Lungs Of Virus-infected Mice Research Articles

Transcriptome Profiles of Highly Pathogenic Pure Avian H7N9 Virus-Infected Lungs of BALB/c Mice

Avian influenza A (H7N9) viruses emerged in China in 2013 and caused a zoonotic disease associated with a high case-fatality ratio of more than 30%. Transcriptional profiles obtained using animal models reveal host responses to the disease, thereby providing insights into disease pathogenesis. Therefore, we aimed to characterize the host responses of the H7N9 virus infected-mouse lungs in this study. First, we isolated an avian-originated H7N9 strain, which was shown to be highly pathogenic to both chickens and mice. Genomic analysis results suggested that a 12-nucleotide-insertion was present at the hemagglutinin cleavage site, and both the hemagglutinin and neuraminidase genes belonged to the Yangtze River Delta lineage. RNA sequencing results revealed 566 differentially expressed genes in the H7N9-infected lungs. Moreover, transcriptome analysis revealed that over-activated antiviral signals and intense interferon-stimulated gene products possibly contributed to the high virulence of the virus in mice. Importantly, lung concentrations of inflammatory cytokines, including interleukin-1β and interleukin-6, interferon-β, and tumor necrosis factor-α, were upregulated in response to H7N9 virus infection. Overall, the present study provided a comprehensive understanding of H7N9 virus pathogenicity and correlated host immune responses.

Quantitative single-cell interactomes in normal and virus-infected mouse lungs.

ABSTRACTMammalian organs consist of diverse, intermixed cell types that signal to each other via ligand-receptor interactions – an interactome – to ensure development, homeostasis and injury-repair. Dissecting such intercellular interactions is facilitated by rapidly growing single-cell RNA sequencing (scRNA-seq) data; however, existing computational methods are often not readily adaptable by bench scientists without advanced programming skills. Here, we describe a quantitative intuitive algorithm, coupled with an optimized experimental protocol, to construct and compare interactomes in control and Sendai virus-infected mouse lungs. A minimum of 90 cells per cell type compensates for the known gene dropout issue in scRNA-seq and achieves comparable sensitivity to bulk RNA sequencing. Cell lineage normalization after cell sorting allows cost-efficient representation of cell types of interest. A numeric representation of ligand-receptor interactions identifies, as outliers, known and potentially new interactions as well as changes upon viral infection. Our experimental and computational approaches can be generalized to other organs and human samples.

C57BL/6J and C57BL/6NJ Mice Are Differentially Susceptible to Inflammation-Associated Disease Caused by Influenza A Virus

Influenza viruses cause seasonal epidemics and sporadic pandemics, and are a major burden on human health. To develop better countermeasures and improve influenza disease outcomes, a clearer understanding of influenza pathogenesis is necessary. Host genetic factors have emerged as potential regulators of human influenza disease susceptibility, and in the mouse model, genetic background has been clearly linked to influenza pathogenicity. Here, we show that C57BL/6J mice are significantly more susceptible to disease caused by a 2009 pandemic H1N1 virus, an H7N9 virus, and a highly pathogenic H5N1 influenza virus compared to the closely related substrain, C57BL/6NJ. Mechanistically, influenza virus infection in C57BL/6J mice results in earlier presentation of edema, increased immune cell infiltration, higher levels of inflammatory cytokines, greater tissue damage, and delayed activation of regenerative processes in infected lung tissues compared to C57BL/6NJ mice. These differences are not dependent on virus replication levels. Six genes with known coding region differences between C57BL/6J and C57BL/6NJ strains exhibit increased transcript levels in influenza virus-infected mouse lungs, suggesting potential contributions to regulation of disease susceptibility. This work uncovers a previously unappreciated difference in disease susceptibility between the closely related C57BL/6J and C57BL/6NJ mice, which may be exploited in future studies to identify host factors and/or specific genetic elements that regulate host-dependent inflammatory mechanisms involved in influenza virus pathogenicity.

In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs

The pathophysiological changes that occur in lungs infected with influenza viruses are poorly understood. Here we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity. This approach allowed us to monitor and correlate several parameters and physiological changes including the spread of infection, pulmonary permeability, pulmonary perfusion speed, number of recruited neutrophils in infected lungs, and neutrophil motion in the lungs of live mice. Several physiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared with those infected with a mouse-adapted human strain. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infections.

Effects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7 Pathway in Influenza Virus Infected Mouse Lungs in a Cold Environment.

Objective We wished to investigate the effects of the traditional Chinese medicine Gui Zhi Ma Huang Ge Ban Tang on controlling influenza A virus (IAV) infection and improving inflammation in mouse lungs. Method Mice were maintained in normal and cold environments and infected with IAV by intranasal application, respectively. Real-time quantitative polymerase chain reaction was used to measure mRNA expression of TLR7, myeloid differentiation primary response 88 (MyD88), and nuclear factor-kappa B (NF-κB)p65 in the TLR7 signaling pathway and virus replication in lungs. Western blotting was used to measure expression levels of TLR7, MyD88, and NF-κB p65 proteins. Flow cytometry was used to detect the proportion of T-helper (Th)1/Th2 and Th17/T-regulatory (Treg) cells. Results Application of Gui Zhi Ma Huang Ge Ban Tang in influenza-infected mice in a cold environment showed (i) downregulation of TLR7, MyD88, and NF-κBp65; (ii) inhibition of transcriptional activities of promoters coding for TLR7, MyD88, and NF-κBp65; (iii) reduction in the proportion of Th1/Th2 and Th17/Treg cells. Conclusions Gui Zhi Ma Huang Ge Ban Tang had a good therapeutic effect on mice infected with IAV, especially in the cold environment. It could reduce lung inflammation in mice significantly and elicit an anti-influenza effect by downregulating expression of the key factors in TLR7 signaling pathway.

The RNA transcriptome of in vivo influenza virus infection reveals unique gene expression profiles in type 2 alveolar epithelial cells.

Abstract Globally, influenza virus outbreaks represent a serious threat to public health. Influenza virus infects type 2 alveolar epithelial cells (AEC) and utilizes cellular machinery in order to produce new infectious virions. During infection, multiple cell types release cytokines and other soluble factors that influence gene expression in both infected and uninfected cells. Here, the unique in vivo gene expression profiles between infected and uninfected bystander AEC from influenza virus infected mice are compared via next generation RNA-sequencing analysis. Mice were infected with PR8-expressing GFP virus (PR8-GFP). On day 3 post infection, GFP positive and negative AEC were FACS sorted from influenza virus infected mouse lungs. Transcriptome next generation RNA-sequencing analysis was performed comparing influenza-infected GFP-positive cells versus uninfected bystander GFP-negative cells from influenza virus-infected mouse lungs. Results from the sequencing data suggest a number of genes and associated pathways that are significantly differentially expressed in GFP-positive versus GFP-negative AEC. Pathways such as interferon signaling and cell death were found to be increased while Wnt signaling was decreased specifically within GFP-positive infected cells. RNA sequencing also identifies unique genes that are upregulated during virus infection, such as heatr9, that have yet to characterized during the response to influenza.

Pleiotropic Effects of Levofloxacin, Fluoroquinolone Antibiotics, against Influenza Virus-Induced Lung Injury.

Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2’-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs.

Differential microRNA expression following infection with a mouse-adapted, highly virulent avian H5N2 virus.

BackgroundMicroRNAs (miRNAs) are known to regulate various biological processes, including expression of cellular gene and virus-induced inflammation. Recently, studies have indicated that some miRNAs could regulate influenza virus replication. Due to differential sensitivities of influenza A virus strains to different species (avian and mammalian), variations in host responses may be observed. Therefore, we investigated and compared the differences in global host miRNA expression in mouse lungs infected with wild type low pathogenicity A/Aquatic bird/Korea/w81/2005 (H5N2) (w81) or mouse-adapted virulent A/Aquatic bird /Korea/ma81/2007 (H5N2) (ma81) virus.ResultsAlthough the mice infected with ma81 exhibited much greater mortality than w81-infected mice, the parental w81 virus induced a higher number of differentially expressed miRNAs compared to the ma81 virus. Between these 2 viruses, a total of 27 and 20 miRNAs were commonly expressed at 1 dpi and 3 dpi, respectively. It is noteworthy that only 9 miRNAs (miR-100-5p, miR-130a-5p, miR-146b-3p, miR-147-3p, miR-151-5p, miR-155-3p, miR-223-3p, miR-301a-3p, and miR-495-3p) were significantly upregulated in both lungs infected with either wild type w81 or the mouse-adapted ma81 strain at both time points. Notably, expression levels of miR-147-3p, miR-151-5p, miR-155-3p, and miR-223-3p were higher in the lungs of mice infected with the ma81 virus than those infected with the w81 virus. To identify potential roles of these miRNAs in regulating influenza virus replication, each group of mice was intranasally treated with each inhibitor of specifically targeting 4 miRNAs, and then challenged with 5 mouse lethal dose 50% (MLD50) of the virulent ma81 virus on the following day. Although the specific miRNA inhibitors could not completely attenuate mortality or reduce viral replication, the miR-151-5p- and miR-223-3p-inhibitors reduced mortality of inoculated mice to 70% and substantially delayed death.ConclusionsOur results suggest that the mammalian adaptation of avian influenza A virus results in a different miRNA expression pattern in lungs of virus-infected mice compared with its parental strain, and use of specific miRNA inhibitors to target genes associated with the immune response or cell death may affect virulence and virus replication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0252-0) contains supplementary material, which is available to authorized users.

Roles of p38 MAPK in the regulation of the inflammatory response to swine influenza virus-induced acute lung injury in mice.

Swine influenza virus (SIV), one of the most important zoonotic agents, is associated with major public health concerns. The current study was conducted to investigate the role of p38 mitogen-activated protein kinase (p38 MAPK) in the regulation of the inflammatory response to acute lung injury (ALI) induced by SIV of H9N2 subtype (H9N2-SIV) in mice. For this purpose, BALB/c mice were intranasally infected with 20 LD(50) of H9N2-SIV (infected group), while non-infected mice served as control (control group). To assess the effect of p38 MAPK, its specific inhibitor SB203580 was employed followed by SIV infection (SB group). At various times after infection, mouse lungs were subjected to pathological and histological observations and detection of inflammatory cytokines tumor necrosis factor α (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 and phosphorylated p38 MAPK. The obtained results showed obvious inflammatory responses, injury and raised levels of inflammatory cytokines and phosphorylated p38 MAPK in the lungs of virus-infected mice. In the mice inoculated with the virus alone, the level of phosphorylated p38 MAPK increased from day 2 and peaked at day 6 post infection (p.i.). However, SB203580 caused lower increases in inflammatory cytokines and phosphorylated p38 MAPK and a milder lung injury. These findings indicate that the activation of p38 MAPK upregulated the inflammatory responses to H9N2-SIV-induced ALI, increased its severity and promoted the production of inflammatory cytokines.

Receptor specificity of the influenza virus hemagglutinin modulates sensitivity to soluble collectins of the innate immune system and virulence in mice

The hemagglutinin (HA) glycoprotein of influenza virus binds to cell surface sialic acid (SA) to initiate infection. In this study, a mutant of influenza A virus strain BJx109 (H3N2) was plaque-purified from the lungs of virus-infected mice that had been depleted of airway macrophages. Sequence analysis identified a single amino acid substitution (S186I) in the vicinity of the receptor-binding site of HA. This substitution was associated with enhanced binding to α(2,3)-Gal-linked SA and an increased ability to infect murine airway epithelial cells. Mutant viruses were less sensitive to neutralization by mouse airway fluids and less efficient in their ability to infect murine macrophages. Moreover, infection of mice with viruses bearing the S186I substitution led to severe disease, characterized by enhanced virus replication, lung pathology and pulmonary edema. Together, these studies confirm that residue 186 of H3 subtype viruses is a critical determinant of virulence in a mouse model of influenza infection.

Enhanced antiviral antibody secretion and attenuated immunopathology during influenza virus infection in nitric oxide synthase-2-deficient mice

NOS2 gene-deficient (NOS2(-/-)) mice are less susceptible than wild-type (NOS2(+/+)) mice to infection with Influenza A virus. Virus titres in the lungs of influenza-infected NOS2(-/-) mice are significantly lower than those in NOS2(+/+) mice, with enhanced viral clearance in NOS2(-/-) mice dependent on gamma interferon (IFN-gamma). The current study was undertaken to ascertain the role of specific components of the immune response in promoting virus clearance in influenza-infected NOS2(-/-) mice. Levels of T cell- and natural killer cell-mediated cytotoxicity in the lungs of virus-infected mice were not significantly different between NOS2(+/+) and NOS2(-/-) mice. However, virus-infected NOS2(-/-) mice produced higher levels of virus-specific IgG2a antibody. Furthermore, more viable B cells and plasmablasts, along with greater levels of IFN-gamma, were found in NOS2(-/-) splenocyte cultures stimulated with B-cell mitogens. In addition to the early reduction in virus titres, clinical symptoms and proinflammatory cytokine production were attenuated in NOS2(-/-) mice. Thus, NOS2(-/-) B cells are capable of responding rapidly to influenza virus infection by proliferating and preferentially producing antibody of the IgG2a subtype. The relationship between viral load and the development of immunopathology is discussed.

Host defence mechanisms against influenza infection. II. Protection of mice with vaccines against A/Port Chalmers/1/73 and B/Hong Kong/5/72

Summary Influenza virus strains A2/Port Chalmers/1/73 (H3N2) and B/Hong Kong/5/72 were adapted to mice by serial passage of virus infected mouse lungs. The adapted viruses were used to challenge mice after they had been immunized with serial dilutions of several bivalent vaccines, containing both A/Port Chalmers and B/Hong Kong antigens. Vaccines included both whole virus and split-product preparations disrupted by Tween 80 and ether (ET) or tri-N-butyl phosphate (TBP). These bivalent vaccines induced antibodies and conferred protection to mice against challenge with approximately 30 LD50 of the adapted A2/Port Chalmers virus. Significant protection against lethality (P<0·01) was observed in groups of mice which developed haemagglutinin-inhibition antibody titres of ≥1:16 following immunization. Protection was not statistically significant in groups of mice which developed antibody titres of ≤1:8 to the A/Port Chalmers/1/73 virus following immunization with higher dilutions of certain vaccines. In a similar fashion, current bivalent vaccines containing B/Hong Kong/5/72 stimulated antibodies and conferred significant protection to immunized mice against lethal challenge with the adapted B/Hong Kong virus. Protection was demonstrated against approximately 30 LD50 of adapted virus in groups of mice which developed mean antibodies of ≥1:16. Groups of mice, which developed antibody titres of <1:8 following immunization with certain dilutions of these vaccines, were not significantly protected. These studies confirm and extend observations made with formerly circulating influenza viruses, i.e. immunization with a specific inactivated vaccine in adequate dosage confers protection against a challenge dose of virus lethal to unimmunized mice.

Further Observations on Red Cell Agglutinating Agent Present in Lungs of Virus-Infected Mice

SummaryThe red cell agglutinating agent present in the lungs of mice infected with pneumonitis virus has been subjected to further study. Maximum agglutination develops after heating the fresh lung extract at 75°C for 5 minutes. The agent is absorbed from active extracts by preparations of mouse red cells and of fresh normal mouse lungs. Mice injected with the heated agent develop active immunity against the living virus and their sera have the capacity of inhibiting red cell agglutination. The sera of rabbits immunized against living pneumonitis virus or against active heated mouse lung extract inhibit red cell agglutination. Hamsters appear to be susceptible to infection with the pneumonitis virus but attempts at continued serial passage were unsuccessful. The agglutinating agent has been purified by precipitation with ammonium sulfate. The purified agent retains its agglutinating and immunizing capacity.