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A question about recombination (could contamination be an issue?)

M

MarkHansen

Member
This is my first post on the forum. I just want to say first that I'm extremely impressed with the work done by Dr. Niman and others here showing evidence for homologous recombination in Influenza. It is ridiculous that this work has apparently not been acknowledged by the Influenza field. The alignments speak for themselves.

I have one question that perhaps Dr. Niman or someone else on the board could clarify. When thinking of possible alternative explanations to the results presented here, the only thing I could come up with is that some contamination may be occurring in Influenza labs, i.e. the cultures from which the viruses to be sequenced are extracted might be infected by more than one strain. Then one could imagine that during the reverse transcription step prior to PCR, the reverse transcriptase jumps between the two different strains, creating a complementary DNA sequence that looks like a homologous recombinant. It's sort of analogous to the presumed mechanism for recombination itself, except the enzyme doing the jumping is the RT that is used to make the cDNA, rather than RNA polymerase.

I'm not sure if the procedures used in virology labs are foolproof against this type of contamination. From my limited experience I'd say virologists can be pretty sloppy...

I would be interested in anyone's thoughts on this.

Mark Hansen
 
Re: A question about recombination (could contamination be an issue?)

Welcome Mark. Thanks for the question.
 
Re: A question about recombination (could contamination be an issue?)

MarkHansen said:
This is my first post on the forum. I just want to say first that I'm extremely impressed with the work done by Dr. Niman and others here showing evidence for homologous recombination in Influenza. It is ridiculous that this work has apparently not been acknowledged by the Influenza field. The alignments speak for themselves.

I have one question that perhaps Dr. Niman or someone else on the board could clarify. When thinking of possible alternative explanations to the results presented here, the only thing I could come up with is that some contamination may be occurring in Influenza labs, i.e. the cultures from which the viruses to be sequenced are extracted might be infected by more than one strain. Then one could imagine that during the reverse transcription step prior to PCR, the reverse transcriptase jumps between the two different strains, creating a complementary DNA sequence that looks like a homologous recombinant. It's sort of analogous to the presumed mechanism for recombination itself, except the enzyme doing the jumping is the RT that is used to make the cDNA, rather than RNA polymerase.

I'm not sure if the procedures used in virology labs are foolproof against this type of contamination. From my limited experience I'd say virologists can be pretty sloppy...

I would be interested in anyone's thoughts on this.

Mark Hansen
Click to expand...
NAMRU-3 human H5N1 sequences are directly from the samples.
 
Re: A question about recombination (could contamination be an issue?)

Mark - are you the co-author of "Retroviral cDNA integration: Stimulation by HMGI Family Proteins"?
 
Re: A question about recombination (could contamination be an issue?)

niman said:
NAMRU-3 human H5N1 sequences are directly from the samples.
Click to expand...

Dr Niman -- thanks for the quick reply! What is the clearest example of recombination coming from the NAMRU-3 H5N1 sequences? Just refer me to an earlier post if you've already discussed this.

By clearest, I mean that some of the examples from e.g. the Canadian pigs, or avian H5N1 in China are very dramatic -- there are very long regions of near sequence identity and then very long regions of divergence. How likely do you think it is that any of these could be due to contamination?

Mark Hansen

P.S. Florida1 -- no, that's not me on that paper.
 
Re: A question about recombination (could contamination be an issue?)

Welcome Mark. Glad to have you here.
 
Re: A question about recombination (could contamination be an issue?)

it's not just one sample. Many different samples from different times
and different labs are very unlikely to show exactly the same sort of contamination with the same other strain.
 
Re: A question about recombination (could contamination be an issue?)

gsgs said:
it's not just one sample. Many different samples from different times
and different labs are very unlikely to show exactly the same sort of contamination with the same other strain.
Click to expand...

Yes, that would pretty much eliminate that possibility.

What's a good example where two different samples from different labs show the same recombination pattern with another strain?
 
Re: A question about recombination (could contamination be an issue?)

your Canadian Swine example has lots of different viruses
isolated at different times, different farms.
Showing that the recombinated strains did persist and evolve.
So it can't just be one virus, one sample which was contaminated.

You could argue that the Vets from the lab infected the swine with
strains from the lab while taking samples or such.

But it should be visible in the sequences, I haven't seen clear
evidence for this. (but haven't searched for it a lot)
Haven't heard about it. Asked Olson about it last year.

But there was the contamination
some years ago with old H2N2 panflu. And 1977 panflu is supposed
to have been a lab-escape.
 
Re: A question about recombination (could contamination be an issue?)

gsgs said:
your Canadian Swine example has lots of different viruses
isolated at different times, different farms.
Showing that the recombinated strains did persist and evolve.
So it can't just be one virus, one sample which was contaminated.

You could argue that the Vets from the lab infected the swine with
strains from the lab while taking samples or such.

But it should be visible in the sequences, I haven't seen clear
evidence for this. (but haven't searched for it a lot)
Haven't heard about it. Asked Olson about it last year.

But there was the contamination
some years ago with old H2N2 panflu. And 1977 panflu is supposed
to have been a lab-escape.
Click to expand...


I think I wasn't being very clear about what I mean by "contamination". I don't necessarily mean that the actual host is infected by different viruses (though of course that's also possible). What I mean is that the contamination might occur in the lab itself, after the samples have been isolated. So the samples could be from different places, times etc. Once they're brought into the lab they have to be cultured (in some cell line, or chicken eggs) before they're sequenced. I'm thinking that if the lab technician is sloppy, viruses from strain A could fly around and infect a culture that was supposed to contain strain B.

So the key piece of evidence that this is not the case would be not samples from different times or places, but submitted by different labs. I think most of the Canadian swine examples are from the same lab, though I could be wrong.

Does this make sense?
 
Re: A question about recombination (could contamination be an issue?)

typically we don't see recombinations with one part of the
segment exactly from one other virus.
There are a few differences usually because the virus continues
to evolve and you won't happen to catch exactly those
strains which then recombine but only relatives of them.

So there are some differences, but still few enough to make
the claim for recombination. You'll find more distant relatives
with more differences in other viruses from other labs.
So, the recombined strains continue to evolve.
This won't happen with your lab-contamination ,
unless the virus escapes the lab.
 
Re: A question about recombination (could contamination be an issue?)

gsgs said:
typically we don't see recombinations with one part of the
segment exactly from one other virus.
There are a few differences usually because the virus continues
to evolve and you won't happen to catch exactly those
strains which then recombine but only relatives of them.

So there are some differences, but still few enough to make
the claim for recombination. You'll find more distant relatives
with more differences in other viruses from other labs.
So, the recombined strains continue to evolve.
This won't happen with your lab-contamination ,
unless the virus escapes the lab.
Click to expand...

That's a good point.

However, I think sometimes mutations are introduced into sequences during the passaging process in the lab. It is interesting to look at examples where what is ostensibly the exact same strain is sequenced more than once. There are often some differences. As an example, look at the sequences corresponding to the PB2 of Hong Kong/213/03, which is a case of recombination from one of your posts here. There are 3 sequences in the NCBI database, with accession numbers AB212050, AB212051, and AY576381. The AB212051 has 2 nt. difference with the other two. I think this is typical.

A different and more dramatic example is this: there are two strains which are both supposed to be A/turkey/Wisconsin/1/1966. Looking again at PB2, one of the sequences has accession number CY014670 and was submitted by Webster, another is DQ067437 submitted by a Chinese lab. They are very different from each other. What's more, the DQ turkey/Wisconsin looks like a recombinant between the CY turkey/Wisconsin and a third strain, turkey/California/189/66 (this is also one of your examples). But even in the part where the two turkey/Wisconsin/1/1966 are close, there are a number of changes.

It's not that surprising, because the virus has to replicate multiple times in culture. So you'd expect new mutations to be introduced. The older the virus, the more times it's probably been passaged. I assume they're not simply sequencing errors, because sequencing as such is supposed to have a very low error frequency.

The Wisconsin example sort of looks like contamination, unless you assume that the turkey was infected by two different strains, one of which was a recombinant, and the two different labs sequenced the two different strains.

Any thoughts?
 
Re: A question about recombination (could contamination be an issue?)

hmm , I'll have to check this and think about it. Takes some time ...
The HK/213 example doesn't look soo strange to me
with just 2 mutations, as you say.
I have no knowledge about this sequencing process, how reliable it is.
The two HK/213 sequences, were it from exactly the same
tissue or swab ?

An interesting example to examine would be the Karo-cluster sequences
from last year. Typically a few mutations between related strains.
E.g. there was a sequence announced with lots of mutations
in the father but apparantly later retracted/corrected.
The reason for the WHO-H2H announcement.
This has never been publically resolved AFAIK.

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

The turkey/Wisconsin example sounds much more unusual.
It was sequenced in USA and China with completely different results ??

See also the 1976 thread here:
http://www.flutrackers.com/forum/showthread.php?t=19246

could have been a contamination of
A/memphis/104/1976 with A/chicken/Dubai/338/2001(H9N2) , PB1 ?

When I reported it to genbank,
they suppressed the sequences, "unable to confirm".

They might retract the Wisconsin sequences too, once you
point this out to them ?!?


more later, when I have checked this...
 
Re: A question about recombination (could contamination be an issue?)

gsgs said:
hmm , I'll have to check this and think about it. Takes some time ...
The HK/213 example doesn't look soo strange to me
with just 2 mutations, as you say.
I have no knowledge about this sequencing process, how reliable it is.
The two HK/213 sequences, were it from exactly the same
tissue or swab ?
Click to expand...

Yes, the HK/213 example isn't strange. In fact I think it's typical.

But the point of this example is that there will often be a few differences when resequencing the same strain. So if you have a situation where strain B looks like a recombinant with respect to strain A, the fact that there are typically a few differences between their similar parts does not in itself prove that the sequence of B is not the result of a lab contamination between A or a close relative of A, and some third strain C.

By the way, there are other examples that are similar to the Wisconsin case. Look at the two HA sequences of A/grey teal/Australia/2/1979. One is CY005672 submitted by Webster in 2006, the other is M25284 submitted also by Webster, but earlier (1990 I think). They are completely different.

What's more, the M25284 grey teal looks like it was contaminated by A/duck/Czechoslovakia/1956 (submitted by Webster in the same sumbission as the early grey teal, M25283). These two are pretty close. But, there are still over 20 nt different!

My guess in this case is that the contamination occurred pretty early on, so the two strains were passaged multiple times and evolved away from each other in the lab after the contamination.

Anyway, the more interesting question is I think the original one -- can it be convincingly shown that the examples of recombination are not due to lab contamination? I'm bringing up these differences between "same strains" to argue that your earlier point about there being some differences in the "similar parts" of apparent recombinants does not automatically preclude the possibility of contamination.
 
Re: A question about recombination (could contamination be an issue?)

below the pictures of the Wisconsin sequences.

Could be, that the DQ was generated in the lab, while experimenting
to do recombinations deliberately ?
The DQ sequences were also only submitted recently.
I haven't yet looked at the other segments...


we have a recombination of type AAAA+BBBB-->ABAB here, which should be pretty rare.


Code: 
>CY014670g,115278262,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2,,,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2
>DQ067437g,66733992,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2,,,A/turkey/Wisconsin/1966,USA,1966,Avian,1,H9N2
------------------------------------..........................................
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>AF156443g,5732354,A/turkey/California/189/1966,USA,1966,Avian,1,H9N2,,,A/Turkey/California/189/1966,USA,1966,Avian,1,H9N2
>DQ067437g,66733992,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2,,,A/turkey/Wisconsin/1966,USA,1966,Avian,1,H9N2
------------------------------------------------------------........o.........
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>AF156443g,5732354,A/turkey/California/189/1966,USA,1966,Avian,1,H9N2,,,A/Turkey/California/189/1966,USA,1966,Avian,1,H9N2
>CY014670g,115278262,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2,,,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2
------------------------------------------------------------........o.........
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>CY005057g,82653629,A/green-winged teal/Alberta/228/1985,Canada,1985,Avian,1,H7N3,,,A/green-winged teal/Alberta/228/1985,Canada,1985,Avian,1,H7N3
>DQ067437g,66733992,A/turkey/Wisconsin/1/1966,USA,1966,Avian,1,H9N2,,,A/turkey/Wisconsin/1966,USA,1966,Avian,1,H9N2
------------------------------------................................o........o
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Re: A question about recombination (could contamination be an issue?)

the Wisconsin-DQ and the Memphis/104 sequences could
have been generated in a lab, but the Canadian swine were not.
There was a paper by Olsen et.al. describing, how they
were collected on different farms and different times.

They had multiple recombinations and identities of long subsequences,
5-10 different viruses with intermediates
 
Re: A question about recombination (could contamination be an issue?)

MarkHansen said:
I think I wasn't being very clear about what I mean by "contamination". I don't necessarily mean that the actual host is infected by different viruses (though of course that's also possible). What I mean is that the contamination might occur in the lab itself, after the samples have been isolated. So the samples could be from different places, times etc. Once they're brought into the lab they have to be cultured (in some cell line, or chicken eggs) before they're sequenced. I'm thinking that if the lab technician is sloppy, viruses from strain A could fly around and infect a culture that was supposed to contain strain B.

So the key piece of evidence that this is not the case would be not samples from different times or places, but submitted by different labs. I think most of the Canadian swine examples are from the same lab, though I could be wrong.

Does this make sense?
Click to expand...
Although all of the Canadian sequences came from the same lab, there are two different factors that would pretty much eliminate contamination. One is the number of different viruses involved. The two best examples are PB2 and PA which involved 1977 Tennessee sequences. However, the Tennesee sequences are from two different isolates (numbers 24 and 26).

PB2 has 24

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

PA has 26 (and 1931 sequences)

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

Moreover, the two Tennessee sequences are not in the other gene segments to any appreciable degree. Moreover, within the two genes above there are 1998, 2002, and 1931 sequences, so the number of contaminating viruses would be large. Again, these sources are predominantly in PB2 and PA.

In addition, the PB1 sequence is human, but from the mid 90's (even though the swine isolates are from 2003 and 2004)

http://www.recombinomics.com/phylo/Canadian_Swine_PB1.html

and the PB1 doesn't have any long stretches of swine sequences (all of the sequences listed above are swine). Moreover, two of the isolates have human H1 and N1, which again do not have "contaminating" swine sequences.

Thus, the multiple recombination events ares quite real. It is in "slow motion", so the long stretches of identity persist, some as long as more than 70 years, showing that the polymerase can copy sequences with a high degreee of fidelity.
 
Re: A question about recombination (could contamination be an issue?)

niman said:
Although all of the Canadian sequences came from the same lab, there are two different factors that would pretty much eliminate contamination. One is the number of different viruses involved. The two best examples are PB2 and PA which involved 1977 Tennessee sequences. However, the Tennesee sequences are from two different isolates (numbers 24 and 26).

PB2 has 24

http://www.recombinomics.com/phylo/Canadian_Swine_PB2.html
Click to expand...

Let's consider the Canadian PB2 alignments. I think they break roughly into 3 groups. The first group is TN/24/77, 48235 and 55383. The alignment has the form

TN A A A
48235 B A B
55383 B A B

the breaks are around nt. 220 and nt. 1930

The second group is NC/98, 23866 and 11112. The alignment looks like

NC A A ?
23866 B B B
11112 B A B

the breaks are at 730 and 1570

The third group is Korea/02, 57561, 56626, 53518. It looks roughly like

Korea A A A
57561 B B B
56626 C B C
53518 C A A

the breaks are at 560 and 1590

I find this last alignment very puzzling. It seems like there are separate recombination events: 56626 got a piece of 57561, and 53518 got a piece of Korea/02. But why do they occur at the exact same place in the sequence?? This doesn't seem to make sense.

The other thing that's puzzling about all three alignments is that there seems to be a pattern where there are always 2 recombination events that occur at roughly complementary positions with respect to each other on the segment. How can this be explained?

It seems hard to believe that there would be three separate contamination events in one lab. But I guess if the lab is a complete mess it's not impossible.

Something that would settle this is simply resequencing the samples. If there is a contamination, one would expect that the resequenced sequences would be very different (a new pattern of RT jumping). But if they're true recombinants, one would expect the same sequence upon resequencing. Is anyone aware whether any of the labs that reported such dramatic cases of apparent recombination has attempted to resequence the strains? Did Olsen try to do that? People in the field are inclined to disbelieve recombination, for whatever reason, so reproducibility is key ("extraordinary claims require extraordinary proof", etc.)
 
Re: A question about recombination (could contamination be an issue?)

MarkHansen said:
Let's consider the Canadian PB2 alignments. I think they break roughly into 3 groups. The first group is TN/24/77, 48235 and 55383. The alignment has the form

TN A A A
48235 B A B
55383 B A B

the breaks are around nt. 220 and nt. 1930

The second group is NC/98, 23866 and 11112. The alignment looks like

NC A A ?
23866 B B B
11112 B A B

the breaks are at 730 and 1570

The third group is Korea/02, 57561, 56626, 53518. It looks roughly like

Korea A A A
57561 B B B
56626 C B C
53518 C A A

the breaks are at 560 and 1590

I find this last alignment very puzzling. It seems like there are separate recombination events: 56626 got a piece of 57561, and 53518 got a piece of Korea/02. But why do they occur at the exact same place in the sequence?? This doesn't seem to make sense.

The other thing that's puzzling about all three alignments is that there seems to be a pattern where there are always 2 recombination events that occur at roughly complementary positions with respect to each other on the segment. How can this be explained?

It seems hard to believe that there would be three separate contamination events in one lab. But I guess if the lab is a complete mess it's not impossible.

Something that would settle this is simply resequencing the samples. If there is a contamination, one would expect that the resequenced sequences would be very different (a new pattern of RT jumping). But if they're true recombinants, one would expect the same sequence upon resequencing. Is anyone aware whether any of the labs that reported such dramatic cases of apparent recombination has attempted to resequence the strains? Did Olsen try to do that? People in the field are inclined to disbelieve recombination, for whatever reason, so reproducibility is key ("extraordinary claims require extraordinary proof", etc.)
Click to expand...
Some of the recombination patterns are due to nested segements. In other cases, more than one isolate has the same origin,

For resequencing, Beijing Genone Institute did that.

http://www.recombinomics.com/News/10120601/H5N1_China_Evolution.html

They submitted a number of H5N1 sequences with obvious recombination. A year later they resequenced and the results were identical.

You are barking up the wrong tree. The recombination is quite real and not due to lab artifact. The reasons the data from the Canadian swine are real have been explained. Trying to argue lab artifact for that many changes really is not very productive. No one is making that argument for the Canadian swine data (which would require 50-100 lab errors to generate the data for the eight gene segments).
 
Re: A question about recombination (could contamination be an issue?)

niman said:
Some of the recombination patterns are due to nested segements. In other cases, more than one isolate has the same origin,

For resequencing, Beijing Genone Institute did that.

http://www.recombinomics.com/News/10120601/H5N1_China_Evolution.html

They submitted a number of H5N1 sequences with obvious recombination. A year later they resequenced and the results were identical.

You are barking up the wrong tree. The recombination is quite real and not due to lab artifact. The reasons the data from the Canadian swine are real have been explained. Trying to argue lab artifact for that many changes really is not very productive. No one is making that argument for the Canadian swine data (which would require 50-100 lab errors to generate the data for the eight gene segments).
Click to expand...

Thanks for the link. Which sequences were resequenced? Following this thread:

http://www.flutrackers.com/forum/showthread.php?t=11309

You say that possibly henan/wu/2004 was a resequencing of henan/210/2004.
Am I missing what the others are?

For what it's worth, my belief is that the recombination is real. The evidence is very impressive. I was simply trying to understand why the Influenza field seems to be ignoring it. Lab contamination is the best objection I could come up with. I'm just looking for the most definitive way to refute it.

I'm curious -- if no one is making the argument of lab artifact, what could they possibly be saying? How do they argue against recombination?
 
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