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

<nobr>Maciej F. Boni^*,</nobr> <nobr>Yang Zhou,</nobr> <nobr>Jeffery K. Taubenberger,</nobr> and <nobr>Edward C. Holmes</nobr> 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
[SIZE=-1]^* To whom correspondence should be addressed. Email: boni@rff.org<script type="text/javascript"><!-- var u = "boni", d = "rff.org"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>.
[/SIZE]


[SIZE=+2] Abstract[/SIZE]
To determine the extent of homologous recombination in humaninfluenza A virus we assembled a data set of 13,852 sequencesrepresenting all eight segments and of both major circulatingsubtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametrictest for mosaic structure, we identified 315 sequences (

2%)in five different RNA segments that, after a multiple comparisonscorrection, had statistically significant mosaic signals compatiblewith homologous recombination. Of these, only two containedrecombinant regions of sufficient length (>100 nt) that theoccurrence of homologous recombination could be verified usingphylogenetic methods, with the rest involving very short sequenceregions (15–30 nt). Although this secondary analysis revealedpatterns of phylogenetic incongruence compatible with the actionof recombination, neither candidate recombinant was stronglysupported. Given our inability to exclude the occurrence ofmixed infection and template switching during amplification,laboratory artifact provides an alternative and likely explanationfor the occurrence of phylogenetic incongruence in these twocases. We therefore conclude that, if it occurs at all, homologousrecombination plays only a very minor role in the evolutionof human influenza A virus.


[SIZE=-1]http://jvi.asm.org/cgi/content/abstract/JVI.02683-07v1
[/SIZE]

 

[gsgs]

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

my list of candidates is here:

http://www.setbb.com/fluwiki2/viewtopic.php?t=243&mforum=fluwiki2

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

 

[HenryN]

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

my list of candidates is here:

http://www.setbb.com/fluwiki2/viewtopic.php?t=243&mforum=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]

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

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]

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

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]

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

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

 

[HenryN]

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

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]

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

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

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

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

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

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

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

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

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

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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​

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

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

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

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

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

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including new antigenic and drug-resistant variants.​

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

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

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

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

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

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

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

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

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

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

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

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recombination.​

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[HenryN]

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

282​

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

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Reservoir, Available from Nature Precedings.​

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

 

[HenryN]

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

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]

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

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]

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

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

 

[HenryN]

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

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]

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.

 

[HenryN]

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

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]

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.

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.

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.