Posted on Thursday 18 March 2010
Recombinomics Commentary
April 23, 2007
The geographical expansion of the Qinghai strain (clade 2.2 (http://www.recombinomics.com/phylo/HA_Vaccine.html)) of H5N1 offers a unique opportunity to monitor the genetic evolution of the strain. Clade 2.2 was first reported at Qinghai Lake in May, 2005 (http://www.recombinomics.com/News/05090502/Geese_Quinhai.html). Initially, 178 bar headed geese were found dead at the nature reserve in central China. The initial OIE report described 519 dead waterfowl (http://www.recombinomics.com/News/05230503/H5N1_Qinghai_519.html). Five species were identified, and the presence of H5N1 in the long range migratory birds was cause for concern. Sequences of the H5N1 indicated the clade was readily distinguished with earlier H5N1 isolates in Asia. In addition to a novel HA cleavage site, GERRRKKR, the isolates also had PB2 E627K (http://www.recombinomics.com/News/07070502/H5N1_Neurotropic_Qinghai.html), a change that was linked to human seasonal flu (http://www.recombinomics.com/News/07090502/Qinghai_E627K_Human.html). All H1N1, H2N2, and H3N2 human isolates dating back to 1918 had E627K. In contrast, no prior H5N1 from birds had E627K.
In July and August, H5N1 outbreaks were reported in Russia, Kazakhstan, and Mongolia. Like the outbreak at Qinghai Lake, migratory waterfowl at Chany Lake (http://www.recombinomics.com/News/08110503/H5N1_Novosibirsk_Qinghai_Lake.html) in Russia and Erhel Lake (http://www.recombinomics.com/News/09290505/H5N1_Erhel_Mongolia_500.html) in Mongolia tested positive for H5N1, and the sequences indicated the H5N1 which was reported for the first time in the three northern countries, were the Qinghai strain.
The presence of H5N1 in the long range waterfowl, which summer in the northern nature reserves signaled subsequent migration to the east in Asia, as well as west to Europe, the Middle East, and Africa. This long range migration offered a unique opportunity to monitor H5N1 evolution, as H5N1 expanded its geographical reach. In 2006, H5N1 spread into over forty countries which had not previously reported H5N1. In all cases, the H5N1 reported was exclusively the Qinghai strain. Sequence analysis indicated the emerging H5N1 had a series of regional markers, which distinguished various sub-groups within the 2.2 sub-clade.
Sequences from Egypt provided a large database of sequences from birds and patients in the 2006 season, as well as the current 2006/2007 season (http://www.recombinomics.com/News/10100601/H5N1_Egypt_Confirmed.html). The more recent isolates were more genetically complex and included a series of polymorphisms which had been previously identified in recent H5N1 isolates in Asia. The Egyptian markers in the isolates from early 2006 were in the isolates from the current season, and these additional polymorphism were overlaid into the Qinghai Egyptian genetic background. These acquisitions were consistent with acquisition via recombination.
The acquisitions were not easily explained by influenza genetics dogma, which maintains that sequences of influenza genes change via random mutations which are selected during escape from immunological recognition, or adaptation to a new host or new region. The polymerases encoded by the influenza genome lacked a proof reading function, which led to rapid evolution.
However, the acquisitions of previously reported polymorphisms suggested that the annual changes were due to recombination (http://www.recombinomics.com/News/04160702/H5N1_Recombination_2007.html), and not de novo “mutations”. The acquisitions in Egypt were found in H5N1 isolates that were easily linked via migratory bird flyways.
Recent simultaneous acquisition (http://www.recombinomics.com/News/04140701/H5N1_Evolution_Egypt_Russia.html) of the same polymorphism, NA G743A, created additional challenges for explanations based on random mutations. The acquisitions were on multiple genetic backgrounds in Egypt and Moscow. Earlier sequences had changes that created a number of new branches on the phylogenetic trees, and G743A was acquired simultaneously on the more recent isolates.
These changes can be seen on the NA phylogram. The top portion of the phylogram is on the left side of the figure (http://www.recombinomics.com/phylo/NA_Phylogram.html). At the bottom are a series of isolates from Germany. These isolates formed a distinct branch on the tree and all isolates had G743A. However, this change was not found in other Qinghai 2006 isolates, including two other branches, which were largely populated by isolates from German. Thus, the isolates from German formed three branches, but G743A was only found in one.
In Egypt, H5N1 in patients and poultry was reported between February and April of 2006. Reported H5N1 infections marked the re-emergence of H5N1 in Egypt in the fall. These isolates had the regional markers that were present in the isolates from early 2006, but the more recent isolates were more genetically complex. This complexity was due to a series of polymorhisms that were previously reported in H5N1 isolates in Asia. The sequences generated by US NAMRU-3 provided a monitor of H5N1 evolution in Egypt.
One example of this added complexity was in isolates from the Gharbiya cluster. The figure has four isolates from the cluster. These isolates were collected 48 hours apart from two of the three cluster members. Although the sequences from the two patients were distinguishable, the sequences were similar and had changes in or near the HA receptor binding domain (V223I and M230I) and had the oseltamivir (Tamiflu) resistance marker, N294S. This marker was present in all four isolates, even though two were collected prior to treatment. Like all of the sequences from Egypt in late 2006 and early 2007, G743A was not detected.
G743A was first detected in three chickens from Gharbiya, collected on February 15, 2007. One of the isolates, A/chicken/Egypt/1892N3-HK49 (http://flu.lanl.gov/search/view_record.html?accession=EF486246&database=fluA)/2007 (see red arrow at the top of tree on right side of figure), had the receptor binding domain changes, as well as a number of additional polymorphisms found in the Gharbiya cluster. However, G743A was appended onto this genetic background.
The two other chicken isolates, (A/chicken/Egypt/1890N3-HK45 (http://flu.lanl.gov/search/view_record.html?accession=EF486244&database=fluA)/2007 and A/chicken/Egypt/1891N2-CLEVB (http://flu.lanl.gov/search/view_record.html?accession=EF486245&database=fluA)/2007, were on another branch with earlier bird isolates from the Nile Delta. Although these earlier isolates also did nit have G743A, the two February 16 isolates also had G743A.
At this time, H5N1 outbreaks were reported in the suburbs of Moscow. As seen in the lower portion of the right hand side of the figure, the 2007 Moscow isolate, A/chicken/Moscow/2 (http://flu.lanl.gov/search/view_record.html?accession=EF474448&database=fluA)/2007, was similar to Azerbaijan isolates from the prior year and the chicken isolate also had G743A (as did a second closely related isolate from Moscow, A/chicken/Russia_Moscow oblast_Odintsovo/1 (http://flu.lanl.gov/search/view_record.html?accession=EF447431&database=fluA)/2007, which is not represented in the figure).
In addition, 2007 isolates from central Egypt formed another branch on the tree. These sequences had a 3 BP deletion that was identical to chicken isolates (http://www.recombinomics.com/News/02140701/H5N1_Fayyoum_Another.html) from Hunan Province in China. Although these sequences were distinct from clade 2.2, the 3 BP deletion (http://www.recombinomics.com/News/02140701/H5N1_Fayyoum_Another.html) was of the same three nucleotides, proving additional compelling data fro acquisition via recombination. The isolates from early 2007 in Egypt did not have G743A, but recent isolates from two siblings from Qena (http://www.recombinomics.com/News/04010701/H5N1_Egypt_Qena_Cluster.html), A/Egypt/2621/2007 and A/Egypt/2629/2007, had the acquisition.
Another recent isolate, A/Egypt/2630/2007 from a patient in Sohag, is on yet another branch, and it too has G743A (see red arrows).
The grouping of this isolates onto separate branches can also be seen in the HA phylogram (http://www.recombinomics.com/phylo/HA_Phylogram.html). The relationships described above on the NA tree is also found on the HA tree, further increasing the genetic differences between the various isolates from Egypt and Russia.
The simultaneous acquisition of G743A onto these diverse genetic backgrounds is most easily explained by recombination with a common source, which is very difficult to explain by random mutations, the cornerstone of current influenza dogma on the mechanism generating genetic drift.
Media sources (http://www.flutrackers.com/forum/showthread.php?t=21879)
Recombinomics Presentations (http://www.recombinomics.com/presentations.html)
http://www.recombinomics.com/News/04230701/H5N1_Dogma_Challenge.html
OK, so help me with the concept of recombination. As I understand reassortment and recombination, the host must be infected with at least two different viruses in order to pick up genetic changes. What I'm struggling with is how the Egyptian H5N1 hosts acquired the P239S change when so few Influenza A viruses of any subtype found anywhere in the world have this genetic marker.
My concern is that these virues are a lot smarter than we give them credit for, and can adopt to their environment in many different ways, including via mutations.
Mutations are largely a wate of time, so the smart approach is to use what has already passed the selection test, which is why recombination is the smart and effecient way to go (as in "elegant evolution").
The sequence database has giant holes and when travel logs jump from 2003 (in Clade 2.1 in Indonesia) to 2006 (in Clade 2.2 in Egypt). it is because of the gaping holes (as in no wild bird H5N1 sequences in Indonesia and very few in China and little low path H5 anywhere).
Dual infections happen all of the time and most changes look like SNPs, but they are really recombination.
If you look at the trees, you will see G743A tacked onto multiple branches formed by earlier isolates that lacked G743A (which is a SILENT change).
This type of change will NOT be explained by random mutation / selection / adaptation.
Most mutations in flu are generated by scientists in labs.
Most polymorphism acquistions in the field are via recombination between natural influenza isolates.
OK, so help me with the concept of recombination. As I understand reassortment and recombination, the host must be infected with at least two different viruses in order to pick up genetic changes. What I'm struggling with is how the Egyptian H5N1 hosts acquired the P239S change when so few Influenza A viruses of any subtype found anywhere in the world have this genetic marker.
My concern is that these virues are a lot smarter than we give them credit for, and can adopt to their environment in many different ways, including via mutations.
Has anyone else found this mutation???
It is also in an H5N1 Indonesian chicken isolate from 2003. The travel log is below
| EF441277 (http://flu.lanl.gov/search/view_record.html?accession=EF441277&database=fluA) | A/chicken/Egypt/1079-NAMRU3/2007 | HA (4) | 1707 | 2007 | H5N1 | |||
| EF469650 (http://flu.lanl.gov/search/view_record.html?accession=EF469650&database=fluA) | A/chicken/Egypt/1129N3-HK9/2007 | HA (4) | 1741 | 2007 | H5N1 | |||
| EF441280 (http://flu.lanl.gov/search/view_record.html?accession=EF441280&database=fluA) | A/chicken/Egypt/1300-NAMRU3/2007 | HA (4) | 1707 | 2007 | H5N1 | |||
| EF469653 (http://flu.lanl.gov/search/view_record.html?accession=EF469653&database=fluA) | A/chicken/Egypt/1889N3-SM26/2007 | HA (4) | 1741 | 2007 | H5N1 | |||
| EF469654 (http://flu.lanl.gov/search/view_record.html?accession=EF469654&database=fluA) | A/chicken/Egypt/1890N3-HK45/2007 | HA (4) | 1741 | 2007 | H5N1 | |||
| EF469659 (http://flu.lanl.gov/search/view_record.html?accession=EF469659&database=fluA) | A/chicken/Egypt/1891N3-CLEVB/2007 | HA (4) | 1738 | 2007 | H5N1 | |||
| EF469660 (http://flu.lanl.gov/search/view_record.html?accession=EF469660&database=fluA) | A/chicken/Egypt/1892N3-HK49/2007 | HA (4) | 1741 | 2007 | H5N1 | |||
| EF441281 (http://flu.lanl.gov/search/view_record.html?accession=EF441281&database=fluA) | A/duck/Egypt/1301-NAMRU3/2007 | HA (4) | 1707 | 2007 | H5N1 | |||
| EF469657 (http://flu.lanl.gov/search/view_record.html?accession=EF469657&database=fluA) | A/duck/Egypt/1888N3-SM25/2007 | HA (4) | 1741 | 2007 | H5N1 | |||
| EF382359 (http://flu.lanl.gov/search/view_record.html?accession=EF382359&database=fluA) | A/Egypt/0636-NAMRU3/2007 | HA (4) | 1749 | 2007 | H5N1 | |||
| ISDN140062 (http://flu.lanl.gov/search/view_record.html?accession=ISDN140062&database=fluA) | A/chicken/Egypt/1/2006 | HA (4) | 1671 | 2006 | H5N1 | |||
| EF441276 (http://flu.lanl.gov/search/view_record.html?accession=EF441276&database=fluA) | A/chicken/Egypt/1078-NAMRU3/2006 | HA (4) | 1707 | 2006 | H5N1 | |||
| EF441278 (http://flu.lanl.gov/search/view_record.html?accession=EF441278&database=fluA) | A/chicken/Egypt/1080-NAMRU3/2006 | HA (4) | 1707 | 2006 | H5N1 | |||
| EF441279 (http://flu.lanl.gov/search/view_record.html?accession=EF441279&database=fluA) | A/chicken/Egypt/1081-NAMRU3/2006 | HA (4) | 1707 | 2006 | H5N1 | |||
| EF042622 (http://flu.lanl.gov/search/view_record.html?accession=EF042622&database=fluA) | A/chicken/Egypt/10845-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF469651 (http://flu.lanl.gov/search/view_record.html?accession=EF469651&database=fluA) | A/chicken/Egypt/12378N3-CLEVB/2006 | HA (4) | 1741 | 2006 | H5N1 | |||
| EF469652 (http://flu.lanl.gov/search/view_record.html?accession=EF469652&database=fluA) | A/chicken/Egypt/12379N3-CLEVB/2006 | HA (4) | 1741 | 2006 | H5N1 | |||
| ISDN140063 (http://flu.lanl.gov/search/view_record.html?accession=ISDN140063&database=fluA) | A/chicken/Egypt/2/2006 | HA (4) | 1700 | 2006 | H5N1 | |||
| CY020645 (http://flu.lanl.gov/search/view_record.html?accession=CY020645&database=fluA) | A/chicken/Egypt/2253-1/2006 | HA (4) | 1734 | 2006 | H5N1 | |||
| DQ862001 (http://flu.lanl.gov/search/view_record.html?accession=DQ862001&database=fluA) | A/chicken/Egypt/2253-1/2006 | HA (4) | 1707 | 2006 | H5N1 | |||
| ISDN140049 (http://flu.lanl.gov/search/view_record.html?accession=ISDN140049&database=fluA) | A/chicken/Egypt/3/2006 | HA (4) | 1698 | 2006 | H5N1 | |||
| DQ837587 (http://flu.lanl.gov/search/view_record.html?accession=DQ837587&database=fluA) | A/chicken/Egypt/5610NAMRU3-F3/2006 | HA (4) | 1595 | 2006 | H5N1 | |||
| DQ837588 (http://flu.lanl.gov/search/view_record.html?accession=DQ837588&database=fluA) | A/chicken/Egypt/5611NAMRU3-AN/2006 | HA (4) | 1613 | 2006 | H5N1 | |||
| DQ837589 (http://flu.lanl.gov/search/view_record.html?accession=DQ837589&database=fluA) | A/chicken/Egypt/5612NAMRU3-S/2006 | HA (4) | 1604 | 2006 | H5N1 | |||
| DQ447199 (http://flu.lanl.gov/search/view_record.html?accession=DQ447199&database=fluA) | A/chicken/Egypt/960N3-004/2006 | HA (4) | 1673 | 2006 | H5N1 | |||
| DQ666146 (http://flu.lanl.gov/search/view_record.html?accession=DQ666146&database=fluA) | A/Djibouti/5691NAMRU3/2006 | HA (4) | 1311 | 2006 | H5N1 | |||
| EF469655 (http://flu.lanl.gov/search/view_record.html?accession=EF469655&database=fluA) | A/duck/Egypt/12380N3-CLEVB/2006 | HA (4) | 1741 | 2006 | H5N1 | |||
| EF469656 (http://flu.lanl.gov/search/view_record.html?accession=EF469656&database=fluA) | A/duck/Egypt/13010N3-CLEVB/2006 | HA (4) | 1715 | 2006 | H5N1 | |||
| CY016899 (http://flu.lanl.gov/search/view_record.html?accession=CY016899&database=fluA) | A/duck/Egypt/2253-3/2006 | HA (4) | 1743 | 2006 | H5N1 | |||
| DQ862002 (http://flu.lanl.gov/search/view_record.html?accession=DQ862002&database=fluA) | A/duck/Egypt/2253-3/2006 | HA (4) | 1707 | 2006 | H5N1 | |||
| EF061116 (http://flu.lanl.gov/search/view_record.html?accession=EF061116&database=fluA) | A/Egypt/12374-NAMRU3/2006 | HA (4) | 1639 | 2006 | H5N1 | |||
| EF200512 (http://flu.lanl.gov/search/view_record.html?accession=EF200512&database=fluA) | A/Egypt/14724-NAMRU3/2006 | HA (4) | 1733 | 2006 | H5N1 | |||
| EF200513 (http://flu.lanl.gov/search/view_record.html?accession=EF200513&database=fluA) | A/Egypt/14725-NAMRU3/2006 | HA (4) | 1745 | 2006 | H5N1 | |||
| EF042614 (http://flu.lanl.gov/search/view_record.html?accession=EF042614&database=fluA) | A/Egypt/2763-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| DQ464377 (http://flu.lanl.gov/search/view_record.html?accession=DQ464377&database=fluA) | A/Egypt/2782-NAMRU3/2006 | HA (4) | 1640 | 2006 | H5N1 | |||
| EF042615 (http://flu.lanl.gov/search/view_record.html?accession=EF042615&database=fluA) | A/Egypt/2783-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042616 (http://flu.lanl.gov/search/view_record.html?accession=EF042616&database=fluA) | A/Egypt/2786-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042617 (http://flu.lanl.gov/search/view_record.html?accession=EF042617&database=fluA) | A/Egypt/2947-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042618 (http://flu.lanl.gov/search/view_record.html?accession=EF042618&database=fluA) | A/Egypt/3105-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042619 (http://flu.lanl.gov/search/view_record.html?accession=EF042619&database=fluA) | A/Egypt/3458-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042620 (http://flu.lanl.gov/search/view_record.html?accession=EF042620&database=fluA) | A/Egypt/5494-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| EF042621 (http://flu.lanl.gov/search/view_record.html?accession=EF042621&database=fluA) | A/Egypt/5614-NAMRU3/2006 | HA (4) | 1596 | 2006 | H5N1 | |||
| ISDN140050 (http://flu.lanl.gov/search/view_record.html?accession=ISDN140050&database=fluA) | A/Egypt/902782/06 | HA (4) | 1692 | 2006 | H5N1 | |||
| ISDN140051 (http://flu.lanl.gov/search/view_record.html?accession=ISDN140051&database=fluA) | A/Egypt/902786/06 | HA (4) | 1686 | 2006 | H5N1 | |||
| EF469658 (http://flu.lanl.gov/search/view_record.html?accession=EF469658&database=fluA) | A/goose/Egypt/13009N3-SM2/2006 | HA (4) | 1715 | 2006 | H5N1 | |||
| CY020653 (http://flu.lanl.gov/search/view_record.html?accession=CY020653&database=fluA) | A/turkey/Egypt/2253-2/2006 | HA (4) | 1745 | 2006 | H5N1 | |||
| DQ837590 (http://flu.lanl.gov/search/view_record.html?accession=DQ837590&database=fluA) | A/turkey/Egypt/5613NAMRU3-T/2006 | HA (4) | 1603 | 2006 | H5N1 | |||
| AM183669 (http://flu.lanl.gov/search/view_record.html?accession=AM183669&database=fluA) | A/chicken/Indonesia/R134/03 | HA (4) | 1482 | 2003 | H5N1 |
Yes, lots of holes. But I am having difficulty in finding the P239S mustation in any other Influenza A virus anywhere in the world. In other words, when I see P239S in the HA segment of an Influenza A virus, I am 99% certain that it is H5N1 and Egyptian. This seems to be a signiture of some sort, but of unknown importance as to the characteristic of the virus. By virute of the fact that it is in the area of the receptor binding domain, I think it is worth investigating. But since I can not find any other virus with this mutation, I don't have a clue as to what to research.
Has anyone else found this mutation???
Other than the Djibouti human isolate and one 2003 Indonesian chicken isolate, no other Influenza A virus appears to reflect this mutation.
There actually are quite a few regional markers. Egypt has a fair number of synonymous and nonsynonymous changes. Many can be traced to low path sequences, and the database for low path H5 has some gaping holes, so full pathways for all acquistions are lacking.
Other than the Djibouti human isolate and one 2003 Indonesian chicken isolate, no other Influenza A virus appears to reflect this mutation.
#If you have any other info about this subject , Please add it free.# |