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Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) previously found in human infections into the NA of three current AIV subtypes of the H5Nx genotype that threaten the poultry industry and human health: highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we showed that a R292K substitution in H5N6 and H5N2 viruses significantly reduced susceptibility to three licenced NAIs: oseltamivir, zanamivir and peramivir. In contrast the mutations E119V, H274Y and N294S had more variable effects with NAI susceptibility being drug- and strain-specific. We measured the replicative fitness of NAI resistant H5N6 viruses and found that they replicated to comparable or significantly higher titres in primary chicken cells and in embryonated hens' eggs as compared to wild type - despite the NA activity of the viral neuraminidase proteins being reduced. The R292K and N294S drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA): Y98F and A189T, respectively (H3 numbering) which reduced receptor binding properties possibly balancing the reduced NA activity seen. Our results demonstrate that the H5Nx viruses can support drug resistance mutations that confer reduced susceptibility to licenced NAIs and that these H5N6 viruses did not show diminished replicative fitness in avian cell cultures. Our results support the requirement for on-going surveillance of these strains in bird populations to include motifs associated with human drug resistance., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Measles virus, a member of the genus Morbillivirus, is highly contagious and still shows considerable mortality with over 100000 deaths annually, although efficient attenuated vaccines exist. Recent studies of measles virus haemagglutinin (MeV-H) and its receptor, including crystallographic and electron microscopic structural analyses combined with functional assays, have revealed how the MeV-H protein recognizes its cognate receptors, SLAM and Nectin-4, and how the glycan shield ensures effective vaccination. In addition, the crystal structure of the MeV-F protein indicated its similarity to those of other paramyxoviruses. Taking into account these data, several models of viral entry/membrane fusion of measles viruses and related paramyxoviruses have been proposed. Furthermore, anti-MeV-F inhibitors targeted to specific regions to inhibit MeV-F protein activation were reported, with potency for preventing MeV infection. The inhibitors targeted for entry events may potentially be applied to treatment of MeV-derived diseases, although escape mutations and drug profiles should be considered., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Camelpox and camel contagious ecthyma are infectious viral diseases of camelids caused by camelpox virus (CMLV) and camel contagious ecthyma virus (CCEV), respectively. Even though, in Ethiopia, pox disease has been creating significant economic losses in camel production, little is known on the responsible pathogens and their genetic diversity. Thus, the present study aimed at isolation, identification and genetic characterization of the causative viruses. Accordingly, clinical case observations, infectious virus isolation, and molecular and phylogenetic analysis of poxviruses infecting camels in three regions and six districts in the country, Afar (Chifra), Oromia (Arero, Miyu and Yabello) and Somali (Gursum and Jijiga) between 2011 and 2014 were undertaken. The full hemagglutinin (HA) and partial A-type inclusion protein (ATIP) genes of CMLV and full major envelope protein (B2L) gene of CCEV of Ethiopian isolates were sequenced, analyzed and compared among each other and to foreign isolates. The viral isolation confirmed the presence of infectious poxviruses. The preliminary screening by PCR showed 27 CMLVs and 20 CCEVs. The sequence analyses showed that the HA and ATIP gene sequences are highly conserved within the local isolates of CMLVs, and formed a single cluster together with isolates from Somalia and Syria. Unlike CMLVs, the B2L gene analysis of Ethiopian CCEV showed few genetic variations. The phylogenetic analysis revealed three clusters of CCEV in Ethiopia with the isolates clustering according to their geographical origins. To our knowledge, this is the first report indicating the existence of CCEV in Ethiopia where camel contagious ecthyma was misdiagnosed as camelpox. Additionally, this study has also disclosed the existence of co-infections with CMLV and CCEV. A comprehensive characterization of poxviruses affecting camels in Ethiopia and the full genome sequencing of representative isolates are recommended to better understand the dynamics of pox diseases of camels and to assist in the implementation of more efficient control measures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology., From the Clinical Editor: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4°C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4°C and room temperature. Therefore, long-term storage at 4°C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens., (Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: The authors had for aim to characterize influenza B strains having circulated in Tunisia to identify new mutations and compare them with reference strains., Methods: The epidemiological surveillance of influenza allowed identifying 19 patients with symptoms related to respiratory infection, who had been infected by influenza B strains isolated in several regions of Tunisia in 2009-2010 and in 2010-2011. Laboratory identification and detection of mutations in the segment encoding hemagglutinin of influenza viruses was performed by real time PCR and sequencing., Results: The two influenza B Tunisian strains of the 2009-2010 season belonged to the Victoria lineage, whereas 2010-2011 season strains belonged to B/Victoria/2/87 and B/Yamagata/16/88 lineages with a dominance of the Yamagata lineage (76%). This study allowed identifying amino acid substitutions: T121A, S150I, N165Y, T181A, G183R, D196N, S229D, M251V and K253R in the B/Yamagata lineage; L58P, N75K, K109N, N165K, S172P and K257R into the B/Victoria lineage. These mutations were specific of Tunisian groups of variants. Most influenza B-Yamagata lineage viruses (69%) were associated with severe cases., Conclusion: Molecular analysis of the various influenza B strains circulating in Tunisia is useful to detect new mutations that can modify the phenotype of influenza strains., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The H1N1 2009 influenza pandemic took the health care workers by surprise in spite of warning about influenza pandemic. Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes by shift or drift. Thus, understanding the evolution of the viruses in human is important for the surveillance and the selection of vaccine strains. A total of 23 pandemic A/H1N1 2009 viral HA gene sequences were downloaded from NCBI submitted during March and May 2010 by NIV and were analysed. Along with that the vaccine strain A/California/07/2009 was also downloaded from NCBI. All the sequences were used to analyse the evolution of the haemagglutinin (HA) by phylogenetic analysis. The HA gene could be divided into four groups with shift from 1 to lV revealing that the HA genes of the influenza A viruses evolved in a sequential way, in comparison to vaccine strain A/California/07/2009. Amino acid sequence analysis of the HA genes of the A/H1N1 2009 isolates, revealed mutations at positions 100, 220 and additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285 .The mutations identified showed the adaptation of the new virus to the host that could lead to genetic changes inherent to the virus resulting in a reassortant which could be catastrophic, hence continuous monitoring of strains is mandatory.

Purpose: Influenza virus is a major cause of human respiratory infections and responsible for pandemics and regional outbreaks around the world. This investigation aims to determine the prevalent influenza genotypes during 2005-2007 outbreaks in Shiraz, the capital city of Fars province, southern Iran and compare the results obtained with those of previous study., Materials and Method: Of the 300 pharyngeal swabs collected from influenza patients, 26 were found to be positive by culture and hemagglutination (HA) assays. Typing and subtyping of the isolates carried out by using multiplex RT-PCR and phylogenetic analysis performed on isolated HA genes using neighbour-joining method., Result: Out of 26 positive isolates 12 and 14 were H1N1 and H3N2 respectively. The phylogenetic and amino acid sequence analyses of our H1N1 isolates showed 99-100% genetic resemblance to A/NewCaledonia/20/99 (H1N1) vaccine strain. Most of the Iranian H3N2 isolates varied form A/California/7/2004 vaccine strain in 20 amino acids of which positions 189,226 and 227 were located in antigenic sites of HA1 molecule. These substitutions were not observed in any of the H3N2 subtypes from the same region reported previously., Conclusion: The H3N2 subtype strains prevalent during the 2005/7 influenza outbreak in southern Iran demonstrated a drastic antigenic variation and differed from A/California/7/2004 vaccine strain. The H1N1 subtypes showed a notable resemblance to A/NewCaledonia/20/99 vaccine strain and therefore were predicted to be capable of conferring sufficient immunity against H1N1 subtypes.

The continuous threat of influenza pandemics determines the urgency and necessity to develop safe and effective vaccines against divergent influenza viruses. This review describes the advancements in the research and development of universal influenza vaccines based on the relatively conserved sequences of M2e, HA, and other proteins of influenza viruses., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.

The wide tissue tropism of the measles virus (MV) suggests that it involves ubiquitously expressed molecules. We have constructed a recombinant MV expressing the enhanced green fluorescent protein (EGFP) (rMV-EGFP) and demonstrated that the rMV-EGFP infected several cell types (HEK-293, HepG2, Hep3B, Huh7, and WRL68 cells) that do not express the human signalling lymphocyte activation molecule (SLAM), which is known as a cellular receptor for morbilliviruses. MV infection of HEK-293 and HepG2 cells was not inhibited in an infectivity-inhibition assay using an anti-SLAM monoclonal antibody, indicating that MV could infect cells without using SLAM. Soluble heparin (HP) inhibited the rMV-EGFP infectivity in SLAM-negative cell lines in a dose-dependent manner. Direct interaction between purified virions and HP was detected in a surface plasmon resonance assay. We also demonstrated that the hemagglutinin (H) protein, but not the fusion (F) protein is responsible for the interaction between the virions and HP. Taken together, our results suggest that HP-like glycosaminoglycans bind to the H protein of MV and play a key role in the infection of SLAM-negative cells.

For the treatment of influenza virus infections, neuraminidase inhibitors (NAIs) that prevent the release of virus particles have been effective against most influenza strains. Several neuraminidase (NA) assays are available for the evaluation of NAIs. To understand the NAI functions under physiological conditions, assays mimicking viral particle release should be useful. We have constructed retrovirus-based reporter viruses that are pseudotyped with hemagglutinin (HA) glycoprotein by transfection of producer cells using plasmids expressing retroviral gag-pol, influenza HA, NA, and firefly luciferase genes. Similarly to the life cycle of influenza viruses, the release of pseudotype viruses also requires neuraminidase functions. This requirement was used to develop an assay to evaluate NAI activities by measuring inhibition of pseudotype virus production at different NAI concentrations. The pseudotype virus release assay was used to determine the IC(50) values of Oseltamivir carboxylate, Zanamivir, and the novel phosphonate congeners of Oseltamivir against N1 group neuraminidases and their H274Y Oseltamivir carboxylate-resistant mutants. The deduced IC(50) values obtained using the release assay correlated with those determined using the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (MUNANA) and also correlated with the infectivity results.

In this study, we calculate the unpredictable portion of amino-acid pairs, which has been developed by us over the last several years, of 1201 hemagglutinins from influenza A viruses dated from 1918 to 2004 in order to compare them with respect to subtypes, species, and years. After noticing the fluctuations of unpredictable portion along the time course, we use the fast Fourier transform to find the mutation periodicity of hemagglutinins. Then we estimate our position at the current cycle of hemagglutinin evolutionary process to determine how many years remain before the next outbreak of influenza and bird flu. Finally, we use the trend line and channel to outlook the hemagglutinins for the next half a century. As our study covers almost all the full-length amino-acid sequences of hemagglutinins from various influenza A viruses, the conclusion will be valid for years until the number of hemagglutinins in protein databank will be significantly increased.

Cranberry juice contains high molecular weight materials (NDM) that inhibit bacterial adhesion to host cells as well as the co-aggregation of many oral bacteria. Because of its broad-spectrum activity, we investigated NDM's potential for inhibiting influenza virus adhesion to cells, and subsequent infectivity. Hemagglutination (HA) of red blood cells (RBC) caused by representatives of both influenza virus A subtypes (H1N1)and H3N2) and the B type was inhibited by NDM at concentrations of 125 microg/ml or lower, which is at least 20-fold lower than that usually found in cranberry juice. A dose-response effect of NDM on HA was demonstrated. The infectivity of the A and B types was significantly reduced by preincubation with NDM (250 microg/ml), as reflected by the lack of cytopathic effect on Madine-Darby canine kidney (MDCK) cells and the lack of HA activity in the media of infected cells. The effect of NDM was also tested after A or B type viruses were allowed to adsorb to and penetrate the cells. Various levels of reduction in virus tissue culture infective dose TCID50 were observed. The effect was most pronounced when NDM was added several times to the infected MDCK cells. Our cumulative findings indicate that the inhibitory effect of NDM on influenza virus adhesion and infectivity may have a therapeutic potential.

Background: Oseltamivir is an effective inhibitor of influenza virus neuraminidase. Although viruses resistant to oseltamivir emerge less frequently than those resistant to amantadine or rimantadine, information on oseltamivir-resistant viruses arising during clinical use of the drug in children is limited. Our aim was to investigate oseltamivir resistance in a group of children treated for influenza., Methods: We analysed influenza A viruses (H3N2) collected from 50 children before and during treatment with oseltamivir. We sequenced the genes for neuraminidase and haemagglutinin and studied the mutant neuraminidases for their sensitivity to oseltamivir carboxylate., Findings: We found neuraminidase mutations in viruses from nine patients (18%), six of whom had mutations at position 292 (Arg292Lys) and two at position 119 (Glu119Val), which are known to confer resistance to neuraminidase inhibitors. We also identified another mutation (Asn294Ser) in one patient. Sensitivity testing to oseltamivir carboxylate revealed that the neuraminidases of viruses that have an Arg292Lys, Glu119Val, or Asn294Ser mutation were about 10(4)-10(5)-fold, 500-fold, or 300-fold more resistant than their pretreatment neuraminidases, respectively. Oseltamivir-resistant viruses were first detected at day 4 of treatment and on each successive day of the study. More than 10(3) infectious units per mL of virus were detected in some of the patients who did not shed drug-resistant viruses, even after 5 days of treatment., Interpretation: Oseltamivir-resistant mutants in children being treated for influenza with oseltamivir arise more frequently than previously reported. Furthermore, children can be a source of viral transmission, even after 5 days of treatment with oseltamivir.

The adhesion molecules involved in the migration and retention of activated effector CD8 T cells in the lung microcirculation and their recruitment into lung tissue are largely unknown. Here, we have analyzed the role of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) on adhesion of influenza hemagglutinin (HA)-specific CD8 T-cell clone D4 under shear conditions in an in vitro binding assay and in an in vivo homing assay to the lungs of naive or transgenic Balb/c mice expressing HA (HA-Tg) by a lung-specific promoter. Blocking LFA-1 or intercellular adhesion molecule 1 (ICAM-1) significantly inhibited adhesion of D4 cells to lung vascular endothelium and parenchyma of lung sections. However, blocking VLA-4 or vascular cell adhesion molecule 1 (VCAM-1) had no effect on cell adhesion. Blocking LFA-1 in vivo significantly delayed lethal injury following adoptive transfer of D4 cells into HA-Tg mice as assessed by weight loss and histology. Residence time of adoptively transferred Indium 111 (111In)-labeled D4 cells in lungs of normal and HA-Tg mice as analyzed by dual modality imaging revealed a significantly shorter transit time of 4 hours for the D4 cells upon in vivo blockade of LFA-1. These results demonstrate a crucial role for LFA-1 in retention of activated CD8 T cells in normal mouse lungs and in the progression of lethal injury in HA-Tg mice.

A procedure described here allows the efficient and rapid purification of histidine-tagged measles virus haemagglutinin that is synthesized under the control of powerful promoters (PSFJ1-10 and PSFJ2-16) of the highly attenuated vaccinia virus (VV) strain LC16mO. A single affinity chromatography step purifies recombinant haemagglutinin proteins from the lysates of cells infected with the recombinant VVs. The recovery and purity are both very high (a yield of 0.5-2.8 mg/10(8) cells and purity of >94-98%), indicating that this procedure is approximately 400 times more efficient than the conventional methods used to prepare haemagglutinin. The haemagglutinins are correctly transported to the cell surface and have haemadsorption activity. Moreover, the recombinant haemagglutinin proteins cooperate with the measles virus fusion protein to elicit cell fusion activity. In addition, the antibody titres against measles virus, as measured by enzyme-linked immunosorbent assay using the purified haemagglutinin as the capture antigen, correlated closely with neutralization test titres (R(2) = 0.84, p < 0.05), indicating the preservation of immunologically relevant antigenicity. Such recombinant haemagglutinin preparations will be useful in diagnostic tests that measure functional anti-measles immunity and investigate the biological functions and structure of the haemagglutinin.

Reversible histone acetylation changes the chromatin structure and can modulate gene transcription. Mammalian histone deacetylase 1 (HDAC1) is a nuclear protein that belongs to a growing family of evolutionarily conserved enzymes catalysing the removal of acetyl residues from core histones and other proteins. Previously, we have identified murine HDAC1 as a growth factor-inducible protein in murine T-cells. Here, we characterise the molecular function of mouse HDAC1 in more detail. Co-immunoprecipitation experiments with epitope-tagged HDAC1 protein reveal the association with endogenous HDAC1 enzyme. We show that HDAC1 can homo-oligomerise and that this interaction is dependent on the N-terminal HDAC association domain of the protein. Furthermore, the same HDAC1 domain is also necessary for in vitro binding of HDAC2 and HDAC3, association with RbAp48 and for catalytic activity of the enzyme. A lysine-rich sequence within the carboxy terminus of HDAC1 is crucial for nuclear localisation of the enzyme. We identify a C-terminal nuclear localisation domain, which is sufficient for the transport of HDAC1 and of reporter fusion proteins into the nucleus. Alternatively, HDAC1 can be shuttled into the nucleus by association with another HDAC1 molecule via its N-terminal HDAC association domain. Our results define two domains, which are essential for the oligomerisation and nuclear localisation of mouse HDAC1., (Copyright 2001 Academic Press.)

To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5-45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830-844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(4)] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L(2)T(2))(4), were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(4)] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.

We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established.

Background: Except for Reye's syndrome, influenza-associated acute encephalopathy or encephalitis is not universally recognised. We did a multicentre study of laboratory and clinical data for patients with influenza-associated acute encephalopathy or encephalitis., Methods: In Nagoya, Japan, ten patients with acute encephalopathy or encephalitis associated with influenza-like illness were admitted to our hospitals between April, 1996, and March, 1997. We collected clinical, laboratory and serological data and assessed cerebrospinal fluid samples by PCR for influenza A and B., Findings: Seven patients, aged 22 months to 4 years, had evidence of recent influenza infection, six with type-A/Hong Kong (H3N2) and one with type B. The first sign in the central nervous system appeared within 2 days of fever in all but one patient. The first sign of involvement of the central nervous system was generalised convulsions in all patients. Two patients died, one had sequelae, and four survived without sequelae. PCR for influenza type A was positive for five patients., Interpretation: The results of PCR suggest that at least part of the influenza type A genome existed in the central nervous system. Influenza-associated acute encephalopathy or encephalitis in young children deserves wider recognition.

An A/PR/8/34 (IHN1) influenza virus hemagglutinin (HA)-specific monoclonal antibody (Z38) was found to react with the solid phase adsorbed influenza virus expressing uncleaved (HA0) molecules but not to bind to virus particles bearing enzymatically cleaved hemagglutinin. Synthetic peptides corresponding to the uncleaved HA0 317-341 intersubunit region of subtypes H1-H3 (IP1-IP3) or comprising either the C-terminal 317-329 amino acids of HA1 (CP1) or the N-terminal 330-341 of HA2 (fusion peptide, FP) subunits of cleaved HA were used to characterize the fine specificity of Z38 mAb. Circular dichroism and Fourier-transform infrared spectroscopy showed that, compared to IP2 and IP3 comprising the H2 and H3 subtype fragments of the intact intersubunit region, IP1 has relatively low helicity but a tendency to adopt beta-turns in trifluoroethanol. The immunological and conformational properties of multiple antigenic peptides (MAPs) containing four copies of CP1 were also studied. Based on the appearance of an infrared component band at 1637 cm-1 (beta-turn band), the CP1 arms of MAPs also contain repeats of beta-turns. However, it is only the MAP1-FP construct comprising also the fusion peptide, which binds Z38 mAb as strongly as IP1 does. This puts emphasis on the role of the fusion region in modifying conformation and consequently the ability of peptides to elicit an antibody response. The results obtained for peptide conformation also suggests a beta-turn(s)/beta-sheet/beta-turn/beta-sheet conformational motif in the recognition by the hemagglutinin subtype-specific Z38 monoclonal antibody or by peptide-induced polyclonal antibodies.

A peptide display library was evaluated as a means to identify peptide binding motifs for class II molecules. Peptides expressed as part of a soluble fusion protein with a maltose binding protein (malE) were produced by Escherichia coli. Constructs containing the high-affinity binding influenza hemagglutinin peptide 307W-319 (mal-HA) or the low-affinity binding tetanus toxoid peptide 830-843 (mal-TT) were used as controls. mal-HA, but not mal-TT, inhibited synthetic biotinylated-HA peptide from binding to purified DR4 Dw4 molecules in a dose-dependent manner. The fusion-peptide presentation system was also evaluated for its ability to induce antigen-specific T cell proliferation. DR4 Dw4+ B cells pulsed with mal-HA, but not mal-TT, induced dose-dependent proliferation of an HA-specific DR4 Dw4-restricted T cell line to the same extent as synthetic HA peptide. Using this type of peptide display library, it may be possible to determine the antigenic specificity of T cell clones isolated from patients with autoimmune diseases.

This study used the nucleotide sequence coding for the HA1 domain of virus haemagglutinin to show that influenza A/Finland/158/91, which represents the H1N1 subtype viruses prevalent in Finland in 1990/91, was a direct descendant of a virus (A/NN/1605/88) isolated during the 1988/89 epidemic season in Japan. The elevated rate of 7.4 x 10(-3) nucleotide substitutions per site per year is discussed. The new branch of H1N1 subtype viruses is characterized by loss of a glycosylation site, which may affect subsequent antigenic drift.

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.

We applied a combination of reverse transcription (RT) with the polymerase chain reaction (PCR) for a rapid detection of influenza virus H1 subtype. We amplified a 441 bp segment of relatively high genetic stability of the hemagglutinin gene. Experimental conditions were established using plasmid DNA and infected cell cultures. The test was applied to 28 nasopharyngeal lavages from patients, two of which were positive for influenza virus H1. When the amplified DNA of a positive sample was sequenced we found 97% homology with the recent strain A/USSR/70.

Cadherins are a family of integral membrane glycoproteins that mediate homophilic, calcium-dependent cell adhesion in vertebrate species. The primary structures of six members of the cadherin family have recently been determined. The extracellular portion of these proteins is composed of five domains, the first of which is the most highly conserved among cadherins. Previous searches of protein sequence databases have revealed little or no sequence homology between cadherins and other proteins. Here we report that the first extracellular domain of cadherins exhibits substantial sequence homology with the amino termini of influenza strain A hemagglutinins. These regions of sequence homology have been shown to be functionally important in both cadherins and hemagglutinins. Our observations suggest that a functional domain of cadherins is conserved among other proteins.

13 adenoviruses (Ad) from the urine of 10 patients with acquired immunodeficiency syndrome (AIDS) were characterised by haemagglutination inhibition and restriction endonuclease analysis of their DNA. The haemagglutinin (HA) for 6 of these isolates was found to be that of Ad34/35. 3 other isolates were found to have Ad7 HA and the remaining 4 viruses were found to have both phenotypes. In contrast, the restriction patterns were more homogeneous than anticipated from the serological analysis. 11 isolates had a SmaI-restriction pattern identical to Ad35, and 2 isolates, which lacked one of the 9 Ad35 SmaI-restriction sites, were identical to Ad34. Analyses with other restriction enzymes reinforced the conclusion that the genomes of all isolates resemble that of Ad35 (and Ad34) more than they resembled the previously isolated Ad7 subtypes. The discrepancy between the restriction endonuclease and the serological analyses is best explained by assuming that some of these new isolates are recombinants between a small part (less than 10%) of the Ad7 genome coding for HA and greater than 90% of the Ad35 genome. It is therefore important to characterise both the genotype and the HA for this potentially important group of pathogens in AIDS patients.