Effects of maoto (ma-huang-tang) on host lipid mediator and transcriptome signature in influenza virus infection.

Maoto, a traditional kampo medicine, has been clinically prescribed for influenza infection and is reported to relieve symptoms and tissue damage. In this study, we evaluated the effects of maoto as an herbal multi-compound medicine on host responses in a mouse model of influenza infection. On the fifth day of oral administration to mice intranasally infected with influenza virus [A/PR/8/34 (H1N1)], maoto significantly improved survival rate, decreased viral titer, and ameliorated the infection-induced phenotype as compared with control mice. Analysis of the lung and plasma transcriptome and lipid mediator metabolite profile showed that maoto altered the profile of lipid mediators derived from ω-6 and ω-3 fatty acids to restore a normal state, and significantly up-regulated the expression of macrophage- and T-cell-related genes. Collectively, these results suggest that maoto regulates the host's inflammatory response by altering the lipid mediator profile and thereby ameliorating the symptoms of influenza.

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.

Of mice and men: the host response to influenza virus infection.

Influenza virus (IV) infections represent a very serious public health problem. At present, no established biomarkers exist to support diagnosis for respiratory viral infections and more importantly for severe IV disease. Studies in animal models are extremely important to understand the biological, genetic, and environmental factors that contribute to severe IV disease and to validate biomarker candidates from human studies. However, mouse human cross-species comparisons are often compromised by the fact that animal studies concentrate on the infected lungs, whereas in humans almost all studies use peripheral blood from patients. In addition, human studies do not consider genetic background as variable although human populations are genetically very diverse. Therefore, in this study, we performed a cross-species gene expression study of the peripheral blood from human patients and from the highly genetically diverse Collaborative Cross (CC) mouse population after IV infection. Our results demonstrate that changes of gene expression in individual genes are highly similar in mice and humans. The top-regulated genes in humans were also differentially regulated in mice. We conclude that the mouse is a highly valuable in vivo model system to validate and to discover gene candidates which can be used as biomarkers in humans. Furthermore, mouse studies allow confirmation of findings in humans in a well-controlled experimental system adding enormous value to the understanding of expression and function of human candidate genes.

Experimental infection of pigs with H1 and H3 influenza A viruses of swine by using intranasal nebulization.

BACKGROUND: Experimental infection of pigs via direct intranasal or intratracheal inoculation has been mainly used to study the infectious process of influenza A viruses of swine (IAVs-S). Nebulization is known to be an alternative method for inoculating pigs with IAVs-S, because larger quantities of virus potentially can be delivered throughout the respiratory tract. However, there is very little data on the experimental infection of pigs by inhalation using nebulizer. In the current study, we used intranasal nebulization to inoculate pigs with 9 different IAVs-S-3 H1N1, 2 H1N2, and 4 H3N2 strains. We then assessed the process of infection by evaluating the clinical signs, nasal and oral viral shedding, and seroconversion rates of the pigs inoculated. RESULTS: Lethargy and sneezing were the predominant clinical signs among pigs inoculated with 7 of the 9 strains evaluated; the remaining 2 strains (1 H1N1 and 1 H1N2 isolate) failed to induce any clinical signs throughout the experiments. Significantly increased rectal temperatures were observed with an H1N1 or H3N2 strains between 1 and 3 days post-inoculation (dpi). In addition, patterns of nasal viral shedding differed among the strains: nasal viral shedding began on 1 dpi for 6 strains, with all 9 viruses being shed from 2 to 5 dpi. The detection of viral shedding was less sensitive from oral samples than nasal secretions. Viral shedding was not detected in either nasal or oral swabs after 10 dpi. According to hemagglutination-inhibition assays, all inoculated pigs had seroconverted to the inoculating virus by 14 dpi, with titers ranging from 10 to 320. CONCLUSIONS: Our current findings show that intranasal nebulization successfully established IAV-S infections in pigs and demonstrate that clinical signs, viral shedding, and host immune responses varied among the strains inoculated.

Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection.

Long-term exposure to PM2.5 lowers influenza virus resistance via down-regulating pulmonary macrophage Kdm6a and mediates histones modification in IL-6 and IFN-ß promoter regions.

Atmospheric particulates, especially PM2.5, not only damage the respiratory system, but also play important roles in pulmonary immunity. China is influenced by atmospheric diffusion conditions, industrial manufacturers, and heating and discharging. PM2.5 levels in the air rise substantially in the winter, which is also a period of flu high-incidence. Although an epidemiological link exists between PM2.5 and flu, we do not understand how long-term PM2.5 inhalation affects pulmonary immunity and the influenza virus response. Our study has prepared an in vivo PM2.5 mouse pharyngeal wall drop-in model and has found that PM2.5 exposure leads to mouse inflammatory injuries and furthers influenza A infection. Our results suggest that short-term exposure to PM2.5 significantly enhances the survival rate of influenza A-contaminated mice, while long-term PM2.5 inhalation lowers the capacity of pulmonary macrophages to secrete IL-6 and IFN-ß. A disorder in the pulmonary innate defense system results in increased death rates following influenza infection. On a macromolecular level, this mechamism involves Kdm6a down-regulation after long-term exposure to PM 2.5 and a resultant increase in H3K4 and H3K9 methylation in IL-6 and IFN-ß promoter regions. In summary, PM2.5 causes injuries of lung tissue cells and downregulates immune defense mechanisms in the lung.

Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor.

The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated nonfunctional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice, derived from wild Mus musculus castaneus, possess a seemingly intact Mx1 gene but are highly susceptible to influenza A virus challenge. To determine whether the enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ mice. Adult animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferon-treated mice carrying the CAST Mx1 allele than in those of mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, the enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life. IMPORTANCE: Although the crystal structure of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occurring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect the enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce the influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity.

Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression.

Induction of microglia activation after infection with the non-neurotropic A/CA/04/2009 H1N1 influenza virus.

Although influenza is primarily a respiratory disease, it has been shown, in some cases, to induce encephalitis, including people acutely infected with the pandemic A/California/04/2009 (CA/09) H1N1 virus. Based on previous studies showing that the highly pathogenic avian influenza (HPAI) A/Vietnam/1203/2004 H5N1 virus was neurotropic, induced CNS inflammation and a transient parkinsonism, we examined the neurotropic and inflammatory potential of the CA/09 H1N1 virus in mice. Following intranasal inoculation, we found no evidence for CA/09 H1N1 virus neurotropism in the enteric, peripheral or central nervous systems. We did, however, observe a robust increase in microglial activity in the brain characterized by an increase in the number of activated Iba-1-positive microglia in the substantia nigra (SN) and the hippocampus, despite the absence of virus in the brain. qPCR analysis in SN tissue showed that the induction of microgliosis was preceded by reduced gene expression of the neurotrophic factors bdnf, and gdnf and increases in the immune modulatory chemokine chemokine (C-C motif) ligand 4 (ccl4). We also noted changes in the expression of transforming growth factor-1 (tgfß1) in the SN starting at 7 days post-infection (dpi) that was sustained through 21 dpi, coupled with increases in arginase-1 (arg1) and csf1, M2 markers for microglia. Given that neuroinflammation contributes to generation and progression of a number of neurodegenerative disorders, these findings have significant implications as they highlight the possibility that influenza and perhaps other non-neurotropic viruses can initiate inflammatory signals via microglia activation in the brain and contribute to, but not necessarily be the primary cause of, neurodegenerative disorders.

Cigarette smoke silences innate lymphoid cell function and facilitates an exacerbated type I interleukin-33-dependent response to infection.

Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease.

Asthma increases susceptibility to heterologous but not homologous secondary influenza.

UNLABELLED: Asthma was the most common comorbidity observed among patients hospitalized with influenza A virus during the 2009 pandemic. However, little remains known about how the asthmatic phenotype influences protective immune responses against respiratory viral pathogens. Using the ovalbumin-induced allergic lung inflammation model, we found that asthmatic mice, unlike nonasthmatic mice, were highly susceptible to secondary heterologous virus challenge. While primary virus infection generated protective memory immune responses against homologous secondary virus challenge in both asthmatic and nonasthmatic mice, full protection against heterologous A/California/04/2009 (CA04) viral infection was observed only in nonasthmatic mice. Significant reductions in CA04-specific IgA, IgG, and IgM levels and in CA04-neutralizing activity of bronchoalveolar lavage fluid (BALF) was observed following secondary CA04 challenge of PR8-immunized asthmatic mice. Furthermore, transfer of immune BALF obtained from nonasthmatic, but not asthmatic, donors following secondary viral infection generated protection against CA04 in naive recipients. Nonspecific B-cell activation by CpG inoculation restored protection in PR8-immunized, CA04-challenged asthmatic mice. These results demonstrate a causal link between defective mucosal antibody responses and the heightened susceptibility of asthmatic mice to influenza infection and provide a mechanistic explanation for the observation that asthma was a major risk factor during the 2009 influenza pandemic. IMPORTANCE: The prevalence of asthma worldwide is increasing each year. Unfortunately, there is no cure for asthma. Asthmatic individuals not only suffer from consistent wheezing and coughing but are also believed to be more prone to serious lung infections that result in bronchitis and pneumonia. However, little is known about the influence of asthma on host mucosal immunity. Here we show that antibody responses during secondary heterologous influenza infections are suboptimal and that this is responsible for the increased mortality in asthmatic mice from viral infections. Understanding the mechanism of increased susceptibility will aid in developing new antiviral therapies for asthmatic patients.

Randomized controlled ferret study to assess the direct impact of 2008-09 trivalent inactivated influenza vaccine on A(H1N1)pdm09 disease risk.

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008-09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008-09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008-09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008-09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.

Influenza A(H1N1)pdm09 virus infection in Norwegian swine herds 2009/10: the risk of human to swine transmission.

Influenza A viruses cause respiratory infection in humans and pigs, and some serotypes can be transmitted between these species. The emergence of influenza A(H1N1)pdm09 virus infections in the spring of 2009 quickly led to a worldwide pandemic in humans, with subsequent introduction of the virus to pig populations. Following a widespread infection in the human population in Norway, influenza A(H1N1)pdm09 virus was introduced to the influenza A naïve Norwegian pig population, and within a few months pigs in more than one third of Norwegian swine herds had antibodies against the virus. A cross-sectional study was performed on all swine nucleus and multiplier herds in Norway to analyze risk factors for introduction of infection, and the preventive effects of recommended biosecurity practices. A surveillance program provided information on infection status of the study herds, and a questionnaire was administered to all 118 nucleus and multiplier herds to collect information on herd variables. The surveillance program revealed that pigs in 42% of the herds had antibodies against influenza A(H1N1)pdm09 virus. The incidence of serologically positive pigs was similar in both multiplier herds (41%) and closed nucleus herds (43%). Multivariable logistic regression showed that presence of farm staff with influenza-like illness (ILI) (OR=4.15, CI 1.5-11.4, p=0.005) and herd size (OR=1.01, CI 1-1.02, p=0.009) were risk factors for infection. The rapid and widespread seroconversion for antibodies against influenza A(H1N1)pdm09 virus in the Norwegian pig population can be explained by the emergence of a novel virus that is readily transmitted between people and swine in a largely susceptible population of humans, and an entirely naïve population of pigs.

A review of influenza viruses in seals and the implications for public health.

With the public health community and general population more concerned with influenza than ever before, it is important to explore all potential vectors for the next pandemic. Potential sources include pinnipeds (seals and sea lions). Between subsistence hunting, managed animals, and shared shoreline habitat, humans are more likely to interact with pinnipeds than any other marine mammal. This paper summarizes documented influenza events and research on Phocidae and Otariidae and discusses the potential challenges they present to public health.

Glutathione peroxidase-1 reduces influenza A virus-induced lung inflammation.

Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus-induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may protect lungs from such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus-induced lung inflammation in vivo. Male wild-type (WT) or GPx-1(-/-) mice were inoculated with HKx31 (H3N2, 1 × 10(4) plaque-forming units), and bronchoalveolar lavage fluid (BALF)/lung compartments were analyzed on Days 3 and 7 after infection for inflammatory marker expression, histology, and viral titer. WT mice infected with HKx31 had significantly more BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 compared with naive WT animals (n = 5-8; P < 0.05). However, infected GPx-1(-/-) mice had significantly more BALF inflammation, which included more total cells, macrophages, and neutrophils, compared with WT mice, and this was abolished by treatment with the GPx mimetic ebselen. BALF inflammation persisted in GPx-1(-/-) mice on Day 10 after infection, and GPx-1(-/-) mice had significantly more influenza-specific CD8(+) T cells in spleen compared with WT mice (n = 3-4; P < 0.05). Infected GPx-1(-/-) mice had greater peribronchial and parenchymal inflammation than WT mice, and viral titer was significantly reduced in GPx-1(-/-) mice at Day 3 (n = 5; P < 0.05). Gene expression analysis revealed that infected GPx-1(-/-) mice had higher whole lung TNF-α, macrophage inflammatory protein (MIP)-1α, MIP-2, KC, and matrix metalloproteinase (MMP)-12 mRNA compared with infected WT mice. GPx-1(-/-) mice had more MIP-2 protein in BALF at Day 3 and more active MMP-9 protease in BALF at Days 3 and 7 than WT mice. These data indicate that GPx-1 reduces influenza A virus-induced lung inflammation.

Dose-response assessment for influenza A virus based on data sets of infection with its live attenuated reassortants.

Reported data sets on infection of volunteers challenged with wild-type influenza A virus at graded doses are few. Alternatively, we aimed at developing a dose-response assessment for this virus based on the data sets for its live attenuated reassortants. Eleven data sets for live attenuated reassortants that were fit to beta-Poisson and exponential dose-response models. Dose-response relationships for those reassortants were characterized by pooling analysis of the data sets with respect to virus subtype (H1N1 or H3N2), attenuation method (cold-adapted or avian-human gene reassortment), and human age (adults or children). Furthermore, by comparing the above data sets to a limited number of reported data sets for wild-type virus, we quantified the degree of attenuation of wild-type virus with gene reassortment and estimated its infectivity. As a result, dose-response relationships of all reassortants were best described by a beta-Poisson model. Virus subtype and human age were significant factors determining the dose-response relationship, whereas attenuation method affected only the relationship of H1N1 virus infection to adults. The data sets for H3N2 wild-type virus could be pooled with those for its reassortants on the assumption that the gene reassortment attenuates wild-type virus by at least 63 times and most likely 1,070 times. Considering this most likely degree of attenuation, 10% infectious dose of H3N2 wild-type virus for adults was estimated at 18 TCID50 (95% CI = 8.8-35 TCID50). The infectivity of wild-type H1N1 virus remains unknown as the data set pooling was unsuccessful.

[Functional morphology of the vagus nerve nuclei changes in the medulla oblongata (N. Ambiguus, N. Dorsalis), induced by influenza a (H(3)N(I)) in experiment].

Morphological changes of the brain cortex IV-V layers, the structures of n. ambiguus, n. dorsalis and n. vagus ganglia on the model of influenza virus A strains (H3NI) MLD50 in number 50 microns intranasal inoculation in mice aged 6-8 weeks were studied. For assessment of virus-induced pathology 2 series of experiments were carried out. Electron microscopy, morphometric and histological methods, including by Nissl stain were used. LD dose, daily loss of body weight with access to the so-called "endpoint" determined previously. Experimental period from 48 hours to 12 days. It is shown that in n.vagus stem structures in the medulla oblongata (n. dorsalis, n. ambiguus) have the mosaic and the polymorphic nature of the changes - signs of influenza virus cytotropic effect, such as swelling, vacuolation, chromatolysis, less pyknosis and hyperchromatosis. In the period of the greatest weight loss and expressed «endpoint¼ irreversible changes in the stem structures associated with n. vagus - apoptotic nuclei and neurons massive edema, lipofuscin accumulation have taken place. Results of the study suggest that the parasympathetic nervous system (n. vagus) may be one of the possible route of influenza A virus (H3NI) genomic structures transnerval invasion in the central nervous system during experimental infection.

Immunopathogenic and antibacterial effects of H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79, and 82.

The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.

Influenza virus aerosol exposure and analytical system for ferrets.

Understanding the transmission ability of newly emerging influenza viruses is central to the development of public health preparedness and prevention strategies. Animals are used to model influenza virus infection and transmission, but the routinely used intranasal inoculation of a liquid virus suspension does not reflect natural infection. We report the development of an inoculation method that delivers an influenza virus aerosol inoculum to ferrets and the characterization of size distribution and viable virus present in aerosols shed from infected ferrets during normal breathing and sneezing. By comparing virus deposition, infectivity, virulence, and transmissibility among animals inoculated intranasally or by aerosols with a human (H3N2) or avian (H5N1) influenza virus, we demonstrate that aerosol inoculations more closely resemble a natural, airborne influenza virus infection and that viable virus is measurable in droplets and droplet nuclei exhaled by infected ferrets. These methods will provide improved risk assessment of emerging influenza viruses that pose a threat to public health.