Antigenic variants of influenza B viruses isolated in Japan during the 2017-2018 and 2018-2019 influenza seasons.

Abstract Background Here, we genetically and antigenically analyzed influenza B viruses (IBVs) isolated in Japan during the 2017‐2018 and 2018‐2019 influenza seasons. Methods A total of 68 IBVs (61 B/Yamagata/16/88‐like [B/Yamagata]‐lineage and 7 B/Victoria/2/87‐like [B/Victoria]‐lineage) were antigenically and genetically characterized by using hemagglutination inhibition (HI) assays and phylogenetic analysis, respectively. The susceptibility of IBVs to neuraminidase (NA) inhibitors was assessed by using a fluorescence‐based NA inhibition assay. Results All 61 B/Yamagata‐lineage isolates were genetically closely related to B/Phuket/3073/2013, the vaccine strain for these two seasons. Eleven B/Yamagata‐lineage isolates tested were antigenically similar to B/Phuket/3073/2013 by the HI test. Seven B/Victoria‐lineage isolates were genetically closely related to B/Texas/02/2013, the WHO‐recommended vaccine strain for the 2017‐2018 season; however, they were antigenically distinct from B/Texas/02/2013 with an eightfold or 16‐fold difference in HI titer. Of these 7 isolates, 4 possessed a two‐amino‐acid deletion at positions 162 and 163 in hemagglutinin (HA) and the other 3 had a three‐amino‐acid deletion at positions 162‐164 in HA. Importantly, the variants with the three‐amino‐acid deletion appeared to be antigenically different from the B/Colorado/06/2017 virus with the two‐amino‐acid deletion, the vaccine strain for the 2018‐2019 season with a fourfold or eightfold difference in HI titer. One B/Yamagata‐lineage isolate carrying a G407S mutation in its NA showed a marked reduction in susceptibility to zanamivir, peramivir, and laninamivir. Conclusions These results highlight the need for continued monitoring for the prevalence of the antigenic variant with the three‐amino‐acid deletion and the variant with reduced NA inhibitor susceptibility.


| INTRODUC TI ON
Influenza is an acute respiratory infectious disease caused by influ-  3,4 Anti-influenza drugs that inhibit the enzymatic activity of NA are available for the treatment and prophylaxis of influenza; however, mutations in the NA active site reduce its susceptibility to NA inhibitor drugs, leading to the emergence of drug-resistant variants. 5,6 During the 2017-2018 influenza season, influenza A/H1N1 2009 pandemic (A/H1N1pdm), A/H3N2, and B viruses co-circulated in Japan. 7 Notably, in Japan, the influenza B epidemic of this season was larger than that of the previous nine seasons. Here, we examined the genetic and antigenic properties of the influenza B viruses isolated in Japan during the winter season. In addition, we characterized the influenza B viruses isolated in Japan during the 2018-2019 season, even though influenza B viruses circulated at a lower level in this season compared with the previous season.

| Clinical specimens
After informed consent was obtained, respiratory specimens were

| Virus isolation and propagation
MDCK, AX4, or hCK cells grown in 12-well plates were inoculated with 0.1 mL per well of the clinical samples and incubated at 33°C for at least 30 minutes. One milliliter of MEM containing 0.3% bovine serum albumin (BSA) and 1 µg/mL TPCK-treated trypsin was then added to cells. The cultures were then incubated for up to 7 days, until cytopathic effects were evident. Cell culture supernatants were harvested, viral RNA was extracted and subjected to RT-PCR, and the viral genes were sequenced (see below). Influenza B viruses were propagated in MDCK, AX4, or hCK cells in MEM containing 1 µg of L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)trypsin/mL at 33°C.

| Reverse genetics
NA inhibitor-sensitive and NA inhibitor-resistant control viruses were generated by using a plasmid-based reverse genetics system as previously described. 10   Quencher-1 (BHQ-1) or 6-carboxytetramethylrhodamine (TAMRA) quencher dye at the 3′ end. A list of the primers and probes used is provided in Table S1.

| RT-PCR and sequencing of viral genes
Viral RNA was extracted from infected cell culture supernatant by using the QIAmp Viral RNA Mini Kit (Qiagen) and reversetranscribed to cDNA by using Superscript III reverse transcriptase (Thermo Fisher Scientific) and the Uni9/FluB1 primer. 12 PCR was performed with primers specific for the HA or NA genes of influenza B virus. The PCR products were purified and directly sequenced by using BigDye Terminator version 3.1 Cycle Sequencing Kits (Thermo Fisher Scientific), and were then analyzed on an ABI Prism 3130xl Genetic Analyzer (Thermo Fisher Scientific). A list of the primers and probes used is provided in Table S1. The nucleotide sequences obtained in this study were submitted to GISAID's EpiFlu™ Database and assigned accession numbers as documented in Table S2.

| Phylogenic analysis
Nucleotide sequences of the HA genes of influenza B viruses were aligned using mega version 7.0.26. 13 The phylogenic tree of the HA nucleotide sequences was built in maximum-likelihood method with 1000 bootstrap replicates using mega version 7.0.26. 13

| Experimental infection of ferrets
Six-to seven-month-old female ferrets (Wuxi Sangosho Biotechnology Co., Ltd.) were used in this study. Ferrets were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg/kg of body weight, respectively) and inoculated intranasally

| HI assay
Ferret sera were treated with receptor-destroying enzyme (RDE II; Denka Seiken Co., Ltd) at 37°C for 20 hours, followed by RDE inactivation at 56°C for 30-60 minutes. The treated sera were serially diluted twofold with PBS in 96-well U-bottom microtiter plates and mixed with the amount of virus equivalent to four hemagglutination units, followed by incubation at room temperature (25°C) for 60 minutes. After addition of 50 µL of 0.5% chicken red blood cells, the mixtures were gently mixed and incubated at 4°C for a further 45 minutes. HI titers are expressed as the inverse of the highest antibody dilution that inhibited hemagglutination.

| NA inhibition assay
Oseltamivir carboxylate, peramivir, and laninamivir were kindly provided by Daiichi Sankyo Inc, Tokyo, Japan. Zanamivir was obtained from GlaxoSmithKline, London, UK. In vitro NA activity of viruses was determined as described previously. 14 were selected for further characterization.

| Antiviral susceptibility
To