Late Phase Of Infection Research Articles

Dynamic Changes of Microglia/Macrophage M1 and M2 Polarization in Theiler's Murine Encephalomyelitis.

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.

Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs.

Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

Immunobiological activity and antiviral regulation efforts of Chinese goose (Anser cygnoides) CD8α during NGVEV and GPV infection

The CD8 molecule is a cell membrane glycoprotein expressed on cytotoxic T lymphocytes, which are involved in the clearance of viruses. However, the functional characterization of goose CD8α is still unclear. The immunobiological characterization of goose CD8α in goose spleen mononuclear cells (MNCs) was examined by real-time quantitative PCR (qPCR). It was shown that CD8α mRNA levels were significantly up-regulated by in vitro treatment of MNCs with phytohemagglutinin (PHA), concanavalin A (ConA), and polyinosinic-polycytidylic acid (poly I:C) in a dose-dependent way, but lipopolysaccharides (LPSs) did not have this same effect. Moreover, the time-course effect of CD8α expression in response to mitogens (PHA, ConA, and poly I:C) was evaluated in MNCs. A significant increase in the transcriptional levels of CD8α was detected in new type gosling viral enteritis virus (NGVEV)–infected goose MNCs at 48 h postinfection (PI) and in goose parvovirus (GPV)–infected MNCs at 72 h PI. Also, the number of CD8α+ cells was significantly increased during viral infection from 72 h on. The seminal changes in mRNA profiles of antiviral cytokines (IFN-α, IFN-γ, and IL-18) were observed and were significantly increased during late phases of NGVEV and GPV infection. Accordingly, our data not only contribute to the understanding of the immune characteristics of goose CD8α, but they also provide new insight into the innate antiviral immunity of geese.

Erythroferrone Represses Hepcidin Expression in a Mouse Model of Malaria

Introduction: Malaria, a mosquito-borne disease caused by a parasite, represents a major global health challenge in developing countries, resulting in over half a million deaths each year. Among the many clinical complications, the multiplication of the parasites in erythrocytes leads to a severe anemia secondary to hemolysis and increased erythrophagocytosis. Malarial anemia is also characterized by insufficient erythropoiesis to compensate for the loss of red blood cells, despite high erythropoietin (EPO) levels. Iron is an essential functional component of erythrocyte hemoglobin, therefore the production of erythrocytes requires the timely delivery of iron to erythroid precursors. The availability of iron for erythropoiesis is controlled by hepcidin-induced endocytosis and degradation of ferroportin, the iron exporter which delivers iron to plasma from absorptive enterocytes and erythrocyte-recycling macrophages. In the late phase of malarial infection, hepcidin is suppressed but the mechanism of suppression is unknown. The erythroid hormone erythroferrone (ERFE) has been recently described as an important regulator of hepcidin expression during increased erythropoietic activity. We assessed hepcidin and erythroferrone expression in mouse malaria and found that ERFE is necessary for hepcidin suppression during malaria infection.Methods: To study the regulation of hepcidin in malaria, we used the rodent malaria parasite Plasmodium berghei K173 (PbK). Mice infected with PbK develop a lethal form of malaria with a high parasitemia and severe anemia and eventually die 18 to 20 days after infection. C57BL/6 mice were challenged intraperitoneally with 106 PbK-parasitized erythrocytes. The parasitemia and the hematologic parameters, were monitored during 18 days (Table 1).TableRBC (106/µL)HGB (g/dL)HCT (%)Parasitemia (%)Controls8.8 +/- 0.615.1 +/- 0.939.8 +/- 3.10Day 77.7 +/- 0.912.6 +/- 1.533.8 +/- 4.02 +/- 1Day 97.0 +/- 0.311.6 +/- 0.632.5 +/- 1.64 +/- 1Day 115.9 +/- 0.59.9 +/- 0.927.8 +/- 1.94 +/- 1Day 133.8 +/- 0.86.6 +/- 1.221.5 +/- 2.320 +/- 5Day 162.0 +/- 0.73.9 +/- 1.214.7 +/- 4.542 +/- 11Day 181.7 +/- 0.43.6 +/- 0.714.8 +/- 2.768 +/- 10Results: Thirteen days after infection, mice showed a high parasitemia (20% of infected red blood cells) and significantly decreased RBC (3.8x106/µL), hemoglobin concentration (6.6 g/dL) and hematocrit (21.5%) despite elevated serum EPO levels (not shown). We examined the time course of liver hepcidin expression and serum hepcidin concentration and found that hepcidin production was profoundly reduced 11 to 18 days after infection. As expected given the increase in EPO production after infection, hepcidin suppression was accompanied by an increase in erythroferrone mRNA expression in the bone marrow and the spleen. To determine whether ERFE plays a role in hepcidin suppression during malaria infection, we studied wild-type and Erfe-deficient mice after PbK infection. Erfe-/- mice failed to adequately suppress hepcidin expression after infection with PbK compared with wild-type mice. [Display omitted] Conclusion: Erythroferrone may be responsible for hepcidin suppression and compensatory iron acquisition during malaria infection.Funded in part by ANR (project ANR-13-BSV3-0015-01) and FRM (project DEQ2000326528) DisclosuresNo relevant conflicts of interest to declare.

Poor growth of human adenovirus-12 compared to adenovirus-2 correlates with a failure to impair PKR activation during the late phase of infection

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-β expression was transient, HAdV-12-infected cells maintained high levels of IFN-β expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections.

Cecropin P1 inhibits porcine reproductive and respiratory syndrome virus by blocking attachment.

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro.ResultsCP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection.ConclusionsCollectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection.

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

Topoisomerases modulate the topological state of DNA during processes, such as replication and transcription, that cause overwinding and/or underwinding of the DNA. African swine fever virus (ASFV) is a nucleo–cytoplasmic double-stranded DNA virus shown to contain an OFR (P1192R) with homology to type II topoisomerases. Here we observed that pP1192R is highly conserved among ASFV isolates but dissimilar from other viral, prokaryotic or eukaryotic type II topoisomerases. In both ASFV/Ba71V-infected Vero cells and ASFV/L60-infected pig macrophages we detected pP1192R at intermediate and late phases of infection, cytoplasmically localized and accumulating in the viral factories. Finally, we used a Saccharomyces cerevisiae temperature-sensitive strain in order to demonstrate, through complementation and in vitro decatenation assays, the functionality of P1192R, which we further confirmed by mutating its predicted catalytic residue. Overall, this work strengthens the idea that P1192R constitutes a target for studying, and possibly controlling, ASFV transcription and replication.

A unique evolution of the s2 gene of equine infectious anemia virus in hosts correlated with particular infection statuses.

Equine infectious anemia virus (EIAV) is a member of the Lentivirus genus in the Retroviridae family that exhibits a genomic structure similar to that of HIV-1. The S2 accessory proteins play important roles in viral replication in vivo and in viral pathogenicity; however, studies on S2 evolution in vivo are limited. This study analyzed the evolutionary characteristics of the S2 gene of a pathogenic EIAV strain, EIAVLN40, in four experimentally infected horses. The results demonstrated that 14.7% (10 of 68 residues) of the stable amino acid mutations occurred longitudinally in S2 during a 150-day infection period. Further analysis revealed that six of the ten mutated residues were positively selected during the infection. Alignment and phylogenetic analyses showed that the S2 gene sequences of viruses isolated from the infected horses at the early stage of EIAVLN40 infection were highly homologous and similar to the vaccine-specific sequence. The S2 gene variants isolated from the febrile episodes and late phase of infection became homologous to the S2 gene sequence of the inoculating EIAVLN40 strain. Our results indicate that the S2 gene evolves in diversity and divergence in vivo in different stages of EIAV infection and that this evolution correlates with the pathogenicity of the virus.

Validation of three splice donor and three splice acceptor sites for regulating four novel low-abundance spliced transcripts of human cytomegalovirus UL21.5 gene locus.

In a previous study, one spliced transcript of human cytomegalovirus (HCMV), named UL21.5 was identified. UL21.5 has been found to be one of the viral transcripts packaged within HCMV particles. The UL21.5 mRNA is translated into a secreted glycoprotein, which is a viral chemokine decoy receptor specifically interacting with regulated upon activation normal T cell expressed and secreted (RANTES). In the present study, four novel low-abundance 3'-coterminal spliced transcripts were identified to be transcribed from the UL21.5 gene region of a low-passage HCMV strain during the late infection phase by cDNA library screening, northern blot hybridization, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR. Three splicing donor and three splicing acceptor sites found in the UL21.5 gene region were validated to be functional in an in vitro expression system. In addition, the determinant regulatory region that is necessary for the splice donor site at nucleotide (nt) 25533 was located in a 9-bp sequence around the site; the regulatory regions for the splice acceptor sites at nt 26597 and nt 26633 were located in a 20-bp sequence upstream of the site at nt 26597 and in a 10-bp sequence from nt 26641 to nt 26650 downstream of the site at nt 26633, respectively.

Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection.

The potent immunoregulatory properties of IL-10 can counteract protective immune responses and, thereby, promote persistent infections, as evidenced by studies of cryptococcal lung infection in IL-10-deficient mice. To further investigate how IL-10 impairs fungal clearance, the current study used an established murine model of C57BL/6J mice infected with Cryptococcus neoformans strain 52D. Our results demonstrate that fungal persistence is associated with an early and sustained expression of IL-10 by lung leukocytes. To examine whether IL-10-mediated immune modulation occurs during the early or late phase of infection, assessments of fungal burden and immunophenotyping were performed on mice treated with anti-IL-10R-blocking Ab at 3, 6, and 9 d postinfection (dpi) (early phase) or at 15, 18, and 21 dpi (late phase). We found that both early and late IL-10 blockade significantly improved fungal clearance within the lung compared with isotype control treatment when assessed 35 dpi. Immunophenotyping identified that IL-10 blockade enhanced several critical effector mechanisms, including increased accumulation of CD4(+) T cells and B cells, but not CD8(+) T cells; specific increases in the total numbers of Th1 and Th17 cells; and increased accumulation and activation of CD11b(+) dendritic cells and exudate macrophages. Importantly, IL-10 blockade effectively abrogated dissemination of C. neoformans to the brain. Collectively, this study identifies early and late cellular and molecular mechanisms through which IL-10 impairs fungal clearance and highlights the therapeutic potential of IL-10 blockade in the treatment of fungal lung infections.

Regulatory effect of interleukin-4 in the innate inflammatory response to Rhodococcus aurantiacus infection in mice.

Interleukin (IL)-4 promotes the regression of granulomas during the late phase of Rhodococcus aurantiacus infection. In this study, the contribution of IL-4 to the initial response against this bacterium was investigated using IL-4-deficient mice. Compared with wild-type (WT) mice, IL-4-deficient mice displayed remarkably lower tumor necrosis factor (TNF)-α and IL-6 secretion in the liver, spleen, and blood at the initial phase of infection, along with improved survival. IL-4-deficient mice also showed diminished IL-10 secretion in the spleen and blood; however, hepatic IL-10 levels were similar to those observed in WT animals, and were concomitant with augmented interferon (IFN)-γ production and decreased bacterial burden in the liver at the early infection phase. Histological examination revealed reduced hepatic granuloma formation in infected IL-4-deficient mice. On challenge with heat-killed R. aurantiacus, IL-4-deficient mouse macrophages showed reduced expression of TNF-α, IL-6, and IL-10 at both the gene and protein levels compared with WT mouse cells. These findings indicate that during the initiation of R. aurantiacus-induced inflammation, IL-4 deficiency attenuates cytokine responses in macrophages, which contributes to amelioration in mouse survival and reduction of granulomatous inflammation, and augments a hepatic IFN-γ response which transiently accelerates bacterial elimination.

Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs), are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR) activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs) and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF) significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms.

Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.

204: Regulatory effect of interleukin-4 in the innate immune and inflammatory responses to Rhodococcus aurantiacus infection in mice

Inoculation of wild-type (WT) mice with gram-positive bacterium Rhodococcus aurantiacus induces Th1-type granulomatous inflammation, in which interleukin (IL)-4 promotes the regression of granulomas during the late phase of infection. As IL-4 plays diverse roles in disease pathogenesis, we investigated the effect of IL-4 in the innate response to this bacterium infection by using IL-4-deficient mice. After intravenous inoculation with 1 × 10 8 CFU of bacteria, mouse survival rates, bacteria load in organs, local and systemic cytokine production, and morphological changes in liver were assessed. Following stimulation with heat-killed R. aurantiacus , cytokine production in mouse peritoneal macrophages was also examined. Compared to WT mice, IL-4-deficient mice showed the decreased production of TNF- α and IL-6 in the spleen, liver and blood during the early phase of infection, as well as low bacterial load in the liver and improvement in survival rate. IL-4-deficient mice also showed diminished IL-10 secretion in the spleen and blood, whereas their hepatic IL-10 levels were similar to those observed in WT mice, which were concomitant with the augmented hepatic IFN-γ production. Moreover, histological studies revealed reduction in hepatic granuloma formation at day 14 post-infection in IL-4-deficient mice. Upon stimulation with heat-killed R. aurantiacus , macrophages from IL-4-deficient mice showed lower expression of TNF-α, IL-6, and IL-10 at both the gene and protein levels than WT mouse cells. These findings indicate that IL-4 deficit attenuates cytokine responses in macrophages but augments the IFN-γ response during the initial stage of R. aurantiacus infection, which together contribute to amelioration of mouse survival, rapid bacterial elimination in liver at the early phase, and reduction of granulomatous inflammation. We previously demonstrated that the early IFN-γ response to R. aurantiacus is induced in mouse natural killer cells. This study thus identifies the essential effect of IL-4 on regulating the activation and function of immunocompetent cells, including macrophages and natural killer cells, during the initial phase of systemic R. aurantiacus infection.

S-18 : Linking pathogen virulence, the microbiota and immunity

The mechanisms that allow pathogens to colonize the intestine and the indigenous microbiota to inhibit pathogen colonization remain unclear. In order to understand the role of the microbiota in controlling pathogen colonization, we have focused our studies on Citrobacter rodentium , a model for human infections by attaching/effacing (A/E) bacteria. These Gram-negative bacteria are food- and waterborne non-invasive pathogens which attach to and colonize the intestinal tract by inducing characteristic A/E lesions on the intestinal epithelium, leading to transient enteritis or colitis in humans. We found that unlike normal mice, germ-free animals are unable to eradicate Citrobacter rodentium from the intestine. The genome of A/E pathogens including Enterohemorragic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC) and C. rodentium harbor the locus of enterocyte effacement (LEE) that is critical for bacterial colonization and the ability to cause pathology. We found that early in infection, LEE virulence genes were expressed and required for pathogen growth in conventionally raised, but not germ-free, mice. LEE virulence gene expression was downregulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was out-competed by commensals. The ability of the microbiota to out-compete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Moreover, we found that dietary carbohydrates can influence the ability of members of the gut microbiota to out-compete the pathogen in the intestine. Our studies indicate the members of the microbiota use metabolic pathways to out-compete pathogens. We will show additional studies that provide an understanding for how the host immune system regulates pathogen virulence and cooperatively acts with the microbiota to control pathogen eradication in the intestine will be discussed.

Human herpesvirus 6 gM/gN complex interacts with v-SNARE in infected cells.

Human herpesvirus 6 (HHV-6) glycoprotein M (gM) is an envelope glycoprotein that associates with glycoprotein N (gN), forming the gM/gN protein complex, in a similar manner to the other herpesviruses. Liquid chromatography-MS/MS analysis showed that the HHV-6 gM/gN complex interacts with the v-SNARE protein, vesicle-associated membrane protein 3 (VAMP3). VAMP3 colocalized with the gM/gN complex at the trans-Golgi network and other compartments, possibly the late endosome in HHV-6-infected cells, and its expression gradually increased during the late phase of virus infection. Finally, VAMP3 was incorporated into mature virions and may be transported with the gM/gN complex.

Marek's disease virus influences the core gut microbiome of the chicken during the early and late phases of viral replication.

Marek's disease (MD) is an important neoplastic disease of chickens caused by the Marek's disease virus (MDV), an oncogenic alphaherpesvirus. In this study, dysbiosis induced by MDV on the core gut flora of chicken was assessed using next generation sequence (NGS) analysis. Total fecal and cecum-derived samples from individual birds were used to estimate the influence of MDV infection on the gut microbiome of chicken. Our analysis shows that MDV infection alters the core gut flora in the total fecal samples relatively early after infection (2-7 days) and in the late phase of viral infection (28-35 days) in cecal samples, corresponding well with the life cycle of MDV. Principle component analyses of total fecal and cecal samples showed clustering at the early and late time points, respectively. The genus Lactobacillus was exclusively present in the infected samples in both total fecal and cecal bird samples. The community colonization of core gut flora was altered by viral infection, which manifested in the enrichment of several genera during the early and late phases of MDV replication. The results suggest a relationship between viral infection and microbial composition of the intestinal tract that may influence inflammation and immunosuppression of T and B cells in the host.

Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural proteins

In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter’s neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.

Crohn's disease-associated Escherichia coli survive in macrophages by suppressing NFκB signaling.

Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD). Crohn's disease-associated Escherichia coli (CDEC) is characterized by their ability to invade epithelial cells and survive and induce high concentration of TNF-α from infected macrophages. However, the molecular mechanisms of CDEC survival in infected macrophages are not completely understood. Intracellular survival of CDEC strain LF82 isolated from inflamed ileum tissue, 13I isolated from inflamed colonic tissue, and control E. coli strains were tested in the murine macrophage cell line, J774A.1 by Gentamicin protection assay. Modulation of intracellular cell signaling pathways by the E. coli strains were assessed by western blot analysis and confocal microscopy. 13I demonstrated increased survival in macrophages with 2.6-fold higher intracellular bacteria compared with LF82, yet both strains induced comparable levels of TNF-α. LF82 and 13I differentially modulated key mitogen-activated protein kinase pathways during the acute phase of infection; LF82 activated all 3 mitogen-activated protein kinase pathways, whereas 13I activated ERK1/2 pathway but not p38 and JNK pathways. Both 13I and LF82 suppressed nuclear translocation of NFκB compared with noninvasive E. coli strains during the acute phase of infection. However, unlike noninvasive E. coli strains, 13I and LF82 infection resulted in chronic activation of NFκB during the later phase of infection. Our results showed that CDEC survive in macrophages by initially suppressing NFκB activation. However, persistence of bacterial within macrophages induces chronic activation of NFκB, which correlates with increased TNF-α secretion from infected macrophages.

Disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different H5N1 highly pathogenic avian influenza viruses.

Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome. Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.