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Antiviral resistance markers in influenza virus sequences in Mexico, 2000–2017

Background Influenza causes high rates of morbidity and mortality. Genetic variability of influenza viruses generates resistance to antivirals, which are of two types, since they act on two different viral targets: adamantanes, which block the M2 ion channel, and the neuraminidase (NA) inhibitors. Methods In Mexico, the available studies on the antiviral resistance of circulating influenza strains are scarce, so this work undertook an analysis of the Mexican sequences reported in public gene banks to perform a systematic analysis of the antiviral resistance markers on both M2 and NA. In all, 284 M2 sequences and 423 NA sequences were retrieved from three genetic databases (sequences from 2000 to 2017 were considered). Results The resistance markers to M2 blockers were present in 100% of H1N1 pdm2009, 83.6% of H3N2, and 5.8% of seasonal H1N1 sequences. Two resistance markers conferring resistance to NA inhibitors were present in seasonal H1N1 sequences, H275Y (50.0%) and N70S (33.3%). None of these viruses had both resistance markers, which are associated with oseltamivir resistance. The more frequent resistance marker in H1N1 pdm2009 NA sequences was H275Y, present in 3.6%, while S247N was present in 0.30%. Only one of the resistance-associated markers (Q136K) in NA (1.5%) was present in the analyzed H3N2 sequences, while sequences of influenza B virus did not present resistance markers to NA inhibitors. Some influenza A H1N1 pdm2009 sequences (1.8%) presented resistance markers to both M2 and NA. Conclusion Based on the present analysis, 7.1% of the all serotypes of influenza virus A sequences analyzed in Mexico from 2000 to 2017 have mutations conferring resistance to NA inhibitors. Because of this, and the limited availability of influenza drugs, it is necessary to increase the epidemiological surveillance, including molecular analysis, which will provide data such as the presence of changes associated with antiviral resistance.


Introduction
Influenza viruses belong to the family Orthomyxoviridae and are classified into three types: A, B, and C. 1 Influenza A and B viruses are associated with high seasonal morbidity and mortality; however, influenza A is a special case because it occasionally causes pandemics and has more rapid evolution than types B and C. 2,3 Influenza A and B viruses cause epidemics each year, associated with a variable number of deaths, absenteeism, hospitalization, and drug costs, as well as other public and private expenditures. 4 To counteract influenza, vaccines and antivirals have been developed; however, in any of the cases, the protection and/or therapeutic capacity are not enough to eradicate or reduce, to the desired degree, the effects of infection in all patients. The failure of vaccines and therapies is largely due to the genetic changes in influenza viruses, which are reflected in phenotypic variations that allow it to escape the host immune response, induce drug resistance, and/or cause pathogenic changes. 5,6 In this regard, surveillance of anti-influenza drug resistance has great global relevance. There are two classes of drugs worldwide approved for treatment of illness caused by influenza virus, M2 ion channel blockers (such as amantadine and rimantadine) and neuraminidase (NA) inhibitors (such as zanamivir, oseltamivir, and peramivir). 7 In recent years, most of the circulating viruses are resistant to adamantanes; therefore, they are not recommended for antiviral treatment. Hence, only NA inhibitors remain as the indicated anti-influenza drugs against the currently circulating viruses. 7,8 In Mexico, the available studies on the drug resistance of circulating influenza strains are scarce, so this study undertook an analysis of sequences reported in public gene banks to perform a systematic analysis of the resistance molecular markers on both M2 and NA proteins.

Methods
Sequences of NA and M2 from influenza A and NA from influenza B viruses were retrieved from Influenza Research Database (www.fludb.org), Influenza Virus Database (https:// www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select. cgi?go=database), and GISAID EpiFlu (http://platform.gisaid. org/epi3/frontend). The inclusion criteria of the study were sequences of human influenza viruses reported from Mexico of any year, any subtype, or lineage, regardless of whether they are complete or partial. Sequences from 2000 to 2017 were considered. The redundant sequences and all sequences that did not contain the antiviral resistance positions were discarded from the analysis of those positions. Results were reported as sequences containing the specific marker with respect to the number of sequences analyzed on the related amino acid position. The amino acid positions related to resistance to M2 and NA inhibitors considered in the present study are summarized in Tables 1 and 2, respectively. The selected sequences were aligned using ClustalW Multiple Sequence Alignment tool (Clustal, Dublin, Ireland) in BioEdit v. 7.2.5 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). 9

Overview of retrieved sequences
After discarding the redundant entries from gene bank entries, we undertook the study with 284 M2 and 423 NA sequences. All these sequences were subclassified as complete or partial,

Resistance to M2 blockers
The analyzed sequences showed a high frequency of the molecular marker S31N, which is associated with resistance to M2 blockers. The most frequent codon for N31 resistance marker in H1N1 pdm09 and H3N2 viruses was AAU, which is present in 100% sequences bearing the resistance marker.

Resistance to na inhibitors
The analysis of amino acid substitutions in NA related to resistance included those markers to well-known drugs Abbreviations: na, neuraminidase; s, sensible; ↓s, decreased sensitivity; R, resistant; ↓R, decreased resistance; ↑R, increased resistance. oseltamivir and zanamivir and the recently approved drugs peramivir and laninamivir (Table 2). Nine amino acid substitutions were analyzed in the H1N1 pdm2009 sequences, and only two changes were present in some of them. The most frequent was H275Y, present in 12/328 (3.6%), while S247N was present in 1/329 (0.30%). These changes and the rest of resistance markers  Table 4. All viruses carrying the marker H275Y had the codon UAC (12/12), while the other viruses had CAC (316/316). The only one virus carrying the marker S247N presented the codon AAU, and the other viruses had AGU (328/328). None of these viruses had both resistance markers, which are associated with oseltamivir resistance. The S247N change is also associated with zanamivir resistance and was the only sequence detected for this drug. No sequence had amino acids changes associated with peramivir and laninamivir resistance.
The analyses of NA resistance markers in H3N2 viruses included eleven positions ( Table 2). Only one of the resistance-associated genotypic markers (Q136K) was present in the 70 (1.5%) analyzed N2 sequences. In the case of influenza B virus, none of the considered eight NA resistance markers was present in the three analyzed sequences. The overall rate of NA resistance markers, including all subtypes of influenza A viruses, was 7.1% (30/423).

combined resistance
Some of viruses presented two or more resistance markers. H1N1 pdm2009 viruses with combined M2 and NA sequences (171) show that all of them were resistant to adamantanes and three (1.8%) were also resistant to oseltamivir.
Seasonal H1N1 and H3N2 viruses had 12 and 61 available sequences to analyze both genes, but none of them had double resistance, neither seasonal H1N1 nor H3N2 had resistance markers to zanamivir.

Discussion
Although vaccination as a form of prevention for influenza is widespread and relatively successful, there is still a high frequency of cases of influenza, which can lead to high severity and present a significant lethality. The NA inhibitors began to be used as specific drugs to reduce viral replication between 1999 and 2000 and have been widely recommended for treatment, especially in severe infections. However, the resistance presented by influenza viruses is significant and represents a global health concern. 10  h275Y  328  12  316  3.6  1  n294s  293  0  293  0  36  I222V  295  0  295  0  34  I222R  295  0  295  0  34  e119g  229  0  229  0  100  e119V  229  0  229  0  100  n325K  292  0  292  0  37  s247n  329  1  328  0.30  0  I117V  229  0  229  0  100 Note: a some of the incomplete sequences did not contain all the sites where the resistance markers are located. Abbreviations: na, neuraminidase; RM, resistance marker. Currently, the NA inhibitors are the antiviral of choice for influenza treatment, although WHO estimates that 2% of circulating strains of influenza virus are resistant to these inhibitors, due to certain characteristic mutations causing of antiviral effectivity of drugs. The predominant resistanceassociated change is H275Y (N1 numbering). 11 Based on the present analysis, 7.1% of the sequences analyzed in Mexico have mutations conferring resistance to NA inhibitors (oseltamivir and zanamivir).
In Mexico, there are few studies reporting oseltamivir resistance in influenza viruses. These reports present values ranging from 0% to 0.33%. [12][13][14][15] The percentage of resistant strains may fluctuate depending on the country and geographical area: in USA, it has been reported at 1.2%, 16 in Brazil 1.4%, 17 in Argentina 0.1%, 18 and in Australia and Japan 18%. 19,20 In the sequences analyzed in this work, the predominant mutation was H275Y in both the H1N1 pdm09 and seasonal H1N1 subtypes, while the H3N2 subtype did not present this mutation (H274Y), and its overall resistance proportion was lower than that of the other two influenza A viruses.
There is a gradual increase in circulating resistant strains worldwide since before 2008: the percentage of resistant strains was <1% 21 and is currently 2% at global level. However, in some countries, high rates, >60%, have been reported. 22 In the present analysis, the sequences of the H1N1 pdm09 during the period 2009-2012 present a higher frequency of H275Y, which could suggest that this mutation is being adapted, maintained, and disseminated in this subtype in Mexico. However, after 2012, none of analyzed NA sequences presented this resistance marker; therefore, it is difficult to predict the behavior of resistance in influenza viruses.
Because of this, and the limited availability of influenza drugs, it is necessary to increase the epidemiological surveillance, including molecular analysis, which will provide data such as the presence of changes associated with antiviral resistance, but can offer other phenotypic-associated features related to sequence changes, such as specificity for receptors, relationship with circulating strains, and antigenicity.

Conclusion
The clinical implications of resistance to antivirals should not be taken lightly. Based on the present analysis, 7.1% of circulating influenza viruses from 2000 to 2017 in Mexico have mutations that confer resistance to NA inhibitors. These resistant viruses increased their presence as of 2009; among them, H275Y (or H274Y) has been maintained and disseminated by different influenza virus subtypes around the world. This, coupled with therapeutic limitations against infection, forces us to establish better epidemiological surveillance systems that include the search for mutations that confer resistance to NA inhibitors, among other biological characteristics such as replication and transmissibility.