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RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a ‘minigenome’ or ‘minireplicon’ assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a ‘first-generation’ plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

Two novel reassortant highly pathogenic avian influenza viruses (H5N1) clade 2.3.4.4b.2 were identified in dead migratory birds in China in November 2021. The viruses probably evolved among wild birds through different flyways connecting Europe and Asia. Their low antigenic reaction to vaccine antiserum indicates high risks to poultry and to public health.

Abstract Background The RNA-dependent RNA polymerase of Influenza A virus is a determinant of viral pathogenicity and host range that is responsible for transcribing and replicating the negative sense segmented viral genome (vRNA). Transcription produces capped and polyadenylated mRNAs whereas genome replication involves the synthesis of an alternative plus-sense transcript (cRNA) with unmodified termini that is copied back to vRNA. Viral mRNA transcription predominates at early stages of viral infection, while later, negative sense genome replication is favoured. However, the "switch" that regulates the transition from transcription to replication is poorly understood. Results We show that temperature strongly affects the balance between plus and minus-sense RNA synthesis with high temperature causing a large decrease in vRNA accumulation, a moderate decrease in cRNA levels but (depending on genome segment) either increased or unchanged levels of mRNA. We found no evidence implicating cellular heat shock protein activity in this effect despite the known association of hsp70 and hsp90 with viral polymerase components. Temperature-shift experiments indicated that polymerase synthesised at 41°C maintained transcriptional activity even though genome replication failed. Reduced polymerase association with viral RNA was seen in vivo and in confirmation of this, in vitro binding assays showed that temperature increased the rate of dissociation of polymerase from both positive and negative sense promoters. However, the interaction of polymerase with the cRNA promoter was particularly heat labile, showing rapid dissociation even at 37°C. This suggested that vRNA synthesis fails at elevated temperatures because the polymerase does not bind the promoter. In support of this hypothesis, a mutant cRNA promoter with vRNA-like sequence elements supported vRNA synthesis at higher temperatures than the wild-type promoter. Conclusion The differential stability of negative and positive sense polymerase-promoter complexes explains why high temperature favours transcription over replication and has implications for the control of viral RNA synthesis at physiological temperatures. Furthermore, given the different body temperatures of birds and man, these finding suggest molecular hypotheses for how polymerase function may affect host range.

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Abstract Background The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. Results Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. Conclusions These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

IntroductionWhile research shows no negative effects of bilingualism on autistic children’s development, due to misconceptions around both autism and bilingualism, bilingual parents and educational/clinical practitioners who advise them often express unfounded concerns that exposing autistic children to more than one language will cause confusion and developmental delays. To understand the reasons that drive these misconceptions, this study focuses on: identifying factors that impact family decisions about (not) raising autistic children bilingually; attitudes toward bilingualism expressed by the community, doctors, family members, and teachers; sources of information about bilingualism and autism available to families.MethodsThrough a mixed-method online survey, we explored these questions in 31 UK-based bilingual families with 34 autistic children (age M = 10.6 years; SD = 7.1).ResultsThe families reported choosing bilingualism for their autistic child primarily so that the child can communicate with family and community members. Attitudes toward bilingualism in their networks were predominantly positive, with a large portion of individuals not having opinions possibly due to lack of information. Only about 1/3 of parents had access to information on bilingualism and autism, mostly found on the internet.DiscussionWe discuss these findings and offer future directions for research, practice, and battling stigmas around bilingualism and autism.

To identify host factors that affect Bovine Herpes Virus Type 1 (BoHV-1) infection we previously applied a genome wide CRISPR knockout screen targeting all bovine protein coding genes. By doing so we compiled a list of both pro-viral and anti-viral proteins involved in BoHV-1 replication. Here we provide further analysis of those that are potentially involved in viral entry into the host cell. We first generated single cell knockout clones deficient in some of the candidate genes for validation. We provide evidence that Polio Virus Receptor-related protein (PVRL2) serves as a receptor for BoHV-1, mediating more efficient entry than the previously identified Polio Virus Receptor (PVR). By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we further demonstrate the significance of HSPG in BoHV-1 entry. Another intriguing cluster of candidate genes, COG1, COG2 and COG4-7 encode six subunits of the Conserved Oligomeric Golgi (COG) complex. MDBK cells lacking COG6 produced fewer but bigger plaques compared to control cells, suggesting more efficient release of newly produced virions from these COG6 knockout cells, due to impaired HSPG biosynthesis. We further observed that viruses produced by the COG6 knockout cells consist of protein(s) with reduced N-glycosylation, potentially explaining their lower infectivity. To facilitate candidate validation, we also detailed a one-step multiplex CRISPR interference (CRISPRi) system, an orthogonal method to KO that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified eight candidates that have been implicated in the synthesis of surface heparan sulfate proteoglycans (HSPGs). In summary, our experiments confirmed the two receptors PVR and PVRL2 for BoHV-1 entry into the host cell and other factors that affect this process, likely through the direct or indirect roles they play during HSPG synthesis and glycosylation of viral proteins.

Abstract Fourth-generation ‘pod’ e-cigarette devices have been driven by technological advances in electronic atomization of the e-liquid. Use of microporous ceramic as a wicking material improves heating efficiency, but how it affects the chemical emissions of these devices is unclear. We assessed the emissions of a pod e-cigarette with innovative ceramic wick-based technology and two flavoured e-liquids containing nicotine lactate and nicotine benzoate (57 and 18 mg mL−1 nicotine, respectively). Among the studied harmful and potentially harmful constituents (HPHCs) listed by the US FDA and/or WHO TobReg, only 5 (acetone, acetaldehyde, formaldehyde, naphthalene and nornicotine) were quantified at levels of 0.14 to 100 ng puff−1. In the combustible cigarette (Kentucky reference 1R6F), levels were from 0.131 to 168 µg puff−1. Nicotine levels ranged 0.10–0.32 mg puff−1 across the 3 study products. From the 19 proposed HPHCs specifically of concern in e-cigarettes, only 3 (glycerol, isoamyl acetate and propylene glycol) were quantified. The low/undetectable levels of HPHCs reflect not only the optimal operating conditions of the e-cigarette, including an efficient supply of e-liquid by the ceramic wick without overheating, but also the potential of the e-cigarettes to be used as an alternative to combustible cigarettes.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Bilingualism changes how people relate to others, and lead their lives. This is particularly relevant in autism, where social interaction presents challenges. Understanding the overlap between the social variations of bilingualism and autism could unveil new ways to support autistic people. This research aims to understand the language learning and social experiences of mono-, bi- and multilingual autistic people. A total of 297 autistic adults (mean age = 32.4 years) completed an online questionnaire including general demographic, language history and social life quality self-rating items. The sample included 89 monolingual English speakers, 98 bilinguals, and 110 multilinguals, with a wide range of language profiles. Regression models were used to analyse how bilingualism variables predicted social life quality ratings. In the full sample, age negatively predicted social life quality scores while the number of languages known positively predicted social life quality scores. In the multilingual subset, age negatively predicted social life quality scores, while third language proficiency positively predicted social life quality scores. This is the first study describing the language history and social experiences of a substantial sample of bilingual and multilingual autistic adults. It provides valuable insight into how autistic people can learn and use a new language, and how their bilingualism experiences shape their social life.

IntroductionHighly pathogenic avian influenza (HPAI) viruses, such as H5N1, continue to pose a serious threat to animal agriculture, wildlife and to public health. Controlling and mitigating this disease in domestic birds requires a better understanding of what makes some species highly susceptible (such as turkey and chicken) while others are highly resistant (such as pigeon and goose). Susceptibility to H5N1 varies both with species and strain; for example, species that are tolerant of most H5N1 strains, such as crows and ducks, have shown high mortality to emerging strains in recent years. Therefore, in this study we aimed to examine and compare the response of these six species, to low pathogenic avian influenza (H9N2) and two strains of H5N1 with differing virulence (clade 2.2 and clade 2.3.2.1) to determine how susceptible and tolerant species respond to HPAI challenge.MethodsBirds were challenged in infection trials and samples (brain, ileum and lung) were collected at three time points post infection. The transcriptomic response of birds was examined using a comparative approach, revealing several important discoveries.ResultsWe found that susceptible birds had high viral loads and strong neuro-inflammatory response in the brain, which may explain the neurological symptoms and high mortality rates exhibited following H5N1 infection. We discovered differential regulation of genes associated with nerve function in the lung and ileum, with stronger differential regulation in resistant species. This has intriguing implications for the transmission of the virus to the central nervous system (CNS) and may also indicate neuro-immune involvement at the mucosal surfaces. Additionally, we identified delayed timing of the immune response in ducks and crows following infection with the more deadly H5N1 strain, which may account for the higher mortality in these species caused by this strain. Lastly, we identified candidate genes with potential roles in susceptibility/resistance which provide excellent targets for future research.DiscussionThis study has helped elucidate the responses underlying susceptibility to H5N1 influenza in avian species, which will be critical in developing sustainable strategies for future control of HPAI in domestic poultry.

Widespread issues in respirator availability and fit have been rendered acutely apparent by the COVID-19 pandemic. This study sought to determine whether personalized 3D printed respirators provide adequate filtration and function for healthcare workers through a Randomized Controlled Trial (RCT). Fifty healthcare workers recruited within NHS Lothian, Scotland, underwent 3D facial scanning or 3D photographic reconstruction to produce 3D printed personalized respirators. The primary outcome measure was quantitative fit-testing to FFP3 standard. Secondary measures included respirator comfort, wearing experience, and function instrument (R-COMFI) for tolerability, Modified Rhyme Test (MRT) for intelligibility, and viral decontamination on respirator material. Of the 50 participants, 44 passed the fit test with the customized respirator, not significantly different from the 38 with the control (p = 0.21). The customized respirator had significantly improved comfort over the control respirator in both simulated clinical conditions (p < 0.0001) and during longer wear (p < 0.0001). For speech intelligibility, both respirators performed equally. Standard NHS decontamination agents were able to eradicate 99.9% of viral infectivity from the 3D printed plastics tested. Personalized 3D printed respirators performed to the same level as control disposable FFP3 respirators, with clear communication and with increased comfort, wearing experience, and function. The materials used were easily decontaminated of viral infectivity and would be applicable for sustainable and reusable respirators.

The SARS-CoV-2 virus is primarily transmitted through virus-laden fluid particles ejected from the mouth of infected people. Face covers can mitigate the risk of virus transmission but their outward effectiveness is not fully ascertained. Objective: by using a background oriented schlieren technique, we aim to investigate the air flow ejected by a person while quietly and heavily breathing, while coughing, and with different face covers. Results: we found that all face covers without an outlet valve reduce the front flow through by at least 63% and perhaps as high as 86% if the unfiltered cough jet distance was resolved to the anticipated maximum distance of 2-3 m. However, surgical and handmade masks, and face shields, generate significant leakage jets that may present major hazards. Conclusions: the effectiveness of the masks should mostly be considered based on the generation of secondary jets rather than on the ability to mitigate the front throughflow.

Evidence suggests that susceptibility to avian influenza A virus in chickens is influenced by host genetics, but the mechanisms are poorly understood. A previous study demonstrated that inbred line 0 chickens are more resistant to low-pathogenicity avian influenza (LPAI) infection than line CB.12 birds based on viral shedding, but the resistance was not associated with higher AIV-specific IFNγ responses or antibody titres. In this study, we investigated the proportions and cytotoxic capacity of T-cell subpopulations in the spleen and the early immune responses in the respiratory tract, analysing the innate immune transcriptome of lung-derived macrophages following in vitro stimulation with LPAI H7N1 or the TLR7 agonist R848. The more susceptible C.B12 line had a higher proportion of CD8αβ+ γδ and CD4+CD8αα+ αVβ1 T cells, and a significantly higher proportion of the CD8αβ+ γδ and CD8αβ+ αVβ1 T cells expressed CD107a, a surrogate marker of degranulation. Lung macrophages isolated from line C.B12 birds expressed higher levels of the negative regulator genes TRIM29 and IL17REL, whereas macrophages from line 0 birds expressed higher levels of antiviral genes including IRF10 and IRG1. After stimulation with R848, the macrophages from line 0 birds mounted a higher response compared to line C.B12 cells. Together, the higher proportion of unconventional T cells, the higher level of cytotoxic cell degranulation ex vivo and post-stimulation and the lower levels of antiviral gene expression suggest a potential role of immunopathology in mediating susceptibility in C.B12 birds.

The induction of antiviral effector proteins as part of a homeostatically controlled innate immune response to infection plays a critical role in limiting the propagation and transmission of respiratory pathogens. However, the prolonged induction of this immune response can lead to lung hyperinflammation, tissue damage, and respiratory failure. We hypothesized that tissues exposed to the constant threat of infection may constitutively express higher levels of antiviral effector proteins to reduce the need to activate potentially harmful innate immune defences. By analysing transcriptomic data derived from a range of human tissues, we identify lung tissue to express constitutively higher levels of antiviral effector genes relative to that of other mucosal and non-mucosal tissues. By using primary cell lines and the airways of rhesus macaques, we show the interferon-stimulated antiviral effector protein TRIM22 (TRIpartite Motif 22) to be constitutively expressed in the lung independently of viral infection or innate immune stimulation. These findings contrast with previous reports that have shown TRIM22 expression in laboratory-adapted cell lines to require interferon stimulation. We demonstrate that constitutive levels of TRIM22 are sufficient to inhibit the onset of human and avian influenza A virus (IAV) infection by restricting the onset of viral transcription independently of interferon-mediated innate immune defences. Thus, we identify TRIM22 to confer a pre-existing (intrinsic) intracellular defence against IAV infection in cells derived from the respiratory tract. Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-existing immune gene expression in the intracellular restriction of IAV from the outset of infection.

Bilingualism is a valuable tool that enriches and facilitates cultural, social and lived experiences for autistic and non-autistic people alike. Research consistently finds no negative effects of bilingualism and highlights the potential for positive effects across cognitive and socio-cultural domains for autistic and non-autistic children. Yet parents of autistic children remain concerned that bilingualism will cause delays in both cognitive and language development and are still frequently advised by practitioners to raise their child monolingually. Evidently, findings from research are not reflected in practice or subsequent advice, and it is essential to identify ways to ensure equal access to additional language learning. We briefly summarise the existing literature on bilingualism and autism, considering perspectives from the bilingual autistic community, and experimental research. We identify the most pertinent barriers to participation for autistic bilingual children in terms of familial, clinical and educational perspectives. We propose novel solutions to promote additional language learning and suggest changes to practice that will contribute to an evidence base for families and practitioners. This commentary makes innovative recommendations at both the individual and societal level to ensure that autistic bilingual people have equal rights and opportunities to language learning and are optimally supported in accessing them.

Background: Concerns over the presence of the diketones 2,4 butanedione (DA) and 2,3 pentanedione (AP) in e-cigarettes arise from their potential to cause respiratory diseases. Their presence in e-liquids is a primary source, but they may potentially be generated by glycerol (VG) and propylene glycol (PG) when heated to produce aerosols. Factors leading to the presence of AP, DA and acetoin (AC) in e-cigarette aerosols were investigated. We quantified direct transfer from e-liquids, examined thermal degradation of major e-liquid constituents VG, PG and 1,3 propanediol (1,3 PD) and the potential for AC, AP and DA production from sugars and flavor additives when heated in e-cigarettes.Method: Transfers of AC, AP and DA from e-liquids to e-cigarette aerosols were quantified by comparing aerosol concentrations to e-liquid concentrations. Thermal generation from VG, PG or 1,3 PD e-liquids was investigated by measuring AC, AP and DA emissions as a function of temperature in an e-cigarette. Thermal generation of AC, AP and DA from sugars was examined by aerosolising e-liquids containing sucrose, fructose or glucose in an e-cigarette. Pyrolytic formation of AP and DA from a range of common flavors was assessed using flash pyrolysis techniques.Results: AC transfer efficiency was >90%, while AP and DA were transferred less efficiently (65%) indicating losses during aerosolisation. Quantifiable levels of DA were generated from VG and PG, and to a lesser extent 1,3 PD at coil temperatures >300°C. Above 350°C AP was generated from VG and 1,3 PD but not PG. AC was not generated from major constituents, although low levels were generated by thermal reduction of DA. Aerosols from e-liquids containing sucrose contained quantifiable (>6 ng/puff) levels of DA at all sucrose concentrations tested, with DA emissions increasing with increasing device power and concentration. 1% glucose, fructose or sucrose e-liquids gave comparable DA emissions. Furanose ring compounds also generate DA and AP when heated to 250°C.Conclusions: In addition to less than quantitative direct transfer from the e-liquid, DA and AP can be present in the e-cigarette aerosol due to thermal decomposition reactions of glycols, sugars and furanonse ring flavors under e-cigarette operating conditions.

Summary: STimulator of INterferon Genes (STING) is an adaptor for cytoplasmic DNA sensing by cGAMP/cGAS that helps trigger innate immune responses (IIRs). Although STING is mostly localized in the ER, we find a separate inner nuclear membrane pool of STING that increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or dsRNA mimic poly(I:C). Immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry revealed a distinct nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of IRF3/7 transcription factors, and testing a subset of these revealed STING partners SYNCRIP, MEN1, DDX5, snRNP70, RPS27a, and AATF as novel modulators of dsDNA-triggered IIRs. Moreover, we find that SYNCRIP is a novel antagonist of the RNA virus, influenza A, potentially shedding light on reports of STING inhibition of RNA viruses.

Background: As e-cigarette popularity has increased, there is growing evidence to suggest that while they are highly likely to be considerably less harmful than cigarettes, their use is not free of risk to the user. There is therefore an ongoing need to characterise the chemical composition of e-cigarette aerosols, as a starting point in characterising risks associated with their use. This study examined the chemical complexity of aerosols generated by an e-cigarette containing one unflavored and three flavored e-liquids. A combination of targeted and untargeted chemical analysis approaches was used to examine the number of compounds comprising the aerosol. Contributions of e-liquid flavors to aerosol complexity were investigated, and the sources of other aerosol constituents sought. Emissions of 98 aerosol toxicants were quantified and compared to those in smoke from a reference tobacco cigarette generated under two different smoking regimes.Results: Combined untargeted and targeted aerosol analyses identified between 94 and 139 compounds in the flavored aerosols, compared with an estimated 72–79 in the unflavored aerosol. This is significantly less complex (by 1-2 orders of magnitude) than the reported composition of cigarette smoke. Combining both types of analysis identified 5–12 compounds over and above those found by untargeted analysis alone. Gravimetrically, 89–99% of the e-cigarette aerosol composition was composed of glycerol, propylene glycol, water and nicotine, and around 3% comprised other, more minor, constituents. Comparable data for the Ky3R4F reference tobacco cigarette pointed to 58–76% of cigarette smoke “tar” being composed of minor constituents. Levels of the targeted toxicants in the e-cigarette aerosols were significantly lower than those in cigarette smoke, with 68.5–>99% reductions under ISO 3308 puffing conditions and 88.4–>99% reductions under ISO 20778 (intense) conditions; reductions against the WHO TobReg 9 priority list were around 99%.Conclusion: These analyses showed that the e-cigarette aerosols contain fewer compounds and at significantly lower concentrations than cigarette smoke. The chemical diversity of an e-cigarette aerosol is strongly impacted by the choice of e-liquid ingredients.

Abstract The avian respiratory tract is a common entry route for many pathogens and an important delivery route for vaccination in the poultry industry. Immune responses in the avian lung have mostly been studied in vivo due to the lack of robust, relevant in vitro and ex vivo models mimicking the microenvironment. Precision-cut lung slices (PCLS) have the major advantages of maintaining the 3-dimensional architecture of the lung and includes heterogeneous cell populations. PCLS have been obtained from a number of mammalian species and from chicken embryos. However, as the embryonic lung is physiologically undifferentiated and immunologically immature, it is less suitable to examine complex host–pathogen interactions including antimicrobial responses. Here we prepared PCLS from immunologically mature chicken lungs, tested different culture conditions, and found that serum supplementation has a detrimental effect on the quality of PCLS. Viable cells in PCLS remained present for ≥ 40 days, as determined by viability assays and sustained motility of fluorescent mononuclear phagocytic cells. The PCLS were responsive to lipopolysaccharide stimulation, which induced the release of nitric oxide, IL-1β, type I interferons and IL-10. Mononuclear phagocytes within the tissue maintained phagocytic activity, with live cell imaging capturing interactions with latex beads and an avian pathogenic Escherichia coli strain. Finally, the PCLS were also shown to be permissive to infection with low pathogenic avian influenza viruses. Taken together, immunologically mature chicken PCLS provide a suitable model to simulate live organ responsiveness and cell dynamics, which can be readily exploited to examine host–pathogen interactions and inflammatory responses.

Here, Li et al. perform a genome-wide CRISPR screen to identify host dependency factors for influenza A virus infection and show that the host mRNA cap methyltransferase CMTR1 is important for viral cap snatching and that it affects expression of antiviral genes.

Respiratory droplets are the primary transmission route for SARS-CoV-2, a principle which drives social distancing guidelines. Evidence suggests that virus transmission can be reduced by face coverings, but robust evidence for how mask usage might affect safe distancing parameters is lacking. Accordingly, we set out to quantify the effects of face coverings on respiratory tract droplet deposition. We tested an anatomically realistic manikin head which ejected fluorescent droplets of water and human volunteers, in speaking and coughing conditions without a face covering, or with a surgical mask or a single-layer cotton face covering. We quantified the number of droplets in flight using laser sheet illumination and UV-light for those that had landed at table height at up to 2 m. For human volunteers, expiratory droplets were caught on a microscope slide 5 cm from the mouth. Whether manikin or human, wearing a face covering decreased the number of projected droplets by less than 1000-fold. We estimated that a person standing 2 m from someone coughing without a mask is exposed to over 10 000 times more respiratory droplets than from someone standing 0.5 m away wearing a basic single-layer mask. Our results indicate that face coverings show consistent efficacy at blocking respiratory droplets and thus provide an opportunity to moderate social distancing policies. However, the methodologies we employed mostly detect larger (non-aerosol) sized droplets. If the aerosol transmission is later determined to be a significant driver of infection, then our findings may overestimate the effectiveness of face coverings.

ABSTRACT Influenza A virus (IAV) causes annual epidemics of respiratory disease in humans, often complicated by secondary coinfection with bacterial pathogens such as Staphylococcus aureus. Here, we report that the S. aureus secreted protein lipase 1 enhances IAV replication in vitro in primary cells, including human lung fibroblasts. The proviral activity of lipase 1 is dependent on its enzymatic function, acts late in the viral life cycle, and results in increased infectivity through positive modulation of virus budding. Furthermore, the proviral effect of lipase 1 on IAV is exhibited during in vivo infection of embryonated hen’s eggs and, importantly, increases the yield of a vaccine strain of IAV by approximately 5-fold. Thus, we have identified the first S. aureus protein to enhance IAV replication, suggesting a potential role in coinfection. Importantly, this activity may be harnessed to address global shortages of influenza vaccines. IMPORTANCE Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease. Secondary bacterial coinfection by organisms such as Staphylococcus aureus is the most common complication of primary IAV infection and is associated with high levels of morbidity and mortality. Here, we report the first identified S. aureus factor (lipase 1) that enhances IAV replication during infection via positive modulation of virus budding. The effect is observed in vivo in embryonated hen’s eggs and greatly enhances the yield of a vaccine strain, a finding that could be applied to address global shortages of influenza vaccines.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV’s major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

After a brief presentation of the main geographical and social characteristics of nomadism in Iran, the article examines the evolution of the treatment of nomadic tribes by the Iranian state, first as partners, under control, in the Qajar dynasty, and then as a problem of security, and/or development by the Pahlavi dynasty. Finally, the Islamic Republic had a pragmatic policy, with mixed and fluctuating results. The conclusion examines the possibilities and conditions for a future mobile livestock farming (nomadic or transhumant) in Iran.

Tomado de Jean-Pierre Digard, "Le tournant obscurantiste en anthropologie", L’Homme [En ligne], 203-204, 2012, mis en ligne le 03 décembre 2014, consulté le 24 février 2017. URL: http://lhomme.revues.org/23292; DOI: 10.4000/lhomme.23292. Traducción de Luis Alfonso Paláu Castaño, Medellín, febrero 26 de 2017. Nota del traductor. La materia de este artículo fue objeto de una comunicación oral en el coloquio internacional organizado en el Colegio de Francia, del 22 al 24 de junio de 2011, por Frédéric Keck y Noëlie Vialles sobre el tema: «¿Un “giro animaliste” en antropología?». Habiéndose quedado a la espera de una eventual publicación en las actas de ese coloquio, he preferido, en razón de la gravedad y de la actualidad del tema, no esperar más tiempo y aprovechar la hospitalidad que la redacción de L’Homme me ha ofrecido amablemente.

ABSTRACT As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients. IMPORTANCE HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

Swine influenza A viruses (IAV-S) found in North American pigs are diverse and the lack of cross-protection among heterologous strains is a concern. The objective of this study was to compare a commercial inactivated A/H1N1/pdm09 (pH1N1) vaccine and two novel subunit vaccines, using IAV M2 ectodomain (M2e) epitopes as antigens, in a growing pig model. Thirty-nine 2-week-old IAV negative pigs were randomly assigned to five groups and rooms. At 3 weeks of age and again at 5 weeks of age, pigs were vaccinated intranasally with an experimental subunit particle vaccine (NvParticle/M2e) or a subunit complex-based vaccine (NvComplex/M2e) or intramuscularly with a commercial inactivated vaccine (Inact/pH1N1). At 7 weeks of age, the pigs were challenged with pH1N1 virus or sham-inoculated. Necropsy was conducted 5 days post pH1N1 challenge (dpc). At the time of challenge one of the Inact/pH1N1 pigs had seroconverted based on IAV nucleoprotein-based ELISA, Inact/pH1N1 pigs had significantly higher pdm09H1N1 hemagglutination inhibition (HI) titers compared to all other groups, and M2e-specific IgG responses were detected in the NvParticle/M2e and the NvComplex/M2e pigs with significantly higher group means in the NvComplex/M2e group compared to SHAMVAC-NEG pigs. After challenge, nasal IAV RNA shedding was significantly reduced in Inact/pH1N1 pigs compared to all other pH1N1 infected groups and this group also had reduced IAV RNA in oral fluids. The macroscopic lung lesions were characterized by mild-to-severe, multifocal-to-diffuse, cranioventral dark purple consolidated areas typical of IAV infection and were similar for NvParticle/M2e, NvComplex/M2e and SHAMVAC-IAV pigs. Lesions were significantly less severe in the SHAMVAC-NEG and the Inact/pH1N1pigs. Under the conditions of this study, a commercial Inact/pH1N1 specific vaccine effectively protected pigs against homologous challenge as evidenced by reduced clinical signs, virus shedding in nasal secretions and oral fluids and reduced macroscopic and microscopic lesions whereas intranasal vaccination with experimental M2e epitope-based subunit vaccines did not. The results further highlight the importance using IAV-S type specific vaccines in pigs.

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.