Influenza Respiratory Viral Infections Research Articles

Incidence and risk factors for acute respiratory illnesses and influenza virus infections in Australian travellers to Asia

BackgroundRespiratory infections including influenza are a common cause of acute short-term morbidity in travellers and yet the risk of these infections is poorly defined. ObjectivesTo estimate the incidence density of and risk factors for acute respiratory infections (ARIs) and influenza in Australian travellers to Asia. Study designTravel-clinic attendees were prospectively identified and completed questionnaires (demographic data, travel itinerary, health and vaccination history) and also provided pre and post-travel serological samples for Influenza A and B (complement fixation test). Returned travellers with an ARI provided nasopharyngeal specimens for RT-PCR identification of respiratory viruses. ResultsIn this cohort (n=387) of predominantly (72%) short-term travellers, 58% were female, the median age was 37 years and 69% were tourists. ARIs occurred in 109 travellers (28%) translating to an incidence of 106.4 ARIs per 10,000 traveller days (95% confidence interval CI 88.6–126.7). The traveller type of missionary or aid worker was a risk factor for acquiring an ARI (p=0.03) and ARIs occurred early (<30 days) in the travel period (p=0.001). Four travellers (1%) acquired influenza A during travel translating to an incidence density of 3.4 infections per 10,000 days of travel (95% CI 1.4–8.6). Influenza vaccination was reported in 49% of travellers with a 3.5-fold higher incidence of influenza in unvaccinated travellers compared to vaccinated travellers (p=0.883). ConclusionsThis is one of the largest prospective studies estimating the incidence of respiratory infections in travellers. These findings have important implications for practitioners advising prospective travellers and for public health authorities.

Integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small RNAs in response to respiratory virus infection.

ABSTRACTWe previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection.

Pandemic and Seasonal Human Influenza Virus Infections in Domestic Cats: Prevalence, Association with Respiratory Disease, and Seasonality Patterns

Domestic cats have several features that make them ideal vehicles for interspecies transmission of influenza viruses; however, they have been largely overlooked as potential reservoirs or bridging hosts. In this study, we conducted serological surveillance to assess the prevalence of novel pandemic H1N1 as well as seasonal human influenza virus infections in domestic cats in Ohio. Four hundred serum samples collected from domestic cats (September 2009 to September 2010) were tested using a hemagglutination inhibition (HI) test. The seroprevalences of pandemic H1N1, seasonal H1N1, and H3N2 were 22.5%, 33%, and 43.5%, respectively. In addition, a significant association between clinical feline respiratory disease and influenza virus infection was documented. In this sample of cats, the prevalence of pandemic H1N1 did not follow the seasonality pattern of seasonal H1N1 or H3N2 influenza, similar to observations in humans. Pandemic H1N1 seroprevalence did not vary in relation to ambient temperature changes, while the seroprevalence of seasonal H3N2 and H1N1 influenza viruses increased with the decline of ambient temperature. Our results highlight the high prevalence of influenza virus infection in domestic cats, a seasonality pattern of influenza virus infection comparable to that in humans, and an association of infection with clinical respiratory disease.

Circulating Human Antibody-Secreting Cells during Vaccinations and Respiratory Viral Infections Are Characterized by High Specificity and Lack of Bystander Effect

Surges of serum Abs after immunization and infection are highly specific for the offending Ag, and recent studies demonstrate that vaccines induce transient increases in circulating Ab-secreting cells (ASCs). These ASCs are highly enriched but not universally specific for the immunizing Ag, suggesting that a fraction of these ASCs could arise from polyclonal bystander stimulation of preexisting memory cells to unrelated Ags. This model is proposed to explain maintenance of long-lived serological memory in the absence of Ag exposure. To test this model, we measure the ability of respiratory syncytial virus and influenza virus infection or immunizations to influenza virus, tetanus toxoid, hepatitis B Ag, and human papillomavirus to stimulate bystander memory cells specific for other major environmental Ags that represent a large fraction of the preexisting memory B compartment. Bystander or nonspecific ASC responses to respiratory syncytial virus and tetanus could not be detected above the background levels in healthy adults, despite the presence of circulating memory B cells specific for the corresponding Ags. Nonspecific ASC responses in the healthy subjects and cord blood samples were similar. In contrast, both vaccination and infection induce massive expansion of circulating Ag-specific ASCs without significant increases in the frequencies of ASCs against unrelated Ags. Hence, nonspecific stimulation of memory B cells is unlikely to contribute to the mechanisms of long-term serological memory against major human pathogens. Additionally, high specificity of circulating ASCs after antigenic challenge highlights the diagnostic value of interrogating ASCs as an ideal single-time-point diagnostic immune surrogate for serology during acute infection.

Microbiota regulates immune defense against respiratory tract influenza A virus infection

Although commensal bacteria are crucial in maintaining immune homeostasis of the intestine, the role of commensal bacteria in immune responses at other mucosal surfaces remains less clear. Here, we show that commensal microbiota composition critically regulates the generation of virus-specific CD4 and CD8 T cells and antibody responses following respiratory influenza virus infection. By using various antibiotic treatments, we found that neomycin-sensitive bacteria are associated with the induction of productive immune responses in the lung. Local or distal injection of Toll-like receptor (TLR) ligands could rescue the immune impairment in the antibiotic-treated mice. Intact microbiota provided signals leading to the expression of mRNA for pro-IL-1β and pro-IL-18 at steady state. Following influenza virus infection, inflammasome activation led to migration of dendritic cells (DCs) from the lung to the draining lymph node and T-cell priming. Our results reveal the importance of commensal microbiota in regulating immunity in the respiratory mucosa through the proper activation of inflammasomes.

Qualitatively Different Memory CD8+ T Cells Are Generated after Lymphocytic Choriomeningitis Virus and Influenza Virus Infections

Viral infections often induce robust T cell responses that are long-lived and protective. However, it is unclear to what degree systemic versus mucosal infection influences the generation of effector and memory T cells. In this study, we characterized memory CD8(+) T cells generated after respiratory influenza virus infection and compared the phenotypic and functional qualities of these cells with memory T cells generated after systemic infection with lymphocytic choriomeningitis virus (LCMV). Using a recombinant influenza virus expressing the LCMV gp(33-41) epitope and TCR transgenic CD8(+) T cells with a fixed TCR, we compared responses to the same Ag delivered by mucosal or systemic viral infection. Memory cells generated postinfection with either virus showed only a few phenotypic differences. Yet, influenza memory T cells produced lower amounts of effector cytokines upon restimulation and displayed reduced proliferation compared with LCMV-induced memory cells. Strikingly, we observed reduced expansion of spleen- and, in particular, lung-derived influenza memory cells after recall in vivo, which correlated with reduced early protection from secondary infection. These findings suggest that qualitatively different memory CD8(+) T cells are generated after respiratory or systemic virus infections.

Temporal and spatial distribution of antigen following influenza virus infection (92.16)

Abstract Memory CD8 T cells generated by vaccines are important for prevention of certain viral infections. However, our understanding of the factors that contribute to the development of memory CD8 T cell remains unclear. We hypothesized that, in response to a localized influenza virus (IAV) infection, CD8 T cells are recruited to the local draining lymph node (dLN) at different times and thus acquire different fates. We therefore studied the kinetics of recruitment and activation of naïve IAV-specific CD8 T cells in the dLN of the respiratory tract following IAV infection. We observed early antigen presentation in the dLN; however, we also observed evidence of antigen encounter in the spleen. To determine the differentiation outcome of CD8 T cells primed in disparate sites following a localized infection we adoptively transferred spleen-primed or dLN-primed cells into naïve mice and determined their survival and ability to respond upon recall. Surprisingly we found that CD8 T cells primed in the spleen differentiate into memory cells of equal longevity and with similar capacity to respond to recall as CD8 T cells that are primed in the dLN. Thus, in a model of respiratory IAV infection, virus-specific memory CD8 T cells are generated in the spleen in addition to the dLN. Antigen presentation in the spleen may therefore serve to ensure the involvement of a greater fraction of the virus-specific CD8 T cell repertoire after a local infection.

Influenza Virus-Induced Glucocorticoids Compromise Innate Host Defense against a Secondary Bacterial Infection

Multicellular organisms are continuously exposed to many different pathogens. Because different classes of pathogens require different types of immune responses, understanding how an ongoing immune response to one type of infection affects the host's ability to respond to another pathogen is essential for a complete understanding of host-pathogen interactions. Here, we used a mouse model of coinfection to gain insight into the effect of respiratory influenza virus infection on a subsequent systemic bacterial infection. We found that influenza infection triggered a generalized stress response leading to a sustained increase in serum glucocorticoid levels, resulting in a systemic suppression of immune responses. However, virus-induced glucocorticoid production was necessary to control the inflammatory response and prevent lethal immunopathology during coinfection. This study demonstrates that activation of the hypothalamic-pituitary-adrenal axis controls the balance between immune defense and immunopathology and is an important component of the host response to coinfection.

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103+ and CD11bhi phenotype, respectively - are the primary DC presenting antigen to naïve CD4+ and CD8+ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103+ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8+ T cells responding to infection into effector cells, and only the CD103+ RDC subset can present to naïve CD8+ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103+ and CD11bhi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.

Seasonal Invasive Pneumococcal Disease in Children: Role of Preceding Respiratory Viral Infection

Our objective was to demonstrate correlations between invasive pneumococcal disease in children and circulating respiratory viruses. This retrospective study included 6 winter respiratory viral seasons (2001-2007) in Intermountain Healthcare, an integrated health system in the Intermountain West, including Primary Children's Medical Center in Salt Lake City, Utah. Children <18 years of age who were hospitalized with either invasive pneumococcal disease in any Intermountain Healthcare facility or culture-confirmed invasive pneumococcal disease at Primary Children's Medical Center were included. We analyzed the correlation between invasive pneumococcal disease and circulating respiratory viruses. A total of 435 children with invasive pneumococcal disease and 203 with culture-confirmed invasive pneumococcal disease were hospitalized in an Intermountain Healthcare facility or Primary Children's Medical Center during the study period. During the same period, 6963 children with respiratory syncytial virus, 1860 with influenza virus, 1459 with parainfluenza virus, and 818 with adenoviruses were evaluated at Primary Children's Medical Center. A total of 253 children with human metapneumovirus were identified during the last 5 months of the study. There were correlations between invasive pneumococcal disease and seasonal respiratory syncytial virus, influenza virus, and human metapneumovirus activity. The correlation with invasive pneumococcal disease was strong up to 4 weeks after respiratory syncytial virus activity. For influenza virus and human metapneumovirus, the correlations were strong at 2 weeks after activity of these viruses. Pneumonia was the most common clinical disease associated with culture-confirmed invasive pneumococcal disease, mostly attributable to serotypes 1, 19A, 3, and 7F. In the post-pneumococcal conjugate vaccine era, seasonal increases in respiratory syncytial virus, influenza virus, and human metapneumovirus infections in children were associated with increased pediatric admissions with invasive pneumococcal disease, especially pneumonia caused by nonvaccine serotypes.

Critical requirement for 4‐1BBL for protection from severe but not mild respiratory Influenza A infection in mice

In view of intensifying concerns about pandemic Influenza virus, there is considerable interest in the mechanisms that control respiratory Influenza virus infection. Here we sought to determine the importance of 4‐1BBL‐mediated costimulation during mild versus severe respiratory Influenza virus infection in mice. During infection with the mildly virulent Influenza A/HKx31 virus, regardless of route of infection or epitope studied, 4‐1BBL is completely dispensable for the primary CD8 T cell response. However, 4‐1BBL is required for maximal recall responses, previously attributed to a requirement for 4‐1BBL in the maintenance of T cell memory. In contrast, during primary intranasal infection with Influenza A/PR8, which leads to severe respiratory disease in mice, the absence of 4‐1BBL results in decreased CD8 T cell responses in the lung, increased viral load and increased mortality. Thus 4‐1BBL plays a key role in protective immunity in the respiratory tract during severe Influenza disease. These results show that costimulation requirements are greater during severe as compared to mild respiratory Influenza virus infection.

A virologic survey of patients admitted to a critical care unit for acute cardiorespiratory failure

ObjectiveTo document the prevalence of respiratory virus infections in patients with chronic cardiac or pulmonary disorders admitted to a critical care unit for acute cardiorespiratory failure.Design, setting, patientsThe study took place in a critical care unit during two consecutive winters. All patients admitted to the unit for acute respiratory or cardiac failure were enrolled. A nasal swab was taken for polymerase chain reaction (PCR) detection of influenza virus, respiratory syncytial virus (RSV), metapneumovirus, rhinovirus, and coronavirus.ResultsOne hundred twenty-two patients were enrolled. Their mean age was 69 years; 42% of the patients were female; the new simplified acute physiology score (SAPS II) score on admission was 35.6; 94% of patients had acute respiratory failure, 14% reported “influenza-like” illness before admission, and 11% of patients died in the unit. Twenty-one patients (17%) tested positive for a respiratory virus. The per-1,000 positivity rates were influenza virus 66, RSV 49, rhinovirus 33, metapneumovirus 16, and coronavirus 8. No link was found between virologic results and clinical outcome. A strong link was found between the rate of influenza virus positivity and the incidence of flu-like illness in the community (p=0.017).ConclusionThese results show that respiratory virus infection—particularly influenza virus infection during epidemic periods—is common among patients hospitalized for acute cardiorespiratory failure.

Interleukin-1 Is Responsible for Acute Lung Immunopathology but Increases Survival of Respiratory Influenza Virus Infection

Interleukin-1alpha (IL-1alpha) and IL-1beta are proinflammatory cytokines, which induce a plethora of genes and activities by binding to the type 1 IL-1 receptor (IL-1R1). We have investigated the role of IL-1 during pulmonary antiviral immune responses in IL-1R1(-/-) mice infected with influenza virus. IL-1R1(-/-) mice showed markedly reduced inflammatory pathology in the lung, primarily due to impaired neutrophil recruitment. Activation of CD4(+) T cells in secondary lymphoid organs and subsequent migration to the lung were impaired in the absence of IL-1R1. In contrast, activation of virus-specific cytotoxic T lymphocytes and killing of virus-infected cells in the lung were intact. Influenza virus-specific immunoglobulin G (IgG) and IgA antibody responses were intact, while the IgM response was markedly reduced in both serum and mucosal sites in IL-1R1(-/-) mice. We found significantly increased mortality in the absence of IL-1R1; however, lung viral titers were only moderately increased. Our results demonstrate that IL-1alpha/beta mediate acute pulmonary inflammatory pathology while enhancing survival during influenza virus infection. IL-1alpha/beta appear not to influence killing of virus-infected cells but to enhance IgM antibody responses and recruitment of CD4(+) T cells to the site of infection.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

DNA vaccination against respiratory influenza virus infection

DNA vaccination using plasmid encoding the hemagglutinin (HA) gene of influenza A/PR/8/34 virus to induce long-lasting protective immunity against respiratory infection was evaluated in this study. Using liposomes as carriers, the efficacy of DNA vaccines was determined using a lethal influenza infection model in mice. Mice immunized intranasally or intramuscularly with liposome-encapsulated pCI plasmid encoding HA (pCI-HA10) were completely protected against an intranasal 5 LD 50 influenza virus challenge. Mice immunized with liposome-encapsulated pCI-HA10, but not naked pCI-HA10, by intranasal administration were found to produce high titers of serum IgA. These results suggest DNA vaccines encapsulated in liposomes are efficacious in inducing complete protective immunity against respiratory influenza virus infection.

Immunoprophylactic strategies against respiratory influenza virus infection

The objective of this report is to evaluate the prophylactic efficacy of liposome-mediated immunotherapy for prevention of respiratory influenza virus infection in mice. Antiviral antibody, interferon-γ and poly (ICLC) were encapsulated in liposomes and they were evaluated for their ability to induce protective immunity against lethal influenza infection. Passive immunization using liposome-encapsulated antiviral antibody was found to offer complete protection against the virus challenge. However, this pretreatment must be administered within 24 h prior to virus challenge to be protective. Pretreatment with liposome-encapsulated interferon-γ was found to stimulate cellular immune responses, but the protection is partial. Immunoprophylaxis using liposome-encapsulated double-stranded (ds) RNA poly (ICLC) provided complete and longer-lasting protection against influenza infection. These results suggest liposome-mediated immunoprophylactic approaches are effective in the prevention of respiratory influenza virus infection.

Different expression of ICAM-1 and LFA-1 alpha by peripheral leukocytes during respiratory syncytial virus and influenza virus infection in young children.

We compared the expression of intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 alpha (alpha) on the surfaces of peripheral immunocompetent cells of young children infected with respiratory syncytial virus (RSV) and influenza virus. Flow cytometric analysis revealed a significantly higher percentage of CD14 + monocytes/macrophages that were strongly ICAM-1-positive in the influenza group than in age-matched controls, whereas the ICAM-1 expression levels of the RSV and control groups did not differ significantly. Analysis of LFA-1alpha expression by CD3 + T lymphocytes showed a significantly higher percentage of strongly positive cells in the influenza group than in the age-matched controls. By contrast, the percentage of cells that were strongly LFA-1alpha-positive was significantly lower in the RSV group than in the age-matched controls. These results suggest that no increase of adhesion molecule expression occurs in vivo in patients with RSV infection, and also that there is little activation of peripheral blood monocytes/macrophages and T lymphocytes in young children with RSV infection.

Liposome-mediated immunotherapy against respiratory influenza virus infection using double-stranded RNA poly ICLC

The use of liposome delivery technology to enhance the antiviral activity of poly ICLC (an immunomodulating dsRNA) while decreasing its intrinsic toxicity is evaluated in this study. The antiviral efficacies of free and liposome-encapsulated poly ICLC were evaluated and compared using a lethal respiratory influenza A virus infection in mice. The toxicity profiles of free and liposome-encapsulated poly ICLC were compared by determining the extent of hypothermia and loss in body weights in mice pretreated with these drugs. Poly ICLC was encapsulated in cationic liposomes prepared by the freeze drying method. To determine the antiviral efficacies of free and liposome-encapsulated poly ICLC, mice were intranasally pretreated with two doses of poly ICLC (free or liposomal, 1 mg/kg/dose) given 48 h apart. At various times post pretreatment, mice were intranasally challenged with 10 LD 50 mouse-adapted influenza A/PR/8 (H1N1) virus. The survival rates of the mice were determined at day 14 post infected and compared to the untreated control mice. Results indicate mice pretreated with liposome-encapsulated poly ICLC within 3 weeks prior to virus challenge were completely protected (100% survival compared to 0% for the untreated control group, p<0.001), while window of protection provided by free unencapsulated poly ICLC was 12 days. When the toxicity profiles of free and liposome-encapsulated poly ICLC were compared, it was found that hypothermia and body weight loss induced by poly ICLC were either completely mitigated or significantly reduced in mice given equivalent doses of poly ICLC in the liposome-encapsulated form. These results suggest that liposomes are an excellent drug carrier for poly ICLC, that liposome-encapsulated poly ICLC may provide a safe and effective immunotherapeutic approach for the prevention of respiratory influenza virus infections.

Alveolar macrophages regulate the induction of primary cytotoxic T-lymphocyte responses during influenza virus infection.

Virus-specific cytotoxic T lymphocytes (CTL) are thought to be responsible for the eradication of respiratory influenza virus infections by direct cytolysis of virus-infected epithelial cells. In this study, we provide evidence for a role for alveolar macrophages (AM) in the regulation of pulmonary virus-specific CTL responses. Prior to infection with influenza virus, AM were selectively eliminated in vivo with a liposome-mediated depletion technique, and virus-specific CTL activities of lung and mediastinal lymph node (MLN) cells were assayed ex vivo and compared with those for normal mice. AM depletion resulted in increased primary CTL responses and changed the kinetics of the CTL response. Flow cytometric analysis of lung and MLN cells showed that the percentage of CD8+ cells was not altered after AM depletion and that lung cells from AM-depleted mice had an increased capacity to lyse virus-infected cells. Upon restimulation in vitro, virus-specific CTL activity in lung cells of normal mice was similar to that in lung cells of AM-depleted mice. Furthermore, elimination of AM resulted in increased virus titers in the lung, but virus clearance as a function of time was not affected. Our results show that AM regulate virus-specific CTL responses during respiratory influenza virus infection by removing viral particles, by downregulating the priming and activity of CTL in MLN cells, and by inhibiting the expansion of virus-specific CTL in the lung.

Comparison of direct immunofluorescence of exfoliated cells (DIF), tissue culture immunofluorescence (TCIF) and conventional virus isolation (CVI) for the diagnosis of respiratory virus infections

Direct immunofluorescent staining (DIF) of exfoliated cells and immunofluorescent staining of infected tissue culture cells (TCIF) are superior to conventional virus isolation (CVI) techniques for the diagnosis of respiratory virus infections. Out of 197 specimens from patients with respiratory syncytial virus (RSV), influenza or parainfluenza virus, measles, adenovirus or cytomegalovirus (CMV) infections, DIF recognised 84%, TCIF 82% and CVI only 42%. DIF and TCIF were particularly useful for the diagnosis of RSV and parainfluenza virus infections, and the optimal specimen for diagnosis was nasopharyngeal aspirate.