Cyclic di-AMP: Small molecule with big roles in bacteria.

Cyclic dinucleotides are second messengers that are present in all the three domains of life, bacteria, archaea, and eukaryotes. These dinucleotides have important physiological and pathophysiological roles in bacteria. Cyclic di-AMP (cdA) is one of the recently discovered cyclic dinucleotides present predominantly in gram-positive bacteria. cdA is synthesized through diadenylate cyclase (DAC) activity from ATP in a two-step process and hydrolyzed to linear dinucleotide pApA (and to 5' AMP in certain cases) by specific phosphodiesterases. cdA regulates various physiological processes like K+ transport and osmotic balance, DNA repair, cell wall homeostasis, drug resistance, central metabolism either by binding directly to the target protein or regulating its expression. It also participates in host-pathogen interaction by binding to host immune receptors ERAdP, RECON, and STING.

DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in Actinobacteria.

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

Nutritional, Phytochemical, and Functional Properties of Four Edible Orchid Species from Malawi.

Terrestrial orchid tubers are an important source of food in some parts of Africa and are traditionally included in the diets of most rural communities in Malawi. However, there is limited information on the nutritional and phytochemical content of the Malawian orchids to substantiate their traditional use. The present study evaluates the nutritional and phytochemical variation of four orchid species: Disa zombica, Satyrium buchananii, Satyrium carsonii, and Satyrium trinerve, collected from the same ecological zone of Malawi. The proximate content, minerals, phytochemicals, and functional properties were analysed using official procedures. Protein ranged from 2.19% to 4.65%, whereas carbohydrate ranged from 65.24% to 80.22%, with S. carsonii and S. trinerve registering the highest protein and carbohydrate contents, respectively. Iron and potassium were highest in D. zombica, while sodium and calcium were highest in S. buchananii. The total phenolics ranged from 228.56 to 500.00 mg GAE/100 g, with D. zombica registering the highest. The water absorption capacity ranged from 4.10 to 10.88 g/g. Despite variable levels, the study provides evidence that orchid species contain essential nutrients and phytochemicals important for human nutrition and health. Furthermore, the functional properties can be utilised in the development of food products, such as baked products.

Influence of spill-over for 99mTc images and the effect of scatter correction for dual-isotope simultaneous acquisition with 99mTc and 18F using small-animal SPECT-PET/CT system.

A dual-isotope simultaneous acquisition (DISA) of 99mTc and 18F affects the image quality of 99mTc by crosstalk and spill-over from 18F. We demonstrated the influence of spill-over and crosstalk on image quality and its correction effect for DISA SPECT with 99mTc and 18F. A fillable cylindrical chamber of 30 mm with NEMA-NU4 image quality phantom was filled with 99mTc only or a mixed 99mTc and 18F solution (C100). Two small-region chambers were filled with 99mTc only or a mixed 99mTc and 18F solution made at half the radioactivity concentration of C100 (C50) and non-radioactive water (C0). The 18F/99mTc ratio for DISA was set at approximately 0.4-12. Two types of 99mTc transverse images with and without scatter correction (SC and nonSC) were created. The 99mTc images of single-isotope acquisition (SIA) were created as a reference. The DISA/SIA ratio and contrast of 99mTc were compared between SIA and DISA. Although the DISA/SIA ratios with nonSC of C100, C50 and C0 gradually increased with increasing 18F/99mTc ratio, it was nearly constant by SC. The contrasts of C100 and C50 were similar to a reference value for both nonSC and SC. In conclusion, DISA images showed lower image quality as the 18F/99mTc ratio increased. The image quality in hot-spot regions such as C100 and C50 was improved by SC, whereas cold-spot regions such as C0 could not completely remove the influence of spill-over even with SC.

The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

BACKGROUND AND AIMS: The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production. METHODS: The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species. KEY RESULTS: Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification. CONCLUSIONS: Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.

Osmotic stress responses and the biology of the second messenger c-di-AMP in Streptomyces.

Streptomyces are prolific antibiotic producers that thrive in soil, where they encounter diverse environmental cues, including osmotic challenges caused by rainfall and drought. Despite their enormous value in the biotechnology sector, which often relies on ideal growth conditions, how Streptomyces react and adapt to osmotic stress is heavily understudied. This is likely due to their complex developmental biology and an exceptionally broad number of signal transduction systems. With this review, we provide an overview of Streptomyces' responses to osmotic stress signals and draw attention to open questions in this research area. We discuss putative osmolyte transport systems that are likely involved in ion balance control and osmoadaptation and the role of alternative sigma factors and two-component systems (TCS) in osmoregulation. Finally, we highlight the current view on the role of the second messenger c-di-AMP in cell differentiation and the osmotic stress responses with specific emphasis on the two models, S. coelicolor and S. venezuelae.

Development of a novel small-animal myocardial phantom can evaluate the image quality of dual-isotope simultaneous acquisition (DISA).

BACKGROUND: Myocardial phantom studies are widely used as a tool to accurately assess the physical phenomenon of dual-isotope simultaneous acquisition (DISA) in the small-animal fields. However, the previous phantom did not reproduce the structures of rats or mice. The aim of this study was to develop a novel myocardial phantom simulating the structure of a small animal that can be evaluated using the image quality of DISA. METHODS: A novel small-animal myocardial phantom that simulated a rat was constructed by the myocardium, liver, lung, spine, and torso. Normal and inferior wall defect myocardial phantoms were filled with 99mTc or 18F solution to simulate single-isotope acquisition (SIA) and DISA. Phantom and small-animal images with no scatter correction (nonSC) and scatter correction (SC) were created. RESULTS: The 99mTc DISA with SC showed a low %CV compared to that with nonSC. Although the 99mTc DISA with nonSC had lower cavity contrast than that of 99mTc SIA with nonSC, the cavity contrast of SC had similar values between SIA and DISA. The minimum %uptake of 99mTc SIA with nonSC was a lower value compared to that of 99mTc DISA with nonSC. The 99mTc DISA was equivalent to the minimum %uptake of 99mTc SIA by SC. CONCLUSION: We have developed a novel myocardial phantom for the rat model to evaluate the image quality for reproducing the physical phenomenon associated with radiation attenuation and scattering. Furthermore, we could demonstrate the usefulness of the novel small-animal myocardial phantom by image quality evaluation of DISA with 99mTc and 18F compared to SIA.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for "European" serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

DisA Limits RecG Activities at Stalled or Reversed Replication Forks.

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3', 5'-diadenosine monophosphate (c­di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography.

All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze c-di-AMP to the linear phosphoadenylate adenosine 5'-pApA or to AMP. Different methods including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and ion exchange chromatography (IEX) can be used to determine activities of c-di-AMP-synthesizing and degrading enzymes. Here, we describe in detail the TLC and IEX methods adapted for characterization of the diadenylate cyclase DisA and the phosphodiesterase AtaC from Streptomyces venezuelae. TLC allows quick and easy separation of radioactive-labeled substrates and products, while IEX avoids utilization of potentially hazardous radioactive substrates and can be used as a good substitute if an HPLC system is not available. Unlike in TLC assays, samples cannot be analyzed in parallel by using the IEX assay, thus it is more time consuming.

The Bluetongue Disabled Infectious Single Animal (DISA) Vaccine Platform Based on Deletion NS3/NS3a Protein Is Safe and Protective in Cattle and Enables DIVA.

The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.

Sexual deception of a beetle pollinator through floral mimicry.

Sexual mimicry is a complex multimodal strategy used by some plants to lure insects to flowers for pollination.1-4 It is notable for being highly species-specific and is typically mediated by volatiles belonging to a restricted set of chemical compound classes.3,4 Well-documented cases involve exploitation of bees and wasps (Hymenoptera)5,6 and flies (Diptera).7-9 Although beetles (Coleoptera) are the largest insect order and are well known as pollinators of both early and modern plants,10,11 it has been unclear whether they are sexually deceived by plants during flower visits.12,13 Here we report the discovery of an unambiguous case of sexual deception of a beetle: male longhorn beetles (Chorothyse hessei, Cerambycidae) pollinate the elaborate insectiform flowers of a rare southern African orchid (Disa forficaria), while exhibiting copulatory behavior including biting the antennae-like petals, curving the abdomen into the hairy lip cleft, and ejaculating sperm. The beetles are strongly attracted by (16S,9Z)-16-ethyl hexadec-9-enolide, a novel macrolide that we isolated from the floral scent. Structure-activity studies14,15 confirmed that chirality and other aspects of the structural geometry of the macrolide are critical for the attraction of the male beetles. These results demonstrate a new biological function for plant macrolides and confirm that beetles can be exploited through sexual deception to serve as pollinators.

Transcriptional coupling (Mfd) and DNA damage scanning (DisA) coordinate excision repair events for efficient Bacillus subtilis spore outgrowth.

The absence of base excision repair (BER) proteins involved in processing ROS-promoted genetic insults activates a DNA damage scanning (DisA)-dependent checkpoint event in outgrowing Bacillus subtilis spores. Here, we report that genetic disabling of transcription-coupled repair (TCR) or nucleotide excision repair (NER) pathways severely affected outgrowth of ΔdisA spores, and much more so than the effects of these mutations on log phase growth. This defect delayed the first division of spore's nucleoid suggesting that unrepaired lesions affected transcription and/or replication during outgrowth. Accordingly, return to life of spores deficient in DisA/Mfd or DisA/UvrA was severely affected by a ROS-inducer or a replication blocking agent, hydrogen peroxide and 4-nitroquinoline-oxide, respectively. Mutation frequencies to rifampin resistance (Rifr ) revealed that DisA allowed faithful NER-dependent DNA repair but activated error-prone repair in TCR-deficient outgrowing spores. Sequencing analysis of rpoB from spontaneous Rifr colonies revealed that mutations resulting from base deamination predominated in outgrowing wild-type spores. Interestingly, a wide range of base substitutions promoted by oxidized DNA bases were detected in ΔdisA and Δmfd outgrown spores. Overall, our results suggest that Mfd and DisA coordinate excision repair events in spore outgrowth to eliminate DNA lesions that interfere with replication and transcription during this developmental period.

Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness.

African Horse Sickness Virus (AHSV) (Orbivirus genus, Reoviridae family) causes high mortality in naïve domestic horses with enormous economic and socio-emotional impact. There are nine AHSV serotypes showing limited cross neutralization. AHSV is transmitted by competent species of Culicoides biting midges. AHS is a serious threat beyond the African continent as endemic Culicoides species in moderate climates transmit the closely related prototype bluetongue virus. There is a desperate need for safe and efficacious vaccines, while DIVA (Differentiating Infected from Vaccinated) vaccines would accelerate control of AHS. Previously, we have shown that highly virulent AHSV with an in-frame deletion of 77 amino acids (aa) in NS3/NS3a is completely safe, does not cause viremia and shows protective capacity. This deletion mutant is a promising DISA (Disabled Infectious Single Animal) vaccine platform, since exchange of serotype specific virus proteins has been shown for all nine serotypes. Here, we show that a prototype NS3 competitive ELISA is DIVA compliant to AHS DISA vaccine platforms. Epitope mapping of NS3/NS3a shows that more research is needed to evaluate this prototype serological DIVA assay regarding sensitivity and specificity, in particular for AHSVs expressing antigenically different NS3/NS3a proteins. Further, an experimental panAHSV PCR test targeting genome segment 10 is developed that detects reference AHSV strains, whereas AHS DISA vaccine platforms were not detected. This DIVA PCR test completely guarantees genetic DIVA based on in silico and in vitro validation, although test validation regarding diagnostic sensitivity and specificity has not been performed yet. In conclusion, the prototype NS3 cELISA and the PCR test described here enable serological and genetic DIVA accompanying AHS DISA vaccine platforms.

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. 'Synthetically derived' virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

Bacillus subtilis DisA helps to circumvent replicative stress during spore revival.

The mechanisms that allow to circumvent replicative stress, and to resume DNA synthesis are poorly understood in Bacillus subtilis. To study the role of the diadenylate cyclase DisA and branch migration translocase (BMT) RadA/Sms in restarting a stalled replication fork, we nicked and broke the circular chromosome of an inert mature haploid spore, damaged the bases, and measured survival of reviving spores. During undisturbed ripening, nicks and breaks should be repaired by pathways that do not invoke long-range end resection or genetic exchange by homologous recombination, after which DNA replication might be initiated. We found that DNA damage reduced the viability of spores that lacked DisA, BMT (RadA/Sms, RuvAB or RecG), the Holliday junction resolvase RecU, or the translesion synthesis DNA polymerases (PolY1 or PolY2). DisA and RadA/Sms, in concert with RuvAB, RecG, RecU, PolY1 or PolY2, are needed to bypass replication-blocking lesions. DisA, which binds to stalled or reversed forks, did not apparently affect initiation of PriA-dependent DNA replication in vitro. We propose that DisA is necessary to coordinate responses to replicative stress; it could help to circumvent damaged template bases that otherwise impede fork progression.

Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence.

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation.

Application of bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2.

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249-398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange.

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA.

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA). BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine. Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses. Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.