Time Course Of Infection Research Articles

Natural Killer Cell Licensing Delineates NK “Helper/Repair” and NK “Effector/Suppressor” Subsets During Viral Infections

Natural killer (NK) cells are innate lymphocytes with anti-viral and anti-tumor capabilities that can be divided into subsets based on differential receptor expression patterns. NK cells that express inhibitory receptors that can bind to the MHC class I molecules present in the host are considered to be “licensed,” fully functional NK cells with normal production of cytokines and cytotoxicity in response to targets. In contrast, “unlicensed” NK cells are unable to strongly bind to host MHC class I molecules and are in turn hyporesponsive to targets in terms of cytotoxicity and cytokine production. Recent data suggest that NK cells also regulate antigen-specific adaptive immune responses during the course of viral infection, playing a significant role in viral clearance and immunopathology. The specific populations of NK cells that may mediate these differential effects during the course of viral infection have not been identified.Here, we demonstrate differential effector and immunoregulatory functions of licensed versus unlicensed NK cells during influenza and murine cytomegalovirus (MCMV) infections in mouse models. We hypothesize that licensed NK cells serve a dual role as both effector and suppressor populations depending on the stage of viral infection. Similarly, unlicensed NK cells serve a dual role as helper and repair populations during the early and late stages of viral infection, respectively. We performed licensed and unlicensed NK cell subset depletions and then infected mice with influenza or MCMV and ascertained effects on: viral titers, antigen-specific T cell responses, and tissue pathology. Our data show that after influenza or MCMV infection, there is a significant reduction in antigen-specific CD4+ and CD8+ T cell populations in the presence of licensed NK cells as determined by tetramer-positive cells. Targeting of these T cells by the NK “effector/suppressor” licensed population appears later in the time course of infection and to be through NKG2D recognition and perforin-mediated lysis based on upregulation of NKG2D ligands Rae-1 and MULT1 on the T cells and the loss of T cell regulation with NKG2D blockade or perforin knockout mice. Depletion of the unlicensed NK “helper/repair” subset reduced the number of DCs in the lymph nodes and reduced total antigen-specific T cells. The unlicensed NK cells were found to home to the lymph node and produce increased levels of GM-CSF early during infection resulting in DC expansion. Additionally, the unlicensed NK cells are the primary producers of IL-22 based on intracellular staining in the damaged tissues in the late stages of viral infection, aiding in tissue regeneration. Adoptive transfer of unlicensed NK cells with IL-22 silencing through siRNA transfection into immunodeficient mice showed increased tissue damage and pathology as compared to transfer of non-IL-22 silenced NK cells.Collectively, these data suggest differential roles of licensed versus unlicensed NK cells that are both tissue and time-specific. At early stages of infection, licensed NK cells serve as direct anti-viral cells at the sites of infection while unlicensed cells promote DC expansion in the lymph nodes promoting antigen-specific T cell responses. Conversely, at the late stages of infection, licensed NK cells serve an immunoregulatory role by lysing antigen-specific T cells at the site of infection and in the lymph nodes, while unlicensed NK cells travel to the sites of injury to aid in tissue repair through production of IL-22. Importantly, a similar functional polarization of resting human NK cells was also observed after PMA/ionomycin stimulation, with a small population of unlicensed NK cells producing IL-22 and a bias towards GM-CSF secretion over IFNγ production by the unlicensed NK cell subset. We conclude that licensed NK cells have an effector/suppressor function while the unlicensed NK cells function as the helper/repair population suggesting distinct roles of NK cell subsets throughout the course of infection. By understanding the functions and characteristics of these NK cell populations, specific subsets can either by adoptively transferred or therapeutically targeted clinically to aid in different stages of immunological response including elimination of the virus, inhibiting the adaptive immune response, or aiding in tissue repair and regeneration. Disclosures:No relevant conflicts of interest to declare.

Changes in xyloglucan endotransglucosylase/hydrolase (XTHs) expression and XET activity during apple fruit infection by Penicillium expansum Link. A

Although the plant cell wall is an extremely effective physical barrier against attack by pathogens, most phytopathogenic microorganisms produce enzymes that are capable of degrading cell wall polymers such as pectin and hemicelluloses. In this work we investigate the possible implication of the cell wall enzyme XET in the host-pathogen interaction as well as the XTH gene regulation during apple fruit infection by Penicillium expansum Link. A. We characterized the time course of fungal infection in apple fruit, and the XET activity and the expression of eleven MdXTHs. The results showed an important decrease in XET specific activity in the fruits after 24, 48 and 72 h of fruit infection (42.9 %, 73.1 % and 95.1 % respectively as compared with the controls). The expression analysis of the MdXTHs showed an important decrease in expression, as the infection progressed, in MdXTH2 and MdXTH10, as well as in MdXTH3, MdXTH4 and MdXTH5, but mainly after 72 h of fruit infection; and in MdXTH1, and MdXTH9 after 24 and 48 h of infection. These results suggested that the decrease in XET activity during infection could be mainly attributed to the decrease in expression of MdXTH2 and MdXTH10, since those were the MdXTHs that showed the highest expression levels during ripening, particularly MdXTH10. These results support the suggestion that inhibition of MdXTHs expression, and consequently the decrease in XET specific activity, could represent a pathogenicity factor, since the potential reconstructing role of the enzyme also decreased and the infection could progress.

Gastric secretion

The review summarizes the past year's literature, basic science and clinical, regarding the neural, paracrine, hormonal, and intracellular regulation of gastric acid secretion. Gastric acid facilitates the digestion of protein as well as the absorption of iron, calcium, vitamin B(12), and certain medications (e.g. thyroxin). It also kills ingested microorganisms and prevents bacterial overgrowth, enteric infection, and possibly spontaneous bacterial peritonitis. Stimulants of acid secretion include histamine, gastrin, acetylcholine, and ghrelin. Inhibitors include somatostatin, nefstatin-1, interleukin-11, and calcitonin gene-related peptide. Helicobacter pylori stimulates or inhibits acid secretion depending upon the time course of infection and the area of the stomach predominantly infected. Acute infection activates calcitonin gene-related peptide sensory neurons coupled to inhibition of histamine and acid secretion. Serum chromogranin A, a marker for neuroendocrine tumors, is elevated in patients taking proton pump inhibitors. Progress continues in our understanding of the regulation of gastric acid secretion in health and disease, as well as the function of gastric neuroendocrine cells. The recognition that gastrin is not only a secretagogue but also a trophic hormone has led to new research into the role of gastrin and its receptor (cholecystokinin-2 receptor) in carcinogenesis and the development of cholecystokinin-2 receptor antagonists.

Dynamics of macronutrient self‐medication and illness‐induced anorexia in virally infected insects

Some animals change their feeding behaviour when infected with parasites, seeking out substances that enhance their ability to overcome infection. This ‘self-medication’ is typically considered to involve the consumption of toxins, minerals or secondary compounds. However, recent studies have shown that macronutrients can influence the immune response and that pathogen-challenged individuals can self-medicate by choosing a diet rich in protein and low in carbohydrates. Infected individuals might also reduce food intake when infected (i.e. illness-induced anorexia).Here, we examine macronutrient self-medication and illness-induced anorexia in caterpillars of the African armyworm (Spodoptera exempta) by asking how individuals change their feeding decisions over the time course of infection with a baculovirus. We measured self-medication behaviour across several full-sib families to evaluate the plasticity of diet choice and underlying genetic variation.Larvae restricted to diets high in protein (P) and low in carbohydrate (C) were more likely to survive a virus challenge than those restricted to diets with a low P: C ratio. When allowed free choice, virus-challenged individuals chose a higher protein diet than controls.Individuals challenged with either a lethal or sublethal dose of virus increased the P: C ratio of their chosen diets. This was mostly due to a sharp decline in carbohydrate intake, rather than an increased intake of protein, reducing overall food intake, consistent with an illness-induced anorexic response. Over time the P: C ratio of the diet decreased until it matched that of controls.Our study provides the clearest evidence yet for dietary self-medication using macronutrients and shows that the temporal dynamics of feeding behaviour depends on the severity and stage of the infection. The strikingly similar behaviour shown by different families suggests that self-medication is phenotypically plastic and not a consequence of genetically based differences in diet choice between families.

MRI and 1H MRS evaluation for the serial bile duct changes in hamsters after infection with Opisthorchis viverrini

A 3T MR scanner was used to investigate the relationship between the alteration of bile duct lesions and the hepatic metabolic changes in hamsters infected with Opisthorchis viverrini by using 3T MRI and 1H MR spectroscopy. Animals were divided into control and infected groups. Five normal hamsters were used as control; fifty-five hamsters were infected with O. viverrini to induce bile duct lesions and hepatic metabolic changes. T2-weighted image sequence in three orthogonal planes were conducted by MRI scans. Single-voxel 1H MRS was performed to obtain the relative choline-to-lipid ratios. The livers and bile ducts were excised for the histologic examination. The progression of bile duct changes by histology and metabolic changes in O. viverrini infected hamsters were co-investigated. In the O. viverrini-infected group, the T2-weighted images revealed the time-dependent intra- and extra-hepatic duct dilatations in the liver. The mean (±SD) choline-to-lipid ratios were 0.11±0.035 in the control group, whereas the ratio in the infected group increased significantly with the progression of time. Histologic grading of hepatic inflammation and fibrosis were correlated well with the MRI grading (Spearman rank correlation test; r=0.746 and p<0.001). The control group showed no dilatation of the bile ducts and showed normal liver patterns. Noninvasive technique, MRI and 1H MRS can demonstrated and applied to evaluate not only the inflammation-related fibrosis in the small bile ducts but also the metabolic changes in the liver induced by O. viverrini infection. A significant increase in the choline-to-lipids ratios were observed in parallel with the time-course of infection. O. viverrini infected in human is detected by stool examination. Hepatobiliary morbidity is detected and followed up by ultrasonography. MRI and MRS can be used in conjunction with ultrasonography for evaluation of progression of the disease.

Contribution of Interleukin-12 p35 (IL-12p35) and IL-12p40 to Protective Immunity and Pathology in Mice Infected with Chlamydia muridarum

The p35 molecule is unique to interleukin-12 (IL-12), while p40 is shared by both IL-12 and IL-23. IL-12 promotes Th1 T cell responses, while IL-23 promotes Th17 T cell responses. The roles of IL-12p35- and IL-12p40-mediated responses in chlamydial infection were compared in mice following an intravaginal infection with Chlamydia muridarum. Mice deficient in either IL-12p35 or p40 both developed similar but prolonged infection time courses, confirming the roles of IL-12-mediated immune responses in clearing primary infection. However, all mice, regardless of genotype, cleared reinfection within 2 weeks, suggesting that an IL-12- or IL-23-independent adaptive immunity is protective against chlamydial infection. All infected mice developed severe oviduct hydrosalpinx despite the increased Th2 responses in IL-12p35- or IL-12p40-deficient mice, suggesting that Th2-dominant responses can contribute to Chlamydia-induced inflammatory pathology. Compared to IL-12p35 knockout mice, the IL-12p40-deficient mice exhibited more extensive spreading of chlamydial organisms into kidney tissues, leading to significantly increased incidence of pyelonephritis, which both confirms the role of IL-12 or IL-23-independent host responses in Chlamydia-induced pathologies and suggests that in the absence of IL-12/IFN-γ-mediated Th1 immunity, an IL-23-mediated response may play an important role in restricting chlamydial organisms from spreading into distal organs. These observations together provide important information for both understanding chlamydial pathogenesis and developing anti-Chlamydia vaccines.

Comparison of a Real-Time PCR Method with Serology and Blood Smear Analysis for Diagnosis of Human Anaplasmosis: Importance of Infection Time Course for Optimal Test Utilization

Anaplasmosis and ehrlichiosis are emerging tick-borne diseases with clinically similar presentations caused by closely related pathogens. Currently, laboratories rely predominantly on blood smear analysis (for the detection of intracellular morulae) and on serologic tests, both of which have recognized limitations, for diagnostic purposes. We compared the performance of a published real-time PCR assay that incorporates melt curve analysis to differentiate Anaplasma and Ehrlichia species with blood smear and serologic methods in an upper Midwest population. Overall, 38.5% of the specimens selected for evaluation had one or more tests that were positive for anaplasmosis. The PCR positivity for all specimens was maximal (21.2%; 29/137) during the early acute phase of illness (0 to 4 days since illness onset) and significantly less frequent (11.5%; 20/174) during later phases (>4 days since illness onset). All positive specimens were Anaplasma phagocytophilum; no Ehrlichia species were identified. The real-time PCR detected 100% of infections that were detected by blood smear analysis (14/14) and broadened the detection window from a maximum of 14 days for smear positivity to 30 days for PCR. Additional infections were detected by real-time PCR in 12.9% (11/85) of smear-negative patients. There was poor agreement between the real-time PCR assay and serologic test results: 19.8% (19/96) and 13.7% (29/212) of seropositive and -negative patients, respectively, were PCR positive. Seropositivity increased with increasing days of illness, demonstrating that serologic detection methods are best utilized during presumed convalescence. Our results indicate that the optimal performance and utilization of laboratory tests for the diagnosis of anaplasmosis require knowledge regarding time of symptom onset or days of illness.

Hematopoietic Stem Cells and HIV Infection

Hematopoietic Stem Cells and HIV Infection

Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar L.) leucocytes after vaccination and during the time course of experimental infection

In the present study, intracellular infectious pancreatic necrosis virus (IPNV) in salmon leucocytes was detected by flow cytometry after experimental cohabitant challenge. IPNV vaccinated, non-vaccinated and intraperitoneally (i.p.) infected salmon (virus shedders) were analysed at different times throughout the period when mortality occurred. Fish that had survived 61 days post challenge (carriers) were also analysed. In particular, we analysed the presence of IPNV in B-cells (C7G7+cells) and in neutrophils (E3D9+ cells) in head kidney leucocytes (HKL) and in peripheral blood leucocytes (PBL).IPNV was present in HKL and PBL from all challenged fish groups at all samplings, including carriers. IPNV was also found intracellular in other leucocytes than B-cells and neutrophils. During the time course of infection there were changes in proportion of B-cells and neutrophils and in proportions of IPNV+ cells. In vaccinated fish, a delay in the changes observed in the proportion of IPNV+ cells and in the proportions of the two subpopulations was identified. The vaccinated fish were protected against disease as no fish died compared to 30.8% of non-vaccinated cohabitant fish. All i.p. infected fish, except one, survived the challenge. This is consistent with previous studies and confirmed that the routes of infection can influence mortality. The analyses in this study could not identify any factors enlightening this absence of mortality in i.p. infected fish, but both flow cytometry and qRT-PCR showed that i.p. infected fish were carriers of IPNV. The present study also found that IPNV was present in both B-cells and neutrophils as well as in other leucocytes in all carriers after cohabitant challenge. These fish had survived 9 weeks post challenge and 4 weeks after mortality has ceased. The fish harbouring virus within their leucocytes might become life long carriers and represent a risk for disease outbreaks, being virus shedders. Such fish are protected from later infections if the virus exposure has resulted in protective immunity. Flow cytometry was found to be very suitable for detection of intracellular virus after in vivo challenge and the sensitivity was demonstrated by the detection of virus in carriers.

Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPI1-mediated gene expression and infectivity

BackgroundSalmonella enterica serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands (SPI), producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell infection: pyruvate formate lyase I (pflB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE). We were therefore interested in examining the role these genes may play in the transmission of Salmonella to humans.MethodsMutant strains of Salmonella with single gene deletions for pflB and adhE were created. Invasion and growth in human HCT-8 intestinal epithelial cells and THP-1 macrophages was examined. Quantitative PCR was performed on 19 SPI-1 genes.ResultsIn HCT-8 cells, both mutant strains had significantly higher intracellular counts than the wild-type from 4 to 48 h post-infection. Various SPI-1 genes in the mutants were up-regulated over the wild-type as early as 1 h and lasting until 24 h post-infection. In THP-1 cells, no significant difference in internal Salmonella counts was observed; however, SPI-1 genes were largely down-regulated in the mutants during the time-course of infection. We also found five SPI-1 genes - hilA, hilC hilD, sicP and rtsA - which were up-regulated in at least one of the mutant strains in log-phase broth cultures alone. We have therefore identified a set of SPI-1 virulence genes whose regulation is effected by the central metabolism of Salmonella.

Host DNA damage response facilitates African swine fever virus infection

Studies with different viral infection models on virus interactions with the host cell nucleus have opened new perspectives on our understanding of the molecular basis of these interactions in African swine fever virus (ASFV) infection. The present study aims to characterize the host DNA damage response (DDR) occurring upon in vitro infection with the ASFV-Ba71V isolate. We evaluated protein levels during ASFV time-course infection, of several signalling cascade factors belonging to DDR pathways involved in double strand break repair - Ataxia Telangiectasia Mutated (ATM), ATM-Rad 3 related (ATR) and DNA dependent protein kinase catalytic subunit (DNA-PKcs). DDR inhibitory trials using caffeine and wortmannin and ATR inducible-expression cell lines were used to confirm specific pathway activation during viral infection. Our results show that ASFV specifically elicits ATR-mediated pathway activation from the early phase of infection with increased levels of H2AX, RPA32, p53, ATR and Chk1 phosphorylated forms. Viral p72 synthesis was abrogated by ATR kinase inhibitors and also in ATR-kd cells. Furthermore, a reduction of viral progeny was identified in these cells when compared to the outcome of infection in ATR-wt. Overall, our results strongly suggest that the ATR pathway plays an essential role for successful ASFV infection of host cells.

Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.

Quantification of viral infection dynamics in animal experiments

Analyzing the time-course of several viral infections using mathematical models based on experimental data can provide important quantitative insights regarding infection dynamics. Over the past decade, the importance and significance of mathematical modeling has been gaining recognition among virologists. In the near future, many animal models of human-specific infections and experimental data from high-throughput techniques will become available. This will provide us with the opportunity to develop new quantitative approaches, combining experimental and mathematical analyses. In this paper, we review the various quantitative analyses of viral infections and discuss their possible applications.

In vitro propagation of hepatopancreatic parvo-like virus (HPV) of shrimp in C6/36 (Aedes albopictus) cell line

Hepatopancreatic parvovirus (HPV) which causes infection in many species of penaeid shrimp is a serious viral pathogen in the young life stages of shrimp. An attempt was made to develop an in vitro system using C6/36 subclone of Aedes albopictus cell line for propagation of HPV. The results revealed that C6/36 cells were susceptible to this virus and the infected cells showed CPE in the form of vacuole formation. The results of PCR, immunocytochemistry and Western blot revealed the HPV-infection in C6/36 cell line. The RT-PCR analysis confirmed the replication of HPV in C6/36 cell line. The HPV load was quantified at different time intervals by ELISA and real time PCR, and the results showed the increase of viral load in C6/36 cell line in time course of infection. HPV propagated in C6/36 cell line was used to infect post-larvae of shrimp and the results showed that the twentieth passage of HPV propagated in C6/36 cell line caused 100% mortality in post-larvae after 6weeks post infection (d.p.i.). The infected post-larvae showed clinical signs of reduced growth, reduced preening, muscle opacity and atrophy of hepatopancreas. The HPV-infection was confirmed by PCR. The results of the present study showed that C6/36 cell line can be used as an in vitro model for HPV replication instead of whole animal.

Dicer-2- and Piwi-Mediated RNA Interference in Rift Valley Fever Virus-Infected Mosquito Cells

Rift Valley fever virus (RVFV) is a Phlebovirus (Bunyaviridae family) transmitted by mosquitoes. It infects humans and ruminants, causing dramatic epidemics and epizootics in Africa, Yemen, and Saudi Arabia. While recent studies demonstrated the importance of the nonstructural protein NSs as a major component of virulence in vertebrates, little is known about infection of mosquito vectors. Here we studied RVFV infection in three different mosquito cell lines, Aag2 cells from Aedes aegypti and U4.4 and C6/36 cells from Aedes albopictus. In contrast with mammalian cells, where NSs forms nuclear filaments, U4.4 and Aag2 cells downregulated NSs expression such that NSs filaments were never formed in nuclei of U4.4 cells and disappeared at an early time postinfection in the case of Aag2 cells. On the contrary, in C6/36 cells, NSs nuclear filaments were visible during the entire time course of infection. Analysis of virus-derived small interfering RNAs (viRNAs) by deep sequencing indicated that production of viRNAs was very low in C6/36 cells, which are known to be Dicer-2 deficient but expressed some viRNAs presenting a Piwi signature. In contrast, Aag2 and U4.4 cells produced large amounts of viRNAs predominantly matching the S segment and displaying Dicer-2 and Piwi signatures. Whereas 21-nucleotide (nt) Dicer-2 viRNAs were prominent during early infection, the population of 24- to 27-nt Piwi RNAs (piRNAs) increased progressively and became predominant later during the acute infection and during persistence. In Aag2 and U4.4 cells, the combined actions of the Dicer-2 and Piwi pathways triggered an efficient antiviral response permitting, among other actions, suppression of NSs filament formation and allowing establishment of persistence. In C6/36 cells, Piwi-mediated RNA interference (RNAi) appeared to be sufficient to mount an antiviral response against a secondary infection with a superinfecting virus. This study provides new insights into the role of Dicer and Piwi in mosquito antiviral defense and the development of the antiviral response in mosquitoes.

P211 Time-Course of Rhinovirus and Bacterial Infection During COPD Exacerbation Recovery

Introduction Airway bacteria and viruses are aetiological triggers of COPD exacerbations. Changes in prevalence of rhinovirus and typical airway bacteria together have not been examined during COPD exacerbation recovery. We compared the prevalence of the clinically relevant microorganisms (CRMs) human rhinovirus and typical pathogenic bacteria ( H. influenzae , M. catarrhalis and S. pneumoniae ) at five time points during COPD exacerbation recovery. Methods London COPD cohort patients recorded any new or increased respiratory symptoms on daily diary cards and contacted the clinical team when symptoms worsened. Exacerbations were defined using our usual symptomatic criteria; an increase in respiratory symptoms for two consecutive days, with at least one symptom being major (dyspnoea, sputum purulence or volume) and the other a major or minor symptom (wheeze, cold, sore throat, cough). Reverse-transcription quantitative PCR was used to detect rhinovirus and real-time quantitative PCR was utilised to identify typical bacteria in sputum samples collected at exacerbation presentation (median 2 days after symptom onset), and at days 3, 7, 14 and 35 post-presentation. Results Nineteen patients with moderate to severe COPD (mean age 68.8 years (SD±8.1); FEV1 48.4% predicted (±19.2%); current smoker 37%; FEV1/FVC 0.46 (SD±0.14); FEV1 1.2L (SD±0.4); male gender 74%) provided 89 of 110 potential sputum samples at 5 time points during 22 exacerbations. Rhinovirus prevalence progressively fell from 71.4% at exacerbation presentation to 0% at day 35 with significant decreases in prevalence between presentation and days 7, 14 and 35 (all p Conclusion The prevalence of CRMs varies during recovery from a COPD exacerbation. Rhinovirus prevalence steadily decreases over 2 weeks whilst bacterial prevalence is more variable, presumably due to the background effects of lower airway bacterial colonisation. This emphasises the importance of rhinovirus as a major exacerbation trigger.

Serum and Egg Yolk Antibody Detection in Chickens Infected with Low Pathogenicity Avian Influenza Virus

Surveillance for low pathogenicity avian influenza virus (LPAIV) infections has primarily relied on labor-intensive collection and serological testing of serum, but for many poultry diseases, easier-to-collect yolk samples have replaced serum for surveillance testing. A time-course LPAIV infection study in layers was performed to evaluate the utility of antibody detection in serum vs. egg yolk samples. Layers inoculated with the LPAIV A/Bobwhite Quail/Pennsylvania/20304/98 (H7N2) were tested for antibody levels in the serum and egg yolk by using the agar gel immunodiffusion test (AGID), hemagglutination-inhibition test (HI), and a commercially available enzyme-linked immunosorbent assay (ELISA). Anti-influenza specific antibodies were detected in the serum as early as 7 days postinoculation (DPI), and the majority of the hens remained positive until 42 DPI. Antibodies in the egg yolk were first detected by AGID at 7 DPI, which was also the first day of detection in serum. However, the majority of the eggs were positive by all techniques at 11 DPI and remained positive until 42 DPI, at which time the number of AGID+ and HI+ samples declined slightly as compared to ELISA+ samples. These results suggest that egg yolk can be an alternative to serum for flock serological surveillance against LPAIV infections, and the three methods (AGID, HI, and ELISA) will give similar results for first 42 days after infection, although AGID may give earlier positive response.

P124 Transcriptional regulation of the human type I IFN locus

Introduction Transcriptional activation of type I interferon (IFN-1) genes (including IFN beta and multiple IFN alpha subtypes) is essential to the immediate cellular response to virus infection and is a key antiviral defense. The induction of IFN-1 causes immunomodulation and serves to prime and coordinate subsequent innate and adaptive immune responses. Virus induction of IFN beta gene expression is well known to involve rapid and dynamic changes in enhancer occupancy, nucleosome positioning, and RNA polymerase recruitment. To better understand the integration of these events and to extrapolate them to the broader type I IFN gene cluster on human chromosome 9 (9p21-22), nucleosome occupancy was examined throughout the locus. Methods Nucleosome protected DNA, generated using micrococcal nuclease digestion, was significantly enriched by a direct genomic selection process and followed by deep sequencing to generate nucleosome maps of the entire locus throughout a time course of Sendai virus infection. In parallel, the early transcriptional initiation events leading to IFN gene activation have been studied by chromatin immunoprecipitation followed by quantitative PCR. Results Nucleosome protected DNA, generated using micrococcal nuclease digestion, was significantly enriched by a direct genomic selection process and followed by deep sequencing to generate nucleosome maps of the entire locus throughout a time course of Sendai virus infection. Results affirm the presence of a positioned nucleosome that obscures the transcriptional start site of the IFN beta gene, and this nucleosome is evicted by 4 h post infection. All of the IFN-1 genes share the presence of a TSS-obscuring nucleosome in an analogous position, which is clearly distinct from the reported +1 nucleosome position of most human genes at steady state. CHIP Results indicate that no pre-existing paused, poised, or otherwise classified Pol II is present at the IFN beta promoter prior to virus infection. Instead de novo recruitment of virus-induced transcription factors, Mediator proteins, RNA Polymerase II (Pol II), and associated Pol II elongation factors to the promoter of the IFN beta gene were observed. Nonetheless, this newly recruited Pol II has features consistent with a paused polymerase, including its phosphorylation on serine-5 of the heptad repeat, its associations with negative elongation factor (NELF), and its sensitivity to a cyclin dependent kinase inhibitor. Conclusion These findings suggest a common mechanism of virus-induced regulation for the entire IFN-1 cluster in which nucleosome obstruction renders the genes quiescent until the convergence of cellular signals that specify nucleosome reorganization in order to achieve Pol II recruitment and robust gene activation.

P128 Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta

Introduction Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. We have shown that the Gram-positive bacterium Listeria monocytogenes stimulates IFN-λ2 transcription during infection of epithelial cells and that this pathogen is able to tightly control the expression of the downstream responsive genes at chromatin level by hijacking a chromatin-silencing complex [1] , [2] . So far, no bacterial species other than L. monocytogenes has been examined for the induction of IFN-λ genes in epithelial cells. Because a wide range of bacterial pathogens colonize epithelia, it was of interest to compare the ability of diverse species to induce these genes in this cell type. Here, we further detailed L. monocytogenes-mediated stimulation of IFN-λ in human epithelial cells and in the mouse placenta. We also analyzed differences between bacterial species in the induction of IFN-λ genes in epithelial cells. Methods To analyze the mechanisms underlying L. monocytogenes stimulation of IFN-III expression, we determined bacterial loads and the kinetics of IFN-λ and IFN-s gene transcription (by qRT-PCR) and IFNs secretion (by ELISA) over a time-course infection with L. monocytogenes in LoVo epithelial cells. We also studied mutants deficient for internalization or phagosomal escape. We next determined whether other bacterial species, either Gram-positive or Gram-negative, could induce type III IFN. Finally, we examined whether type III IFN could be induced during in vivo infection with Listeria at the placental barrier. Results We report that IFN-III are the major interferons produced and secreted by epithelial cells in response to L. monocytogenes infection, by mechanisms involving both the internalization and cytosolic phases of infection. Other major bacterial pathogens, Mycobacterium tuberculosis and the Gram-positive cocci Staphylococcus aureus, also stimulate type III IFN expression in epithelial cells. In contrast, Gram-negative pathogens Salmonella enterica, Shigella flexneri and Chlamydia trachomatis have no or only weak effects. We also provide evidence for in vivo induction of these genes during infection of the placenta by L. monocytogenes in the mouse, and show that the feto-placental tissue is responsive to IFN-λ2. Conclusion Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues. Most pathogenic bacteria target epithelia, in particular in skin, throat, gut, liver, lung, genital mucosa or placenta. We propose that some bacterial species allow epithelial cells to become a source of IFN-λ,acting as paracrine immunomodulators of mucosal surfaces.

Hepatitis E virus: neutralizing sites, diagnosis, and protective immunity

There have been increased attentions on HEV and its associated diseases in recent years as a result of an increased number of reports on autochthonous patients from many developed countries. Vaccine development and better disease management are expected from protective immunity with increased knowledge on the pathogenesis and virology of HEV. This review summarizes the current understanding of the HEV virology, the key neutralization sites (epitopes) on the surface of the viral capsid, the host humoral immune responses for HEV infection, and the protective immunity conferred by natural infection and vaccination. Recombinant VLPs were prepared to mimic the protective and neutralizing epitopes on the virion surface, thus being capable of eliciting protective immunity when injected to nonhuman primates or human volunteers during preclinical tests and clinical trials. Four markers-viral RNA, anti-HEV IgM, anti-HEV IgG, and low avidity of anti-HEV IgG-are important in the diagnosis of HEV infection, particularly for patients presenting with acute hepatitis symptoms. This toolbox of genomic and immunological assays is valuable in furthering our understanding of the time course of HEV infection and the subsequent hepatitis during preclinical and clinical development of an efficacious vaccine. Two vaccine candidates had shown good tolerability, high immunogenicity, and high efficacy against symptomatic and/or asymptomatic HEV infection. One of them has been licensed in China recently. However, many issues need to be resolved before new technological progresses can benefit the people who need them most.