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

Development of pseudotyped virus assays for the serological study of Japanese encephalitis flavivirus

Japanese encephalitis virus (JEV) is one of the primary global causes of viral encephalitis, with approximately 68,000 clinical cases and 20,000 deaths attributed to the virus annually. Between 30% and 50% of survivors suffer from debilitating neurological sequelae. Despite being a vaccine-preventable disease, no antiviral treatments are licensed and commercially available to counteract JEV infection. In order to quantify the neutralising antibody response raised against antigenic epitopes on flavivirus prME glycoproteins, conventional serological assays such as the plaque reduction neutralisation test (PRNT) can be employed. However, these assays often necessitate the handling of pathogenic wild-type virus in expensive high-biosafety laboratories, restricting the scope of their application, particularly in resource-deprived areas. Chimeric, replication-deficient pseudotype viruses can offer a solution to this problem, as they mimic wild-type virus entry mechanisms, enabling their use in pseudotype virus neutralisation assays (PVNAs). PVNAs bypass high biosafety requirements and permit vaccine evaluation and serosurveillance studies with no risk of inadvertent infection. This project focuses on the production of functional pseudotype viruses displaying the prM and E surface glycoproteins of the JEV flavivirus, for utilisation in serological neutralisation assays. Subcloning of the prME gene into an appropriate eukaryotic expression vector and insertion mutagenesis to produce prME with 15- and 24-residue upstream signal peptides are shown, before production of JEVpp with either HIV or MLV cores is attempted, via the conventional multi-plasmid co-transfection approach or the utilisation of constitutive gag-pol expressing cell lines. The impact of additional plasmid-derived furin protease expression and low glucose culture medium, as well as the construction of JEV/VSV chimeric prME glycoproteins and the introduction of Kozak consensus sequences upstream of the prME gene, to enhance the efficiency of JEVpp generation is also explored. Finally, the infectivity of lentiviral pseudotype viruses following lyophilisation, storage and reconstitution is confirmed, thus enabling their affordable global distribution

The prevalences of Salmonella Genomic Island 1 variants in human and animal Salmonella Typhimurium DT104 are distinguishable using a Bayesian approach

Throughout the 1990s, there was an epidemic of multidrug resistant Salmonella Typhimurium DT104 in both animals and humans in Scotland. The use of antimicrobials in agriculture is often cited as a major source of antimicrobial resistance in pathogenic bacteria of humans, suggesting that DT104 in animals and humans should demonstrate similar prevalences of resistance determinants. Until very recently, only the application of molecular methods would allow such a comparison and our understanding has been hindered by the fact that surveillance data are primarily phenotypic in nature. Here, using large scale surveillance datasets and a novel Bayesian approach, we infer and compare the prevalence of Salmonella Genomic Island 1 (SGI1), SGI1 variants, and resistance determinants independent of SGI1 in animal and human DT104 isolates from such phenotypic data. We demonstrate differences in the prevalences of SGI1, SGI1-B, SGI1-C, absence of SGI1, and tetracycline resistance determinants independent of SGI1 between these human and animal populations, a finding that challenges established tenets that DT104 in domestic animals and humans are from the same well-mixed microbial population

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses

Detection of Rare Antimicrobial Resistance Profiles by Active and Passive Surveillance Approaches.

Antimicrobial resistance (AMR) surveillance systems are generally not specifically designed to detect emerging resistances and usually focus primarily on resistance to individual drugs. Evaluating the diversity of resistance, using ecological metrics, allows the assessment of sampling protocols with regard to the detection of rare phenotypes, comprising combinations of resistances. Surveillance data of phenotypic AMR of Canadian poultry Salmonella Heidelberg and swine Salmonella Typhimurium var. 5- were used to contrast active (representative isolates derived from healthy animals) and passive (diagnostic isolates) surveillance and assess their suitability for detecting emerging resistance patterns. Although in both datasets the prevalences of resistance to individual antimicrobials were not significantly different between the two surveillance systems, analysis of the diversity of entire resistance phenotypes demonstrated that passive surveillance of diagnostic isolates detected more unique phenotypes. Whilst the most appropriate surveillance method will depend on the relevant objectives, under the conditions of this study, passive surveillance of diagnostic isolates was more effective for the detection of rare and therefore potentially emerging resistance phenotypes.AEM was supported by the William Stewart Fellowship whilst at the University of Glasgow, and is currently supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/ M014088/1; RR is supported by BBSRC grant BB/ E010326/1 and BB/L004070/1; LM is supported by BB/K01126X/1, BB/L004070/1, BB/F015313/1, National Science Foundation DEB1216040 and an EU-funded Marie Curie Initial Training Network (MCITN) program (NEMATODE SYSTEM HEALTH project (FP7-PEOPLE-2010-ITN- ID:264639)).This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pone.015851

Geomorphology on geologic timescales: Evolution of the late Cenozoic Pacific paleosurface in Northern Chile and Southern Peru

publisher: Elsevier articletitle: Geomorphology on geologic timescales: Evolution of the late Cenozoic Pacific paleosurface in Northern Chile and Southern Peru journaltitle: Earth-Science Reviews articlelink: http://dx.doi.org/10.1016/j.earscirev.2017.04.004 content_type: article copyright: © 2017 Elsevier B.V. All rights reserved

Estimating the Relative Sociolinguistic Salience of Segmental Variables in a Dialect Boundary Zone

One way of evaluating the salience of a linguistic feature is by assessing the extent to which listeners associate the feature with a social category such as a particular socioeconomic class, gender, or nationality. Such ‘top–down’ associations will inevitably differ somewhat from listener to listener, as a linguistic feature – the pronunciation of a vowel or consonant, for instance – can evoke multiple social category associations, depending upon the dialect in which the feature is embedded and the context in which it is heard. In a given speech community it is reasonable to expect, as a consequence of the salience of the linguistic form in question, a certain level of intersubjective agreement on social category associations. Two metrics we can use to quantify the salience of a linguistic feature are (a) the speed with which the association is made, and (b) the degree to which members of a speech community appear to share the association. Through the use of a new technique, designed as an adaptation of the Implicit Association Test, this paper examines levels of agreement among 40 informants from the Scottish/English border region with respect to the associations they make between four key phonetic variables and the social categories of ‘Scotland’ and ‘England.’ Our findings reveal that the participants exhibit differential agreement patterns across the set of phonetic variables, and that listeners’ responses vary in line with whether participants are members of the Scottish or the English listener groups. These results demonstrate the importance of community-level agreement with respect to the associations that listeners make between social categories and linguistic forms, and as a means of ranking the forms’ relative salience

Comparing Biological Motion Perception in Two Distinct Human Societies

Cross cultural studies have played a pivotal role in elucidating the extent to which behavioral and mental characteristics depend on specific environmental influences. Surprisingly, little field research has been carried out on a fundamentally important perceptual ability, namely the perception of biological motion. In this report, we present details of studies carried out with the help of volunteers from the Mundurucu indigene, a group of people native to Amazonian territories in Brazil. We employed standard biological motion perception tasks inspired by over 30 years of laboratory research, in which observers attempt to decipher the walking direction of point-light (PL) humans and animals. Do our effortless skills at perceiving biological activity from PL animations, as revealed in laboratory settings, generalize to people who have never before seen representational depictions of human and animal activity? The results of our studies provide a clear answer to this important, previously unanswered question. Mundurucu observers readily perceived the coherent, global shape depicted in PL walkers, and experienced the classic inversion effects that are typically found when such stimuli are turned upside down. In addition, their performance was in accord with important recent findings in the literature, in the abundant ease with which they extracted direction information from local motion invariants alone. We conclude that the effortless, veridical perception of PL biological motion is a spontaneous and universal perceptual ability, occurring both inside and outside traditional laboratory environments