Lyophilisation of lentiviral pseudotypes for the development and distribution of virus neutralisation assay kits for rabies, Marburg and influenza viruses

Purpose: Some conventional serological assays can accurately quantify neutralising antibody responses raised against epitopes on virus glycoproteins, enabling mass vaccine evaluation and serosurveillance studies to take place. However, these assays often necessitate the handling of wild-type virus in expensive high biosafety laboratories, which restricts the scope of their application, particularly in resource-deprived areas. A solution to this issue is the use of lentiviral pseudotype viruses (PVs)—chimeric, replication-deficient virions that imitate the binding and entry mechanisms of their wild-type equivalents. Pseudotype virus neutralisation assays (PVNAs) bypass high biosafety requirements and yield comparable results to established assays. This study explores the potential for using lyophilisation of pseudotypes as a cost-effective, alternative means for production, distribution and storage of a PVNAbased diagnostic kit. Methods & Materials: Rabies, Marburg and H5 subtype Influenza virus pseudotypes were each suspended in cryoprotectant solutions of various molarities and subjected to freeze-drying before incubation at a variety of temperatures, humidities and time periods. Samples were then employed in antibody neutralisation assays using specific sera. Results: High levels of PV titre were retained post-lyophilisation, with acceptable levels of virus activity maintained even after medium-term storage in tropical conditions. Also, the performance of PVs in neutralisation assays was not affected by the lyophilisation process. Conclusion: These results confirm the viability of a freeze-dried PVNA-based diagnostic kit, which could considerably facilitate in-field serology for a number of clinically important viruses

Communicating and Thinking Through Drawing

The author considers the behavioural link which exists between curiosity, local processing, visuo-spatial drawing ability and functional changes which might occur as a consequence of intensive drawing practice in the individual. Drawing skill varies enormously in the population and access to creative subjects in education particularly since the introduction of the new baccalaureate continues to challenge the creative sector. How we value drawing as a process matters in respect of culture, economic potential and use. Many children have a burning need to draw the world around them. While making a mark remains essential in public language drawing skill at a certain age becomes less useful except to those associated to have natural talent alone outside of the rigour of core subjects. Drawing could be considered a physical activity promoted by the way in which our minds reach out to the external domain in order to intellectually disseminate, interpret and express, all advanced critical academic activities stimulated by our common ability to visually interpret the world. There has been some academic debate which suggests drawing accuracy is not a result of better perception, veridical or motor coordination but an ability to construct in the mind a robust internal representation of object structure in visual memory by discriminating between the relative spatial position of object segments, in both broad and fine detail. Is it possible then that drawing can be used across the modern curriculum as a critical form of inquiry in support of STEM

Measuring community attitudes and awareness towards the Great Barrier Reef 2007

One of the Great Barrier Reef Marine Park Authority’s (GBRMPA) corporate goals is to promote understanding of the Great Barrier Reef and the issues affecting its health and management. In line with this, GBRMPA regularly conducts a community survey to measure attitudes and awareness towards the Great Barrier Reef. Colmar Brunton Social Research (CBSR) was commissioned by GBRMPA to conduct a community survey with Queensland coastal regions and southern capital cities and six focus groups with residents from five Great Barrier Reef regions. This report presents the findings of the qualitative and quantitative research

Day-length is central to maintaining consistent seasonal diversity in marine bacterioplankton

Marine bacterial diversity is vast, but seasonal variation in diversity is poorly understood. Here we present the longest bacterial diversity time series consisting of monthly (72) samples from the western English Channel over a 6 year period (2003-2008) using 747,494 16SrDNA-V6 amplicon-pyrosequences. Although there were characteristic cycles for each phylum, the overall community cycle was remarkably stable year after year. The majority of taxa were not abundant, although on occasion these rare bacteria could dominate the assemblage. Bacterial diversity peaked at the winter solstice and showed remarkable synchronicity with day-length, which had the best explanatory power compared to a combination of other variables (including temperature and nutrient concentrations). Day-length has not previously been recognised as a major force in structuring microbial communities

The production and development of H7 Influenza virus pseudotypes for the study of humoral responses against avian viruses

In recent years, high pathogenicity avian influenza (HPAI) virus, H5N1, low pathogenicity avian influenza (LPAI) virus, H9N2, and both HPAI and LPAI H7 viruses have proved devastating for the affected economies reliant on poultry industry, and have posed serious public health concerns. These viruses have repeatedly caused zoonotic disease in humans, raising concerns of a potential influenza pandemic. Despite the focus on the HPAI H5N1 outbreak in 1997 some H7 strains have also shown to be occasionally adaptable to infecting humans. Therefore, applying knowledge of the H5 virus evolution and spread to the development of sensitive serological methods is likely to improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses, present within the avian populations, with the potential to infect humans and other species. In the present study we describe the construction and production of lentiviral pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza viruses, which have been responsible for several outbreaks in the past decade. The H7 pseudotypes were evaluated in pseudotype-based neutralization (pp-NT) assays in order to detect and quantify the presence of neutralizing antibodies in avian sera, which were confirmed H7 positive by inhibition of haemagglutination (HI) test. Overall, our results substantiate influenza virus pseudotype neutralization as a robust tool for influenza sero-surveillance

Bias in culture-independent assessments of microbial biodiversity in the global ocean

On the basis of 16S rRNA gene sequencing, the SAR11 clade of marine bacteria has almost universal distribution, being detected as abundant sequences in all marine provinces. Yet SAR11 sequences are rarely detected in fosmid libraries, suggesting that the widespread abundance may be an artefact of PCR cloning and that SAR 11 has a relatively low abundance. Here the relative abundance of SAR11 is explored in both a fosmid library and a metagenomic sequence data set from the same biological community taken from fjord surface water from Bergen, Norway. Pyrosequenced data and 16S clone data confirmed an 11-15% relative abundance of SAR11 within the community. In contrast not a single SAR11 fosmid was identified in a pooled shotgun sequenced data set of 100 fosmid clones. This under-representation was evidenced by comparative abundances of SAR11 sequences assessed by taxonomic annotation; functional metabolic profiling and fragment recruitment. Analysis revealed a similar under-representation of low-GC Flavobacteriaceae. We speculate that the fosmid bias may be due to DNA fragmentation during preparation due to the low GC content of SAR11 sequences and other underrepresented taxa. This study suggests that while fosmid libraries can be extremely useful, caution must be used when directly inferring community composition from metagenomic fosmid libraries

An optimised method for the production of MERS-CoV spike expressing viral pseudotypes

The production and use of pseudotyped viral particles is widely established for many viruses, and applications in the fields of serology and vaccine development are manifold. Viral pseudotypes have proven to be powerful tools to study the effects of viral evolution on serological outcomes, viral tropism and immunogenicity studies. Pseudotyped viruses are chimeric constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral “core” of another virus. Pseudotypes allow for accurate, sequence-directed, sensitive antibody neutralization assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use. The protocol outlined here represents a rapid and reliable method for the generation of high-titre pseudotype viral particles with the MERS-CoV spike protein on a lentiviral core, and is adapted from previously published protocols. This protocol is optimised for transfection in a 100mm petri dish with 7ml of supernatant harvested, however it can be readily scaled to different production volumes. This protocol has a number of advantages including. 1. Use of readily available reagents 2. Consistent, high virus titres 3. Rapid generation of novel glycoproteins for research into strain variatio

Mechanisms of soil carbon storage in experimental grasslands

International audienceWe investigated the fate of root and litter derived carbon into soil organic matter and dissolved organic matter in soil profiles, in order to explain unexpected positive effects of plant diversity on carbon storage. A time series of soil and soil solution samples was investigated at the field site of The Jena Experiment. In addition to the main biodiversity experiment with C3 plants, a C4 species (Amaranthus retroflexus L.) naturally labeled with 13C was grown on an extra plot. Changes in organic carbon concentration in soil and soil solution were combined with stable isotope measurements to follow the fate of plant carbon into the soil and soil solution. A split plot design with plant litter removal versus double litter input simulated differences in biomass input. After 2 years, the no litter and double litter treatment, respectively, showed an increase of 381 g C m?2 and 263 g C m?2 to 20 cm depth, while 71 g C m?2 and 393 g C m?2 were lost between 20 and 30 cm depth. The isotopic label in the top 5 cm indicated that 11 and 15% of soil organic carbon were derived from plant material on the no litter and the double litter treatment, respectively. Without litter, this equals the total amount of carbon newly stored in soil, whereas with double litter this corresponds to twice the amount of stored carbon. Our results indicate that litter input resulted in lower carbon storage and larger carbon losses and consequently accelerated turnover of soil organic carbon. Isotopic evidence showed that inherited soil organic carbon was replaced by fresh plant carbon near the soil surface. Our results suggest that primarily carbon released from soil organic matter, not newly introduced plant organic matter, was transported in the soil solution and contributed to the observed carbon storage in deeper horizons

Hemagglutinin sequence conservation guided stem immunogen design from influenza A H3 subtype

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential ‘universal’ vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, ‘isoleucine-zipper’ or ‘foldon’. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up

Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field sero-surveillance and vaccine studies in resource-limited regions worldwide