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Flu News: What is the significance of D225G?

Swine Flu News: What is the significance of D225G?

By Ilene

Dr. Henry L. NimanWith the current rush of news about the swine flu virus morphing into more aggressive lung-shredding and tamiflu-resistant mutants, there is some confusion as to why these changes are being seen in people in "hotspots" around the world, with no clear connection to each other. Officials at the WHO and CDC suggest that the same mutations are arising spontaneously in multiple locations but this doesn’t quite make sense.  To better understand how changes in the swine flu virus may be occurring, I contacted Dr. Henry L. Niman, founder and president of Recombinomics.  Dr. Niman has been an active researcher in the evolution of flu virus.  His latest thoughts on the ongoing progression of the flu pandemic may be found at his website, Recombinomics.   

For a little background, the D222G mutation or D225G mutation (same mutation, different numbering system) was found in three cases in Norway ("Norway" mutation), and in other countries, including Brazil, China, Japan, Mexico, Ukraine, the United States, and more recently Hong Kong. The change in a single nucleotide results in an amino acid change in the virus’s receptor binding protein. This has the effect of allowing the virus to bind receptors in the lung tissue, rather than the more usual binding to cells in the upper airways. Theoretically, this may confer greater virulence to the virus, potentially leading to more severe disease as the infection invades deeper in the respiratory tract. This change was also seen in the 1918 flu pandemic, in some (but not all) cases. 


The name of Dr. Niman’s company "Recombinomics" is taken from the word "recombine" or "recombination" – the driver of rapid molecular evolution and the emergence of infectious agents. Recombination* is a mechanism whereby small bits of genetic information pass between viruses so that a virus may quickly acquire a genetic change that evolved previously over the years in other viruses. Recombination is similar to reassortment, but with less genetic material being exchanged. 

Sporadic mutations do not usually lead to successful adaptive changes – often they have no effect or prove to be non-adaptive, with the mutation failing to be further replicated.  In contrast, recombination allows viruses to quickly alter their characteristics by acquiring genetic material that already exists in the viral reservoir (i.e., the pool of viruses circulating in a population). This genetic material has already survived the trial and error period of natural selection. The viral reservoir consists of wild-type viruses (the predominant viruses) and a low levels of variants carrying a variety of different sequences (genetic “polymorphisms”).  

Dr. Niman explained that recombination is not the favored theory regarding how the flu viruses evolve.  Nevertheless, Dr. Niman’s theory has led to accurate predictions about the swine flu’s course during this pandemic.  CDC and WHO officials may be slow to understand the changes we are seeing in the swine flu virus, but as this disease progresses, the consensus view on how viruses evolve may also change.

My questions to Dr. Niman about recombination may have prompted him to write the following article explaining how the D225G marker, which is a genetic change that enables the virus to invade and replicate in the lungs, may begin to appear in many geographical locations at once. The process of recombination explains this phenomenon better than the competing theory of sporadic mutation, which assumes that these genetic changes are occurring independently as copy errors in multiple places at the same time. 

Spontaneous Mutation Media Myth
Recombinomics Commentary
November 24, 2009

The mutations appear to occur sporadically and spontaneously. To date, no links between the small number of patients infected with the mutated virus have been found and the mutation does not appear to spread.  [WHO briefing]

The above comment from the WHO briefing on D225G (aka D222G) in Norway describes how the "mutations appear".  However, this appearance is based on an outdated view of influenza evolution, which maintains that all newly acquired drift "mutations" are based on copy errors.  For D225G, this would require the same copy error to occur again and again on multiple [genetic] backgrounds, which simply is not reality based. 

Although the "random mutation" explanation is one of the basic tenets of the WHO and CDC view of influenza evolution, this explanation is only viable in the absence of data.  Extensive influenza sequence data moved this hypothesis into the indefensible category years ago, but it remains at the core of WHO explanations of drift variants, such as the comments above.

The "random mutation" and failure to spread would require each detection to be an independent event. Thus, in Norway, the same copy error would have been made in each of the three patients with D225G.  Similarly, the same error would be required for each of the four fatal cases in Ukraine.  Moreover, the same error would be made in the vaccine target, A/California/7/2009, because one of the [candidate clones for the flu mist vaccine had D225G].  As the number of sequences with D225G increases, the likelihood that the same error happens again and again, among a very small number of differences… becomes untenable…

For D225G, the change was present in one of the earliest isolates [viral material isolated from a sick patient] in the United States.  It could jump from one [genetic] background [virus] to another via recombination between sequences that are closely related.  As a result, the new acquisitions lead to a new single nucleotide polymorphism, which looked like a point mutation, but was really recombination between closely related sequences.

[D225G] moves from one genetic background [virus] to another via recombination. A new spontaneous mutation is not required for each isolate [viruses isolated from a patient] and the same sequence in a given area is just due to clonal expansion [growth] of an isolate….

Thus, the movement of the same polymorphism via recombination is common. It explains the sudden appearance of the same marker on multiple genetic backgrounds, and forms a basis for predicting changes.

However, the reliance on a "random mutation" produces "surprise after surprise" among influenza "experts" and creates "appearances" such as spontaneous mutations and lack of transmission which are not based on reality.


The D225G Marker

According to Dr. Niman, the most likely explanation for the concurrent emergence of the D225G variant in multiple regions is that the "strain" of swine flu virus circulating is not a homogeneous strain but consists of a predominant (wild-type) strain with a variety of less common variants, including viruses with the D225G genetic marker and viruses that have the genetic sequences conferring tamiflu resistance. Dr. Niman believes viruses with the D225G marker are not adequately represented in the flu database because this variant is not easily detected in nasopharyngeal swabs.  However, there is enough of the D225G variant in the viral reservoir to act as "donor sequences" so the D225G change can jump from one virus to another, leading to its detection in many locations around the same time. The reason detection increases is that the viral reservoir, along with numbers of D225G variants, expands as flu season progresses.  As the viral reservoir grows (more viruses, greater numbers of people infected), greater numbers of D225G viruses begin to show up in more and more flu cases.

The background presence of the D225G mutation in the H1N1 virus strain explains why cases with the mutation are found throughout the world and why these mutations have been found in mild as well as severe cases. The presence of virus with the D225G marker should not be seen as an all-or-nothing phenomenon.  Theoretically, if a D225G subclone takes hold in the lungs and expands, it can cause a more severe flu. While the D225G marker may increase the virulence of the virus, the receptor binding profile is only one of a number of factors influencing the severity and outcome of an infection. Other factors include the viral load (how much virus a patient is exposed to), the patient’s immune system (does the patient have antibodies against the virus?), and other characteristics of the infecting viruses (e.g., how transmissible is the virus?).

Supporting the suggestion that viruses with the D225G marker are more virulent than the wild-type virus is the finding of D225G sequences in isolates from the 1918 Spanish flu pandemic. There is also evidence that this marker may have become more common in the Ukraine, where the swine flu seems to be particularly severe. Nevertheless, further evidence is needed to draw conclusions. Research is needed to answer questions such as what proportion of flu isolates contain the D225G marker? Is the D225G variant more prevalent in countries such as Ukraine where the flu seems to be more aggressive--is the increased severity of the flu in fact due to the presence of the D225G genetic change? Is the ratio of wild-type virus to D225G virus different in different geographical regions? Does this ratio differ in mild vs. severe cases? Are viruses with the D225 marker found in lung tissue of patients with mild flues? Is the ratio of D225G variant to the wild-type variant changing?

Answers to these questions will greatly add to our understanding of the mechanisms by which flu viruses change their genetics to become more (or less) virulent and develop resistance to anti-viral agents. Solving the mystery of how the flu virus so quickly evolves may help us stay one step ahead with our yearly flu vaccines, anticipating changes, rather than chasing them. Hopefully, Dr. Niman will continue to make predictions and present evidence for his theory that the flu virus is able to change so quickly because it recombines with other flu viruses, exchanging small bits of genetic information with its viral neighbors. In time, perhaps the WHO and CDC will pay attention. 

For more detailed discussions, see: 

Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor

A silent change appearing at the same time on multiple backgrounds:
A paper on tamiflu resistance (H274Y):
Detailed description of D225G, made 9 days BEFORE the Ukraine sequences were released:   
More on D225G, before the sequences were released  
Here is the Oct 22, 2005 prediction on S227N 
*Note on definitions of recombination and reassortment: Flu Trackers blog.  As Dr. Niman uses the term "recombination," the genetic material does not undergo a reciprocal exchange, but rather, a double infection in the same cell results in the potential for a sequence in one virus to be replaced during copying with a sequence from another virus. – Ilene


Dr. Henry Niman earned a PhD in biochemistry at the University of Southern California in 1978. His dissertation focused on feline retroviral expression in tumors in domestic cats.  Working on his post-doctorate at the Scripps Clinic and Research Foundation, Dr. Niman developed monoclonal antibody technology.  He later accepted a staff position at Scripps, and subsequently had a joint appointment as an Instructor in Surgery at Harvard/Massachusetts General Hospital and as a Research Associate at the Shriner’s Burn Center across the street from Mass General. (These were research positions – he did not teach or do surgery.)

In 1982, Dr. Niman developed the flu monoclonal antibody, which is widely used throughout the pharmaceutical, biotech, and research industries.  He also produced a broad panel of monoclonal antibodies against synthetic peptides of oncogenes and growth factors.  The technology developed by Dr. Niman was used to form ProgenX, a cancer diagnostic company that became Ligand Pharmaceuticals. More recently, he became interested in infectious diseases.  He founded Recombinomics and has been studying viral evolution.

As a coincidence, I also discovered that Dr. Niman and I worked in the same pathology/biochemistry lab at the University of Southern California in Los Angeles, separated only by about ten years.


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