Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

A downstream processing was examined for Vero cell-derived human influenza virus (H1N1) grown in serum free medium. Vero cell banks were established in serum free medium and characterized according to regulatory requirements. Serum free Vero cells were grown on Cytodex 3 microcarriers in 5L bioreactor and infected with influenza A virus (A/New Caledonia/99/55). The harvests were processed with the sequence of inactivation, clarification, anion exchange chromatography (DEAE FF), Cellufine Sulfate Chromatography (CSC) and size exclusion chromatography (Sepharose 6FF). Host cell DNA (hcDNA) was mainly removed with DEAE FF column and CSC by 40 and 223 fold, respectively. Most of Vero cell proteins were eliminated in CSC and Sepharose 6FF unit operation by about 13 fold. The overall scheme resulted in high recovery of hemagglutinin (HA) activity and the substantial removal of total protein, host protein and DNA. The total protein content and DNA content per 15 μg HA protein in final product was 89 μg and 33 pg, respectively, which complied with regulatory requirements for single strain influenza vaccines. SDS-PAGE analysis and Western blotting confirmed the purity of the final product. In conclusion, the suggested downstream process is suitable for the purification of microcarrier-based cell-derived influenza vaccine.     

Influenza and the Vocal Performer: Update on Prevention and Treatment

SUMMARY: Upper respiratory tract infections (URIs) are a major cause of morbidity among vocal arts professionals, both from their acute impairment of the vocal mechanism and their predisposing influence for the development of serious vocal sequelae. In this review, we present some of the salient features of currently available treatments effective against influenza, the virus family responsible for the most serious form of URI. At present, these include an inactivated vaccine and four antiviral drugs, each approved in the United States and many other countries for the prevention and treatment of influenza. A live attenuated vaccine is also available, and other vaccines and antiviral drugs are under development. This review details the current options available for treating both influenza and noninfluenza related URIs in the professional voice user.     

Synthesis of Lacto‐ and Neolacto‐series Ganglioside Analogs Containing N‐Glycolylneuraminic Acid: Probes for Investigation of Specific Receptor Structures Recognized by Influenza A Viruses

Sialic acids are essential components of host‐cell surface receptors for infection of influenza virus. To investigate the specific receptor structures recognized by various influenza A viruses, a series of lacto‐ and neolacto‐series ganglioside analogs containing N‐glycolylneuraminic acid (Neu5Gc) have been synthesized. The pentasaccharide structures of Neu5Gc‐α‐(2→3)/(2→6)‐lactotetraose (IV³⁽⁶⁾Neu5GcLcOse) and Neu5Gc‐α‐(2→3)/(2→6)‐neolactotetraose (IV³⁽⁶⁾Neu5GcnLcOse) were constructed by glycosylation of the suitably protected trisaccharide acceptors (2A and 2B) with the Neu5Gc‐α‐(2→3)/(2→6)‐Gal trichloroacetimidate donors (1 and 21), respectively. Transformation of the 2‐(trimethylsilyl)ethyl group at the reducing end in 4, 11, 23, and 30 into the trichloroacetimidate group gave a series of Neu5Gc‐α‐(2→3)/(2→6)‐lacto‐ and neolactotetraose donors (7, 13, 26, and 33), which were coupled with 2‐(tetradecyl)hexadecanol (8), to give the corresponding glycolipids (9, 14, 27, and 34). Finally, the complete removal of the O‐acyl groups and saponification of the methyl ester group gave the desired ganglioside analogs (10, 15, 28, and 35).     

Quantifying the transmission potential of pandemic influenza

This article reviews quantitative methods to estimate the basic reproduction number of pandemic influenza, a key threshold quantity to help determine the intensity of interventions required to control the disease. Although it is difficult to assess the transmission potential of a probable future pandemic, historical epidemiologic data is readily available from previous pandemics, and as a reference quantity for future pandemic planning, mathematical and statistical analyses of historical data are crucial. In particular, because many historical records tend to document only the temporal distribution of cases or deaths (i.e. epidemic curve), our review focuses on methods to maximize the utility of time-evolution data and to clarify the detailed mechanisms of the spread of influenza.

First, we highlight structured epidemic models and their parameter estimation method which can quantify the detailed disease dynamics including those we cannot observe directly. Duration-structured epidemic systems are subsequently presented, offering firm understanding of the definition of the basic and effective reproduction numbers. When the initial growth phase of an epidemic is investigated, the distribution of the generation time is key statistical information to appropriately estimate the transmission potential using the intrinsic growth rate. Applications of stochastic processes are also highlighted to estimate the transmission potential using similar data. Critically important characteristics of influenza data are subsequently summarized, followed by our conclusions to suggest potential future methodological improvements.     

Theoretical analysis of binding specificity of influenza viral hemagglutinin to avian and human receptors based on the fragment molecular orbital method

The hemagglutinin (HA) protein of the influenza virus binds to the host cell receptor in the early stage of viral infection. A change in binding specificity from avian 2-3 to human 2-6 receptor is essential for optimal human-to-human transmission and pandemics. Therefore, it is important to reveal the key factors governing the binding affinity of HA-receptor complex at the molecular level for the understanding and prediction of influenza pandemics. In this work, on the basis of ab initio fragment molecular orbital (FMO) method, we have carried out the interaction energy analysis of HA-receptor complexes to quantitatively elucidate the binding specificity of HAs to avian and human receptors. To discuss the binding property of influenza HA comprehensively, a number of HAs from human H1, swine H1, avian H3 and avian H5 viruses were analyzed. We performed detailed investigations about the interaction patterns of complexes of various HAs and receptor analogues, and revealed that intra-molecular interactions between conserved residues in HA play an important role for HA-receptor binding. These results may provide a hint to understand the role of conserved acidic residues at the receptor binding site which are destabilized by the electrostatic repulsion with sialic acid. The calculated binding energies and interaction patterns between receptor and HAs are consistent with the binding specificities of each HA and thus explain the receptor binding mechanism. The calculated results in the present analysis have provided a number of viewpoints regarding the models for the HA-receptor binding specificity associated with mutated residues. Examples include the role of Glu190 and Gln226 for the binding specificity of H5 HA. Since H5 HA has not yet been adapted to human receptor and the mechanism of the specificity change is unknown, this result is helpful for the prediction of the change in receptor specificity associated with forthcoming possible pandemics.     

Synthesis and Biological Evaluation of Analogs of Methyl Ursolate 3-O-β-Chacotrioside as H5N1 Viral Entry Inhibitors

The earliest stage of influenza virus infection is the viral entry to the host cell. In our previous work, we discovered the first three small molecule H5N1 viral entry inhibitors 1–3. Here, based on saponin 3, methyl ursolate 3-O-β-chacotrioside, several analogs were synthesized and evaluated to understand the structure-activity relationships of this type of compound on the H5N1 viral entry inhibitory activity. The preliminary studies demonstrated that unlike saponins 1 and 2, it is possible to reduce the 3-O-chacotriosyl residue of compound 3 to a disaccharide without affecting the viral entry activities significantly. The results obtained will render new clues to the understanding of the antiviral profile for these types of compounds.