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

[Sally Furniss]

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

Maciej F. Boni^*, Yang Zhou, Jeffery K. Taubenberger, and Edward C. Holmes Resources for the Future, Washington, DC 20036, Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544; Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania State University, State College, PA, 16802; Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. USA; Fogarty International Center, National Institutes of Health, Bethesda, MD 20892. USA
^* To whom correspondence should be addressed. Email: boni@rff.org.



Abstract
To determine the extent of homologous recombination in human influenza A virus we assembled a data set of 13,852 sequences representing all eight segments and of both major circulating subtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametric test for mosaic structure, we identified 315 sequences (2%) in five different RNA segments that, after a multiple comparisons correction, had statistically significant mosaic signals compatible with homologous recombination. Of these, only two contained recombinant regions of sufficient length (>100 nt) that the occurrence of homologous recombination could be verified using phylogenetic methods, with the rest involving very short sequence regions (15–30 nt). Although this secondary analysis revealed patterns of phylogenetic incongruence compatible with the action of recombination, neither candidate recombinant was strongly supported. Given our inability to exclude the occurrence of mixed infection and template switching during amplification, laboratory artifact provides an alternative and likely explanation for the occurrence of phylogenetic incongruence in these two cases. We therefore conclude that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human influenza A virus.


http://jvi.asm.org/cgi/content/abstract/JVI.02683-07v1

[gsgs]

my list of candidates is here:

http://www.setbb.com/fluwiki2/viewto...forum=fluwiki2

about 40000 sequences were considered
only homologuous recombinations in influenza A
with one cut per segment were considered (AA+BB-->AB)

[HenryN]

Originally Posted by gsgs

my list of candidates is here:

http://www.setbb.com/fluwiki2/viewto...forum=fluwiki2

about 40000 sequences were considered
only homologuous recombinations in influenza A
with one cut per segment were considered (AA+BB-->AB)

Why limit to one cut?

[HenryN]

It is worth noting that the group publishing this paper have been denying recombination for some time. The recombination in the 1918 sequence was said to be due to "differential evolution". Now its "lab error" and use of phylogentic analysis to mask origins.

[gsgs]

Originally Posted by niman

Why limit to one cut?

because it's faster. With many sequences, this routine is
time-critical. If you have spare computer time, we might
run it with 2 cuts...

Most recombinations are of this 1-cut-sort or are detected
as recombination by the 1-cut-routine.

[gsgs]

I'm curious ... did you read the whole paper ?
Is their list of 315 somehow secret,copyright
(for 6 months) ?

[HenryN]

Originally Posted by gsgs

because it's faster. With many sequences, this routine is
time-critical. If you have spare computer time, we might
run it with 2 cuts...

Most recombinations are of this 1-cut-sort or are detected
as recombination by the 1-cut-routine.

Please. Influenza knows how to recombine and there is no "one cut" rule (especially since sequences represent recombination events over a long period. Influenza doesn't replicate once and then stop, and co-infections are common).

[HenryN]

More controversial, however, is the occurrence of homologous recombination in

67

influenza viruses, most likely involving copy-choice (template-switching) replication of RNA

68

molecules that co-infect a single cell. Although bioinformatic evidence for homologous

recombination has been suggested (13, 19), these results remain unsubstantiated, 69 with

70

extensive lineage-specific rate variation a likely source of a false-positive signal for at least

71

some putative recombination events (24, 31). Indeed, because the genomic RNA generated

72

during replication is rapidly packaged with ribonucleoprotein, which will act to prevent the

73

occurrence of template-switching that is central to copy-choice replication, homologous RNA

74

recombination is thought to occur rarely, if at all, in both influenza viruses (17), and negative75

strand RNA viruses in general (8). In particular, a comprehensive phylogenetic analysis of

76

recombination in negative-sense RNA viruses found only sporadic evidence for recombination,

77

and not among influenza viruses (8), although the process was recently demonstrated in Zaire

78

ebolavirus, an unsegmented negative-sense single-stranded RNA virus (30). If proven to occur,

79

homologous recombination would facilitate two evolutionary processes in influenza virus: the

80

purging of deleterious mutations and the rapid generation of novel genotypes, potentially

81

including new antigenic and drug-resistant variants.

82

To assess whether homologous recombination has played a role in shaping the genetic

83

diversity of human influenza A virus we compiled a data set of 13,852 sequences representing

84

all eight RNA segments of isolates of A/H1N1 and A/H3N2 subtypes. Using an exhaustive

85

search method (4), we statistically assessed the possibility of every potential two-breakpoint

86

homologous recombination event, considering each sequence as a possible recombinant and

87

searching over all possible parents and all possible breakpoints. In our data set, this translated

88

to considering over seven billion sequence triplets, where two of the sequences in each triplet

89

are posited to have recombined to form the third sequence in the triplet. For those sequences

90

identified by this method to contain putative recombinant sections longer than 100 nucleotides

91

(nt), we used more stringent phylogenetic methods to further verify that they contained an

92

evolutionary signal (i.e. phylogenetic incongruence) compatible with the action of homologous

93

recombination.

94

[HenryN]

282

19. Niman, H. 2007. Swine Influenza A Evolution via Recombination – Genetic Drift

283

Reservoir, Available from Nature Precedings.

284 <http://hdl.handle.net/10101/npre.2007.385.1>.

[HenryN]

Originally Posted by niman

282

19. Niman, H. 2007. Swine Influenza A Evolution via Recombination – Genetic Drift

283

Reservoir, Available from Nature Precedings.
284 <http://hdl.handle.net/10101/npre.2007.385.1>.

Active link

http://precedings.nature.com/documents/385/version/1

[HenryN]

Briefly, the authors found many examples of short sequences, but couldn't verify with phylogenetic analysis (which doesn't work for short segments). Although they cited the swine data, they didn't address the swine data. For two human sequences they assumed that they had to be artifacts because the the parents were from the distant past.

[Malcolm]

We need a translation in everyday terms, why they found homologous recombination only rarely.

[HenryN]

Originally Posted by Malcolm

We need a translation in everyday terms, why they found homologous recombination only rarely.

They were looking for the "obvious" recombination and ignored small regions (or couldn't verfiy them). However, their program also appears to have missed obvious examples in Korea, which suggest there are some systematic errors in their search. They cite the swine paper, but don't address the swine data in the paper or avian data, which have more obvious examples. They also ignore movement of single nucleotide polymorphisms, which is the most common form of recombination.

[HenryN]

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.

[HenryN]

It is therefore possible that homologous recombination, should it occur in
218 influenza A virus, more commonly involves the transfer of very short sections of RNA, a process
219 that would be undetectable by the majority of other methods devised to detect recombination. If
220 homologous recombination of short segments is determined to be a relevant process in
221 influenza A virus evolution, the basis of our more frequent observation of mosaicism in A/H3N2 viruses compared to A/H1N1 viruses will need to be investigated further. However,
222 by far the
223 strongest signal in the influenza A virus sequence data analyzed here is that of strict clonality,
224 supporting most models of influenza virus evolution proposed to date.

[HenryN]

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]

Originally Posted by niman

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]

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]

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]

Originally Posted by Oracle

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]

Originally Posted by gsgs

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="MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMC
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]

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

[HenryN]

Originally Posted by gsgs

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]

maybe they didn't check HA

> in five different RNA segments

[HenryN]

Originally Posted by gsgs

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]

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]

Originally Posted by gsgs

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]

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]

Originally Posted by gsgs

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]

235

REFERENCES

236

1. Aaskov, J., K. Buzacott, E. Field, K. Lowry, A. Berlioz-Arthaud, and E. C. Holmes.

237

2007. Multiple recombinant dengue type 1 viruses in an isolate from a dengue patient. J.

238

Gen. Virol. 88:3334-3340.

239

2. Aoki, F. Y., G. Boivin, and N. Roberts. 2007. Influenza virus susceptibility and

240

resistance to oseltamivir. Antiviral Therapy 12:603-616.

241

3. Bergmann, M., A. Garcia-Sastre, and P. Palese. 1992. Transfection-mediated

242

recombination of influenza A virus. J.Virol. 66:7576-7580.

243

4. Boni, M. F., D. Posada, and M. W. Feldman. 2007. An exact nonparametric method for

244

inferring mosaic structure in sequence triplets. Genetics 176:1035-1047.

245

5. Both, G. W., M. J. Sleigh, N. J. Cox, and A. P. Kendal. 1983. Antigenic drift in

246

influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and

247

sequential amino acid changes at key antigenic sites. J. Virol. 48:52-60.

248

6. Bush, R. M., W. M. Fitch, C. A. Bender, and N. J. Cox. 1999. Positive selection on the

249

H3 hemagglutinin gene of human influenza virus A. Mol. Biol. Evol. 16:1457-1465.

250

7. CDC. 2007. Deaths: Final Data for 2004. National Vital Statistics Reports 55.

251

8. Chare, E. R., E. A. Gould, and E. C. Holmes. 2003. Phylogenetic analysis reveals a

252

low rate of homologous recombination in negative-sense RNA viruses. J. Gen. Virol.

253

84:2691-2703.

254

9. Deyde, V. M., X. Xu, R. A. Bright, M. Shaw, C. B. Smith, Y. Zhang, Y. Shu, L. V.

255

Gubareva, N. J. Cox, and A. I. Klimov. 2007. Surveillance of resistance to

256

adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J.

257

Infect. Dis. 196:249-257.

258

10. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and

259 high throughput. Nucleic Acids Res. 32:1792-1797.

11.

Ferguson, N. M., A. P. Galvani, and R. M. Bush. 2003. Ecological 260 and immunological

261

determinants of influenza evolution. Nature 422:428-433.

262

12. Fitch, W. M., R. M. Bush, C. A. Bender, and N. J. Cox. 1997. Long term trends in the

263

evolution of H(3) HA1 human influenza type A. Proc. Nat. Acad. Sci. USA 94:7712-7718.

264

13. Gibbs, M. J., J. S. Armstrong, and A. J. Gibbs. 2001. Recombination in the

265

Hemagglutinin Gene of the 1918 "Spanish Flu". Science 293:1842.

266

14. Holmes, E. C., E. Ghedin, N. Miller, J. Taylor, Y. M. Bao, K. St George, B. T.

267

Grenfell, S. L. Salzberg, C. M. Fraser, and D. J. Lipman. 2005. Whole-genome

268

analysis of human influenza A virus reveals multiple persistent lineages and

269

reassortment among recent H3N2 viruses. PLoS Biol. 3:e300.

270

15. Kawaoka, Y., S. Krauss, and R. G. Webster. 1989. Avian-to-human transmission of the

271

PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J. Virol. 63:4603-

272

4608.

273

16. Khatchikian, D., M. Orlich, and R. Rott. 1989. Increased viral pathogenicity after

274

insertion of a 28 S ribosomal RNA sequence into the haemagglutinin gene of an

275

influenza virus. Nature 340:156-157.

276

17. Kilbourne, E. D. 1978. Molecular epidemiology – influenza as archetype. Harvey

277

Lectures 73:225-258.

278

18. Nelson, M. I., L. Simonsen, C. Viboud, M. A. Miller, J. Taylor, K. S. George, S. B.

279

Griesemer, E. Ghedin, N. A. Sengamalay, D. J. Spiro, I. Volkov, B. T. Grenfell, D. J.

280

Lipman, J. K. Taubenberger, and E. C. Holmes. 2006. Stochastic processes are key

281

determinants of short-term evolution in influenza A virus. PLoS Pathog. 2:e125.

282

19. Niman, H. 2007. Swine Influenza A Evolution via Recombination – Genetic Drift

283

Reservoir, Available from Nature Precedings.

284 <http://hdl.handle.net/10101/npre.2007.385.1>.

20.

Orlich, M., H. Gottwald, and R. Rott. 1994. Nonhomologous recombination 285 between

286

the hemagglutinin gene and the nucleoprotein gene of an influenza virus. Virology

287

204:462-5.

288

21. Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA

289

substitution. Bioinformatics 14:817-818.

290

22. Scholtissek, C., W. Rohde, V. Von Hoyningen, and R. Rott. 1978. On the origin of the

291

human influenza virus subtypes H2N2 and H3N2. Virology 87:13-20.

292

23. Smith, D. J., A. S. Lapedes, J. C. de Jong, T. M. Bestebroer, G. F. Rimmelzwaan, A.

293

D. M. E. Osterhaus, and R. A. M. Fouchier. 2004. Mapping the antigenic and genetic

294

evolution of influenza virus. Science 305:371-376.

295

24. Strimmer, K., K. Forslund, B. Holland, and V. Moulton. 2003. A novel exploratory

296

method for visual recombination detection. Genome Biol. 4:R33.

297

25. Suarez, D. L., D. A. Senne, J. Banks, I. H. Brown, S. C. Essen, C. W. Lee, R. J.

298

Manvell, C. Mathieu-Benson, V. Moreno, and J. C. Pedersen. 2004. Recombination

299

resulting in virulence shift in avian influenza outbreak, Chile. Emerg. Infect. Dis. 10:693-

300

699.

301

26. Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other

302

methods). Version 4. Sinauer Associates, Sunderland, MA.

303

27. Taubenberger, J. K., and D. M. Morens. 2006. 1918 Influenza: the mother of all

304

pandemics. Emerg. Infect. Dis. 12:15-22.

305

28. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J. Anderson,

306

and K. Fukuda. 2003. Mortality associated with influenza and respiratory syncytial virus

307

in the United States. JAMA 289:179-186.

308

29. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992.

309 Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152-179.

30.

Wittmann, T. J., R. Biek, A. Hassanin, P. Rouquet, P. Reed, P. Yaba, 310 X. Pourrut, L.

311

A. Real, J.-P. Gonzalez, and E. M. Leroy. 2007. Isolates of Zaire ebolavirus from wild

312

apes reveal genetic lineage of recombinants. Proc. Nat. Acad. Sci. USA 104:17123-

313

17127.

314

31. Worobey, M., A. Rambaut, O. G. Pybus, and D. L. Robertson. 2002. Questioning the

315

evidence for genetic recombination in the 1918 "Spanish Flu” Virus. Science 296:211-

316

211.

317

318