An H5N1 influenza DNA vaccine for South Africa

The highly pathogenic avian influenza virus H5N1 is a potent pandemic threat because of its frequent transmission from birds to humans and the increasing possibility of human to human transmission. During the 2009 H1N1 pandemic it was clear that rapid influenza vaccine production is a problem worldwide. Additionally, developing countries like South Africa generally cannot produce their own influenza vaccines because the traditional egg-based vaccine production method currently employed is too lengthy and too difficult to establish. As part of an exercise aimed at exploring the feasibility of producing emergency response influenza vaccines, we investigated an experimental DNA vaccine to the H5N1 influenza virus. We focused on the virion haemagglutinin, because it elicits the primary neutralising immune response following infection. Accordingly, we developed an H5N1 DNA vaccine with full-length and truncated versions of the haemagglutinin gene, to match previously developed protein candidates. Vaccinated mice developed a strong antibody response to the haemagglutinin protein. In addition, the full-length H5 gene elicited high haemagglutination inhibition titres in mice, indicating that it has potential as a candidate pandemic vaccine for South Africa

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries

Evaluation of influenza vaccine effectiveness and description of circulating strains in outpatient settings in South Africa, 2014

The effectiveness of the trivalent seasonal influenza vaccine during the 2014 season in South Africa was assessed using a test-negative case–control study design including 472 cases and 362 controls. Influenza A(H3N2) was the dominant strain circulating. The overall vaccine effectiveness estimate, adjusted for age and underlying conditions, was43.1% (95% CI: 26.8-74.5). 2014 H3N2 viruses from South Africa were mainly in sublineage 3C.3 with accumulation of amino acid changes that differentiate them from the vaccine strain in 3C.1

Mechanisms for doped PEDOT:PSS electrical conductivity improvement

Due to their good electrical conductivity and versatility, conductive polymers (CPs), in particular poly(3,4- ethylene dioxythiophene) (PEDOT):poly(styrene sulphonate) (PSS) have recently attracted considerable research interests in bioelectronics application. This research aims to provide an insight into the mechanisms in PEDOT:PSS to increase electrical conductivity. As such, the preparation of doped PEDOT:PSS using distinctive approaches such as undergoing treatment and using secondary dopant is focused primarily from the perspective of improving its electrical efficiency. It also systematically addresses various primary parameters, which have a significant effect on its conductivity. Finally, we present the potential of doped PEDOT:PSS for many promising applications, such as bioelectronics, through an in-depth analysis of the most stupendous works recorded from various research groups over the past decade. Therefore, this review is expected to be significantly helpful in promoting more studies as well as covering the way to increased qualification and productivity for future revolutions of organic CP material

Comparative studies on the electrical properties of PEDOT:PSS doped SNP films and hydrogels for medical electrode applications

Poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) is a promising conductive polymer to be the next-generation electrode for medical purposes. However, PEDOT:PSS exhibits low conductivity (~1×10−3 S cm−1); hence, incorporating silver nanoparticles (SNP) with PEDOT:PSS will improve the electrical conductivity. This paper aims to investigate the electrical properties differences between PEDOT:PSS doped SNP-based films and hydrogels. The two different states of PEDOT:PSS/SNP serves its particular purpose as an electrode. Initially, the PEDOT:PSS/SNP solution was prepared by homogeneously mixing at constant stirring. Then, the solution was drop-casting onto a glass substrate to produce a film, while another part of the solution was undergoing a freeze-thaw method to produce hydrogel. Surface resistance measurement exhibits lower resistance values for a film (0.11 kΩ) than hydrogel (0.59 kΩ). A scanning electron microscope (SEM) was utilized to observe the morphology of the films, while an optical microscope (OM) observed the surface of the hydrogel since they are in different states. Fourier Transform Infrared (FTIR) spectra display prominent peaks that described the successful blending between PEDOT:PSS and SNP for both films and hydrogels. These findings demonstrate that varying processing methods of preparing PEDOT:PSS/SNP in films or hydrogels may influence its properties like the electrode, which should provide a valuable contribution to the bioelectronic areas

Effectiveness and knowledge, attitudes and practices of seasonal influenza vaccine in primary healthcare settings in South Africa, 2010-2013

OBJECTIVES : Influenza vaccine effectiveness (VE) and coverage data for sub-Saharan Africa are scarce. Using a test-negative case–control design, we estimated influenza VE annually among individuals with influenza-like illness presenting to an outpatient sentinel surveillance programme in South Africa from 2010 to 2013. A knowledge, attitudes and practices (KAP) influenza vaccine survey of programme clinicians was conducted in 2013. SAMPLE : In total, 9420 patients were enrolled in surveillance of whom 5344 (56.7%) were included in the VE analysis: 2678 (50.1%) were classified as controls (influenza test-negative) and 2666 (49.9%) as cases (influenza test-positive). RESULTS : Mean annual influenza vaccine coverage among controls was 4.5% for the four years. Annual VE estimates adjusted for age, underlying medical conditions and seasonality for 2010-2013 were 54.2% (95% confidence interval (CI): 2.4–78.6%), 57.1% (95% CI: 15.5–78.2%), 38.4% (95% CI: 71.7–78.1%) and 87.2% (95% CI: 67.2–95.0%), respectively. The KAP survey showed that >90% of clinicians were familiar with the indications for and the benefits of influenza vaccination. CONCLUSIONS : Our study showed that the vaccine was significantly protective in 2010, 2011 and 2013, but not in 2012 when the circulating A(H3N2) strain showed genetic drift. Vaccine coverage was low despite good clinician knowledge of vaccination indications. Further studies are needed to investigate the reason for the low uptake of influenza vaccine.The programme forms part of the National Institute for Communicable Diseases core function and is not funded by external bodies.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1750-2659hb2016Medical Virolog

Epidemiology of influenza B/Yamagata and B/Victoria lineages in South Africa, 2005-2014

BACKGROUND : Studies describing the epidemiology of influenza B lineages in South Africa are lacking. METHODS : We conducted a prospective study to describe the circulation of influenza B/Victoria and B/ Yamagata lineages among patients of all ages enrolled in South Africa through three respiratory illness surveillance systems between 2005 and 2014: (i) the Viral Watch (VW) program enrolled outpatients with influenza-like illness (ILI) from private healthcare facilities during 2005±2014; (ii) the influenza-like illnesses program enrolled outpatients in public healthcare clinics (ILI/PHC) during 2012±2014; and (iii) the severe acute respiratory illnesses (SARI) program enrolled inpatients from public hospitals during 2009±2014. Influenza B viruses were detected by virus isolation during 2005 to 2009 and by real-time reverse transcription polymerase chain reaction from 2009±2014. Clinical and epidemiological characteristics of patients hospitalized with SARI and infected with different influenza B lineages were also compared using unconditional logistic regression. RESULTS : Influenza viruses were detected in 22% (8,706/39,804) of specimens from patients with ILI or SARI during 2005±2014, of which 24% (2,087) were positive for influenza B. Influenza B viruses predominated in all three surveillance systems in 2010. B/Victoria predominated prior to 2011 (except 2008) whereas B/Yamagata predominated thereafter (except 2012). B lineages co-circulated in all seasons, except in 2013 and 2014 for SARI and ILI/PHC surveillance. Among influenza B-positive SARI cases, the detection of influenza B/Yamagata compared to influenza B/Victoria was significantly higher in individuals aged 45±64 years (adjusted odds ratio [aOR]: 4.2; 95% confidence interval [CI]: 1.1±16.5) and 65 years (aOR: 12.2; 95% CI: 2.3±64.4) compared to children aged 0±4 years, but was significantly lower in HIV-infected patients (aOR: 0.4; 95% CI: 0.2±0.9). CONCLUSION : B lineages co-circulated in most seasons except in 2013 and 2014. Hospitalized SARI cases display differential susceptibility for the two influenza B lineages, with B/Victoria being more prevalent among children and HIV-infected persons.The National Institute for Communicable Diseases (NICD) (http://www.nicd.ac.za/) and the US Centers for Disease Control and Prevention (https://www.cdc. gov/) grant number 5U51/IP000155.http://www.plosone.orgam2017Medical Virolog

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.publishedVersio

Sequencing and analysis of globally obtained human parainfluenza viruses 1 and 3 genomes

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003–2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10−4 mutations/ site/year (95% highest posterior density 4.55 × 10−4 to 5.38 × 10−4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10−4 mutations/site/year, 95% highest posterior density 3.26 × 10−4 to 3.94 × 10−4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.S1 Text. HPIV-1 Sanger sequencing primers.S2 Text. HPIV-3 Sanger sequencing primers.S1 Table. The sequence information of the 40 HPIV-1 genomes.S2 Table. The sequence information of the 75 HPIV-3 genomes.S3 Table. MEME episodic selection results for HPIV-1 and HPIV-3.The National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract number HHSN272200900007C and grant numbers U19AI110819, with the sub-project directed by HAL, and grants U01AI070428 and U01AI077988 awarded to KJH.http://www.plosone.orgam2019Medical Virolog

Soluble Guanylate Cyclase Generation of cGMP Regulates Migration of MGE Neurons

Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2−/− and Lhx6−/− mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the α subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2−/− mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3−/− mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity