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Homologous Recombination

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  • Homologous Recombination

    Dr. Niman, et al..

    Please explain this concept a bit better and expound on Webster's acknowledging that it may be happening in the H5N1 virus. How does it change it's virulence? In layman's terms... what does this *mean*??

    "If you're not living on the edge, you're wasting space."

  • #2
    Re: Homologous Recombination

    Sara see post #18 in this thread

    I am sure Dr.Niman could do a lot better but it gives a brief description.

    If you want a more detailed description PM me.


    • #3
      Re: Homologous Recombination

      Layman's terms ???, I'll try... if I can.

      It's a general theory about the way virus evolves.
      It does not specifically specify anything about the changes in virulence.

      Historically, two majors ways was know to drive the evolution of virus

      -genetic drift (accumulation of successives singles point mutation in a strain)
      genetic drift is driven by a random process.

      -genetic shift (sudden change of a whole gene from the 8 that contain
      genetic shift depend of co-infection but the rules that made it possible are complex and depend mainly of the viability of the new strain.

      Dr. Niman scream above all roofs since years now that there is an other major way virus evolves.

      He call that homologous recombination or elegant evolution.
      From that theory,
      When two virus infect a host, there is a chance that the polymerase that replicate the virus jump to and from an other strain while copying.

      This way, genes that are hybrids from the two previous strain are produce.
      Polymerase need an homologous region to realised that.
      That's why it is called homologous recombination.

      So homologous recombination are changes inside genes that came from other strain.

      Dr.Niman even goes farter, He claim that most of the so-called point mutation are in fact small homologous recombination.

      He also claim to being able to analyse a database to predict the chance of recombination between the majors clades of virus in presence.

      He had a patent about this.

      I hope this is understaindable and accurate.
      Correct me anyone if there is mistake.



      • #4
        Re: Homologous Recombination

        Thanks Mingus and JJackson.

        When the term homologous recombination is used, does it consider a viral mixing vessel (humans, pigs, birds, etc...) containing different viruses (Orthomyxoviridae, Togaviridae, and/or Flaviviridae) with similiar genetic characteristics? ... not so much the general hemagluttinin or neurimidase.... but the internal genetic structures within the virus?

        All are single stranded RNA virus of similar size and genetic makeup... two positive and one negative.
        "Predictable is Preventable" by Safety Expert Dr. Gordon Graham.


        • #5
          Re: Homologous Recombination

          The Niman's patent cover all posibility of recombination including between two diferent virus and different gene that are not the same.

          But in fact, the more similar they are the more likely recombination will occur because they have more homologous region.

          In fact probably this "polymerase step" recombination occur between similar strain very often but we don't see them because they are already similar at these places.


          • #6
            Re: Homologous Recombination

            Below you will find a post of mine from the Dr Niman Discussion Thread in the Experts Forum with a response form Dr. Niman.

            Firstly I think the second sentence of is reply should be read as "Most examples in flu are not like the one above ....." (Henry please correct if I am wrong). Typos aside my point was that the linked article gives as elegant and detailed example of the recombination process. As Dr Niman points out in vivo normally there would be two flu viruses (A & B) and in the 'cut & paste' process a section from residue x to residue y of strain A would be inserted to replace the equivalent section in strain B forming a recombinant strain C.

            I would like to thank Mellie for her link to
            posted earlier in this thread. While this is not why she posted it turns out to be perfect for this tread.

            As a layman trying to get to grips with the mechanism of mutation (Recombination vs Reassortment/point mutation) and its consequences for base sequences leading to Amino acid sequence leading to 3D protein shape and so to clinical effects ? infectivity & virulence this little story has it all.

            Why do I like it so much?
            1] It is free. The full text is available without subscription.
            2] It is easy to follow even without any virology degrees
            3] It is a slam dunk for Recombination.

            In brief a farm had two chicken sheds, in shed 1 the flock seemed a little under the weather and then they had a massive die off in shed 2 so they culled all the birds, two of the cullers then became ill (not too seriously). The researchers had sequences for both workers and each shed. Shed 1 had an LPAI infection, shed 2 had the same HA strand but with a new insertion making it longer that it?s ?parent?, the two cullers both had the extended HA strand and a few point mutations and were slightly different from each other and shed 2. The inserted strand exactly matched a sequence of bases from one of the virus? other strands (21 bases from M).

            While I am convinced by the sheer weight of examples of recombinant insertions listed on Dr Niman?s site we are not talking about a clear linkage that can be followed in parent/child/grandchild sense. It is more likely that we observe an atypical 30 base sequence in a Turkish duck and when we scan for that sequence in the literature we find an exact, or close, match in some Swan which died 3 years ago on a different continent. The linkage is not obvious but statistically sound due to the lower probability of that 30 base sequence recurring as 30 consecutive point mutations.
            Where this example wins out is we have four sequences from the same location over the course of a few days. We can almost see the mutations occurring in real-time and we do not even need a second virus for the donor sequence it is provided from within the Matrix sequence. The short time scale makes the argument for accumulated point mutations fatuous. Once the mechanism is demonstrated - as I believe it is here ? then it is difficult to argue it does not occur elsewhere. The authors then go on to show how the insertion effects the structure of the HA protein and why that may account for the change from LPAI to HPAI.
            and Dr Niman's Response

            The above description is for non-homolgous recombination, which happens on RARE occasion. Most examples in flu are like the one above in which a new HA cleave site is created. The new cleavage site has many basic amino acids, so the virus with the new cleavage site has a major advantage because it can then infect many cell types.

            The major driver of influenza evolution is HOMOLOGOUS recombination, which involves using stretches of sequence identity to copy part of one gene and then switch to another.

            Homologous recombination happens when the polymerase is using one gene as a template. The newly created RNA the hops off the template 1 and lands on template 2 (the gene from the other virus in a dual infection).

            Because the region (i.e the last 10 nucleotides) that was just copied is the same sequence on the second template, the newly created RNA finds the right place to bind to template 2 and then the polykmerase starts copying the gene again. However, now the new sequence is directed by template 2 so the new RNA has some genetic information from temoplate 1 and some from template 2. The new RNA is a recombinant.

            This type of dual infectoion and jumping happens most often between the same serotype, because the two copies have a lot of identity so it is easy for the new RNA to find the correct spot on template 2. If template 2 was grossly different than 1, the new RNA would just find copies of template 1 and the new gene would be a copy of 1 and not have 2.

            If the template 2 differs from 1 by just 1%, then most of the "new" information from 2 will just look like a point mutation.

            That is why the newly released Indonesian H5N1 has so many changes that look like point mutations, but match other H5N1 sequences from nearby locations. The dual infections involve two closley related H5N1's, and the recombinant only has a few chnages from the new parent. However, dual infections are common, so there are many changes tracing back to several parents.

            These changes are NOT random mutations. They are polymorphisms that can be easily found on other H5N1's.

            In some cases, like the Canadian swine, the number of dual infections is limited, so there may be only one cross-over over a long time period. Therefore the two parents are exact matches over a large portions of the gene. This identity can be found in isolates more than 25 years apart, showing that copy errors are RARE, and the vast majority of changes are due to HOMOLOGOUS recombination, not random mutations.


            • #7
              Re: Homologous Recombination

              Apologies I just tried my link in post #2 and found I did not end up where I expected.

              Why it matters.

              Recombinations may be occurring at a fixed rate but the returning strains will increase the apparent rate.

              Consider what happens during recombination. A host cell is co-infected with two different strains. A section of the RNA segment from strain 1 is copied ready to be build up in a new RNA strand for a new viron. Due to an administrative error it ends up being inserted into a new strand of strain 2 and a recombined virus is born. One thing to note at this point is that the insertion is more likely to be viable if it is inserted in the same position on the wrong strand.

              There are some ducks on a pond and they have flu, one bird bought it in and it spread through the population. One of the later ducks to catch it ? unknown to him ? had picked up the infection from three different ducks and some of his cells were co-infected. A sequence of x residues was inserted at the right place on the wrong strand in a recombination event but there was no mutation. Because the two viruses involved could trace a shared ancestor only a few generations back their RNA was substantially the same, the random section that was shuffled was in fact identical so no change occurred. There is no reason to believe this is not the norm and a sequence is the exception, particularly if x is small.

              A flight of migratory geese arrive, they have been away for several months and the strain they bring with them has evolved and now has many more divergent residues. Had the same event occurred between duck and goose strains a visible change would have occurred and we would count it as a recombination. So more apparent recombinations, if only one residue changed we could not distinguish it from a point mutation, if it was a sequence we could match it to the goose strain and call it recombination. If it occurred in a critical section of the fragment, like a binding or cleavage site, then it may be extremely significant.

              The moral of the story is ? Be ware the returning migratory fowl.


              • #8
                Re: Homologous Recombination

                Thanks Mingus and JJackson.

                So, if they have similar genetic substrates and characteristics, and are from different but closely related viral families, are they "potentially" homologous?.. or are they considered to be "non-homolgous recombination, which happens on RARE occasion"?.
                "Predictable is Preventable" by Safety Expert Dr. Gordon Graham.


                • #9
                  Re: Homologous Recombination


                  Theoretically they could possibly undergo either homologous or non-homologous recombination. As I understand it (someone correct me if I'm wrong), it's "easier" for a replicating virus --ie, one of two viruses co-infecting a host-- to begin copying one virus strand, shift to copying the other where they're tangled in the cell "soup" and then shift back (copy choice) --> homologous recombination. Both offspring are the same length. What one looses, the other gains. One might read YYYYYZZZYYY and the other ZZZZZYYYZZZ. In non-homologous recombination, one can be long and the other shorter, for example. Are non-homologous less frequent because the offspring are consistently less viable?

                  I don't know that anyone has determined or demonstrated if homologous recombination has happened in any particular case involving flu and some other similar RNA virus. I haven't been able to find reference to any scientific research on it in any case.


                  • #10
                    Re: Homologous Recombination

                    .....if homologous recombination has happened in any particular case involving flu
                    On Recombinomics, it claims there is homologous recombination in the following flu cases:

                    Acquisition of H5N1Polymorphisms by H5N2 Bavarian Mallard

                    S227N and Changes in the H5N1 Receptor Binding Domain

                    Homologous Recombination in PB2 in Korean Swine

                    Homologous Recombination in NP in Korean Chicken

                    Homologous Recombination in PB2 in Korean Chicken

                    More Recombination in PB1 Genes of H5N1 Henan Chickens

                    Recombination in NA Gene of H5N1 Guangdong Wild Bird

           name a few referenced there.

                    "The next major advancement in the health of American people will be determined by what the individual is willing to do for himself"-- John Knowles, Former President of the Rockefeller Foundation


                    • #11
                      Re: Homologous Recombination

                      Originally posted by sara.smiles
                      Dr. Niman, et al..

                      Please explain this concept a bit better and expound on Webster's acknowledging that it may be happening in the H5N1 virus. How does it change it's virulence? In layman's terms... what does this *mean*??

                      Sara, It is not clear that Webster's position had dramatically changed. Although he mentions homologous recombination in his abstract, his slide actually indicates he couldn't find it, which is quite remarkable, since sequences he has published have some pretty glaring examples.

                      Recent swine sequences from Canada however have some of the most dramatic examples because two of the genes have very long stretches of sequences that are exact matches of two different 1977 isolates. These examples raise very serious questions about the frequency of "random mutations", which is the conventional wisdom on how a flu gene changes.




                      The swine data indicate the change is almost exclusively by recombination, because the regions that do change are exact matches of isolates from other areas and much earlier times. Thus, a 2003 isolate has an exact match with a 1977 and 1998 virus. This indicates that the changes are not by small mutations because the matches with the earlier genes are EXACT, meaning that there are NO differences, even though the genes have been copied for 5 or 25 years without ANY mistakes.

                      The data also contains nested regions, where the sequence is 1998-1977-1998 which not only show recombination, but shows recombination from a reservoir of 1977 sequences (which can also be demonstrated in seasonal flu).

                      Thus, the "old genes" are tucked away in a non-human reservoir and are cut and pasted into a human gene to create a new gene via recombination.

                      Since this concept represents a paradigm shift, those that are trying to keep the convention wisdom on track have tried to defend it with comments by Webster which say that it is rare and can't be easily found.

                      Once there is a concession, the slope becomes VERY slippery, because recombination is easiest and most frequent in viruses that are very similar (because some sequence identity is required to reposition the gene on the new template and this repositioning is easiest with more regions that are identical).

                      Thus, conceptually, it is MUCH easier to swap tiny bits of information, such as single nucleotide changes, which are called "random mutations" but are additional examples of recombination.

                      These small changes, called genetic drift, happen every year in influenza, which is why annual flu shots are given. These small changes quickly accumulate and evolve away from the immunizing virus, so immunizations have to be repeated with a more current version.

                      Recombination is between two existing viruses, so the new sequence can be predicted or identified earlier, allowing immunizations to be more effective because they are a closer match to the emerging sequence.
                      Last edited by HenryN; June 15, 2006, 07:45 AM.


                      • #12
                        Re: Homologous Recombination

                        The data pretty much shows why the "random mutation" explanation really is dead in the water:

                        A/swine/Ontario/11112/04(H1N1) identity between 721-1319
                        A/swine/Ontario/23866/04(H1N1) identity between 992-1745
                        A/swine/Ontario/48235/04(H1N2) identity between 150-2016
                        A/swine/Ontario/53518/03(H1N1) identity between 25-1469
                        A/swine/Ontario/55383/04(H1N2) identity between 589-1787
                        A/swine/Ontario/57561/03(H1N1) identity between 541-1817

                        A/Swine/Tennessee/24/77 (H1N1)

                        The above lists the six swine sequences that have various regions of identity with the 1977 sequences. The numbers represent the location of the region that is exactly the same as the 1977 sequence. Thus, for the first isolate, a stretch of 599 letters are an exact match of the 1977 sequences. The next sequence has 754 letters in a row that are exactly the same, but the identity is shifted and is at a region farther into the PA gene sequence. The third example has 1867 letters in a row that are exact matches with the 1977 sequence.

                        Since each of the 2003/2004 isolates has a different region that is an exact match, the argument that the sequence is essential doesn't work, because the sequence is replaced in other isolates. Thus, the six isolates were able to replace portions of the gene with other sequences, but still keep a large portion exactly the same. If the changes were due to random mutations, these LARGE regions of identity would not be maintained for 26 years. If however, the changed regions were changed by recombination, large segments could be swapped out and the changes would be clustered and not spread across the gene.

                        The changes in the swine sequences are easier to identify because swine sequences in general change more slowly. If fact these same isolates have a human PB1 gene but the sequence of the human gene isn't like the sequence of a human PB1 gene form 2003 or 2004, but it is like the human gene in the mid 90's. Thus, the human gene in swine evolves more slowly, which is due in part because the recombination rate is slower, which could be due to selection pressures or the frequency of dual infections.

                        As mentioned earlier, the swine acts as a reservoir, so pieces of the 1996 gene can disappear from humans, only to reappear later, which is what happened in the swine sequences.

                        Thus, the recombination not only affects swine flu sequences, but also human flu sequences.

                        Recombination is the name of the game, and this will become increasingly obvious as the pillars of influenza genetic crumble under the weight of HARD data.


                        • #13
                          Re: Homologous Recombination

                          Thanks for the attention to detail, Alaska Denise.

                          In my post a couple back, I was meaning that to my knowledge there is no research or physical demonstration of sequences recombining between flu virus and a virus like West Nile virus, which is one of KC's special interests. He's been asking for information on that question ever since he joined FluTrackers. I was speaking to the specific question I know he's asked repeatedly.

                          Early on it was clear to me that homologous recombination occurres between flu viruses.

                          There are some good posts by Toaster2 that GaudiaRay posted early on that explain at a very basic building-block level how recombination works with sequences if anyone wants to search on that for those interested.