The transient nature of bunyamwera orthobunyavirus NSs protein expression : effects of increased stability of NSs protein on virus replication

The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.Peer reviewe

Knockdown of piRNA pathway proteins results in enhanced Semliki forest virus production in mosquito cells

The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production

Following acute encephalitis, Semliki Forest virus is undetectable in the brain by infectivity assays but functional virus RNA capable of generating infectious virus persists for life

Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the brain, by standard infectivity assays, for around ten days. As we have shown previously, in severe combined immunodeficient (SCID) mice, infectious virus is detectable for months in the brain. Here we show that in MHC-II-/- mice, with no functional CD4 T-cells, infectious virus is also detectable in the brain for long periods. In contrast, in the brains of CD8-/- mice, virus RNA persists but infectious virus is not detectable. In SCID mice infected with SFV, repeated intraperitoneal administration of anti-SFV immune serum rapidly reduced the titer of infectious virus in the brain to undetectable, however virus RNA persisted. Repeated intraperitoneal passive transfer of immune serum resulted in maintenance of brain virus RNA, with no detectable infectious virus, for several weeks. When passive antibody transfer was stopped, antibody levels declined and infectious virus was again detectable in the brain. In aged immunocompetent mice, previously infected with SFV, immunosuppression of antibody responses many months after initial infection also resulted in renewed ability to detect infectious virus in the brain. In summary, antiviral antibodies control and determine whether infectious virus is detectable in the brain but immune responses cannot clear this infection from the brain. Functional virus RNA capable of generating infectious virus persists and if antibody levels decline, infectious virus is again detectable

Novel reporter systems to study Semliki Forest virus pathogenesis

Semliki Forest virus (SFV) has been much used to study the cell biology and molecular pathogenesis of RNA viruses, particularly virus encephalitis. The genome encodes nine functional proteins in two open-reading frames (ORFs). The 5' ORF carries information for synthesis of four non-structural replicase proteins (nsPl - nsP4). The 3' third of the genome encodes the structural proteins. The aim of this study was to develop novel reporter systems to study the pathogenesis of SFV in vivo. Two different types of recombinant viruses each carrying one of two foreign genes, enhanced green fluorescent protein (eGFP) or Cre recombinase, were constructed based on the SFV4 backbone. In the first type of construct the transgene was inserted in the non-structural ORF, between the coding sequences for nsP3 and nsP4, flanked by processing sites recognised by the nsP2 proteinase. In the second type of constructs the 2A sequence from foot-and-mouth disease virus was added to the Cterminus of the foreign gene and this was placed between the capsid and the p62 protein of SFV4 (structural ORF). The in vitro and in vivo phenotypes of the resulting viruses were assessed and compared to SFV4.All recombinant viruses constructed were viable and able to replicate in vitro. eGFP expressing viruses reached titres similar to those of wild-type virus whereas Cre expressing viruses were slightly attenuated. For viruses with the marker gene inserted in the non-structural ORF, western blotting showed that the processing pattern of the non-structural polyprotein was similar to that of SFV4 and verified the expression of both foreign genes. In vivo, following intracerebral inoculation, all viruses caused encephalitis. Viruses expressing the foreign gene as a cleavable component of the structural ORF induced disease slower than SFV4 or viruses carrying the transgene in the replicase ORF. eGFP fluorescence was stronger and occurred later in infection when expressed in the structural ORF than in the replicase ORF.eGFP expression from the replicase ORF marked only recently infected cells; a property useful in pathogenesis studies. eGFP expressing viruses demonstrated the same cell tropism as SFV4 with infection principally of neurons and IX oligodendrocytes. None of the mice infected intraperitoneal^ with SFV4 or the recombinant viruses succumbed to infection demonstrating poor neuroinvasiveness. The powerful suppression of alphavirus replication by the interferon system was demonstrated in IFN a/p receptor knockout mice. The true tropism and the potential of SFV4 was revealed in the absence of a functional IFN system.These studies demonstrated that foreign genes can be inserted into the non-structural or the structural ORF of SFV4 without destroying virus infectivity or major changes in phenotype. These viruses are likely to be highly valuable for in vivo pathogenesis studies

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Advances in dissecting mosquito innate immune responses to arbovirus infection

Arthropod-borne viruses – arboviruses – are a significant threat to public health. Whilst there is considerable knowledge about arbovirus interactions with vertebrate immunity, relatively little is known about how vectors such as mosquitoes control arbovirus infections. In this review, we discuss novel findings in the field of mosquito antiviral responses to arboviruses, in particular RNA interference, the up-and-coming field of general immune-signalling pathways, and cell death/apoptosis

Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyse CHIKV nsP1 functionality, several mutations that severely reduced, but did not completely block, CHIKV replicase activity were identified: (i) tagging the N-terminus of nsP1 with eGFP; (ii) mutations D63A and Y248A blocking the RNA capping; (iii) mutation R252E affecting nsP1 membrane anchoring. In contrast, a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirms that this novel system provides a valuable tool to study CHIKV replicase, RNA replication and virus-host interactions

Τεχνικοτακτικοί παράγοντες επιτυχίας των νικητριών ομάδων της Μπουντεσλίγκα για τον δεύτερο γύρο 2022-2023

Η παρούσα εργασία είχε ως στόχο την εξέταση και την ανάλυση των τεχνικοτακτικών παραγόντων που επηρεάζουν την απόδοση στο ποδόσφαιρο, ειδικά σε ομάδες υψηλού επιπέδου. Ο βασικός στόχος ήταν η καλύτερη κατανόηση των παραγόντων που συνδέονται με την επιτυχία των επιτυχημένων ομάδων σε αυτό το αθλητικό πεδίο. Το δείγμα της παρούσας εργασίας αποτέλεσαν 114 αγώνες (228 ομάδες), εκ των οποίων οι 114 αφορούσαν τις νικήτριες και οι 114 τις ηττημένες ομάδες. Τα δεδομένα συλλέχθηκαν από επίσημο έγγραφο με αναφορά της FIFA και από το διαδικτυακό ιστότοπο whoscored.com. Κατά την εξέταση των μεταβλητών μελετήθηκαν τα τέρματα που επιτεύχθηκαν, οι προσπάθειες εντός εστίας, οι προσπάθειες στο στόχο που αποκρούστηκαν, η κατοχή της μπάλας, οι μεταβιβάσεις και η ευστοχία τους, όπως και διαφορετικές μεταβλητές ενός αγώνα, που αποτελούνται από τις σέντρες, τα τάκλιν και τα φάουλ, αλλά και οι συνολικές προσπάθειες από οργανωμένη επίθεση και από αντεπίθεση. Πραγματοποιήθηκε σύγκριση των ανεξάρτητων μεταβλητών μέσω ανάλυσης διακύμανσης (ANOVA). Ορίστηκε στατιστικά σημαντικό το επίπεδο στο sig<0.05. Από τις κύριες μεταβλητές που εξετάστηκαν, είναι εμφανής η σημαντική διαφορά στον μέσο όρο των γκολ μεταξύ των νικητριών (2,71±1,210) και των ηττημένων ομάδων (0,73±0,768). Επίσης, παρατηρήθηκε ότι οι επιτυχημένες ομάδες είχαν σημαντικά υψηλότερη απόδοση στον μέσο όρο των συνολικών σουτ και των σουτ στον στόχο σε σχέση με τις αποτυχημένες (14,24±5,531 vs 11,65±4,458 και 6,15±2,73 vs 3,60±1,917 αντίστοιχα), όπως σημαντικές διαφορές φάνηκαν εξίσου στις σέντρες και στις συνολικές προσπάθειες από αντεπιθέσεις (16,22±6,828 vs 18,36±7,991 και 0,89±1,051 vs 0,37±0,756 αντίστοιχα). Παράλληλα, στην κατοχή της μπάλας, στις μεταβιβάσεις και στα τάκλιν δεν σημειώθηκαν σημαντικές διαφορές μεταξύ νικητριών και ηττημένων ομάδων (49,53±11,001 vs 50,00±10,987, 368,75±137,83 vs 371,13±115,523 και 64,47±12,014 vs 62,61±12,686 αντιστοίχως). Συνοψίζοντας, οι παράγοντες που διαμορφώνουν την επιτυχία ή την αποτυχία μιας ομάδας είναι κρίσιμοι παράγοντες που απαιτούν προσεκτική εξέταση από προπονητές και αθλητές. Η ανάλυση αυτών των παραγόντων είναι απαραίτητη προκειμένου να επιτευχθούν παρόμοιες επιδόσεις με εκείνες των επιτυχημένων ομάδων.The present study aimed to examine and analyze the technical-tactical factors influencing performance in football, especially in high-level teams. The primary objective was to gain a better understanding of the factors associated with the success of teams in this sport. The sample of this study consisted of 114 matches (228 teams), with 114 involving the winning teams and 114 the losing teams. Data were collected from an official FIFA document and the website whoscored.com. When examining variables, goals scored, attempts on target, saved attempts, ball possession, passes, accuracy, as well as other match variables such as crosses, tackles, and fouls, were studied. Statistical measures such as mean, minimum, maximum, frequency, and standard deviation were utilized. Additionally, analysis of variance (ANOVA) was performed to compare independent variables, with a statistically significant level set at sig<0.05. From the main variables examined, a significant difference is evident in the average number of goals between the winning (2.71±1.21) and losing teams (0.73±0.77). Furthermore, successful teams displayed significantly higher performance in the average of total shots and shots on target compared to the unsuccessful ones (14.24±5.531 vs 11.65±4.458 and 6.15±2.73 vs 3.60±1.917, respectively). Significant differences were also observed in crosses and total attempts from counterattacks (16.22±6.828 vs 18.36±7.991 and 0.89±1.051 vs 0.37±0.756, respectively). However, no significant differences were noted in ball possession, passes, and tackles. In summary, the factors shaping the success or failure of a team are crucial elements requiring careful analysis by coaches and players. Understanding these factors is essential to achieve performances similar to successful teams

Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster premiRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes. </p

Generation of antibodies against foot-and-mouth-disease virus capsid protein VP4 using hepatitis B core VLPs as a Scaffold

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the eco-nomically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neu-tralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.</p