H1N1 Infection Research Articles

Integrated analysis of miRNA-mRNA expression of newly emerging swine H3N2 influenza virus cross-species infection with tree shrews

BackgroundCross-species transmission of zoonotic IAVs to humans is potentially widespread and lethal, posing a great threat to human health, and their cross-species transmission mechanism has attracted much attention. miRNAs have been shown to be involved in the regulation of IAVs infection and immunity, however, few studies have focused on the molecular mechanisms underlying miRNAs and mRNAs expression after IAVs cross-species infection.MethodsWe used tree shrews, a close relative of primates, as a model and used RNA-Seq and bioinformatics tools to analyze the expression profiles of DEMs and DEGs in the nasal turbinate tissue at different time points after the newly emerged swine influenza A virus SW2783 cross-species infection with tree shrews, and miRNA-mRNA interaction maps were constructed and verified by RT-qPCR, miRNA transfection and luciferase reporter assay.Results14 DEMs were screened based on functional analysis and interaction map, miR-760-3p, miR-449b-2, miR-30e-3p, and miR-429 were involved in the signal transduction process of replication and proliferation after infection, miR-324-3p, miR-1301-1, miR-103-1, miR-134-5p, miR-29a, miR-31, miR-16b, miR-34a, and miR-125b participate in negative feedback regulation of genes related to the immune function of the body to activate the antiviral immune response, and miR-106b-3p may be related to the cross-species infection potential of SW2783, and the expression level of these miRNAs varies in different days after infection.ConclusionsThe miRNA regulatory networks were constructed and 14 DEMs were identified, some of them can affect the replication and proliferation of viruses by regulating signal transduction, while others can play an antiviral role by regulating the immune response. It indicates that abnormal expression of miRNAs plays a crucial role in the regulation of cross-species IAVs infection, which lays a solid foundation for further exploration of the molecular regulatory mechanism of miRNAs in IAVs cross-species infection and anti-influenza virus targets.

Myositis in H1N1 Infection Compounds to Myasthenic Crisis.

Infection is an important trigger of myasthenic crisis (MC), and those infections manifest with pneumonia and muscle involvement may result in more frequent MC. We report two myasthenia gravis (MG) patients with H1N1 infection, and highlight the reasons for deterioration. Two patients with MG had H1N1 infection. The diagnosis of MG was confirmed by neostigmine, repetitive nerve stimulation, and anti-acetylcholine receptor antibody tests. H1N1 was confirmed by nucleic acid detection study, and myositis by creatinine kinase. The patient with pneumonia and myositis had MC needing mechanical ventilation for 10 days, and the other patient without myositis did not have MC. They were treated with oseltamivir 75 mg twice daily for 5 days, and the patients with MC received ceftriaxone intravenously. Both the patients were on prednisolone and azathioprine, and none received prior H1N1 vaccination. The lady with MC with myositis was discharged on day 27 in wheelchair bound state, and the other one patient without myositis or MC was discharged on 6th day with full recovery. These patients highlight the need for evaluation for myositis along with pneumonia in the MG patients with H1N1 infection. Vaccination in MG patients on immunosuppression may be useful.

Kako se pripremiti za pandemiju ptičjeg gripa H5N1 - pouke iz pandemije influence H1N1 iz 1918

In this paper, we highlight the significant challenges encountered during the influenza H1N1 pandemic of 1918. These lessons are being utilized to prepare for potential avian influenza H5N1 pandemic outbreaks, as this virus will impact both humans and food-serving animals. Preparation for this threat is crucial because H5N1 infections are already affecting the United States and many other countries.

Identification of dihydroorotate dehydrogenase inhibitor, vidofludimus, as a potent and novel inhibitor for influenza virus.

Influenza A virus (IAV) infection causes respiratory disease. Recently, infection of IAV H5N1 among mammals are reported in farmed mink. Therefore, to discover antivirals against IAV, we screened a compound library by using the RNA-dependent RNA polymerase (RdRp) assay system derived from H5N1 IAV including a drug-resistant PA mutant (I38T) and a viral polymerase activity enhancing PB2 mutant (T271A). Upon screening, we found vidofludimus can be served as a potential inhibitor for IAV. Vidofludimus an orally active inhibitor for dihydroorotate dehydrogenase (DHODH), a key enzyme for the cellular de novo pyrimidine biosynthesis pathway. We found that vidofludimus exerted antiviral activity against wild-type and drug-resistant mutant IAV, with effective concentrations (EC50 ) of 2.10 and 2.11 μM, respectively. The anti-IAV activity of vidofludimus was canceled by the treatment of uridine or cytidine through pyrimidine salvage synthesis pathway, or orotic acid through pyrimidine de novo synthesis pathway. This indicated that the main target of vidofludimus is DHODH in IAV RdRp expressing cells. We also produced recombinant seasonal IAV H1N1 virion and influenza B virus (IBV) RdRp assay system and confirmed vidofludimus also carried highly antiviral activity against seasonal IAV and IBV. Vidofludimus is a candidate drug for the future threat of IAV H5N1 infection among humans as well as seasonal influenza virus infection.

Highly pathogenic avian influenza virus H5N1 infection in skua and gulls in the United Kingdom, 2022.

The reemergence of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the United Kingdom in 2021-2022 has caused unprecedented epizootic events in wild birds and poultry. During the summer of 2022, there was a shift in virus transmission dynamics resulting in increased HPAIV infection in seabirds, and consequently, a profound impact on seabird populations. To understand the pathological impact of HPAIV in seabirds, we evaluated the virus antigen distribution and associated pathological changes in the tissues of great skua (Stercorarius skua, n = 8), long-tailed skua (Stercorarius longicaudus, n = 1), European herring gull (Larus argentatus, n = 5), and black-headed gull (Chroicocephalus ridibundus, n = 4), which succumbed to natural infection of HPAIV during the summer of 2022. Cases were collected from Shetland, including Scatness (mainland), No Ness (mainland), Clumlie (mainland), Hermaness (island), Fair Isle (island), Noss (island), and the West Midlands, South East, and South West of England. Grossly, gizzard ulceration was observed in one great skua and pancreatic necrosis was observed in 4 herring gulls, with intralesional viral antigen detected subsequently. Microscopical analysis revealed neuro-, pneumo-, lymphoid-, and cardiomyotropism of HPAIV H5N1, with the most common virus-associated pathological changes being pancreatic and splenic necrosis. Examination of the reproductive tract of the great skua revealed HPAIV-associated oophoritis and salpingitis, and virus replication within the oviductal epithelium. The emergence of HPAIV in seabirds Stercorariidae and Laridae, particularly during summer 2022, has challenged the dogma of HPAIV dynamics, posing a significant threat to wild bird life with potential implications for the reproductive performance of seabirds of conservation importance.

A TiO2-adenine nanocomposite as modification material for screen-printed gold electrode to detect H1N1 (swine flu) virus: A disposable genosensor

A genosensor based on TiO2-adenine nanocomposites (NCs) modified screen-printed gold electrode surface (TA-SPGE) was immobilized with haemagglutinin (HA) gene specific probe containing 5′ amine group and investigated for swine flu virus (H1N1). Bare SPGE surface was modified by drop coating of the nanocomposites. The NCs and precursors were characterized structurally and morphologically by adopting various techniques such as Fourier Transform Infrared (FTIR) spectra, X-ray Diffraction (XRD) spectra, UV–Visible spectra, Raman spectra, Zeta sizer, Field-Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive spectra (EDS) and High-Resolution Transmission Electron Microscopy (HR-TEM) with Selected Area Electron Diffraction (SAED) pattern. Electrochemical techniques termed as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) were employed for analytical detection of single-stranded viral genome in phosphate buffer saline (pH-7.2) by using methylene blue (MB) redox indicator. Moreover, the performance of designed electrode was optimized at different concentration, pH, and scan rates. The proposed genosensor revealed high sensitivity 10.6 μA/ng.cm2, lower detection limit (LOD) of 6.7 × 10-8 ng/mL and wide linear range of 3 × 10-8-3 × 10-3 ng/mL with regression coefficient of R2 = 0.989 towards swine flu virus DNA. Additionally, the modified genosensor revealed good stability and specificity against human DNA and various pathogens such as H3N2 influenza virus, Bacillus subtilis, Neisseria meningitidis, Staphylococcus aureus and Streptococcus pyogenes. The proposed genosensor is based on relatively simple methodology, quite amicable and could promptly be used as point-of-care test for swine flu virus detection.

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

Different routes of infection of H5N1 lead to changes in infecting time

Influenza virus infection can result in a wide range of clinical outcomes from asymptomatic infection to severe disease and death. While there are undoubtedly many factors that contribute to the severity of disease, one possible contributing factor that needs more investigation is the route of infection. In this study, we use previously published data from cynomolgus macaques infected with A/Vietnam/1203/04 (H5N1) via either aerosol (with and without bronchoalveolar lavages (BAL)) or a combined intrabronchial, oral, and intranasal route. We fit a mathematical model of within host viral kinetics to the data and find that when the macaques are infected via the aerosol route with subsequent BAL, the infecting time is significantly lower than for the other two groups. A lower infecting time indicates that the virus spreads from cell to cell more rapidly for aerosol infection with BAL than for the combined deposition or aerosol deposition alone. This study helps elucidate the mechanism behind different infection outcomes caused by differences in routes of infection.

First Report of Low Pathogenic Avian Influenza Subtype H9N2 in African Houbara Bustards (Chlamydotis undulata undulata) and Gamebirds in Morocco: Clinico-Pathological Findings, Molecular Characterization, and Associated Coinfections.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

HIF-1α promotes virus replication and cytokine storm in H1N1 virus-induced severe pneumonia through cellular metabolic reprogramming

The mortality of patients with severe pneumonia caused by H1N1 infection is closely related to viral replication and cytokine storm. However, the specific mechanisms triggering virus replication and cytokine storm are still not fully elucidated. Here, we identified hypoxia inducible factor-1α (HIF-1α) as one of the major host molecules that facilitates H1N1 virus replication followed by cytokine storm in alveolar epithelial cells. Specifically, HIF-1α protein expression is upregulated after H1N1 infection. Deficiency of HIF-1α attenuates pulmonary injury, viral replication and cytokine storm in vivo. In addition, viral replication and cytokine storm were inhibited after HIF-1α knockdown in vitro. Mechanistically, the invasion of H1N1 virus into alveolar epithelial cells leads to a shift in glucose metabolism to glycolysis, with rapid production of ATP and lactate. Inhibition of glycolysis significantly suppresses viral replication and inflammatory responses. Further analysis revealed that H1N1-induced HIF-1α can promote the expression of hexokinase 2 (HK2), the key enzyme of glycolysis, and then not only provide energy for the rapid replication of H1N1 virus but also produce lactate, which reduces the accumulation of the MAVS/RIG-I complex and inhibits IFN-α/β production. In conclusion, this study demonstrated that the upregulation of HIF-1α by H1N1 infection augments viral replication and cytokine storm by cellular metabolic reprogramming toward glycolysis mainly through upregulation of HK2, providing a theoretical basis for finding potential targets for the treatment of severe pneumonia caused by H1N1 infection.

Erratum: Investigation of risk factors for introduction of highly pathogenic avian influenza H5N1 infection among commercial turkey operations in the United States, 2022: a case-control study

Erratum: Investigation of risk factors for introduction of highly pathogenic avian influenza H5N1 infection among commercial turkey operations in the United States, 2022: a case-control study

A Review on H3N2 Influenza Virus

The aim of this review is to discuss the influenza A i.e H3N2 influenza virus with their characteristics. Here we present the outline of the treatment regarding the H3N2 infection, precautions to be taken. The review focused on the symptoms and prevention of H3N2 influenza virus infection. It also highlites on the recent changes in new H3N2 influenza virus and focused on vaccinations against influenza, which researchers are currently developing to study these virus. Moreover this review states that the H3N2 influenza virus are rapidly altered themselves in different ways. The study also shows that the biology of influenza and adaptation acquired by new H3N2 influenza A virus create difficulties in future and current also to predicting the changes. Study of viral growth and their transmition. This review provides support and guide the researcher in the study and better understanding of influenza viruses.

A Clinicoradiological Profile of H3N2 Outbreak in Western India

Abstract Introduction: Although H3N2 outbreaks were once rare, they have become more common in recent years. With a significant toll on health-care resources and the ability to cripple any society, the epidemiological significance of this disease is paramount. While most of the previous studies on influenza outbreaks have reported H1N1 disease, there is a scarcity of literature regarding the H3N2 clinicoradiological profile. Hereby, we present the clinicoradiological profile of a series of H3N2 cases from western India. Materials and Methods: This is a retrospective chart-based review of clinicoradiological profile of a series of cases that were reported to a tertiary care center in western India between February 2023 and March 2023. Results: A total of 10 patients had tested positive for H3N2. All patients had a history of fever lasting an average of 4.7 days, and six had varying degrees of dyspnea. One patient had gastrointestinal symptoms, and six developed tachypnea with hypoxemia requiring oxygen supplementation. One patient with multiple comorbidities required invasive mechanical ventilation and had a complicated course with a superadded bacterial infection. Out of the four patients with radiographic findings, two had atypical pneumonia/acute infective etiology. Conclusion: This study provides valuable insights into the clinical presentation and management of H3N2 infections. The findings highlight the importance of influenza vaccination and early detection of H3N2 infections to prevent severe complications. The successful outcomes of the patients in this study demonstrate the effectiveness of prompt intervention and appropriate treatment in managing H3N2 infections.

Targeting H3N2 influenza virus RNA dependent RNA polymerase dependent inhibitory activity by principal components from latex of Calotropis gigantean

The H3N2 influenza virus is spiking dramatically, which is a major concern worldwide and in India. The multifunctional hetero-trimer influenza virus RNA-dependent RNA polymerase (RdRP) is involved in the generation of viral mRNA and is crucial for viral infectivity, which is directly related to the virus’s ability to survive. The goal of the current work was to use molecular docking to determine how the RdRP protein might be affected by powerful bioactive chemicals found in Calotropis gigantia latex. By applying CB-dock 2 analysis and 2D interactions, an in-silico docking study was conducted using a GC-FID (gas chromatography with flame-ionization detection) based composition profile. Tocospiro A (15%), Amyrin (7%), and Gombasterol A were found by GC-FID to be the main phytocompounds in the latex of Calotropis gigantia. The docking result showed that ligands were effectively bound to RdRP. According to interaction studies, RdRP/ligand complexes create hydrogen bonds, van der Waals forces, pi-alkyl bonds, alkyl bonds, and pi-Sigma bonds. Therefore, it was suggested that Calotropis gigantia latex may represent a possible herbal remedy to attenuate H3N2 infections based on the above findings of the fragrance profile and docking.

Intradermal Vaccination against Influenza with a STING-Targeted Nanoparticle Combination Adjuvant Induces Superior Cross-Protective Humoral Immunity in Swine Compared with Intranasal and Intramuscular Immunization.

The development of cross-protective vaccines against the zoonotic swine influenza A virus (swIAV), a potential pandemic-causing agent, continues to be an urgent global health concern. Commercially available vaccines provide suboptimal cross-protection against circulating subtypes of swIAV, which can lead to worldwide economic losses and poor zoonosis deterrence. The limited efficacy of current swIAV vaccines demands innovative strategies for the development of next-generation vaccines. Considering that intramuscular injection is the standard route of vaccine administration in both human and veterinary medicine, the exploration of alternative strategies, such as intradermal vaccination, presents a promising avenue for vaccinology. This investigation demonstrates the first evaluation of a direct comparison between a commercially available multivalent swIAV vaccine and monovalent whole inactivated H1N2 swine influenza vaccine, delivered by intradermal, intranasal, and intramuscular routes. The monovalent vaccines were adjuvanted with NanoST, a cationic phytoglycogen-based nanoparticle that is combined with the STING agonist ADU-S100. Upon heterologous challenge, intradermal vaccination generated a stronger cross-reactive nasal and serum antibody response in pigs compared with intranasal and intramuscular vaccination. Antibodies induced by intradermal immunization also had higher avidity compared with the other routes of vaccination. Bone marrow from intradermally and intramuscularly immunized pigs had both IgG and IgA virus-specific antibody-secreting cells. These studies reveal that NanoST is a promising adjuvant system for the intradermal administration of STING-targeted influenza vaccines.

Mendelian randomization study on the causal effect of serum IgA levels on H7N9 avian influenza A virus susceptibility.

Avian influenza A viruses (IAVs) that cross the species barrier to infect humans have the potential to initiate a new pandemic. However, the host factors influencing avian IAV infection remain poorly understood. To address this knowledge gap, we conducted a two-sample Mendelian randomization (MR) analysis by integrating our in-house genome-wide association study (GWAS) of avian IAV H7N9 susceptibility (with 217 cases and 116 controls) with the largest GWAS of serum IgA levels to date (sample size 41 263). Using the inverse-variance weighted (IVW) method, we discovered that genetically decreased serum IgA levels were associated with an increased risk of H7N9 infection (β = -2.528, 95% confidence interval [CI]: -4.572 to -0.484; p = 0.015). Consistent results were obtained from three other MR methods, including robust IVW estimation (β = -2.506, 95% CI: -4.109 to -0.902; p = 0.002), generalized summary-data-based MR (GSMR) (β = -2.238, 95% CI: -4.106 to -0.602; p = 0.019), and MR-pleiotropy residual sum and outlier (MR-PRESSO) (β = -2.528, 95% CI: -4.396 to -0.892; p = 0.026). In conclusion, our analysis provided compelling evidence support a causal relationship between genetically predicted serum IgA levels and avian IAV H7N9 susceptibility.

Heterologous prime-boost H1N1 vaccination exacerbates disease following challenge with a mismatched H1N2 influenza virus in the swine model

Influenza A viruses (IAVs) pose a significant threat to both human and animal health. Developing IAV vaccine strategies able to elicit broad heterologous protection against antigenically diverse IAV strains is pivotal in effectively controlling the disease. The goal of this study was to examine the immunogenicity and protective efficacy of diverse H1N1 influenza vaccine strategies including monovalent, bivalent, and heterologous prime-boost vaccination regimens, against a mismatched H1N2 swine influenza virus. Five groups were homologous prime-boost vaccinated with either an oil-adjuvanted whole-inactivated virus (WIV) monovalent A/swine/Georgia/27480/2019 (GA19) H1N2 vaccine, a WIV monovalent A/sw/Minnesota/A02636116/2021 (MN21) H1N1 vaccine, a WIV monovalent A/California/07/2009 (CA09) H1N1, a WIV bivalent vaccine composed of CA09 and MN21, or adjuvant only (mock-vaccinated group). A sixth group was prime-vaccinated with CA09 WIV and boosted with MN21 WIV (heterologous prime-boost group). Four weeks post-boost pigs were intranasally and intratracheally challenged with A/swine/Georgia/27480/2019, an H1N2 swine IAV field isolate. Vaccine-induced protection was evaluated based on five critical parameters: (i) hemagglutination inhibiting (HAI) antibody responses, (ii) clinical scores, (iii) virus titers in nasal swabs and respiratory tissue homogenates, (iv) BALf cytology, and (v) pulmonary pathology. While all vaccination regimens induced seroprotective titers against homologous viruses, heterologous prime-boost vaccination failed to enhance HAI responses against the homologous vaccine strains compared to monovalent vaccine regimens and did not expand the scope of cross-reactive antibody responses against antigenically distinct swine and human IAVs. Mismatched vaccination regimens not only failed to confer clinical and virological protection post-challenge but also exacerbated disease and pathology. In particular, heterologous-boosted pigs showed prolonged clinical disease and increased pulmonary pathology compared to mock-vaccinated pigs. Our results demonstrated that H1-specific heterologous prime-boost vaccination, rather than enhancing cross-protection, worsened the clinical outcome and pathology after challenge with the antigenically distant A/swine/Georgia/27480/2019 strain.

Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells.

The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

Dissection of key factors correlating with H5N1 avian influenza virus driven inflammatory lung injury of chicken identified by single-cell analysis.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-β, IL1β, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 influenza A viruses on Korean swine farms, obtained using a newly developed one-step multiplex RT-qPCR assay.

Genotype 4 (G4) Eurasian avian-like lineage swine H1N1 influenza A viruses, which are reassortants containing sequences from the pandemic 2009 H1N1 virus lineage, triple-reassortant-lineage internal genes, and EA-lineage external genes, have been reported in China since 2013. These have been predominant in pig populations since 2016 and have exhibited pandemic potential. In this study, we developed a one-step multiplex RT-qPCR assay targeting the M, HA1, and PB2 genes to detect G4 and related EA H1N1 viruses, with detection limits of 1.5 × 101 copies/μL and 1.15 × 10-2 ng/μL for the purified PCR products and RNA templates, respectively. The specificity of the detection method was confirmed using various influenza virus subtypes. When the one-step multiplex RT-qPCR assay was applied to swine respiratory samples collected between 2020 and 2022 in Korea, a virus related to G4 EA H1N1 strains was detected. Phylogenetic analysis based on portions of all eight genome segments showed that the positive sample contained HA, NA, PB2, NS, and NP genes closely related to those of G4 EA H1N1 viruses, confirming the ability of our assay to accurately detect G4 EA H1N1 viruses in the field.