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H5N1 Clade 2.2 Polymorphism Tracing Identifies Influenza Recombination and Potential Vaccine Targets
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- Recombinomics, Inc.
- US Navy Medical Research Unit #3 (NAMRU-3)
- Ministry of Health, Arabic Republic of Egypt
- Central Laboratory for Veterinary Quality Control, Giza
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- Document Type:
- Manuscript
- Date:
- Received 27 July 2007 11:41 UTC; Posted 27 July 2007
- Subjects:
- Biotechnology, Evolution and Ecology, Genetics, Microbiology, Bioinformatics
- Abstract:
Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province in 1996, followed by human cases in Hong Kong in 1997 1. The number of confirmed human cases now exceeds 300 and the associated Case Fatality Rate exceeds 60% 2. The genetic diversity of the serotype continues to increase. Four distinct clades or sub-clades have been linked to human cases 3.4. The gradual genetic changes identified in the sub-clades have been attributed to copy errors by viral encoded polymerases that lack an editing function, thereby resulting in antigenic drift 5. We traced polymorphism acquisition in Clade 2.2 sequences. We report here the concurrent acquisition of the same polymorphism by multiple, genetically distinct, Clade 2.2 sub-clades in Egypt, Russia and Ghana. These changes are not easily explained by the current theory of “random mutation” through copy error, and are more easily explained by recombination with a common source. This conclusion is supported by additional polymorphisms shared by Clade 2.2 isolates in Egypt, Nigeria and Germany including aggregation of regional polymorphisms from each of these areas into a single Nigerian human hemagglutinin gene.
Discussion
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21 votes
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6 comments
Plaque purification of virus from a given sample gives a indication of the diversity, which is markedly higher than is seen in public sequences.
In the HK49 sample, there were two distinct populations. Clones were about 7 to 1 in favor of the public sequence (the minor sequence matched HK45). However, even though there were 11 differences between the two NA sequences, only 2 of the 11 positions gave mixed signals in the consensus sequence.
Dual (or multiple) infections by H5N1 are much more common than many realize.
Hints of dual infections come from sequences with mixed signals, but the frequency is actually much higher, because consensus sequences miss minor species. These are identified through plaque purification and the results can be quite revealing.
Cloning of the HK49 sequence, which is closely related to the sequences from relatives from Gharbiya in the Nile Delta showed that the chicken sequence, also from Gharbiya had two distinct sequences. One matched the HK49 sequence, while the other matched related sequences from two other chickens from Gharbiya. In addition to the sequences matching the parental strains, there were clones that had recombined the parental sequences.
Recombination is the major driver of antigenic drift involving SNPs.
In figure 2 there are several isolates that share three polymorphisms with the human Nigerian HA sequence, which include
A/great crested grebe/Germany/R1226/06
A/duck/Cote d’Ivoire/1787-18/2006
A/hooded vulture/Burkina Faso/2/2006
A/chicken/Burkina Faso/01.03/2006
A/chicken/Burkina Faso/13.1/2006
A/chicken/Nigeria/641/2006
A/turkey/Ivory Coast/4372-4/2006
A/turkey/Ivory Coast/4372-3/2006
A/turkey/Ivory Coast/4372-2/2006
A/chicken/Ghana/3160-NAMRU-3/2007
A/chicken/Ghana/3159-NAMRU-3/2007
A/chicken/Ghana/3158-NAMRU-3/2007
A/hooded vulture/Burkina Faso/1/2006
A/chicken/Cote d’Ivoire/1787-34/2006
A/chicken/Cote d’Ivoire/1787-35/2006
A/chicken/Sudan/2115-12/2006
A/chicken/Sudan/2115-9/2006
A/chicken/Sudan/1784-10/2006Sequences sharing four polymorphisms are
A/chicken/Sudan/1784-8/2006
A/chicken/Sudan/1784-7/2006
A/chicken/Sudan/2115-10/2006Today 10 clade 2.2 HA sequences from Switzerland were released (see below). Like the three previous Swiss sequences, these new sequences are similar to a subset from Germany (see figure 2 from doi:10.1038/npre.2007.553.1).
The 10 new sequences all have G296A and C1480T, which has been acquired by the human sequence from Nigeria.
These new sequences support the aggregation of regional polymorphisms into the Nigerian sequence.
A/mallard/Switzerland/V537/2006
A/duck/Switzerland/V426/2006
A/little grebe/Switzerland/V330/2006
A/common pochard/Switzerland/V592/2006
A/mute swan/Switzerland/V68/2006
A/common pochard/Switzerland/V505/2006
A/common pochard/Switzerland/V762/2006
A/mallard/Switzerland/V558/2006
A/duck/Switzerland/V389/2006
A/duck/Switzerland/V487/2006There are additional indications of frequent dual infections. NIAID has an influenza sequencing program and details of isolates are posted. Samples submitted by Ohio State University list the serotype of the sample as well as the serotype of the sequenced plaque purified clones. Frequently, the serotypes do not match, indicating the birds are infected by at least two distinct serotypes. This is in addition to co-infections by closely related viruses of the same serotype.
Dual infections in birds are quite common.
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Additional information
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- This document is licensed to the public under the Creative Commons Attribution 2.5 License
- How to cite this document:
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Niman, Henry, Saad, Magdi, Boynton, Bruce, Tjaden, Jeffery, Earhart, Kenneth, Mansour, Moustafa, ElSayed, Nasr, Nayei, A, Abdelghani, A, Essmat, Hala, Labib, Elassai, Ayoub, E, Aly, Mona, Arafa, A-SA, and Monteville, Marshall. H5N1 Clade 2.2 Polymorphism Tracing Identifies Influenza Recombination and Potential Vaccine Targets. Available from Nature Precedings <http://dx.doi.org/10.1038/npre.2007.553.1> (2007)
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Other versions of this document in Nature Precedings
Version number Document title Date v2 Posted 07 August 2007 Other versions of this document elsewhere on the web
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Alice Campbell on 27 July 2007 22:56 UTC
Acquisition of polymorphisms by recombination implies simultaneous infections by each of the parent strains. Unfortunately, the current protocol for obtaining isolates assumes a single strain is present, and so has an inherent bias against detecting multiple parent strains in the same patient. Splitting a blood sample and performing multiple isolations might shed some interesting light on how homogeneous the original viral infection was. It seems like this would be a relatively simple way to show that there is substantial sampling bias against detection of multiple infections, and to show the extent of viral population diversity in a small outbreak. Both of these things seem important in understanding the biology of H5N1.