Seneca Valley virus 3Cpro antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals.

IMPORTANCE: Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3Cpro) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3Cpro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3Cpro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Targeting GBM with an Oncolytic Picornavirus SVV-001 alone and in combination with fractionated Radiation in a Novel Panel of Orthotopic PDX models.

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.

Standardized blood volume changes monitored by capnodynamic hemodynamic variables: An experimental comparative study in pigs.

BACKGROUND: The capnodynamic method, based on Volumetric capnography and differential Fick mathematics, assess cardiac output in mechanically ventilated subjects. Capnodynamic and established hemodynamic monitoring parameters' capability to depict alterations in blood volume were investigated in a model of standardized hemorrhage, followed by crystalloid and blood transfusion. METHODS: Ten anesthetized piglets were subjected to controlled hemorrhage (450 mL), followed by isovolemic crystalloid bolus and blood re-transfusion. Intravascular blood volume, and all hemodynamic variables, were determined twice after each intervention. The investigated hemodynamic variables were: cardiac output and stroke volume for capnodynamics and pulse contour analysis, respectively, pulse pressure and stroke volume variability and mean arterial pressure. One-way ANOVA and Tukey's test for multiple comparisons were used to identify significant changes. Trending was assessed by correlation and concordance. RESULT: Concordance against intravascular volume changes for capnodynamic cardiac output and stroke volume were 96 and 94%, with correlations r = .78 and .68, (p < .0001) with significant changes for 6 and 5 of the 6 measuring points, respectively. Mean arterial pressure and pulse pressure variation had a concordance of 85% and 87%, r = .67 (p < .0001) and r = -.45 (p < .0001), respectively, and both changed significantly for 3 of 6 measuring points. Pulse contour stroke volume variation, stroke volume and cardiac output, showed concordance and correlation of 76%, r = -.18 (p = .11), 63%, r = .28 (p = .01) and 50%, r = .31 (p = .007), respectively and significant change for 1, 1 and 0 of the measuring points, respectively. CONCLUSION: Capnodynamic cardiac output and stroke volume did best depict the changes in intravascular blood volume. Pulse contour parameters did not follow volume changes in a reliable way.

Effect of goal-directed fluid therapy based on plasma colloid osmotic pressure on the postoperative pulmonary complications of older patients undergoing major abdominal surgery.

BACKGROUND: As an important component of accelerated rehabilitation surgery, goal-directed fluid therapy (GDT) is one of the optimized fluid therapy strategies and is closely related to perioperative complications and mortality. This article aimed to study the effect of combining plasma colloid osmotic pressure (COP) with stroke volume variation (SVV) as a target for intraoperative GDT for postoperative pulmonary complications in older patients undergoing major abdominal surgery. METHODS: In this study, older patients (n = 100) undergoing radical resection of gastroenteric tumors were randomized to three groups: Group C (n1 = 31) received a conventional infusion regimen, Group S1 (n2 = 34) received GDT based on SVV, and Group S2 (n3 = 35) received GDT based on SVV and COP. The results were recorded, including the lung injury score (LIS); PaO2/FiO2 ratio; lactic acid value at the times of beginning (T0) and 1 h (T1), 2 h (T2), and 3 h (T3) after liquid infusion in the operation room; the total liquid infusion volume; infusion volumes of crystalline and colloidal liquids; urine production rate; pulmonary complications 7 days after surgery; and the severity grading of postoperative pulmonary complications. RESULTS: The patients in the S2 group had fewer postoperative pulmonary complications than those in the C group (P < 0.05) and the proportion of pulmonary complications of grade 1 and higher than grade 2 in S2 group was significantly lower than that in C group (P <0.05); the patients in the S2 group had a higher PaO2/FiO2 ratio than those in the C group (P < 0.05), lower LIS than those in the S1 and C groups (P < 0.05), less total liquid infusion than those in the C group (P < 0.05), and more colloidal fluid infusion than those in the S1 and C groups (P < 0.05). CONCLUSION: The findings of our study show that intraoperative GDT based on COP and SVV can reduce the incidence of pulmonary complications and conducive to shortening the hospital stay in older patients after gastrointestinal surgery. TRIAL REGISTRATION: Chinese Clinical Trial. no. ChiCTR2100045671. Registry at www.chictr.org.cn on April 20, 2021.

Reliability of stroke volume or pulse pressure variation as dynamic predictors of fluid responsiveness in laparoscopic surgery: a systematic review.

The reliability of stroke volume variation (SVV) and pulse pressure variation (PPV) in predicting fluid responsiveness during laparoscopic surgery remains unclear. We conducted the present systematic review to summarize the current evidence. We reviewed studies that investigated the reliability of SVV and PPV in laparoscopic surgery. Seven studies were included in the final analysis. Two studies demonstrated that the area under the receiver operating characteristic curve (AUROC) for SVV was less than 0.8, and five studies reported that the AUROC was > 0.8. The pooled AUROC for SVV and PPV was more than 0.8 with high heterogeneities between the included studies. Most individual studies have suggested that SVV and PPV are sufficiently reliable for predicting fluid responsiveness during laparoscopic surgery. However, the limited number of patients, varied apparatus used to define fluid responsiveness, diverse definitions of fluid responsiveness, and different fluids used to perform fluid challenges in the included studies render firm conclusions about SVV's and PPV's reliability impossible.

An enzyme-linked immunosorbent assay (ELISA) to determine Simian Varicella Virus antibody titers in infected rhesus monkeys (Macaca mulatta).

BACKGROUND: Simian varicella virus (SVV) is a primate herpesvirus that causes a natural varicella-like disease in Old World monkeys and may cause epizootics in facilities housing nonhuman primates. SVV infection of nonhuman primates is used as an experimental model to investigate varicella pathogenesis and to develop antiviral strategies. METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect SVV antibodies in infected rhesus macaque monkeys. RESULTS: An ELISA determined SVV antibody titers following experimental infection. SVV IgG was detected by day 14 post-infection and remained elevated for at least 84 days. CONCLUSIONS: The SVV ELISA is a safe and rapid approach to confirm SVV seropositivity and to determine SVV antibody titers in naturally and experimentally SVV-infected monkeys. In addition to being a useful diagnostic assay to rapidly confirm acute disease or past SVV infection, the SVV ELISA is a valuable epidemiological tool to determine the incidence of SVV in non-human primate facilities.

A virulent and pathogenic infectious clone of Senecavirus A.

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.

Pathogenicity of Seneca Valley virus in pigs and detection in Culicoides from an infected pig farm.

BACKGROUND: Porcine vesicular disease is caused by the Seneca Valley virus (SVV), it is a novel Picornaviridae, which is prevalent in several countries. However, the pathogenicity of SVV on 5-6 week old pigs and the transmission routes of SVV remain unknown. METHODS: This research mainly focuses on the pathogenicity of the CH-GX-01-2019 strain and the possible vector of SVV. In this study, 5-6 week old pigs infected with SVV (CH-GX-01-2019) and its clinical symptoms (including rectal temperatures and other clinical symptoms) were monitored, qRT-PCR were used to detect the viremia and virus distribution. Neutralization antibody assay was set up during this research. Mosquitoes and Culicoides were collected from pigsties after pigs challenge with SVV, and SVV detection within mosquitoes and Culicoides was done via RT-PCR. RESULTS: The challenged pigs presented with low fevers and mild lethargy on 5-8 days post infection. The viremia lasted more than 14 days. SVV was detected in almost all tissues on the 14th day following the challenge, and it was significantly higher in the hoofs (vesicles) and lymph nodes in comparison with other tissues. Neutralizing antibodies were also detected and could persist for more than 28 days, in addition neutralizing antibody titers ranged from 1:128 to 1:512. Mosquitoes and Culicoides were collected from the pigsty environments following SVV infection. Although SVV was not detected in the mosquitoes, it was present in the Culicoides, however SVV could not be isolated from the positive Culicoides. CONCLUSIONS: Our work has enriched the knowledge relating to SVV pathogenicity and possible transmission routes, which may lay the foundation for further research into the prevention and control of this virus.

Static and dynamic otolith reflex function in people with Parkinson's disease.

PURPOSE: Parkinson's disease (PD) is a neurodegenerative disorder with possible vestibular system dysfunction. This study reports the transient and sustained functions of the otoliths and their reflex pathways in PD compared to healthy controls (HC) and determines if otolith function relates to previous fall history. METHODS: Forty participants with PD and 40 HC had their otolith function assessed. Transient saccular and utricular-mediated reflexes were assessed by cervical and ocular vestibular evoked myogenic potentials (cVEMPs and oVEMPs, respectively) elicited by air-conducted stimulus (clicks) and bone-conducted vibration (light tendon hammer taps). Static otolith function was assessed by the Curator Subjective Visual Vertical (SVV) test. RESULTS: Compared to HC, the PD group had significantly more absent cVEMP responses to both clicks (47.5% vs. 30%, respectively, p = 0.03) and taps (21.8% vs. 5%, respectively, p = 0.002). Only the PD group had bilaterally absent tap cVEMPs, this was related to previous falls history (p < 0. 001). In both groups, click oVEMPs were predominantly absent, and tap oVEMPs were predominantly present. The PD group had smaller tap oVEMP amplitudes (p = 0.03) and recorded more abnormal SVV responses (p = 0.01) and greater error on SVV compared to HC, p < 0.001. SVV had no relationship with VEMP responses (p = 0.14). CONCLUSIONS: PD impacts on cVEMP reflex pathways but not tap oVEMP reflex pathways. Bone-conducted otolith stimuli (taps) are more robust than air-conducted sound stimuli (clicks) for both o and cVEMPs. A lack of association between SVV and VEMP responses suggest that static and dynamic otolith functions are differentially affected in PD.

Frontal scalp potentials foretell perceptual choice confidence.

When making decisions, people naturally ask two implicit questions: how soon can I make a decision, and how certain am I? In perception, people's confidence (how certain?) shows a nonmonotonic relationship with response time (how soon?), such that choice confidence can either increase or decrease with response time. Although a frontoparietal network has been implicated as a neural substrate that binds choice confidence and action (e.g., response time), the dynamic interplay between choice behaviors within such a network has not been clarified. Here, we show that frontal event-related potentials (ERPs) reflect choice confidence before a decision. Specifically, we report a second positive peak of the stimulus-locked frontal ERP at ~500 ms that scales with confidence but not stimulus level, whereas the centroparietal ERP amplitude covaries inversely with response time. This frontal ERP component occurs before the response, which helps explain the inverse relationship between choice confidence and response time (i.e., higher confidence for shorter response time) when choice accuracy is emphasized over speed. Our findings provide the first early neural representation of confidence, consistent with the temporal precedence for its causal role in the current decision-making task: "I decided earlier because I am confident."NEW & NOTEWORTHY We report novel neural correlates of predecisional choice confidence in frontal scalp potential in humans. In conjunction with the centroparietal choice-action event-related potential component, this new frontal choice confidence component further elucidates the dynamics of the frontoparietal decision-making neural circuitry.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets.

Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.

Management of Primary Small-Vessel Vasculitis.

Small-vessel vasculitides (SVV) are a group of disorders that occur due to primarily systemic inflammation or as sequelae of an infection, malignancy, or other rheumatic disease. Arising in any organ including the skin, the clinical features of SVV encompass a variety of manifestations. A comprehensive diagnostic assessment should be performed as management protocols widely differ. Although rare, physicians should be familiar with the common types of SVV to ensure prompt management and prevention of severe, life-threatening end-organ damage. Given the variable manifestations and associated etiologies of SVV, the following review aims to discuss the pathogenesis of more prevalent SVVs, highlight distinguishing features to aid in patient evaluation and diagnosis, and examine evidence-based management options for treatment and care.

Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques.

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

Adaptive Immune Responses following Senecavirus A Infection in Pigs.

Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of αß T cells, especially CD4+ T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8+ and double-positive CD4+ CD8+ T cells (effector/memory T cells) expressing interferon gamma (IFN-γ) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host.IMPORTANCE Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4+ T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.

Pathogenicity of two Chinese Seneca Valley virus (SVV) strains in pigs.

Seneca Valley virus (SVV) has been identified as the causative agent of SVV-associated vesicular disease (SAVD). To investigate the pathogenicity of two newly isolated SVV strains (GD-S5/2018 and GD04/2017) in China, experimental infections of pigs were performed. In pig experiments, both SVV strains successfully infected all animals, evidenced by presence of virus shedding and robust protective antibody responses. SVV GD-S5/2018 infection resulted in characteristic clinical signs, and ulcerative lesions on the tongue and gums. However, SVV GD04/2017 did not cause any clinical symptoms except depression in pigs during the experiment. Taken together, these results demonstrate that SVV GD-S5/2018 is a virulent strain for pigs, whereas SVV GD04/2017 is nearly avirulent. The established animal models for SVV infection will be utilized to dissect the immunity and pathogenesis, and develop vaccines and antivirals.

The bucket test differentiates patients with MRI confirmed brainstem/cerebellar lesions from patients having migraine and dizziness alone.

BACKGROUND: Amongst the most challenging diagnostic dilemmas managing patients with vestibular symptoms (i.e. vertigo, nausea, imbalance) is differentiating dangerous central vestibular disorders from benign causes. Migraine has long been recognized as one of the most common causes of vestibular symptoms, but the clinical hallmarks of vestibular migraine are notoriously inconsistent and thus the diagnosis is difficult to confirm. Here we conducted a prospective study investigating the sensitivity and specificity of combining standard vestibular and neurological examinations to determine how well central vestibular disorders (CVD) were distinguishable from vestibular migraine (VM). METHOD: Twenty-seven symptomatic patients diagnosed with CVD and 36 symptomatic patients with VM underwent brain imaging and clinical assessments including; 1) SVV bucket test, 2) ABCD2, 3) headache/vertigo history, 4) presence of focal neurological signs, 5) nystagmus, and 6) clinical head impulse testing. RESULTS: Mean absolute SVV deviations measured by bucket testing in CVD and VM were 4.8 ± 4.1° and 0.7 ± 1.0°, respectively. The abnormal rate of SVV deviations (> 2.3°) in CVD was significantly higher than VM (p < 0.001). Using the bucket test alone to differentiate CVD from VM, sensitivity was 74.1%, specificity 91.7%, positive likelihood ratio (LR+) 8.9, and negative likelihood ratio (LR-) 0.3. However, when we combined the SVV results with the clinical exam assessing gaze stability (nystagmus) with an abnormal focal neurological exam, the sensitivity (92.6%) and specificity (88.9%) were optimized (LR+ (8.3), LR- (0.08)). CONCLUSION: The SVV bucket test is a useful clinical test to distinguish CVD from VM, particularly when interpreted along with the results of a focal neurological exam and clinical exam for nystagmus.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus.

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells. METHODS: In this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-ß and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay. RESULTS: Gene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-ß and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-ß and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection. CONCLUSION: Altogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development.