Local spread of influenza A (H1N1) viruses without a mutation for the maximum duration of an epidemic season in Japan

Close observation of the local transmission of influenza A(H1N1) viruses enabled an estimate of the length of time the virus was transmitted without a mutation. Of 4,448 isolates from 11 consecutive years, 237 isolates could be categorized into 57 strain groups with identical hemagglutinin genes, which were monitored for the entire duration of an epidemic season. In addition, 35 isolates with identical sequences were identified at the study site and in other countries within 147 days. Consequently, it can be postulated that once an influenza virus enters a temperate region, the strain rarely mutates before the end of the season.

The Global Burden of Disease Study estimated that influenza-related lower respiratory tract infections (LRTIs) were responsible for an estimated 145,000 deaths among people of all ages in 2017. The influenza LRTI mortality rate was highest among adults older than 70 years. This study estimated that influenza LRTIs accounted for 9,459,000 hospitalizations and 81,536,000 hospital days and that 11.5% of LRTI cases were attributable to influenza. This study also showed the substantial annual effect of influenza on global health and advised that vaccine use should be encouraged [5].
Influenza viruses are capable of evading antibodymediated immunity induced by previous infections or vaccinations by gradually accumulating mutations in their hemagglutinin and neuraminidase segments. This process, termed antigenic drift, necessitates frequent updates of influenza vaccines to ensure sufficient antigenic relatedness between the vaccine and emerging virus variants [1]. Frequent updates should also be done to reduce the risk of drug resistance. For example, the emergence of oseltamivir resistance has become a particular concern for influenza A H1N1 viruses because it has happened in the past; in 2007, resistant influenza A H1N1 viruses began to circulate and quickly became dominant by the 2008-2009 season [6].
An earlier study identified 100 countries with one influenza peak season (85%), 13 with two influenza peak seasons (11%), and five with year-round influenza activity (4%) [10]. Many tropical populations seem to have significant influenza activity year-round, in contrast to temperate regions. Therefore, the question of whether influenza virus persists in temperate regions all year long or is reintroduced from the tropics each year at the beginning of winter is important in relation to vaccine composition and influenza control in the tropics as well as in temperate regions. The influenza vaccination strategy may have to be tailored to each individual tropical region, based on local prevalence, timing, cycling of influenza strains, and how closely the local strains match the strains circulating in temperate areas [17]. It has been hypothesized that new lineages are seeded from a persistent influenza virus reservoir in the tropics to sink populations in temperate regions [15]. Studies on the global dynamics of A/H3N2 viruses have provided further evidence of local extinction of viruses between epidemics and have identified East and Southeast (E-SE) Asia as the global source of A/ H3N2 [2]. Previously, we carried out a 3-year study in which we isolated 288 strains, successfully traced 38 groups of strains through frequent isolations, and found a strain transmitted without a mutation for a maximum duration of 94 days. Two strains were found to be identical to those isolated in Mongolia and Thailand in the same epidemic season [20]. The present study enlarges the scale of the previous study and continues over an additional 8 years in order to estimate the length of time the virus can spread without mutations.
After written informed consent was obtained, nasal swabs and/or aspirated nasal discharges were collected from influenza-virus-antigen-positive individuals and patients with suspected influenza living in Tottori Prefecture, Japan, during 11 consecutive winters from 2009 to 2020. Each swab or aspirate was kept in 3 ml of viral transport medium (2% Difco™ veal infusion broth, 0.4% gelatin, 0.06% bovine serum albumin, 200 units of penicillin G per ml, and 100 µg of streptomycin per ml in Dulbecco's modified Eagle's medium) at 4 °C prior to virus isolation. The Institutional Review Board of Tottori University Faculty of Medicine approved the study protocol (no. 1981).
The procedure for influenza virus isolation was based on the manual issued by the World Health Organization, with slight modifications [18]. Briefly, an aliquot (500 µl) of diluted sample in transport medium was inoculated onto Madin-Darby canine kidney (MDCK) cells, which were then incubated for 1 hour. The culture was maintained in Dulbecco's modified Eagle's medium supplemented with 5 µg of trypsin (Difco™ Trypsin 250, Becton Dickinson, Tokyo, Japan) per ml, 0.2% heat-inactivated bovine serum albumin, 4 mM L-glutamine, 200 units of penicillin G per ml, and 100 μg of streptomycin per ml at 34 °C in a 5% CO 2 /95% air atmosphere until the appearance of a cytopathic effect. The culture supernatant was kept frozen at -80 °C as a virus stock for subsequent experiments.
Total RNA was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo, Japan). Eight RNA segments of influenza A virus were simultaneously reverse transcribed and amplified using a SuperScript™III One Step RT-Polymerase Chain Reaction (PCR) System (Life Technologies, Tokyo, Japan) with the MBTuni-12/MBTuni-13 primer pair [21]. The first-round PCR products were subjected to a second PCR in order to amplify 266 bases of A(H1N1) for subtyping [9].
The nucleotide sequences of the amplified hemagglutinin fragments were determined using a BigDye ® Terminator v3.1 Cycle Sequencing Kit in accordance with the manufacturer's instructions (Life Technologies). M13 and PCR primers were used for the sequencing reaction. Nucleotide sequences excluding the primer regions (1,701 bases of A(H1N1)) were aligned with sequences obtained from the International Nucleotide Sequence Database. Inspection, manual modification, and evolutionary analysis of the sequences were conducted in Molecular Evolutionary Genetics Analysis Version X (MEGA X). A phylogenetic tree was constructed using the neighbor-joining method (1,000 bootstrap replications) in MEGA X. An estimate of the mean evolutionary diversity was also conducted using MEGA X [7]. The influenza A(H1N1) virus clade was determined using the Influenza Research Database.
The sequences described in this study have been deposited in the International Nucleotide Sequence Database under accession numbers LC637996 to LC638404.
Isolation of influenza A(H1N1) viruses was carried out for 11 consecutive seasons in the study area, Tottori Prefecture, Japan, and sequences from these isolates were used to make a phylogenetic tree with A/South Carolina/1/1918(H1N1) as an out-group. The A(H1N1) tree was composed of 409 strains. The cluster of influenza A (H1N1) viruses from each year is shown in a shaded square. None of the clusters overlapped in the phylogenetic tree, although the epidemic source was unknown (Fig. 1).
Sequential isolations of epidemic influenza viruses were carried out during the epidemic seasons in Tottori Prefecture from 2009 to 2020, and these isolates were analyzed together with those from the previous study (2009)(2010)(2011)288 isolates). The total number of isolates was 4,448. Close observation of influenza A viruses from patients admitted at two clinics and a hospital in the study region enabled the identification of 57 transmission chains of influenza A viruses with 100% sequence identity in the coding region of the hemagglutinin segment (1.7 kb). The transmission chains could be observed for up to 94 days (Fig. 2).
The nucleotide sequences of influenza A (H1N1) virus hemagglutinin (1.7 kb) were compared with those in the International Nucleotide Sequence Databases. Strains showing 100% sequence identity were found in the USA (n = 28), Canada (n = 2), Thailand (n = 2), Mongolia (n = 1), Taiwan (n = 1), and Italy (n = 1). The longest time gap between the time of virus isolation in the present study and identification in another country was 147 days (Table 1).
It has been estimated that influenza A virus mutates at a rate of 2 × 10 -6 to 2 × 10 -4 nucleotides per site per genome replication [3,[12][13][14]19]. The value is equivalent to 2.6 × 10 -3 mutations per site per year according to Nobusawa et al. [11], and it takes about 83 days for the virus to mutate at one site. This value fits well with the observations in the current study.
Influenza viruses disappear in most areas in Japan after the epidemic season in winter and re-appear as variants in the following season, except for the southern part of the country [4,16]. At the end of winter, the spread of the current strain may be terminated in Japan, but transmission might continue in susceptible populations in other countries [2,8]. The stable transmission of influenza viruses in Japan and other countries with a temperate climate suggests that antigenic drift of influenza viruses is less likely to occur within an influenza epidemic season in temperate regions. However, the source of these variants is unknown. A seasonal influenza epidemic in a temperate region may occur with the introduction of variants originating in variant-prevalent areas or as the result of global circulation. Therefore, it is indispensable to compare the nucleotide sequences of epidemic strains from temperate regions with those from tropical regions each year.
Of the 57 influenza A strain groups identified here, one was identified 11 times in the local study area in Japan in 94 days. It can therefore be postulated that once an influenza virus enters a temperate region, it rarely mutates before the end of the epidemic season. The long-term stable transmission of influenza A viruses may be useful for identifying possible epidemic strains in advance of the  following epidemic season in countries with a temperate climate. Table 1 Influenza A (H1N1) viruses with identical hemagglutinin sequences that were also isolated in other countries/regions Accession numbers of the 409 strains included in the present study: LC637996-LC638404 *: Time between isolation in the study area and isolation in another area of the world **: The collection date was not documented.