EID Journal: Emergence of D225G Variant A/H1N1, 2013–14 Flu Season, Florida

 

Flu Virus binding to Receptor Cells – Credit CDC

 

# 9775

 

Although we’ve barely seen any A/H1N1 infections in North America during this latest flu season – last year was a different story, with H1N1 dominating  the flu scene. 

 

Today’s EID Journal carries a dispatch describing the detection of a small number of unusually severe H1N1 cases in Florida last year, which they have linked to the emergence of a D225G mutation that increased the virus’s ability to bind deep into the lungs.

 

If this sounds vaguely familiar, it is because we’ve been down this road before during the H1N1 pandemic. Since we have a bit of history with this variant, a quick review:

 

In November 2009 the Norwegian Institute of Public Health (see Norway Reports An H1N1 Mutation) announced the discovery of a mutation that “could possibly make the virus more prone to infect deeper in the airways and thus cause more severe disease."

Word of the `Norway’ or D222G (D225G in influenza H3 Numbering) mutation immediately sent researchers around the world on a hunt for similar changes in the virus, and over the following months several variations on a theme were discovered; D222N, D222E, and D222A.

 

The D222G mutation involves a single amino acid change in the HA1 gene at position 222 from aspartic acid (D) to glycine (G).

The pdmH1N1 virus carrying this mutation appeared to bind more readily to receptor cells (α2-3) found deep in the lungs, whereas unmutated seasonal flu strains bind preferentially to the (α2-6) receptor cells found in the upper airway.

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.

(A Very Simplified Illustration of RBDs)

Like a key into a padlock, the RBD must `fit’ in order to open the cell to infection.

Similarly, D222A changes the HA1 gene at position 222 to Alanine, while D222E changes the gene to Glutamic acid and D222N changes to Asparagine.

This D222G mutation had actually been detected months earlier, and in several other countries, but Norway was the first country to announce a possible link between that mutation and greater virulence.

 

While a worrisome genetic change, it appeared that it was a fairly rare variant. 

 

In January of 2010, the World Health Organization’s Weekly Epidemiological Record (No. 4, 2010, 85, 21–28) provided a detailed overview of what was then known about this mutation.  While stating that more study was needed, the WHO pointed out:

• 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.
• They reported on the occurrence of  two other mutations at this amino acid position, D222E and D222N, although their significance is unclear.
• And concluded `Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.’

 

The debate over the significance (and origins) of the D222G mutation have continued since then. You can revisit some of those studies in the following blogs:

 

Eurosurveillance: Analysis Of Fatal H1N1 Cases In The UK)

Eurosurveillance: Debating The D222G/N Mutation In H1N1

D222G And Deep Lung Infections

 

In 2013, in EuroSurveillance: Revisiting The D222G Mutation In A/H1N1pdm09, we saw a study that suggested viruses with this mutation don’t transmit well in the wild, and that most of the time this variant comes about through a spontaneous mutation in the host after the host has been infected.  

 

The concern is that viruses can change, and co-mutations could occur that could make the D222G mutation more transmissible.

 

Fast forward to today’s EID report, and we learn of the discovery of the D224G mutation in a small number of unusually severe flu cases in Florida last year.

 

Volume 21, Number 4—April 2015

Dispatch

Severity of Influenza A(H1N1) Illness and Emergence of D225G Variant, 2013–14 Influenza Season, Florida, USA

Nicole M. Iovine , J. Glenn Morris, Kristianna Fredenburg, Kenneth Rand, Hassan Alnuaimat, Gloria Lipori, Joseph Brew, and John A. Lednicky

Author affiliations: University of Florida, Gainesville, Florida, USA (N.M. Iovine, J.G. Morris Jr., K. Fredenburg, K. Rand, H. Alnuaimat, G. Lipori, J.A. Lednicky); Florida Department of Health, Gainesville (J. Brew)

 

Abstract

Despite a regional decline in influenza A(H1N1)pdm09 virus infections during 2013–14, cases at a Florida hospital were more severe than those during 2009–10. Examined strains had a hemagglutinin polymorphism associated with enhanced binding to lower respiratory tract receptors. Genetic changes in this virus must be monitored to predict the effect of future pandemic viruses.

<SNIP >

The molecular basis for the ability of the H1N1 viruses to cause severe lower respiratory tract disease was first noted with the 1918 pandemic H1N1 virus (10). A single amino acid change of aspartic acid at position 225 to glycine (D225G) enabled binding to α2-3- and α2-6-sialylglycans (10,11). The association of this polymorphism with severe and lower respiratory tract disease was also noted with the A(H1N1)pdm09 virus from small subsets of patients during the 2009–10 influenza season (12–14). To determine if this polymorphism existed in the H1N1 viruses isolated in our study, we sequenced viral RNA corresponding to the coding regions of all 8 influenza virus genomic segments from viruses isolated from or detected in 7 patient samples (GenBank sequences KJ645758–KJ645765 and KJ645774–KJ645791) (8). The consensus sequences were similar to those of key American A(H1N1)pdm09 virus isolates, and like a subset of those isolates, our 7 H1N1 isolates harbored the D225G polymorphism.

None of the 7 isolates harbored the H275Y neuraminidase polymorphism that confers oseltamivir resistance (15), and in vitro test results confirmed oseltamivir susceptibility (data not shown). To determine whether polymorphisms in the 3′ and 5′ untranslated regions were associated with the 2013 H1N1 virus, we sequenced 1 isolate by using rapid amplification of cDNA ends (FirstChoice RLM-RACE; Ambion, Bleiswijk, the Netherlands). No substantial differences were detected between this virus and the original A(H1N1)pdm09 virus or circulating contemporary H1N1 viruses.

Our work was subject to certain limitations. First, the use of ICD-9 codes for diagnostic information is imprecise. To mitigate this, we considered only 2 ICD-9 codes for influenza. Second, it is not known whether our findings extend beyond our region. Last, changes in health-seeking behavior in 2009–10 versus 2013–14 were not addressed.

Conclusions

We hypothesize that the emergence of an influenza virus variant bearing the D225G polymorphism enabled the 2013 H1N1 virus to infect lower and upper respiratory tract cells, thereby contributing to the increased severity of the 2013–14 influenza season in our region. Our findings highlight the importance of monitoring genetic changes in the 2013 H1N1 virus to predict the effect of future influenza viruses.

 

As we’ve discussed before (see When Influenza Goes Rogue & CHP CDW Report On Antiviral Resistant Influenza In Hong Kong), even seasonal influenza needs to be watched carefully, as it is capable of dramatically changing its behavior should it pick up the right mutations. 

EuroSurveillance: Revisiting The D222G Mutation In A/H1N1pdm09

 

 

Flu Virus binding to Receptor Cells – Credit CDC 

 

 

# 6865

 

One of the unresolved  mysteries of the 2009 H1N1 pandemic is that while most people saw a relatively mild illness - for a small percentage of the population - the virus proved unusually severe and sometimes deadly. 

 

We saw the burden of the disease shift to younger adults and adolescents, a cohort that normally endures influenza infection pretty well. In Study: Years Of Life Lost Due To 2009 Pandemic, researchers calculated the mean age of death from the pandemic virus to be half that of seasonal flu, or 37.4 years.

 

And in September of 2011 we saw research indicating the H1N1pdm virus was more likely to exacerbate an S. pneumoniae co-infection (in mice, anyway) than was seasonal H1N1 (see mBio: Lethal Synergism of H1N1 Pandemic Influenza & Bacterial Pneumonia)

 

During November of 2009, news of a small change in the novel H1N1 virus came by way of the Norwegian Institute of Public Health (see Norway Reports An H1N1 Mutation) who announced the discovery of a mutation that “could possibly make the virus more prone to infect deeper in the airways and thus cause more severe disease."

 

The announcement of the `Norway’ or D222G (D225G in influenza H3 Numbering) mutation immediately sent researchers around the world on a hunt for similar changes in the virus, and over the following months several variations on a theme were discovered; D222N, D222E, and D222A.

 

The D222G mutation involves a single amino acid change in the HA1 gene at position 222 from aspartic acid (D) to glycine (G).

 

The pdmH1N1 virus carrying this mutation appeared to bind more readily to receptor cells (α2-3) found deep in the lungs, whereas unmutated seasonal flu strains bind preferentially to the (α2-6) receptor cells found in the upper airway.

 

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.

(A Very Simplified Illustration of RBDs)

Like a key into a padlock, the RBD must `fit’ in order to open the cell to infection.

Similarly, D222A changes the HA1 gene at position 222 to Alanine, while D222E changes the gene to Glutamic acid and D222N changes to Asparagine.

 

This D222G mutation had actually been detected months earlier, and in several other countries, but Norway was the first country to announce a possible link between that mutation and greater virulence.

 

In January of 2010, the World Health Organization’s  Weekly Epidemiological Record (No. 4, 2010, 85, 21–28) provided a detailed overview of what was then known about this mutation. 

While stating that more study was needed, the WHO pointed out the lack of apparent ongoing transmission of this mutation, and stated that:

 

`Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.’

 

Later in 2010, in Study: Receptor Binding Changes With H1N1 D222G Mutation, we saw more evidence of preferential binding to deep lung cells by viruses with the D222G mutation.

 

The debate over the significance (and origins) of the D222G mutation have continued since then. You can revisit some of those studies in the following blogs:

 

Eurosurveillance: Analysis Of Fatal H1N1 Cases In The UK)

Eurosurveillance: Debating The D222G/N Mutation In H1N1

D222G And Deep Lung Infections

 

All of which serves as prelude to a new study on the D222G mutation – again from Norwegian Institute of Public Health – that appeared yesterday in the journal Eurosurveillance.

 

 

Eurosurveillance, Volume 18, Issue 3, 17 January 2013

Within-patient emergence of the influenza A(H1N1)pdm09 HA1 222G variant and clear association with severe disease, Norway

R Rykkvin, A Kilander, S G Dudman, O Hungnes

---

Date of submission: 29 December 2012

---

ABSTRACT (reparagraphed for readability)

The association between a particular mutation in the HA1 subunit of the influenza virus haemagglutinin, D222G, and severe and fatal disease in cases of influenza A(H1N1)pdm09 in Norway during the 2009 pandemic was investigated using pyrosequencing.

 

The prevalence of the variant among fatal cases was 8/26 and among severe non-fatal cases 5/52. No D222G mutations were found among the 381 mild cases.

 

This difference could not be attributed to sampling differences, such as body location of sampling, or duration of illness. In cases with mutant virus where clinical specimens from different days of illness were available, transition from wild-type to mutant virus was commonly observed (4/5), indicating that the mutant virus emerged sporadically in individual patients.

 

In patients with paired samples from both the upper and lower respiratory tract (n=8), the same viral genotypes were detected in both locations. In most of the D222G cases (11/13), the mutant virus was found as a quasispecies.

 

 

This a long, and fairly technical report with a lot to digest, and I’m sure many of you will want to read it in its entirety.

 

But in short the authors present several findings, which they summarize in the discussion portion of the paper:

 

In the present study, we provide further epidemiological evidence of the association between the D222G mutation in HA1 of influenza A(H1N1)pdm09 virus and severe or fatal clinical course.

 

Furthermore, we present evidence that the mutated viruses emerge in individual patients after the onset of illness and demonstrate the presence of mutant virus in both the upper and lower respiratory tract. We also address some potential biases that could conceivably confound the analysis.

 

The Norwegian cases of infection with HA1 222G genotype viruses have occurred sporadically and do not cluster epidemiologically or in phylogenetic analysis.

<SNIP>

 

The 222G viruses appear to be rare among circulating strains, but are still quite frequent in patients with severe disease, who are not epidemiologically linked. A likely explanation is that the presence of mutant viruses in these particular individuals experiencing severe disease is due to selective upgrowth of mutant genomes during infection.

 

 

In other words, this mutation appears to occur spontaneously after a person is infected by the H1N1pdm virus, and then, only rarely. But when that happens, the patient appears more likely to experience a more severe illness.

 

As previously reported, It does not appear to transmit efficiently in the wild.

 

The concern here is that viruses can change, and co-mutations could occur that make the D222G mutation more transmissible in the future. 

 

We’ve seen that happen before.

 

In 2006 we saw a smattering of oseltamivir (Tamiflu ®) resistant seasonal H1N1 cases, almost always attributed to `spontaneous mutations’ within a patient receiving the drug.  While of concern to the patient afflicted, it appeared to be poorly transmissible.

 

In the 2006-2007 flu season, laboratories found no resistant strains in Europe or Japan, and in less than 1% of samples from the United States.

 

This resistance was caused by a mutation called H275Y, where a single amino acid substitution (histidine (H) to tyrosine (Y)) occurs at the neuraminidase position 275.

 

(Note: some scientists use 'N2 numbering' (H274Y) and some use 'N1 numbering' (H275Y))

 

The following year, during the 2007-2008 flu season, oseltamivir resistant viruses suddenly took flight, and by the spring of 2008 roughly 25% of European samples tested showed the H275Y mutation (see Increased Tamiflu Resistance In Seasonal Influenza).   

 

By the end of the year, resistant seasonal H1N1 was pretty much the norm around the world.

 

Influenza viruses are both unpredictable and constantly changing. So we watch subtle mutations like the D222G carefully, with the knowledge that the limited threat it poses today (due to its poor transmissibility) may not necessarily hold true tomorrow.

Eurosurveillance: Debating The D222G/N Mutation In H1N1

 

 

 

# 5263

 

 

For well over a year there has been considerable debate among virologists, and other researchers, over the impact of an amino acid substitution seen in a small percentage of 2009 H1N1 samples.

 

The `Norway’ or D222G/N (D225G/N in influenza H3 Numbering) mutation was first linked to more severe disease by Norwegian Scientists in November 2009, although patients carrying these strains can have mild illness as well. 

 

While we’ve covered this territory a number of times over the past year, a brief (and hopefully simple) review is in order. If you are up to speed on receptor binding, and the history of the D222G/N variant, feel free to skip the next section.

 

 

This mutation involves a single amino acid change in the HA1 gene at position 222 from aspartic acid (D) to glycine (G) (or asparagine (N)).

 

The pdmH1N1 virus carrying this mutation appears to bind more readily to receptor cells (α2-3) found deeper in the lungs, whereas unmutated seasonal flu strains bind preferentially to the (α2-6) receptor cells found in the upper airway.

 

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.

(A Very Simplified Illustration of RBDs)

Like a key into a padlock, the RBD must `fit’ in order to open the cell to infection.

 

 

The evidence for the D222G/N  amino acid substitution driving increased virulence has been mixed, with the World Health Organization, the CDC, and the HPA continuing to investigate. 

 

During the first week of January, Eurosurveillance  printed a study looking at fatal and non-fatal cases of influenza in the UK (see Eurosurveillance: Analysis Of Fatal H1N1 Cases In The UK).

 

Ellis et al. reported that almost all of the virus samples tested in fatal and non-fatal cases during the early wave of the 2010/11 influenza season showed aspartic acid (D) at position 222.

 

In other words, no `Norway’ mutation.

Today, Eurosurveillance published a letter from an Italian researcher who has found a high percentage of D222G/N mutations in severely ill patients (43%)  – particularly when taking virus samples from the lower respiratory tract (lungs).

 

You can read the entire letter at the link below.

 

Eurosurveillance, Volume 16, Issue 4, 27 January 2011

Letters

Letter to the editor. Virological analysis of fatal influenza cases in the United Kingdom during the early wave of influenza in winter 2010/11

F Baldanti

 

 

The point being, that if the UK researchers were only taking nasal (or upper respiratory) swabs, they might be missing some D222G/N mutations.

 

In a reply, the authors of the original study concede that in many cases, only upper respiratory swabs were available for this analysis, and that when possible, samples from the lower respiratory system would be useful.

 

 

Eurosurveillance, Volume 16, Issue 4, 27 January 2011

Letters

Authors’ reply. Virological analysis of fatal influenza cases in the United Kingdom during the early wave of influenza in winter 2010/11

J Ellis , M Galiano, R Pebody, A Lackenby, CI Thompson, A Bermingham, E McLean, H Zhao, S Bolotin, O Dar, J M Watson, M Zambon

 

 

This scholarly debate isn’t over, of course. As Ellis et al. state in their reply:

 

The selection and emergence of the D222G mutation as a cause or consequence of more severe lower respiratory tract infection is still to be resolved.

 

Emergence of this mutant is likely to exacerbate severity of disease, but by itself, may be neither necessary nor sufficient to account for a severe disease outcome, which is invariably a balance between virus virulence factors and host immune response capability.

 

It will take more samples, more research, and more time to determine the truth in the matter. 

 

And even if this mutation should eventually be linked to higher virulence, its ultimate impact on public health will ride on how just prevalent this D222G/N is among the H1N1 viruses in circulation.

 

Stay tuned.   There’s a lot left for us to discover.

Eurosurveillance: More On H1N1 Mutations

 

 

 

# 5018

 

 

From Eurosurveillance yesterday, another report on mutations in the novel H1N1 virus that may be associated with increased virulence.

 

The operative word here being `may’.

 

The main thrust of this report is on the rare, so-called `Norway’ mutation  (D222G), but it also looks at a more common mutation; (D222E).

 

The D222G mutation involves a single amino acid change in the HA1 gene at position 222 from aspartic acid (D) to glycine (G).

 

The D222E mutation is the switching to glutamic acid (E) from aspartic acid (D) in the same location.

 

First the link, then some excerpts and discussion.  The entire report is worth reading.

 

 

Molecular surveillance of pandemic influenza A(H1N1) viruses circulating in Italy from May 2009 to February 2010: association between haemagglutinin mutations and clinical outcome

Puzelli S, Facchini M, De Marco MA, Palmieri A, Spagnolo D, Boros S, Corcioli F, Trotta D, Bagnarelli P, Azzi A, Cassone A, Rezza G, Pompa MG, Oleari F, Donatelli I, the Influnet Surveillance Group for Pandemic A(H1N1) 2009 Influenza Virus in Italy.

Euro Surveill. 2010;15(43):pii=19696.

 

A daunting title to be sure, but once you get past that, it gets a bit easier.  Here then is the Abstract:

 

Haemagglutinin sequences of pandemic influenza A(H1N1) viruses circulating in Italy were examined, focusing on amino acid changes at position 222 because of its suggested  pathogenic relevance.

 

Among 169 patients, the D222G substitution was detected in three of 52 (5.8%) severe cases and in one of 117 (0.9%) mild cases, whereas the D222E mutation was more frequent and evenly distributed in mild (31.6%) and severe cases (38.4%).

 

A cluster of D222E viruses among school children confirms reported human-to-human transmission of viruses mutated at amino acid position 222.

 

Influenza viruses are inherently unstable, and are  therefore prone to replication errors.

 

Amino acids can get substituted somewhere in the virus’s protein, and that can affect virulence, receptor binding, sensitivity to antivirals, and transmission.

 

Most mutations go nowhere.

 

They do nothing to enhance the virus, and may actually decrease its ability to replicate, transmit, or compete with other strains.

 

Quite frankly, mutations happen all the time and are often of little consequence.

 

Most are evolutionary dead-ends.

 

But with millions of infected hosts sporting trillions and trillions of (sometimes badly) replicated viruses, this genetic roulette wheel has ample opportunities to test out new and potentially viable amino acid combinations.

 

Mutations can happen spontaneously in an infected host, and only affect that individual.  Or . . .  if the mutated virus is `biological fit’ and easily transmitted – it may move onto other hosts as well. 

 

Complicating matters - viruses can have multiple amino acid changes – and the combination of these changes can unpredictably (at least for now) alter the virus’s behavior. 

 

Thus far, the D222G mutation has only shown up rarely (less than 2% of samples), and serious questions over its ability to transmit easily from human-to-human remain.

 

Recent studies have suggested that it may be linked to more severe pulmonary infections (see D222G And Deep Lung Infections), and that it may have a binding affinity for α2-3 receptor cells found in the lower respiratory system.

 

In this most recent study of 169 patients from Italy, the D222G substitution was found  in 3 of 52 (5.8%) of severe cases and in only  1 of 117 (0.9%) of mild cases.

 

 

While suspiciously more common in severely ill patients, the authors state that these results (and the limited dataset) are too small to conclude that they are statistically significant.

 

It should be noted that the one mild case with this mutation also had a concurrent G155E mutation.  Whether this was a contributing factor to D222G appearing in a mildly ill patient is unknown.

 

The D222E mutation was far more common, and fairly evenly divided between severe cases (38.5%) and mild cases (31.6%).   All of which means that the clinical impact (if any) of this mutation remains unknown.

 

This increased prevalence, along with the detection of a cluster of D222E mutations in a group of mildly ill high school students (described in this paper) suggests some degree of inter-human transmission.

 

The authors wrap up their report with this:

 

Finally, our data suggest that the D222G substitution is overall rather infrequent, even among severe cases. However, we confirm that it occurs with a higher frequency in severe cases.

 

Whether this association is indicative of higher virulence or is the consequence of receptor-specific adaptive mutation needs to be further investigated.

 

All of which means that while we continue to get more and very useful data - we don’t have a lot of answers yet.

 

Stay tuned. 

 

For more on pdmH1N1 mutations, you may wish to revisit:

 

Eurosurveillance On Recently Isolated H1N1 Mutations

Study: Receptor Binding Changes With H1N1 D222G Mutation

WER Review: D222G Mutation In H1N1

D222G And Deep Lung Infections

 

 

 

# 5002

 

 

 

This morning we’ve a joint study from Imperial College London and the University of Marburg that may shed some light on why at least some cases of pandemic H1N1 proved severe (or fatal) while the great majority remained mild.

 

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.

 

Although we’ve covered this territory a number of times over the past year, a brief (and hopefully simple) review is in order. If you are up to speed on receptor binding, and the history of the D222G variant, feel free to skip the next section.

 

 

The D222G mutation had actually been detected months earlier, and in several 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 pdmH1N1 virus carrying this mutation appeared to bind more readily to receptor cells (α2-3) found deep in the lungs, whereas unmutated seasonal flu strains bind preferentially to the (α2-6) receptor cells found in the upper airway.

 

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.

 

 

(A Very Simplified Illustration of RBDs)

 

Like a key into a padlock, the RBD must `fit’ in order to open the cell to infection.

 

For some deeper background you may wish to read Looking For the Sweet Spot, and a follow-up blog called Receptor Binding Domains:Take Two.

 

The World Health Organization’s take on this mutation has been that 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 all may sound like fairly damning evidence, it should be noted that mild cases have been detected with this D222G mutation in other studies, and many severe and fatal cases of pandemic H1N1 that have been examined did not have this mutation.

 

Some recent blogs on this mutation include:

 

Study: Receptor Binding Changes With H1N1 D222G Mutation

Eurosurveillance On Recently Isolated H1N1 Mutations

Referral: Virology Blog On D225G Mutation

 

 

Today’s study, which appears in the Journal of Virology, is called:

 

Altered receptor specificity and cell tropism of D222G haemagglutinin mutants from fatal cases of Pandemic A(H1N1) 2009 influenza

Yan Liu, Robert A. Childs, Tatyana Matrosovich, Stephen Wharton, Angelina S. Palma, Wengang Chai, Rodney Daniels, Victoria Gregory, Jennifer Uhlendorff, Makoto Kiso, Hans-Dieter Klenk, Alan Hay, Ten Feizi*, and Mikhail Matrosovich*

 

 

Admittedly a daunting title, but the abstract is a bit easier to follow.  I’ve re-paragraphed, and added a couple of highlights to it for readability.

 

Abstract

Mutations in the receptor-binding site of the haemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease.

 

Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than viruses with 222D or 222E which targeted mainly non-ciliated cells.

 

Carbohydrate microarray analyses showed that 222G variants bind a broader range of {alpha}2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung.

 

These features of 222G mutants may contribute to exacerbation of disease.

 

 

The discovery that D222G enhances the binding to ciliated cells is important because cilia are motile hair-like protuberances that line the airway and help move mucus (and debris) up and out of the lungs.

 

SEM micrograph of the cilia projecting from respiratory epithelium in the lungs

If you infect (and impair) the lung’s cilia, you (theoretically, at least) increase the odds of that person developing pneumonia.

 

In this study, researchers tested 6 different variants of the pdmH1N1 virus, including 3 (Lvi, Nor, Ham-E) with the D222G mutation.  

 

The `money quote’ from the study is:

 

The viruses with  222D  (Mol and Ham) and 222E  (Dak) showed a pattern of cell tropism typical of seasonal influenza A and B viruses  infecting predominantly non-ciliated cells known to be rich in α2-6 Sia sequence: less than 5% of infected cells were ciliated.

 

By contrast, the three viruses with 222G, Lvi, Nor and Ham-e, infected both ciliated and non-ciliated cells, and  20% or more of infected cells were ciliated, known to express α2-3 Sia sequences.

 

This change in the cell tropism, with a 5-10 fold increase in infection of ciliated cells, thus correlated with the presence of the D222G substitution in the HA, and other amino acid  differences, in particular D222E, had little or no effect.

 

 

Where then, does all of this leave us?

Well, the authors state that:

 

Whether the selection of the D222G mutation is a cause or a consequence of more severe lower respiratory tract infection has still to be resolved. It is evident, however, that its emergence is likely to exacerbate the severity of disease.

 

Luckily, this mutation has been slow to spread. 

 

It has been detected in less than 2% of the samples tested, and that suggests that (right now, anyway) it may be less fit for transmission than other competing strains.  

 

The fact that it tends to promote deep lung infections, and reduces the ability to expel mucus (and therefore cough productively), may help inhibit its spread.

 

A scenario not unlike what we’ve seen with the H5N1 (bird flu) virus, which as an avian virus, binds even more preferentially to α2-3 receptor cells. 

 

What is true today, however, may not hold true tomorrow. Influenza viruses are capable of swift and sometimes dramatic mutations. 

 

This research shows that even a seemingly mild strain of influenza can easily pick up virulence, and if it can retain transmissibility, could spark a serious public health hazard.

 

Which is why continued influenza research, the monitoring of this and other influenza strains, and the maintaining of pandemic readiness remain vital even after the pandemic of 2009 has passed.

Study: Receptor Binding Changes With H1N1 D222G Mutation

 

 

 

# 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

Dissecting the Influenza Pathogenesis Study Pt. 2

Dissecting the Influenza Pathogenesis Study Pt. 3

Referral: Virology Blog On D225G Mutation

 

 

# 4447

 

 

There has been a lot of speculation regarding the D225G mutation first announced in Norway last November, suggesting that it may increase the virulence of the novel H1N1 virus.

 

This mutation, sometimes called D222G (or D225G in influenza H3 numbering) had actually been detected months earlier, and in many other countries.  Norway was simply the first country to announce a possible connection 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.

 

Not being a virologist (or scientist of any flavor), I don’t have a pathogen in this fight. Therefore, I’m not offering any theories of my own.

 

The best I can do is offer up the opinions of scientists and agencies that I view as credible (admittedly a subjective evaluation), along with the caveat that we are still learning about such things.

 

Nothing is writ in stone.

 

That said, today I’ll refer you to Vincent Racaniello’s excellent Virology Blog, where he takes us on a tour of some of the possibilities that surround this D225G mutation.  

 

A bit technical at times, granted.  But well worth reading, even if you don’t get every reference.   

 

The upshot here being that the interaction between pathogen and host is very complex, incompletely understood, and that simplistic models are likely to fall short of the mark.

 

Read Vincent Racaniello’s:

 

The D225G change in 2009 H1N1 influenza virus

CIDRAP Report On The H1N1 Mutation Debate

 

 

# 4506

 

 

If you’ve followed the H1N1 story much over the past six months, you are no doubt aware of the concerns over the so called `Norwegian mutation’ – a single amino acid change in the HA1 gene at position 222 (225 in H3 numbering) from aspartic acid (D) to glycine (G).

 

While this mutation had been seen before and in other countries, it was in Norway last November that scientists announced a suspected link between this mutation and the virulence of the pandemic virus (see Norway Reports An H1N1 Mutation).

 

Since then we’ve seen conflicting evidence and a good deal of debate regarding the significance this mutation. 

 

In a  World Health Organization’s recent (No. 4, 2010, 85, 21–28) Weekly Epidemiological Record (WER), we get an update on this mutation. 

 

While noting that it is worthy of further study (along with other mutations to the virus), for now the WHO’s position is that `Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.’

 

That is not to say that the book has been closed on this mutation. Certainly not.  Only that – at this point in time – WHO scientists find insufficient evidence to link this mutation to greater virulence in the H1N1 virus.

 

Once again, scientists from Norway have announced new findings that they claim suggest a link between the severity of the virus and this mutation.  Not all scientists share their in their conclusions, however.

 

Robert Roos from CIDRAP news sorts all of this out for us (as best as can be done at this stage, anyway), with a terrific piece on both sides of this growing debate.

 

 

H1N1 mutation's proposed link to severe illness debated

Robert Roos News Editor

Mar 4, 2010 (CIDRAP News) – Norwegian scientists today reported a pandemic H1N1 virus mutation that appears to be associated with severe disease, but a leading US flu expert said global data on the mutation don't show a clear connection with severe illness.

 

A team from the Norwegian Institute of Public Health in Oslo reports that it found the mutation in 11 of 61 severe illness cases that were analyzed between July and December 2009. The mutation was not found in any of 205 mild cases that were analyzed between May 2009 and January 2010.

 

"This difference is statistically significant and our data are consistent with a possible relationship between this mutation and the clinical outcome," says the report by A. Kilander and colleagues, published today in Eurosurveillance. "To our knowledge, this is the first identification of a change in the pandemic virus that correlates with a severe clinical outcome."

 

However, Dr. Nancy Cox, director of the Influenza Division at the US Centers for Disease Control and Prevention (CDC), said global H1N1 data so far do not show a clear association between the mutation and severe illness.

 

"If you look globally you can see that this mutation is neither necessary nor sufficient for a severe or fatal outcome," Cox told CIDRAP News.

(Continue . . . )

WER Review: D222G Mutation In H1N1

 

# 4280

 

 

It seems that every month or so we are confronted with a new aspect of the H1N1 (or H5N1) virus that generates a certain amount of consternation in the blogosphere.  

 

In very early November it was the Ukraine story, which had morphed into wild tales of Russian bio-weapons, `pneumonic plague’, and a UN cover up of massive numbers of fatalities.

 

On November 14th, I wrote of this spectacularly unbridled speculation In Ukraine And The Internet Rumor Mill.

 

Scarcely a week later we were hit by two stories, one revolving around an announced mutation in the pandemic H1N1 virus by scientists in Norway, and another concerning a pair of Tamiflu resistant clusters of novel H1N1.

 

Both have launched their fair share of media reports, and no lack of speculation.  Of the two, the `Norway Mutation’ has captured perhaps the most attention.

 

While announced by Norwegian scientists (see Norway Reports An H1N1 Mutation) , this mutation D222G (or D225G in influenza H3 numbering) had actually been detected months earlier, and in many other countries.   

 

Norway was simply the first country to announce a possible connection 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).

 

 

Over the past couple of months, much has been made over the potential dangers of this mutation – mostly by those in the `new media’.   Little direct evidence of this mutation posing a public health threat has emerged over that time, however.

 

In the The World Health Organization’s latest (No. 4, 2010, 85, 21–28) Weekly Epidemiological Record (WER), we get an update on this mutation. 

 

To my loyal reader in France, who emailed me this link overnight: Merci beaucoup, Anne.

 

While noting that it is worthy of further study (along with other mutations to the virus), for now the WHO’s position is that `Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.’

That is not to say that the book has been closed on this mutation. Certainly not.  Only that – at this point in time – WHO scientists find insufficient evidence to link this mutation to greater virulence in the H1N1 virus.

 

Will D222G prove to be a public health concern down the road?

 

Perhaps.

 

But it may well require other mutations in the virus to occur - in lieu of, or in concert with D222G - to produce a `Frankenswine’ virus.

 

And those changes may never happen.  Or we could start seeing them tomorrow . . .

 

Influenza is (quite literally) an evolving story.

 

Stay tuned.

 

 

Preliminary review of D222G amino acid substitution in the haemagglutinin of pandemic influenza A (H1N1) 2009 viruses

Summary

Since the first appearance of pandemic influenza A (H1N1) 2009 viruses, certain mutations, including those leading to the D222G substitution in the haemagglutinin (HA) protein and the K340N substitution in the polymerase basic protein 2 (PB2), have appeared sporadically. These substitutions in HA and/or PB2 have been reported in viruses obtained from cases of mild to severe to fatal illness but such viruses have neither formed distinct phylogenetic clusterings nor been associated with consistent changes in virus antigenicity.

 

Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue. However, the WHO Global Influenza Surveillance Network (GISN) and its partners will continue to closely monitor pandemic viruses for the D222G and other amino acid substitutions and continually assess  associated risks.

 

Background

Influenza viruses are known for their high evolutionary rate and tendency to acquire point mutations at different positions in their genomes. Some mutations can result in amino acid  substitutions at key locations in proteins, such as antigenic sites or the receptor binding site of the HA, and can alter properties such as those associated with the virus antigenicity or  pathogenecity.

 

Recently, the D222G substitution was observed in the HA of pandemic (H1N1) 2009 viruses isolated from fatal cases in several countries. WHO organized a global  teleconference with experts from GISN laboratories, external research institutions and WHO regional offices to assess the public health significance. This review is based on those data and other information provided by GISN laboratories.

 

Detection of D222G substitution by GISN

The D222G substitution has been detected in virus isolates from around 20 countries, areas and territories in the Americas, Asia, Europe and Oceania. These changes have been  found since April 2009 but have not been associated with temporal or geographical clustering, strongly suggesting the mutation in these viruses has occurred sporadically as  opposed to the emergence and sustained transmission of a variant virus.

Based on currently available data shared with WHO, the prevalence of D222G substitution is <1.8% (52 detections among >2755 HA sequences). Of 364 fatal cases analysed to date, viruses from 26 cases (7.1%) had the D222G substitution.

 

The clinical information about potential underlying medical conditions in these cases is limited. Surveillance and laboratory analysis efforts to study this substitution have given priority to specimens from hospitalized and severely ill patients, leading to potential biases in the data. Additionally, a study done by the WHO Collaborating Centre for Reference and Research on Influenza (WHOCC) in Atlanta – located in the United States Centers for Disease Control and Prevention (CDC) – found the D222G substitution in 14 virus isolates but not in viruses in the original clinical specimens indicating the D222G substitution in these 14 virus isolates occurred after growth in the laboratory.

 

These observations have made determining the clinical relevance of this substitution difficult. Otherwise, the pandemic (H1N1) 2009 viruses with D222G substitution have been antigenically similar to the A/California/7/2009 (H1N1) virus, the WHO-recommended vaccine virus. Three of the D222G variant viruses carry the H275Y substitution in the neuraminidase (NA) associated with oseltamivir resistance.

 

Other substitutions of potential public health significance WHO has been monitoring several other reported substitutions in the HA (D222E and D222N) and K340N substitution in PB2. The clinical significance of these substitutions remains uncertain.

 

Ongoing studies

Preliminary results from in vitro studies suggest that D222G substitution in the HA may increase binding to a2-3 sialic acid (avian-like) cell receptors. Ferret studies have shown that viruses with D222G substitution, whose virulence is similar to wild-type viruses lacking this mutation, can be transmitted efficiently. Studies using mice and guinea-pigs are ongoing to better characterize the receptor-binding specificity, replication fitness, transmissibility and pathogenicity of viruses with this D222G substitution alone or in combination with other substitutions.

 

More detailed information on clinical, epidemiological and viral features is needed to assess the future public health significance of these viruses.

 

 

Since I’m not a scientist, I have no theory in this fight.   All I can do is review the literature and listen to those experts that I have faith in (admittedly, a purely subjective criteria on my part), and report what they currently believe.  

 

Conducting good science takes time. 

 

What scientists believe today may change tomorrow.  In fact, you can almost count on it.

 

We call that progress.

 

This proves somewhat problematic for the media (both new & traditional), however.  They would much prefer that all science be complete, definitive, and wrapped up with a tidy bow instead of riddled with caveats.  

 

Like Harry Truman - who once said what he wanted most as President was a one-armed economist -  I’m sure we all wish for the day when scientists couldn’t follow every statement with:

 

“But . . . on the other hand . . . “

 

I just wouldn’t hold my breath.