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RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

A 7‐month‐old, spayed, female, mixed breed dog was referred for a 3‐day duration of elevated liver enzymes, azotaemia, anorexia and lethargy following ovariohysterectomy. Bloodwork showed non‐regenerative anaemia, inflammatory leukogram, moderate thrombocytopenia, severe azotaemia, severe mixed hepatopathy, hyperbilirubinemia and hypoalbuminemia. Point‐of‐care ultrasound identified free fluid and a heterogeneous mass in the mid‐ventral abdomen. Septic peritonitis was confirmed on cytology, and abdominal exploratory with left medial liver lobectomy was performed. Histopathology identified submassive hepatic necrosis and bridging fibrosis with biliary reaction, confirming a liver lobe torsion. The dog recovered perioperatively, but remained hospitalised to receive dialysis for severe renal dysfunction. The dog was transitioned to outpatient dialysis for several treatments. Unfortunately, kidney function continued to decline after dialysis was discontinued, and humane euthanasia was performed approximately 2 months postoperatively. To the authors' knowledge, this is the first reported case of liver lobe torsion following routine ovariohysterectomy in a dog. [ABSTRACT FROM AUTHOR]

OBJECTIVE To evaluate the role of simulation models and previous surgical experience on subjective and objective stress levels of students performing their 1st elective surgery within the veterinary curriculum. SAMPLE 141 third-year veterinary students METHODS Using a pre–post experimental design, salivary alpha-amylase, and cortisol were evaluated as markers of physiologic stress response before students’ first elective surgery. Student self-reported State-Trait Anxiety Inventory (STAI) scores and quantitative measures of experience were correlated to biomarker results. RESULTS No association was found for change in salivary biomarkers of stress, alpha-amylase, and cortisol, between baseline and presurgical samples accounting for gender, age, type of elective surgery performed, previous surgical experience, or simulation model use. Salivary cortisol levels were markedly elevated falling between the 66th and 99th percentile compared to an age and gender-matched population. Salivary alpha-amylase levels were also 2 to 3 times higher than those recorded by other health professionals. Veterinary student STAI scores were high falling between the 65th and 73rd percentile compared to working adults in the general population. CLINICAL RELEVANCE Veterinary students’ salivary cortisol, alpha-amylase, and STAI scores fell into the upper 2/3rds of the general population, demonstrating a high level of stress. Simulation models and previous surgical experience were not associated with decreased stress. Further evaluation of the implementation of high-fidelity simulation models and the role of stress on performance is indicated. [ABSTRACT FROM AUTHOR]

Currently, intraoperative tumour margin imaging is not routinely utilized in veterinary medicine. Optical coherence tomography (OCT) allows for real‐time assessment of tissue morphology of 1–2 mm depth. The aims of this study were (1) to compare the histologic and OCT features of excised canine skin and subcutaneous specimens, and (2) to determine the diagnostic accuracy of OCT for surgical margin evaluation. The authors hypothesized that OCT imaging would correlate well with histopathology and that OCT would be sensitive for detection of incomplete margins. Eighty dogs were prospectively enrolled. Tumours were excised, and the surgical margins were imaged using a spectral domain OCT system. The tumour type and completeness of excision were determined by histopathology. Nine blinded observers received training in OCT image interpretation and were then given a set of OCT images and videos. The observers assigned each image/video a grade from 1 (no tumour) to 4 (tumour) and the results were compared to histopathology. The overall median sensitivity and specificity of OCT imaging for detection of incomplete margins were 86.7% and 84.6%, respectively. A potential limitation is that observers had varied experience with OCT image interpretation, ranging from no prior experience to participating in a previous OCT project. OCT is sensitive for detection of incomplete margins and could be a promising real‐time surgical margin imaging modality. Further study is needed to evaluate intraoperative applications of OCT and its impact on tumour recurrence and long‐term outcome. [ABSTRACT FROM AUTHOR]

Viral signaling through retinoic acid-inducible gene-I (RIG-I) and its adaptor protein, IFN promoter-stimulator 1 (IPS-1), activates IFN regulatory factor-3 (IRF-3) and the host IFN-[alpha]/[beta] response that limits virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves IPS-1 to block RIG-I signaling, but how this regulation controls the host response to HCV is not known. Moreover, endogenous IPS-1 cleavage has not been demonstrated in the context of HCV infection in vitro or in vivo. Here, we show that HCV infection transiently induces RIG-I- and IPS-1-dependent IRF-3 activation. This host response limits HCV production and constrains cellular permissiveness to infection. However, HCV disrupts this response early in infection by NS3/4A cleavage of IPS-1 at C508, releasing IPS-1 from the mitochondrial membrane. Cleavage results in subcellular redistribution of IPS-1 and loss of interaction with RIG-I, thereby preventing downstream activation of IRF-3 and IFN-[beta] induction. Liver tissues from chronically infected patients similarly demonstrate subcellular redistribution of IPS-1 in infected hepatocytes and IPS-1 cleavage associated with a lack of ISG15 expression and conjugation of target proteins in vivo. Importantly, small-molecule inhibitors of NS3/4A prevent cleavage and restore RIG-I signaling of IFN-[beta] induction. Our results suggest a dynamic model in which early activation of IRF-3 and induction of antiviral genes are reversed by IPS-1 proteolysis and abrogation of RIG-I signaling as NS3/4A accumulates in newly infected cells. HCV protease inhibitors effectively prevent IPS-1 proteolysis, suggesting they may be capable of restoring this innate host response in clinical practice. CARDIF | MAVS | VISA

Objective: To evaluate outcomes of dogs with parathyroid carcinoma (PTC) treated by surgical excision and to describe the incidence of postoperative hypocalcemia, degree of hypocalcemia, duration of hospitalization, duration of calcium supplementation, and survival time. Animals: 100 client-owned dogs with PTC admitted to academic, referral veterinary institutions. Procedures: In a retrospective multi-institutional study, medical records of dogs undergoing surgical excision of PTC between 2010 to 2019 were reviewed. Signalment, relevant medical history, clinical signs, clinicopathologic testing, imaging, surgical findings, intraoperative complications, histologic examination, and survival time were recorded. Results: 100 dogs with PTC were included, and 96 dogs had clinical or incidental hypercalcemia. Common clinical signs included polyuria (44%), polydipsia (43%), hind limb paresis (22%), lethargy (21%), and hyporexia (20%). Cervical ultrasonography detected a parathyroid nodule in 91 of 91 dogs, with a single nodule in 70.3% (64/91), 2 nodules in 25.3% (23/91), and ≥ 3 nodules in 4 (4/91)% of dogs. Hypercalcemia resolved in 89 of 96 dogs within 7 days after surgery. Thirty-four percent of dogs developed hypocalcemia, on the basis of individual analyzer ranges, within 1 week after surgery. One dog had metastatic PTC to the prescapular lymph node, and 3 dogs were euthanized for refractory postoperative hypocalcemia. Estimated 1-, 2-, and 3-year survival rates were 84%, 65%, and 51% respectively, with a median survival time of 2 years. Conclusions and clinical relevance: Excision of PTC results in resolution of hypercalcemia and excellent long-term tumor control. Surgical excision of PTC is recommended because of resolution of hypercalcemia and a good long-term prognosis. Future prospective studies and long-term follow-up are needed to further assess primary tumor recurrence, metastasis, and incidence of postoperative hypocalcemia. [ABSTRACT FROM AUTHOR]

Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways. [ABSTRACT FROM AUTHOR]

Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod vectors and vertebrate hosts. While barriers limiting arbovirus population diversity have been observed in mosquitoes, whether barriers exist in vertebrate hosts is unclear. To investigate whether arboviruses encounter bottlenecks during dissemination in the vertebrate host, we infected immunocom-petent mice and immune-deficient mice lacking alpha/beta interferon (IFN-a/P) receptors (IFNAR-/- mice) with a pool of genetically marked viruses to evaluate dissemination and host barriers. We used the live attenuated vaccine strain YFV-17D, which contains many mutations compared with virulent YFV. We found that intramuscularly injected immunocompetent mice did not develop disease and that viral dissemination was restricted. Conversely, 32% of intramuscularly injected IFNAR-/- mice developed disease. By following the genetically marked viruses over time, we found broad dissemination in IFNAR-/- mice followed by clearance. The patterns of viral dissemination were similar in mice that developed disease and mice that did not develop disease. Unlike our previous results with polio virus, these results suggest that YFV-17D encounters no major barriers during dissemination within a vertebrate host in the absence of the type IIFN response. [ABSTRACT FROM AUTHOR]

Viral signaling through retinoic acid-inducible gene-I (RIG-I) and its adaptor protein, IFN promoter-stimulator 1 (IPS-1), activates IFN regulatory factor-3 (IRF-3) and the host lFN-α/β response that limits virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves IPS-1 to block RIG-I signaling, but how this regulation controls the host response to HCV is not known. Moreover, endogenous IPS-1 cleavage has not been demonstrated in the context of HCV infection in vitro or in vivo. Here, we show that HCV infection transiently induces RIG-I- and IPS-1-dependent IRF-3 activation. This host response limits HCV production and constrains cellular permissiveness to infection. However, HCV disrupts this response early in infection by N53/4A cleavage of IPS-1 at C508, releasing IPS-1 from the mitochondrial membrane. Cleavage results in subcellular redistribution of IPS-1 and loss of interaction with RIG-I, thereby preventing downstream activation of IRF-3 and IFN-β induction. Liver tissues from chronically infected patients similarly demonstrate subcellular redistribution of IPS-1 in infected hepatocytes and IPS-1 cleavage associated with a lack of ISG15 expression and conjugation of target proteins in vivo. Importantly, small-molecule inhibitors of NS3/4A prevent cleavage and restore RIG-I signaling of IFN-β induction. Our results suggest a dynamic model in which early activation of lRF-3 and induction of antiviral genes are reversed by IPS-1 proteolysis and abrogation of RIG-I signaling as N53/4A accumulates in newly infected cells. HCV protease inhibitors effectively prevent IPS-1 proteolysis, suggesting they may be capable of restoring this innate host response in clinical practice. [ABSTRACT FROM AUTHOR]

The article presents information on a research study that was conducted to identify factors that influence the regulation of interferon production and innate antiviral immunity through translational control of interferon regulatory factor (IRF)-7. The study also demonstrates the importance of translational control mechanisms in regulation of the host response to viral infection.

Coxsackievirus typically infects humans via the gastrointestinal tract, which has a large number of microorganisms collectively referred to as the microbiota. To study how the intestinal microbiota influences enteric virus infection, several groups have used an antibiotic regimen in mice to deplete bacteria. These studies have shown that bacteria promote infection with several enteric viruses. However, very little is known about whether antibiotics influence viruses in a microbiota-independent manner. In this study, we sought to determine the effects of antibiotics on coxsackievirus B3 (CVB3) using an in vitro cell culture model in the absence of bacteria. We determined that an aminoglycoside antibiotic, neomycin, enhanced the plaque size of CVB3 strain Nancy. Neomycin treatment did not alter viral attachment, translation, or replication. However, we found that the positive charge of neomycin and other positively charged compounds enhanced viral diffusion by overcoming the negative inhibitory effect of sulfated polysaccharides present in agar overlays. Neomycin and the positively charged compound protamine also enhanced plaque formation of reovirus. Overall, these data provide further evidence that antibiotics can play noncanonical roles in viral infections and that this should be considered when studying enteric virus-microbiota interactions. IMPORTANCE Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown the bacteria promote infection with a variety of enteric viruses. However, it is possible that antibiotics have microbiota-independent effects on viruses. Here we show that an aminoglycoside antibiotic, neomycin, can influence quantification of coxsackievirus in cultured cells in the absence of bacteria., (Copyright © 2019 Woods Acevedo et al.)

The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high. IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations., (Copyright © 2017 Aguilera et al.)

The host and viral factors that influence disease outcome during flavivirus infections are not fully understood. Using the live attenuated yellow fever virus (YFV) vaccine strain 17D as a model system we evaluated how viral dose, inoculation route and immunopathogenesis contributed to disease outcome in mice deficient in the type I IFN response. We found that YFV-17D infection of IFN-α/β receptor knockout mice resulted in three distinct disease outcomes: no clinical signs of disease, fatal viscerotropic disease or fatal neurotropic disease. Interestingly, viral load at disease onset did not correlate with disease outcome. However, we found increased immune infiltrates in the brain tissues of mice that developed neurotropic disease. Additionally, mice that developed viscerotropic disease, as characterized by liver and spleen pathology and/or intestinal haemorrhage, had significantly elevated levels of alanine aminotransferase, monocyte chemotactic protein and IFN-inducible protein (IP)-10 as compared with mice with no clinical signs of disease or neurotropic disease. Furthermore, mice treated with recombinant IP-10 throughout YFV-17D infection showed increased mortality and an increased percentage of mice with viscerotropic disease. Our results demonstrated that viral load did not correlate with pathogenesis, and the host immune response played a pivotal role in disease outcome and contributed to YFV-17D pathogenesis in mice., (© 2015 The Authors.)

Background: Current treatments for chronic hepatitis C virus (HCV) employing pegylated interferon (PEG-IFN) plus ribavirin are successful in approximately 50% of patients. Consensus IFN (CIFN) is a recombinant type I IFN that has demonstrated efficacy where conventional therapy has failed. We evaluated the host cell antiviral response and anti-HCV actions induced by IFN-alpha2a, PEG-IFN-alpha2b or CIFN on cultured immortalized human hepatocytes, Huh7 human hepatoma cells and Huh7 cells that harboured genetically distinct HCV RNA replicons or were infected with HCV 2a., Methods: Cultured cells were treated with each IFN at relevant dosing based upon the pharmacological attainable in vivo serum maximum IFN concentrations. Gene expression and antiviral properties were measured using protein, RNA and virus quantification assays., Results: CIFN treatment maximally triggered Janus kinase signal transducer and activator of transcription signalling in association with enhanced IFN-stimulated gene (ISG) expression. Increased antiviral potency of CIFN was associated with enhancement of IFN-induced blockade upon viral protein synthesis, protection of the cellular IFN promoter stimulator-1 (IPS-1) protein from HCV proteolysis and reduced replication of an IFN-resistant HCV replicon variant. Microarray analyses revealed that CIFN treatment induced a distinct pattern of ISG expression in cultured hepatocytes compared with other IFNs., Conclusions: CIFN exhibits increased anti-HCV potency over IFN-alpha2a and PEG-IFN through maximal and distinct induction of ISG expression and enhanced intracellular innate antiviral response, while protecting IPS-1 from HCV proteolysis. CIFN might offer a treatment regimen imparting translational control programmes and restoration of the retinoic acid-inducible gene-1/IPS-1 pathway and could be considered for previous treatment failures.