Influenza B Virus Infection Research Articles

Xiao-Er-Kang-Du capsules regulate autophagy against the influenza B virus (Victoria strain) through the mTOR/ULK1/Beclin1/VPS34 pathway

Ethnopharmacological relevanceXiao-er-kang-du (XEKD) capsule is a Chinese herbal formula used for treatment of upper respiratory tract infection caused by various viruses in pediatric patients in China. XEKD is used clinically for the treatment of influenza-like symptoms, including fever, chills, cough, stuffy and runny nose, headache, and sore throat, with remarkable efficacy. However, the pharmacologic mechanism of XEKD against influenza B virus (IBV) infection is unclear. Aim of the studyThe main purpose of the present work is to explore the curative effect as well as possible mechanisms of XEKD against influenza B virus (IBV) (Victoria strain). Materials and methodsBoth in vivo and in vitro experiments were performed to confirm the antiviral properties of XEKD. High-performance liquid chromatography was used to analyze the active components and assess the stability of XEKD. In addition, the mechanism of action of XEKD against IBV (Victoria) was investigated by western blot, immunofluorescence, and immunohistochemical analyses, in addition to confocal fluorescence microscopy. ResultsThe findings revealed that XEKD demonstrated antiviral effects against IBV infection in both in vivo and in vitro via the mTOR/ULK1/Beclin1/VPS34 pathway and promote cellular autophagy to mitigate IBV-induced lung tissue damage. The results of this work are expected to lead to a deeper understanding of the mechanism underlying the effect of the XEKD capsule against IBV infections. ConclusionsIBV infection was found to inhibit autophagy, which exacerbated inflammatory damage. XEKD regulates autophagy through the mTOR/ULK1/Beclin1/VPS34 pathway and exerts antiviral effects, thereby laying a foundation for further development of XEKD as a potential therapeutic against IBV infection.

Natural History of Influenza B Virus-Current Knowledge on Treatment, Resistance and Therapeutic Options.

Influenza B virus (IBV) significantly impacts the health and the economy of the global population. WHO global health estimates project 1 billion flu cases annually, with 3 to 5 million resulting in severe disease and 0.3 to 0.5 million influenza-related deaths worldwide. Influenza B virus epidemics result in significant economic losses due to healthcare expenses, reduced workforce productivity, and strain on healthcare systems. Influenza B virus epidemics, such as the 1987-1988 Yamagata lineage outbreak and the 2001-2002 Victoria lineage outbreak, had a significant global impact. IBV's fast mutation and replication rates facilitate rapid adaptation to the environment, enabling the evasion of existing immunity and the development of resistance to virus-targeting treatments. This leads to annual outbreaks and necessitates the development of new vaccination formulations. This review aims to elucidate IBV's evolutionary genomic organization and life cycle and provide an overview of anti-IBV drugs, resistance, treatment options, and prospects for IBV biology, emphasizing challenges in preventing and treating IBV infection.

Fc-Effector-Independent in vivo Activity of a Potent Influenza B Neuraminidase Broadly Neutralizing Antibody.

Influenza B virus (IBV) contributes to substantial influenza-mediated morbidity and mortality, particularly among children. Similar to influenza A viruses (IAV), the hemagglutinin (HA) and neuraminidase (NA) of IBV undergo antigenic drift, necessitating regular reformulation of seasonal influenza vaccines. NA inhibitors, such as oseltamivir, have reduced activity and clinical efficacy against IBV, while M2 channel inhibitors are only effective against IAV, highlighting the need for improved vaccine and therapeutics for the treatment of seasonal IBV infections. We have previously described a potent human monoclonal antibody (hMAb), 1092D4, that is specific for IBV NA and neutralizes a broad range of IBVs. The anti-viral activity of MAbs can include direct mechanisms such as through neutralization and/or Fc-mediated effector functions that are dependent on accessory cells expressing Fc receptors and that could be impacted by potential host-dependent variability. To discern if the in vivo efficacy of 1092D4 was dependent on Fc-effector function, 1092D4 hMAb with reduced ability to bind to Fc receptors (1092D4-LALAPG) was generated and tested. 1092D4-LALAPG had comparable in vitro binding, neutralization, and inhibition of NA activity to 1092D4. 1092D4-LALAPG was effective at protecting against a lethal challenge of IBV in mice. These results suggest that hMAb 1092D4 in vivo activity is minimally dependent on Fc-effector functions, a characteristic that may extend to other hMAbs that have potent NA inhibition activity.

Streptococcus pneumoniaecoinfection results in increased disease in a murine model of influenza B virus infection

Abstract Secondary Streptococcus pneumoniae (Spn) infection is commonly associated with increased hospitalization and death following seasonal influenza virus infection. The animal models of influenza A virus (IAV) and secondary Spn coinfection have described the increased morbidity and mortality leading to several proposed mechanisms of enhanced disease. Influenza B virus (IBV) causes 25% of seasonal influenza cases and is associated with bacterial coinfection, yet little is known about the immune response to IBV and Spn coinfection. Here, we established a mouse model of IBV and Spn coinfection to study disease in singly and coinfected animals and define mechanism(s) of enhanced disease. Like IAV and Spn coinfection, infection with IBV followed by Spn inoculation resulted in increased morbidity and mortality compared to IBV or Spn infection alone. In IBV primed mice, Spn titers were increased early following bacterial inoculation and Spn rapidly migrated to the lung. Analysis of cytokines in the lung showed increased pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) responses with secondary Spn infection, which may be associated with enhanced disease. These data suggest that IBV infection initiates innate immune responses that are rapidly enhanced with Spn infection, leading to worsening disease. Ongoing studies include analysis of infiltrating cells during coinfection and histopathologic analysis to complement the innate response data to define mediators of clinical disease. NIH CIDER Contract #75N93021C00018

Asymmetric Cross-Protection is Elicited by Distinct Influenza B Lineages

Abstract The immune response to influenza B virus (IBV) infection is understudied compared to influenza A virus (IAV), despite IBV accounting for 20% of influenza-related hospitalizations annually. Influenza B was first isolated in the 1940s and subsequently diverged into two distinct lineages, Victoria (B/Vic) and Yamagata (B/Yam), defined by changes in the hemagglutinin (HA) and basic polymerase (PB1 and PB2) gene segments. The two IBV lineages are antigenically distinct, resulting in the development of influenza vaccines containing both B/Vic and B/Yam in addition to H1N1 and H3N2 IAVs. Differential epidemiologic dynamics of the IBV lineages and the recent proposed extinction of B/Yam suggests the lineages do not elicit equivalent immunity. Human studies have shown B/Yam-infected persons are susceptible to reinfection with the same lineage virus, suggesting B/Yam-elicited immunity is insufficient compared to B/Vic-elicited immunity, which prevents homotypic reinfection. Here we established a murine model of contemporary IBV infection that supports observational studies. We demonstrated immune responses elicited by B/Yam infection is insufficient to protect against a heterolineal infection (B/Vic challenge). Conversely, B/Vic-elicited immunity protected against homologous and heterolineal (B/Yam) challenge. We assessed virus-specific serum antibody (Ab) responses following primary infection and challenge. Serological data suggest B/Yam infection failed to elicit cross-reactive Ab, allowing B/Vic infection, while B/Vic elicited serum Ab against both lineages. Ongoing studies seek to define the virus-specific B-cell response to B/Yam and B/Vic infection and to confirm these results in a ferret model of IBV infection. Supported by grants from NIH Contract 75N93021C00018

Modeling the effects of cigarette smoke extract on influenza B virus infections in mice.

Influenza B virus (IBV) is a major respiratory viral pathogen. Due to a lack of pandemic potential for IBV, there is a lag in research on IBV pathology and immunological responses compared to IAV. Therefore, the impact of various lifestyle and environmental factors on IBV infections, such as cigarette smoking (CS), remains elusive. Despite the increased risk and severity of IAV infections with CS, limited information exists on the impact of CS on IBV infections due to the absence of suitable animal models. To this end, we developed an animal model system by pre-treating mice for two weeks with cigarette smoke extract (CSE), then infected them with IBV and monitored the resulting pathological, immunological, and virological effects. Our results reveal that the CSE treatment decreased IBV specific IgG levels yet did not change viral replication in the upper airway/the lung, and weight recovery post infection. However, higher concentrations of CSE did result in higher mortality post infection. Together, this suggests that CS induced inflammation coupled with IBV infection resulted in exacerbated disease outcome.

Oxidative profile, inflammatory responses and δ-aminolevulinate dehydratase enzyme activity in influenza B virus infection.

determine the activity of the delta-aminolevulinate dehydratase (δ-ALA-D) enzyme, oxidative stress biomarkers and the expression of cytokines in those infected with influenza B virus (IBV). to evaluate the activity of the δ-ALA-D enzyme, lipid peroxidation estimated as levels of thiobarbituric acid reactive substances (TBARS), protein (PSH), non-protein (NPSH) thiol groups, ferric reducing antioxidant power (FRAP), vitamin C concentration (VIT C) and cytokine levels in IBV infected individuals (n = 50) and a control group (n = 30). δ-ALA-D activity was significantly lower in IBV-infected compared to controls, as well as levels of thiols, vitamin C and FRAP. Lipid peroxidation and cytokine levels IL-6, IL-10, IL-17A and IFN-y were statistically higher in the IBV group. we found evidenced of the generation of oxidants, the depletion of the antioxidant system, the decrease in the activity of the δ-ALA-D enzyme and the increase in the synthesis of cytokines, thus contributing to a better understanding of oxidative and inflammatory pathways during IBV infection.

Cellular glycan modification by B3GAT1 broadly restricts influenza virus infection

Communicable respiratory viral infections pose both epidemic and pandemic threats and broad-spectrum antiviral strategies could improve preparedness for these events. To discover host antiviral restriction factors that may act as suitable targets for the development of host-directed antiviral therapies, we here conduct a whole-genome CRISPR activation screen with influenza B virus (IBV). A top hit from our screen, beta-1,3-glucuronyltransferase 1 (B3GAT1), effectively blocks IBV infection. Subsequent studies reveal that B3GAT1 activity prevents cell surface sialic acid expression. Due to this mechanism of action, B3GAT1 expression broadly restricts infection with viruses that require sialic acid for entry, including Victoria and Yamagata lineage IBVs, H1N1/H3N2 influenza A viruses (IAVs), and the unrelated enterovirus D68. To understand the potential utility of B3GAT1 induction as an antiviral strategy in vivo, we specifically express B3GAT1 in the murine respiratory epithelium and find that overexpression is not only well-tolerated, but also protects female mice from a lethal viral challenge with multiple influenza viruses, including a pandemic-like H1N1 IAV. Thus, B3GAT1 may represent a host-directed broad-spectrum antiviral target with utility against clinically relevant respiratory viruses.

Cross-Protection Induced by Virus-like Particles Derived from the Influenza B Virus.

The mismatch between the circulating influenza B virus (IBV) and the vaccine strain contributes to the rapid emergence of IBV infection cases throughout the globe, which necessitates the development of effective vaccines conferring broad protection. Here, we generated influenza B virus-like particle (VLP) vaccines expressing hemagglutinin, neuraminidase, or both antigens derived from the influenza B virus (B/Washington/02/2019 (B/Victoria lineage)-like virus, B/Phuket/3073/2013 (B/Yamagata lineage)-like virus. We found that irrespective of the derived antigen lineage, immunizing mice with the IBV VLPs significantly reduced lung viral loads, minimized bodyweight loss, and ensured 100% survival upon Victoria lineage virus B/Colorado/06/2017 challenge infection. These results were closely correlated with the vaccine-induced antibody responses and HI titer in sera, IgG, IgA antibody responses, CD4+ and CD8+ T cell responses, germinal center B cell responses, and inflammatory cytokine responses in the lungs. We conclude that hemagglutinin, neuraminidase, or both antigen-expressing VLPs derived from these influenza B viruses that were circulating during the 2020/21 season provide cross-protections against mismatched Victoria lineage virus (B/Colorado/06/2017) challenge infections.

Influenza A and B Virus-Triggered Epithelial–Mesenchymal Transition Is Relevant to the Binding Ability of NA to Latent TGF-β

Epithelial–mesenchymal transition (EMT) is an important mechanism of lung tissue repair after injury, but excessive EMT may lead to pulmonary fibrosis, respiratory failure, and even death. The EMT triggered by influenza A virus (IAV) and influenza B virus (IBV) is not well understood. We hypothesized that there was difference in EMT induced by different influenza virus strains. Here we discovered that both IAV [A/WSN/1933 (H1N1), WSN] and IBV (B/Yamagata/16/88, Yamagata) infection caused EMT in mouse lung and A549 cells, and more EMT-related genes were detected in mice and cells infected with WSN than those infected with Yamagata. Neuraminidase (NA) of IAV is able to activate latent TGF-β and the downstream TGF-β signaling pathway, which play a vital role in EMT. We observed that IAV (WSN) triggered more activated TGF-β expression and stronger TGF-β/smad2 signaling pathway than IBV (Yamagata). Most importantly, WSN NA combined more latent TGF-β than Yamagata NA in A549 cells. Collectively, these data demonstrate that both IAV and IBV induce TGF-β/smad2 signaling pathway to promote EMT, which might depend on the binding ability of NA to latent TGF-β.

FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines.

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.

Epidemiology of influenza B infection in the state of Rio Grande do Sul, Brazil, from 2003 to 2019.

Influenza B virus (IBV) causes respiratory tract infections with mild, moderate, or life-threatening symptoms. This study describes the epidemiology of IBV infection in Rio Grande do Sul (RS), Brazil, over 17 years. Nasopharyngeal samples were collected from outpatients presenting acute respiratory illness (ARI) between 2003 and 2019, and from inpatients with severe acute respiratory infection (SARI) from 2009 to 2019. IBV was detected by immunofluorescence assay or quantitative real-time polymerase chain reaction; demographic and clinical data were analyzed.In total, 48,656 cases of respiratory infection were analyzed, of which 20.45% were ARI, and 79.46% were SARI. Respiratory viruses accounted for 22.59% and 37.47% of the cases of ARI and SARI, respectively. Considering respiratory viral infections, 17.10% of ARI and 3.06% of SARI were associated with IBV. IBV circulated year-round in RS, with an increase in autumn and winter, peaking in July (p = .005). IBV infection showed an association with age, and most outpatients positive for IBV were between 10 and 49 years old, whereas IBV infection in SARI affected mainly individuals ≤ 1 year or ≥ 60 years old. No significant association was found between sex and IBV infection. Coryza, sore throat, and myalgia were associated with ARI (p < .001). Moreover, 3.18% of the deaths associated with respiratory virus infection were positive for IBV; notably, cardiopathy (p < .001), metabolic disease (p < .001), and smoking (p = .003) were associated to fatality in IBV infection.IBV is an important cause of severe respiratory infections, and the fatality risk is high in individuals with cardiopathy and metabolic diseases.

Cortisol and perceived stress are associated with cytokines levels in patients infected with influenza B virus

Influenza B virus (IBV) causes respiratory infectious disease. Cytokines are important immune mediators during infectious diseases. Cortisol and stress have been related to respiratory infection susceptibility and cytokine regulation. Little is known about systemic cytokines, cortisol, and perceived stress in the early stages of IBV infection. We researched the systemic cytokines and cortisol, as well as the perceived stress and blood cell count in patients infected with IBV. The diagnosis was established using the Luminex xTAG RVP kit and confirmed with qRT-PCR for IBV viral load. The perceived stress was evaluated using the perceived stress scale (PSS-10). Twenty-five plasma cytokines were determined using multiplex immunoassay and cortisol by ELISA. The leukocyte differential count was measured with a standard laboratory protocol. Th1, Th17, and IL-10 cytokines were higher in IBV infected patients (P < 0.05). Leukocytes and neutrophil count negatively correlated with viral load (P < 0.05). Perceived stress had a negative effect on monocyte and systemic cytokines in IBV infected patients (P < 0.05). Cortisol was higher in patients infected with IBV and correlated positively with CCL20 (P < 0.05). Cortisol showed a positive effect on most of the systemic cytokines (P < 0.05). In conclusion, a cytokine pattern was found in IBV infected patients, as well as the possible role of leukocyte counts in the control of IBV. Our results suggest the importance of cortisol and perceived stress on systemic cytokines in patients infected with IBV, but more studies are needed to understand their role in cytokine production in respiratory infectious disease.

Impact of Porcine Arterivirus, Influenza B, and Their Coinfection on Antiviral Response in the Porcine Lung.

Interferon (IFN) cytokines induce an autonomous antiviral state in cells of the infected site to restrict virus spreading and critically regulate overall antiviral response. The antiviral state leads to host protection through expression of hundreds of IFN-stimulated genes that restrict viral infection through multiple mechanisms, for example, directly in viral genome degradation and indirectly through cellular metabolic inhibition. Young pigs were split into four treatment groups: control, porcine reproductive and respiratory syndrome virus (PRRSV, also known as porcine arterivirus) infected, influenza B virus (IBV) infected, and IBV/PRRSV coinfection. Lung tissue was collected at 3, 5, and 7 days post infection (dpi) for control, PRRSV and IBV/PRRSV coinfection, and at 3 and 5 dpi for IBV. Transcriptomic analysis, using usegalaxy.org tools, was performed against the S.scrofa 11.1 reference genome. Differentially expressed gene (DEG) analysis was carried out using DeSeq2 based on the model treatment + dpi + treatment:dpi + E. Downstream analysis examined the interaction of DEG at each dpi for over-enriched gene ontology (G.O.) terms and pathways. Comparisons of the infected groups vs. the controls yielded a total of (n = 1412) DEGs for the PRRSV group and (n = 1578) for the IBV/PRRSV group across all timepoints. The IBV group had (n = 64) total DEGs across 3 and 5 dpi. Expression data were considered statistically significant based on false discovery rate (FDR) ⫹ 0.1. Venn diagram comparisons of the DEGs across dpi showed that groups shared only 16 DEGs at 3 dpi, no DEGs were shared at 5 dpi, and for 7 dpi, only the PRRSV and IBV/PRRSV groups were compared and shared a total of 43 DEGs. Across the comparisons, differential expression was observed in antiviral genes such as IRF1, MX1, and OAS2. The IBV and IBV/PRRSV groups showed higher expression of antiviral genes at earlier dpi than the PRRSV group. Additionally, downregulated genes from the comparisons clustered around Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways effecting lung development and cellular integrity. Early expression of host IFN and antiviral genes may lead to viral RNA degradation, and assembly and transcription inhibition in the IBV infections. In comparison, expression of antiviral genes in the PRRSV group decreased across time. The decrease may explain why PRRSV infections persist, while IBV clears. Moreover, all infected groups showed prolonged upregulation in neutrophil degranulation pathway activity, possibly exacerbating symptomatic lung lesion pathology seen in these respiratory infections.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus invitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

Comparative Transcriptomic Analysis of Rhinovirus and Influenza Virus Infection.

Rhinovirus (RV) and influenza virus are the most frequently detected respiratory viruses among adult patients with community acquired pneumonia. Previous clinical studies have identified major differences in the clinical presentations and inflammatory or immune response during these infections. A systematic transcriptomic analysis directly comparing influenza and RV is lacking. Here, we sought to compare the transcriptomic response to these viral infections. Human airway epithelial Calu-3 cells were infected with contemporary clinical isolates of RV, influenza A virus (IAV), or influenza B virus (IBV). Host gene expression was determined using RNA-seq. Differentially expressed genes (DEGs) with respect to mock-infected cells were identified using the overlapping gene-set of four different statistical models. Transcriptomic analysis showed that RV-infected cells have a more blunted host response with fewer DEGs than IAV or IBV-infected cells. IFNL1 and CXCL10 were among the most upregulated DEGs during RV, IAV, and IBV infection. Other DEGs that were highly expressed for all 3 viruses were mainly genes related to type I or type III interferons (RSAD2, IDO1) and chemokines (CXCL11). Notably, ICAM5, a known receptor for enterovirus D68, was highly expressed during RV infection only. Gene Set Enrichment Analysis (GSEA) confirmed that pathways associated with interferon response, innate immunity, or regulation of inflammatory response, were most perturbed for all three viruses. Network analysis showed that steroid-related pathways were enriched. Taken together, our data using contemporary virus strains suggests that genes related to interferon and chemokine predominated the host response associated with RV, IAV, and IBV infection. Several highly expressed genes, especially ICAM5 which is preferentially-induced during RV infection, deserve further investigation.

Epidemiological Study of Influenza A, Influenza B, Enterovirus 71, and Coxsackievirus A16 Among Children in the Central Region of Zhejiang Province, China.

Influenza A virus (IAV), influenza B virus (IBV), enterovirus 71 (EV71), and coxsackievirus A16 (CVA16) are common pathogens for viral infection in children. In order to investigate the epidemiology of these four viral infections in the central region of Zhejiang province, China, 10,638 respiratory secretion samples previously tested for IAV and IBV, and 6427 whole blood samples previously tested for EV71 and CVA16 detection were analyzed retrospectively. The present data shows that viral infections with these four viruses featured with distinct seasonal patterns. Both IAV and IBV infections more frequently occurred in winter, while infections with the two enteroviruses peaked in summer with high positive rates in other months. The most susceptible ages for IAV, IBV, EV71, and CVA16 were 2-7years old, 4-6years old, 1-3years old, and 1-2years old, respectively. It was recommended that children in the central region of Zhejiang Province should be vaccinated for influenza by the end of October every year, especially between the ages of 2 and 7years old and children in age from 1 to 3years old should be paid more attention all year round for EV71 and CVA16 infection. Moreover, the female gender appeared to be a risk factor only for IBV infection, while CVA16 inflicted more infection in young children. This study revealed that season, age, and gender should be taken into consideration when devising vaccination schedules for children in the central region of Zhejiang.

Robust induction of interferon and interferon-stimulated gene expression by influenza B/Yamagata lineage virus infection of A549 cells.

Influenza B virus (IBV) belongs to the Orthomyxoviridae family and generally causes sporadic epidemics but is occasionally deadly to individuals. The current research mainly focuses on clinical and pathological characteristics of IBV. However, to better prevent or treat the disease, one must determine the strategies developed by IBV to invade and disrupt 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 able to infect alveolar epithelial cells (A549) cells, with good potential for replication. To identify host cellular responses against IBV infection, differentially expressed genes (DEGs) were obtained using RNA sequencing. The GO and KEGG pathway term enrichment analyses with the DEGs were performed, and we found that the DEGs were primary involved in metabolic processes and cellular function, which may be related to the host response, including the innate immune response against the virus. Our transcriptome analysis results demonstrated robust induction of interferon and interferon-stimulated gene expression by IBV in human cells during the early stages of infection, providing a foundation for further studies focused on antiviral drug development and interactions between the virus and host.

Next-Generation Sequencing Analysis of Cellular Response to Influenza B Virus Infection.

Influenza B virus (IBV) is a respiratory pathogen that infects humans and causes seasonal influenza epidemics. However, cellular response to IBV infection in humans and mechanisms of host-mediated restriction of IBV replication are not thoroughly understood. In this study, we used next-generation sequencing (NGS) to perform transcriptome profiling of IBV-infected human lung epithelial A549 cells at 0, 6, 12, and 24 h post infection (hpi) and characterized the cellular gene expression dynamics. We observed that more than 4000 host genes were differentially regulated during the study period, which included up regulation of genes encoding proteins, having a role in the innate antiviral immune responses, immune activation, cellular metabolism, autophagy, and apoptosis, as well as down regulation of genes involved in mitosis and cell proliferation. Further analysis of RNA-Seq data coupled with RT-qPCR validation collectively showed that double-strand RNA recognition pathways, including retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3), were substantially activated following IBV infection. Taken together, these results provide important initial insights into the intimate interaction between IBV and lung epithelial cells, which can be further explored towards elucidation of the cellular mechanisms in restriction or elimination of IBV infections in humans.

Establishment of RIG-I knockout 293T cell line and its effect on the replication of influenza B virus

The CRISPR/Cas9 gene editing technology directs Cas9 protein to recognize, bind and cleave the target site specifically by using artificial single-guide RNA (sgRNA), through non-homologous end joining or homologous end-recombinant repair mechanisms of cells, which can be engineered to knockout or knock-in of genomes. RIG-I is a pattern recognition receptor that recognizes the 5'-triphosphate-containing RNA in the cytoplasm and activates IRF3/7 and NF-κB by interacting with the downstream signaling molecule MAVS, thus initiating the expression of type I interferons and inflammatory factors. Previous studies found that influenza B virus (IBV) can up-regulate the expression of RIG-I. In the present study, to explore whether RIG-I is the major receptor for IBV to active the antiviral innate immune response and its effect on IBV replication, RIG-I gene in 293T cells was knocked out by CRISPR-Cas9 system, and a stable RIG-I knockout 293T (RIG-I(-/-) 293T) cell line was screened by puromycin pressure. The results of Western blotting showed that RIG-I was not expressed in this cell line after IBV or Sendai virus (SeV) infection, indicating that the RIG-I(-/-) 293T cell line was successfully constructed. The transcription levels of interferons, inflammatory factors and interferon-stimulated genes in RIG-I(-/-) 293T cells which were infected by IBV decreased significantly compared with those in wild-type 293T cells. Moreover, the phosphorylation of p65 and IRF3 were not detected in IBV or SeV infected RIG-I(-/-) 293T cells. It is indicated that the expression of cytokines mainly depends on the RIG-I-mediated signaling pathway at the early stage of IBV infection. Furthermore, the multi-step growth curves of IBV in the wild type and RIG-I(-/-) 293T cells showed that RIG-I inhibited the replication of IBV. Collectively, the RIG-I knockout 293T cell line was successfully constructed. We found that RIG-I is the main receptor for IBV to active the antiviral innate immune response and is critical for inhibiting IBV replication, which lays the foundation for further study of IBV infection mechanism.