Severe and Fatal Influenza Cases in Russia in 2014-2015

Corresponding Author: Tatyana N. Ilyicheva Vector State Research Center of Virology and Biotechnology, Koltsovo, Novosibirsk Region, Russia Email: ilyichev@mail.ru Abstract: This paper aims to characterize herd immunity of the population inhabiting Asian part of Russia before influenza epidemic and to describe influenza viruses isolated from severe cases including cases with fatal outcomes in the 2014-2015 epidemic season. HI test enabled us to study 3888 serum samples from healthy individuals including 1939 samples collected from poultry farm workers. We showed that none of the 3888 samples produced positive results with the antigens A/H5N1, А(H5N8) and A/H7N9. 41% of the samples are positive to A/California/07/09(H1N1pdm09), 36% of the samples are positive to A/Texas/50/2012 (H3N2), 40% of the samples are positive to B/Brisbane/60/2008 (Victoria lineage) and 47% of the samples are positive to B/Massachusetts/2/2012 (Yamagata lineage). In the 2014-2015 epidemic season 25 clinical and 19 autopsy samples were collected from individuals with severe flu-like infection. Fifteen influenza A(H3N2), two influenza A(H1N1pdm09) and one influenza B (Yamagata) virus strains were isolated in Madin-Darby Canine Kidney cell culture. All viruses exhibited normal inhibition by oseltamivir and zanamivir. A/KMAO/1/2015 and A/Kurgan/149/2015 were antigenically characterized as A/California/07/2009like. Their hemagglutinin (HA) gene sequences fell into the predominant genetic group 6B and were similar to other recent H1N1pdm09 viruses circulating in Asian region. Eight H3N2 isolated viruses (A/Omsk/160/2015, A/Krasnoyarsk/324/2015, A/NizhnyNovgorod/788/2015, A/Omsk/141/2015, A/Buryatia/19/2015, A/Komi/99/2015, A/Novosibirsk/122/2015 and A/Chelyabinsk/160/2015) were characterized as A/Hong Kong/4801/2014-like and seven viruses (A/Irkutsk/88/2015, A/Krasnoyarsk/365/2015, A/Blagoveshchensk/19/2015, A/Kemerovo/2/2015, A/Chelyabinsk/160/2015, A/Chelyabinsk/192/2015 and A/Novosibirsk/64/2015) as A/Switzerland/9715293/2013-like. Their HA gene sequences belong to 3C.2a and 3C.3a genetic groups, respectively. B/Yekaterinburg/155/2015 virus was antigenically similar to B/Phuket/3073/2013 with HA sequence belonging to Y3 genetic group. Obtained findings are important for adjustment of public health measures and vaccine strategy in Russia.


Introduction
Since 2009 seasonal influenza has been caused by viruses A(H1N1pdm09), A(H3N2), influenza B (Victoria lineage) and influenza B (Yamagata lineage) (WHO, 2016). In the 2014-2015 epidemic season A(H3N2) was prevalent among influenza A viruses and strains of Yamagata lineage predominated among influenza B viruses. Thus, in Canada 81% of confirmed cases were caused by influenza A and 19% by influenza B viruses. The majority of circulating influenza A viruses (41.5%) belonged to H3N2 subtype, while a share of A(H1N1pdm09) viruses was only 0.3% (there were no subtyping for 58.2% of the cases) (FluWatch, 2015). In the USA similar data were registered (Appiah et al., 2015). In Europe 67% of approximately 16000 positive results belonged to influenza A viruses (of them 21.7% influenza A(H1N1pdm09), 72.1% influenza A(H3N2) and the rest untyped) and 33% belonged to influenza B (of them 0.6% Victoria, 25.2% Yamagata lineage and the rest untyped) (ECDC, 2015a).
In Russia the epidemic rise of influenza incidence began during the 5th calendar week in 2015 (25 January-01 February); the peak of epidemic was observed during the 8th week (16 February-22 February) and decreased activity was registered up to the 13th week. More than 59% of all isolated strains in Russia accounted for influenza A(H3N2) viruses; approximately 37% -for influenza B viruses and share of A(H1N1pdm) viruses were <4% (Rospotrebnadzor, 2015).
This research pursues an objective to isolate influenza virus strains from autopsy and clinical materials obtained from people who suffered from a severe flu-like disease, study their antigenic and biomolecular features and analyze anti-neuraminidase drug sensitivity. Besides, herd immunity just before epidemic was investigated. This paper continues our previous studies devoted to monitoring of influenza in Russia (Ilyicheva et al., 2011;2013;2016).
In total 3888 serum samples from healthy individuals were tested including 1939 samples collected from poultry farm workers. Sera were collected in various regions of Russia in October-November, 2014 ( Fig. 1).

Influenza Virus Isolates from Autopsy and Clinical Materials
Vector State Research Center of Virology and Biotechnology received the autopsy materials (pieces of the bronchi, trachea and lungs) from individuals who died presumably from influenza and clinical materials from people with severe disease. Figure 1 shows regions where the materials were collected. All samples were transferred in tubes placed into thermal containers with cold pack. In total 25 clinical and 19 autopsy samples were collected from individuals with severe flu-like infection. The isolates were recovered in Madin-Darby Canine Kidney (MDCK) cell culture (London line) as described previously (Ilyicheva et al., 2016).
Studying sensitivity to anti-neuraminidase drugs was carried out using fluorescent method according to WHO recommendations (WHO, 2009).

Investigation of the Herd Immunity to Influenza
None of the 3888 samples produced positive results with the antigens A/H5N1, А(H5N8) and A/H7N9 even at dilution 1:10.
The HI results with the antigens A/H1pdm09, A/H3N2, B/Victoria and B/Yamagata are shown in Table 1.
Analysis of the data from Table 1 shows that 41% of the samples were positive to A/California/07/09(H1N1pdm09), 36% of the samples were positive to A/Texas/50/2012 (H3N2), 40% of the samples were positive to B/Brisbane/60/2008 (Victoria lineage) and 47% of the samples were positive to B/Massachusetts/2/2012 (Yamagata lineage). In addition, 22% of the samples reacted in HI with all antigens with the reciprocal titer lower than 40, i.e., they were negative to all studied antigens while 10% of the samples were positive to all the antigens.

Influenza Virus Isolates from Autopsy and Clinical Materials
Fifteen influenza A(H3N2), two A(H1N1pdm09) and one influenza B (Yamagata) virus strains were isolated from autopsy and clinical materials. Table 2 demonstrates the data concerning isolated strains.       Antigenic features of isolated strains were studied in HI test with reference sera. Results are shown in Table 3-5. A virus is considered "reference virus-like" if its HI titer is equal to or within a 4-fold difference to the homologous HI titer of the reference strain. A virus is considered as low to the reference virus if there is an 8fold or greater reduction in the HI titer when compared to the homologous HI titer of the reference strain.   All isolated strains were sensitive to anti-neuraminidase drugs oseltamivir and zanamivir (Table 2). We conducted sequence analysis of HA and NA genes of all isolated strains (Table 2) and built phylogenetic tree for H3 (Fig. 2).

Discussion
In 2014-2015 more than 59% of all isolated strains in Russia accounted for influenza A(H3N2) viruses; approximately 37% -for influenza B viruses and share of A(H1N1pdm) viruses were <4% (Rospotrebnadzor, 2015). These data can be easily explained with regard to previously studied conditions of herd immunity before epidemics. Thus, adaptive immunity insufficiently protected Russian population against influenza A and B. New variant of influenza A(H3N2) virus was prevalent and due to antigenic drift was able to evade specific immunity targeting previous epidemic and vaccine virus strains. United States Centers for Disease Control and Prevention suggested that the 2014-2015 influenza vaccine strain A/Texas/50/2012 was essentially ineffective against the circulating A(H3N2) strains (CDC, 2015). The phylogenetic tree for the HA gene of H3N2 viruses can be divided into 7 genetic groups based on shared amino acid changes (compared to previous vaccine strain A/Perth/16/2009); with only group 3 viruses currently circulating. Group 3 viruses share HA amino acid change V223I and are further divided into subgroups 3A, 3B and 3C. Subgroup 3A viruses share amino acid changes of N144D (loss of a glycosylation site), N145S and D487N. Subgroup 3B and 3C viruses share amino acid changes of A198S and N312S. Subgroup 3B viruses also have amino acid changes of N145S and D487N (Stucker et al., 2015).
Since 2013, genetic group 3C has been the dominant subgroup circulating worldwide. Viruses from group 3C share amino acid changes of S45N (gain of a glycosylation site) and T48I. Group 3C has further diverged into three genetic subgroups (3C.1, 3C.2 and 3C.3). Genetic subgroups 3C.2 and 3C.3 share amino acid changes of Q33R, N145S and N278K. Genetic subgroup 3C.2 viruses share amino acid change D489N. Genetic subgroup 3C.3 viruses share additional amino acid changes T128A (loss of a glycosylation site) and R142G in the HA gene. Despite the genetic divergences, these three subgroups of H3N2 viruses were antigenically similar (ECDC, 2015b).
In this study, seven H3N2 isolated viruses were characterized as A/Switzerland/9715293/2013-like and eight viruses as A/Hong Kong/4801/2014-like. We presented a phylogenetic tree of the HA genes of these viruses with HA sequences belonging to 3C.2a and 3C.3a genetic groups. All H3N2 viruses exhibited normal inhibition by oseltamivir and zanamivir.
Influenza A(H1N1pdm09) viruses have continued to circulate worldwide since their emergence in 2009. In 2014-2015, A(H1N1pdm09) activity was variable with notable widespread outbreaks in the Indian subcontinent and in parts of Africa (Parida et al., 2016;Takashita et al., 2016). The phylogenetic tree for the HA gene of H1N1pdm09 viruses can be divided into nine major genetic groups, although recently isolated viruses belong to group 6. Genetic group 6 is represented by viruses circulating worldwide and shares amino acid changes D97N, S185T, S203T, E374K and S451N in the HA. Group 6 can be divided into three subgroups -6A, 6B  (Pan et al., 2015). Group 3 isolates are antigenically similar to B/Phuket/3073/2013-like viruses, the recommended B vaccine component for the 2015 Southern Hemisphere and 2015-2016 Northern Hemisphere seasons. Recently within group 3 there were two separate reassortment events leading to viruses with the HA from B/Yamagata genetic group 3 and the NA from B/Victoria genetic groups 1A or 4. These reassortant viruses remain antigenically similar to the recommended Group 3 B/Yamagata component in the vaccine (Oong et al., 2015).
This paper presents one B/Yamagata lineage virus isolated from a fatal influenza case. It was tested by HI and antigenically similar to B/Phuket/3073/2013. We also sequenced HA and NA genes of this virus, its HA sequence belongs to Y3 genetic group. This B virus exhibited normal inhibition by oseltamivir and zanamivir.

Conclusion
Thus, in 2014-2015 in Russia A(H3N2) viruses belonging to 3C.2a and 3C.3a genetic groups predominated among influenza A viruses. Fifteen influenza A(H3N2) virus strains were isolated from people who died presumably from influenza and individuals with severe flulike infection. All viruses differed from vaccine strain of the 2014-2015 season (A/Texas/50/2012) and were similar to A/Switzerland/9715293/2013 and A/Hong Kong/4801/2014. Although there were less than 4% of A(H1N1pdm09) viruses among all circulated influenza viruses in Russia, we isolated two influenza A/California/07/2009-like virus strains from autopsy and clinical materials. Besides, one influenza B/Phuket/3073/2013-like virus strain was isolated from a fatal case. All isolated strains were sensitive to antineuraminidase drugs.
Obtained findings are important for adjustment of public health measures and vaccine strategy in Russia.