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

[gsgs]

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

ahh yes, genbank gives them as partial.
Although 1650 is pretty long for HA

 

[HenryN]

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

For those not used to looking at sequences, GSGS posted a comparison of two HA sequences from 2002/2003. A dash means the sequences are the same at the represented position and the 0's are positions that differ. Thus, the clustering of the 0's alone pretty much eliminates the basic tenet of influenza genetics, which was repeated multiple time in the paper - genetic drift is due to copy errors. Thus, the basic tenet would hold that the polymerase got very stupid in the middle of the gene and made a series of errors, but was generally error free at the beginning and end of the gene.

However, the HA sequences from the early 90's show that the 0's are not due to a stupid polymerase, because the 0's match the sequence of the 1991 isolate. Thus, the 0's were created by swapping the middle of the 1991 sequence for the middle of the 2002 sequence to create a recombinant withe the sequence 2002-1991-2002.

The paper maintains that this rarely or never happens in human influenza, yet the example posted and five other isolates from various locations in South Korea in 2002 show that it does happen, and includes slight variations on the theme.

The six South Korean human HA recombinants (from four distinct locations) are:

A/Cheonnam/323/2002(H3N2)
A/Cheonnam/338/2002(H3N2)
A/Cheonnam/340/2002(H3N2)
A/Kyongbuk/320/2002(H3N2)
A/Daejeon/258/2002(H3N2)
A/Incheon/260/2002(H3N2)

 

[gsgs]

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

the paper's method considering triples is too slow, so they unreasonably
limit their dataset to human H1N1 and human H3N2, but most genbank-
recombinations are in H5N1 and H9N2 and in swine or birds.
They apparantly also only find recombinations where all 2 parents are available,
but you can already have strong evidence of recombination when you have only one parent
and the recombinant.They also exclude partial sequences.
So they miss e.g. the pairs

HA : A/TW/4845/99(H1N1) , A/HK/1131/98(H1N1)
NA : A/Ft.Monmouth/1/47(H1N1) , A/Rhodes/47(H1N1)
PB2: A/HK/498/97(H3N2) , A/Albany/1/76(H3N2)
HA : A/Daejeon/258/02(H3N2) , A/Habana/26/05(H3N2)
NP : A/HK/498/97(H3N2) , A/NY/136/02(H3N2)

Their conclusion, that there are only few recombinations in H1N1 and H3N2
(e.g. as compared with reassortments or as compared with H5N1) however looks correct.
I see no evidence for increased frequency of small recombinations.
We should see increased frequency of nearby differences then, which is not observed.
(I had tested this earlier)

The work should be redone with a larger dataset and by looking at pairs
insteat of triples.Also some statistics to test for frequency of small
recombinations. And compare with other viruses or random data.

Another idea is to compare flu-databases with databases of other viruses (which ?),
where recombination is known to be frequent.

 

[HenryN]

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

the paper's method considering triples is too slow, so they unreasonably
limit their dataset to human H1N1 and human H3N2, but most genbank-
recombinations are in H5N1 and H9N2 and in swine or birds.
They apparantly also only find recombinations where all 2 parents are available,
but you can already have strong evidence of recombination when you have only one parent
and the recombinant.They also exclude partial sequences.
So they miss e.g. the pairs

HA : A/TW/4845/99(H1N1) , A/HK/1131/98(H1N1)
NA : A/Ft.Monmouth/1/47(H1N1) , A/Rhodes/47(H1N1)
PB2: A/HK/498/97(H3N2) , A/Albany/1/76(H3N2)
HA : A/Daejeon/258/02(H3N2) , A/Habana/26/05(H3N2)
NP : A/HK/498/97(H3N2) , A/NY/136/02(H3N2)

Their conclusion, that there are only few recombinations in H1N1 and H3N2
(e.g. as compared with reassortments or as compared with H5N1) however looks correct.
I see no evidence for increased frequency of small recombinations.
We should see increased frequency of nearby differences then, which is not observed.
(I had tested this earlier)

The work should be redone with a larger dataset and by looking at pairs
insteat of triples.Also some statistics to test for frequency of small
recombinations. And compare with other viruses or random data.

Another idea is to compare flu-databases with databases of other viruses (which ?),
where recombination is known to be frequent.

Their analysis of full human sequences pretty much limits the bulk of sequences to a few locations (US and Australia) and most co-infections will involve closely related sequences and the recombination will look like point mutations. They would call the recombination in Egypt in H5N1 point mutations also, which won't explain the sudden appearance of a silent change on multiple genetic backgrounds at the same time, as was seen for G743A (and the same mechanism is in play for Tamiflu resistance -H274Y in H1N1).

Once homologous recombination is acknowledged, the slope gets VERY slippery, becasue recombination would be most common between closely related sequences (due to frequency of co-infections and ability to switch templates), which is why single nucleotide polymorphisms are really due to homologous recombination.

 

[Oracle]

Which viruses to look at other than Orthomyxoviridae

Which viruses to look at other than Orthomyxoviridae

That would mean looking at positive, rather than negative-sense ss-RNA viruses.

The stellar case would be the Coronaviridae. Recombination has been shown to occur between coronaviruses for many years.

Caution is advised, however, in comparison of these viruses, as coronavirus has a large, complex genome and it's recombination is driven by a very different mechanism than the one demonstrated by your analysis, GS. Leader sequence misalignment is not an apparent issue in influenza viral replication.

I think you should stay on topic to avoid confusion.

 

[HenryN]

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

Commentary

http://www.recombinomics.com/News/03260801/Human_Influenza_Recombination.html

 

[gsgs]

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

Commentary

Recombination Analysis In Human Influenza

Recombinomics Commentary 18:48
March 26, 2008

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.

More controversial, however, is the occurrence of homologous recombination in influenza viruses, most likely involving copy-choice (template-switching) replication of RNA molecules that co-infect a single cell. Although bioinformatic evidence for homologous recombination has been suggested (13, 19), these results remain unsubstantiated, with extensive lineage-specific rate variation a likely source of a false-positive signal for at least some putative recombination events (24, 31).

The above comments from the upcoming paper, “Homologous Recombination is Very Rare or Absent in Human Influenza A Virus”, describe the detection of small stretches of genetic information consistent with homologous recombination. The comments also note the presence of much longer regions of recombination in Canadian swine, although the possibility of differential evolution within a gene is cited as an explanation for the differential patterns of polymorphisms.

However, the differential evolution was discounted in the swine paper, because much of the divergence exactly matched early swine isolates and the crossover points varied, signaling independent recombination events.

The paper on the human influenza sequences however, failed to find longer stretches of recombination. Only two examples were found, and the authors suggest those two examples may have been due to contamination and the recombinant sequences may have been generated during amplification.

However, the detection of longer examples of recombination was limited by restrictions on the dataset being tested. Only full human sequences were used, which excluded sequences with clear examples of recombination. Moreover, the requirement of full sequences eliminated one set of parental sequences for the recombinants

The clear recombination involved a series of six HA sequences from Korea in 2002. The first and last third of the gene match contemporary H3N2 sequences, but the middle third of the genes match HA from sequences from isolates collected a decade earlier. The earlier origin, and the small differences in the recombined region of the six isolates reduced the likelihood that the recombined region was due to laboratory contamination.

In addition, the analysis did not include swine or avian sequences, which are frequent donor sequences for seasonal flu. Since the study focused on the recombinant as well as both parental sequences, the exclusion of swine and avian sequences would further reduce the number of recombinants identified using the criteria in the paper.

Similarly, the paper did not analyze recombination in avian or swine influenza.

 

[gsgs]

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

they don't talk much about those short recombination signals,
how strong they were or if there were more than statistically
expected. (they should have told us)

There is quite some diversity in human flu and the flu travels
around the globe, so the concentration on US and NZ
sequences is not so severe.
And even with the Korean sequences, there is still much
less recombination in human flu than in swine or birds.
Why ?
Is the same true for reassortment ?

 

[HenryN]

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

they don't talk much about those short recombination signals,
how strong they were or if there were more than statistically
expected. (they should have told us)

There is quite some diversity in human flu and the flu travels
around the globe, so the concentration on US and NZ
sequences is not so severe.
And even with the Korean sequences, there is still much
less recombination in human flu than in swine or birds.
Why ?
Is the same true for reassortment ?

I posted the P values. For NA in H3N2 there were 240 examples. The p value was 1.2 X 10 to the -10 (the likelihood that the distribution was due to chance was 10 billion to 1).

 

[HenryN]

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

they don't talk much about those short recombination signals,
how strong they were or if there were more than statistically
expected. (they should have told us)

There is quite some diversity in human flu and the flu travels
around the globe, so the concentration on US and NZ
sequences is not so severe.
And even with the Korean sequences, there is still much
less recombination in human flu than in swine or birds.
Why ?
Is the same true for reassortment ?

H3N2 recombination frequencies and p values

http://www.flutrackers.com/forum/showpost.php?p=143846&postcount=26

 

[HenryN]

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

they don't talk much about those short recombination signals,
how strong they were or if there were more than statistically
expected. (they should have told us)

There is quite some diversity in human flu and the flu travels
around the globe, so the concentration on US and NZ
sequences is not so severe.
And even with the Korean sequences, there is still much
less recombination in human flu than in swine or birds.
Why ?
Is the same true for reassortment ?

I would put the recombination frequency much higher than reported. Almost all polymorphisms are in the database with regions of identity to allow for recombination.

That is what the travel logs are. They take a given polymorphisms and show its distribution in the database.

The name of the game is recombination, regardless of what it is called (mosaics or short regions, etc).

This isn't Kansas. Influenza knows how to rapidly evolve. When the recombiantion is between closely related sequences, the acquistions look like point mutations (or single nucleotide polymorphisms).

Most evolution is recombination and it is well controled, which is why I call it "elegant evolution".

 

[HenryN]

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

they don't talk much about those short recombination signals,
how strong they were or if there were more than statistically
expected. (they should have told us)

There is quite some diversity in human flu and the flu travels
around the globe, so the concentration on US and NZ
sequences is not so severe.
And even with the Korean sequences, there is still much
less recombination in human flu than in swine or birds.
Why ?
Is the same true for reassortment ?

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

 

[gsgs]

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

I found this:
http://www.3seq.com/sample-output.html
http://www.rff.org/~boni/3seq_manual.pdf
and sent email to M.Boni


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

http://www.rff.org/~boni/boni_etal_genetics_2007.pdf
http://darwin.uvigo.es/people/dposada.html

 

[HenryN]

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

Commentary

http://www.recombinomics.com/News/03270802/Influenza_Recombination_Short.html

 

[gsgs]

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

the two-breakpoint-recombinations, are they supposed
to happen

during one and the same replication

or

by two consecutive recombinations in different cells ?

 

[HenryN]

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

the two-breakpoint-recombinations, are they supposed
to happen

during one and the same replication

or

by two consecutive recombinations in different cells ?

Either is possible. In the same cell, if there is one recombination event, there really is no reason why there cannot be a second event. However, co-infections are common, so a second event can involve another host as well as another donor sequence.

When influenza is isolated, it comes with a history, which can involve multiple recombination events with multiple donor sequences.

Look at PB2 in the Canadian swine

http://www.recombinomics.com/phylo/Canadian_Swine_PB2.html

The North Carolina sequence is at position 755-1594 in three isolates (11112, 57561, 56626)

In remains intact in 11112, but in 57561 and 56626 the Tennessee sequence is nested in the middle. Moreover, in 56626 it also has the 53518 sequence which has the same breakpoint.

Thus, the 2003/2004 Canadian swine isolates reflect a series on well defined (because they exactly match earlier isolates) recombination events.

 

[gsgs]

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

how many recombination events ?
how many swine were involved ?
how many different (original,un-recombined) viruses were involved ?
(minimum)


since long recombined sequences are pretty rare we won't expect many
aaa+bbb+ccc ---> abc
(except maybe in Canadian swine,who are special)

 

[HenryN]

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

how many recombination events ?
how many swine were involved ?
how many different (original,un-recombined) viruses were involved ?
(minimum)


since long recombined sequences are pretty rare we won't expect many
aaa+bbb+ccc ---> abc
(except maybe in Canadian swine,who are special)

Yes, the Candain swine represnt slow motion recombination. It is infrequent enough, so the long stretches remain, and are easily identified and confirmed.

Most of the time however, recombination is between closely related sequences, so the new acquistions just look like point mutations. However, when they are traced, the parental sequences can be identified.

That is why the travel logs form clear patterns and representing distribution routes. That is also why the same change appears on multiple backgrounds, like G743A on H5N1 in Egypt, Russia, Kuwait, Ghana and Nigeria at the same time.

 

[camster]

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

what is the proposed method of recombination for influenza viruses?

 

[HenryN]

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

Commentary

http://www.recombinomics.com/News/03270804/Human_Swine_Avian.html