# 4909
Although it’s nestled behind a pay wall at the Journal of Virology, the abstract from a study published ahead of print this week in the Journal of Virology gives us a tantalizing glimpse at research conducted on the D222G mutation that has been found in some isolates of the pandemic H1N1 (pdmH1N1) virus.
While we’ve discussed the D222G mutation before, this is an obscure enough subject as to make a review helpful. I’ll keep it simple (essential so that I can follow, as well), so real scientists may wish to skim or skip ahead.
The `Norway’ or D222G (D225G in influenza H3 Numbering) mutation first announced by Norwegian Scientists last November has sparked repeated speculation that it might be associated with increased virulence.
This mutation had actually been detected months earlier, and in many other countries, but Norway was the first country to announce a possible link between that mutation and greater virulence.
This mutation involves a single amino acid change in the HA1 gene at position 222 from aspartic acid (D) to glycine (G).
The World Health Organization’s take on this mutation has been pretty consistent. It is worth following, and studying, but there is no evidence (as yet) that it poses a substantial public health hazard.
In January, in a blog entitled WER Review: D222G Mutation In H1N1, I quoted the latest WHO report that stated:
`Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.’
This view is not universally held, however. There are some who have maintained that that the WHO is underestimating the impact of this mutation.
In March of this year, researchers from the Norwegian Institute of Public Health in Oslo reported that they found the mutation in 11 of 61 severe illness cases that they analyzed, but that it was not found in any of the 205 mild cases they looked at (see CIDRAP Report On The H1N1 Mutation Debate).
The WHO WER Review reported that the overall prevalence of D222G was <1.8% (52 detections among >2755 HA sequences) in contrast to a rate of 7.1% in fatal cases. The WHO paper also reported on the occurrence of two other mutations at this amino acid position, D222E and D222N, although their significance is unclear.
While this may sound like fairly damning evidence, it should be noted that mild cases have been detected with this D222G mutation in other studies, and most of the severe and fatal cases of pandemic H1N1 that have been examined did not have this mutation.
Which brings us to today’s study, which features an impressive pedigree and some very familiar names including Ab Osterhaus and Ron Fouchier of the Erasmus Medical Center in Rotterdam.
This study was also supported by researchers from the Mt. Sinai School of Medicine in New York, the NIH, the University of Cambridge, the University of Maryland . . . among others.
First the abstract (hat tip Tetano on FluTrackers) slightly reformatted for readability, then a little discussion.
Virulence-associated substitution D222G in hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding.
Chutinimitkul S, Herfst S, Steel J, Lowen AC, Ye J, van Riel D, Schrauwen EJ, Bestebroer TM, Koel B, Burke DF, Sutherland-Cash KH, Whittleston CS, Russell CA, Wales DJ, Smith DJ, Jonges M, Meijer A, Koopmans M, Rimmelzwaan GF, Kuiken T, Osterhaus AD, Garcia-Sastre A, Perez DR, Fouchier RA.
Abstract
The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities.
Here, D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated.
pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs.
The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands.
Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding.
Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
Testing here was done on mice, ferrets, guinea pigs, and on human cells in vitro, and each demonstrated (sometimes small) pathogenic differences between the D222G-engineered and regular pdmH1N1 virus.
In mice and ferrets, the D222G virus showed no increase in virulence with the exception of `ocular disease’ in mice (I’m guessing conjunctivitis, but without access to the full article, I can’t be certain).
Given the low incidence of the D222G mutation in the wild (less than 1.8%), it has been suggested that this mutation might render the virus less contagious, but ferret and guinea pig studies showed it retained transmissibility via aerosols and respiratory droplets.
The increased binding to type II pneumocytes in the alveoli (in vitro) is a particularly interesting finding, given that this was also observed in the Baskin Study of H5N1 vs human H1N1 viruses.
Seasonal H1N1 viruses, when they invade the lungs, are more likely to attack type I pneumocytes which handle the gas exchange (02 and C02) between the lungs and the blood stream.
Type II pneumocytes are responsible for the production of surfactant with antimicrobial, immunomodulatory, and anti-inflammatory properties, and are the lung’s primary mechanism for repairing damaged cells.
Damaging them can significantly degrade the lung’s ability to recover from injury.
Which brings us to the last major finding, that D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids.
Familiar territory to regular readers of this blog, but at the risk of repeating myself:
A virus’s ability to bind to specific cells is controlled by its RBD or Receptor Binding Domain; an area of its genetic code that allows it to attach to, and infect, specific types of host cells.
(Very Simplified Illustration of RBDs)
Like a key into a padlock, the RBD must `fit’ in order to open the cell to infection.
Avian adapted influenza viruses bind preferentially to Alpha 2,3 receptor cells, which are commonly found in the digestive tract of birds.
Human adapted viruses have an affinity for the alpha 2,6 receptor cell, which populate the upper airway and lungs.
Humans have some avian-like alpha 2,3 receptor cells, particularly deep in the lungs.
This has been suggested as the reason that when humans contract H5N1, it is usually a deep lung infection. It has also been postulated that H5N1’s deeper lung infections may reduce human-to-human transmission, as sneezing is a less common symptom.
This duel receptor affinity with the D222G mutation may help explain why some patients that contract it also develop more serious lung infections.
The operative word here being `may’.
The bottom line here is that so far, whatever pathogenic differences this mutation may spark, it has had a relatively small effect on the overall mortality and morbidity of this virus.
That could change, of course, if this mutation were to become more common, or if complementary concurrent changes to the genetic structure of the virus were to further enhance its virulence.
All in all, a fascinating piece of research, and one that advances our knowledge of this mutation considerably. No, it doesn’t answer the `big question’, of whether this mutation will end up becoming a significant public health threat.
But scientific knowledge is gained incrementally.
So stay tuned.
For more on the Baskin Study (Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus by Carole Baskin et. al. that appeared PNAS), which looked at the comparative pathogenesis of seasonal H1N1, a 1918-like H1N1, and the H5N1 virus, you may enjoy my 3-part series available at the following links:
Dissecting the Influenza Pathogenesis Study Pt. 1
