PLOS ONE RESEARCH ARTICLE Robust induction of interferon and interferon- stimulated gene expression by influenza B/ Yamagata lineage virus infection of A549 cells Pengtao Jiao1,2☯, Wenhui Fan2☯, Ying Cao2, He Zhang2, Lu Tian2,3, Lei Sun2,3, 1 1,2,3,4 2,3 Tingrong Luo *, Wenjun Liu *, Jing LiID * 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, Guangxi, China, 2 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of a1111111111 Microbiology, Chinese Academy of Sciences, Beijing, China, 3 University of Chinese Academy of Sciences, a1111111111 Beijing, China, 4 Institute of Microbiology, Center for Biosafety Mega-Science, Chinese Academy of a1111111111 Sciences, Beijing, China a1111111111 a1111111111 ☯ These authors contributed equally to this work. * [email protected] (JL); [email protected] (WJL); [email protected] (TRL) Abstract OPEN ACCESS Influenza B virus (IBV) belongs to the Orthomyxoviridae family and generally causes spo- Citation: Jiao P, Fan W, Cao Y, Zhang H, Tian L, Sun L, et al. (2020) Robust induction of interferon radic epidemics but is occasionally deadly to individuals. The current research mainly and interferon-stimulated gene expression by focuses on clinical and pathological characteristics of IBV. However, to better prevent or influenza B/Yamagata lineage virus infection of treat the disease, one must determine the strategies developed by IBV to invade and disrupt A549 cells. PLoS ONE 15(4): e0231039. https:// doi.org/10.1371/journal.pone.0231039 cellular proteins and approach to replicate itself, to suppress antiviral innate immunity, and understand how the host responds to IBV infection. The B/Shanghai/PD114/2018 virus was Editor: Kui Li, University of Tennessee Health Science Center, UNITED STATES able to infect alveolar epithelial cells (A549) cells, with good potential for replication. To iden- tify host cellular responses against IBV infection, differentially expressed genes (DEGs) Received: December 23, 2019 were obtained using RNA sequencing. The GO and KEGG pathway term enrichment analy- Accepted: March 13, 2020 ses with the DEGs were performed, and we found that the DEGs were primary involved in Published: April 8, 2020 metabolic processes and cellular function, which may be related to the host response, Copyright: © 2020 Jiao et al. This is an open including the innate immune response against the virus. Our transcriptome analysis results access article distributed under the terms of the demonstrated robust induction of interferon and interferon-stimulated gene expression by Creative Commons Attribution License, which IBV in human cells during the early stages of infection, providing a foundation for further permits unrestricted use, distribution, and reproduction in any medium, provided the original studies focused on antiviral drug development and interactions between the virus and host. author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Introduction Funding: This work was supported by grants from the National Science and Technology Major Project Influenza viruses are single-stranded negative-sense RNA viruses belonging to the family (2018ZX10101004) (W.H.F), and the National Key Orthomyxoviridae, and are phylogenetically grouped into four virus genera: influenza A, B, C, R&D Program of China (2017YFD051105, and D viruses [1, 2]. Influenza B virus (IBV) generally causes local mild epidemics, but it may 2016YFD0500206) (W.J.L), the National Natural Science Foundation of China (Grant No. 31970153, be fatal to individuals, especially infants, pregnant woman, the elderly, and people with 31630079) (J.L, W.J.L), and the Youth Innovation impaired immune systems [3]. Since the 1980s, IBV have two genetically and antigenically dif- Promotion Association of CAS (2019091) (J.L). ferentiated lineages, termed B/Yamagata lineage and B/Victoria lineage, and the antigenic drift PLOS ONE | https://doi.org/10.1371/journal.pone.0231039 April 8, 2020 1 / 18 PLOS ONE Robust induction of IFNs and ISGs expression by IBV infection of A549 cells The funders had no role in study design, data rate is slower compared with influenza A viruses[4]. IBV is a pathogen with very limited host collection and analysis, decision to publish, or range, and its natural host is mainly humans with fever and cold symptoms. However, seasonal preparation of the manuscript. pandemics can break out, such as from the winter of 2017 to the spring of 2018, when the Yama- Competing interests: The authors have declared gata lineage strain became the dominant pandemic strain alongside IAV (H1N1 and H3N2) [5]. that no competing interests exist. When influenza virus invades a host cell, the innate immune system is activated against the virus. During the infection, viruses are recognized by pattern recognition receptors, the innate response is triggered, and interferons (IFNs) are secreted to limit early viral proliferation [6]. This identification leads to an appropriate antiviral response and to the activation of inflam- matory response and adaptive immune responses [7]. While the reasons for the limited host range of IBV remain unclear, one of explanations is that the interaction between IBV and the innate immune system imbues the virus with distinct characteristics, such as the role of ISG15, a typical interferon-induced antiviral gene for the innate immune response [8, 9]. Existing studies describe the production of host cytokines, including IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines against influenza virus infection, as integral parts of the process of the cellular antiviral response [10±13]. We have also reported that type I IFNs, ISGs, and proinflammatory cytokines can be induced by IBV virions at the early stage of virus infection [7, 14]. The interaction between influenza virus infection and the host anti-viral response has been investigated in depth [15, 16], and the regulatory mechanism of IBV infection is also under investigation. Previous research has already shown that IBV ribonucleoprotein rapidly acti- vates TLR signaling pathways [14]. Nevertheless, the overall circumstances of the innate immune response, the pathways and types of IFNs that are dominant in innate immunity against IBV infection, and how they are regulated remain unclear. As has been demonstrated, the potential cytokines after viral infection can be directly or indirectly identified with the aid of transcriptome analysis [17]. RNA sequencing (RNA-seq) technology has been used to identify differences in gene regulation, which links transcriptional activators to the general transcriptional machinery [18]. In the present study, we investigated the host immune responses against IBV infection, using the B/Shanghai/PD114/2018 strain as a model, to study the anti-viral capability of IFNs and ISGs, which released in response to IBV infections. These results give us a glimpse into the host innate immunity and antiviral responses, particularly, multiple signaling pathways and antiviral factors that collectively limit viral replication. Materials and methods Cell lines, antibodies, and virus preparation Human lung carcinoma epithelial cells (A549) and Madin-Darby Canine Kidney cells (MDCK) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown at 37ÊC in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. A rabbit polyclonal antibody against NP was purchased from Thermo-Fisher Scientific. Goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The mouse anti-GAPDH mono- clonal antibody and anti-GFP antibody were purchased from Santa Cruz Biotechnology. HRP goat anti-mouse IgG was purchased from Beyotime. Genes encoding full-length ISGs, including the IFIT family (IFIT1, IFIT2, IFIT3, and IFIT5), IFITM family (IFITM1, IFITM2, IFITM3, and IFITM5), ISG15, ISG20, LAMP3, and RSAD2, were synthesized by GENEWIZ and then cloned into the pcDNA3.1-GFP vector. PLOS ONE | https://doi.org/10.1371/journal.pone.0231039 April 8, 2020 2 / 18 PLOS ONE Robust induction of IFNs and ISGs expression by IBV infection of A549 cells The B/Shanghai/PD114/2018 (B-SH; Yamagata lineage) virus was isolated from patients in Shanghai, China, and was a gift from Prof. Dayan Wang of the Chinese Center for Disease Control and Prevention. The virus stock was propagated in the allantoic cavities of 9-day-old specific-pathogen-free embryonated chicken eggs, and the virus titer was determined by 10-fold serial titration in MDCK cells according to plaque assays. The virus was stored at −80ÊC before use. Plaque assay Plaque assays were performed as previously described [19]. Briefly, MDCK cells were plated on 6-well culture plates and infected by serially diluted B-SH virus in serum-free DMEM sup- plemented with 2 μg/mL of TPCK-treated trypsin for 1 h at 37ÊC, 5% CO2. Then, the virus was removed, and the cells were washed with sterile phosphate-buffered saline (PBS). Next, the cells were overlaid with phenol red free-DMEM medium containing TPCK-treated trypsin (2 μg/mL), 0.6% low melting point agarose and incubated at 37ÊC with 5% CO2. Visible pla- ques were counted 72 h post infection to determine the virus titer. Data of all assays were obtained from at least three independent experiments, and expressed as means ± SD. Immunofluorescence assay A549 cells were seeded in 24-well culture plates overnight. The cells were proliferated on glass coverslips, infected with B-SH (MOI = 0.1) until a monolayer of cells spread to > 80% of the bottom of the culture glass coverslips, incubated for 1 h, and then the inoculum was displaced with DMEM for continued incubation at 37ÊC, 5% CO2. Cells were collected at 0, 4, 8, and 12 h, and the specific steps of immunofluorescence assay, as described by Jing et al [7] and Cao et al [15].