Exploring the Potential of a Behavior Theory-Informed Digital Intervention for Infant Fall Prevention: Mixed Methods Longitudinal Study.

BACKGROUND: Falls are the most common hospitalized injury mechanism in children aged ≤1 years, and currently, there are no targeted prevention interventions. The prevention of falls in children of this age requires changes in the behavior of their caregivers, and theoretically informed digital behavior change interventions (DBCIs) may provide a unique mechanism for achieving effective intervention. However, user acceptance and the ability of DBCIs to effect the required changes in behavior are critical to their likelihood of success. OBJECTIVE: This study aims to evaluate a behavior theory-informed digital intervention developed following a user-centered approach for user experience, the potential for this intervention to prevent infant falls, and its impact on behavioral drivers underpinning fall risk in young children. METHODS: Parents of infants aged <1 year were recruited and asked to use the intervention for 3 months. A pre-post longitudinal design was used to examine the change in the potential to reduce the risk of falls after a 3-month exposure to the intervention. Postintervention data on behavioral drivers for fall prevention, user acceptability, and engagement with the app were also collected. Interviews were conducted to explore user experiences and identify areas for further improvement of the intervention. RESULTS: A total of 62 parents participated in the study. A statistically significant effect on the potential to reduce falls was observed after the intervention. This effect was higher for new parents. Parents agreed that the intervention targeted most of the target behavior drivers. The impact of behavior drivers and intervention on the potential for fall prevention had a positive correlation. The intervention demonstrated good levels of acceptability. Feedback from participants was mostly positive, and the primary area identified for further improvement was widening the scope of the intervention. CONCLUSIONS: This study demonstrated the promise of a newly developed digital intervention to reduce the risk of infant falls, particularly among new parents. It also showed a positive influence of the DBCI on the drivers of parental behaviors that are important for fall reduction among infants. The acceptability of the app was high, and important insights were gained from users about how to further improve the app.

Updated population-level estimates of child restraint practices among children aged 0-12 years in Australia, 10 years after introduction of age-appropriate restraint use legislation.

OBJECTIVE: Optimal child passenger protection requires use of a restraint designed for the age/size of the child (appropriate use) that is used in the way the manufacturer intended (correct use).This study aimed to determine child restraint practices approximately 10 years after introduction of legislation requiring correct use of age-appropriate restraints for all children aged up to 7 years. METHODS: A stratified cluster sample was constructed to collect observational data from children aged 0-12 years across the Greater Sydney region of New South Wales (NSW). Methods replicated those used in a similar 2008 study. Population weighted estimates for restraint practices were generated, and logistic regression used to examine associations between restraint type, and child age with correct use accounting for the complex sample. RESULTS: Almost all children were appropriately restrained (99.3%, 95% CI 98.4% to 100%). However, less than half were correctly restrained (no error=27.3%, 95% CI 10.8% to 43.8%, no serious error=43.8%, 95% CI 35.0% to 52.7%). For any error, the odds of error decreased by 39% per year of age (OR 0.61, 95% CI 0.46 to 0.81) and for serious error by 25% per year (OR 0.75, 95% CI 0.60 to 0.93). CONCLUSION: The findings demonstrate high levels of appropriate restraint use among children across metropolitan Sydney approximately 10 years after introduction of legislation requiring age-appropriate restraint use until age 7, however, errors in the way restraints remain common. IMPLICATIONS FOR PUBLIC HEALTH: Given the negative impact incorrect use has on crash protection, continuing high rates of incorrect use may reduce effectiveness of legislative change on injury reduction.

Retrieval fluency inflates perceived preparation for difficult problems.

When faced with a difficult problem, people often rely on past experiences. While remembering clearly helps us reach solutions, can retrieval also lead to misperceptions of our abilities? In three experiments, participants encountered "worst case scenarios" they likely had never experienced and that would be difficult to navigate without extensive training (e.g., bitten by snake). Learning brief tips improved problem-solving performance later, but retrieval increased feelings of preparation by an even larger margin. This gap occurred regardless of whether people thought that tips came from an expert or another participant in the study, and it did not reflect mere familiarity with the problems themselves. Instead, our results suggest that the ease experienced while remembering, or retrieval fluency, inflated feelings of preparation.

Differences in participant characteristics and observed child restraint use between population-based and restraint fitting service samples.

OBJECTIVE: To compare characteristics and restraint use between a population-based and fitting service sample of child restraint users. METHOD: Characteristics of the two samples were compared using chi-squared tests. Differences in errors in restraint use observed in the two samples were modeled using logistic regression. RESULTS: There were significant differences in child age (p < 0.001), and restraint types (p < 0.001) between the two samples, with more younger children in the fitting service sample. Controlling for differences in restraint type, the odds that adult participants were female were 61% less in the fitting service sample than in the population-based sample (OR 0.39, 95%CI 0.21-0.71). The odds that adult participants perceived a large risk associated with restraint misuse (OR 3.62, 95%CI 1.33-9.84), had a household income in the highest bracket (OR 3.89, 95%CI 1.20-12.62) and were living in areas of highest socioeconomic advantage (OR 2.72, 95%CI 1.22-6.06) were approximately three times higher in the fitting service sample. Overall, more participants had errors in restraint use in the population-based sample (p = 0.021). However, after controlling for restraint type, securing errors were three times more likely (OR 3.34, 95%CI 1.12-10.2), and serious installation errors were almost twice as likely (OR 1.91, 95% CI 1.09-3.39) in the fitting service sample. CONCLUSIONS: While less resource intensive, convenience and/or fitting service samples may be less representative than population-based samples. Given the need for efficiency, methods that combine randomized population-based invitations to participate in restraint fitting check day events across geographically representative areas may be useful for ongoing surveillance of child restraint use.

Toward a Behavior Theory-Informed and User-Centered Mobile App for Parents to Prevent Infant Falls: Development and Usability Study.

BACKGROUND: Falls account for approximately 50% of infant injury hospitalizations, and caretaker behavior is central to preventing infant falls. Behavior theory-informed interventions for injury prevention have been suggested, but to date, few have been reported. The potential of using smartphones for injury prevention intervention delivery is also underexploited. OBJECTIVE: This study aims to develop a behavior theory- and evidence-based as well as user-centered digital intervention as a mobile app for parents to prevent infant falls following agile development practices. METHODS: Infant falls while feeding was selected as the fall mechanism to demonstrate the approach being taken to develop this intervention. In phase 1, the Behaviour Change Wheel was used as a theoretical framework supported by a literature review to define intervention components that were then implemented as a mobile app. In phase 2, after the person-based approach, user testing through think-aloud interviews and comprehension assessments were used to refine the content and implementation of the intervention. RESULTS: The target behaviors identified in phase 1 were adequate rest for the newborn's mother and safe feeding practices defined as prepare, position, and place. From behavioral determinants and the Behaviour Change Wheel, the behavior change functions selected to achieve these target behaviors were psychological capability, social opportunity, and reflective motivation. The selected behavior change techniques aligned with these functions were providing information on health consequences, using a credible source, instruction on performing each behavior, and social support. The defined intervention was implemented in a draft Android app. In phase 2, 4 rounds of user testing were required to achieve the predefined target comprehension level. The results from the think-aloud interviews were used to refine the intervention content and app features. Overall, the results from phase 2 revealed that users found the information provided to be helpful. Features such as self-tracking and inclusion of the social and environmental aspects of falls prevention were liked by the participants. Important feedback for the successful implementation of the digital intervention was also obtained from the user testing. CONCLUSIONS: To our knowledge, this is the first study to apply the Behaviour Change Wheel to develop a digital intervention for child injury prevention. This study provides a detailed example of evidence-based development of a behavior theory-informed mobile intervention for injury prevention refined using the person-based approach.

Identification and characterization of bisbenzimide compounds that inhibit human cytomegalovirus replication.

The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study. We generated analogues of RO-90-7501 and found that one compound, MRT00210423, had increased anti-HCMV activity compared to RO-90-7501. Using a combination of compound analogues, microscopy and biochemical assays we found RO-90-7501 and MRT00210423 interacted with DNA. In single molecule microscopy experiments we found RO-90-7501, but not MRT00210423, was able to compact DNA, suggesting that compaction of DNA was non-obligatory for anti-HCMV effects. Using bioinformatics analysis, we found that there were many putative bisbenzimide binding sites in the HCMV DNA genome. However, using western blotting, quantitative PCR and electron microscopy, we found that at a concentration able to inhibit HCMV replication our compounds had little or no effect on production of certain HCMV proteins or DNA synthesis, but did have a notable inhibitory effect on HCMV capsid production. We reasoned that these effects may have involved binding of our compounds to the HCMV genome and/or host cell chromatin. Therefore, our data expand our understanding of compounds with anti-HCMV activity and suggest targeting of DNA with bisbenzimide compounds may be a useful anti-HCMV strategy.

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques.

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

Prophylactic intranasal administration of a TLR2/6 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model.

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.

Maintained Hand Perfusion in the Setting of Radial and Ulnar Artery Transections: A Report of 2 Cases.

CASE: Two otherwise healthy male patients presented with lacerations to the volar distal forearm. Both patients had capillary refill at the fingertips and intact Doppler signals of the palmar arch. Computed tomography angiography revealed cessation of flow in the major forearm arteries at the level of the lacerations, with distal reconstitution from the anterior interosseous artery in both cases and from a branch off the ulnar artery in 1 case. The patients underwent operative exploration, where complete transections of the radial and ulnar arteries were found and repaired. CONCLUSIONS: In patients without arterial disease, contributions from minor forearm arteries can be sufficient for hand perfusion in the setting of radial and ulnar artery transection.

Dysregulation of Hypothalamic Gene Expression and the Oxytocinergic System by Soybean Oil Diets in Male Mice.

Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil (high in linoleic acid, LA), a genetically modified, low-LA soybean oil (Plenish), and coconut oil (high in saturated fat, containing no LA). The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling. Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications.

User-driven design of child restraint information to reduce errors in use: a pilot randomised controlled trial.

BACKGROUND: Incorrect use of child restraints is a long-standing problem that increases the risk of injury in crashes. We used user-centred design to develop prototype child restraint instructional materials. The objective of this study was to evaluate these materials in terms of comprehension and errors in the use of child restraints. The relationship between comprehension and errors in use was also explored. METHODS: We used a parallel-group randomised controlled trial in a laboratory setting. The intervention group (n=22) were provided with prototype materials and the control group (n=22) with existing instructional materials for the same restraint. Participants installed the restraint in a vehicle buck, secured an appropriately sized mannequin in the restraint and underwent a comprehension test. Our primary outcome was overall correct use, and our secondary outcomes were (1) comprehension score and (2) percent errors in the installation trial. RESULTS: There was 27% more overall correct use (p=0.042) and a higher mean comprehension score in the intervention group (mean 17, 95% CI 16 to 18) compared with the control group (mean 12, 95% CI 10 to 14, p<0.001). The mean error percentage in the control group was 23% (95% CI 16% to 31%) compared with 14% in the intervention group (95% CI 8% to 20%, p=0.056). For every one point increase in comprehension, there was an almost 2% (95% CI -2.7% to -1.0%) reduction in errors (y=45.5-1.87x, p value for slope <0.001). CONCLUSIONS: Consumer-driven design of informational materials can increase the correct use of child restraints. Targeting improved comprehension of informational materials may be an effective mechanism for reducing child restraint misuse.

TGF-ß Superfamily Regulation of Follicle-Stimulating Hormone Synthesis by Gonadotrope Cells: Is There a Role for Bone Morphogenetic Proteins?

Bone morphogenetic proteins (BMPs) are pleiotropic ligands in the TGF-ß superfamily. In the early to mid-2000s, several BMPs, including BMP2, were shown to regulate FSH synthesis alone and in synergy with activins in immortalized gonadotrope-like cell lines and primary pituitary cultures. Activins are also TGF-ß family members, which were identified and named based on their abilities to stimulate FSH production selectively. Mechanistic analyses suggested that BMP2 promoted expression of the FSHß subunit gene (Fshb) via at least two nonmutually exclusive mechanisms. First, BMP2 stimulated the production of the inhibitor of DNA-binding proteins 1, 2, and 3 (Id1, Id2, and Id3), which potentiated the stimulatory actions of homolog of Drosophila mothers against decapentaplegic 3 (SMAD3) on the Fshb promoter. SMAD3 is an intracellular signaling protein that canonically mediates the actions of activins and is an essential regulator of Fshb production in vitro and in vivo. Second, BMP2 was shown to activate SMAD3-dependent signaling via its canonical type IA receptor, BMPR1A (also known as ALK3). This was a surprising result, as ALK3 conventionally activates distinct SMAD proteins. Although these initial results were compelling, they were challenged by contemporaneous and subsequent observations. For example, inhibitors of BMP signaling did not specifically impair FSH production in cultured pituitary cells. Of perhaps greater significance, mice lacking ALK3 in gonadotrope cells produced FSH normally. Therefore, the physiological role of BMPs in FSH synthesis in vivo is presently uncertain.

Identification of lead anti-human cytomegalovirus compounds targeting MAP4K4 via machine learning analysis of kinase inhibitor screening data.

Chemogenomic approaches involving highly annotated compound sets and cell based high throughput screening are emerging as a means to identify novel drug targets. We have previously screened a collection of highly characterized kinase inhibitors (Khan et al., Journal of General Virology, 2016) to identify compounds that increase or decrease expression of a human cytomegalovirus (HCMV) protein in infected cells. To identify potential novel anti-HCMV drug targets we used a machine learning approach to relate our phenotypic data from the aforementioned screen to kinase inhibition profiling of compounds used in this screen. Several of the potential targets had no previously reported role in HCMV replication. We focused on one potential anti-HCMV target, MAPK4K, and identified lead compounds inhibiting MAP4K4 that have anti-HCMV activity with little cellular cytotoxicity. We found that treatment of HCMV infected cells with inhibitors of MAP4K4, or an siRNA that inhibited MAP4K4 production, reduced HCMV replication and impaired detection of IE2-60, a viral protein necessary for efficient HCMV replication. Our findings demonstrate the potential of this machine learning approach to identify novel anti-viral drug targets, which can inform the discovery of novel anti-viral lead compounds.

Burkholderia multivorans: A rare yet emerging cause of bacterial meningitis.

Burkholderia multivorans is a member of the Burkholderia cepacia complex. Although it is usually associated with infections in patients with cystic fibrosis, chronic granulomatous disease, and immunosuppression, central nervous infections are not commonly reported. Moreover, management of these infections is difficult due to multiple mechanisms of bacterial resistance to antimicrobial agents. We report a 55-year-old-man who developed Burkholderia multivorans meningitis after two episodes of central line-associated bloodstream infections. The patient was successfully treated with intravenous trimethoprim/sulfamethoxazole. Burkholderia multivorans is an emerging cause of meningitis with limited antibacterial treatment options. However, trimethoprim/sulfamethoxazole remains an effective agent with excellent penetration into the central nervous system. To our knowledge, this is the first case reported of Burkholderia cepacia complex meningitis identified to the species level as Burkholderia multivorans.

Short report: Follow-up of Bahamian women with a BRCA1 or BRCA2 mutation.

PURPOSE: We sought to determine to what extent the knowledge of carrying a BRCA1 or BRCA2 mutation influences the uptake of preventive surgeries in Bahamian women, including bilateral salpingo-oophorectomy and bilateral mastectomy. PATIENTS AND METHODS: The study population consisted of 78 female residents of the Bahamas for whom a BRCA1 or BRCA2 mutation had been detected between 2004 and 2014. The mean age of the 78 participants at the time of genetic testing was 46 years (age range 22-73 years). The mean time of follow-up was 4.4 years. RESULTS: Of the 78 study participants, 19 women had a bilateral salpingo-oophorectomy (24%). Seven out of 37 patients who had unilateral breast cancer chose to remove the unaffected contralateral breast (19%). Three of 13 patients with no history of breast cancer chose to have a prophylactic bilateral mastectomy (23%). CONCLUSION: Preventive surgery is an acceptable option for a significant proportion of Bahamian women with a BRCA1 or BRCA2 mutation. It will be important to identify and reduce barriers to preventive surgery in the Bahamas in order that the benefit of getting testing can be fully realized.

High-throughput screening of a GlaxoSmithKline protein kinase inhibitor set identifies an inhibitor of human cytomegalovirus replication that prevents CREB and histone H3 post-translational modification.

To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets. We focused our study on a top 'hit' in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibited polypharmacology and was an inhibitor of several proteins from the AGC and CMCG kinase groups. Using Western blotting, we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (cAMP response element-binding protein and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identified SB-734117 as a novel anti-HCMV compound and found that inhibition of AGC and CMCG kinase proteins during productive HCMV replication was associated with inhibition of viral protein production and prevented post-translational modification of cellular factors associated with viral protein production.

Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins.

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production.