A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing ...surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.
The first cluster of patients suffering from coronavirus disease 2019 (COVID-19) was identified on December 21, 2019, and as of July 29, 2020, severe acute respiratory syndrome coronavirus 2 ...(SARS-CoV-2) infections have been linked with 664,333 deaths and number at least 16,932,996 worldwide. Unprecedented in global societal impact, the COVID-19 pandemic has tested local, national, and international preparedness for viral outbreaks to the limits. Just as it will be vital to identify missed opportunities and improve contingency planning for future outbreaks, we must also highlight key successes and build on them. Concomitant to the emergence of a novel viral disease, there is a 'research and development gap' that poses a threat to the overall pace and quality of outbreak response during its most crucial early phase. Here, we outline key components of an adequate research response to novel viral outbreaks using the example of SARS-CoV-2. We highlight the exceptional recent progress made in fundamental science, resulting in the fastest scientific response to a major infectious disease outbreak or pandemic. We underline the vital role of the international research community, from the implementation of diagnostics and contact tracing procedures to the collective search for vaccines and antiviral therapies, sustained by unique information sharing efforts.
Hazara nairovirus (HAZV) is a member of the family
in the order
and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome ...comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3' and 5' NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3' NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5' NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent
-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed.
Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3' and 5' ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3' and 5' NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.
Summary
The ability of T7 RNA polymerase (RNAPT7) fusions to cytosine deaminases (CdA) for entering C➔T changes in any DNA segment downstream of a T7 promoter was exploited for hyperdiversification ...of defined genomic portions of Pseudomonas putida KT2440. To this end, test strains were constructed in which the chromosomally encoded pyrF gene (the prokaryotic homologue of yeast URA3) was flanked by T7 transcription initiation and termination signals and also carried plasmids expressing constitutively either high‐activity (lamprey's) or low‐activity (rat's) CdA‐RNAPT7 fusions. The DNA segment‐specific mutagenic action of these fusions was then tested in strains lacking or not uracil‐DNA glycosylase (UDG), that is ∆ung/ung+ variants. The resulting diversification was measured by counting single nucleotide changes in clones resistant to 5‐fluoroorotic acid (5FOA), which otherwise is transformed by wild‐type PyrF into a toxic compound. Although the absence of UDG dramatically increased mutagenic rates with both CdA‐RNAPT7 fusions, the most active variant – pmCDA1 – caused extensive appearance of 5FOA‐resistant colonies in the wild‐type strain not limited to C➔T but including also a range of other changes. Furthermore, the presence/absence of UDG activity swapped cytosine deamination preference between DNA strands. These qualities provided the basis of a robust system for continuous evolution of preset genomic portions of P. putida and beyond.
This work reports adaptation to Pseudomonas putida of a genetic diversification system based on fusions of the T7 RNA polymerase to cytosine deaminases. This platform achieves continuous mutagenesis of defined genomic segments in living cells, enabling evolution of genes of interest.
Fingolimod is an immunomodulatory sphingosine-1-phosphate (S1P) analogue approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). The identification of biomarkers of clinical ...responses to fingolimod is a major necessity in MS to identify optimal responders and avoid the risk of disease progression in non-responders. With this aim, we used RNA sequencing to study the transcriptomic changes induced by fingolimod in peripheral blood mononuclear cells of MS-treated patients and their association with clinical response. Samples were obtained from 10 RRMS patients (five responders and five non-responders) at baseline and at 12 months of fingolimod therapy. Fingolimod exerted a vast impact at the transcriptional level, identifying 7155 differentially expressed genes (DEGs) compared to baseline that affected the regulation of numerous signaling pathways. These DEGs were predominantly immune related, including genes associated with S1P metabolism, cytokines, lymphocyte trafficking, master transcription factors of lymphocyte functions and the NF-kB pathway. Responder and non-responder patients exhibited a differential transcriptomic regulation during treatment, with responders presenting a higher number of DEGs (6405) compared to non-responders (2653). The S1P, NF-kB and TCR signaling pathways were differentially modulated in responder and non-responder patients. These transcriptomic differences offer the potential of being exploited as biomarkers of a clinical response to fingolimod.
Despite being simultaneously male and female, hermaphrodites may still need to assume the male or female sexual role in a mating encounter, with the option to swap roles afterwards. For the great ...pond snail,
Lymnaea stagnalis,
deciding which sexual role to perform has important consequences, since sperm transfer and male reproductive success can be decreased. We hypothesised that detecting cues that indicate a possible mating encounter could help them to adapt their mating behaviour. Therefore, we experimentally assessed whether signalling the presence of a conspecific with an odour can affect the sexual role of
Lymnaea stagnalis
. The results showed that learning resulted in either an increased ability to mate as a male or in faster mating compared to the control group. These findings reveal that learning shapes the mating dynamics of
Lymnaea stagnalis
, thus showing that cognitive processes not only affect mating in separate-sexed species but also in hermaphrodites.
Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against ...rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.
In November 1630 the Treaty of Madrid was signed between Philip IV, king of Spain, and Charles I, king of England. During the peace negotiations several pamphlets critical of the pro-Spanish policy ...of Charles I were printed in England. These publications contributed to the development of an increasing criticism of Charles I, by identifying his neutral European policy with pro-Spanish policies. The anti-Spanish and anti-Catholic rhetoric, which formed the basis of this criticism, was used to promote an aggressive foreign policy and consequently an active opposition to the king. The following pamphlets are discussed in this paper: Considerations T
ouching a W
arre with Spaine; The M
erchandises of Popish P
riests; The English Spanish Pilgrim; Further O
bservations of the English Spanish Pilgrim; The P
ractise of P
rinces; The P
resent E
state of Spayne; and Three S
everall T
reatises C
oncerning the T
ruce.
•Decision-making is a characteristic of intelligence not previously observed in snails.•We demonstrate that when hungry enough snails detect and eat novel substances but reject familiar ones if ...sated.•This species can therefore be used as a model for studying the cognitive capacities of higher species.•The fact that snails decide whether to eat a given food or not implies that they may be misled.•This could be helpful for a more efficient deployment of baits that are currently used to protect crops.
The foraging behaviour of gastropod molluscs usually involves complex decisions that provide a model for the study of high-order cognitive processes. Land snails tested for food-finding in the laboratory, however, have shown an invariable feeding pattern: novel foods are mostly missed (i.e. just found by chance) whilst familiar foods, due to a type of conditioned attraction, are always located and ingested. This effect, known as Food-attraction conditioning, has led to the conclusion that, regardless of their hunger level, land snails are both willing to eat anything at any moment and also blind to the odours of novel foods. An alternative account of these findings emerges from the fact that the snails are usually tested whilst in a moderate state of hunger, so that they benefit from feeding on known foods but not from taking the risk of feeding on those that are unknown. The present experiments suggest that it is the case. Snails of the invasive species Theba pisana were tested for food-finding according to their seasonal cycle in a laboratory located in their native Mediterranean region. Subjects collected at the beginning of their aestivation period succeed in locating novel food items after being deprived for a long period (45 days), but ignored a conditioned food when they were sated with this food at the end of their lethargy. The results allow us to conclude that the feeding behaviour of snails is the product of a complex cost-benefit analysis in which their motivational state and the stimuli they perceive (and the memory of such stimuli), are evaluated. Finally, we anticipate that these results will be of use in increasing the efficiency of current baits employed for the protection of crops.
The
order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of ...multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies.
Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.
PDF
