Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.     

A novel pathogenic mechanism of highly pathogenic avian influenza H5N1 viruses involves hemagglutinin mediated resistance to serum innate inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.     

Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity

There are many peptides known that inhibit the entry of enveloped viruses into cells, including one peptide that is successfully being used in the clinic as a drug. In this review, we discuss the discovery, antiviral activity and mechanism of action of such peptides. While peptide entry inhibitors have been discovered by a wide variety of approaches (structure-based, accidental, intentional, rational and brute force) we show here that they share a common physical chemical property: they are at least somewhat hydrophobic and/or amphipathic and have a propensity to interact with membrane interfaces. We propose that this propensity drives a shared mechanism of action for many peptide entry inhibitors, involving direct interactions with viral and cellular membranes, as well as interactions with the complex hydrophobic protein/lipid interfaces that are exposed, at least transiently, during virus–cell fusion. By interacting simultaneously with the membrane interfaces and other critical hydrophobic surfaces, we hypothesize that peptide entry inhibitors can act by changing the physical chemistry of the membranes, and the fusion protein interfaces bridging them, and by doing so interfere with the fusion of cellular and viral membranes. Based on this idea, we propose that an approach that focuses on the interfacial hydrophobicity of putative entry inhibitors could lead to the efficient discovery of novel, broad-spectrum viral entry inhibitors. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Discovery of highly potent small molecule Hepatitis C Virus entry inhibitors

Novel, highly potent small molecule HCV entry inhibitors are reported. The SAR exploration of a hit molecule identified from screening of a compound library led to the identification of highly potent compounds with IC(50) values of <5 nM in the tissue culture HCV infectious assay.     

Rapid screening of highly efficient vaccine candidates by immunoproteomics

Diseases caused by microorganisms can be controlled by vaccines, which require neutralizing antigens. Therefore, it is very important to identify highly efficient immunogens for immune prevention. By combining immunoproteomics and bacterial challenge after immunization, we developed a rapid method for screening protected antigens of pathogenic bacteria in aquaculture. Our approach may be divided into three consecutive steps. First, dominant immunogens of outer membrane proteins are screened by immunoproteomics. Second, proteins with the ability to induce production of neutralizing antibodies are identified from the immunogens by virulent bacterium challenge following vaccination. Third, vaccine candidates are determined by evaluation of neutralizing abilities. Information on the candidates has been obtained for further gene cloning by mass spectrometry. Our results indicate that highly efficient protected antigens were identified from the outer membrane proteome of Aeromonas hydrophila, in which an immunogen showed 71.4% protective ability with multivalent functions to A. hydrophila and Aeromonas sobria. In summary, we have developed a high-throughout, accurate, rapid and highly efficient method which will play an active role in immune prevention for microbiological diseases.     

Characterization of low-pathogenic H5 subtype influenza viruses from Eurasia: implications for the origin of highly pathogenic H5N1 viruses

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.     

Rapid whole-cell sensing chip for low-level arsenite detection

A novel whole-cell sensing chip system consisted of a micro-concentrator, a set of electrochemical detection electrodes, and a microfluidic channel was developed for rapid detection of arsenite in water. Firstly, the E. coli cells transformed with arsenited-regulated reporter plasmids were incubated with solution contained arsenite. Under this condition, the level of reporter protein, β-galactosidase, expressed by E. coli cells is dependent on the concentration of arsenite. Using the dielectrophoretic force, the micro-concentrator continuously enriched the E. coli cells into a small area above the embedded detection electrodes. And then the relative expression levels of β-galactosidase were obtained using the electrochemical method to measure the amount of p-aminophenol (PAP) which converted from the p-aminophenyl-β-D-galactopyranoside (PAPG) by β-galactosidase. From the result, it indicates this device can detect as low as 0.1 ppm of arsenite within 30 min. Compared with other traditional detection methods, our new device provides better performance like higher sensitivity, shorter analysis time, and lower cost in detecting the arsenite.     

Antigenic diversity of H5 highly pathogenic avian influenza viruses of clade 2.3.4.4 isolated in Asia

H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1‐7/2014 (H5N8), belonging to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 2.3.4.4 had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade 2.3.4.4 were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade 2.3.4.4 differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains.     

Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses

Genetic reassortment of H5N1 highly pathogenic avian influenza viruses (HPAI) with currently circulating human influenza A strains is one possibility that could lead to efficient human-to-human transmissibility. Domestic pigs which are susceptible to infection with both human and avian influenza A viruses are one of the natural hosts where such reassortment events could occur. Virological, histological and serological features of H5N1 virus infection in pigs were characterized in this study. Two- to three-week-old domestic piglets were intranasally inoculated with 10(6) EID(50) of A/Vietnam/1203/04 (VN/04), A/chicken/Indonesia/7/03 (Ck/Indo/03), A/Whooper swan/Mongolia/244/05 (WS/Mong/05), and A/Muscovy duck/Vietnam/ 209/05 (MDk/VN/05) viruses. Swine H3N2 and H1N1 viruses were studied as a positive control for swine influenza virus infection. The pathogenicity of the H5N1 HPAI viruses was also characterized in mouse and ferret animal models. Intranasal inoculation of pigs with H5N1 viruses or consumption of infected chicken meat did not result in severe disease. Mild weight loss was seen in pigs inoculated with WS/Mong/05, Ck/Indo/03 H5N1 and H1N1 swine influenza viruses. WS/Mong/05, Ck/Indo/03 and VN/04 viruses were detected in nasal swabs of inoculated pigs mainly on days 1 and 3. Titers of H5N1 viruses in nasal swabs were remarkably lower compared with those of swine influenza viruses. Replication of all four H5N1 viruses in pigs was restricted to the respiratory tract, mainly to the lungs. Titers of H5N1 viruses in the lungs were lower than those of swine viruses. WS/Mong/05 virus was isolated from trachea and tonsils, and MDk/VN/05 virus was isolated from nasal turbinate of infected pigs. Histological examination revealed mild to moderate bronchiolitis and multifocal alveolitis in the lungs of pigs infected with H5N1 viruses, while infection with swine influenza viruses resulted in severe tracheobronchitis and bronchointerstitial pneumonia. Pigs had low susceptibility to infection with H5N1 HPAI viruses. Inoculation of pigs with H5N1 viruses resulted in asymptomatic to mild symptomatic infection restricted to the respiratory tract and tonsils in contrast to mouse and ferrets animal models, where some of the viruses studied were highly pathogenic and replicated systemically.     

Characterization of highly pathogenic H5N1 avian influenza A viruses isolated from South Korea

An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.     

The role of nuclear NS1 protein in highly pathogenic H5N1 influenza viruses

The non-structural protein (NS1) of influenza A viruses (IAV) performs multiple functions during viral infection. NS1 contains two nuclear localization signals (NLS): NLS1 and NLS2. The NS1 protein is located predominantly in the nucleus during the early stages of infection and subsequently exported to the cytoplasm. A nonsense mutation that results in a large deletion in the carboxy-terminal region of the NS1 protein that contains the NLS2 domain was found in some IAV subtypes, including highly pathogenic avian influenza (HPAI) H7N9 and H5N1 viruses. We introduced different mutations into the NLS domains of NS1 proteins in various strains of IAV, and demonstrated that mutation of the NLS2 region in the NS1 protein of HPAI H5N1 viruses severely affects its nuclear localization pattern. H5N1 viruses expressing NS1 protein that is unable to localize to the nucleus are less potent in antagonizing cellular antiviral responses than viruses expressing wild-type NS1. However, no significant difference was observed with respect to viral replication and pathogenesis. In contrast, the replication and antiviral defenses of H1N1 viruses are greatly attenuated when nuclear localization of the NS1 protein is blocked. Our data reveals a novel functional plasticity for NS1 proteins among different IAV subtypes.     

Collections of pathogenic viruses for addressing general biologic problems

Development of the theory and practice of building and maintenance of collections of reference cultures of pathogens strains is actual in conservation of a biodiversity, and as for maintenance with standardized superfine starting materials fundamental and applied researches in fields of microbiology, an immunology, biotechnology, ecology, and biosafety. Various approaches to taxonomy of viruses, and also definition of concepts "strain" and "species" are discussed in the article. Formulations of the concepts most conforming to museum collections activity are given. The methodology of collecting pathogens, including fifteen-year experience of maintenance of the National collection of hemorrhagic fever viruses belonging to group I of pathogenicity, is summarized. The systemic approach is suggested as a basis of methodology of collecting. Selection of pathogen species and their specific strains according to purpose and on the basis of complex study of their biological and other characteristics, as well as standardization and unification of methods of pathogens maintenance and preparing on their basis working materials, diagnostic kits and other preparations, create a real basis of optimization, unification and continuity of the subsequent applied researches. The offer about formation at the state level of a new independent direction--pathogens collecting with establishment of federal centers based on pathogens' classes has been made.     

Structure-based virtual screening of highly potent inhibitors of the nematode chitinase CeCht1

Nematode chitinases play vital roles in various physiological processes, including egg hatching, larva moulting, and reproduction. Small-molecule inhibitors of nematode chitinases have potential applications for controlling nematode pests. On the basis of the crystal structure of CeCht1, a representative chitinase indispensable to the eggshell chitin degradation of the model nematode Caenorhabditis elegans, we have discovered a series of novel inhibitors bearing a (R)-3,4-diphenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one scaffold by hierarchical virtual screening. The crystal structures of CeCht1 complexed with two of these inhibitors clearly elucidated their interactions with the enzyme active site. Based on the inhibitory mechanism, several analogues with improved inhibitory activities were identified, among which the compound PP28 exhibited the most potent activity with a K_i value of 0.18 μM. This work provides the structural basis for the development of novel nematode chitinase inhibitors.     

Discovery of 9-phenylacridinediones as highly selective butyrylcholinesterase inhibitors through structure-based virtual screening

Butyrylcholinesterase (BuChE) is considered a promising drug target as it plays an important role in the progression of late stage Alzheimer’s disease (AD). Two compound libraries were selected and 64 124 amine containing moieties were screened using a hierarchical virtual screening protocol to discover new selective BuChE inhibitors. From these and subsequent docking experiments, 9-phenylacridinedione (9-PAD) was identified as a promising scaffold for selective inhibition of BuChE. Selected top dock scored 9-PADs were assayed and compounds 3 and 6 exhibited potent and highly selective human BuChE inhibition (IC₅₀: 98 nM and 142 nM, respectively). Both molecules were also predicted to show sufficient brain permeability, not have any substantial toxicities, especially hepatotoxicity, and no significant in vitro cytotoxicity against SH-SY5Y neuroblastoma cells at concentrations up to 100 µM. These findings indicate that 9-PAD is a promising lead structure for the development of agents able to treat late stage AD.     

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.