Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

Porcine reproductive and respiratory syndrome virus: phylogenetic analysis of circulating strains in the Republic of Ireland from 2016 to 2017.

In order to investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains currently circulating in the Republic of Ireland (ROI), the ORF5 gene from 17 field strains originating from four vaccinating commercial herds was sequenced and phylogenetically analysed. High genetic variability was observed between farms at the nucleotide (86.3-95.2%) and amino acid (85.5-96%) levels. Phylogenetic analysis confirmed that all field strains belonged to the European species (type 1) and clustered into three separate groups within the subtype 1 subgroup. This variation may pose challenges for diagnosis and prophylactic control of PRRSV through vaccination in the ROI.

Receptor binding by H10 influenza viruses.

H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.

Receptor binding by an H7N9 influenza virus from humans.

Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.

Receptor binding by a ferret-transmissible H5 avian influenza virus.

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.

Edited course of biomedical research: leaping forward with CRISPR.

Within the short few years since the report of its application in precise genome editing, CRISPR technology has become the method of choice to modify and modulate gene expression in biomedical research and therapeutic development. Subsequently, a variety of research, diagnostic, and therapeutic tools have been developed based upon CRISPR's mechanism of action. Such tools have helped to deepen the understanding of fundamental biology and broaden the horizon in the search for treatments for diseases that have been considered hard or impossible to cure. As CRISPR technology advances closer to clinical applications, its short comings are becoming more apparent, thus creating opportunities to improve the technology's efficacy, specificity, and safety profile in this setting. We will summarize the current status of CRISPR technology and discuss its future impact in this review.

Recent evolution of equine influenza and the origin of canine influenza.

In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222 → Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30 → Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.

Genetic Conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.).

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam, and Australia. Each of 4 strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from 2 independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mg L(-1) for a 48-h exposure at 25 °C. Thus, the mechanism of high-level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera).

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain×Weak-R, S-strain×Strong-R and Weak-R×Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25°C and 55% RH for 72h. Weak-R and Strong-R showed resistance factors of 6.3× and 505× compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of -0.48 and -0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE=0.89) whereas there were two genes controlling strong resistance (nE=1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~80×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.

UNLABELLED: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment. METHODS: Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed. RESULTS: The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir. CONCLUSIONS: Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.

Biology and management of psocids infesting stored products.

Previously regarded as minor nuisance pests, psocids belonging to the genus Liposcelis now pose a major problem for the effective protection of stored products worldwide. Here we examine the apparent biological and operational reasons behind this phenomenon and why conventional pest management seems to be failing. We investigate what is known about the biology, behavior, and population dynamics of major pest species to ascertain their strengths, and perhaps find weaknesses, as a basis for a rational pest management strategy. We outline the contribution of molecular techniques to clarifying species identification and understanding genetic diversity. We discuss progress in sampling and trapping and our comprehension of spatial distribution of these pests as a foundation for developing management strategies. The effectiveness of various chemical treatments and the availability and potential of nonchemical control methods are critically examined. Finally, we identify research gaps and suggest future directions for research.

H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis.

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants.

The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.

Two-tiered approach identifies a network of cancer and liver disease-related genes regulated by miR-122.

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design.

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.

The ets-related transcription factor GABP directs bidirectional transcription.

Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous ets-family transcription factor GA-binding protein (GABP). Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.

[Impact of avian influenza virus H5N1 neuraminidase mutations on the activity of neuraminidase and the sensibility to neuraminidase inhibitors].

OBJECTIVE: To study the impact of avian influenza virus H5N1 neuraminidase mutations I117V, I314V and I117V + I314V on the sensibility of neuraminidase inhibitors (NAIs) and the activity of neuraminidase (NA). METHODS: The mutations were introduced into NA genes of virus strain A/Vietnam/1203/04 (H5N1) by site-directed mutagenesis. With the A/WSN/33 (H1N1) background, recombinant influenza viruses containing NA mutations were rescued by reverse genetics. After viral propagation in chicken embryos, fluorimetric assays were conducted to assess the sensibility to NAIs and NA activity (IC(50), Km & Ki). RESULTS: Compared to the wild-type virus VN1203, the mutation I117V decreased the susceptibility to oseltamivir (17-fold increment of IC(50) value, 20-fold increment of Ki value) and the NA activity (23-fold increment of Km value) while there was little impact on zanamivir sensitivity (2-fold increment of IC(50) value, 3-fold increment of Ki value). The mutation I314V had no marked influence on either the NA activity or the NAIs susceptibility. CONCLUSION: It appears that the NA mutations of I117V and I314V can not cause NAIs resistance. Oseltamivir or zanamivir may still be prescribed for anti-viral treatment.

Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction.

Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L (AAVR), we confirmed the role of two genes, GPR108 and TM9SF2, in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes.

Galactose-conjugates of the oseltamivir pharmacophore--new tools for the characterization of influenza virus neuraminidases.

We describe the synthesis of mimetics of the alpha2-3 and alpha2-6 sialogalactoside substrates of influenza neuraminidase which include the oseltamivir pharmacophore, and report the sub-nanomolar affinities for these novel neuraminidase inhibitors. The challenge of synthesizing a Phospha-Oseltamivir/Tamiphosphor monoester involving the secondary 3-hydroxy group of galactose required to mimic the alpha2-3 sialogalactoside has been overcome by palladium-promoted coupling of the oseltamivir-derived vinyl iodide with a protected galactose-3-phosphonate. The difference in binding of these two inhibitors to a given influenza neuraminidase should be a function of its alpha2-3/alpha2-6-selectivity, an important, but not yet fully understood factor in the adaptation of highly pathogenic avian influenza viruses to human hosts.