Influenza A(H3N2) virus variants and patient characteristics during a summer influenza epidemic in Taiwan, 2017

We report a summer influenza epidemic caused by co-circulation of multiple influenza A(H3N2) variants in clade 3C.2a. Compared with other clades, a putative clade 3C.2a.3a was more commonly isolated from severely ill patients; 3C.2a.4 was more commonly isolated in outbreak cases. Time from vaccination to illness onset was significantly shorter in severely ill patients infected with clade 3C.2a.3; characteristics and outcomes of patients infected with different clades were similar. No resistance to antiviral medications was found.

We report a summer influenza epidemic caused by co-circulation of multiple influenza A(H3N2) variants in clade 3C.2a. Compared with other clades, a putative clade 3C.2a.3a was more commonly isolated from severely ill patients; 3C.2a.4 was more commonly isolated in outbreak cases. Time from vaccination to illness onset was significantly shorter in severely ill patients infected with clade 3C.2a.3; characteristics and outcomes of patients infected with different clades were similar. No resistance to antiviral medications was found.
Recently, influenza A(H3N2) virus variants carrying substitutions N121K, S144K and T135K have been reported in Canada, Denmark, Israel and the United Kingdom (UK), causing outbreaks during the northern hemisphere 2016/17 influenza season and suboptimal vaccine effectiveness (VE) [1][2][3][4][5]. Information on clinical characteristics of patients infected with these variants is lacking, and the impact on public health practice remains unknown.
In Taiwan, the influenza season usually starts from December, and peaks in January to February of the following year [6]. The 2016/17 influenza season in Taiwan has been characterised by an unusual summer peak, which started in mid-May, peaked in late June and returned to baseline in August, with predominant circulation of influenza A(H3N2) viruses [7]. We performed a phylogenetic analysis of the variants and analysed the characteristics of patients with severe illness to fill the gap between knowledge about virological characteristics and their possible implications for public health practice.

Collection and testing of respiratory samples
The Taiwan Centers for Disease Control (TCDC) in Taipei has a national influenza laboratory surveillance network collecting respiratory specimens from three different sources to monitor changes in the circulating influenza strains. Samples from outpatients with influenza-like illness (ILI), defined as fever ≥ 38.0 °C plus respiratory symptoms and at least one of the following: myalgia, headache, general malaise, are collected by sentinel doctors (general practitioners in local clinics, or doctors in medical centers collecting specimens from their outpatients). Samples from patients involved in suspected influenza outbreaks are collected by public health workers in 22 city/county health bureaus. Severe influenza requiring intensive care is a notifiable disease in Taiwan; samples from severe cases are collected by physicians in the hospitals. All specimens obtained by the three different sources are sent to one of the eight contract laboratories or TCDC central laboratory for real-time RT-PCR and viral culture [8]. All viruses isolated by cell culture are sent to the central laboratory for genotyping, antigenic characterisation, and antiviral resistance testing. Detailed methods have been previously described [8].     (Figure 3). None of the tested influenza A(H3N2) virus isolates showed mutations associated with neuraminidase inhibitor resistance.

Discussion
We described the emergence of variant influenza A(H3N2) viruses in Taiwan     rates of patients infected with different clades were similar. Among a group of severely ill patients, mostly with pre-existing medical conditions, the clade of viruses they were infected with did not seem to change their outcome. However, although the proportion of severely ill cases receiving 2016/17 influenza vaccination was comparable, the time elapsed between vaccination and illness onset was significantly shorter among patients infected with clades 3C.2a.3 and other 3C.2a variants. This finding could be explained by the possible lower VE and shorter duration of protection against these two clades compared with other circulating ones. Nevertheless, all the described variants were susceptible to neuraminidase inhibitors, including oseltamivir and zanamivir, and current guidance continues to recommend antiviral treatment as early as possible for any patient with confirmed or suspected influenza who has severe illness or is at higher risk for complications [12].
A major concern related to the observed amino acid substitutions is whether they could cause major antigenic change and increase the potential of a large epidemic. Substitutions responsible for major antigenic change are located exclusively in HA antigenic sites A and B [13]. The substitutions we found in the putative clades 3C.2a.3a (T135K, R150K) and 3C.2a.4 (R142G, S144R) were all in antigenic site A. Position 135 has been identified as a site of accessory substitutions. Although no additional antigenic distance is acquired by addition of an accessory substitution, it corrects for directionality towards the subsequent antigenic phenotype in the antigenic maps [13]. Position 144 is associated with glycosylation of influenza A(H3N2) viruses and is adjacent to the receptor binding site, where most of the major cluster-transition substitutions are located. Because any amino acid changes in the 140-146 region of HA have been shown to be characteristic for antigenically distinct viruses of epidemic significance [3], the 3C.2a.4 variant carrying R142G and S144R substitutions may have the potential for antigenic change that warrants further monitoring in the following seasons. We found more 3C.2a.4 isolates from patients involved in outbreaks; whether these antigenic substitutions are related to virus transmission also needs further investigation.
The findings in this report are subject to at least three limitations. First, the isolates from patients in outbreaks and sentinel outpatients were collected by convenient, instead of systematic sampling, and only 32.3% (720/2,228) of all virus isolates were sequenced. Therefore, the results might not represent the circulating strains among the population as a whole. Second, clinical information was only available for patients with severe influenza therefore the results may not be applied to less severely ill patients. Finally, we did not collect data on 'test-negative' patients to calculate VE against influenza A(H3N2), and the impact of annual influenza vaccination campaign was not evaluated.  to monitor the evolution of circulating clade 3C.2a viruses and the impact on transmission and vaccine protection.