Announcement

**tetano** · July 16, 2013, 11:57 AM

Re: mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

H7N9 Influenza Strain Resistant to Antivirals, but Tests Fail to Identify Resistance

July 16, 2013 ? Some strains of the avian H7N9 influenza that emerged in China this year have developed resistance to the only antiviral drugs available to treat the infection, but testing for antiviral resistance can give misleading results, helping hasten the spread of resistant strains.

The authors of a study published in mBio?, the online open-access journal of the American Society for Microbiology, characterized viruses taken from the first person known to be stricken with H7N9 influenza and found that 35% of those viruses are resistant to oseltamivir (commercially known as Tamiflu) and zanamivir (Relenza), front line drugs used for treating H7N9 infections. However, lab testing of the viruses, which detects the activity of a viral enzyme, fails to detect that these strains are resistant, so monitoring for the development of resistance using this technique would prove futile.

"If H7N9 does acquire human-to-human transmissibility, what do we have to treat it with until we have a vaccine? Oseltamivir. We would be in big trouble," says corresponding author Robert Webster of St. Jude Children's Research Hospital in Memphis, Tennessee. Resistant strains of H7N9 can flourish in patients treated with oseltamivir or zanamivir, he says, inadvertently leading to the spread of resistant infections.

...

http://www.sciencedaily.com/releases/2013/07/130716080024.htm

**sharon sanders** · July 16, 2013, 12:28 PM

Re: mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

mBio: Antiviral Resistance In H7N9

# 7485

Early reports of H7N9 out of China have indicated a small, but worrisome number of oseltamivir (Tamiflu ?) resistant strains detected among those infected during the first wave (see The Lancet: Antiviral Resistance In Two H7N9 Patients).

In most cases, patients were believed to have developed resistance after being placed on the antiviral medication.

What is commonly called `spontaneous resistance?.

The Lancet study found that a mutation R292K (Arginineto Lysine at position 292 in the NA) ? also known as Arg292Lys ? already known to confer antiviral resistance to seasonal flu (see Resistant influenza A viruses in children treated with oseltamivir: descriptive study), appeared in two patients after several days of oseltamivir therapy.

Today we?ve a new study appearing in the open access journal mBio that finds that the standard laboratory tests for antiviral susceptibility can miss `mixed population? infections (comprised of both resistant and susceptible strains), and that antiviral treatment could suppress the susceptible strains while allowing resistant strains to flourish.

And this particular mutation can provide resistance not only to oseltamivir, but to to zanamivir and peramivir as well.

The corresponding author of the study is Dr. Robert Webster of St. Jude Children?s Hospital, considered the dean of influenza virology. In a press releasefrom the American Society for Microbiology, we get a summary of what this study discovered.

In the mBio study, the authors tested antiviral susceptibility of an H7N9 strain isolated from the first confirmed human case of avian H7N9 influenza using a method that tests the activity of the neuraminidase enzyme. The reassuring results were, unfortunately, misleading: the enzyme-based test indicated that the flu strain was susceptible to NA inhibiting antiviral drugs, but it is not.

A closer look at the viral isolate revealed it is actually made up of two distinct types of H7N9 viruses. Roughly 35% of the viruses carry the R292K mutation, making them resistant to NA inhibitors, and 65% are sensitive to these same drugs. The enzyme-based testing gave misleading results, says Webster, because the functioning wild-type enzymes masked the presence of the non-functioning mutant enzymes.

Using NA inhibitors to treat a patient infected with a resistant strain of H7N9 only encourages the virus to proliferate and can lead to enhanced spread of the resistant strain. The authors write that these results prove that it is crucial to use a gene-based surveillance technique that can detect these resistant influenza strains in a mixed infection.
<SNIP>

But the news isn't all bad. Webster also points out that antiviral resistance is something of a burden for influenza viruses, and that fitter wild-type H7N9 strains may eventually win out over resistant strains. In the absence of a drug like Tamiflu, Webster says, it seems unlikely that these resistant viruses would acquire epidemic characteristics.
<SNIP>

"The great need at the moment are additional drugs aimed at additional sites in the influenza genome. There are some [drugs] in the pipeline, but they are still under testing at the moment," says Webster. "We'd better get some vaccine seed stocks up and ready. The antiviral option for controlling H7N9 isn't too good."
(Continue . . . )

Here is a link to the mBio study, after which I?ll be back with a cautionary tale about our previous experiences with evolving antiviral resistance and seasonal flu.

Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population
H.-L. Yen, J. L. McKimm-Breschkin, K.-T. Choy, D. D. Y. Wong, P. P. H. Cheung, J. Zhou, I. H. Ng, H. Zhu, R. J. Webby, Y. Guan, R. G. Webster and J. S. M. Peiris
doi:10.1128/mBio.00396-13
(EXCERPT)

Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay.

IMPORTANCE The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants.
We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir.
Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.
(Full Text . . .)

While it is true that resistant strains of influenza A tend to be less transmissible than their susceptible `wild type? counterparts, we have some examples where resistant strains have been `fit? enough spread globally.

By 2005, nearly all of the influenza samples around the globe had developed resistant to the older M2 ion channel blockers like Amantadine, leaving oseltamivir and zanamivir (neuraminidase inhibitors) as the two remaining treatments.

In 2006 we saw a smattering of oseltamivir resistant seasonal H1N1 cases, almost always attributed to `spontaneous mutations? within a patient receiving the drug. While of concern to the patient being treated, 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 in this viral strain was mostly 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 December of 2008, the CDC was forced to issue major new guidance for the use of antivirals for the second time in just three years (see CIDRAP article With H1N1 resistance, CDC changes advice on flu drugs).

The `replacement? 2009 pandemic virus, that supplanted the largely resistant seasonal strain the following spring, was fortunately sensitive to Tamiflu. While we?ve seen some scattered cases of resistance in the new H1N1 strain, so far they are relatively few, and highly scattered (see Antiviral Resistance In 2009 H1N1 Influenza A Strain).

Granted, H7N9 is a different influenza A strain, and it carries a different mutation than the old H1N1 virus, but this bit of history shows how quickly our pharmaceutical options can evaporate.

The concern with today?s study is the use of antivirals on patients who have both susceptible and resistant viruses could help promote the replication and spread of resistant strains.

Which could inadvertently help drive the evolution of the H7N9 virus towards a predominantly resistant strain.

Posted by Michael Coston at <a class="timestamp-link" href="http://afludiary.blogspot.ca/2013/07/mbio-antiviral-resistance-in-h7n9.html" rel="bookmark" title="permanent link"><abbr class="published" title="2013-07-16T11:50:00-04:00">11:50 AM</abbr>

**Giuseppe** · July 16, 2013, 01:57 PM

Re: mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

Despite the concerning results of the above mentioned study, it is Worth to note that the WHO stated since the beginning of the H7N9 crisis that at least in some cases, the virus possessed the R292K mutation conferring neuraminidase inhibitors resistance. However the position of the Agency in relation to antiviral treatment of suspected, probable and confirmed H7N9 human cases is the following:

[Source: World Health Organization, full PDF document: http://www.who.int/influenza/human_a...ew_31May13.pdf - Excerpt.]

Overview of the emergence and characteristics of the avian influenza A(H7N9) virus, 31 May 2013

(...)

7. Antiviral therapy

Based on the sequence of the M2 protein, H7N9 viruses are predicted to be resistant to adamantane antiviral drugs (Gao et al. 2013) which are therefore not recommended for use.

In accord with the NA (neuraminidase) sequencing data, testing of the A/Anhui/1/2013 virus in the neuraminidase inhibition assay indicates that this virus is susceptible to neuraminidase inhibitor antiviral drugs oseltamivir and zanamivir (CDC 2013b) (Tables 3a and 3b).

The arginine (R) to lysine (K) substitution at residue 292 (N2 numbering), which is likely to diminish efficacy of oseltamivir and zanamivir (McKimm-Breschkin 2013, Gubareva et al. 1997) (Tables 3a and 3b), was detected initially in the A/Shanghai/1/2013 virus (Gao et al. 2013).

However, testing of A/Shanghai/1/2013 virus in the neuraminidase inhibition assay generated discrepant results, which may be attributed to a mixture of R and K at 292 residue of the virus (Table 3b).

The clinical specimen containing Shanghai/1/2013 was collected two days after commencement of oseltamivir therapy (Gao et al. 2013).

The previously mentioned study by Hu et al (2013) on the hospitalised pneumonia patients found that reduction of viral load following antiviral treatment correlated with improved outcome.

The R292K mutants were detected from two of the three poor responders to the neuraminidase inhibitor (NAI) antiviral therapy with persistently high viral load in the throat.

In one of the two patients the NA had 292R on day 2 of antiviral therapy and 292K on day 9 suggesting selection of the resistant virus to dominate the infection.

While no data are available regarding early inhibitor treatment of persons infected with H7N9 virus, the potential severity of H7N9-associated illness warrants recommending that all confirmed cases, probable cases, and H7N9 cases under investigation receive antiviral treatment with a neuraminidase inhibitor drug as early as possible.

(...)

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**Giuseppe** · July 17, 2013, 01:33 AM

Re: mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

The extremely limited medical arsenal against influenza should suggests further research for a better understanding of already marketed drugs options for treatment as suggested by this paper by David Fedson:

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Antiviral Res. 2013 Jul 3. pii: S0166-3542(13)00184-8. doi: 10.1016/j.antiviral.2013.06.018. [Epub ahead of print]

Treating Influenza with Statins and Other Immunomodulatory Agents.

Fedson DS.

Source: 57, chemin du Lavoir, 01630 Sergy Haut, France. Electronic address: dfedson@wanadoo.fr.

Abstract

Statins not only reduce levels of LDL-cholesterol, they counteract the inflammatory changes associated with acute coronary syndrome and improve survival. Similarly, in patients hospitalized with laboratory-confirmed seasonal influenza, statin treatment is associated with a 41% reduction in 30-day mortality. Most patients of any age who are at increased risk of influenza mortality have chronic low-grade inflammation characteristic of metabolic syndrome. Moreover, differences in the immune responses of children and adults seem responsible for the low mortality in children and high mortality in adults seen in the 1918 influenza pandemic and in other acute infectious and non-infectious conditions. These differences probably reflect human evolutionary development. Thus the host response to influenza seems to be the major determinant of outcome. Outpatient statins are associated with reductions in hospitalizations and deaths due to sepsis and pneumonia. Inpatient statins are also associated with reductions in short-term pneumonia mortality. Other immunomodulatory agents - ACE inhibitors (ACEIs), angiotensin receptor blockers (ARBs), PPARγ and PPARα agonists (glitazones and fibrates) and AMPK agonists (metformin) - also reduce mortality in patients with pneumonia (ACEIs, ARBs) or in mouse models of influenza (PPARγ and AMPK agonists). In experimental studies, treatment has not increased virus replication. Thus effective management of influenza may not always require targeting the virus with vaccines or antiviral agents. Clinical investigators, not systems biologists, have been the first to suggest that immunomodulatory agents might be used to treat influenza patients, but randomized controlled trials will be needed to provide convincing evidence that they work. To guide the choice of which agent(s) to study, we need new types of laboratory research in animal models and clinical and epidemiological research in patients with critical illness. These studies will have crucial implications for global public health. During the 2009 H1N1 influenza pandemic, timely and affordable supplies of vaccines and antiviral agents were unavailable to more than 90% of the world's people. In contrast, statins and other immunomodulatory agents are currently produced as inexpensive generics, global supplies are huge, and they would be available to treat patients in any country with a basic health care system on the first pandemic day. Treatment with statins and other immunomodulatory agents represents a new approach to reducing mortality caused by seasonal and pandemic influenza. This article forms part of a symposium in Antiviral Research on ''Treatment of influenza: targeting the virus or the host.''

Copyright ? 2013 Elsevier B.V. All rights reserved.

KEYWORDS:

Immunomodulatory agents, Influenza, Metabolic syndrome, Public health, Statins

PMID: 23831494 [PubMed - as supplied by publisher]

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Announcement

mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

mBio. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

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