paper (and authors) are now fair game, and deserve the attention they will shortly receive.

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

I have this in my NP-file :

NP:
p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+ 95 A/Christchurch/14/2004(H3N2) A/quail/Shantou/1218/2003(H6N1)

but it involves H6N1.
I'll check later.

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

 
p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+  95    >A/Christchurch/14/04(H3N2)     >A/Qa/Shantou/1218/03(H6N1)
p=0.0000089305815657764 1445  345 l:1496 cut: 300 dif:  51=  24+  27    >A/Christchurch/14/04(H3N2)     >A/HK/498/97(H3N2)
p=0.0000245520022496002 1445  481 l:1001 cut: 750 dif: 205= 130+  75    >A/Christchurch/14/04(H3N2)     >A/Dk/Shantou/2088/01(H9N2)
p=0.0000422254888616796 1445  334 l:1496 cut: 750 dif: 275= 108+ 167    >A/Christchurch/14/04(H3N2)     >A/Sw/Nebraska/209/98(H3N2)

Below is what is currently at Genbank, and I don't see any recombination

LOCUS CY002925 1565 bp ss-RNA linear VRL 15-SEP-2005
DEFINITION Influenza A virus (A/Christchurch/14/2004(H3N2)) segment 5,
complete sequence.
ACCESSION CY002925
VERSION CY002925.1 GI:75750237
KEYWORDS .
SOURCE Influenza A virus (A/Christchurch/14/2004(H3N2))
ORGANISM Influenza A virus (A/Christchurch/14/2004(H3N2))
Viruses; ssRNA negative-strand viruses; Orthomyxoviridae;
Influenzavirus A.
REFERENCE 1 (bases 1 to 1565)
AUTHORS Ghedin,E., Spiro,D., Sengamalay,N., Zaborsky,J., Feldblyum,T.,
Subbu,V., Sparenborg,J., Groveman,L., Halpin,R., Shumway,M.,
Sitz,J., Katzel,D., Koo,H., Salzberg,S.L., Jennings,L., Smit,M.,
Wells,V., Bao,Y., Bolotov,P., Dernovoy,D., Kiryutin,B., Lipman,D.J.
and Tatusova,T.
TITLE The NIAID Influenza Genome Sequencing Project
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1565)
CONSRTM The NIAID Influenza Genome Sequencing Consortium
TITLE Direct Submission
JOURNAL Submitted (15-SEP-2005) on behalf of TIGR/Canterbury Health
Laboratories, NZ/NCBI, National Center for Biotechnology
Information, NIH, Bethesda, MD 20894, USA
FEATURES Location/Qualifiers
source 1..1565
/organism="Influenza A virus
(A/Christchurch/14/2004(H3N2))"
/mol_type="genomic RNA"
/strain="A/Christchurch/14/2004"
/serotype="H3N2"
/isolation_source="gender:M; age:6y"
/specific_host="human"
/db_xref="taxon:345301"
/segment="5"
/lab_host="MDCK2 passage(s)"
/country="New Zealand: Christchurch"
/collection_date="08/07/2004"
gene 46..1542
/gene="NP"
CDS 46..1542
/gene="NP"
/codon_start=1
/product="nucleocapsid protein"
/protein_id="ABA26737.1"
/db_xref="GI:75750238"
/translation="MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIG RFYIQMC
TELKLSDHEGRLIQNSLTIEKMVLSAFDERRNKYLEEHPSAGKDPKKTGG PIYRRVDG
KWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWHSNLNDATYQRTR ALVRTGMD
PRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGE NGRKTRSA
YERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRG SVAHKSCL
PACAYGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKS QLVWMACH
SAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNMGSSTLELRSG YWAIRTRS
GGNTNQQRASAGQTSVQPTFSVQRNLPFEKSTIMAAFTGNTEGRTSDMRA EIIRMMEG
AKPEEVSFRGRGVFELSDEKAANPIVPSFDMSNEGSYFFGDNAEEYDN"
ORIGIN
1 agcaaaagca gggttaataa tcactcactg agtgacatca aaatcatggc gtcccaaggc
61 accaaacggt cttatgaaca gatggaaact gatggggatc gccagaatgc aactgagatt
121 agggcatccg tcgggaagat gattgatgga attgggagat tctacatcca aatgtgcact
181 gaacttaaac tcagtgatca tgaagggcga ttaatccaga acagcttgac aatagagaaa
241 atggtgctct ctgcttttga tgaaagaagg aataaatacc tggaagaaca ccccagcgcg
301 gggaaagatc ccaagaaaac tgggggaccc atatacagga gagtagatgg aaaatggatg
361 agggaactcg tcctttatga caaagaagaa ataaggcgaa tctggcgcca agccaacaat
421 ggtgaggatg cgacagctgg tctaactcac ataatgatct ggcattccaa tttgaatgat
481 gcaacatacc agaggacaag agctcttgtt cgaactggaa tggatcccag aatgtgctct
541 ctgatgcagg gctcgactct ccctagaagg tccggagctg caggtgctgc agtcaaagga
601 atcgggacaa tggtgatgga actgatcaga atggtcaaac gggggatcaa cgatcgaaat
661 ttctggagag gtgagaatgg gcggaaaaca agaagtgctt atgagagaat gtgcaacatt
721 cttaaaggaa aatttcaaac agctgcacaa agagcaatgg tggatcaagt gagagaaagt
781 cggaacccag gaaatgctga gatcgaagat ctcatatttt tggcaagatc tgcattgata
841 ttgagagggt cagttgctca caaatcttgc ctacctgcct gtgcgtatgg acctgcagta
901 tccagtgggt acgacttcga aaaagaggga tattccttgg tgggaataga ccctttcaaa
961 ctacttcaaa atagccaaat atacagccta atcagaccta acgagaatcc agcacacaag
1021 agtcagctgg tgtggatggc atgccattct gctgcatttg aagatttaag attgttaagc
1081 ttcatcagag ggacaaaagt atctcctcgg gggaaactgt caactagagg ggtacaaatt
1141 gcttcaaatg agaacatgga taatatggga tcgagcactc ttgaactgag aagcgggtac
1201 tgggccataa ggaccaggag tggaggaaac actaatcaac agagggcctc cgcaggccaa
1261 accagtgtgc aacctacgtt ttctgtacaa agaaacctcc catttgaaaa gtcaaccatc
1321 atggcagcat tcactggaaa tacggaggga agaacttcag acatgagggc agaaatcata
1381 agaatgatgg aaggtgcaaa accagaagaa gtgtcattcc gggggagggg agttttcgag
1441 ctctcagacg agaaggcagc gaacccgatc gtgccctctt ttgatatgag taatgaagga
1501 tcttatttct tcggagacaa tgcagaagag tacgacaatt aaggaaaaat acccttgttt
1561 ctact

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

The OBVIOUS HA sequences from Korea are below (recombination is between 575-1008 of submitted sequences)

A/Cheonnam/323/2002
A/Cheonnam/338/2002
A/Cheonnam/340/2002
A/Kyongbuk/320/2002
A/Daejeon/258/2002
A/Incheon/260/2002

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

No, they check all eight gene segments, but didn't find anything in HA.

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

TABLE 1. Results of recombination analysis of ten A/H3N2 influenza virus data sets.

Segment Number
sequences
(distinct)
Alignment
length (nt)
Variable
Sites
3SEQ

p-

value
Number
?recombinant?
Number
?recombinant?
> 100 nt
Phylogenetic
signal for
recombination?

PB2 1086 (912) 2347 898 10

-3 24 1 Weak
PB1 878 (715) 2341 849 0.49 0 0 No
PA 1365 (1156) 2242 947 2.6 ? 10-6 28 0 No
HA 1365 (1154) 1772 918 1 0 0 No
413 (336) 1711 518 1 0 0 No
NP 1256 (938) 1570 620 2.9 ? 10-6 6 1 Weak
NA 1365 (1059) 1475 754 1.2 ? 10-10 240 0 No
413 (274) 1407 438 1 0 0 No
MP 1250 (682) 1028 366 0.04 1 0 No

NS 630 (344) 906 318 1 0 0 No

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

Mosaic just means recombinant (sequences from two seperate sources). They looked at the nucleotide level.

I think they use a phylogentic tree to "confirm" the recombination, so they need the various pieces of genes to be 100 nt to create a tree to "prove" the recombination.

Of course most of teh recombination is between closely related sequences and the recombination is common, so its hard to fit their definition, which really only works for OBVIOUS recombination. i.e. the virus knows what it is doing, but the programmer doesn't.

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

16

TABLE 2. Results of the recombination analysis of eight A/H1N1 influenza virus data sets.

Segment Number
sequences
(distinct)
Alignment
length (nt)
Variable
Sites
3SEQ

p-

value
Number
?recombinant?
Number
?recombinant?
> 100 nt
Phylogenetic signal
for recombination?

PB2 478 (421) 2346 871 1 0 0 No
PB1 478 (399) 2341 870 0.95 0 0 No
PA 478 (380) 2238 776 0.96 0 0 No
HA 482 (440) 1781 804 0.82 0 0 No
NP 478 (332) 1566 544 1 0 0 No
NA 481 (405) 1464 657 10

-5 16 0 No
MP 478 (248) 1027 296 1 0 0 No

NS 478 (297) 890 336 1 0 0 No

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

I think the Korea sequences are about 1300 BP (out of 1700) so they didn't use them (although the recombination is in the center of the sequence).

This is a good example of the recombination not existing because the submitted sequences don't fit the program, so they don't exist!

It's like no H2H in Pakistan because the sample wasn't collected from the dead brother, and all of the other positives degraded so there is only one confirmed case.

However influenza really doesn't care about the sample integrity, the press releases, the peer reviewed papers, or the programming requirements for the obvious recombination.

This paper was an answer in search of a publication.

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

235

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