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.

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.

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.

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.

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.

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.

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

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.

Classification of a new member of the TBE flavivirus subgroup by its immunological, pathogenetic and molecular characteristics: identification of subgroup-specific pentapeptides.

The antigenic, pathogenic and molecular characteristics of Turkish sheep encephalitis (TSE) virus, strain TTE80, were compared with other members of the tick-borne encephalitis (TBE) virus complex. Monoclonal antibodies with defined specificity for the flavivirus envelope glycoprotein distinguished TSE virus from louping ill (LI), western or far eastern TBE, Langat and Powassan virus in indirect immunofluorescence, haemagglutination-inhibition and neutralization tests. On the other hand, TSE virus, which produces an LI-like disease in sheep, resembled LI virus in mouse neurovirulence tests. Molecular homology data of all the structural genes of TSE virus compared with other tick-borne flaviviruses demonstrated that TSE virus is a distinct member in the TBE virus subgroup. The data are consistent with the conclusion that TSE virus has evolved by a separate evolutionary pathway as compared with the close antigenic relatives, western European, far eastern TBE viruses and LI virus. By aligning the encoded amino acids in the viral envelope glycoprotein of mosquito- and tick-borne flaviviruses, we have also identified subgroup-specific pentapeptide motifs for the tick-borne encephalitis, Japanese encephalitis and dengue subgroup viruses of the genus Flavivirus. These pentapeptides have important implications for the evolution, classification and diagnosis of flaviviruses.

The virus causing encephalomyelitis in sheep in Spain: a new member of the tick-borne encephalitis group.

The nucleotide and deduced primary amino acid sequence of the envelope gene of two virus isolates from the brains of Spanish sheep with encephalomyelitis, were determined and compared with those of other flaviviruses. The amino acid alignments showed that the Spanish viruses shared 95 to 96 per cent homology with the envelope protein of louping ill virus and western European tick-borne encephalitis virus. In comparison, the maximum variation in amino acid identities among strains of louping ill virus from the British Isles is 1.8 per cent. The Spanish isolates were distinguishable from all other known flaviviruses by the presence of a unique tripeptide sequence (AQR) at amino acid positions 232 to 234 in the E protein, the position at which a genetic marker for distinct flavivirus species has been identified. Other genetic markers, viz DSGHD (amino acids 320 to 324) and EHLPTA (amino acids 207 to 212), which identify the tick-borne encephalitis group within the genus Flavivirus, were present in the amino acid sequences of the Spanish virus. It is concluded that the cause of sheep encephalomyelitis in Spain is a distinct species in the tick-borne encephalitis virus group.

Molecular variation, evolution and geographical distribution of louping ill virus.

Following the demonstration that the tick-borne encephalitis (TBE) subgroup viruses are distributed as a cline across the Northern Hemisphere (Zanotto et al., 1995), we have analyzed the dispersal pattern of louping ill (LI) virus, the most westerly located member in the cline. A total number of 21 LI or LI-related virus E gene sequences have been used for a detailed molecular analysis of the evolution, phylogeny and geographical distribution of LI virus in the British Isles and Ireland. The results show that LI virus is genetically stable in general but minor differences enable its separation into four genetically distinct subtypes (genotypes) with clear geographical correlation, designated Type 1 in Scotland and England, Type 2 in Scotland, Type 3 in Wales and Type 4 in Ireland. These data demonstrate that geographically independent evolution of LI viruses has occurred. The molecular systematics and substitutional parameters analyses combined with the clinal distribution of the TBE virus complex allow the assignment of the origin for both Negishi (NEG) virus and a Norwegian isolate to the British Isles. Moreover, proposals for the classification of LI and LI-like viruses which cause encephalomyelitis in sheep, goat or cattle are presented.

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.

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.

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.

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.

Identification of naturally occurring monoclonal antibody escape variants of louping ill virus.

Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelope-specific and five non-structural protein NS1-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.

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.

Tracing the origins of louping ill virus by molecular phylogenetic analysis.

The nucleotide and deduced amino acid sequences of louping ill (LI) virus isolates, collected from representative regions of the British Isles and Norway, were determined for either the entire envelope gene (20 isolates) or for a portion of the envelope gene that spans a hypervariable region and includes an LI virus specific marker sequence (53 isolates). Phylogenetic analysis reveals the presence of three major geographical populations of LI virus in the British Isles, viz. Irish, Welsh and British LI viruses, which all cause encephalomyelitis in animals, predominantly sheep, and co-habit the same tick population. British LI virus occurs throughout Scotland, England, Ireland and Norway. Irish and Welsh LI viruses occur only in Ireland and Wales, respectively. Phylogenetic analysis also predicts that LI virus initially emerged in Ireland and that a descendant was introduced into Great Britain via Wales and was subsequently transported to the borders of Scotland, from where it was dispersed throughout Scotland, northern England and Norway. More recently, the British LI virus was reintroduced into Ireland and also into south-west England. Dates of lineage divergence, calculated from the synonymous substitution rate, indicate that LI virus emerged in the British Isles less than 800 years ago and most LI virus dispersal occurred during the last 300 years. By combining these data with historical records it appears that livestock movement can be implicated in the dispersal of LI virus.