Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic


The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104–1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.

Influenza A virus subtypes H1N1 (A/H1N1) and H3N2 (A/H3N2) and influenza B viruses circulate in the human population every winter in temperate countries. They account for 3–5 million cases of severe illness and about 290,000–650,000 respiratory deaths annually ( In addition to the social burden, disease severity is a serious concern, measured by hospitalization and fatality rates [22].

The viral load plays a key role in assessing the prognosis of influenza and the probability of viral transmission. Viral loads typically peak with maximal symptoms approximately 48 hours after inoculation, after which pathological damage, such as lung injury, can occur as a result of immune reactions [6, 18]. Viral growth capability may be one of the factors contributing to the viral load. However, few studies have been conducted on the viral growth capability of epidemic strains, even though several host factors are known to be required for replication of influenza viruses [7].

Our previous study showed that emerging pandemic viruses consisted of only highly replicative strains, but more-slowly replicating strains were observed soon in the following 2010–2011 seasons [21]. This demonstrated that strain-dependent differences play a role in replication capability. However, further observation is needed to evaluate the clinical significance of these strain-dependent differences. Therefore, we performed a study examining the growth capability of influenza viruses, including another subtype A/H3N2 isolate and type B isolates from six consecutive seasons, and the correlation between growth kinetics and the nasopharyngeal viral load was investigated.

Nasal swabs and nasopharyngeal aspirates were collected from influenza A and B virus antigen-positive patients and those with suspected influenza who were admitted to a hospital and three clinics for medical consultation in Tottori prefecture of Japan during the six consecutive winter seasons of 2009–2015 (n = 2,746). The procedures for influenza virus isolation were based on a manual ( with slight modifications. One of the frozen aliquots was used as an inoculum to minimize bias in experiments.

Total RNA was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN K.K., Tokyo, Japan). The eight RNA segments of the influenza A and B viruses were reverse transcribed and simultaneously amplified using a SuperScript™ III One Step RT-Polymerase Chain Reaction (PCR) System with Platinum® Taq DNA Polymerase (Thermo Fisher Scientific K.K., Tokyo, Japan) and the primer pairs MBTuni-12/MBTuni-13 and MBTuni-12b/MBTuni-13b [11, 23]. Two primer pairs, PH1F/PH1R for A/H1N1 and SH3F/SH3R for A/H3N2, were used in second-round PCR for typing and subtyping of influenza A viruses [13]. Similarly, the influenza B virus type-specific primer pair BHAB-5/BHACII [15] was used for second-round PCR.

To synthesize control RNA for generating a standard curve in real-time RT-PCR, a portion of the matrix gene of the influenza A and B viruses was targeted as described previously [1, 9]. A SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Thermo Fisher Scientific) was used for the one-step real-time RT-PCR reaction in accordance with the manufacturer’s instructions with slight modifications. Briefly, either serially diluted control RNA (108 to 102 copies in 5 µL) or sample RNA (5 µL) was mixed with 20 µL of buffer solution containing the primers FLUA-MAT-F/FLUA-MAT-R [1] or BMLF/BMLR [9] (250 nM each), a reverse transcriptase/Taq polymerase mixture, and ROX reference dye. The reaction was initiated from the first reverse transcription step at 50°C for 30 minutes and was followed by 40 cycles of denaturing (95°C for 15 seconds) and annealing/elongation (60°C for 30 seconds) steps.

The growth capability of seasonal influenza viruses was assessed by measuring virus production in cultures of MDCK cells and A549 human lung epithelial cells inoculated with the seasonal isolates at 3,300 and 106 copies/mL and culturing for 72 hours.

The correlation between growth capability and another factor was analyzed using Pearson’s correlation coefficient. A P -value less than 0.05 was considered significant.

In the A/H1N1 pandemic season (2009–2010), all 21 A/H1N1 isolates exhibited high growth capability and reached more than 108 copies/mL in the cultures. Influenza A/H3N2 and B viruses were not detected in this season at all. In the next season (2010–2011), A/H1N1 retained its high replication capability (n = 17, more than 108 copies/mL), and strains with lower replication capability (106–108 copies/mL) were also recognized. In the following four consecutive epidemic seasons of 2011–2012, 2012–2013, 2013–2014, and 2014–2015 in Japan, all detected strains of A/H1N1, A/H3N2, and type B showed a wide range of growth in MDCK cell cultures in vitro (104–1011, Fig. 1).

Fig. 1

Growth capability of epidemic influenza viruses from Japan, 2009–2015. Viral RNA copies produced in MDCK cell cultures were measured to assess the growth capability of epidemic influenza A/H1N1 (2009–2010, n = 21; 2010–2011, n = 20; 2013–2014, n = 11; total, n = 52), A/H3N2 (2010-11, n = 4; 2011–2012, n = 10; 2012–2013, n = 7; 2014–2015, n = 21; total, n = 42), and B (2010–2011, n = 12; 2011–2012, n = 11; 2012–2013, n = 17; 2013–2014, n = 22; total, n = 62) viruses circulating in Tottori prefecture, Japan, during the six consecutive seasons of 2009–2015

One reference strain (A/Puerto Rico/8/1934(H1N1), hereinafter referred to as PR8) and five epidemic strains (A/Tottori/ST215/2009(H1N1), ST215; A/Tottori/ST777/2011(H3N2), ST777; A/Tottori/ST1349/2014(H3N2), ST1349; A/Tottori/ST1705/2014(H3N2), ST1705; A/Tottori/ST1890/2015(H3N2), ST1890) were selected for growth kinetics analysis in MDCK cell culture. A difference in viral growth levels was clearly recognizable at 24 hours post-inoculation and became more obvious at 48 and 72 hours. Although highly replicative strains (PR8 and ST215) produced progeny viruses at levels of 108 and 1010 copies/mL in MDCK cell cultures, no virus production was observed after inoculation with the strain ST777 at the 24- and 48-hour time points. Cell viability decreased to different levels in a strain-specific manner. The largest decrease was observed with the strain PR8, which had the highest growth capability, and the least was observed with ST777, which had the lowest growth capability. The cell viability was related inversely to the viral growth in MDCK cell culture (r = 0.96, Fig. 2). In A549 cell cultures, strain-specific differences in progeny virus production were less obvious than in MDCK cell cultures. However, there was still 105 and 108 copies/mL of difference between the levels of progeny viruses of PR8 and ST777 (Fig. 2).

Fig. 2

Viral growth and cell viability. The growth of five selected strains (● ST1349, △ ST215, ▲ ST1890, □ ST777, ■ ST1705) and a laboratory strain (○PR8) was examined in MDCK and A549 cells after inoculation with 3,000 and 106 copies/mL. Viable cells were quantitated using a WST-8 assay with a linear standard curve. Cell viability decreased to different levels in a strain-specific manner (MDCK cell culture, r = 0.96, P <0.05)

There was a correlation between viral growth capability and nasopharyngeal viral load, although the Pearson’s correlation coefficient value was not high (r = 0.53, Fig. 3).

Fig. 3

Correlation between viral growth in MDCK cell culture and nasopharyngeal viral load. A/H1N1, n = 27, r = 0.40, P <0.05; A/H3N2, n = 24, r = 0.45, P < 0.05; B, n = 25, r = 0.51, P < 0.05; total, n = 76, r = 0.53, P < 0.05

The viral load has been reported to be an important surrogate marker for assessing the prognosis of influenza [2, 4, 5, 10, 14, 19, 20]. Patients with a high viral load more often show abnormal findings in chest X-rays. However, these findings are not always associated with a significantly worse prognosis [8, 19]. Although relationships were observed between viral growth and the cell death rate, virus-induced cell death was obvious only in MDCK cell culture, with a significant amount of virus production, but it was rather mild in A549 cell culture, with relatively modest virus production in the present study. The growth levels of 109–1010 copies/mL observed in MDCK cell culture may not be common in immunocompetent patients and occur only in cases of immunodeficiency, such as AIDS, with low CD4 cell counts [16]. Indeed, the maximum viral loads in viral transport medium have been reported to be 104–106 copies/mL in cases with immunocompetent hosts [3, 4, 10, 12].

Although a strong relationship of viral load levels to the severity of influenza has been recognized [12], wide variations in viral load are frequently observed [17]. The variations in viral load appear to be due to the difficulties associated with sample collection procedures, particularly with obtaining precise sample volumes from nasal and pharyngeal mucous membranes and the absence of a suitable internal control for estimating the original sample volume. Because the values for growth capability and viral load of epidemic influenza viruses correlated with each other in the present study, the growth capability appears to be one of the major factors contributing to the prognosis, apart from the viral load. However, the current methodology for measuring growth capability takes time and cannot give an immediate result after specimen collection. Therefore, additional surrogate markers are required for high-throughput detection of strains with high growth capability.

The present study demonstrates that epidemic influenza viruses can have a wide range of growth capacity. The combination of nasopharyngeal viral load, growth capability, and immunological factors may be useful for establishing a prognosis for influenza illness.


  1. 1.

    Chidlow G, Harnett G, Williams S, Levy A, Speers D, Smith DW (2010) Duplex real-time reverse transcriptase PCR assays for rapid detection and identification of pandemic (H1N1) 2009 and seasonal influenza A/H1, A/H3, and B viruses. J Clin Microbiol 48:862–866

    CAS  Article  Google Scholar 

  2. 2.

    Clark TW, Ewings S, Medina MJ, Batham S, Curran MD, Parmar S, Nicholson KG (2016) Viral load is strongly associated with length of stay in adults hospitalised with viral acute respiratory illness. J Infect 73:598–606

    Article  Google Scholar 

  3. 3.

    Duchamp MB, Casalegno JS, Gillet Y, Frobert E, Bernard E, Escuret V, Billaud G, Valette M, Javouhey E, Lina B, Floret D, Morfin F (2010) Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR: is viral quantification useful? Clin Microbiol Infect 16:317–321

    CAS  Article  Google Scholar 

  4. 4.

    Granados A, Peci A, McGeer A, Gubbay JB (2017) Influenza and rhinovirus viral load and disease severity in upper respiratory tract infections. J Clin Virol 86:14–19

    Article  Google Scholar 

  5. 5.

    Hara M, Morihara M, Takao S, Fukuda S, Shimazu Y, Tanizawa Y, Matsuo T (2012) Influenza viral load and rapid influenza diagnostic tests in children and adults. Diagn Microbiol Infect Dis 73:99–100

    Article  Google Scholar 

  6. 6.

    Herold S, Becker C, Ridge KM, Budinger GR (2015) Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J 45:1463–1478

    CAS  Article  Google Scholar 

  7. 7.

    König R, Stertz S, Zhou Y, Inoue A, Hoffmann HH, Bhattacharyya S, Alamares JG, Tscherne DM, Ortigoza MB, Liang Y, Gao Q, Andrews SE, Bandyopadhyay S, De Jesus P, Tu BP, Pache L, Shih C, Orth A, Bonamy G, Miraglia L, Ideker T, García-Sastre A, Young JA, Palese P, Shaw ML, Chanda SK (2010) Human host factors required for influenza virus replication. Nature 463:813–817

    Article  Google Scholar 

  8. 8.

    Lalueza A, Folgueira D, Muñoz-Gallego I, Trujillo H, Laureiro J, Hernández-Jiménez P, Moral-Jiménez N, Castillo C, Ayuso B, Díaz-Pedroche C, Torres M, Arrieta E, Arévalo-Cañas C, Madrid O, Lumbreras C (2019) Influence of viral load in the outcome of hospitalized patients with influenza virus infection. Eur J Clin Microbiol Infect Dis 38:667–673

    Article  Google Scholar 

  9. 9.

    Lambert SB, Whiley DM, O’Neill NT, Andrews EC, Canavan FM, Bletchly C, Siebert DJ, Sloots TP, Nissen MD (2008) Comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction. Pediatrics 122:e615-620

    Article  Google Scholar 

  10. 10.

    Lee N, Chan PK, Hui DS, Rainer TH, Wong E, Choi KW, Lui GC, Wong BC, Wong RY, Lam WY, Chu IM, Lai RW, Cockram CS, Sung JJ (2009) Viral loads and duration of viral shedding in adult patients hospitalized with influenza. J Infect Dis 200:492–500

    Article  Google Scholar 

  11. 11.

    Lee YS, Seong BL (1998) Nucleotides in the panhandle structure of the influenza B virus virion RNA are involved in the specificity between influenza A and B viruses. J Gen Virol 79(Pt 4):673–681

    CAS  Article  Google Scholar 

  12. 12.

    Li CC, Wang L, Eng HL, You HL, Chang LS, Tang KS, Lin YJ, Kuo HC, Lee IK, Liu JW, Huang EY, Yang KD (2010) Correlation of pandemic (H1N1) 2009 viral load with disease severity and prolonged viral shedding in children. Emerg Infect Dis 16:1265–1272

    Article  Google Scholar 

  13. 13.

    Mahony JB, Chong S, Luinstra K, Petrich A, Smieja M (2010) Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses. J Clin Virol 49:277–282

    CAS  Article  Google Scholar 

  14. 14.

    Ngaosuwankul N, Noisumdaeng P, Komolsiri P, Pooruk P, Chokephaibulkit K, Chotpitayasunondh T, Sangsajja C, Chuchottaworn C, Farrar J, Puthavathana P (2010) Influenza A viral loads in respiratory samples collected from patients infected with pandemic H1N1, seasonal H1N1 and H3N2 viruses. Virol J 7:75

    Article  Google Scholar 

  15. 15.

    Pechirra P, Nunes B, Coelho A, Ribeiro C, Goncalves P, Pedro S, Castro LC, Rebelo-de-Andrade H (2005) Molecular characterization of the HA gene of influenza type B viruses. J Med Virol 77:541–549

    CAS  Article  Google Scholar 

  16. 16.

    Sheth AN, Patel P, Peters PJ (2011) Influenza and HIV: lessons from the 2009 H1N1 influenza pandemic. Curr HIV/AIDS Rep 8:181–191

    Article  Google Scholar 

  17. 17.

    Spencer S, Thompson MG, Flannery B, Fry A (2019) Comparison of respiratory specimen collection methods for detection of influenza virus infection by reverse transcription-PCR: a literature review. J Clin Microbiol 57:e00027–19

  18. 18.

    Taubenberger JK, Morens DM (2008) The pathology of influenza virus infections. Annu Rev Pathol 3:499–522

    CAS  Article  Google Scholar 

  19. 19.

    To KK, Chan KH, Li IW, Tsang TY, Tse H, Chan JF, Hung IF, Lai ST, Leung CW, Kwan YW, Lau YL, Ng TK, Cheng VC, Peiris JS, Yuen KY (2010) Viral load in patients infected with pandemic H1N1 2009 influenza A virus. J Med Virol 82:1–7

    Article  Google Scholar 

  20. 20.

    Tsou TP, Shao PL, Lu CY, Chang LY, Kao CL, Lee PI, Yang PC, Lee CY, Huang LM (2012) Viral load and clinical features in children infected with seasonal influenza B in 2006/2007. J Formos Med Assoc 111:83–87

    Article  Google Scholar 

  21. 21.

    Tsuneki A, Itagaki A, Tsuchie H, Tokuhara M, Okada T, Narai S, Kasagi M, Tanaka K, Kageyama S (2013) Reduced replication capacity of influenza A(H1N1)pdm09 virus during the 2010–2011 winter season in Tottori, Japan. J Med Virol 85:1871–1877

    Article  Google Scholar 

  22. 22.

    Wong JY, Kelly H, Cheung CM, Shiu EY, Wu P, Ni MY, Ip DK, Cowling BJ (2015) Hospitalization fatality risk of influenza A(H1N1)pdm09: a systematic review and meta-analysis. Am J Epidemiol 182:294–301

    Article  Google Scholar 

  23. 23.

    Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, Wentworth DE (2009) Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses. J Virol 83:10309–10313

    CAS  Article  Google Scholar 

Download references


The authors thank Dr. Kurozawa for his suggestion on the statistical analysis, and Ms. Naohara and Ms. Komatsu for their technical assistance.


This work was supported in part by JSPS KAKENHI (Grant numbers 15K19228 and 18K17353), a Grant-in-Aid for Environmental Health Research from Tottori prefecture, and a Grant-in-Aid for Scientific Research on Infection Control and Prevention by the International Platform for Dryland Research and Education, Tottori University.

Author information



Corresponding author

Correspondence to Seiji Kageyama.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

The present study was conducted with the approval and under the control of the Institutional Review Board of the Faculty of Medicine, Tottori University, Japan (no. 1981). All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Sample collection was conducted after obtaining informed consent from patients in collaborating medical facilities consisting of a hospital and three clinics. Samples were then shipped to the laboratory without patient identification.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Handling Editor: William G Dundon.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tsuneki-Tokunaga, A., Kanai, K., Itagaki, A. et al. Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic. Arch Virol 166, 1193–1196 (2021).

Download citation