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.

A strong quantitative trait locus for wing length on chromosome 2 in a wild population of great reed warblers

Wing length is a key character for essential behaviours related to bird flight such as migration and foraging. In the present study, we initiate the search for the genes underlying wing length in birds by studying a long-distance migrant, the great reed warbler (Acrocephalus arundinaceus). In this species wing length is an evolutionary interesting trait with pronounced latitudinal gradient and sex-specific selection regimes in local populations. We performed a quantitative trait locus (QTL) scan for wing length in great reed warblers using phenotypic, genotypic, pedigree and linkage map data from our long-term study population in Sweden. We applied the linkage analysis mapping method implemented in GRIDQTL (a new web-based software) and detected a genome-wide significant QTL for wing length on chromosome 2, to our knowledge, the first detected QTL in wild birds. The QTL extended over 25 cM and accounted for a substantial part (37%) of the phenotypic variance of the trait. A genome scan for tarsus length (a bodysize-related trait) did not show any signal, implying that the wing-length QTL on chromosome 2 was not associated with body size. Our results provide a first important step into understanding the genetic architecture of avian wing length, and give opportunities to study the evolutionary dynamics of wing length at the locus level. This journal is© 2010 The Royal Society.

Epidemiologic characteristics of cases for influenza A(H7N9) virus infections in China

China's National Health and Family Planning Commission announced 3 deaths caused by avian-origin influenza A(H7N9) virus in March, which was the first time that the H7N9 strain has been found in humans [1]. This is of major public health significance and raises urgent questions and global concerns [2, 3]. To explore epidemic characteristics of human infections with H7N9 virus, data on individual cases from 19 February 2013 (onset date of first case) to 14 April 2013 were collected from the China Information System for Disease Control and Prevention, which included information about sex; age; occupation; residential address; and day of symptom onset, diagnosis, and outcome for each case. The definition of an unconfirmed probable H7N9 case is a patient with epidemiologic evidence of contact …

A systematic review of community-based interventions for emerging zoonotic diseases in Southeast Asia

Background Southeast Asia has been at the epicentre of recent epidemics of emerging and re-emerging zoonotic diseases. Community-based surveillance and control interventions have been heavily promoted but the most effective interventions have not been identified. Objectives This review evaluated evidence for the effectiveness of community-based surveillance interventions at monitoring and identifying emerging infectious disease; the effectiveness of community-based control interventions at reducing rates of emerging infectious disease; and contextual factors that influence intervention effectiveness. Inclusion criteria Participants Communities in Brunei, Cambodia, Indonesia, Laos, Malaysia, Myanmar, the Philippines, Singapore, Thailand and Viet Nam. Types of intervention(s) Non-pharmaceutical, non-vaccine, and community-based surveillance or prevention and control interventions targeting rabies, Nipah virus , dengue, SARS or avian influenza. Types of outcomes Primary outcomes: measures: of infection or disease; secondary outcomes: measures of intervention function. Types of studies Original quantitative studies published in English. Search strategy Databases searched (1980 to 2011): PubMed, CINAHL, ProQuest, EBSCOhost, Web of Science, Science Direct, Cochrane database of systematic reviews, WHOLIS, British Development Library, LILACS, World Bank (East Asia), Asian Development Bank. Methodological quality Two independent reviewers critically appraised studies using standard Joanna Briggs Institute instruments. Disagreements were resolved through discussion. Data extraction A customised tool was used to extract quantitative data on intervention(s), populations, study methods, and primary and secondary outcomes; and qualitative contextual information or narrative evidence about interventions. Data synthesis Data was synthesised in a narrative summary with the aid of tables. Meta-analysis was used to statistically pool quantitative results. Results Fifty-seven studies were included. Vector control interventions using copepods, environmental cleanup and education are effective and sustainable at reducing dengue in rural and urban communities, whilst insecticide spraying is effective in urban outbreak situations. Community-based surveillance interventions can effectively identify avian influenza in backyard flocks, but have not been broadly applied. Outbreak control interventions for Nipah virus and SARS are effective but may not be suitable for ongoing control. Canine vaccination and education is more acceptable than culling, but still fails to reach coverage levels required to effectively control rabies. Contextual factors were identified that influence community engagement with, and ultimately effectiveness of, interventions. Conclusion Despite investment in community-based disease control and surveillance in Southeast Asia, published evidence evaluating interventions is limited in quantity and quality. Nonetheless this review identified a number of effective interventions, and several contextual factors influencing effectiveness. Identification of the best programs will require comparative evidence of effectiveness acceptability, cost-effectiveness and sustainability.

Geographic co-distribution of influenza virus subtypes H7N9 and H5N1 in humans, China

Human infection with a novel low pathogenicity influenza A(H7N9) virus in eastern China has recently raised global public health concerns (1). The geographic sources of infection have yet to be fully clarified, and confirmed human cases from 1 province have not been linked to those from other provinces. While some studies have identified epidemiologic characteristics of subtype H7N9 cases and clinical differences between these cases and cases of highly pathogenic influenza A(H5N1), another avian influenza affecting parts of China (2–4), the spatial epidemiology of human infection with influenza subtypes H7N9 and H5N1 in China has yet to be elucidated. To test the hypothesis of co-distribution of high-risk clusters of both types of infection, we used all available data on human cases in mainland China and investigated the geospatial epidemiologic features...

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

The duty to report disease outbreaks : of interest or value? Lessons from H5N1

Since the severe acute respiratory syndrome outbreak in 2003, it has been argued that there has been a substantial revision to the norm dictating the behaviour of states in the event of a disease outbreak. This article examines the evolution of the norm to ‘report and verify’ disease outbreaks and evaluates the extent to which this revised norm has begun to guide state behaviour. Examination of select East Asian countries affected by human infections of the H5N1 (avian influenza) virus strain reveals the need to further understand the mutually constitutive relationship between the value attached to prompt reporting against the capacity to report, and how states manage both in fulfilling their duty to report.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Evidence for an Auditory Fovea in the New Zealand Kiwi (Apteryx mantelli).

Kiwi are rare and strictly protected birds of iconic status in New Zealand. Yet, perhaps due to their unusual, nocturnal lifestyle, surprisingly little is known about their behaviour or physiology. In the present study, we exploited known correlations between morphology and physiology in the avian inner ear and brainstem to predict the frequency range of best hearing in the North Island brown kiwi. The mechanosensitive hair bundles of the sensory hair cells in the basilar papilla showed the typical change from tall bundles with few stereovilli to short bundles with many stereovilli along the apical-to-basal tonotopic axis. In contrast to most birds, however, the change was considerably less in the basal half of the epithelium. Dendritic lengths in the brainstem nucleus laminaris also showed the typical change along the tonotopic axis. However, as in the basilar papilla, the change was much less pronounced in the presumed high-frequency regions. Together, these morphological data suggest a fovea-like overrepresentation of a narrow high-frequency band in kiwi. Based on known correlations of hair-cell microanatomy and physiological responses in other birds, a specific prediction for the frequency representation along the basilar papilla of the kiwi was derived. The predicted overrepresentation of approximately 4-6 kHz matches potentially salient frequency bands of kiwi vocalisations and may thus be an adaptation to a nocturnal lifestyle in which auditory communication plays a dominant role.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Rapid-response vaccines - does DNA offer a solution?

In responding to future influenza pandemics and other infectious agents, plasmid DNA overcomes many of the limitations of conventional vaccine production approaches.

Weather variability and influenza A (H7N9) transmission in Shanghai, China: A Bayesian spatial analysis

Abstract Background A novel avian influenza A (H7N9) virus was first found in humans in Shanghai, and infected over 433 patients in China. To date, very little is known about the spatiotemporal variability or environmental drivers of the risk of H7N9 infection. This study explored the spatial and temporal variation of H7N9 infection and assessed the effects of temperature and rainfall on H7N9 incidence. Methods A Bayesian spatial conditional autoregressive (CAR) model was used to assess the spatiotemporal distribution of the risk of H7N9 infection in Shanghai, by district and fortnight for the period 19th February–14th April 2013. Data on daily laboratory-confirmed H7N9 cases, and weather variability including temperature (°C) and rainfall (mm) were obtained from the Chinese Information System for Diseases Control and Prevention and Chinese Meteorological Data Sharing Service System, respectively, and aggregated by fortnight. Results High spatial variations in the H7N9 risk were mainly observed in the east and centre of Shanghai municipality. H7N9 incidence rate was significantly associated with fortnightly mean temperature (Relative Risk (RR): 1.54; 95% credible interval (CI): 1.22–1.94) and fortnightly mean rainfall (RR: 2.86; 95% CI: 1.47–5.56). Conclusion There was a substantial variation in the spatiotemporal distribution of H7N9 infection across different districts in Shanghai. Optimal temperature and rainfall may be one of the driving forces for H7N9.

Integration over song classification replicates: song variant analysis in the hihi

Human expert analyses are commonly used in bioacoustic studies and can potentially limit the reproducibility of these results. In this paper, a machine learning method is presented to statistically classify avian vocalizations. Automated approaches were applied to isolate bird songs from long field recordings, assess song similarities, and classify songs into distinct variants. Because no positive controls were available to assess the true classification of variants, multiple replicates of automatic classification of song variants were analyzed to investigate clustering uncertainty. The automatic classifications were more similar to the expert classifications than expected by chance. Application of these methods demonstrated the presence of discrete song variants in an island population of the New Zealand hihi (Notiomystis cincta). The geographic patterns of song variation were then revealed by integrating over classification replicates. Because this automated approach considers variation in song variant classification, it reduces potential human bias and facilitates the reproducibility of the results.

Connecting soundscape to landscape: Which acoustic index best describes landscape configuration?

Soundscape assessment has been proposed as a remote ecological monitoring tool for measuring biodiversity, but few studies have examined how soundscape patterns vary with landscape configuration and condition. The goal of our study was to examine a suite of published acoustic indices to determine whether they provide comparable results relative to varying levels of landscape fragmentation and ecological condition in nineteen forest sites in eastern Australia. Our comparison of six acoustic indices according to time of day revealed that two indices, the acoustic complexity and the bioacoustic index, presented a similar pattern that was linked to avian song intensity, but was not related to landscape and biodiversity attributes. The diversity indices, acoustic entropy and acoustic diversity, and the normalized difference soundscape index revealed high nighttime sound, as well as a dawn and dusk chorus. These indices appear to be sensitive to nocturnal biodiversity which is abundant at night in warm, subtropical environments. We argue that there is need to better understand temporal partitioning of the soundscape by specific taxonomic groups, and this should involve integrated research on amphibians, insects and birds during a 24 h cycle. The three indices that best connected the soundscape with landscape characteristics, ecological condition and bird species richness were acoustic entropy, acoustic evenness and the normalized difference soundscape index. This study has demonstrated that remote soundscape assessment can be implemented as an ecological monitoring tool in fragmented Australian forest landscapes. However, further investigation should be dedicated to refining and/or combining existing acoustic indices and also to determine if these indices are appropriate in other landscapes and for other survey purposes.