Improving access to chronic pain care with central referral and triage: The 6-year findings from a single-entry model.

Background: Despite the established efficacy of multidisciplinary chronic pain care, barriers such as inflated referral wait times and uncoordinated care further hinder patient health care access. Aims: Here we describe the evolution of a single-entry model (SEM) for coordinating access to chronic pain care across seven hospitals in Toronto and explore the impact on patient care 6 years after implementation. Methods: In 2017, an innovative SEM was implemented for chronic pain referrals in Toronto and surrounding areas. Referrals are received centrally, triaged by a clinical team, and assigned an appointment according to the level of urgency and the most appropriate care setting/provider. To evaluate the impact of the SEM, a retrospective analysis was undertaken to determine referral patterns, patient characteristics, and referral wait times over the past 6 years. Results: Implementation of an SEM streamlined the number of steps in the referral process and led to a standardized referral form with common inclusion and exclusion criteria across sites. Over the 6-year period, referrals increased by 93% and the number of unique providers increased by 91%. Chronic pain service wait times were reduced from 299 (±158) days to 176 (±103) days. However, certain pain diagnoses such as chronic pelvic pain and fibromyalgia far exceed the average. Conclusions: The results indicate that the SEM helped reduce wait times for pain conditions and standardized the referral pathway. Continued data capture efforts can help identify gaps in care to enable further health care refinement and improvement.

Contexte: Malgré l'efficacité établie des soins multidisciplinaires dans le traitement de la douleur chronique, des obstacles tels que des délais d'attente prolongés et l'absence de coordination des soins entravent davantage l'accès des patients aux services de santé.Objectifs: Nous décrivons ici l'évolution d'un modèle à entrée unique visant à coordonner l'accès aux soins pour la douleur chronique dans sept hôpitaux de Toronto. Nous examinons également l'effet de ce modèle sur les soins aux patients six ans après sa mise en œuvre.Méthodes: En 2017, un modèle à entrée unique novateur a été mis en place pour orienter les patients souffrant de douleur chronique à Toronto et dans les régions avoisinantes. Les patients sont reçus de manière centralisée, triés par une équipe clinique et un rendez-vous leur est attribué en fonction du degré d'urgence et de l'établissement de soins ou du prestataire le plus approprié.Pour évaluer l'impact du modèle à entrée unique, une analyse rétrospective a été entreprise afin de déterminer les schémas de consultation, les caractéristiques des patients et les temps d'attente pour les demandes de consultation au cours des six dernières années.Résultats: La mise en œuvre d'un modèle à entrée unique a permis de rationaliser le nombre d'étapes du processus de demande de consultation et a conduit à l'élaboration d'un formulaire de demande de consultation normalisé comprenant des critères d'inclusion et d'exclusion communs à tous les sites. Au cours de la période de six ans, le nombre de demandes de consultation a augmenté et le nombre de prestataires uniques a augmenté de 91 %.Les temps d'attente pour les services de traitement de la douleur chronique ont diminué de 299 (±158) jours à 176 (±103) jours. Cependant, certains diagnostics de douleur, comme la douleur pelvienne chronique et la fibromyalgie, dépassent de loin la moyenne.Conclusions: Les résultats indiquent que le modèle à entrée unique a contribué à réduire les temps d'attente pour les affections douloureuses et à normaliser le parcours de consultation. La poursuite des efforts de collecte des données peut aider à recenser les lacunes dans les soins, permettant ainsi une amélioration continue des soins de santé.

Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus.

Introduction: Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host. Methods: In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies. Results: The absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7. Discussion: This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

The Effect of Temperature on the Stability of African Swine Fever Virus BA71V Isolate in Environmental Water Samples.

African swine fever virus (ASFV) is known to be very stable and can remain infectious over long periods of time especially at low temperatures and within different matrices, particularly those containing animal-derived organic material. However, there are some gaps in our knowledge pertaining to the survivability and infectivity of ASFV in groundwater. This study aims to determine the stability and infectivity of the cell culture-adapted ASFV strain BA71V by plaque assay after incubation of the virus within river water samples at three different environmentally relevant temperatures (4 °C, 15 °C, and 21 °C) over the course of 42 days. The results from this study indicate that ASFV can remain stable and infectious when maintained at 4 °C in river water for more than 42 days, but as incubation temperatures are increased, the stability is reduced, and the virus is no longer able to form plaques after 28 days and 14 days, respectively, when stored at 15 °C and 21 °C. Characterizing the survivability of ASFV in groundwater can allow us to develop more appropriate inactivation and disinfection methods to support disease control and mitigate ASFV outbreaks.

A Systematic Review of Optogenetic Vision Restoration: History, Challenges, and New Inventions from Bench to Bedside.

Blindness due to rod-cone dystrophies is a significant comorbidity and cause of reduced quality of life worldwide. Optogenetics uses adeno-associated viral (AAV) vectors to bypass lost photoreceptors and transfect remnant cell populations of the degenerated retina aiming to restore vision via the ectopic expression of opsins. The optogenetic targeting of retinal ganglion cells (RGCs) has been remarkably successful and several studies have advanced to clinical trials over the recent years. The inner retina and specifically ON bipolar cells represent even more appealing targets due to their intrinsically coded tasks in parallel processing and fine-tuning of visual signals before reaching the output: RGCs. However, present success with pursuing inner and outer retinal cells for optogenetic vision restoration is limited by multiple factors, including AAV tropism, promoter specificity, and retinal morphofunctional remodeling. Here we provide a review of the evolution of optogenetics, its greatest challenges, and solutions from bench to bedside.

Evaluation of the Delivery of a Live Attenuated Porcine Reproductive and Respiratory Syndrome Virus as a Unit Solid Dose Injectable Vaccine.

Solid dose vaccine formulation and delivery systems offer potential advantages over traditional liquid vaccine formulations. In addition to enhanced thermostability, needle-free delivery of unit solid dose injectable (USDI) vaccines offers safe, rapid, and error-free administration, with applicability to both human and animal health. Solid dose formulation technologies can be adapted for delivery of different vaccine formats including live attenuated vaccines, which remain the 'gold standard' for many disease targets. Porcine reproductive and respiratory syndrome viruses (PRRSV) cause one of the most economically important diseases affecting the global pig industry. Despite several shortcomings, live attenuated vaccines are widely used to control PRRSV. We optimised a freeze-dried USDI formulation of live attenuated PRRSV-1, which fully retained infectious titre, and evaluated its immunogenicity in comparison to virus delivered in liquid suspension via intramuscular and subcutaneous needle inoculation. Pigs vaccinated with the USDI formulation displayed vaccine viraemia, and PRRSV-specific antibody and T cell responses comparable to animals immunised with the liquid vaccine. The USDI vaccine formulation was stable for at least 6 months when stored refrigerated. These data demonstrate the potential for a solid dose vaccine delivery system as an alternative to conventional needle-syringe delivery of live attenuated PRRSV vaccines.

Gene therapy for inherited retinal disease: long-term durability of effect.

The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the pre-clinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases, was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with pre-clinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention.

A random priming amplification method for whole genome sequencing of SARS-CoV-2 virus.

BACKGROUND: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. METHODS: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. RESULTS: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. CONCLUSIONS: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

Photopharmacology for vision restoration.

Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.

Laboratory Diagnosis and Quantification of African Swine Fever Virus Using Real-Time Polymerase Chain Reaction.

Real-time polymerase chain reaction (PCR) for the detection of African swine fever virus (ASFV) is the tool of choice for the diagnostic laboratory and is a robust and easily scalable method for the researcher analyzing viral replication both in vitro and in vivo. In this chapter, we describe protocols for both quantitative real-time polymerase chain reactions (qPCR) and non-quantitative real-time polymerase chain reactions (real-time PCR) for the detection of African swine fever virus genome in a range of samples.

Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.

SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.

A novel strain of lumpy skin disease virus causes clinical disease in cattle in Hong Kong.

Lumpy skin disease virus (LSDV) is an emerging poxviral pathogen of cattle that is currently spreading throughout Asia. The disease situation is of high importance for farmers and policy makers in Asia. In October 2020, feral cattle in Hong Kong developed multi-focal cutaneous nodules consistent with lumpy skin disease (LSD). Gross and histological pathology further supported the diagnosis and samples were sent to the OIE Reference Laboratory at The Pirbright Institute for confirmatory testing. LSDV was detected using quantitative polymerase chain reaction (qPCR) and additional molecular analyses. This is the first report of LSD in Hong Kong. Whole genome sequencing (WGS) of the strain LSDV/Hong Kong/2020 and phylogenetic analysis were carried out in order to identify connections to previous outbreaks of LSD, and better understand the drivers of LSDV emergence. Analysis of the 90 core poxvirus genes revealed LSDV/Hong Kong/2020 was a novel strain most closely related to the live-attenuated Neethling vaccine strains of LSDV and more distantly related to wildtype LSDV isolates from Africa, the Middle East and Europe. Analysis of the more variable regions located towards the termini of the poxvirus genome revealed genes in LSDV/Hong Kong/2020 with different patterns of grouping when compared to previously published wildtype and vaccine strains of LSDV. This work reveals that the LSD outbreak in Hong Kong in 2020 was caused by a different strain of LSDV than the LSD epidemic in the Middle East and Europe in 2015-2018. The use of WGS is highly recommended when investigating LSDV disease outbreaks.

A field study evaluating the humoral immune response in Mongolian sheep vaccinated against sheeppox virus.

Sheeppox is a transboundary disease of small ruminants caused by infection with the capripoxvirus sheeppox virus. Sheeppox is found in Africa, the Middle East and Asia and is characterized by fever, multifocal cutaneous raised lesions and death. Vaccination with live attenuated capripoxvirus (CPPV) strains is an effective and widely used strategy to contol sheeppox outbreaks; however, there are few reports of post-vaccination field surveillance studies. This study used a commercially available enzyme-linked immunosorbent assay (ELISA) to examine quantitative and temporal features of the humoral response of sheep vaccinated with a live-attenuated CPPV strain in Mongolia. Four hundred samples were tested using the ELISA commercial kit, and a subset of 45 samples were also tested with a virus neutralization test (VNT). There was substantial agreement between the VNT and ELISA tests. Antibodies to CPPV were detected between 40 and 262 days post-vaccination. There was no significant difference between serological status (positive/negative) and sex or age; however, an inverse correlation was found between the length of time since vaccination and serological status. Animals between 90 and 180 days post-vaccination were more likely to be positive than animals greater than 180 days post-vaccination. Our results show that a commercial CPPV ELISA kit is a robust and reliable assay for post-CPPV vaccination surveillance in resource-restricted settings and provide temporal parameters to be considered when planning sheeppox post-vaccination monitoring programmes.

Development and Initial Evaluation of Psychometric Properties of a Pain Competence Assessment Tool (PCAT).

Competency-based education is now considered the best approach for pain educational programs provided for pre and postgraduate healthcare providers (HCPs). To demonstrate learners' progression, an assessment tool that aligns with this educational approach and targets different HCPs is needed. A Pain Competence Assessment Tool (PCAT) was developed based on the pain management core competencies that align with the International Association for the Study of Pain interprofessional pain curriculum. The PCAT is an online competency-based assessment tool for HCPs that consists of 5 case scenarios followed by 17 key-feature questions. HCPs and trainees completed the PCAT through a series of studies to assess its psychometric properties. The preliminary evaluation suggested that the PCAT had adequate content validity. Apart from 6 questions, the PCAT questions demonstrated homogeneity and acceptable reliability, and substantial stability. No ceiling or floor effect was found. A significant difference was detected between the HCPs' and trainees' scores. The PCAT scores strongly correlated with other variables reflecting different competence levels. The PCAT scores showed significant changes in the baseline scores compared to scores after attending an educational intervention. The PCAT offers a first-of-its-kind tool for assessing HCPs' competence (ie, knowledge and its application) in managing chronic pain. Future research is needed for further validation and adaptation of the PCAT. PERSPECTIVE: The Pain Competence Assessment Tool (PCAT) offers a first-of-its-kind tool for assessing clinicians' core competencies that overlap between different professions and support the clinicians' capacity to successfully manage chronic pain in the real world focusing on the patient-centered perspective rather than the profession-specific perspective.

Positive impact of a telemedicine education program on practicing health care workers during the COVID-19 pandemic in Ontario, Canada: A mixed methods study of an Extension for Community Healthcare Outcomes (ECHO) program.

INTRODUCTION: In addition to shifting and expanding clinical responsibilities, rapidly evolving information and guidelines during the COVID-19 pandemic has made it difficult for health care workers (HCW) to synthesise and translate COVID-19 information into practice. This study evaluated whether a COVID-19-specific telemedicine education program (ECHO COVID) would impact health care workers' self-efficacy and satisfaction in the management of patients with COVID-19. METHODS: A prospective mixed methods parallel-design study was conducted among ECHO COVID participants using pre-post questionnaires and a focus group discussion. Questionnaire results were examined for changes in health care workers' self-efficacy and satisfaction. Focus group discussion data were analysed to explore health care workers' experience in ECHO COVID and the context of their practice during the COVID-19 pandemic. RESULTS: 239 health care workers registered in ECHO COVID and 114 (47.7%) completed questionnaires and attended at least one ECHO COVID session. Median self-efficacy scores increased from 5 (IQR 4-6) to 6 (IQR 6-6) (p < 0.0001), independent of profession, years in practice, age group, or practice environment. Participants were highly satisfied with ECHO COVID sessions with a median score of 4 (IQR 4-5). Focus group discussion data indicated that health care workers gained knowledge through ECHO COVID and revealed facilitators for ECHO COVID program success, including the transition to virtual care, the practicability of knowledge provided, and a 'perspective from the trenches.' DISCUSSION: This study demonstrated that a telemedicine education program aimed to support health care workers in managing patients with COVID-19 had a positive impact on health care workers' self-efficacy and satisfaction. This impact was specifically mediated by the ECHO COVID program.

Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.

scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research.

Gene therapy is an experimental approach to treating disease that involves altering faulty genes or replacing them with new, working copies. Most often, the new genetic material is delivered into cells using a modified virus that no longer causes disease, called a viral vector. Virus-mediated gene therapies are currently being explored for degenerative eye diseases, such as retinitis pigmentosa, and neurological disorders, like Alzheimer's and Parkinson's disease. A number of gene therapies have also been approved for treating some rare cancers, blood disorders and a childhood form of motor neuron disease. Despite the promise of virus-mediated gene therapy, there are significant hurdles to its widespread success. Viral vectors need to deliver enough genetic material to the right cells without triggering an immune response or causing serious side effects. Selecting an optimal vector is key to achieving this. A type of viruses called adeno-associated viruses (AAV) are prime candidates, partly because they can be easily engineered. However, accurately comparing the safety and efficacy of newly engineered AAVs is difficult, due to variation between test subjects and the labor and cost involved in careful testing. Öztürk et al. addressed this issue by developing an experimental pipeline called scAAVengr for comparing gene therapy vectors head-to-head. The process involves tagging potential AAV vectors with unique genetic barcodes, which can then be detected and quantified in individual cells using a technique called single-cell RNA sequencing. This means that when several vectors are used to infect lab-grown cells or a test animal at the same time, they can be tracked. The vectors can then be ranked on their ability to infect specific cell types and deliver useful genetic material. Using scAAVengr, Öztürk et al. compared viral vectors designed to target the light-sensitive cells of the retina, which allow animals to see. First, a set of promising viral vectors were evaluated using the scAAVengr pipeline in the eyes of marmosets and macaques, two small primates. Precise levels and locations of gene delivery were quantified. The top-performing vector was then identified and used to deliver Cas9, a genome editing tool, to primate retinas. Öztürk et al. also used scAAVengr to compare viral vectors in mice, analysing the vectors' ability to deliver their genetic cargo to the brain, heart, and liver. These experiments demonstrated that scAAVengr can be used to evaluate vectors in multiple tissues and in different organisms. In summary, this work outlines a method for identifying and precisely quantifying the performance of top-performing viral vectors for gene therapy. By aiding the selection of optimal viral vectors, the scAAVengr pipeline could help to improve the success of preclinical studies and early clinical trials testing gene therapies.

Outcomes of progranulin gene therapy in the retina are dependent on time and route of delivery.

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit.

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/µl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against the putative novel genotypes. RT-qPCR assays targeting BTV segment 9 and 10 detected both strains (SPvvvv/02 and SPvvvv/03) whereas a BTV segment 1 RT-qPCR assay was unable to detect either BTV strain. The work presented here expands upon the current repertoire of RT-qPCR assays for BTV genotype determination.