[show abstract][hide abstract] ABSTRACT: Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction.
[show abstract][hide abstract] ABSTRACT: A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.
[show abstract][hide abstract] ABSTRACT: Evolution of H1N1 influenza A outbreaks of the past 100 years is interesting and significantly complex and details of H1N1 genetic drift remains unknown. Here we investigated the clinical characteristics and immune cross-reactivity of significant historical H1N1 strains. We infected ferrets with H1N1 strains from 1943, 1947, 1977, 1986, 1999, and 2009 and showed each produced a unique clinical signature. We found significant cross-reactivity between viruses with similar HA sequences. Interestingly, A/FortMonmouth/1/1947 antisera cross-reacted with A/USSR/90/1977 virus, thought to be a 1947 resurfaced virus. Importantly, our immunological data that didn't show cross-reactivity can be extrapolated to failure of past H1N1 influenza vaccines, ie. 1947, 1986 and 2009. Together, our results help to elucidate H1N1 immuno-genetic alterations that occurred in the past 100 years and immune responses caused by H1N1 evolution. This work will facilitate development of future influenza therapeutics and prophylactics such as influenza vaccines.
[show abstract][hide abstract] ABSTRACT: The novel re-assortment A influenza H7N9 (nrH7N9) emerged in humans in the Shanghai and surrounding provinces of China in late February and early March. Three infected index patients developed severe viral pneumonia with acute respiratory distress syndrome (ARDS) and resulted in fatal outcome. As of 15 April 2013 there were reported 60 confirmed nrH7N9 infections with 13 fatalities. Human-to-human transmission has not been observed, but zoonotic infections of nrH7N9 from birds to humans appear to be associated with live poultry markets. Elderly patients greater than 60 years of age accounted for 61% of the cases, indicating that the elderly may be at high risk for severe disease.
The Journal of Infection in Developing Countries 04/2013; 7(4):302-307. · 1.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Curious anomalies in vaccine effectiveness during the 2012/2013 flu season The 2012/2013 influenza season is winding down now in North America, Europe and other Northern hemisphere locations, and interim estimates for vaccine effectiveness are now available from multiple sources [1,2,3,4]. The most striking statistic reported [3,4] is that the vaccine displayed extremely low effectiveness against A/H3N2 in the elderly. This data has significant implications for future vaccine design, influenza surveillance, and treatment of the elderly during influenza outbreaks and epidemics. In the early months of each year, the Centers for Disease Control in Atlanta and the World Health Organization make recommendations for the vaccine strains of influenza to be used by vaccine producers for manufacturing influenza vaccines  in the northern hemisphere for use in the following influenza season. In the absence of a pandemic, usually three strains of influenza are chosen for vaccine production: two influenza A viruses, one from the H1N1 subtype and one from the H3N2 subtype, and one B influenza virus. In early 2012 the three chosen viruses for the 2012/2013 flu season were A/California/7/2009 (H1N1)pdm09-like virus, A/Victoria/361/2011 (H3N2)-like virus, and B/Wisconsin/1/2010-like virus . The 2012/2013 influenza season was early and moderately severe in the United States . The predominant circulating strains were antigenically similar to A/Victoria/2011-like (H3N2) and B/Wisconsin/1/2010-like (69% of the B viruses detected, Yamagata lineage) and B/Victoria (31% of the B viruses detected, Victoria lineage). The early flu season allowed for interim vaccine effectiveness case-control studies to be determined for Canada , the United Kingdom , Denmark  and the United States . The studies from Canada, the United States, and the United Kingdom have similar overall vaccine effectiveness for all influenza strains across the entire population (see Table 1). The effectiveness ranged from 38% to 49% for A viruses and 46% to 50% for all influenza viruses. B virus effectiveness was 52% and 67% respectively for the United Kingdom and the United States. These numbers for the general population are not so surprising and appear on the surface to be in line with what has been reported for previous years. The disturbing statistic is evident when effectiveness is broken down for age (Table 2). The studies from the United States  and Denmark  both show that vaccine effectiveness in the elderly (> 65 years of age) is extremely low (see last row of Table 2). What is even more perplexing is that the younger age groups in the American study show between 46% and 58% vaccine effectiveness against H3N2, validating that the vaccine can elicit protective responses, at least in people younger than 65 years of age. Furthermore, the elderly group in both the American study and the Danish studies show that vaccine effectiveness against B viruses was relatively high at 69% and 67% for the two countries (Table 2).
The Journal of Infection in Developing Countries 03/2013; 7(3):299-301. · 1.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Severe influenza remains a major public health threat and is responsible for thousands of deaths annually. Increasing antiviral resistance and limited effectiveness of current therapies highlight the need for new approaches to influenza treatment. Extensive pre-clinical data have shown that mesenchymal stromal (stem) cell (MSC) therapy can induce anti-inflammatory effects and enhance repair of the injured lung. We hypothesized that MSC therapy would improve survival, dampen lung inflammation and decrease acute lung injury (ALI) in a murine model of severe influenza.
C57Bl/6 mice were infected with influenza A/PuertoRico/8/34 (mouse-adapted H1N1) or influenza A/Mexico/4108/2009 (swine-origin pandemic H1N1) and administered human or mouse MSCs via the tail vein, either pre- or post- infection. MSC efficacy was evaluated as both an independent and adjunctive treatment strategy in combination with the antiviral agent, oseltamivir. Weight loss and survival were monitored. Inflammatory cells, cytokine/chemokines (IFN-γ, CXCL10, CCL2 and CCL5) and markers of ALI (total protein and IgM), were measured in bronchoalveolar lavage fluid and lung parenchyma.
Administration of murine MSCs or human MSCs in a prophylactic or therapeutic regimen failed to improve survival, decrease pulmonary inflammation/inflammatory cell counts or prevent ALI in influenza virus-infected mice. MSCs administered in combination with oseltamivir also failed to improve outcomes.
Despite similarities in the clinical presentation and pathobiology of ALI and severe influenza, our findings suggest that MSC therapy may not be effective for prevention and/or treatment of acute severe influenza.
PLoS ONE 01/2013; 8(8):e71761. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ferrets have become an indispensable tool in the understanding of influenza virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza "infectome" during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals were subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pdmH1N1 influenza infection were analyzed by Digital Gene Expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days post-infection, the innate immune gene expression was replaced by the adaptive immune response which correlates with viral clearance. Reinfection with homologous pdm influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.
[show abstract][hide abstract] ABSTRACT: Young children are typically considered a high risk group for disease associated with influenza infection. Interestingly, recent clinical reports suggested that young children were the smallest group of cases with severe pandemic 2009 H1N1 (H1N1pdm) influenza infection. Here we established a newly weaned ferret model for the investigation of H1N1pdm infection in young age groups compared to adults. We found that young ferrets had a significantly milder fever and less weight loss in comparison to adult ferrets, which paralleled the mild clinical symptoms in the younger age humans. Although there was no significant difference in viral clearance, disease severity was associated with pulmonary pathology where newly weaned ferrets had an earlier pathology improvement. We examined the immune responses associated with protection of young age group during H1N1pdm infection. We found that interferon and regulatory IL-10 responses were more robust in the lungs of young ferrets. In contrast, myeloperoxidase and major histocompatibility complex responses were persistently higher in the adult lungs, as well, numbers of inflammation-prone granulocytes were highly elevated in the adult peripheral blood. Importantly, we observed H1N1pdm infection triggered formation lung structures that resembled inducible bronchus-associated lymphoid tissues (iBALT) in young ferrets which were associated with high levels of homeostatic chemokines, CCL19 and CXCL13, not seen in the adult ferrets with severe disease. These results may be extrapolated to a mild disease model seen in human children. Furthermore, these mechanistic analyses provide significant new insight into the developing immune system and effective intervention and vaccination strategies against respiratory viruses.
[show abstract][hide abstract] ABSTRACT: Background: It is widely recognized that the introduction of saliva of bloodsucking arthropods at the site of pathogen transmission might play a central role in vector-borne infections. However, how the interaction between salivary components and the host immune system takes place and which physiological processes this leads to has yet to be investigated. Armigeres subalbatus is one of the prominent types of mosquitoes involved in the transmission of parasitic and viral diseases in humans and animals. Methodology/Principal Findings: Using murine peritoneal macrophages and lymphocytes, and human peripheral mononuclear cells (PBMCs), this study shows that saliva of the female Ar. subalbatus induces apoptosis via interaction with the Fas receptor within a few hours but without activating caspase-8. The process further activates downstream p38 MAPK signaling, a cascade that leads to the induction of apoptosis in capase-3 dependent manner. We further illustrate that Ar. subalbatus saliva suppresses proinflammatory cytokines without changing IL-10 levels, which might happen as a result of apoptosis. Conclusions: Our study shows for the first time that saliva-induced apoptosis is the leading phenomenon exerted by Ar. subalbatus that impede immune cells leading to the suppression of their effecter mechanism.
PLoS ONE 07/2012; 7(7):e41145. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Understanding the pathogenesis of influenza infection is a key factor leading to the prevention and control of future outbreaks. Pandemic 2009 Influenza H1N1 infection, although frequently mild, led to a severe and fatal form of disease in certain cases that make its virulence nature debatable. Much effort has been made toward explaining the determinants of disease severity; however, no absolute reason has been established.
This study presents the heterogeneous virulence of clinically similar strains of pandemic 2009 influenza virus in human alveolar adenocarcinoma cells and mice. The viruses were obtained from patients who were admitted in a local hospital in China with a similar course of infection and recovered. The A/Nanchang/8002/2009 and A/Nanchang/8011/2009 viruses showed efficient replication and high lethality in mice while infection with A/Nanchang/8008/2009 was not lethal with impaired viral replication, minimal pathology and modest proinflammatory activity in lungs. Sequence analysis displayed prominent differences between polymerase subunits (PB2 and PA) of viral genomes that might correlate with their different phenotypic behavior.
The study confirms that biological heterogeneity, linked with the extent of viral replication, exists among pandemic H1N1 strains that may serve as a benchmark for future investigations on influenza pathogenesis.
[show abstract][hide abstract] ABSTRACT: CXCL10 is part of the group of interferon-stimulated genes and it plays an important role during different viral infections by inducing cell activation, chemotaxis and lymphocyte priming toward the Th1 phenotype. In this study, we investigated in vitro the effects of CXCL10 in activated human primary T lymphocytes in terms of apoptosis or survival, and delineated the signaling pathways that are involved. CXCL10, in combination with IL-2 and/or IFNα, induces apoptosis in T lymphocytes. Moreover, CXCL10-induced activation of CXCR3 also triggers pro-survival signals that can be blocked by pertussis toxin. The analysis of the downstream signaling kinases shows that apoptosis is p38 MAPK-dependent and the pro-survival signals rely on the sustained activation of PI3K and the transient activation of Akt. On the other hand, the transient activation of p44/p42 ERK did not have an impact on T lymphocyte survival. We propose an immunological model in which CXCL10, together with other co-stimulating cytokines, participates in the activation of T lymphocytes, promotes survival and expansion of certain lymphocyte subsets, and induces chemotaxis toward the infected tissues. On the other hand, CXCL10 might contribute to the triggering of apoptosis in other subsets of T lymphocytes, including those lymphocytes that were transiently activated but later lacked the appropriate sets of specific co-stimulating signals to ensure their survival.
[show abstract][hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infection is characterized by persistent viral replication in the context of CD4(+) T cell depletion and elevated immune activation associated with disease progression. In contrast, simian immunodeficiency virus (SIV) infection of African-origin sooty mangabeys (SM) generally does not result in simian AIDS despite high viral loads and therefore affords a unique model in which to study the immunologic contributions to a nonpathogenic lentiviral disease outcome. A key feature of these natural SIV infections is the maintenance of low levels of immune activation during chronic infection. Our goal was to delineate the contribution of monocytes to maintaining low levels of immune activation in SIV-infected SM. Utilizing an ex vivo whole-blood assay, proinflammatory cytokine production was quantified in monocytes in response to multiple Toll-like receptor (TLR) ligands and a specific, significant reduction in the tumor necrosis factor alpha (TNF-α) response to lipopolysaccharide (LPS) was observed in SIV-infected SM. In contrast, monocytes from hosts of pathogenic infections (HIV-infected humans and SIV-infected Asian macaques) maintained a robust TNF-α response. In SIV-infected SM, monocyte TNF-α responses to low levels of LPS could be augmented by the presence of plasma from uninfected control animals. The impact of LPS-induced TNF-α production on immune activation was demonstrated in vitro, as TNF-α blocking antibodies inhibited downstream CD8(+) T cell activation in a dose-dependent manner. These data demonstrate an association between nonpathogenic SIV infection of SM and a reduced monocyte TNF-α response to LPS, and they identify a role for monocytes in contributing to the suppressed chronic immune activation observed in these natural hosts.
Journal of Virology 05/2012; 86(14):7605-15. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays.
Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested.
Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing.
Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply.
The Journal of Infectious Diseases 03/2012; 205(6):886-94. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)(3)-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.
PLoS ONE 01/2012; 7(9):e45842. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Wild migratory birds are global distributors of pathogens. Sardinia, Italy, is the second largest Island in the Mediterranean and is a land bridge between Europe and Africa.
We designed a surveillance protocol to investigate wild migratory birds for presence, frequency, and type of avian influenza viruses. We collected over 4,000 avian samples and compared three sampling methods, fecal, cloacal, and tracheal, to determine the most productive for virus identification. To determine frequency of infection, RNA was extracted and RT-PCRs for avian influenza virus genes were run. Positive samples were cultivated for live virus, sub typed and sequenced.
Forty-four samples were positive for influenza nucleoprotein gene. We identified two previously unidentified H3 subtype strains and found cloacae to have the highest rate of virus identification and fecal sampling to provide quality RNA and repeatable results for determination of virus presence.
Our investigation provides information on the frequency of Mediterranean avian influenza viruses, and validates the initiation of an avian influenza surveillance protocol. Taken together with global avian influenza findings, these results give insight into infectious disease distributions which is important for viral pandemic monitoring and design of preventative measures.
The Journal of Infection in Developing Countries 01/2012; 6(11):786-97. · 1.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.
PLoS ONE 01/2012; 7(6):e38214. · 3.73 Impact Factor