Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N- or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

During May 20-October 13, 2018,* low levels of influenza activity were reported in the United States, with a mix of influenza A and B viruses circulating. Seasonal influenza activity in the Southern Hemisphere was low overall, with influenza A(H1N1)pdm09 predominating in many regions. Antigenic testing of available influenza A and B viruses indicated that no significant antigenic drift in circulating viruses had emerged. In late September, the components for the 2019 Southern Hemisphere influenza vaccine were selected and included an incremental update to the A(H3N2) vaccine virus used in egg-based vaccine manufacturing; no change was recommended for the A(H3N2) component of cell-manufactured or recombinant influenza vaccines. Annual influenza vaccination is the best method for preventing influenza illness and its complications, and all persons aged ≥6 months who do not have contraindications should receive influenza vaccine, preferably before the onset of influenza circulation in their community, which often begins in October and peaks during December-February. Health care providers should offer vaccination by the end of October and should continue to recommend and administer influenza vaccine to previously unvaccinated patients throughout the 2018-19 influenza season (1). In addition, during May 20-October 13, a small number of nonhuman influenza "variant" virus infections† were reported in the United States; most were associated with exposure to swine. Although limited human-to-human transmission might have occurred in one instance, no ongoing community transmission was identified. Vulnerable populations, especially young children and other persons at high risk for serious influenza complications, should avoid swine barns at agricultural fairs, or close contact with swine.§.

A highly sensitive self-enzyme immunoassay with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed for the first time to quantify influenza A (H1N1) virus. A specific capture antibody, capable of capturing hemagglutinin (HA) subtypes of H1N1, was immobilized on the surface of the magnetic Au-Fe3O4 nanocomposite. Neuraminidase (NA) subtype of H1N1 was acts as an enzyme in the self-enzyme immunoassay. A sample mixed with HA antibodies immobilized on the surface of magnetic Au-Fe3O4 nanocomposites was incubated for 1 h at 37 °C. After the incubation, magnetic Au-Fe3O4 nanocomposites separated with a magnetic bar were washed 3 times using phosphate buffered saline with Tween-20 (PBST). Then, 4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA), a fluorogenic substrate of NA, was added and incubated for 10 min to produce 4-Methylumbelliferone (4-MU) from the enzyme reaction between MUNANA and NA of H1N1. After the incubation, the solution containing 4-MU emitted bright light with the addition of ODI-CL reagents (e.g., H2O2 and ODI). The relative CL intensity of 4-MU was proportionally enhanced with the increase of H1N1. Also, the brightness and color of 4-MU light emitted from the self-enzyme immunoassay was dependent on pH. The self-enzyme immunoassay was able to analyze trace levels of influenza A (H1N1) virus with good accuracy, precision, reproducibility and excellent selectivity. The limit of detection (LOD = 3σ/slope) was as low as 0.19 U/ml. We expect that the self-enzyme immunoassay can be applied as a cost-effective and rapid method capable of selectively quantifying a specific influenza A virus such as H1N1, H3N2, and H5N1.

Treatment of drug resistant protozoa, bacteria, and viruses requires new drugs with alternative chemotypes. Such compounds could be found from Southeast Asian medicinal plants. The present study examines the cytotoxic, antileishmanial, and antiplasmodial effects of 11 ethnopharmacologically important plant species in Malaysia. Chloroform extracts were tested for their toxicity against MRC-5 cells and Leishmania donovani by MTT, and chloroquine-resistant Plasmodium falciparum K1 strain by Histidine-Rich Protein II ELISA assays. None of the extract tested was cytotoxic to MRC-5 cells. Extracts of Uvaria grandiflora, Chilocarpus costatus, Tabernaemontana peduncularis, and Leuconotis eugenifolius had good activities against L. donovani with IC50 < 50 μg/mL. Extracts of U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities against P. falciparum K1 with IC50 < 10 μg/mL. Pinoresinol isolated from C. costatus was inactive against L. donovani and P. falciparum. C. costatus extract and pinoresinol increased the sensitivity of Staphylococcus epidermidis to cefotaxime. Pinoresinol demonstrated moderate activity against influenza virus (IC50 = 30.4 ± 11 μg/mL) and was active against Coxsackie virus B3 (IC50 = 7.1 ± 3.0 μg/mL). β-Amyrin from L. eugenifolius inhibited L. donovani with IC50 value of 15.4 ± 0.01 μM. Furanodienone from C. aeruginosa inhibited L. donovani and P. falciparum K1 with IC50 value of 39.5 ± 0.2 and 17.0 ± 0.05 μM, respectively. Furanodienone also inhibited the replication of influenza and Coxsackie virus B3 with IC50 value of 4.0 ± 0.5 and 7.2 ± 1.4 μg/mL (Ribavirin: IC50: 15.6 ± 2.0 μg/mL), respectively. Our study provides evidence that medicinal plants in Malaysia have potentials as a source of chemotypes for the development of anti-infective leads.

In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that a reassortment event may lead to highly pathogenic influenza strains. H1N1pdm09 and H5N1 are able to infect the same target cells of the lower respiratory tract. To investigate the capacity of the emergence of reassortant viruses, we characterized viruses obtained from the co-infection of cells with H5N1 (A/Turkey/13/2006) and H1N1pdm09 (A/Lyon/969/2009 H1N1). In our analysis, all the screened reassortants possessed the PB2, HA, and NP segments from H5N1 and acquired one or two of the H1N1pdm09 segments. Moreover, the in vivo infections showed that the acquisition of the NS segment from H1N1pdm09 increased the virulence of H5N1 in mice. We conclude, therefore, that reassortment can occur between these two viruses, even if this process has never been detected in nature.

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA-detergent micelles with full-length HA-Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.