A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

The Application of CRISPR/Cas Technology to Efficiently Model Complex Cancer Genomes in Stem Cells.

CRISPR/Cas gene editing technologies have emerged as powerful tools in the study of oncogenic transformation. The system's specificity, versatility, and ease of implementation allow researchers to identify important molecular markers and pathways which grant cancers stem cell like properties. This technology has already been applied to researching specific cancers, but has seen restricted therapeutic applications due to inherent ethical and technical limitations. Active development and adaptation of the CRISPR/Cas system has produced new methods to take advantage of both non-homologous end joining and homologous recombination repair mechanisms in attempts to remedy these limitations and improve the versatility of gene edits that can be created. Nonetheless, until issues with specificity and in vivo efficiency are resolved, utilization of CRISPR/Cas systems would be best employed in the modeling and study of various cancer genes. While it may have potential therapeutic applications to targeted cancer therapies in the future, presently CRISPR/Cas is a remarkable technique that can be utilized for easy and efficient gene editing when it comes to cancer research. J. Cell. Biochem. 119: 134-140, 2018. © 2017 Wiley Periodicals, Inc.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

Adherence to Tympanostomy Tube Clinical Practice Guidelines in an Advanced Practice Provider Clinic.

OBJECTIVE: We compared adherence rates by attending otolaryngologists (OTOs) and advanced practice providers (APPs) to the 2013 American Academy of Otolaryngology-Head and Neck Surgery Foundation clinical practice guideline (CPG) for children with recurrent acute otitis media (RAOM) undergoing bilateral myringotomy and tympanostomy tube placement (BMT). METHODS: Patients aged 6 months to 12 years old undergoing BMT for RAOM who had a pre-operative visit with an independent APP or OTO were reviewed. Patients satisfied CPG criteria if middle ear effusion was identified at the pre-operative visit (pre-op) or if they did not have effusion but met exception criteria based on their risk for developmental difficulties and contraindications to medical therapy. Adherence rates between APPs and OTOs were compared. Agreement between pre-op and time-of-surgery middle ear effusion identification was assessed. RESULTS: Nine hundred twenty-three patients were included. Six hundred one patients were seen by OTOs and 322 by APPs. Middle ear effusion was identified at pre-op in 84% of APP patients and in 76% of OTO patients (P = .005). Eight percent of APP patients and 11% of OTO patients met exception criteria (P = .138). Overall, 87% of OTO patients and 92% of APP patients met either CPG or exception criteria for BMT (P = .037). A logistic regression model demonstrated that pre-op provider type did not significantly impact rates of agreement between pre-op visit and time-of-surgery middle ear effusion identification. CONCLUSIONS: Independent APP-led clinics can reliably and effectively deliver evidence-based care for prevalent conditions such as RAOM at similar rates of adherence to CPGs as OTOs.

Effect of Insurance Type on Postoperative Tympanostomy Tube Follow-up.

OBJECTIVE: Bilateral myringotomy with tube insertion (BMT) is a common procedure performed in children. Appropriate follow-up is necessary to ensure management of postoperative sequalae. The objectives are to investigate (1) the relationship between insurance type and postoperative follow-up attendance and (2) the effect of follow-up on need for further care after BMT. STUDY DESIGN: Retrospective cohort study. SETTING: The study included patients <3 years of age undergoing BMT for recurrent acute otitis media at a tertiary care children's hospital within a single year and followed for 3 years. Patients were excluded if they had received a prior BMT; underwent a concurrent otolaryngologic procedure; or had a syndromic diagnosis, craniofacial abnormality, or any significant cardiac or respiratory comorbidity. METHODS: Number of follow-up appointments, demographics, socioeconomic status, and postoperative outcomes were analyzed. RESULTS: A total of 734 patients were included with mean (SD) age of 1.4 years (0.50). The majority of patients had private insurance (520/734, 70.8%). Patients with public insurance attended fewer postoperative appointments (1.5 vs 1.8, P < .001) and had a higher incidence of BMT-related emergency department (ED) visits (10.3% vs 3.8%, P = .001). There was no significance found when different insurance providers were compared. An adjusted multivariate regression analysis showed that patients with private insurance were more likely to attend postoperative appointments (odds ratio, 3.52 [95% CI, 2.12-5.82]; P < .001) and less likely to have a BMT-related ED visit (odds ratio, 0.42 [95% CI, 0.20-0.89]; P = .024). CONCLUSION: Insurance type is related to outcomes after the treatment of recurrent acute otitis media with BMT. Future studies that survey individuals will help identify barriers that contribute to patient absence at follow-ups and need for subsequent ED visits.

Advanced Practice Provider Clinics: Expediting Care For Children Undergoing Tympanostomy Tube Placement.

OBJECTIVE/HYPOTHESIS: Advanced practice provider (APP) employment is becoming common in pediatric otolaryngology practices, though few studies have evaluated the consequences that APP-led clinics have on access to care. The objectives of this study were: 1) to investigate whether access to bilateral myringotomy with tympanostomy tube placement (BMT) for recurrent acute otitis media (RAOM) differed between patients seen in otolaryngologist and APP-led clinics 2) to compare clinical characteristics of patients seen by provider type. METHODS: Retrospective cohort study at an academic, tertiary care pediatric otolaryngology practice. All children were <18 years old and underwent evaluation for RAOM followed by BMT. We compared time in days from scheduling pre-operative appointment to appointment date and time from appointment to BMT between patients seen by APPs and otolaryngologists using Mann-Whitney U tests and multivariate linear regression models. We compared clinical characteristics by provider type using Mann-Whitney U tests and Fisher exact tests. RESULTS: A total of 957 children were included. Children seen by APPs had significantly shorter wait times for appointments (median 19 vs. 39 days, P < .001) and shorter times from preoperative appointment to BMT (median 25 vs. 37 days, P < .001). Patients seen by otolaryngologists had increased prevalence of craniofacial abnormalities, Down Syndrome, hearing loss, history of otologic surgery, and higher ASA physical status classification. CONCLUSIONS: Children seen by APPs received care more quickly than those seen by otolaryngologists. Patients seen by otolaryngologists tended to be more medically complex. Implementation of independent APP clinics may expedite and improve access to BMT for children with RAOM. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:2133-2140, 2021.