RESUMEN
Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1ß upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought.
Asunto(s)
Animales Lactantes/virología , Interacciones Huésped-Parásitos/fisiología , Glándulas Mamarias Animales/virología , Glándulas Mamarias Humanas/virología , Infecciones por Orthomyxoviridae/transmisión , Animales , Animales Recién Nacidos , Western Blotting , Línea Celular , Modelos Animales de Enfermedad , Femenino , Hurones , Humanos , Inmunohistoquímica , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/virología , Lactancia , Glándulas Mamarias Animales/patología , Microscopía Confocal , Leche/virología , Madres , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Orthomyxoviridae/patología , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Influenza B viruses have become increasingly more prominent during influenza seasons. Influenza B infection is typically considered a mild disease and receives less attention than influenza A, but has been causing 20 to 50â% of the total influenza incidence in several regions around the world. Although there is increasing evidence of mid to lower respiratory tract diseases such as bronchitis and pneumonia in influenza B patients, little is known about the pathogenesis of recent influenza B viruses. Here we investigated the clinical and pathological profiles of infection with strains representing the two current co-circulating B lineages (B/Yamagata and B/Victoria) in the ferret model. Specifically, we studied two B/Victoria (B/Brisbane/60/2008 and B/Bolivia/1526/2010) and two B/Yamagata (B/Florida/04/2006 and B/Wisconsin/01/2010) strain infections in ferrets and observed strain-specific but not lineage-specific pathogenicity. We found B/Brisbane/60/2008 caused the most severe clinical illness and B/Brisbane/60/2008 and the B/Yamagata strains instigated pathology in the middle to lower respiratory tract. Importantly, B/Brisbane/60/2008 established efficient lower respiratory tract infection with high viral burden. Our phylogenetic analyses demonstrate profound reassortment among recent influenza B viruses, which indicates the genetic make-up of B/Brisbane/60/2008 differs from the other strains. This may explain the pathogenicity difference post-infection in ferrets.
Asunto(s)
Virus de la Influenza B/fisiología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/veterinaria , Infecciones del Sistema Respiratorio/patología , Infecciones del Sistema Respiratorio/virología , Animales , Modelos Animales de Enfermedad , Hurones , Humanos , Virus de la Influenza B/aislamiento & purificación , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virologíaRESUMEN
Young children are typically considered a high-risk group for disease associated with influenza virus infection. Interestingly, recent clinical reports suggested that young children were the smallest group of cases with severe pandemic 2009 H1N1 (H1N1pdm) influenza virus 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 than adult ferrets, which paralleled the mild clinical symptoms in the younger 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 the young age group during H1N1pdm infection. We found that interferon and regulatory interleukin-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, the numbers of inflammation-prone granulocytes were highly elevated in the adult peripheral blood. Importantly, we observed that H1N1pdm infection triggered formation of lung structures that resembled inducible bronchus-associated lymphoid tissues (iBALTs) in young ferrets which were associated with high levels of homeostatic chemokines CCL19 and CXCL13, but these were not seen in the adult ferrets with severe disease. These results may be extrapolated to a model of the mild disease seen in human children. Furthermore, these mechanistic analyses provide significant new insight into the developing immune system and effective strategies for intervention and vaccination against respiratory viruses.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/inmunología , Gripe Humana/patología , Interferones/biosíntesis , Animales , Anticuerpos Antivirales/biosíntesis , Hurones , Humanos , Gripe Humana/virología , Interleucina-10/biosíntesis , Pulmón/metabolismo , Tejido Linfoide/inmunología , Tejido Linfoide/virologíaRESUMEN
During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Antígenos CD8/inmunología , Protección Cruzada , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Embrión de Pollo , Femenino , Hurones , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/epidemiología , Gripe Humana/virología , Masculino , México/epidemiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PandemiasRESUMEN
The major burden of influenza morbidity resides within the elderly population. The challenge managing influenza-associated illness in the elderly is the decline of immune function, where mechanisms leading to immunological senescence have not been elucidated. To better represent the immune environment, we investigated clinical morbidity and immune function during sequential homologous and heterologous H1N1 influenza infection in an aged ferret model. Our findings demonstrated experimentally that aged ferrets had significant morbidity during monosubtypic heterologous 2° challenge with significant weight loss and respiratory symptoms. Furthermore, increased clinical morbidity was associated with slower and shorter hemagglutinin antibody generation and attenuated type 1 T-cell gene responses in peripheral blood. These results revealed dampened immune activation during sequential influenza infection in aged ferrets. With the presence of an aged model, dissecting clinical morbidity, viral dynamics and immune response during influenza infection will aid the development of future prophylactics such as age specific influenza vaccines.
Asunto(s)
Envejecimiento/inmunología , Inmunidad Heteróloga , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Factores de Edad , Anciano , Animales , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , Masculino , Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
Bronquios/citología , Citocinas/genética , Células Epiteliales/inmunología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/genética , Gripe Humana/inmunología , Fusión de Membrana , Bronquios/inmunología , Células Cultivadas , Citocinas/inmunología , Células Epiteliales/virología , Humanos , Mediadores de Inflamación/inmunología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/epidemiología , Gripe Humana/virología , PandemiasRESUMEN
The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX(-/-)) model and found that survival after infection was significantly greater in GX(-/-) mice than in GX(+/+) mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX(-/-) mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX(-/-) mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.
Asunto(s)
Fosfolipasas A2 Grupo X/deficiencia , Subtipo H1N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Animales , Linfocitos B/inmunología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Fosfolipasas A2 Grupo X/genética , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
Hurones/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunidad Innata/inmunología , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas Virales/administración & dosificación , Animales , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Biomarcadores/sangre , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Interleucina-6/sangre , Infecciones por Orthomyxoviridae/sangre , Animales , Femenino , Subtipo H1N1 del Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pandemias , Carga ViralRESUMEN
Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.