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Viruses compartmentalize their replication and assembly machinery to both evade detection and concentrate the viral proteins and nucleic acids necessary for genome replication and virion production. Accumulating evidence suggests that diverse RNA and DNA viruses form replication organelles and nucleocapsid assembly sites using phase separation. In general, the biogenesis of these compartments is regulated by two types of viral protein, collectively known as antiterminators and nucleocapsid proteins, respectively. Herein, we discuss how RNA viruses establish replication organelles and nucleocapsid assembly sites, and the evidence that these compartments form through phase separation. While this review focuses on RNA viruses, accumulating evidence suggests that all viruses rely on phase separation and form biomolecular condensates important for completing the infectious cycle.

Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or ‘riboswitch’, allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5′ untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including selection of adaptive mutations and emergence of new variant lineages. Here we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ∼⅔ were plausibly introduced into these animal species from local human populations, while the remaining ∼⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health.ImportanceSARS-CoV-2, the causative agent of COVID-19, can infect many animal species, making eradication difficult because it can be reseeded from different reservoirs. When viruses replicate in different species, they may be faced with different evolutionary pressures and acquire new mutations, with unknown consequences for transmission and virulence in humans. Here we analyzed SARS-CoV-2 genome sequences from cats, dogs, deer, and mink to estimate transmission between each of these species and humans. We found several transmission events from humans to each animal, but relatively few detectable transmissions from animals back to humans, with the exception of mink. We also identified three mutations more likely to be found in mink than humans, and 26 in deer. These mutations could help the virus adapt to life in these different species. Ongoing surveillance of SARS-CoV-2 from animals will be important to understand their potential impacts on both human and animal health.

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is widely used to quantify viral RNA genomes for diagnostics and research, yet conventional RT-qPCR protocols are unable to accurately distinguish between the different viral RNA species that exist during infection. Here we show that false-priming and self-priming occur during reverse transcription with several published Zika virus (ZIKV) primer sets. We developed a RT-qPCR assay using tagged primers and thermostable reverse transcriptase, which greatly reduced the occurrence of nonspecific cDNA products. Furthermore, we optimized the assay for use in multiplex qPCR which allows for simultaneous quantitative detection of positive-strand and negative-strand ZIKV RNA along with an internal control from both human and mosquito cells. Importantly, this assay is sensitive enough to study early stages of virus infection in vitro. Strikingly, using this assay, we detected ZIKV negative-strand RNA as early as 3 h post-infection in mammalian cell culture, at a time point prior to the onset of positive-strand RNA synthesis. Overall, the strand-specific RT-qPCR assay developed herein is a valuable tool to quantify ZIKV RNA and to study viral replication dynamics during infection. The application of these findings has the potential to increase accuracy of RNA detection methods for a variety of viral pathogens.HighlightsSelf-primed cDNA is amplified by widely-used ZIKV qPCR primer setsUse of tagged primers and thermostable RT increases strand-specificity for RT-qPCRMultiplexed qPCR allows for simultaneous quantitation of (+) and (-) strand viral RNAs, and an internal controlStrand-specific RT-qPCR can detect fewer than one copy of viral RNA per cell in human and mosquito cells

Poly(rC)-binding protein 2 (PCBP2) was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, we found that PCBP2 does not directly affect viral entry, translation, genome stability, replication, or virion egress. Rather, our data suggests that endogenous PCBP2 normally limits virion assembly, thereby indirectly promoting translation and replication by increasing the translating/replicating pool of viral RNAs. Additionally, we found that an alternative RNA conformation (SLIIalt) was important for efficient virion assembly, but functions in a PCBP2-independent manner. The latter may explain why the Japanese fulminant hepatitis 1 isolate is able to produce infectious particles in cell culture, while other HCV isolates are lost in translation. Taken together, our results suggest that PCBP2 and SLIIalt independently modulate HCV genome packaging and alter the balance of viral RNAs in the translating/replicating pool and those engaged in virion assembly.Graphical Abstract

Significance MicroRNA (miRNA)–based drugs are quickly taking the clinic by storm. Herein, we analyzed resistance-associated variants (RAVs) to the first miRNA inhibitors to make it to the clinic, namely miR-122 inhibitors for chronic hepatitis C virus (HCV) infection. We uncovered three distinct resistance mechanisms based on unique alterations to the structure of the viral RNA. Specifically, RAVs altered the structure of the viral RNA in a manner that promotes riboswitch activity, genome stability, or positive-strand viral RNA synthesis. Our findings support recent models of miR-122–mediated HCV RNA accumulation and provide mechanism(s) of resistance to antiviral therapy. These early insights into the mechanism(s) of resistance to miRNA-based therapies may be of importance as more miRNA-targeted therapies enter into the clinic., Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5′ untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor–based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122–mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

Background The leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV). Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis. Methodology/Principal findings In vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV’s hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone. Conclusion/Significance Overall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness., Author summary The leishmaniases are a group of chronic infectious diseases caused by parasites of the genus Leishmania. The disease poses a great public health risk; millions of people are affected annually. In addition, leishmaniasis is characterized by widely varying clinical manifestations, for which the cause is still unknown. Parasites of the genus Leishmania are the ethological agents of the disease and are vectored by phlebotomine sandflies. While examining sections of the sandfly midgut, we noted viral entities, later identified as phleboviruses. Interestingly, there is a large overlap in the geographic distribution of L. major and Sandfly Fever Sicilian Virus (SFSV); this led us to hypothesize that viral presence could influence disease severity. With the use of various wild-type and knock-out mouse models, we deduced that co-infection with SFSV indeed affected disease severity, leading to exacerbated skin lesions and higher parasite numbers. However, this was not paralleled by the modulation of immunomodulatory molecules. Our findings demonstrate that viral presence may facilitate parasite entry into host cells. Overall, our research constitutes novel findings that contribute toward a better understanding of the mechanisms underlying leishmaniasis infections.

Objective:To circumvent the need for rationing personal protective equipment (PPE), we explored whether germicidal ultraviolet light (GUV) could be used to inactivate human coronaviruses on PPE, enabling safe reuse.Design:We performed a laboratory study to assess the ability of 2 commercially available portable GUV devices to inactivate 2 common cold coronaviruses (HCoV-229E and HCoV-OC43) and severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), on the surface of whole N95 respirators and coupons cut from those respirators. We experimentally contaminated N95 respirators with coronavirus cultures and then assessed viral inactivation after GUV exposure by plaque assay, the median tissue culture infectious dose (TCID50) assay, and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR).Results:We found that GUV could efficiently inactivate coronaviruses on the surface of N95 masks, with an average reduction in viral titers of 5-log for HCoV-229E, 3-log for HCoV-OC43, and 5-log for SARS-CoV-2. In addition, the GUV susceptibility of HCoV-229E was similar on coupons and whole N95 respirators.Conclusions:We demonstrate that diverse human coronaviruses, including SARS-CoV-2, are susceptible to GUV inactivation, and 2 scalable portable GUV devices were effective in inactivating coronaviruses on N95 respirators. Thus, GUV treatment with commercially scalable devices may be an effective method to decontaminate PPE, allowing their safe reuse.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5′ untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5′ UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1–117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5′ terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5′ UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5′ terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES–40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5′ terminus and promote formation of the HCV IRES.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to two adjacent sites (S1 and S2) located at the 5' end of the viral RNA genome. This interaction promotes HCV RNA accumulation by stabilising the viral RNA and resulting in alteration of the secondary structure of the viral genome. In addition to S1 and S2, the HCV genome contains several other putative miR-122 binding sites, one in the IRES region, three in the NS5B coding region, and one in the 3' UTR. We investigated and compared the relative contributions of the S1, S2, IRES, NS5B (NS5B.1, 2 and 3) and 3' UTR sites on protein expression, viral RNA accumulation, and infectious particle production by mutational analysis and supplementation with compensatory mutant miR-122 molecules. We found that mutations predicted to alter miR-122 binding at the IRES and NS5B.2 sites lead to reductions in HCV core protein expression and viral RNA accumulation; with a concomitant decrease in viral particle production for the NS5B.2 mutant. However, supplementation of miR-122 molecules with compensatory mutations did not rescue these site mutants to wild-type levels, suggesting that mutation of these sequences likely disrupts an additional interaction important to the HCV life cycle, beyond direct interactions with miR-122. Thus, S1 and S2 play a predominant role in viral RNA accumulation, while miR-122 interactions with the IRES, NS5B and 3' UTR regions have negligible contributions to viral protein expression, viral RNA accumulation, and infectious particle production.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread., SUMMARY The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

Virion assembly is an important step in the life cycle of all viruses. For viruses of the Flavivirus genus, a group of enveloped positive-sense RNA viruses, the assembly step represents one of the least understood processes in the viral life cycle. While assembly is primarily driven by the viral structural proteins, recent studies suggest that several nonstructural proteins also play key roles in coordinating the assembly and packaging of the viral genome. This review focuses on describing recent advances in our understanding of flavivirus virion assembly, including the intermolecular interactions between the viral structural (capsid) and nonstructural proteins (NS2A and NS2B-NS3), host factors, as well as features of the viral genomic RNA required for efficient flavivirus virion assembly.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a human-liver-specific microRNA, termed miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) of the viral genome, and this interaction promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been demonstrated to be efficacious in reducing HCV titers in chronic HCV-infected patients. However, the precise mechanism(s) of miR-122-mediated viral RNA accumulation have remained elusive. We have used biophysical analysis and assays for viral replication in cell culture to understand the interactions between the human Argonaute 2 (hAgo2):miR-122 complex and the HCV genome. In addition, we have analyzed several resistance-associated variants which were isolated from patients who underwent miR-122 inhibitor-based therapy in order to shed light on novel mechanisms of antiviral resistance. Our results provide a new model for miR-122:HCV RNA interactions and demonstrate that miR-122 plays at least three roles in the HCV life cycle: (1) miR-122 acts as an RNA chaperone to suppress an energetically favorable secondary structure and allows the viral internal ribosomal entry site (IRES) to form; (2) miR-122 binding to the 5' terminus protects the genome from the activity of cellular pyrophosphatases (DOM3Z and DUSP11) and subsequent exonuclease-mediated decay; and (3) the Argonaute (Ago) protein at Site 2 makes direct contact with the HCV IRES, enhancing viral translation. In addition, analyses of several resistance-associated variants that were isolated from patients that underwent miR-122 inhibitor-based therapy suggests that mutations in the 5' terminus alter the structure of the 5' UTR in a manner that promotes RNA chaperone activity or viral genome stability, even in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and suggest new mechanisms of antiviral resistance which are mediated by changes in RNA structure.

Hepatitis C virus (HCV) affects approximately 250,000 Canadians. Although safe and effective (>95% cure rates) antiviral therapies have become available within the past 5 years, chronic HCV infection still remains a major driver of end-stage liver disease and liver transplantation. Both the Canadian Institute for Health Research and the Public Health Agency of Canada recognize the impact of HCV-related liver diseases and support the Canadian Network for Hepatitis C (CanHepC), a National network for the scientific study of hepatitis C that organizes an annual symposium as part of its knowledge translation mandate. At the 8th Canadian Symposium on Hepatitis C Virus in May 2019, basic scientists, clinicians, epidemiologists, social scientists, and community members came together to share their work under the theme of “Improving diagnosis and linkage to care”. This symposium also marked the launch of the Blueprint to inform hepatitis C elimination efforts in Canada, a policy framework that outlines specific targets, suggested activities, and evidence-based best practices to guide provincial, territorial and federal organizations developing their own HCV elimination strategies.

Estimates are that more than 250,000 people in Canada are chronically infected with hepatitis C virus (HCV), and many more are unaware of their infection status. If untreated, chronic HCV infection can lead to cirrhosis and subsequent complications such as hepatocellular carcinoma. The Canadian Network on Hepatitis C, supported by the Public Health Agency of Canada and the Canadian Institutes of Health Research, has been committed to the scientific study of chronic hepatitis C and to supporting the advocacy work to improve diagnosis and access to HCV care in Canada. Although the treatment of HCV infection has been greatly advanced with direct-acting antivirals, with cure rates as high as 95%, many challenges remain in the implementation of HCV care. These issues include the lack of an effective vaccine, infection screening, treatment failure or resistance, post-cure health issues, limitations of treatment access despite increased provincial subsidization, complex needs of at-risk populations (ie, injection drug users, societal obstacles). At the 6th Canadian Symposium on HCV in March 2017, the theme “Delivering a Cure for Hepatitis C Infection: What Are the Remaining Gaps?” provided a framework in which basic scientists, clinicians, epidemiologists, social scientists, and community members interested in HCV research in Canada could showcase how they are working to address these ongoing challenges.

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Health care–associated hepatitis C virus outbreaks from contaminated medication vials continue to be reported even though most practitioners deny reusing needles or syringes. The hypothesis was that when caring for hepatitis C virus–infected patients, healthcare providers may inadvertently contaminate the medication vial diaphragm and that subsequent access with sterile needles and syringes can transfer hepatitis C virus into the medication, where it remains stable in sufficient quantities to infect subsequent patients. Methods A parallel-arm lab study (n = 9) was performed in which contamination of medication vials in healthcare settings was simulated using cell culture–derived hepatitis C virus. First, surface-contaminated vials were accessed with sterile needles and syringes, and then hepatitis C virus contamination was assessed in cell culture. Second, after contaminating several medications with hepatitis C virus, viral infectivity over time was assessed. Last, surface-contaminated vial diaphragms were disinfected with 70% isopropyl alcohol to determine whether disinfection of the vial surface was sufficient to eliminate hepatitis C virus infectivity. Results Contamination of medication vials with hepatitis C virus and subsequent access with sterile needles and syringes resulted in contamination of the vial contents in sufficient quantities to initiate an infection in cell culture. Hepatitis C virus remained viable for several days in several commonly used medications. Finally, a single or 2- to 3-s wipe of the vial diaphragm with 70% isopropyl alcohol was not sufficient to eliminate hepatitis C virus infectivity. Conclusions Hepatitis C virus can be transferred into commonly used medications when using sterile single-use needles and syringes where it remains viable for several days. Furthermore, cleaning the vial diaphragm with 70% isopropyl alcohol is not sufficient to eliminate the risk of hepatitis C virus infectivity. This highlights the potential risks associated with sharing medications between patients.

The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2′-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2′-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.

Hepatitis C virus (HCV) affects approximately 268,000 Canadians and results in more years of life lost than any other infectious disease in the country. Both the Canadian Institutes of Health Research (CIHR) and the Public Health Agency of Canada (PHAC) have identified HCV-related liver disease as a priority and supported the establishment of a National Hepatitis C Research Network. In 2015, the introduction of new interferon- (IFN-) free therapies with high cure rates (>90%) and few side effects revolutionized HCV therapy. However, a considerable proportion of the population remains undiagnosed and treatment uptake remains low in Canada due to financial, geographical, cultural, and social barriers. Comprehensive prevention strategies, including enhanced harm reduction, broader screening, widespread treatment, and vaccine development, are far from being realized. The theme of the 2016 symposium, “We’re not done yet: remaining challenges in Hepatitis C,” was focused on identifying strategies to enhance prevention, diagnosis, and treatment of HCV to reduce disease burden and ultimately eliminate HCV in Canada.

Hepatitis C virus (HCV) affects at least 268,000 Canadians and causes greater disease burden than any other infectious disease in the country. The Canadian Institutes of Health Research (CIHR) and the Public Health Agency of Canada (PHAC) have identified HCV-related liver disease as a priority. In 2015, the release of well-tolerated, short course treatments (~12 weeks) able to cure the majority of treated HCV patients revolutionized HCV therapy. However, treatment is extremely costly and puts a significant burden on the Canadian healthcare system. Thus, managing treatment costs and improving treatment engagement in those most in need will be a key challenge. Diagnosis and treatment uptake are currently poor in Canada due to financial, geographical, cultural, and social barriers. The United States, Australia, and Scotland all have National Action Plans to prevent, diagnose, and treat HCV in order to efficiently reduce the burden and costs associated with HCV-related liver disease. The theme of the 4th annual symposium held on Feb 27, 2015, “Strategies to Manage HCV Infection in Canada: Moving towards a National Action Plan,” was aimed at identifying strategies to maximize the impact of highly effective therapies to reduce HCV disease burden and ultimately eliminate HCV in Canada.

MicroRNAs (miRNA) are emerging as important regulators of immune function due to their fast action and ability to regulate programs of gene expression. Dendritic cell (DC) responses to stimuli involves a rapid transition from steady-state an activated state, leading to numerous phenotypic changes underpinned by changes in gene expression. We have found that microRNA-9 (miR-9) expression is rapidly increased upon LPS stimulation. Pathway analysis on predicted miR-9 targets show a significant enrichment of negative regulators of gene expression. We investigated whether miR-9 promotes DC activation through targeting negative regulators. We found that DCs overexpressing miR-9 showed an increased activation phenotype whereas DCs sequestering miR-9 showed blunted activation. Co-culture of miR-9 sequestering DCs with T cells led to decreased T-cell activation, whereas miR-9 overexpressing DCs promoted T cell activation. Mice immunized with miR-9 overexpressing DCs following injection with B16-OVA melanoma cells displayed decreased tumour volume compared to controls. This work demonstrates that miR-9 promotes the activation and function of DCs, adding to the growing evidence that miRNAs are involved in the regulation of immune responses.

Summary Dendritic cells, cells of the innate immune system, are found in a steady state poised to respond to activating stimuli. Once stimulated, they rapidly undergo dynamic changes in gene expression to adopt an activated phenotype capable of stimulating immune responses. We find that the microRNA miR-9 is upregulated in both bone marrow-derived DCs and conventional DC1s but not in conventional DC2s following stimulation. miR-9 expression in BMDCs and conventional DC1s promotes enhanced DC activation and function, including the ability to stimulate T cell activation and control tumor growth. We find that miR-9 regulated the expression of several negative regulators of transcription, including the transcriptional repressor Polycomb group factor 6 (Pcgf6). These findings demonstrate that miR-9 facilitates the transition of DCs from steady state to mature state by regulating the expression of several negative regulators of DC function in a cell-type-specific manner.

Hepatitis C virus (HCV) affects more than 268,000 people in Canada. Both the Canadian Institutes of Health Research and the Public Health Agency of Canada recognize the significant impact of HCV-related liver diseases and supported the establishment of a national hepatitis C research network, the Canadian Network on Hepatitis C (CanHepC). Interferon-free direct-acting antiviral regimens lead to more than 95% cure rates in almost all patients with well-tolerated short-course therapy. However, the goal of eliminating HCV in Canada cannot be fully realized until we overcome the financial, geographical, cultural, and social barriers that affect the entire continuum of care from diagnosis and linkage to care through treatment and prevention of new and reinfections. Current practices face difficulties in reversing HCV-induced immunological defects, expanding treatment to neglected communities, combating reinfections and co-infections, and expediting and simplifying the processes of diagnosis and treatment. As part of its knowledge translation mandate, CanHepC has organized the annual Canadian symposium on hepatitis C since 2012. The theme of this year’s symposium, “Toward Elimination of HCV: How to Get There?” focused on identifying the requirements of our therapeutic strategies and health policies for the elimination of HCV in Canada.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5’ and 3’ untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease but the molecular mechanisms underlying neurodegenerative aspects of the disease are poorly understood. microRNAs (miRNAs) are powerful regulators of gene expression that regulate numerous mRNAs simultaneously and can thus regulate programs of gene expression. Here, we describe miRNA expression in neurons captured from mice subjected to experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) inflammation. Lumbar motor neurons and retinal neurons were laser captured from EAE mice and miRNA expression was assessed by next-generation sequencing and validated by qPCR. We describe 14 miRNAs that are differentially regulated in both neuronal subtypes and determine putative mRNA targets though in silico analysis. Several upregulated neuronal miRNAs are predicted to target pathways that could mediate repair and regeneration during EAE. This work identifies miRNAs that are affected by inflammation and suggests novel candidates that may be targeted to improve neuroprotection in the context of pathological inflammation.

BACKGROUND: Approximately 200 million individuals worldwide are infected by hepatitis C virus (HCV). MicroRNA-122 (miR-122) is a highly abundant liver-specific miRNA shown to interact at two miRNA-binding sites in the 5’ end of the HCV genome. This unusual interaction promotes HCV RNA accumulation in both HCV-infected cells and the livers of infected patients. Previous investigation of the stabilization of HCV RNA by miR-122 shows a slowed rate of decay in cells supplemented with miR-122 duplexes. Recent findings demonstrate that miR-122 protects HCV RNA from degradation by exoribonucleases. These results support a model whereby miR-122 acts to shield the 5’ terminus of the viral RNA, preventing its degradation or recognition by nucleases or cellular sensors of RNA. Protein kinase R (PKR) is activated mainly by long dsRNA, but short RNA stem-loops can activate PKR in a 5’ triphosphate-dependent manner, suggesting that the 3’ overhang created by miR-122 binding to the HCV 5’ end may also prevent recognition of HCV by PKR. In addition, the LGP2 protein is another RIG-I-like receptor that binds to dsRNA and acts as an on/off switch for RIG-I signaling. AIMS: We hypothesize that miR-122 forms a distinct complex with host and/or viral proteins that together protect the HCV 5’ terminus from recognition by cellular sensors of RNA, such as PKR and LGP2. Herein, we are investigating a protective role for miR-122 against these cellular sensors of RNA. METHODS: We are inhibiting PKR and LGP2 expression by siRNA knockdown in Huh7.5 cells, in the presence or absence of miR-122. To investigate the stabilization of the viral RNA in this context, we are montitoring viral RNA accumulation by luciferase assay and northern blot analyses. To investigate the contribution of miR-122, we are using miR-122 site mutants or sequestering miR-122 using an antisense locked nucleic acid inhibitor. RESULTS: We demonstrate that LGP2 expression is increased early during HCV infection in Huh7.5 cells. Knockdown of PKR or LGP2 in the presence of miR-122 has no significant effect on HCV RNA accumulation. Our current focus is on elucidating the effect of PKR and LGP2 knockdown on HCV RNA accumulation in miR-122 site mutants under limited miR-122 conditions or during miR-122 sequestration. CONCLUSIONS: We expect that the results will reveal whether miR-122 binding to the 5’ terminus of HCV is protective against recognition by the cellular sensors of RNA, PKR and LGP2 and together with our collaborators in the Wilson lab, we are investigating the role of several other sensors of RNA, including IFIT-1, IFIT-5, RIG-I and MAVS. These results will provide insights into whether miR-122 binding to the HCV genome protects the viral RNA from recognition by cellular sensors of RNA and has implications for the mechanisms of miR-122 mediated promotion of HCV RNA accumulation. FUNDING AGENCIES: CIHRCanHepC, FRSQ

Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.

Recommendations for safe medication injection practices to eliminate the risk of patient-to-patient transmission of blood-borne infections have been in place for many years. The purpose of our study was to evaluate the medication administration practices of Canadian anesthesiologists relative to current safe practice guidelines. An anonymous 17-question online survey was sent to all members of the Canadian Anesthesiologists’ Society (CAS) via the membership email list. Data pertaining to respondent demographics, practice characteristics, and medication preparation and administration practices were collected and analyzed descriptively using frequencies and percentages as well as Fisher’s exact tests followed by post hoc multiple comparisons. Of the 2,656 CAS members, 546 (21%) responded. The practice of reusing needles (2%) and/or syringes (7%) between patients is reported by only a minority of practitioners; however, sharing a medication vial between more than one patient using new needles and syringes is still widely practiced with 83% doing this sometimes or routinely. The main reasons for sharing medications include the desire to reduce medication waste and the associated costs. Sharing medication vials between multiple patients is common practice in Canada, with new needles and syringes used for each patient. Unfortunately, a small minority of anesthesiologists continue to reuse needles and/or syringes between patients, and this may pose a significant risk of patient-to-patient infection transmission.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to the 5′ end of its genome. This interaction promotes viral RNA accumulation, but the precise mechanism(s) remain incompletely understood. Previous studies suggest that miR-122 is able to protect the HCV genome from 5′ exonucleases (Xrn1/2), but this protection is not sufficient to account for the effect of miR-122 on HCV RNA accumulation. Thus, we investigated whether miR-122 was also able to protect the viral genome from innate sensors of RNA or cellular pyrophosphatases. We found that miR-122 does not play a protective role against recognition by PKR, RIG-I-like receptors, or IFITs 1 and 5. However, we found that knockdown of both the cellular pyrophosphatases, DOM3Z and DUSP11, was able to rescue viral RNA accumulation of subgenomic replicons in the absence of miR-122. Nevertheless, pyrophosphatase knockdown increased but did not restore viral RNA accumulation of full-length HCV RNA in miR-122 knockout cells, suggesting that miR-122 likely plays an additional role(s) in the HCV life cycle, beyond 5′ end protection. Overall, our results support a model in which miR-122 stabilizes the HCV genome by shielding its 5′ terminus from cellular pyrophosphatase activity and subsequent turnover by exonucleases (Xrn1/2).

RNA–protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA–protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA–protein recognition across the diversity of human and pathogen RNAs.

Hepatitis C virus (HCV) currently infects approximately 250,000 individuals in Canada and causes more years of life lost than any other infectious disease in the country. In August 2011, new therapies were approved by Health Canada that have achieved higher response rates among those treated, but are poorly tolerated. By 2014/2015, short-course, well-tolerated treatments with cure rates >95% will be available. However, treatment uptake is poor due to structural, financial, geographical, cultural and social barriers. As such, ‘Barriers to access to HCV care in Canada’ is a crucial topic that must be addressed to decrease HCV disease burden and potentially eliminate HCV in Canada. Understanding how to better care for HCV-infected individuals requires integration across multiple disciplines including researchers, clinical services and policy makers to address the major populations affected by HCV including people who inject drugs, baby boomers, immigrants and Aboriginal and/or First Nations people. In 2012, the National CIHR Research Training Program in Hepatitis C organized the 1st Canadian Symposium on Hepatitis C Virus (CSHCV) in Montreal, Quebec. The 2nd CSHCV was held in 2013 in Victoria, British Columbia. Both symposia were highly successful, attracting leading international faculty with excellent attendance leading to dialogue and knowledge translation among attendees of diverse backgrounds. The current article summarizes the 3rd CSHCV, held February 2014, in Toronto, Ontario.

The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5′ end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3′ noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

Zika virus (ZIKV) is an emerging arthropod-borne pathogen that has recently gained notoriety due to its rapid and ongoing geographic expansion and its novel association with neurological complications. Reports of ZIKV-associated Guillain–Barre syndrome as well as fetal microcephaly place emphasis on the need to develop preventative measures and therapeutics to combat ZIKV infection. Thus, it is imperative that models to study ZIKV replication and pathogenesis and the immune response are developed in conjunction with integrated vector control strategies to mount an efficient response to the pandemic. This paper summarizes the current state of knowledge on ZIKV, including the clinical features, phylogenetic analyses, pathogenesis, and the immune response to infection. Potential challenges in developing diagnostic tools, treatment, and prevention strategies are also discussed.

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5′ terminus of the hepatitis C virus (HCV) RNA with 3′ overhanging nucleotides, masking the 5′ terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3′ terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5′ terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Through this activity, they are implicated in almost every cellular process investigated to date. Hence, it is not surprising that miRNAs play diverse roles in regulation of viral infections and antiviral responses. Diverse families of DNA and RNA viruses have been shown to take advantage of cellular miRNAs or produce virally encoded miRNAs that alter host or viral gene expression. MiRNA-mediated changes in gene expression have been demonstrated to modulate viral replication, antiviral immune responses, viral latency, and pathogenesis. Interestingly, viruses mediate both canonical and non-canonical interactions with miRNAs to downregulate specific targets or to promote viral genome stability, translation, and/or RNA accumulation. In this review, we focus on recent findings elucidating several key mechanisms employed by diverse virus families, with a focus on miRNAs at the host–virus interface during herpesvirus, polyomavirus, retroviruses, pestivirus, and hepacivirus infections.

SummaryHepatitis C virus (HCV) is a global health problem. Designing therapeutic agents that target HCV's RNA genome remains challenging. HCV genomic RNA is large and highly structured with long-range genome-scale ordered RNA structures. Predicting the secondary- and tertiary-structure elements that reveal the accessibility of target sites within HCV RNA is difficult because of the abundance of long-range interactions. Target site accessibility remains a significant barrier to the design of effective therapeutics such as small interfering RNAs (siRNAs) against different strains of HCV. Here we developed two methods that interrogate the folding of HCV RNA, an approach involving viral RNA microarrays (VRMs) and an HCV viral RNA-coated magnetic bead-based (VRB) assay. VRMs and VRBs were used to determine target site accessibility for siRNAs designed against the HCV genome. Both methods predicted potency of siRNAs in cell-culture models for HCV replication that are not easily predicted by informatic means.

Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.

Carnation Italian Ringspot virus (CIRV) has evolved a protein called p19 that acts as a suppressor of RNA silencing in the host cell and aids in viral persistence. This protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering RNA (siRNA). To determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. Variants of the p19 protein were created at locations C110, C134 and C160 where the cysteines were replaced by an inert amino acid such as serine or isoleucine. The results from activity measurements of the purified mutant p19 proteins indicate that the mutants maintain the ability to bind siRNAs with nanomolar affinity, however, their stabilities, as measured by circular dichroism (CD), vary. Functional studies in the presence of the cysteine alkylating agent N-ethylmaleimide (NEM) indicated that p19's ability to bind siRNAs and act as a suppressor of RNA silencing is sensitive to alkylation at all three cysteine residues with the maximum effects occurring when C110 and C134 are both alkylated. These results suggest that the role of the cysteine amino acid conservation is likely to preserve the overall structural integrity of p19 for optimal thermostability and subsequent siRNA-binding activity. We find that p19 function is maximally compromised at high levels of thiol alkylation or in an oxidizing environment.