Breastfeeding infants transmit influenza virus to the mammary glands during feeding [Dataset Part I]
ABSTRACT: Influenza virus transmission between mothers and nursing-infants has not been investigated although mothers and infants often develop severe disease. Ferrets are considered the most appropriate model for influenza studies. We investigated influenza transmission in infant and nursing-mother ferrets. Influenza infected infants transmitted virus to mother mammary glands leading to live virus excretion in milk and influenza virus positive mammary gland epithelial cells. Global gene expression analysis showed down-regulation of milk production and induction of breast involution and oncogenesis pathways. Our results provide insight into influenza transmission between mothers and infants which may impact fields of infectious disease, maternal/infant health and neoplasm etiology. Total RNA was obtained from nursing mother ferret mammary glands at days 3/4 and 6/7 post-intranasal kit infection with 10^5 EID50 A/California/07/2009 (H1N1). Total RNA was also collected from uninfected control nursing mother mammary gland tissues (n = 3). Changes in gene expression relative to uninfected tissue controls were then investigated.
Project description:Influenza virus transmission between mothers and nursing-infants has not been investigated although mothers and infants often develop severe disease. Ferrets are considered the most appropriate model for influenza studies. We investigated influenza transmission in infant and nursing-mother ferrets. Influenza infected infants transmitted virus to mother mammary glands leading to live virus excretion in milk and influenza virus positive mammary gland epithelial cells. Global gene expression analysis showed down-regulation of milk production and induction of breast involution and oncogenesis pathways. Our results provide insight into influenza transmission between mothers and infants which may impact fields of infectious disease, maternal/infant health and neoplasm etiology. Total RNA was obtained from ferret lungs at days 3 and 6 post-intranasal infection with 10^5 EID50 A/California/07/2009 (H1N1) (n = 3/time-point). Total RNA was also collected from uninfected control lung tissues (n = 3). Changes in gene expression relative to uninfected tissue controls were then investigated.
Project description:The determinants of influenza transmission remain poorly understood. Swine influenza viruses preferentially attach to receptors found in the upper airways; however, most swine influenza viruses fail to transmit efficiently from swine to humans, and from human-to-human. The pandemic 2009 H1N1 (H1N1pdm) virus was a rare exception of a swine virus that acquired efficient transmissibility from human-to-human, and is reflected in efficient respiratory droplet transmission in ferrets. We hypothesize that virus-induced host responses in the upper airways correlate with airborne transmission in ferrets. To address this question, we used the H1N1pdm virus and swine influenza A/swine/Hong Kong/201/2010 (HK201) virus that has comparable titre in the ferret nasopharynx, but it exhibits differential transmissibility in ferrets via respiratory droplet route. We performed a transcriptomic analysis of tissues from the upper and lower respiratory tract from ferrets infected with either H1N1pdm or HK201 viruses using ferret-specific Agilent oligonucleotide arrays. We found differences in the kinetics of the innate immune response elicited by these two viruses that varied across tissues. Overall design: Ferrets were mock inoculated with 0.5mL media or inoculated with 105 TCID50 of the A/CA/04/09 (H1N1) or A/Sw/HK/ 210/10 (H1N1) influenza viruses intra-nasally. There are a total of 9 ferrets per virus group and six ferrets for the mock group. Ferrets were euthanized at day 1 (n = 4 per virus group) and day 2 (n = 5 per virus group) post-inoculation (p.i.). Three mock inoculated ferrets were euthanized at days 1 and 2 p.i. Tissues from nasal turbinate, soft palate, pharynx, trachea, bronchus, and lungs were collected at necropsy, placed in RNAlater overnight at 4°C and conserved at -80°C. RNA was extracted and gene expression was analyzed by custom 4x180K ferret-specific oligonucleotide arrays (Agilent Cat# G4862A, design ID 48472)
Project description:A global genomics approach was used to identify patterns of immune dysregulation during H5N1 influenza virus infection as the host response, in particular hyperchemokinemia, is thought to contribute to the extreme pathology associated with this disease. Keywords: time course Ferrets were inoculated intranasally with 10(6) EID50 of either A/Vietnam/1203/04 (H5N1) or A/Panama/2007/99 (H3N2). At 2, 4 and 6 days post-infection (DPI), ferrets were euthanized and lung tissue was excised for RNA purification and subsequent gene expression analysis.
Project description:Different respiratory viruses induce virus-specific gene expression in the host. Recent evidence, including those presented here, suggests that genetically related isolates of influenza virus induce strain specific host gene regulation in several animal models. Here, we identified systemic strain-specific gene expression signatures in ferrets infected with pandemic influenza A/California/07/2009, A/Mexico/4482/2009 or seasonal influenza A/Brisbane/59/2007. Using uncorrelated shrunken centroid classification, we were able to accurately identify the infecting influenza strain with a combined gene expression profile of 10 selected genes, independent of the severity of disease. Another gene signature, consisting of 7 genes, could classify samples based on lung pathology. Furthermore, we identified a gene expression profile consisting of 31 probes that could classify samples based on both strain and severity of disease. Thus, we show that expression-based analysis of non-infected tissue enables distinction between genetically related influenza viruses as well as lung pathology. These results open for development of alternative tools for influenza diagnostics. Blood derived total RNA from 63 ferrets. 30 ferrets were infected with A/California/07/2009 (15 with 10E6 TCID50/ml and 15 with 10E4 TCID50/ml, 15 with A/Mexico/4482/2009 and 12 with A/BN/2007. 6 animals were mock infected with PBS and used as controls. Three animals per group were euthanized at days 1, 2, 3, 5 and 7.
Project description:Background: Type I interferons (IFNs) are essential to the clearance of viral diseases, in part by initiating upregulation of IFN regulated genes (IRGs). A clear distinction between genes upregulated directly by virus and genes upregulated by secondary IFN production has not been made. Here we investigated the genes regulated by IFN-a2b compared to the genes regulated by SARS-CoV infection in ferrets. Methods: We characterized early host immune responses in peripheral blood and lung necropsies of ferrets injected with IFN-a2b or infected with SARS-CoV/Tor 2 strain, using microarray analysis on the Affymetrix platform. Results: We identified a common IRG signature that was upregulated in both SARS-CoV infected ferrets as well as in ferrets injected with IFN-a2b. We also identified unique patterns of gene expression for leukocyte activation, cell adhesion and complement pathways between IFN-a2b injection and SARS-CoV infection. Conclusions: Our results define the effects of IFN-a2b on the immune system of ferrets highlighting genes regulated by IFN during SARS-CoV infection. We have shown the similarities and differences of top funcional gene groups as well as pathways that play key roles in early immune responses in ferrets in response to IFN-a2b or SARS-CoV. Key words: ferret, gene expression, SARS, interferon. Keywords: time course In experiments with IFN-a2b, for peripheral blood, 15 ferrets were randomly allocated to 3 groups: Day 0, 5 ferrets (no IFN injection), day 1, 6 ferrets (injected), and day 2, 4 ferrets (injected). For lung necropsies of injected ferrets with IFN-a2b, we used 12 ferrets in 3 groups: 4 ferrets, day 0 (no IFN injection), 4 ferrets, day 1 (injected) and 4 ferrets, day 2 (injected). Experimental groups for SARS-CoV infection was as follows: For peripheral blood, 3 and 4 ferrets for day 0 (no infection) and day 2 (infection) respectively. For lung neceropsies, a total of 9 ferrets in 3 groups, each with 3 replicates for day 0 (no infection), day 1 (infection) and day 2 (infection).
Project description:Background: Pandemic H1N1 influenza A is a newly emerging strain of human influenza that is easily transmitted between people and has spread globally to over 116 countries. Human infection leads to symptoms ranging from mild to severe with lower respiratory complications observed in a small but significant number of infected individuals. Little is currently known about host immunity and Pandemic H1N1 influenza infections. Methods: We examined the pathogenic potential of the pandemic influenza A vaccine strain, A/California/07/2009 (H1N1), in ferrets, and characterized the host immune responses using microarray analysis. Gene expression profiles in lung tissue were compared with those from ferrets infected with A/Brisbane/59/2007. Results: Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of ISGs were expressed early, correlated to lung pathology, and abruptly decreased expression in 5 days. Interestingly, the drop in innate immune gene expression was replaced by a significant increase in the expression of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-pandemic influenza H1N1 antibodies were also observed on day 7, commensurate with the elimination of viral load. Conclusions: We propose that the innate phase of host immunity causes lung pathology and a delay or failure to effectively switch to the adaptive phase contributes to morbidity and mortality during severe human pandemic H1N1 influenza A infections. Keywords: influenza, immune response, cytokines, chemokines, lung infection, time course In the experiment with influenza A/California/07/2009 (H1N1),15 ferrets were randomly allocated to 5 groups: Day 0 (before infection), and Day 3, 5, 7 and 14 (post infection) with 3 biological replicates for each group. Likewise, a second experiment with A/Brisbane/59/2007 (H1N1) was carried out using the same experimental groups, except for a group in Day 2, instead Day 3. Ferrets were euthanized and lung tissue was excised for RNA purification on the scheduled date. The subsequent gene expression analysis was performed with Affymetrix GeneChip Canine Genome 2.0 Array. Day 0 groups were used as control.
Project description:Pandemic H1N1 influenza A Human infection leads to symptoms ranging from mild to severe with lower respiratory complications observed in a small but significant number of infected individuals. Microarray analysis of the lymph nodes from ferrets infected with A/California/07/2009 shows intense gene upregulation during days 3 and 5 post-infection, and followed by marked downregulation during days 7 and 14 post infection. Gene expression profiles during the upregulation phase show intense chemokine activity, cell replication and activation of the lymphocyte-related signaling pathways. 15 ferrets were randomly allocated to 5 groups: Day 0 (before infection), and Day 3, 5, 7 and 14 (post infection) with 3 biological replicates for each group. Infection was performed with 10E6 EID50 of influenza A/California/07/2009 (H1N1). Ferrets were euthanized and lymph nodes were excised for RNA purification on the scheduled date. The subsequent gene expression analysis was performed with Affymetrix GeneChip Canine Genome 2.0 Array. Day 0 groups were used as control.
Project description:The 2009 H1N1 influenza pandemic has prompted a significant need for the development of efficient, single-dose, adjuvanted vaccines. Here we investigated the adjuvant potential of CpG oligodeoxynucleotide (ODN) when used with a human seasonal influenza virus vaccine in ferrets. We found that the CpG ODNadjuvanted vaccine effectively increased antibody production and activated type I interferon (IFN) responses compared to vaccine alone. Based on these findings, pegylated IFN- 2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and complete Freund’s adjuvant (CFA). Our results showed that all three vaccines with adjuvant added prevented seasonal human A/Brisbane/59/2007 (H1N1) virus replication more effectively than did vaccine alone. Gene expression profiles indicated that, as well as upregulating IFN-stimulated genes (ISGs), CpG ODN enhanced B-cell activation and increased Toll-like receptor 4 (TLR4) and IFN regulatory factor 4 (IRF4) expression, whereas PEG-IFN augmented adaptive immunity by inducing major histocompatibility complex (MHC) transcription and Ras signaling. In contrast, the use of CFA as an adjuvant induced limited ISG expression but increased the transcription of MHC, cell adhesion molecules, and B-cell activation markers. Taken together, our results better characterize the specific molecular pathways leading to adjuvant activity in different adjuvant-mediated influenza virus vaccinations. In this experiment, 3 ferrets in each group immunizied with different adjuvanted human seasonal vaccines of CFA plus vaccine, CpG plus vaccine, pegylated IFN-alpha plus vaccine and vaccine alone (PBS plus vaccine) and 4 ferrets from control group (PBS only) were anesthetized at day 1 post vaccination. The whole blodd was collected for RNA extraction and purification on the scheduled date. The subsequent gene expression analysis was performed with Affymetrix GeneChip Canine Genome 2.0 Array.
Project description:The infected ferrets depleted of alveolar macrophages showed up-regulation of inflammatory related genes compared to the infected ferrets which has alveolar macrophage. There are three group of ferret. One is the infected ferrets depleted of alveolar macrophages. Another is the infected ferret non-depleted of alveolar macrophages. The other is mock Overall design: This is the data of ferrets infected with influenza virus. After challenge influenza virus to animals, I collected total RNA from lung of ferret to compare their gene expression level which is related with inflammation. Control is the uninfected mock ferret. CL is the ferret which is treated with some medicine and challenged with influenza virus. PBS is the ferret untreated and challenged with influenza virus. In this study I just want to compare gene expression level of control, CL and PBS. These are all RNA of ferrets.
Project description:To further investigate the underlying mechanisms of severe acute respiratory syndrome (SARS) pathogenesis and evaluate the therapeutic efficacy of potential drugs and vaccines it is necessary to use an animal model that is highly representative of the human condition in terms of respiratory anatomy, physiology and clinical sequelae. The ferret, Mustela putorius furo, supports SARS-CoV replication and displays many of the symptoms and pathological features seen in SARS-CoV-infected humans. We have recently established a SARS-CoV infection-challenge ferret platform for use in evaluating potential therapeutics to treat SARS. The main objective of the current study was to extend our previous results and identify early host immune responses upon infection and determine immune correlates of protection upon challenge with SARS-CoV in ferrets. Keywords: time course This study is a simple time course (58 day) examination of host responses in 35 SARS-CoV (TOR2) infected ferrets with the addition of a challenge inoculation of SARS CoV (TOR2) at day 29 post infection. Three mock-infected ferrets are included as negative controls. Due to the unavailability of ferret microarrays, Affymetrix Canine 2.0 oligonucleotide arrays were chosen following sequence analysis of our ferret cDNA library (~5000 clones) and demonstration of high levels of homology (>80%) between dog and ferret.