Physical activity in out of school hours care: an observational study.

BACKGROUND: Opportunities for physical activity within out of school hours care (OSHC) are not well documented in Australia. This study explored factors associated with children (5-12 years) meeting 30 min of moderate to vigorous physical activity (MVPA) while attending OSHC in the afternoon period. METHODS: A cross-sectional study, conducted in 89 OSHC services in New South Wales, Australia, serving 4,408 children. Each service was visited twice between 2018-2019. Physical activity promotion practices were captured via short interviews and System for Observing Staff Promotion of Physical Activity and Nutrition (SOSPAN). Physical activity spaces was measured (m2) and physical activity of 3,614 child days (42% girls), were collected using Acti-Graph accelerometers. Association between program practices and children accumulation of MVPA was tested using mixed effects logistic regression, adjusted by OSHC service and child. RESULTS: Twenty-six percent of children (n = 925) accumulated 30 min or more of MVPA. Factors associated with children reaching MVPA recommendations included: services scheduling greater amounts of child-led free play, both 30-59 min (OR 2.6, 95%CI 1.70, 3.98) and ≥ 60 min (OR 6.4, 95%CI 3.90, 10.49); opportunities for staff-led organised play of ≥ 30 min (OR 2.3, 95%CI 1.47, 3.83); and active games that engaged the majority of children (OR 1.7, 95%CI 1.11, 2.61). Children were less likely to meet MVPA recommendations if services played games with elimination components (OR 0.56, 95%CI 0.37, 0.86). CONCLUSION: Improvements to service-level physical activity promotion practices, specifically the type of physical activity scheduled and the structure of games, may be an effective strategy to increase MVPA of children attending OSHC afterschool in NSW, Australia.

A Multicomponent mHealth-Based Intervention (SWAP IT) to Decrease the Consumption of Discretionary Foods Packed in School Lunchboxes: Type I Effectiveness-Implementation Hybrid Cluster Randomized Controlled Trial.

BACKGROUND: There is significant opportunity to improve the nutritional quality of foods packed in children's school lunchboxes. Interventions that are effective and scalable targeting the school and home environment are therefore warranted. OBJECTIVE: This study aimed to assess the effectiveness of a multicomponent, mobile health-based intervention, SWAP IT, in reducing the energy contribution of discretionary (ie, less healthy) foods and drinks packed for children to consume at school. METHODS: A type I effectiveness-implementation hybrid cluster randomized controlled trial was conducted in 32 primary schools located across 3 local health districts in New South Wales, Australia, to compare the effects of a 6-month intervention targeting foods packed in children's lunchboxes with those of a usual care control. Primary schools were eligible if they were not participating in other nutrition studies and used the required school communication app. The Behaviour Change Wheel was used to co-design the multicomponent SWAP IT intervention, which consisted of the following: school lunchbox nutrition guidelines, curriculum lessons, information pushed to parents digitally via an existing school communication app, and additional parent resources to address common barriers to packing healthy lunchboxes. The primary outcome, mean energy (kilojoules) content of discretionary lunchbox foods and drinks packed in lunchboxes, was measured via observation using a validated school food checklist at baseline (May 2019) and at 6-month follow-up (October 2019). Additional secondary outcomes included mean lunchbox energy from discretionary foods consumed, mean total lunchbox energy packed and consumed, mean energy content of core lunchbox foods packed and consumed, and percentage of lunchbox energy from discretionary and core foods, all of which were also measured via observation using a validated school food checklist. Measures of school engagement, consumption of discretionary foods outside of school hours, and lunchbox cost were also collected at baseline and at 6-month follow-up. Data were analyzed via hierarchical linear regression models, with controlling for clustering, socioeconomic status, and remoteness. RESULTS: A total of 3022 (3022/7212, 41.90%) students consented to participate in the evaluation (mean age 7.8 years; 1487/3022, 49.22% girls). There were significant reductions between the intervention and control groups in the primary trial outcome, mean energy (kilojoules) content of discretionary foods packed in lunchboxes (-117.26 kJ; 95% CI -195.59 to -39.83; P=.003). Relative to the control, the intervention also significantly reduced secondary outcomes regarding the mean total lunchbox energy (kilojoules) packed (-88.38 kJ; 95% CI -172.84 to -3.92; P=.04) and consumed (-117.17 kJ; 95% CI -233.72 to -0.62; P=.05). There was no significant difference between groups in measures of student engagement, consumption of discretionary foods outside of school hours, or cost of foods packed in children's lunchboxes. CONCLUSIONS: The SWAP IT intervention was effective in reducing the energy content of foods packed for and consumed by primary school-aged children at school. Dissemination of the SWAP IT program at a population level has the potential to influence a significant proportion of primary school-aged children, impacting weight status and associated health care costs. TRIAL REGISTRATION: Australian Clinical Trials Registry ACTRN12618001731280; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376191&isReview=true. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s12889-019-7725-x.

Protocol for an effectiveness- implementation hybrid trial to assess the effectiveness and cost-effectiveness of an m-health intervention to decrease the consumption of discretionary foods packed in school lunchboxes: the 'SWAP IT' trial.

BACKGROUND: At a population level, small reductions in energy intake have the potential to contribute to a reduction in the prevalence of childhood obesity. In many school systems, there is the potential to achieve a reduction in energy intake through modest improvements in foods packed in children's school lunchboxes. This study will assess the effectiveness and cost-effectiveness of a multi-component intervention that uses an existing school-based communication application to reduce the kilojoule content from discretionary foods and drinks consumed by children from school lunchboxes whilst at school. METHODS: A Type I hybrid effectiveness-implementation cluster randomised controlled trial will be conducted in up to 36 primary schools in the Hunter New England, Central Coast and Mid North Coast regions of New South Wales, Australia. Designed using the Behaviour Change Wheel, schools will be randomly allocated to receive either a 5-month (1.5 school terms) multi-component intervention that includes: 1) school lunchbox nutrition guidelines; 2) curriculum lessons; 3) information pushed to parents via an existing school-based communication application and 4) additional parent resources to address common barriers to packing healthy lunchboxes or a control arm (standard school practices). The study will assess both child level dietary outcomes and school-level implementation outcomes. The primary trial outcome, mean energy (kJ) content of discretionary lunchbox foods packed in children's lunchboxes, will be assessed at baseline and immediately post intervention (5 months or 1.5 school terms). Analyses will be performed using intention to treat principles, assessing differences between groups via hierarchical linear regression models. DISCUSSION: This study will be the first fully powered randomised controlled trial internationally to examine the impact of an m-health intervention to reduce the mean energy from discretionary food and drinks packed in the school lunchbox. The intervention has been designed with scalability in mind and will address an important evidence gap which, if shown to be effective, has the potential to be applied at a population level. TRIAL REGISTRATION: Australian Clinical Trials Registry ACTRN:12618001731280 registered on 17/10/2018. Protocol Version 1.

In Vitro Metabolite Formation in Human Hepatocytes and Cardiomyocytes and Metabolism and Tissue Distribution in Monkeys of the 2'-C-Methylguanosine Prodrug BMS-986094 : Potential Role in Clinical Cardiovascular Toxicity.

BMS-986094, a 2'-C-methylguanosine prodrug for the treatment of chronic hepatitis C virus infection, was withdrawn from phase 2 clinical trials because of unexpected cardiac and renal toxicities. To better understand these toxicities, the in vitro metabolism of BMS-986094 in human hepatocytes (HHs) and human cardiomyocytes (HCMs) and the measurement of BMS-986094 and selected metabolites in monkey plasma and tissues were assessed. BMS-986094 was extensively metabolized by HHs and HCMs, resulting in more efficient formation and accumulation of the active triphosphorylated metabolite, INX-09114, and less efficient efflux of metabolites in HCMs. The predominant metabolism pathway (hydrolysis) in HHs and HCMs was not associated with the formation of reactive metabolites or oxidative stress. In cynomolgus monkeys dosed with BMS-986094 of 15 or 30 mg/kg/d for 3 weeks, the nucleoside metabolite M2 was the major plasma analyte (66%-68% of the combined area under the curve). INX-09114 was the highest drug-related species in the heart and kidney (2,610-4,280 ng/mL [males]; ∼2-420× the concentration of other analytes). Other analytes increased dose dependently, with BMS-986094 highest in diaphragm (≤4,400 ng/mL) followed by M2 in liver and kidney (≤1,360 ng/mL), and M7 and M8 in other tissues (≤124 ng/mL). Three weeks after the last dose, INX-09114 remained high in the heart and kidney (≤1,870 ng/mL), with low M2 (≤37 ng/mL) in plasma and tissues. Persistent high concentrations of INX-09114 in the heart and kidney appeared to correlate with toxicities in these tissues in monkeys.

D-α-tocopheryl polyethylene glycol 1000 succinate-containing vehicles provide no detectable chemoprotection from oxidative damage.

The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H(2)O(2), with or without TS (0.5, 5, 50 or 500 µm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals.

Genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type I interferon response.

Viruses must be able to resist host innate responses, especially the type I interferon (IFN) response. They do so by preventing the induction or activity of IFN and/or by resisting the antiviral effectors that it induces. Poxviruses are no exception, with many mechanisms identified whereby mammalian poxviruses, notably, vaccinia virus (VACV), but also cowpox and myxoma viruses, are able to evade host IFN responses. Similar mechanisms have not been described for avian poxviruses (avipoxviruses). Restricted for permissive replication to avian hosts, they have received less attention; moreover, the avian host responses are less well characterized. We show that the prototypic avipoxvirus, fowlpox virus (FWPV), is highly resistant to the antiviral effects of avian IFN. A gain-of-function genetic screen identified fpv014 to contribute to increased resistance to exogenous recombinant chicken alpha IFN (ChIFN1). fpv014 is a member of the large family of poxvirus (especially avipoxvirus) genes that encode proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. By binding the Skp1/cullin-1 complex, the F box in such proteins appears to target ligands bound by the ANKs for ubiquitination. Mass spectrometry and immunoblotting demonstrated that tandem affinity-purified, tagged fpv014 was complexed with chicken cullin-1 and Skp1. Prior infection with an fpv014-knockout mutant of FWPV still blocked transfected poly(I·C)-mediated induction of the beta IFN (ChIFN2) promoter as effectively as parental FWPV, but the mutant was more sensitive to exogenous ChIFN1. Therefore, unlike the related protein fpv012, fpv014 does not contribute to the FWPV block to induction of ChIFN2 but does confer resistance to an established antiviral state.

Genetic screen of a mutant poxvirus library identifies an ankyrin repeat protein involved in blocking induction of avian type I interferon.

Mammalian poxviruses, including vaccinia virus (VACV), have evolved multiple mechanisms to evade the host type I interferon (IFN) responses at different levels, with viral proteins targeting IFN induction, signaling, and antiviral effector functions. Avian poxviruses (avipoxviruses), which have been developed as recombinant vaccine vectors for permissive (i.e., poultry) and nonpermissive (i.e., mammals, including humans) species, encode no obvious equivalents of any of these proteins. We show that fowlpox virus (FWPV) fails to induce chicken beta IFN (ChIFN2) and is able to block its induction by transfected poly(I·C), an analog of cytoplasmic double-stranded RNA (dsRNA). A broad-scale loss-of-function genetic screen was used to find FWPV-encoded modulators of poly(I·C)-mediated ChIFN2 induction. It identified fpv012, a member of a family of poxvirus genes highly expanded in the avipoxviruses (31 in FWPV; 51 in canarypox virus [CNPV], representing 15% of the total gene complement), encoding proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. Under ectopic expression, the first ANK of fpv012 is dispensable for inhibitory activity and the CNPV ortholog is also able to inhibit induction of ChIFN2. FWPV defective in fpv012 replicates well in culture and barely induces ChIFN2 during infection, suggesting that other factors are involved in blocking IFN induction and resisting the antiviral effectors. Nevertheless, unlike parental and revertant viruses, the mutants induce moderate levels of expression of interferon-stimulated genes (ISGs), suggesting either that there is sufficient ChIFN2 expression to partially induce the ISGs or the involvement of alternative, IFN-independent pathways that are also normally blocked by fpv012.

Improving implementation of school-based healthy eating and physical activity policies, practices, and programs: a systematic review.

Although best practice recommendations exist regarding school-based healthy eating and physical activity policies, practices, and programs, research indicates that implementation is poor. As the field of implementation science is rapidly evolving, an update of the recent review of strategies to improve the implementation of healthy eating and physical activity interventions in schools published in the Cochrane Library in 2017 was required. The primary aim of this review was to examine the effectiveness of strategies that aim to improve the implementation of school-based policies, practices, or programs to address child diet, physical activity, or obesity. A systematic review of articles published between August 31, 2016 and April 10, 2019 utilizing Cochrane methodology was conducted. In addition to the 22 studies included in the original review, eight further studies were identified as eligible. The 30 studies sought to improve the implementation of healthy eating (n = 16), physical activity (n = 11), or both healthy eating and physical activity (n = 3). The narrative synthesis indicated that effect sizes of strategies to improve implementation were highly variable across studies. For example, among 10 studies reporting the proportion of schools implementing a targeted policy, practice, or program versus a minimal or usual practice control, the median unadjusted effect size was 16.2%, ranging from -0.2% to 66.6%. Findings provide some evidence to support the effectiveness of strategies in enhancing the nutritional quality of foods served at schools, the implementation of canteen policies, and the time scheduled for physical education.

The eIF4A inhibitor silvestrol sensitizes T-47D ductal breast carcinoma cells to external-beam radiotherapy.

PURPOSE: eIF4A is an RNA helicase that forms part of the machinery of translation initiation.Proteomic analysis demonstrated eIF4A expression to be at least two-fold greater in a radioresistant derivative of T-47D breast cancer cells compared to parental cells.Inhibition of eIF4A has previously been shown to re-sensitize lymphomas to chemotherapeutic agents that cause DNA damage.The objective of this work is to investigate whether inhibition of eIF4A using silvestrol sensitizes breast cancer cells to radiotherapy in tissue culture, using T-47D as a model system. METHODS AND MATERIALS: T-47D cells were incubated in medium containing 0 nM to 1 nM silvestrol either for 24 h prior to irradiation at 0 Gy to 10 Gy, delivered by linear accelerator (LINAC) or continually for six days post irradiation. MTT viability and clonogenic assays were used to quantify response. RESULTS: Pre-treatment of T-47D cells with 1 nM silvestrol caused a 34% reduction (p = 0.014) in viability on irradiation at 2 Gy compared to treatment with a DMSO control, as assessed by MTT assay.Maintenance of cells in 1 nM silvestrol for six days following irradiation at 2 Gy caused a 58% reduction (p = <0.001) in tumor cell viability.Clonogenic assays performed on cells maintained in 1 nM silvestrol following irradiation showed a dose modifying factor (DMF) of 1.4 (p = <0.001, one-way ANOVA). CONCLUSIONS: Low concentrations of silvestrol sensitize T-47D breast cancer cells to radiation with minimal effects on unirradiated cells. This highlights the possible usefulness of eIF4A inhibition in potentiating radiation-induced damage at the tumor site without causing systemic toxicity.

Healthy eating and physical activity environments in out-of-school hours care: an observational study protocol.

INTRODUCTION: Childcare settings have been widely identified as important venues for promoting healthy lifestyles to children. Out-of-school hours care (OSHC) is a rapidly growing childcare service, yet there has been limited research reported on healthy eating and physical activity (HEPA) environments within the Australian OSHC setting. This research aims to describe the HEPA environments related to foods and beverages served, staff behaviours and child physical activity levels across two local health districts within New South Wales, Australia. This study will provide evidence to support future interventions and policies in Australian OSHC settings. METHODS AND ANALYSIS: A cross-sectional study design will be used to describe the food and beverages provided and child activity levels, and report on environmental correlates. OSHC programmes will be visited on non-consecutive weekdays between 2018 and 2020. The frequency of foods and beverages offered will be observed and categorised into food groups aligned to the Australian Dietary Guidelines. Children's physical activity will be measured using ActiGraph wGT3X-BT accelerometers. Staff behaviour will be captured via direct observation and the System for Observing Staff Promotion of Activity and Nutrition. Short interviews with programme directors will gather contextual information about OSHC practices and policies. ETHICS AND DISSEMINATION: Findings will be disseminated through peer-reviewed scientific journals, conference presentations and individualised feedback to each participating service. Ethical approval was granted by the University of Wollongong Human Research Ethics Committee (HE17/490).

From the Cover: Investigative Nonclinical Cardiovascular Safety and Toxicology Studies with BMS-986094, an NS5b RNA-Dependent RNA Polymerase Inhibitor.

BMS-986094, a 2'-C-methylguanosine prodrug that was in development for treatment of chronic hepatitis C infection was withdrawn from Phase 2 clinical trials because of unexpected cardiac and renal adverse events. Investigative nonclinical studies were conducted to extend the understanding of these findings using more comprehensive endpoints. BMS-986094 was given orally to female CD-1 mice (25 and 150 mg/kg/d) for 2 weeks (53/group) and to cynomolgus monkeys (15 and 30 mg/kg/d) for up to 6 weeks (2-3/sex/group for cardiovascular safety, and 5/sex/group for toxicology). Endpoints included toxicokinetics; echocardiography, telemetric hemodynamics and electrocardiography, and tissue injury biomarkers (monkey); and light and ultrastructural pathology of heart, kidney, and skeletal muscle (mouse/monkey). Dose-related and time-dependent findings included: severe toxicity in mice at 150 mg/kg/d and monkeys at 30 mg/kg/d; decreased left ventricular (LV) ejection fraction, fractional shortening, stroke volume, and dP/dt; LV dilatation, increased QTc interval, and T-wave flattening/inversion (monkeys at ≥ 15 mg/kg/d); cardiomyocyte degeneration (mice at 150 mg/kg/d and monkeys at ≥ 15 mg/kg/d) with myofilament lysis/myofbril disassembly; time-dependent proteinuria and increased urine ß-2 microglobulin, calbindin, clusterin; kidney pallor macroscopically; and tubular dilatation (monkeys); tubular regeneration (mice 150 mg/kg/d); and acute proximal tubule degeneration ultrastructurally (mice/monkeys); and skeletal muscle degeneration with increased urine myoglobin and serum sTnI. These studies identified changes not described previously in studies of BMS-986094 including premonitory cardiovascular functional changes as well as additional biomarkers for muscle and renal toxicities. Although the mechanism of potential toxicities observed in BMS-986094 studies was not established, there was no evidence for direct mitochondrial toxicity.

Effects of BMS-986094, a Guanosine Nucleotide Analogue, on Mitochondrial DNA Synthesis and Function.

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection.

A reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) has been described in which a full-length cDNA, corresponding to the IBV (Beaudette-CK) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cDNAs [Casais, R., Thiel, V., Siddell, S.G., Cavanagh, D., Britton, P., 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75, 12359-12369]. The method has subsequently been used to generate a recombinant IBV expressing a chimaeric S gene [Casais, R., Dove, B., Cavanagh, D., Britton, P., 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77, 9084-9089]. Use of vaccinia virus as a vector for the full-length cDNA of the IBV genome has the advantage that modifications can be made to the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. We describe the use of homologous recombination as a method for modifying the Beaudette full-length cDNA, within the vaccinia virus genome, without the requirement for in vitro assembly of the IBV cDNA. To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs.

Differential distribution of WC1(+) gammadelta TCR(+) T lymphocyte subsets within lymphoid tissues of the head and respiratory tract and effects of intranasal M. bovis BCG vaccination.

BCG vaccination of neonatal calves induces significant protection against bovine tuberculosis. The enhanced protection observed in neonatal calves may be linked to an enhanced capacity for IFNgamma production by innate cells, including WC1(+) gammadelta T cells, which constitute a major population in young cattle. Intranasal BCG vaccination of mice induces high levels of IFNgamma in the lungs, which may enhance protection against subsequent challenge with virulent strains of mycobacteria. We used an intranasal BCG vaccination model in calves to study the effect on the distribution of WC1(+) gammadelta T cells expressing two alternate forms of WC1: WC1.1 and WC1.2. These subsets of WC1(+) gammadelta T cells have previously been shown to have a differential capacity for IFNgamma secretion. Our results indicate that there is a selective expansion/recruitment of gammadelta T cells expressing the IFNgamma-associated WC1.1 isoform in tissues of the lungs and upper respiratory tract following intranasal BCG vaccination.

Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins.

Infectious bronchitis coronavirus (IBV) is the cause of the single most economically costly infectious disease of domestic fowl in the UK--and probably so in many countries that have a developed poultry industry. A major reason for its continued dominance is its existence as many serotypes, determined by the surface spike protein (S), cross-protection being poor. Although controlled to some degree by live and inactivated vaccines, a new generation of IB vaccines is called for. Reverse genetic or 'infectious clone' systems, which allow the manipulation of the IBV genome, are key to this development. New vaccines would ideally be: genetically stable (i.e. maintain a stable attenuated phenotype); administered in ovo; and be flexible with respect to the source of the spike protein gene. Rational attenuation of IBV requires the identification of genes that are simultaneously not essential for replication and whose absence would reduce pathogenicity. Being able to modify a 'core' vaccine strain to make it applicable to a prevailing serotype requires a procedure for doing so, and the demonstration that 'spike-swapping' is sufficient to induce good immunity. We have demonstrated that four small IBV proteins, encoded by genes 3 and 5, are not essential for replication; failure to produce these proteins had little detrimental affect on the titre of virus produced. Our current molecularly cloned IBV, strain Beaudette, is non-pathogenic, so we do not know what effect the absence of these proteins would have on pathogenicity. That said, plaque size and composition of various gene 3/5 recombinant IBVs in cell culture, and reduced output and ciliostasis in tracheal organ cultures, shows that they are less aggressive than the wild-type Beaudette. Consequently these genes remain targets for rational attenuation. We have recently obtained evidence that one or more of the 15 proteins encoded by gene 1 are also determinants of pathogenicity. Hence gene 1 is also a target for rational attenuation. Replacing the S protein gene of Beaudette with that from the pathogenic M41 strain resulted in a recombinant virus that was still non-pathogenic but which did induce protection against challenge with M41. We have since made other 'spike-swapped' recombinants, including ones with chimaera S genes. Uniquely, our molecular clone of Beaudette is benign when administered to 18-day-old embryos, even at high doses, and induces immunity after this route of vaccination. Taken together, our results point to the creation of a new generation of IB vaccines, based on rational modification of the genome, as being a realisable objective.

Gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication.

The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.

An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge.

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

Poliovirus type 1 in working stocks of typed human rhinoviruses.

After eradication of circulating polioviruses, reintroduction might arise from cultured laboratory stocks, or from collections of patients' or environmental samples. In an example of a further class of risk, we recorded poliovirus type 1 in at least four working stocks of what had been identified as various serotypes of human rhinovirus. This finding emphasises the need to urgently assess the possibility of presence of poliovirus, even in apparently well-characterised stocks of other viruses, to screen for the virus where necessary, and to destroy materials in which risk outweighs potential benefit.