NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2.

1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host's immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied.2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h).3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection.4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron.

Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP(+)-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.

Clinical severity of human infections with avian influenza A(H7N9) virus, China, 2013/14.

Assessing the severity of emerging infections is challenging because of potential biases in case ascertainment. The first human case of infection with influenza A(H7N9) virus was identified in China in March 2013; since then, the virus has caused two epidemic waves in the country. There were 134 laboratory-confirmed cases detected in the first epidemic wave from January to September 2013. In the second epidemic wave of human infections with avian influenza A(H7N9) virus in China from October 2013 to October 2014, we estimated that the risk of death among hospitalised cases of infection with influenza A(H7N9) virus was 48% (95% credibility interval: 42-54%), slightly higher than the corresponding risk in the first wave. Age-specific risks of death among hospitalised cases were also significantly higher in the second wave. Using data on symptomatic cases identified through national sentinel influenza-like illness surveillance, we estimated that the risk of death among symptomatic cases of infection with influenza A(H7N9) virus was 0.10% (95% credibility interval: 0.029-3.6%), which was similar to previous estimates for the first epidemic wave of human infections with influenza A(H7N9) virus in 2013. An increase in the risk of death among hospitalised cases in the second wave could be real because of changes in the virus, because of seasonal changes in host susceptibility to severe infection, or because of variation in treatment practices between hospitals, while the increase could be artefactual because of changes in ascertainment of cases in different areas at different times.

Genetic tuning of the novel avian influenza A(H7N9) virus during interspecies transmission, China, 2013.

A novel avian influenza A(H7N9) virus causing human infection emerged in February 2013 in China. To elucidate the mechanism of interspecies transmission, we compared the signature amino acids of avian influenza A(H7N9) viruses from human and non-human hosts and analysed the reassortants of 146 influenza A(H7N9) viruses with full genome sequences. We propose a genetic tuning procedure with continuous amino acid substitutions and reassorting that mediates host adaptation and interspecies transmission. When the early influenza A(H7N9) virus, containing ancestor haemagglutinin (HA) and neuraminidase (NA) genes similar to A/Shanghai/05 virus, circulated in waterfowl and transmitted to terrestrial poultry, it acquired an NA stalk deletion at amino acid positions 69 to 73. Then, receptor binding preference was tuned to increase the affinity to human-like receptors through HA G186V and Q226L mutations in terrestrial poultry. Additional mammalian adaptations such as PB2 E627K were selected in humans. The continual reassortation between H7N9 and H9N2 viruses resulted in multiple genotypes for further host adaptation. When we analysed a potential association of mutations and reassortants with clinical outcome, only the PB2 E627K mutation slightly increased the case fatality rate. Genetic tuning may create opportunities for further adaptation of influenza A(H7N9) and its potential to cause a pandemic.

Molecular characterization and phylogenetics of a reassortant H13N8 influenza virus isolated from gulls in Mongolia.

Double reassortant H13N8 influenza A virus was isolated from gull in Mongolia. The basic virological characteristics were studied. Complete genome sequence analysis indicated the complicated evolutionary history. The PA gene belongs to classical Avian-like lineage and more likely originated from non-gull avian virus pool. Data confirm the state of extensive geographic mosaicism in AIV from gulls in the Northern Hemisphere.

Serological surveillance of influenza A virus infection in swine populations in Fujian province, China: no evidence of naturally occurring H5N1 infection in pigs.

Several highly pathogenic H5N1 avian influenza viruses were isolated from swine populations in Fujian Province, China, since 2001. Because it is thought that H5N1 infection in pigs might result in virus adaptation to humans, we surveyed swine populations in Fujian Province in 2004 and 2007 for serological evidence of the infection. Twenty-five pig farms covering all nine administrative districts of Fujian Province were sampled and a total of 1407 serum specimens were collected. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection and only a few cases of H9 infection. The negative results for H5 infection were further verified by micro-neutralization tests. By contrast, H1 influenza virus infections were prevalent in swine in both surveys according to the results of enzyme-linked immunosorbent assay (ELISA). The H3 infection rate was reduced dramatically in 2007 compared with 2004, when examined by HI and ELISA. In summary, the results imply that the swine populations in Fujian Province had not been affected greatly by the H5N1 avian influenza virus, given that there is no serological evidence that H5N1 influenza virus has infected the pig populations. The reported isolates represent only sporadic cases.

Recurrence of human Streptococcus suis infections in 2007: three cases of meningitis and implications that heterogeneous S. suis 2 circulates in China.

Emergence of fatal Streptococcus suis serotype 2 (S. suis 2, SS2) in China, 2005, has greatly challenged the public health, raising serious concerns about its global circulation. Whole genome sequencing of the representative strains has identified a potential pathogenicity island (PAI), named 89K. Here, we reported the recurrence of human S. suis meningitis cases (two in Shenzhen City and one in Sichuan Province) in China, in 2007. Multiple lines of evidence supported that these human cases are caused by highly pathogenic SS2 isolates. Pulsed-field gel electrophoresis analysis indicated that these re-emerging SS2 isolates (07CQH01, 07SZH01 and 07SZH02) can be featured with different genotypes. Molecular dissection demonstrated that they all share 89K, the newly proposed PAI that is common to 2005 prevalent strains in China. Further mapping of 89K revealed that gene losses/deletions occur in 07CQH01 and 07SZH01, except 07SZH02. These findings suggested that heterogeneous SS2 circulates in China, with the capability of causing endemic with life-threatening danger.

Highly pathogenic H5N1 influenza virus infection in migratory birds.

H5N1 avian influenza virus (AIV) has emerged as a pathogenic entity for a variety of species, including humans, in recent years. Here we report an outbreak among migratory birds on Lake Qinghaihu, China, in May and June 2005, in which more than a thousand birds were affected. Pancreatic necrosis and abnormal neurological symptoms were the major clinical features. Sequencing of the complete genomes of four H5N1 AIV strains revealed them to be reassortants related to a peregrine falcon isolate from Hong Kong and to have known highly pathogenic characteristics. Experimental animal infections reproduced typical highly pathogenic AIV infection symptoms and pathology.

Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase.

In order to further the present knowledge of the emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 486 different specimens from 54 patients with a clinical diagnosis of SARS were investigated for the presence of viral RNA, and 314 plasma specimens of 73 patients were examined for IgM and IgG antibodies specific against SARS-CoV using an indirect ELISA. Viral RNA was detectable in 28 of the 54 patients tested. Cumulative data showed that 67 of the 73 SARS patients demonstrated seroconversion by week 5 of illness. In contrast, only 1 of 278 healthy subjects enrolled in the study was found to be positive for the IgG antibody. Coexistence of viral RNA in plasma and specific antibodies was simultaneously observed over three consecutive weeks in two critical cases. In three convalescent patients in particular, cultivable SARS-CoV was detected in stool or urine specimens for longer than 4 weeks (29-36 days). These findings suggest that SARS-CoV may remain viable in the excretions of convalescent patients.

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus.

Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials.

HBV-specific peptide associated with heat-shock protein gp96.

Glycoprotein 96 (gp96), a member of the heat-shock protein family, can elicit priming of antigen-specfic cytotoxic T lymphocytes, when bound to antigenic viral or tumour peptides. We used direct peptide isolation from purified gp96 and microsequencing to show that a virus-specific peptide is bound to gp96 derived from liver tissues of patients with hepatitis B virus (HBV)-induced hepatocellular carcinoma. This virus-specific peptide has potential for engineering tumour vaccines against hepatocellular carcinoma and chronic HBV infection.

Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor.

In recent years, substantial progress has been made towards understanding the molecular basis for CD8 binding to class I MHC and the coreceptor's role in cytotoxic T-cell activation. Here, we review the structural, mechanistic and functional studies that point to a model of coordination of T-cell receptor and CD8 signaling that might provide the key to cytotoxic T-cell activation.

Macrophage-tropic HIV induces and exploits dendritic cell chemotaxis.

Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell-derived factor (SDF)-1alpha. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell-tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC-T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell-specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual.

Gene expression profiling in the human hypothalamus-pituitary-adrenal axis and full-length cDNA cloning.

The primary neuroendocrine interface, hypothalamus and pituitary, together with adrenals, constitute the major axis responsible for the maintenance of homeostasis and the response to the perturbations in the environment. The gene expression profiling in the human hypothalamus-pituitary-adrenal axis was catalogued by generating a large amount of expressed sequence tags (ESTs), followed by bioinformatics analysis (http://www.chgc.sh.cn/ database). Totally, 25,973 sequences of good quality were obtained from 31,130 clones (83.4%) from cDNA libraries of the hypothalamus, pituitary, and adrenal glands. After eliminating 5,347 sequences corresponding to repetitive elements and mtDNA, 20,626 ESTs could be assembled into 9, 175 clusters (3,979, 3,074, and 4,116 clusters in hypothalamus, pituitary, and adrenal glands, respectively) when overlapping ESTs were integrated. Of these clusters, 2,777 (30.3%) corresponded to known genes, 4,165 (44.8%) to dbESTs, and 2,233 (24.3%) to novel ESTs. The gene expression profiles reflected well the functional characteristics of the three levels in the hypothalamus-pituitary-adrenal axis, because most of the 20 genes with highest expression showed statistical difference in terms of tissue distribution, including a group of tissue-specific functional markers. Meanwhile, some findings were made with regard to the physiology of the axis, and 200 full-length cDNAs of novel genes were cloned and sequenced. All of these data may contribute to the understanding of the neuroendocrine regulation of human life.

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha.

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties.

Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.

TCR binding to peptide-MHC stabilizes a flexible recognition interface.

The binding of TCRs to their peptide-MHC ligands is characterized by a low affinity, slow kinetics, and a high degree of cross-reactivity. Here, we report the results of a kinetic and thermodynamic analysis of two TCRs binding to their peptide-MHC ligands, which reveal two striking features. First, significant activation energy barriers must be overcome during both association and dissociation, suggesting that conformational adjustments are required. Second, the low affinity of binding is a consequence of highly unfavorable entropic effects, indicative of a substantial reduction in disorder upon binding. This is evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding. Such conformational flexibility, which may also be a feature of primary antibodies, is likely to contribute to cross-reactivity in antigen recognition.

T cell receptor and coreceptor CD8 alphaalpha bind peptide-MHC independently and with distinct kinetics.

The T cell surface glycoprotein CD8 enhances T cell antigen recognition by binding to MHC class I molecules. We show that human CD8 alphaalpha binds to the MHC class I molecule HLA-A2 with an extremely low affinity (Kd approximately 0.2 mM at 37 degrees C) and with kinetics that are between 2 and 3 orders of magnitude faster than reported for T cell receptor/peptide-MHC interactions. Furthermore, CD8 alphaalpha had no detectable effect on a T cell receptor (TCR) binding to the same peptide-MHC class I complex. These binding properties provide an explanation as to why the CD8/MHC class I interaction is unable to initiate cell-cell adhesion and how it can enhance TCR recognition without interfering with its specificity.