Assessing the Efficacy of VLP-Based Vaccine against Epstein-Barr Virus Using a Rabbit Model.

Epstein-Barr virus (EBV) is etiologically associated with a number of malignant and non-malignant conditions. Thus, a prophylactic vaccine against this virus could help to reduce the burden of many EBV-associated diseases. Previously, we reported that an EBV virus-like particle (VLP) vaccine was highly immunogenic and produced a strong humoral response in mice. However, since EBV does not infect mice, the efficacy of the VLP in preventing EBV infection could not be addressed. Here we examined, for the first time, the efficacy of the EBV-VLP vaccine using a novel rabbit model of EBV infection. Animals vaccinated with two doses of VLP elicited higher antibody responses to total EBV antigens compared to animals receiving one dose. Vaccinated animals also elicited both IgM and IgG to EBV-specific antigens, VCA and EBNA1. Analysis of peripheral blood and spleen for EBV copy number indicated that the viral load in both of these compartments was lower in animals receiving a 2-dose vaccine. However, the VLP vaccine was ineffective in preventing EBV infection. With several other EBV vaccine candidates currently at various stages of development and testing, we believe that the rabbit model of EBV infection could be a great platform for evaluating potential candidates.

What do animal models tell us about the role of EBV in the pathogenesis of multiple sclerosis?

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS), marked primarily by demyelination, inflammation, and neurodegeneration. While the prevalence and incidence rates of MS are on the rise, the etiology of the disease remains enigmatic. Nevertheless, it is widely acknowledged that MS develops in persons who are both genetically predisposed and exposed to a certain set of environmental factors. One of the most plausible environmental culprits is Epstein-Barr virus (EBV), a common herpesvirus asymptomatically carried by more than 90% of the adult population. How EBV induces MS pathogenesis remains unknown. A comprehensive understanding of the biology of EBV infection and how it contributes to dysfunction of the immune system and CNS, requires an appreciation of the viral dynamics within the host. Here, we aim to outline the different animal models, including nonhuman primates (NHP), rodents, and rabbits, that have been used to elucidate the link between EBV and MS. This review particularly focuses on how the disruption in virus-immune interaction plays a role in viral pathogenesis and promotes neuroinflammation. We also summarize the effects of virus titers, age of animals, and route of inoculation on the neuroinvasiveness and neuropathogenic potential of the virus. Reviewing the rich data generated from these animal models could provide directions for future studies aimed to understand the mechanism(s) by which EBV induces MS pathology and insights for the development of prophylactic and therapeutic interventions that could ameliorate the disease.

Primary Peripheral Epstein-Barr Virus Infection Can Lead to CNS Infection and Neuroinflammation in a Rabbit Model: Implications for Multiple Sclerosis Pathogenesis.

Epstein-Barr virus (EBV) is a common herpesvirus associated with malignant and non-malignant conditions. An accumulating body of evidence supports a role for EBV in the pathogenesis of multiple sclerosis (MS), a demyelinating disease of the CNS. However, little is known about the details of the link between EBV and MS. One obstacle which has hindered research in this area has been the lack of a suitable animal model recapitulating natural infection in humans. We have recently shown that healthy rabbits are susceptible to EBV infection, and viral persistence in these animals mimics latent infection in humans. We used the rabbit model to investigate if peripheral EBV infection can lead to infection of the CNS and its potential consequences. We injected EBV intravenously in one group of animals, and phosphate-buffered saline (PBS) in another, with and without immunosuppression. Histopathological changes and viral dynamics were examined in peripheral blood, spleen, brain, and spinal cord, using a range of molecular and histopathology techniques. Our investigations uncovered important findings that could not be previously addressed. We showed that primary peripheral EBV infection can lead to the virus traversing the CNS. Cell associated, but not free virus in the plasma, correlated with CNS infection. The infected cells within the brain were found to be B-lymphocytes. Most notably, animals injected with EBV, but not PBS, developed inflammatory cellular aggregates in the CNS. The incidence of these aggregates increased in the immunosuppressed animals. The cellular aggregates contained compact clusters of macrophages surrounded by reactive astrocytes and dispersed B and T lymphocytes, but not myelinated nerve fibers. Moreover, studying EBV infection over a span of 28 days, revealed that the peak point for viral load in the periphery and CNS coincides with increased occurrence of cellular aggregates in the brain. Finally, peripheral EBV infection triggered temporal changes in the expression of latent viral transcripts and cytokines in the brain. The present study provides the first direct in vivo evidence for the role of peripheral EBV infection in CNS pathology, and highlights a unique model to dissect viral mechanisms contributing to the development of MS.

Uncovering early events in primary Epstein-Barr virus infection using a rabbit model.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of several malignant and non-malignant conditions. However, a number of fundamental aspects about the biology of EBV and the mechanism(s) by which this virus induces pathology remain unknown. One major obstacle has been the lack of a suitable animal model for EBV infection. In this study, using our recently established rabbit model of EBV infection, we examined the early events following primary EBV infection. We show that, both immunocompetent and immunosuppressed animals were readily susceptible to EBV infection. However, immunosuppressed animals showed marked splenomegaly and widespread infection. Following EBV infection, the virus primarily targeted naïve IgM+, CD20+, CD21+ and CD79a+ B cells. Infected cells expressed varying sets of viral latent/lytic gene products. Notably, co-expression of latent and lytic proteins in the same cell was not observed. Infected cells in type 0/1 latency (EBERs+), were small and proliferating (Ki67+). By contrast, cells in type 2/3 latency (LMP1+), were large, non-proliferating (Ki-67-) and p53+. Although infected B-cells were widely present in splenic follicles, they did not express germinal center marker, BCL-6. Taken together, this study shows for the first time, some of the early events following primary EBV infection.

Human-Animal Interaction and the Emergence of SARS-CoV-2.

The COVID-19 pandemic has affected all sectors of society, from health and economics to socialization and travel. The level and extent of this impact are unprecedented. Although the cause of COVID-19 was quickly identified to be a new coronavirus (SARS-CoV-2), the world was poorly prepared for preventing its spread. One important pillar of preparedness is surveillance of the sources of emerging pathogens and responding appropriately to prevent their spread in the human population. The ever-increasing interaction between humans and animals is one leading factor in facilitating the emergence of new pathogens. In this viewpoint, we discuss the possibility of the zoonotic origin of SARS-CoV-2, highlight the importance of understanding human-animal interaction to improve preparedness for future outbreaks, and outline recommendations for prevention.

Epstein-Barr Virus and miRNAs: Partners in Crime in the Pathogenesis of Multiple Sclerosis?

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression post transcriptionally. In healthy individuals, miRNAs contribute to maintaining gene expression homeostasis. However, the level of miRNAs expressed is markedly altered in different diseases, including multiple sclerosis (MS). The impact of such changes is being investigated, and thought to shape the immune system into the inflammatory autoimmune phenotype. Much is yet to be learned about the contribution of miRNAs in the molecular pathology of MS. Epstein-Barr virus (EBV) infection is a major risk factor for the development of MS. EBV encodes more than 40 miRNAs, most of which have been studied in the context of EBV associated cancers. These viral miRNAs regulate genes involved in cell apoptosis, antigen presentation and recognition, as well as B cell transformation. If EBV infection contributes to the pathology of MS, it is plausible that EBV miRNAs may be involved. Unfortunately, there are limited studies addressing how EBV miRNAs are involved in the pathogenesis of MS. This review summarizes what has been reported regarding cellular and viral miRNA profiles in MS and proposes possible interactions between the two in the development of MS.

The Effect of Chronic Consumption of Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Antagonists on Blood Pressure and Inotrope Consumption After Separation from Cardiopulmonary Bypass.

BACKGROUND: Chronic use of renin-angiotensin system (RAS) antagonists (angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor antagonists (ARAS)) can cause hypotension during anesthesia. In some studies hemodynamic instability, including hypotension and its effects on the clinical outcome in patients treated with these drugs during coronary artery bypass graft (CABG) and need to excessive vasoactive drugs in these patient population, has been described. The aim of this study was to evaluate the effect of chronic consumption of ACEIs and ARAS on blood pressure and inotrope consumption during coronary artery bypass graft under cardiopulmonary bypass. METHODS: A total of 200 patients undergoing coronary artery bypass graft surgery, who were treated with either ARAS or ACEIs (n = 100) over at least 2 months, or who were not treated with any RAS antagonists (control group, n = 100) were enrolled. The mean arterial blood pressure, central venous pressure, and need for vasoactive drugs, were measured after induction of anesthesia (T1) before cardiopulmonary bypass (T2) and after separation from (CPB), (T3). RESULTS: There were no significant differences regarding the mean arterial pressure (case group: T1: 84 ± 7 mmHg, T2: 77 ± 6 mmHg, T3: 83 ± 8 mmHg), (control group: T1: 85 ± 7 mmHg, T2: 81 ± 7 mmHg, T3:84 ± 6 mmHg) between two groups (P > 0.05). Also there were no significant differences regarding mean central venous pressure, mean heart rate, and vasoactive drug consumption between the two groups during the time of intervals. CONCLUSIONS: We found that preoperative (RAS) antagonist's continuation have not profound hemodynamic changes during coronary artery bypass graft under cardiopulmonary bypass and so we conclude that omitting these drugs before surgery did not have a sufficient advantage to be recommended routinely.

Epstein-Barr virus is present in the brain of most cases of multiple sclerosis and may engage more than just B cells.

Multiple sclerosis (MS) is a chronic neuroinflammatory condition of the central nervous system (CNS). It is a major cause of neurological disability in young adults, particularly women. What triggers the destruction of myelin sheaths covering nerve fibres is unknown. Both genetic and infectious agents have been implicated. Of the infectious agents, Epstein-Barr virus (EBV), a common herpesvirus, has the strongest epidemiological and serological evidence. However, the presence of EBV in the CNS and demonstration of the underlying mechanism(s) linking EBV to the pathogenesis of MS remain to be elucidated. We aimed at understanding the contribution of EBV infection in the pathology of MS. We examined 1055 specimens (440 DNA samples and 615 brain tissues) from 101 MS and 21 non-MS cases for the presence of EBV using PCR and EBER-in situ hybridization (EBER-ISH). EBV was detected by PCR and/or EBER-ISH in 91/101 (90%) of MS cases compared to only 5/21 (24%) of non-MS cases with other neuropathologies. None of the samples were PCR positive for other common herpesviruses (HSV-1, CMV, HHV-6). By quantitative PCR, EBV viral load in MS brain was mainly low to moderate in most cases. However, in 18/101 (18%) of MS cases, widespread but scattered presence of EBV infected cells was noted in the affected tissues by EBER-ISH. Immunohistochemical analysis of EBV gene expression in the 18 heavily infected cases, revealed that the EBV latent protein EBNA1, and to a lesser extent the early lytic protein BZLF1 were expressed. Furthermore, using double-staining we show for the first time that astrocytes and microglia, in addition to B-cells can also be infected. To the best of our knowledge, this is the most comprehensive study demonstrating that EBV is present and transcriptionally active in the brain of most cases of MS and supports a role for the virus in MS pathogenesis. Further studies are required to address the mechanism of EBV involvement in MS pathology.

No evidence for the presence of Epstein-Barr virus in squamous cell carcinoma of the mobile tongue.

Squamous cell carcinoma of the head and neck (SCCHN) comprises a large group of cancers in the oral cavity and nasopharyngeal area that typically arise in older males in association with alcohol/tobacco usage. Within the oral cavity, the mobile tongue is the most common site for tumour development. The incidence of tongue squamous cell carcinoma (TSCC) is increasing in younger people, which has been suggested to associate with a viral aetiology. Two common human oncogenic viruses, human papilloma virus (HPV) and Epstein-Barr virus (EBV) are known causes of certain types of SCCHN, namely the oropharynx and nasopharynx, respectively. EBV infects most adults worldwide through oral transmission and establishes a latent infection, with sporadic productive viral replication and release of virus in the oral cavity throughout life. In view of the prevalence of EBV in the oral cavity and recent data indicating that it infects tongue epithelial cells and establishes latency, we examined 98 cases of primary squamous cell carcinoma of the mobile tongue and 15 cases of tonsillar squamous cell carcinoma for the presence of EBV-encoded RNAs (EBERs), EBV DNA and an EBV-encoded protein, EBNA-1. A commercially available in situ hybridisation kit targeting EBER transcripts (EBER-ISH) showed a positive signal in the cytoplasm and/or nuclei of tumour cells in 43% of TSCCs. However, application of control probes and RNase A digestion using in-house developed EBER-ISH showed identical EBER staining patterns, indicating non-specific signals. PCR analysis of the BamH1 W repeat sequences did not identify EBV genomes in tumour samples. Immunohistochemistry for EBNA-1 was also negative. These data exclude EBV as a potential player in TSCC in both old and young patients and highlight the importance of appropriate controls for EBER-ISH in investigating EBV in human diseases.

Advanced 3D movement analysis algorithms for robust functional capacity assessment.

OBJECTIVES: We developed a novel system for in home functional capacities assessment in frail older adults by analyzing the Timed Up and Go movements. This system aims to follow the older people evolution, potentially allowing a forward detection of motor decompensation in order to trigger the implementation of rehabilitation. However, the pre-experimentations conducted on the ground, in different environments, revealed some problems which were related to KinectTM operation. Hence, the aim of this actual study is to develop methods to resolve these problems. METHODS: Using the KinectTM sensor, we analyze the Timed Up and Go test movements by measuring nine spatio-temporal parameters, identified from the literature. We propose a video processing chain to improve the robustness of the analysis of the various test phases: automatic detection of the sitting posture, patient detection and three body joints extraction. We introduce a realistic database and a set of descriptors for sitting posture recognition. In addition, a new method for skin detection is implemented to facilitate the patient extraction and head detection. 94 experiments were conducted to assess the robustness of the sitting posture detection and the three joints extraction regarding condition changes. RESULTS: The results showed good performance of the proposed video processing chain: the global error of the sitting posture detection was 0.67%. The success rate of the trunk angle calculation was 96.42%. These results show the reliability of the proposed chain, which increases the robustness of the automatic analysis of the Timed Up and Go. CONCLUSIONS: The system shows good measurements reliability and generates a note reflecting the patient functional level that showed a good correlation with 4 clinical tests commonly used. We suggest that it is interesting to use this system to detect impairment of motor planning processes.

A simple procedure for the extraction of DNA from long-term formalin-preserved brain tissues for the detection of EBV by PCR.

Long-term formalin fixed brain tissues are potentially an important source of material for molecular studies. Ironically, very few protocols have been published describing DNA extraction from such material for use in PCR analysis. In our attempt to investigate the role of Epstein-Barr virus (EBV) in the pathogenesis of multiple sclerosis (MS), extracting PCR quality DNA from brain samples fixed in formalin for 2-22 years, proved to be very difficult and challenging. As expected, DNA extracted from these samples was not only of poor quality and quantity, but more importantly, it was frequently found to be non-amplifiable due to the presence of PCR inhibitors. Here, we describe a simple and reproducible procedure for extracting DNA using a modified proteinase K and phenol-chloroform methodology. Central to this protocol is the thorough pre-digestion washing of the tissues in PBS, extensive digestion with proteinase K in low SDS containing buffer, and using low NaCl concentration during DNA precipitation. The optimized protocol was used in extracting DNA from meninges of 26 MS and 6 non-MS cases. Although the quality of DNA from these samples was generally poor, small size amplicons (100-200 nucleotides) of the house-keeping gene, ß-globin could be reliably amplified from all the cases. PCR for EBV revealed positivity in 35% (9/26) MS cases, but 0/6 non-MS cases. These findings indicate that the method described here is suitable for PCR detection of viral sequences in long-term formalin persevered brain tissues. Our findings also support a possible role for EBV in the pathogenesis of MS.

Kinematic analysis of motor strategies in frail aged adults during the Timed Up and Go: how to spot the motor frailty?

OBJECTIVE: The purpose of this work was to analyze and compare the movement kinematics of sit-to-stand (STS) and back-to-sit (BTS) transfers between frail aged adults and young subjects, as well as to determine the relationship between kinematic changes and functional capacities. METHODS: We analyzed the Timed Up and Go (TUG) movements by using a 3D movement analysis system for real-time balance assessment in frail elderly. Ten frail aged adults (frail group [FG]) and ten young subjects (young group [YG]) performed the TUG. Seven spatiotemporal parameters were extracted and compared between the two groups. Moreover, these parameters were plotted with TUG test duration. RESULTS: The experiments revealed that there were significant differences between FG and YG in trunk angle during both STS and BTS, and in TUG duration. The trunk angle of the young subjects was more than two times higher than that of the FG. As expected, the TUG duration was higher in the FG than in YG. Trunk angles during both transfers were the most different parameters between the groups. However, the BTS trunk angle and STS ratio were more linked to functional capacities. CONCLUSION: There was a relationship between kinematic changes, representing the motor planning strategies, and physical frailty in these aged adults. These changes should be taken into account in clinical practice.