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

    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

  • #2
    Re: A question about recombination (could contamination be an issue?)

    Welcome Mark. Thanks for the question.

    Comment


    • #3
      Re: A question about recombination (could contamination be an issue?)

      Originally posted by MarkHansen View Post
      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
      NAMRU-3 human H5N1 sequences are directly from the samples.

      Comment


      • #4
        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"?

        Comment


        • #5
          Re: A question about recombination (could contamination be an issue?)

          Originally posted by niman View Post
          NAMRU-3 human H5N1 sequences are directly from the samples.
          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.

          Comment


          • #6
            Re: A question about recombination (could contamination be an issue?)

            Welcome Mark. Glad to have you here.

            Comment


            • #7
              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.
              I'm interested in expert panflu damage estimates
              my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

              Comment


              • #8
                Re: A question about recombination (could contamination be an issue?)

                Originally posted by gsgs View Post
                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.
                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?

                Comment


                • #9
                  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.
                  I'm interested in expert panflu damage estimates
                  my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                  Comment


                  • #10
                    Re: A question about recombination (could contamination be an issue?)

                    Originally posted by gsgs View Post
                    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.

                    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?

                    Comment


                    • #11
                      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.
                      I'm interested in expert panflu damage estimates
                      my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                      Comment


                      • #12
                        Re: A question about recombination (could contamination be an issue?)

                        Originally posted by gsgs View Post
                        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.
                        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?

                        Comment


                        • #13
                          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:


                          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...
                          I'm interested in expert panflu damage estimates
                          my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                          Comment


                          • #14
                            Re: A question about recombination (could contamination be an issue?)

                            Originally posted by gsgs View Post
                            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 ?
                            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.

                            Comment


                            • #15
                              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
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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
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                              -----------------------------------------------------------------------
                              I'm interested in expert panflu damage estimates
                              my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                              Comment

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