Examining the potential for porcine-derived islet cells to harbour viral pathogens

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal-derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture

COVID-19: The environmental implications of shedding SARS-CoV-2 in human faeces

First paragraph: The ongoing COVID-19 pandemic is having significant public health repercussions, with a global response to limit the predicted mortality associated with this outbreak. The virus, ‘severe acute respiratory syndrome coronavirus 2’ (SARS-CoV-2), is a respiratory virus disseminated though droplets from coughs and sneezes from an infected person or from fomites (Hellewell et al., 2020). Therefore, many countries have put ‘social distancing’ measures in place to reduce person-to-person spread of the disease. However, recently it has been confirmed that infectious virions can also be present in human faeces (Ling et al., 2020), and there are reports that viral RNA can be persistently shed in faeces for a maximum of 33 days after the patient has tested negative for respiratory viral RNA (Wu et al 2020). Although it remains unclear whether SARS-CoV-2 can be transmitted via the faecal-oral route (Xu et al., 2020), viral shedding from the digestive system can last longer than shedding from the respiratory tract. As such, faecal-oral transmission may be an important, but as yet unquantified, pathway for increased exposure during the current outbreak (Wu et al., 2020). Therefore, safely managing faecal wastes from infected, recovering and recovered patients poses a significant nosocomial challenge. For example, during the SARS outbreak of 2002–2003, the closely related SARS-CoV-1 was detected in sewage discharged by two hospitals (Wang et al., 2005), which emphasises the care needed when handling such faecal wastes. However, these challenges are not limited to hospital wastes, as it has been predicted that most of the population will experience only mild symptoms of COVID 19 and convalesce at home, whilst others, including children, can carry the virus asymptomatically, and are still capable of shedding the virus in their faeces (Kam et al., 2020, Tang et al., 2020). This means that the virus could soon become widespread throughout wastewater systems (Naddeo and Liu, 2020). Whilst a lack of testing for the majority of the population makes it difficult to predict the spatially-distributed volume of potentially infectious faeces delivered through the sewerage infrastructure to wastewater treatment works (WWTWs), wastewater surveillance may be a useful tool to indicate where the virus is circulating in the human population (Lodder and de Roda Husman, 2020). However, whilst knowingly infected individuals can take steps to increase their level of hygiene, asymptomatic carriers do not change their behaviour, and can anonymously spread enteric pathogens within the community (Quilliam et al., 2013)

Characterization of <i>Aedes aegypti</i> innate-immune pathways that limit Chikungunya virus replication

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways

The Development of Videoconference-Based Support for People Living With Rare Dementias and Their Carers:Protocol for a 3-Phase Support Group Evaluation

BACKGROUND: People living with rarer dementias face considerable difficulty accessing tailored information, advice, and peer and professional support. Web-based meeting platforms offer a critical opportunity to connect with others through shared lived experiences, even if they are geographically dispersed, particularly during the COVID-19 pandemic. OBJECTIVE: We aim to develop facilitated videoconferencing support groups (VSGs) tailored to people living with or caring for someone with familial or sporadic frontotemporal dementia or young-onset Alzheimer disease, primary progressive aphasia, posterior cortical atrophy, or Lewy body dementia. This paper describes the development, coproduction, field testing, and evaluation plan for these groups. METHODS: We describe a 3-phase approach to development. First, information and knowledge were gathered as part of a coproduction process with members of the Rare Dementia Support service. This information, together with literature searches and consultation with experts by experience, clinicians, and academics, shaped the design of the VSGs and session themes. Second, field testing involved 154 Rare Dementia Support members (people living with dementia and carers) participating in 2 rounds of facilitated sessions across 7 themes (health and social care professionals, advance care planning, independence and identity, grief and loss, empowering your identity, couples, and hope and dementia). Third, a detailed evaluation plan for future rounds of VSGs was developed. RESULTS: The development of the small groups program yielded content and structure for 9 themed VSGs (the 7 piloted themes plus a later stages program and creativity club for implementation in rounds 3 and beyond) to be delivered over 4 to 8 sessions. The evaluation plan incorporated a range of quantitative (attendance, demographics, and geography; pre-post well-being ratings and surveys; psycholinguistic analysis of conversation; facial emotion recognition; facilitator ratings; and economic analysis of program delivery) and qualitative (content and thematic analysis) approaches. Pilot data from round 2 groups on the pre-post 3-word surveys indicated an increase in the emotional valence of words selected after the sessions. CONCLUSIONS: The involvement of people with lived experience of a rare dementia was critical to the design, development, and delivery of the small virtual support group program, and evaluation of this program will yield convergent data about the impact of tailored support delivered to geographically dispersed communities. This is the first study to design and plan an evaluation of VSGs specifically for people affected by rare dementias, including both people living with a rare dementia and their carers, and the outcome of the evaluation will be hugely beneficial in shaping specific and targeted support, which is often lacking in this population. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/3537

Effects of pathway stimulation on CHIKVRep(3F)RLuc-SG-FFLuc replication.

<p>Aag2 cells were pre-stimulated with heat inactivated bacteria, followed by transfection of CHIKVRep(3F)RLuc-SG-FFLuc replicon RNA. Replicon replication was assessed 12 h post pathway stimulation by FFLuc activity. PBS, PBS added to cells containing CHIKVRep(3F)RLuc-SG-FFLuc replicon; <i>E. coli</i>, heat inactivated bacteria added to cells containing CHIKVRep(3F)RLuc-SG-FFLuc replicon; <i>B. subtilis</i>, heat inactivated bacteria added to cells containing CHIKVRep(3F)RLuc-SG-FFLuc replicon. The dark grey bar shows CHIKVRep(3F)RLuc-SG-FFLuc replication after JAK/STAT and IMD pathway stimulation with heat inactivated <i>E. coli</i>. Light grey bar shows CHIKVRep(3F)RLuc-SG-FFLuc replication after Toll pathway stimulation with heat inactivated <i>B. subtilis</i>. Replicon replication after bacterial stimulation is calculated relative to replicon replication after stimulation with PBS (black bar). The graph represents the mean of three independent experiments performed in triplicate and error bars represent the standard error of mean.</p

Table of all primer sequences.

<p>All primer sequences are shown reading from 5′ to 3′. Bases in bold indicate the T7 promoter sequence. The use of each primer pair is also indicated.</p

Infectious viral particles in midguts and heads of <i>Ae. aegypti</i> mosquitoes injected with Ago-2-specific dsRNA and infected with CHIKV.

<p>Two days after injection of 500-meal at a titer of 10⁷ PFU/ml. The expression of Ago-2 in midguts (A) and heads (C) was calculated relative to the expression of internal control S7 gene at each day post infection (pi) by real-time quantitative PCR. At indicated days pi, PFUs as measure of virus replication were determined by plaque assay of (B) midguts and (D) heads. 10 individuals were analyzed at each day pi. Controls were FFluc-specific dsRNA-injected, PBS-injected and non-injected (NINJ) mosquitoes. Bars indicate standard deviations. Significance (*) was determined by using Kruskall-Wallis test (p<0.05).</p

Replication of CHIKVRep(3F)RLuc-SG-FFLuc in Aag2 cells.

<p>(<b>A</b>) Schematic representation of CHIKV replicons. RLuc was inserted into the C-terminal region of the nsP3 protein and sequence encoding for FFLuc was cloned in place of the structural open reading frame under the control of the subgenomic promoter to give CHIKVRep(3F)RLuc-SG-FFLuc. A second replicon with no reporter in the non-structural open reading frame and coding sequence of eGFP cloned under control of the subgenomic promoter was created, CHIKVRep-SG-eGFP. (<b>B</b>) Aag2 cells were transfected with <i>in vitro</i> transcribed CHIKVRep(3F)RLuc-SG-FFLuc RNA and RLuc activity measured 24, 48 or 72 h post transfection (hpt). Graph shows the mean RLuc readings from a representative single experiment carried out in triplicate. Error bars show the standard error of mean. Three independent experiments have been carried out in triplicate.</p

CHIKV inhibition of JAK/STAT, IMD and Toll signaling pathways.

<p>Signaling assays were performed as previously described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002994#pntd.0002994-Fragkoudis2" target="_blank">[23]</a>. Aag2 cells were co-transfected with reporter plasmids and <i>in vitro</i> transcribed CHIKVRep-SG-eGFP RNA, followed by stimulation of immune pathways by heat inactivated bacteria. Signaling activity was measured by activity of FFluc. Internal transfection control (RLuc) is shown to indicate the levels of gene expression. PBS, PBS added to cells containing only the pathway reporter and internal transfection control; <i>E. coli</i>, heat inactivated bacteria added to cells containing only the pathway reporter and internal transfection control; <i>B. subtilis</i>, heat inactivated bacteria added to cells containing only the pathway reporter and internal transfection control; CHIKV, PBS added to cells containing pathway reporter, internal transfection control and CHIKVRep-SG-eGFP replicon RNA; CHIKV+<i>E. coli</i>, heat inactivated <i>E. coli</i> added to cells containing pathway reporter, internal transfection control and CHIKVRep-SG-eGFP replicon; CHIKV+<i>B. subtilis</i>, heat inactivated <i>B. subtilis</i> added to cells containing pathway reporter, internal transfection control and CHIKVRep-SG-eGFP replicon. Data is shown after subtraction of background luciferase levels and relative to PBS control. (<b>A</b>) JAK/STAT pathway stimulation with heat inactivated <i>E. coli</i> in the presence or absence of CHIKV replicon. (<b>B</b>) IMD pathway stimulation with heat inactivated <i>E. coli</i> in the presence or absence of CHIKV replicon. (<b>C</b>) Toll pathway stimulation with heat inactivated <i>B. subtilis</i> in the presence or absence of CHIKV replicon. (<b>D</b>) Co-transfection of pACT-<i>Renilla</i> with either CHIKVRep-SG-eGFP replicon RNA or control FFLuc expressing RNA. (<b>E</b>) Co-transfection of control RNA in the presence and absence of CHIKVRep-SG-eGFP replicon RNA. Graphs represent the mean of three independent experiments performed in triplicate and error bars represent the standard error of mean. *, significantly different using a general linear mixed model.</p

CHIKV infection in <i>Ae. aegypti</i>.

<p>Disseminated infection rates (DIR) were determined by estimating the proportion of mosquitoes with disseminated infection (where infection has spread beyond the midgut and led to infection of secondary organs including the salivary glands) among tested females. Three viral titers were tested: 10⁶, 10⁷ and 10⁸ PFU/ml.</p