Relevant Viruses Research Articles

Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease.

Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.

Hooking on Viral Glycoproteins with Single Molecule Force Spectroscopy to Study Single and Multiple Bond Formations

The influenza A virus (IAV) interacts with sialic acid (SA) receptors on host cell membranes upon multivalent interactions of its major spike glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA mediates the viral attachment to cells, while NA enables the release of newly formed virus particles by destroying SA receptors. Since both proteins target the same receptor, but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral spread. Here dynamic single molecule force spectroscopy (SMFS) was employed to determine bond specific kinetics, characterized by the off-rate and on-rate of virus binding, the rupture length as well as the related free energy barrier ΔG. Measurements were conducted on two relevant virus systems, differing in the HA/NA type, and by using a synthetic SA-displaying receptor attached to the SFM cantilever. The bond strength was tested to immobilized HA and NA of the IAV strains California/H1N1, and Aichi/H3N2 as well as intact virus particles of the latter. SMSF at variable force loading rates revealed most probable rupture forces of the protein-SA bond being in the range of 10-100 pN. Measurements of the binding probability at increasing contact time between the SFM force probe, and the surface allowed an estimation of KD, which suggests a stronger interaction for NA-SA than for HA-SA. A systematic study of multiple bonds connected in parallel demonstrated a rapid formation of multivalent interactions of both proteins increasing strongly the bond life-time (msec-sec). In conclusion, we obtained new insights for the orchestrated role of HA and NA in the viral binding and release process. In particular, our results provide strong evidence for involvement of NA into viral attachment to the host cell.

Virus⁻Host Interactions Involved in Lassa Virus Entry and Genome Replication.

Lassa virus (LASV) is the causative agent of Lassa fever, a human hemorrhagic disease associated with high mortality and morbidity rates, particularly prevalent in West Africa. Over the past few years, a significant amount of novel information has been provided on cellular factors that are determinant elements playing a role in arenavirus multiplication. In this review, we focus on host proteins that intersect with the initial steps of the LASV replication cycle: virus entry and genome replication. A better understanding of relevant virus–host interactions essential for sustaining these critical steps may help to identify possible targets for the rational design of novel therapeutic approaches against LASV and other arenaviruses that cause severe human disease.

Validation of a site-specific recombination cloning technique for the rapid development of a full-length cDNA clone of a virulent field strain of vesicular stomatitis New Jersey virus

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.

An integrated device for the rapid and sensitive detection of the influenza hemagglutinin.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600 000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 μL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 102 TCID50 per mL on device.

Advanced oxidation technology for the development of a next-generation inactivated West Nile virus vaccine

West Nile virus (WNV) is the most frequent mosquito-borne disease reported in the continental United States and although an effective veterinary vaccine exists for horses, there is still no commercial vaccine approved for human use. We have previously tested a 3% hydrogen peroxide (H2O2)-based WNV inactivation approach termed, HydroVax, in Phase I clinical trials and the vaccine was found to be safe and modestly immunogenic. Here, we describe an advanced, next-generation oxidation approach (HydroVax-II) for the development of inactivated vaccines that utilizes reduced concentrations of H2O2 in combination with copper (cupric ions, Cu2+) complexed with the antiviral compound, methisazone (MZ). Further enhancement of this oxidative approach included the addition of a low percentage of formaldehyde, a cross-linking reagent with a different mechanism of action that, together with H2O2/Cu/MZ, provides a robust two-pronged approach to virus inactivation. Together, this new approach results in rapid virus inactivation while greatly improving the maintenance of WNV-specific neutralizing epitopes mapped across the three structural domains of the WNV envelope protein. In combination with more refined manufacturing techniques, this inactivation technology resulted in vaccine-mediated WNV-specific neutralizing antibody responses that were 130-fold higher than that observed using the first generation, H2O2-only vaccine approach and provided 100% protection against lethal WNV infection. This new approach to vaccine development represents an important area for future investigation with the potential not only for improving vaccines against WNV, but other clinically relevant viruses as well.

Current Knowledge on Porcine circovirus 3 (PCV-3): A Novel Virus With a Yet Unknown Impact on the Swine Industry.

Porcine circovirus 3 (PCV-3) is a recently described virus belonging to the family Circoviridae. It represents the third member of genus Circovirus able to infect swine, together with PCV-1, considered non-pathogenic, and PCV-2, one of the most economically relevant viruses for the swine worldwide industry. PCV-3 was originally found by metagenomics analyses in 2015 in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. The lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that PCV-3 might etiologically be involved in disease occurrence. Subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that PCV-3 was already present in pigs by early 1990s. In fact, current evidence suggests that PCV-3 is a rather widespread virus worldwide. Recently, the virus DNA has also been found in wild boar, expanding the scope of infection susceptibility among the Suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. Phylogenetic studies with available PCV-3 partial and complete sequences from around the world have revealed high nucleotide identity (>96%), although two main groups and several subclusters have been described as well. Moreover, it has been proposed the existence of a most common ancestor dated around 50 years ago. Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. Therefore, the present review will update the current knowledge and future trends of research on PCV-3.

Chaperoning the Mononegavirales: Current Knowledge and Future Directions.

The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals.

Detection of Viruses in Clinical Samples by Use of Metagenomic Sequencing and Targeted Sequence Capture.

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.

ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasis.

Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.

Virus-Bacteria Interactions: Implications for Prevention and Control of Human Enteric Viruses from Environment to Host.

Human enteric viruses, specifically human norovirus (hNoV), are the most common cause of foodborne illness boasting a wide range of transmission routes. These include person to person, contact with contaminated fomites, as well as ingestion of contaminated water and food. Because of this, the control and prevention of enteric viruses in food and other relevant environments have been a research focus over the past few decades. Interestingly, viruses as well as many other pathogens are often studied in isolation even though it is known that microorganisms do not occur in isolation but rather as part of complex microbial communities-both external from the host and within the host. Therefore, the overall goal of this review is to present the current evidence on virus-microbe interactions as these relate to the infectivity as well as the control and prevention of epidemiologically relevant foodborne viruses (such as hNoV) within our food systems. Therefore, this review is divided into in vivo, in situ, and in vitro implications of virus-microbe interactions through discussion of studies investigating the complex relationships between human enteric viruses and microbial cohabitants, specifically hNoV and bacteria.

A Flow Cytometry-Based Method to Determine the Titer of Adenoviruses Expressing an Extraneous Gene.

In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP)50 titer as a measure of viral quality. The GFP50 titer (GFP50/mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP50/mL and tissue culture infectious dose (TCID50/mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP50/mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.

DETECTION OF LYMPHOPROLIFERATIVE DISEASE VIRUS IN CANADA IN A SURVEY FOR VIRUSES IN ONTARIO WILD TURKEYS ( MELEAGRIS GALLOPAVO).

The successful reintroduction of Wild Turkeys ( Meleagris gallopavo) to Ontario, Canada, has led to established populations in southern portions of the province and currently allows for biannual hunting seasons. These populations geographically overlap Domestic Turkey farms, an important sector of the provincial agri-food industry. Potential pathogen transmission between Wild Turkeys and Domestic Turkeys ( Meleagris gallopavo) is a concern, because they are susceptible to infection with many of the same pathogens and have direct and indirect contact in outdoor or open farm settings and contaminated environmental substrates. However, data concerning potential poultry pathogens in Wild Turkeys in Canada are scarce. Thus, we assessed the prevalence and geographic distribution of geographically relevant viruses in Ontario Wild Turkeys. Oropharyngeal and cloacal swabs were tested for avian influenza viruses (AIV) by real-time reverse transcriptase (RT)-PCR ( n=207), pooled tissues for lymphoproliferative disease virus (LPDV; n=183) and reticuloendotheliosis virus ( n=119) by PCR, and gross skin lesions by real-time RT-PCR for avian poxvirus ( n=8). We sequenced a fragment of the gag polyprotein (p31) gene of LPDV on a subset ( n=10) of LPDV-positive samples for phylogenetic analysis and tested additional upland game bird species ( n=39) and domestic fowl for LPDV ( n=17). To the best of our knowledge, we document the first detection of LPDV in Wild Turkeys in Canada, with a prevalence of 65% (119/183). Phylogenetic analysis revealed that LPDV sequences from Ontario were genetically similar to other North American strains and did not group into separate clades. Reticuloendotheliosis virus was detected in 4% (5/119) of LPDV-positive Wild Turkeys. Grossly evident skin lesions from five Wild Turkeys tested positive for poxvirus, and all turkeys tested negative for AIV. This study provides evidence of LPDV circulation in Canada and provides a baseline for comparison with future Wild Turkey pathogen surveillance and monitoring in Ontario and elsewhere.

Synthesis of a 3′-C-ethynyl-β-d-ribofuranose purine nucleoside library: Discovery of C7-deazapurine analogs as potent antiproliferative nucleosides

A focused nucleoside library was constructed around a 3′-C-ethynyl-d-ribofuranose sugar scaffold, which was coupled to variously modified purine nucleobases. The resulting nucleosides were probed for their ability to inhibit tumor cell proliferation, as well as for their activity against a panel of relevant human viruses. While C6-aryl substituted purine nucleosides were found to be weakly active, several C7-substituted 7-deazapurine nucleosides elicited potent antiproliferative activity. Their activity spectrum was evaluated in the NCI-60 tumor cell line panel indicating activity against several solid tumor derived cell lines. Analog 32, equipped with a 7-deaza 7-chloro-6-amino-purin-9-yl base was evaluated in a metastatic breast tumor (MDA-MB-231-LM2) xenograft model. It inhibited both tumor growth and reduced the formation of lung metastases as revealed by BLI analysis. The dideazanucleoside analog 66 showed interesting activity against hCMV. These results highlight the potential advantages of recombining known sugar and nucleobase motifs as a library design strategy to discover novel antiviral or antitumor agents.

Protein Nanoparticles Made of Recombinant Viral Antigens: A Promising Biomaterial for Oral Delivery of Fish Prophylactics.

In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as “nanopellets” (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism.

The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8+ cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses.

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.

Myocarditis associated with influenza infection in five children.

Myocarditis is an inflammatory condition located mainly in the myocardium. It is caused by a variety of bacterial and viral infections. Influenza is one of the most common relevant viruses that cause myocarditis. We attempted to share our experiences about clinical and laboratory findings, cardiac evaluation, and treatment of children with influenza myocarditis. This retrospective study was performed by the Department of Pediatric Infectious Diseases at the Faculty of Medicine, Hacettepe University in Turkey. The medical records of patients diagnosed with myocarditis associated with an influenza infection between January 2014 and January 2017 were systematically reviewed. Vaccination seems likely to be an important protection strategy for both influenza infections and complications.

Nanoparticle vaccines against viral infections.

Despite numerous efforts, we still do not have prophylactic vaccines for many clinically relevant viruses, such as HIV, hepatitis C virus, Zika virus, and respiratory syncytial virus. Several factors have contributed to the current lack of effective vaccines, including the high rate of viral mutation, low immunogenicity of recombinant viral antigens, instability of viral antigenic proteins administered in vivo, sophisticated mechanisms of viral immune evasion, and inefficient induction of mucosal immunity by vaccine models studied to date. Some of these obstacles could be partially overcome by the use of vaccine adjuvants. Nanoparticles have been intensively investigated as vaccine adjuvants because they possess chemical and structural properties that improve immunogenicity. The use of nanotechnology in the construction of immunization systems has developed into the field of viral nanovaccinology. The purpose of this paper is to review and correlate recent discoveries concerning nanoparticles and specific properties that contribute to the immunogenicity of viral nanoparticle vaccines, bio-nano interaction, design of nanoparticle vaccines for clinically relevant viruses, and future prospects for viral nanoparticle vaccination.

Recombination events and clusters of ADAR1‐like hypermutations increase genome diversity of measles virus, a negative strand RNA virus

Genome diversity is key to viral fitness and adaptability, especially for RNA viruses, which generate it through both mutation and recombination. Recombination occurs frequently in positive strand RNA virus infections, where the RNA‐dependent RNA polymerase can jump from one genomic template to another. On the other hand, template exchange events have been rarely detected in infections of negative strand RNA viruses even when sensitive functional assays were used. However, while characterizing defective interfering RNAs (DI RNA) generated by an innate immunity‐stimulating measles virus (MeV) mutant, we documented an unexpectedly high number of recombination events (J. Virol. 89, 7735‐47, 2015). We asked here how frequently these events occur in the 15,894 bases genome of a MeV strain with intact innate immunity control functions. Towards this we purified MeV ribonucleocapsids and performed unbiased deep sequencing analyses. We detected more than 3000 recombination events while covering 32,000 genomes, or about one event every ten genomes. Mapping of breakpoints and re‐initiation sites indicated that recombination occurred along the entire genome, rather than being restricted to either end, where most DI RNAs were previously mapped. Recombination was phased neither by encapsidation (6‐base periodicity) nor by ribonucleocapsid helical turns (approximately 75‐base periodicity). Rather, most recombination events joined sequences located more than 1000 bases apart. Jump‐back events were prominent, occurring about 1.5 times more frequently than deletion or copy‐back events. Most events were unique, but a few short jump‐back events retaining 6‐base periodicity were detected up to 40 times, suggesting that the corresponding genomes were amplified. At new junctions, deletions or insertions were rare (<5%). Analyses of 10 nucleotides on each side of the new junctions suggested that base pairing guided about one third of the re‐initiation events. In addition to the recombination events, we detected clusters of adenosine deaminase acting on RNA 1 (ADAR1)‐like hypermutations in about 4% of the genomes. ADAR1 de‐stabilizes base pairing on double‐strand RNA, catalyzing C‐6 deamination of adenosine to produce inosine until double‐strand regions are unwound. Indeed, we document that in an infection with a MeV mutant known to generate many DI RNAs, the frequency of ADAR1‐like hypermutations was even higher than in standard infections. In summary, we show here that significant levels of RNA recombination occur even in standard lytic infections of a prototypic negative strand RNA virus. Even if some recombinant genomes may form double‐strand RNA, infections remain productive, probably due to the unwinding/modification activity of ADAR1. We discuss our data in the context of published observations indicating that recombination events, as well as ADAR1‐like hypermutations, contribute to the adaptation of other medically relevant negative strand RNA viruses to new hosts and environments.Support or Funding InformationR21 AI 128037; Mayo Clinic Graduate School of Biomedical SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.