Case Series of Jamestown Canyon Virus Infections with Neurologic Outcomes, Canada, 2011-2016.

Jamestown Canyon virus (JCV) is a mosquitoborne orthobunyavirus in the California serogroup that circulates throughout Canada and the United States. Most JCV exposures result in asymptomatic infection or a mild febrile illness, but JCV can also cause neurologic diseases, such as meningitis and encephalitis. We describe a case series of confirmed JCV-mediated neuroinvasive disease among persons from the provinces of British Columbia, Alberta, Quebec, and Nova Scotia, Canada, during 2011-2016. We highlight the case definitions, epidemiology, unique features and clinical manifestations, disease seasonality, and outcomes for those cases. Two of the patients (from Quebec and Nova Scotia) might have acquired JCV infections during travel to the northeastern region of the United States. This case series collectively demonstrates JCV's wide distribution and indicates the need for increased awareness of JCV as the underlying cause of meningitis/meningoencephalitis during mosquito season.

Does hepatitis delta virus have a preference for hepatitis B virus genotype? A systematic review of the literature.

Hepatitis delta virus (HDV) is a deficient virus that requires the surface proteins of Hepatitis B virus (HBV) to complete its replication. HDV is thus only found in those already infected with HBV (~5% worldwide). There are eight different HDV genotypes (1-8) and 10 HBV genotypes (A-J), each having fairly distinct geographic distributions. While their pairings may be coincidental based on epidemiological occurrence, some evidence exists regarding possible virologic basis for genotype dominance and patterns. Here we sought to determine which HBV genotype is most often linked with active HDV infection and speculate on whether this may represent a viral 'preference'. Electronic databases with OVID Medline were comprehensively searched for studies published between 1977 and 2022 indexing the word 'genotype' and all permutations of 'HDV' (hepatitis D virus, hepatitis delta, etc.). Primary studies of patient samples reporting genotype data for either or both of HDV and HBV were tabulated. The initial search revealed 419 articles and was narrowed to 133 studies reporting genotype data for either or both HBV and HDV. We limited our search to cases with detectable HDV RNA. These represented over 5800 samples from over 70 countries. Of these, 1947 samples had paired genotype data for both viruses. The most common pairing was HDV-1 with HBV-D, but it remains unclear whether this represents a viral 'preference' or mere co-endemicity of the two viruses. Determining if there is a virologic link between HBV and HDV genotypes may have important implications for emerging HDV and HBV research.

Special Issue of the 4th Symposium of the Canadian Society for Virology 2022.

In this Special Issue of Viruses, we showcase some of the fascinating and diverse virology being undertaken in Canada that was presented at the 4th Symposium of the Canadian Society for Virology 2022 [...].

The domestication of SARS-CoV-2 into a seasonal infection by viral variants.

Introduction: The COVID-19 pandemic was caused by the zoonotic betacoronavirus SARS-CoV-2. SARS-CoV-2 variants have emerged due to adaptation in humans, shifting SARS-CoV-2 towards an endemic seasonal virus. We have termed this process 'virus domestication'. Methods: We analyzed aggregate COVID-19 data from a publicly funded healthcare system in Canada from March 7, 2020 to November 21, 2022. We graphed surrogate calculations of COVID-19 disease severity and SARS-CoV-2 variant plaque sizes in tissue culture. Results and Discussion: Mutations in SARS-CoV-2 adapt the virus to better infect humans and evade the host immune response, resulting in the emergence of variants with altered pathogenicity. We observed a decrease in COVID-19 disease severity surrogates after the arrival of the Delta variant, coinciding with significantly smaller plaque sizes. Overall, we suggest that SARS-CoV-2 has become more infectious and less virulent through viral domestication. Our findings highlight the importance of SARS-CoV-2 vaccination and help inform public policy on the highest probability outcomes during viral pandemics.

G4-quadruplex-binding proteins: review and insights into selectivity.

There are over 700,000 putative G4-quadruplexes (G4Qs) in the human genome, found largely in promoter regions, telomeres, and other regions of high regulation. Growing evidence links their presence to functionality in various cellular processes, where cellular proteins interact with them, either stabilizing and/or anchoring upon them, or unwinding them to allow a process to proceed. Interest in understanding and manipulating the plethora of processes regulated by these G4Qs has spawned a new area of small-molecule binder development, with attempts to mimic and block the associated G4-binding protein (G4BP). Despite the growing interest and focus on these G4Qs, there is limited data (in particular, high-resolution structural information), on the nature of these G4Q-G4BP interactions and what makes a G4BP selective to certain G4Qs, if in fact they are at all. This review summarizes the current literature on G4BPs with regards to their interactions with G4Qs, providing groupings for binding mode, drawing conclusions around commonalities and highlighting information on specific interactions where available.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Prevalence and genetic variability of occult hepatitis B virus in a human immunodeficiency virus positive patient cohort in Gondar, Ethiopia.

BACKGROUND: Occult hepatitis B (OHB) is a major concern in HIV infected patients as it associates with a high risk of HBV reactivation and disease progression. However, data on the prevalence of OHB among HIV positive patients in Ethiopia is lacking. This study aims to determine the prevalence of OHB in HBV/HIV co-infected patients from Gondar, Ethiopia. METHODS: A total of 308 consented HIV positive patients were recruited from the University of Gondar Teaching Hospital, Ethiopia. Clinical and demographic data of the participants were recorded. Plasma was tested for HBsAg and anti-HBc using commercial assays (Abbott Architect). In HBsAg negative anti-HBc positive patient samples, total DNA was isolated and amplified using nested PCR with primers specific to HBV polymerase, surface and pre-core/core regions, followed by Sanger sequencing and HBV mutational analysis using MEGA 7.0. RESULTS: Of the total study subjects, 62.7% were female, median age 38.4 years, interquartile range (IQR): 18-68, and 208 (67.5%) had lifestyle risk factors for HBV acquisition. Two hundred and ninety-one study subjects were HIV+/HBsAg-, out of which 115 (39.5%) were positive for anti-HBc. Occult hepatitis B was detected in 19.1% (22/115) of anti-HBc positive HIV patients. HBV genotype D was the predominant genotype (81%) among OHB positive patients. Mutations associated with HBV drug resistance, HBV reactivation, and HCC risk were detected in 23% (5/22), 14% (3/22) and 45.5% (10/22) of patients, respectively. CONCLUSION: This study found a high rate of occult hepatitis B in HIV patients. Further, high rates of mutations associated with HBV reactivation, drug resistance, and HCC risk were detected in these patients. These data highlighted the need for integrating OHB screening for proper management of liver diseases in HIV patients.

Host Transcription Factors in Hepatitis B Virus RNA Synthesis.

The hepatitis B virus (HBV) chronically infects over 250 million people worldwide and is one of the leading causes of liver cancer and hepatocellular carcinoma. HBV persistence is due in part to the highly stable HBV minichromosome or HBV covalently closed circular DNA (cccDNA) that resides in the nucleus. As HBV replication requires the help of host transcription factors to replicate, focusing on host protein-HBV genome interactions may reveal insights into new drug targets against cccDNA. The structural details on such complexes, however, remain poorly defined. In this review, the current literature regarding host transcription factors' interactions with HBV cccDNA is discussed.

Prevalence and molecular characterization of occult hepatitis B virus in pregnant women from Gondar, Ethiopia.

Background: The greatest risk of chronic hepatitis B (CHB) is from mother-to-child transmission. Approximately 20% of individuals in sub-Saharan Africa are hepatitis B virus (HBV) surface antigen-positive (HBsAg+), but the prevalence of occult hepatitis B (OHB) is unknown. Aim: This study investigated CHB and OHB prevalence and viral variants in a cohort of pregnant women in Gondor, Ethiopia. Methods: Patients were prospectively recruited from the University of Gondar Hospital (N = 200; median age 27 [inter-quartile range] 8.3y) from March through June 2016. Data were collected using an investigator-administered questionnaire. Plasma was tested for HBsAg and HBV core antibody (anti-HBc), and HBV genotype and presence of HBV variants (ie, vaccine escape mutants [VEMs]) were determined by polymerase chain reaction, Sanger sequencing, and phylogenetic analysis. Results: Of women tested, 1% (2/200) were HBsAg+; 26.8% (47/182) of HBsAg-negative patients were anti-HBc+, of whom 37/47 (78.7%) had detectable HBV DNA. The overall rate of OHB was 20.3%. Both HBsAg+ cases were HBV genotype D, and 36/37 (97.3%) of OHB individuals were genotype D. None carried VEM, but both HBsAg+ cases and 32/37 (86.5%) of the OHB cases showed lamivudine-resistant mutations. Conclusions: Twenty-seven percent of pregnant women in this cohort showed evidence of CHB or prior HBV exposure (ie, HBsAg+ or anti-HBc+) and clinically relevant HBV variants. Data from this single-centre study suggests high HBV prevalence, reinforcing the World Health Organization's recommendation for universal prenatal HBV screening and infant vaccination.

Current approaches for RNA-labelling to identify RNA-binding proteins.

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.

Targeted Polo-like Kinase Inhibition Combined With Aurora Kinase Inhibition in Pediatric Acute Leukemia Cells.

BACKGROUND: Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. MATERIALS AND METHODS: We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. RESULTS: Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. DISCUSSION: This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.

Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutations.

Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytes' nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (A⁻H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virus' viability. Such findings may have key implications for designing antivirals to target these areas.

DEAD-box helicases: the Yin and Yang roles in viral infections.

Viruses hijack the host cell machinery and recruit host proteins to aid their replication. Several host proteins also play vital roles in inhibiting viral replication. Emerging class of host proteins central to both of these processes are the DEAD-box helicases: a highly conserved family of ATP-dependent RNA helicases, bearing a common D-E-A-D (Asp-Glu-Ala-Asp) motif. They play key roles in numerous cellular processes, including transcription, splicing, miRNA biogenesis and translation. Though their sequences are highly conserved, these helicases have quite diverse roles in the cell. Interestingly, often these helicases display contradictory actions in terms of the support and/or clearance of invading viruses. Increasing evidence highlights the importance of these enzymes, however, little is known about the structural basis of viral RNA recognition by the members of the DEAD-box family. This review summarizes the current knowledge in the field for selected DEAD-box helicases and highlights their diverse actions upon viral invasion of the host cell. We anticipate that through a better understanding of how these helicases are being utilized by viral RNAs and proteins to aid viral replication, it will be possible to address the urgent need to develop novel therapeutic approaches to combat viral infections.

The HIV protease inhibitor, nelfinavir, as a novel therapeutic approach for the treatment of refractory pediatric leukemia.

PURPOSE: Refractory pediatric leukemia remains one of the leading causes of death in children. Intensification of current chemotherapy regimens to improve the outcome in these children is often limited by the effects of drug resistance and cumulative toxicity. Hence, the search for newer agents and novel therapeutic approaches are urgently needed to formulate the next-generation early-phase clinical trials for these patients. MATERIALS AND METHODS: A comprehensive library of antimicrobials, including eight HIV protease inhibitors (nelfinavir [NFV], saquinavir, indinavir, ritonavir, amprenavir, atazanavir, lopinavir, and darunavir), was tested against a panel of pediatric leukemia cells by in vitro growth inhibition studies. Detailed target modulation studies were carried out by Western blot analyses. In addition, drug synergy experiments with conventional and novel antitumor agents were completed to identify effective treatment regimens for future clinical trials. RESULTS: Several of the HIV protease inhibitors showed cytotoxicity at physiologically relevant concentrations (half-maximal inhibitory concentration values ranging from 1-24 µM). In particular, NFV was found to exhibit the most potent antileukemic properties across all cell lines tested. Mechanistic studies show that NFV leads to the induction of autophagy and apoptosis possibly through the induction of endoplasmic reticulum stress. Furthermore, interference with cell signaling pathways, including Akt and mTOR, was also noted. Finally, drug combination studies have identified agents with potential for synergy with NFV in its antileukemic activity. These include JQ1 (BET inhibitor), AT101 (Bcl-2 family inhibitor), and sunitinib (TK inhibitor). CONCLUSION: Here, we show data demonstrating the potential of a previously unexplored group of drugs to address an unmet therapeutic need in pediatric oncology. The data presented provide preclinical supportive evidence and rationale for future studies of these agents for refractory leukemia in children.

In Vitro Sensitivity Profiling of Neuroblastoma Cells Against A Comprehensive Small Molecule Kinase Inhibitor Library to Identify Agents for Future Therapeutic Studies.

BACKGROUND: Neuroblastoma (NB) constitutes about 8% of all childhood tumors, yet accounts for more than 15% of deaths, with an unacceptable overall survival rate. These rates are despite the current multimodal therapeutic approaches involving surgery, radiation, chemotherapy and myeloablation with hematopoietic stem cell rescue. Hence, efforts have intensified to identify new targets and novel therapeutic approaches to improve cure rates in these children. Numerous new agents for adult malignancies are developed and evaluated for cancer each year, providing an invaluable resource, with the added advantage of available pharmacologic and toxicity data for consideration. METHODS: To identify potential therapeutic targets, we screened a small molecule library of 151 small kinase inhibitors against NB cell lines. Based on our initial screening data, we further examined the potential of Bcr-Abl targeting small molecule inhibitors to affect the growth and survival of NB cells. RESULTS: There is diverse activity among the currently available Bcr-Abl inhibitors, possibly reflecting the molecular heterogeneity and off-target activity in each combination. In depth analyses of ponatinib, an oral multi-target kinase inhibitor and effective agent in the treatment of refractory Philadelphia chromosome (Ph) positive leukemia, show growth inhibition at sub-micromolar concentrations. In addition, we also identified the potential of this agent to interfere with insulin-like growth factor-1 receptor (IGF-1R) signaling pathways and Src activity, inhibit cell migration and induce apoptosis. CONCLUSION: Our findings provide initial data on ponatinib's potential to target key growth regulatory pathways and provide the rationale for further studies and evaluation in future early phase clinical trials for the treatment of refractory NB.

Effects of a pharmacy-driven perisplenectomy vaccination program on vaccination rates and adherence to guidelines.

BACKGROUND: Overwhelming postsplenectomy infection is a serious potential outcome for patients who have undergone resection of the spleen and is associated with a high mortality rate. The most common bacterial causes are the encapsulated organisms Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae type B, all of which are vaccine-preventable. Current guidelines recommend vaccination against these 3 bacteria, but adherence to these guidelines is less than ideal. In 2007, a "perisplenectomy vaccination kit" was introduced at the authors' institution to improve compliance with immunization guidelines by making the vaccines and necessary information for patients and providers more readily available. OBJECTIVE: To evaluate and compare vaccination rates for patients who underwent splenectomy before and after introduction of the perisplenectomy vaccination kit and, secondarily, to identify any characteristics unique to those who did not receive appropriate perisplenectomy vaccinations. METHODS: In this observational study, performed at the QEII Health Sciences Centre of Capital Health in Halifax, Nova Scotia, data were reviewed for patients who underwent splenectomy between 2008 and 2011. Vaccination rates and other descriptive statistics were calculated and compared with data for a 3-year period before implementation of the program. RESULTS: Vaccination rates in the 3-year period following implementation of the perisplenectomy vaccination kit were 100% against S. pneumoniae, 97% against N. meningitidis, and 93% against H. influenzae type B. The corresponding rates in the 3 years before introduction of the kit were 91%, 75%, and 68%, respectively. No characteristics predicting inadequate immunization were identified in univariate or multivariate analysis. CONCLUSION: Introduction of a pharmacy-driven perisplenectomy vaccination kit program improved rates of appropriate vaccination for patients who underwent splenectomy.

CONTEXTE: L'infection fulminante post-splénectomie, une complication potentielle sérieuse chez les patients ayant subi une ablation de la rate, présente un taux de mortalité élevé. Les causes bactériennes les plus fréquentes de cette infection sont les bactéries encapsulées Streptococcus pneumoniae, Neisseria meningitidis et Haemophilus influenzae de type b, qui peuvent toutes être prévenues par un vaccin. Les lignes directrices actuelles recommandent une vaccination contre ces trois espèces de bactéries, mais le respect de ces lignes directrices est loin d'être idéal. En 2007, une « trousse de vaccination périsplénectomie ¼ a été adoptée dans l'établissement des auteurs. Cette trousse avait pour but d'améliorer le degré de conformité aux lignes directrices sur l'immunisation en facilitant l'accès aux vaccins et aux renseignements nécessaires pour les patients et les professionnels de la santé. OBJECTIF: Déterminer quels étaient les taux de vaccination des patients ayant subi une splénectomie avant et après la mise en place de la « trousse de vaccination périsplénectomie ¼, puis comparer ces valeurs; et identifier toute caractéristique propre à ceux qui n'ont pas reçu les vaccins nécessaires durant la période entourant la splénectomie. MÉTHODES: Dans cette étude observationnelle menée au QEII Health Sciences Centre de Capital Health à Halifax (Nouvelle-Écosse), on a procédé à l'analyse des données concernant les patients ayant subi une splénectomie entre 2008 et 2011. Les taux de vaccination ainsi que d'autres statistiques descriptives ont été calculés et comparés à des données d'une période de trois ans avant la mise en œuvre du programme d'utilisation de la « trousse de vaccination périsplénectomie ¼. RÉSULTATS: Les taux de vaccination de la période de trois ans qui a suivi la mise en œuvre de la « trousse de vaccination périsplénectomie ¼ étaient de 100 % pour S. pneumoniae, 97 % pour N. meningitidis et 93 % pour H. influenzae de type b. Les taux correspondants des trois années précédant cette mise en œuvre étaient respectivement de 91 %, 75 % et 68 %. Aucun prédicteur d'une immunisation insuffisante n'a été cerné par l'analyse univariée ou multivariée. CONCLUSION: L'implantation d'un programme de « trousse de vaccination périsplénectomie ¼ mené par les pharmaciens a permis d'accroître les taux d'administration des vaccins qui sont indiqués chez les patients ayant subi une splénectomie. [Traduction par l'éditeur].