A targeted single mutation in influenza A virus universal epitope transforms immunogenicity and protective immunity via CD4+ T cell activation.

CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.

Serological evidence of virus infection in Eidolon helvum fruit bats: implications for bushmeat consumption in Nigeria.

Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.

Protease-Independent Production of Poliovirus Virus-like Particles in Pichia pastoris: Implications for Efficient Vaccine Development and Insights into Capsid Assembly.

The production of enterovirus virus-like particles (VLPs) that lack the viral genome have great potential as vaccines for a number of diseases, such as poliomyelitis and hand, foot, and mouth disease. These VLPs can mimic empty capsids, which are antigenically indistinguishable from mature virions, produced naturally during viral infection. Both in infection and in vitro, capsids and VLPs are generated by the cleavage of the P1 precursor protein by a viral protease. Here, using a stabilized poliovirus 1 (PV-1) P1 sequence as an exemplar, we show the production of PV-1 VLPs in Pichia pastoris in the absence of the potentially cytotoxic protease, 3CD, instead using the porcine teschovirus 2A (P2A) peptide sequence to terminate translation between individual capsid proteins. We compare this to protease-dependent production of PV-1 VLPs. Analysis of all permutations of the order of the capsid protein sequences revealed that only VP3 could be tagged with P2A and maintain native antigenicity. Transmission electron microscopy of these VLPs reveals the classic picornaviral icosahedral structure. Furthermore, these particles were thermostable above 37°C, demonstrating their potential as next generation vaccine candidates for PV. Finally, we believe the demonstration that native antigenic VLPs can be produced using protease-independent methods opens the possibility for future enteroviral vaccines to take advantage of recent vaccine technological advances, such as adenovirus-vectored vaccines and mRNA vaccines, circumventing the potential problems of cytotoxicity associated with 3CD, allowing for the production of immunogenic enterovirus VLPs in vivo. IMPORTANCE The widespread use of vaccines has dramatically reduced global incidence of poliovirus infections over a period of several decades and now the wild-type virus is only endemic in Pakistan and Afghanistan. However, current vaccines require the culture of large quantities of replication-competent virus for their manufacture, thus presenting a potential risk of reintroduction into the environment. It is now widely accepted that vaccination will need to be extended posteradication into the foreseeable future to prevent the potentially catastrophic reintroduction of poliovirus into an immunologically naive population. It is, therefore, imperative that novel vaccines are developed which are not dependent on the growth of live virus for their manufacture. We have expressed stabilized virus-like particles in yeast, from constructs that do not require coexpression of the protease. This is an important step in the development of environmentally safe and commercially viable vaccines against polio, which also provides some intriguing insights into the viral assembly process.

A safe and just operating space for human identity: a systems perspective.

A safe and just operating space for socioecological systems is a powerful bridging concept in sustainability science. It integrates biophysical earth-system tipping points (ie, thresholds at which small changes can lead to amplifying effects) with social science considerations of distributional equity and justice. Often neglected, however, are the multiple feedback loops between self-identity and planetary boundaries. Environmental degradation can reduce self-identification with nature, leading to decreased pro-environmental behaviours and decreased cooperation with out-groups, further increasing the likelihood of transgressing planetary boundaries. This vicious cycle competes with a virtuous one, where improving environmental quality enhances the integration of nature into self-identity and improves health, thereby facilitating prosocial and pro-environmental behaviour. These behavioural changes can also cascade up to influence social and economic institutions. Given a possible minimum degree of individual self-care to maintain health and prosperity, there would seem to exist an analogous safe and just operating space for self-identity, for which system stewardship for planetary health is crucial.

Identification of Host Factors Differentially Induced by Clinically Diverse Strains of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is an important human arthropod-borne virus that causes tick-borne encephalitis (TBE) in humans. TBEV acutely infects the central nervous system (CNS), leading to neurological symptoms of various severity. No therapeutics are currently available for TBEV-associated disease. Virus strains of various pathogenicity have been described, although the basis of their diverse clinical outcome remains undefined. Work with infectious TBEV requires high-level biocontainment, meaning model systems that can recapitulate the virus life cycle are highly sought. Here, we report the generation of a self-replicating, noninfectious TBEV replicon used to study properties of high (Hypr) and low (Vs) pathogenic TBEV isolates. Using a Spinach2 RNA aptamer and luciferase reporter system, we perform the first direct comparison of Hypr and Vs in cell culture. Infectious wild-type (WT) viruses and chimeras of the nonstructural proteins 3 (NS3) and 5 (NS5) were investigated in parallel to validate the replicon data. We show that Hypr replicates to higher levels than Vs in mammalian cells, but not in arthropod cells, and that the basis of these differences map to the NS5 region, encoding the methyltransferase and RNA polymerase. For both Hypr and Vs strains, NS5 and the viral genome localized to intracellular structures typical of positive-strand RNA viruses. Hypr was associated with significant activation of IRF-3, caspase-3, and caspase-8, while Vs activated Akt, affording protection against caspase-mediated apoptosis. Higher activation of stress-granule proteins TIAR and G3BPI were an additional early feature of Vs but not for Hypr. These findings highlight novel host cell responses driven by NS5 that may dictate the differential clinical characteristics of TBEV strains. This highlights the utility of the TBEV replicons for further virological characterization and antiviral drug screening. IMPORTANCE Tick-borne encephalitis virus (TBEV) is an emerging virus of the flavivirus family that is spread by ticks and causes neurological disease of various severity. No specific therapeutic treatments are available for TBE, and control in areas of endemicity is limited to vaccination. The pathology of TBEV ranges from mild to fatal, depending on the virus genotype. Characterization of TBEV isolates is challenging due to the requirement for high-containment facilities. Here, we described the construction of novel TBEV replicons that permit a molecular comparison of TBEV isolates of high and low pathogenicity.

Towards Reverse Vaccinology for Bovine TB: High Throughput Expression of Full Length Recombinant Mycobacterium bovis Proteins.

Bovine tuberculosis caused by Mycobacterium bovis, is a significant global pathogen causing economic loss in livestock and zoonotic TB in man. Several vaccine approaches are in development including reverse vaccinology which uses an unbiased approach to select open reading frames (ORF) of potential vaccine candidates, produce them as recombinant proteins and assesses their immunogenicity by direct immunization. To provide feasibility data for this approach we have cloned and expressed 123 ORFs from the M. bovis genome, using a mixture of E. coli and insect cell expression. We used a concatenated open reading frames design to reduce the number of clones required and single chain fusion proteins for protein pairs known to interact, such as the members of the PPE-PE family. Over 60% of clones showed soluble expression in one or the other host and most allowed rapid purification of the tagged bTB protein from the host cell background. The catalogue of recombinant proteins represents a resource that may be suitable for test immunisations in the development of an effective bTB vaccine.

SARS-CoV-2 Virus-like Particles Produced by a Single Recombinant Baculovirus Generate Anti-S Antibody and Protect against Variant Challenge.

Coronavirus Disease 2019 (COVID-19), caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous ~100 nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein and reduced virus shedding and protected against disease associated weight loss following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant). Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection.

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Identification of a Membrane Binding Peptide in the Envelope Protein of MHV Coronavirus.

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.

The Effects of Photoperiod on Diapause Induction in Hypena opulenta (Lepidoptera: Erebidae), a Biological Control Agent Against Invasive Swallow-Worts in North America.

Many insects exhibit a short-day diapause response, whereby diapause is induced when daylength falls below a critical threshold. This response is an adaptation to ensure synchrony between periods of insect activity, and the availability of resources, but it can cause problems when organisms are moved to new locations, where early or late-induced diapause can prove a barrier to establishment. We explored the role of photoperiod in diapause induction in Hypena opulenta, a recently introduced classical biological control agent for invasive swallow-worts in North America. We conducted four experimental cage releases as well as a growth chamber experiment to determine the threshold photoperiod for diapause induction in H. opulenta. We determined that the critical photoperiod for inducing diapause in 50% of H. opulenta is 15 h 35 min, which the moth only experiences in the Ottawa release site around summer solstice. This may lead to univoltinism, premature diapause, and poor establishment at some North American release sites. Our results can inform practical aspects of the biological control program for H. opulenta, such as fine-tuning methodologies for stockpiling diapausing pupae in the laboratory and narrowing down the optimal time window for releases at a given location. Additionally, our results will be important for the development of a temperature-based phenology model to more accurately predict voltinism in H. opulenta across the invasive range of swallow-worts in North America.

A Fusion Peptide in the Spike Protein of MERS Coronavirus.

Coronaviruses represent current and emerging threats for many species, including humans. Middle East respiratory syndrome-related coronavirus (MERS-CoV) is responsible for sporadic infections in mostly Middle Eastern countries, with occasional transfer elsewhere. A key step in the MERS-CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within the MERS S protein has not been precisely mapped. We used isolated peptides and giant unilamellar vesicles (GUV) to demonstrate membrane binding for a peptide located near the N-terminus of the S2 domain. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S-mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS-CoV.

Wind speed predicts population dynamics of the eriophyid mite Floracarus perrepae on invasive Old World climbing fern (Lygodium microphyllum) in a shade house colony.

Lygodium microphyllum is one of the most noxious invasive plants in Florida, USA, smothering native vegetation in cypress swamps, pine flatwoods, and Everglades tree islands and altering fire regimes. The eriophyid mite Floracarus perrepae was introduced from Australia to help control L. microphyllum infestations. While F. perrepae exhibits high population growth rates in its native range, its population dynamics in Florida are unknown, particularly the dynamics that occur within the leaf roll galls the mite induces on the margins of leaves. Here, we monitored a shade house colony of F. perrepae in south Florida for 2 years to identify seasonal patterns and potential climate drivers of within-gall mite density. Gall dissections of mite-infested colony plants were performed monthly. Mite density within galls exhibited two cycles per year: a strong cycle that boomed in spring and busted in summer, and a weak cycle that moderately increased mite density in fall and declined in winter. Climate variables, particularly those related to wind speed, were positively associated with higher mite density. Our study sheds light on the within-gall dynamics of F. perrepae and suggests that the highest within-gall mite densities occur in the spring and fall.

Membrane binding proteins of coronaviruses.

Coronaviruses (CoVs) infect many species causing a variety of diseases with a range of severities. Their members include zoonotic viruses with pandemic potential where therapeutic options are currently limited. Despite this diversity CoVs share some common features including the production, in infected cells, of elaborate membrane structures. Membranes represent both an obstacle and aid to CoV replication - and in consequence - virus-encoded structural and nonstructural proteins have membrane-binding properties. The structural proteins encounter cellular membranes at both entry and exit of the virus while the nonstructural proteins reorganize cellular membranes to benefit virus replication. Here, the role of each protein in membrane binding is described to provide a comprehensive picture of their role in the CoV replication cycle.

Correction: The Influence of Host Plant Extrafloral Nectaries on Multitrophic Interactions: An Experimental Investigation.

[This corrects the article DOI: 10.1371/journal.pone.0138157.].

Unpicking the determinants of amide NHO[double bond, length as m-dash]C hydrogen bond strength with diphenylacetylene molecular balances.

Hydrogen bonding plays an essential part in dictating the properties of natural and synthetic materials. Secondary amides are well suited to cross-strand interactions through the display of both hydrogen bond donors and acceptors and are prevalent in polymers such as proteins, nylon, and Kevlar™. In attempting to measure hydrogen bond strength and to delineate the stereoelectronic components of the interaction, context frequently becomes vitally important. This makes molecular balances - systems in which direct comparison of two groups is possible - an appealing bottom up approach that allows the complexity of larger systems to be stripped away. We have previously reported a family of single molecule conformational switches that are responsive to diverse stimuli including Brønsted and Lewis acids, anions, and redox gradients. In this work we assess the ability of the scaffold, based on a 2,6-disubstituted diphenylacetylene, to measure accurately the difference in hydrogen bond strength between variously functionalised amides. In all of the examples investigated hydrogen bond strength closely correlate to measures of Brønstead acidity suggesting that the scaffold is well-suited as a platform for the accurate determination of bond strength in variously substituted systems.

Multiple viral infections in Agaricus bisporus - Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing.

Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3' motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interactive viral ecosystem with sequence variability ranging over 2 orders of magnitude and evidence of recombination, horizontal gene transfer and variable fragment numbers. Large numbers of viral RNAs were detected in multiple Agaricus samples; up to 24 in samples symptomatic for disease and 8-17 in asymptomatic samples, suggesting adaptive strategies for co-existence. The viral composition of growing cultures was dynamic, with evidence of gains and losses depending on the environment and included new hypothetical viruses when compared with the current transcriptome and EST databases. As the non-cellular transmission of mycoviruses is rare, the founding infections may be ancient, preserved in wild Agaricus populations, which act as reservoirs for subsequent cell-to-cell infection when host populations are expanded massively through fungiculture.

A Comparison of Deformed Wing Virus in Deformed and Asymptomatic Honey Bees.

Deformed wing virus (DWV) in association with Varroa destructor is currently attributed to being responsible for colony collapse in the western honey bee (Apis mellifera). The appearance of deformed individuals within an infested colony has long been associated with colony losses. However, it is unknown why only a fraction of DWV positive bees develop deformed wings. This study concerns two small studies comparing deformed and non-deformed bees. In Brazil, asymptomatic bees (no wing deformity) that had been parasitised by Varroa as pupae had higher DWV loads than non-parasitised bees. However, we found no greater bilateral asymmetry in wing morphology due to DWV titres or parasitisation. As expected, using RT-qPCR, deformed bees were found to contain the highest viral loads. In a separate study, next generation sequencing (NGS) was applied to compare the entire DWV genomes from paired symptomatic and asymptomatic bees from three colonies on two different Hawaiian islands. This revealed no consistent differences between DWV genomes from deformed or asymptomatic bees, with the greatest variation seen between locations, not phenotypes. All samples, except one, were dominated by DWV type A. This small-scale study suggests that there is no unique genetic variant associated with wing deformity; but that many DWV variants have the potential to cause deformity.

Moku virus; a new Iflavirus found in wasps, honey bees and Varroa.

There is an increasing global trend of emerging infectious diseases (EIDs) affecting a wide range of species, including honey bees. The global epidemic of the single stranded RNA Deformed wing virus (DWV), driven by the spread of Varroa destructor has been well documented. However, DWV is just one of many insect RNA viruses which infect a wide range of hosts. Here we report the full genome sequence of a novel Iflavirus named Moku virus (MV), discovered in the social wasp Vespula pensylvanica collected in Hawaii. The novel genome is 10,056 nucleotides long and encodes a polyprotein of 3050 amino acids. Phylogenetic analysis showed that MV is most closely related to Slow bee paralysis virus (SBPV), which is highly virulent in honey bees but rarely detected. Worryingly, MV sequences were also detected in honey bees and Varroa from the same location, suggesting that MV can also infect other hymenopteran and Acari hosts.