Rapid spread of influenza A(H1N1)pdm09 viruses with a new set of specific mutations in the internal genes in the beginning of 2015/2016 epidemic season in Moscow and Saint Petersburg (Russian Federation)

A dramatic increase of influenza activity in Russia since week 3 of 2016 significantly differs from previous seasons in terms of the incidence of influenza and acute respiratory infection (ARI) and in number of lethal cases. We performed antigenic analysis of 108 and whole‐genome sequencing of 77 influenza A(H1N1)pdm09 viruses from Moscow and Saint Petersburg. Most of the viruses were antigenically related to the vaccine strain. Whole‐genome analysis revealed a composition of specific mutations in the internal genes (D2E and M83I in NEP, E125D in NS1, M105T in NP, Q208K in M1, and N204S in PA‐X) that probably emerged before the beginning of 2015/2016 epidemic season.


Introduction
Influenza viruses are constantly evolving posing a significant threat for public health, therefore constant monitoring of genetic and antigenic properties is required for detection of newly emerging strains and annual revision of influenza vaccine composition. WHO-recognized National Influenza Centers (NIC) in Moscow and Saint Petersburg collect and analyze information on the influenza activity in Russian Federation and perform antigenic and genetic analysis of circulating strains.
2015/2016 epidemic season in Russia is characterized by rapid increase of influenza and acute respiratory infection (ARI) morbidity with exceeding the epidemic thresholds in all federal districts in a short time period ( Figure 1A).
An increase of influenza activity was registered from week 3 of 2016 (11-17Á01Á2016). Increases in influenza-like illness (ILI) and ARI incidence and hospitalization rates were registered up to week 5 when the epidemic peak was reached. These indicators were similar to those observed during the 2009 pandemic and epidemic in 2010-2011 and were much higher than in other seasons ( Figure 1B).
The number of cases with rRT-PCR confirmed influenza A (H1N1)pdm09 increased from 28Á5% in week 3 to 37Á2% in week 5 of 2016. Influenza A(H3N2) and influenza B cases were detected sporadically ( Figure 1C).

Antigenic analysis of influenza A(H1N1) pdm09 viruses
Specimens used for virus isolation and sequencing were collected from 25Á11Á2015 (when the first PCR-positive clinical samples were obtained) to 18Á01Á2016.
About 108 strains were characterized antigenically in both WHO NICs in Moscow and Saint Petersburg using WHO diagnostic kit and ferret and rat polyclonal antisera to the reference strains 1 . Most of the tested viruses reacted within two-to fourfold difference with antisera raised against the A/ California/07/2009 virus.
Detailed antigenic analysis was performed for strains isolated in the NIC in Saint Petersburg (see Table 1). The hemagglutination inhibition (HI) test showed that all tested viruses were antigenically closely related to the vaccine strain as well as with the reference strains such as A/Hong Kong/ 5659/12 (cell-derived) and A/Bolivia/559/13 (egg-derived). They also reacted within two-to fourfold difference with antisera raised against contemporary reference influenza A (H1N1)pdm09 viruses isolated in Russia in 2009-2014. These results clearly demonstrate that most of the A(H1N1) pdm09 viruses isolated in the beginning of the epidemic season in Russia are antigenically A/California/07/2009-like, for example, similar to the vaccine strain.
Antigenic cartography was used for the analysis and visualization of the antigenic data 2 . The antigenic map (see Figure 2) based on the HI test demonstrates that the antigenic distance between the vaccine strain and the strains tested does not exceed the fourfold difference in homologous titer.

Genetic characterization of influenza A (H1N1)pdm09 viruses
Full-genome amplification of influenza A(H1N1)pdm09 viruses from 55 isolated strains (41 from Moscow, 13 from Saint Petersburg, 1 from Tomsk) and 22 clinical specimens (including 4 autopsy specimens) was performed according to Zhou et al. 3 Nextera XT sample preparation was used to obtain libraries for next-generation sequencing; full-length genome sequences were obtained using Illumina MiSeq. All sequences were submitted to Epiflu GISAID database (isolates Acc. Nos. EPI_ISL_205809, 207503-207574, 208228, 208230-208232) (for detailed information, see Supplement 2).    substitutions found in HA (S84N and S162N (CHO+), I216T) and NA (V264I, N270K, and N386K (CHO-)) do not lead to significant changes in antigenic properties in the HI assay. Detailed analysis of substitutions in the proteins encoded by internal genes showed that all viruses from Saint Petersburg and Moscow bear gene constellation, represented by specific amino acid changes: D2E and M83I in NEP, E125D in NS1, M105T in NP, Q208K in M1, and N204S in PA-X ( Figure 5). E125D in NS1 is known to be one of the key substitutions involved in shutdown of host mRNA transport, restoring inherent disability of A(H1N1)pdm09 virus to efficiently control human cell gene expression 5 . NS1 of all seasonal human influenza viruses (H1N1 seasonal and H3N2) contains D125 that interacts with cellular cleavage and polyadenylation factor 30 (CPSF30) 6 . Interaction with CPSF30 is absent in most animal-adapted strains, so E125D substitution can be considered a milestone in host adaptation of influenza A(H1N1)pdm09 virus. Likewise NS1 protein, PA-X is involved in host antiviral response shutoff 7,8 , but the role of N204S substitution in PA-X has not been studied before. Possible functional significance of other substitutions remains elusive.

Set of specific mutations in internal genes
It was a tempting idea to trace back this constellation of substitutions in internal genes to the strain in which it was detected for the first time. An EpiFlu GISAID database search showed that the first influenza strain bearing all the mentioned substitutions was A/New York/61/2015 isolated in August 2015. The evolutionary origin and stability of this constellation require further phylogenetic analysis. The observed rapid spread of influenza A(H1N1)pdm09 viruses with no significant antigenic changes in HA can be speculatively explained by increased transmissibility, as well as by increased virulence or by combination of both. The possible link between transmissibility or virulence and described internal gene constellation in influenza A(H1N1) pdm09 viruses awaits experimental proof.  performed epidemiological analysis and data visualization and participated in the writing of the manuscript. Elena Burtseva, Anna Sominina, Mikhail Eropkin, and Mikhail Grudinin revised and approved the final manuscript.