Viruses, Volume 13, Issue 6 (June 2021) – 237 articles

Historically, adeno-associated virus (AAV)-defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double-stranded DNA genome in a “snapback” configuration. The 5′- snapback genomes (SBGs) include the P5 promoters and partial rep gene sequences. The 3′-SBGs contains the capsid region. The molecular configuration of 5′-SBGs theoretically may allow double-stranded RNA transcription in their dimer configuration. Our studies demonstrated that 5-SBG regulated AAV rep expression and improved AAV packaging. In contrast, 3′-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5′-SBGs and 3′-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5′-SBGs and 3′-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of a viral genome and encodes additional essential functions. Full article

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested. Full article

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes. Full article

Successful joint replacement is a life-enhancing procedure with significant growth in the past decade. Prosthetic joint infection occurs rarely; it is a biofilm-based infection that is poorly responsive to antibiotic alone. Recent interest in bacteriophage therapy has made it possible to treat some biofilm-based infections, as well as those caused by multidrug-resistant pathogens, successfully when conventional antibiotic therapy has failed. Here, we describe the case of a 61-year-old woman who was successfully treated after a second cycle of bacteriophage therapy administered at the time of a two-stage exchange procedure for a persistent methicillin-sensitive Staphylococcus aureus (MSSA) prosthetic knee-joint infection. We highlight the safety and efficacy of both intravenous and intra-articular infusions of bacteriophage therapy, a successful outcome with a single lytic phage, and the development of serum neutralization with prolonged treatment. Full article

Antibody responses are crucial for the control of virus infection. Understanding of the mechanism of antibody induction is important for the development of a vaccine eliciting effective anti-virus antibodies. Virus-specific B cell receptor (BCR)/antibody repertoires are different among individuals, but determinants for this difference remain largely unclear. We have recently reported that a germline BCR immunoglobulin (IgG) gene polymorphism (VH3.33_ET or VH3.33_VI) in rhesus macaques is the determinant for induction of potent B404-class anti-simian immunodeficiency virus (SIV) neutralizing antibodies in neutralization-sensitive SIVsmH635FC infection. In the present study, we examined whether neutralization-resistant SIVsmE543-3 infection can induce the anti-SIV neutralizing antibodies associated with the germline VH3.33 polymorphism. Anti-SIVsmE543-3 neutralizing antibodies were induced in all the macaques possessing the VH3.33_ET allele, but not in those without VH3.33_ET, in the chronic phase of SIVsmE543-3 infection. Next generation sequencing analysis of BCR VH genes found B404-class antibody sequences only in those with VH3.33_ET. These results indicate that anti-SIVsmE543-3 neutralizing antibody induction associated with the germline BCR IgG gene polymorphism can be triggered by infection with neutralization-resistant SIVsmE543-3. This animal model would be useful for the elucidation of the mechanism of potent antibody induction against neutralization-resistant viruses. Full article

Mature male mice produce a particularly high concentration of major urinary proteins (MUPs) in their scent marks that provide identity and status information to conspecifics. Darcin (MUP20) is inherently attractive to females and, by inducing rapid associative learning, leads to specific attraction to the individual male’s odour and location. Other polymorphic central MUPs, produced at much higher abundance, bind volatile ligands that are slowly released from a male’s scent marks, forming the male’s individual odour that females learn. Here, we show that infection of C57BL/6 males with LCMV WE variants (v2.2 or v54) alters MUP expression according to a male’s infection status and ability to clear the virus. MUP output is substantially reduced during acute adult infection with LCMV WE v2.2 and when males are persistently infected with LCMV WE v2.2 or v54. Infection differentially alters expression of darcin and, particularly, suppresses expression of a male’s central MUP signature. However, following clearance of acute v2.2 infection through a robust virus-specific CD8 cytotoxic T cell response that leads to immunity to the virus, males regain their normal mature male MUP pattern and exhibit enhanced MUP output by 30 days post-infection relative to uninfected controls. We discuss the likely impact of these changes in male MUP signals on female attraction and mate selection. As LCMV infection during pregnancy can substantially reduce embryo survival and lead to lifelong infection in surviving offspring, we speculate that females use LCMV-induced changes in MUP expression both to avoid direct infection from a male and to select mates able to develop immunity to local variants that will be inherited by their offspring. Full article

The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC—EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies. Full article

Viruses are pathogens that have evolved to hijack the cellular machinery to replicate themselves and spread to new cells. During the course of evolution, viruses developed different strategies to overcome the cellular defenses and create new progeny. Among them, some RNA and many DNA viruses require access to the nucleus to replicate their genome. In non-dividing cells, viruses can only access the nucleus through the nuclear pore complex (NPC). Therefore, viruses have developed strategies to usurp the nuclear transport machinery and gain access to the nucleus. The majority of these viruses use the capsid to manipulate the nuclear import machinery. However, the particular tactics employed by each virus to reach the host chromatin compartment are very different. Nevertheless, they all require some degree of capsid remodeling. Recent notions on the interplay between the viral capsid and cellular factors shine new light on the quest for the nuclear entry step and for the fate of these viruses. In this review, we describe the main components and function of nuclear transport machinery. Next, we discuss selected examples of RNA and DNA viruses (HBV, HSV, adenovirus, and HIV) that remodel their capsid as part of their strategies to access the nucleus and to replicate. Full article

Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome characterized by thrombocytopenia, increased capillary leakage, and acute kidney injury (AKI). As glucosuria at hospital admission predicts the severity of PUUV infection, we explored how plasma glucose concentration associates with disease severity. Plasma glucose values were measured during hospital care in 185 patients with PUUV infection. They were divided into two groups according to maximum plasma glucose concentration: P-Gluc < 7.8 mmol/L (n = 134) and P-Gluc ≥ 7.8 mmol/L (n = 51). The determinants of disease severity were analyzed across groups. Patients with P-Gluc ≥7.8 mmol/L had higher hematocrit (0.46 vs. 0.43; p < 0.001) and lower plasma albumin concentration (24 vs. 29 g/L; p < 0.001) than patients with P-Gluc < 7.8 mmol/L. They presented with higher prevalence of pulmonary infiltrations and pleural effusion in chest radiograph, higher prevalence of shock and greater weight change during hospitalization. Patients with P-Gluc ≥ 7.8 mmol/L were characterized by lower platelet count (50 vs. 66 × 10⁹/L; p = 0.001), more severe AKI (plasma creatinine 272 vs. 151 µmol/L; p = 0.001), and longer hospital treatment (8 vs. 6 days; p < 0.001) than patients with P-Gluc < 7.8 mmol/L. Plasma glucose level is associated with the severity of capillary leakage, thrombocytopenia, inflammation, and AKI in patients with acute PUUV infection. Full article

The wild boar is the most important reservoir of zoonotic HEV-3 strains among different wildlife species. The aim of the study was subtype identification of wild boar HEV-3 strains circulating in Poland. Wild boar liver was used in the study in the form of homogenates prepared from 57 samples positive for HEV in a real-time RT-PCR. These samples were collected from juvenile and adult wild boars hunted in the jurisdictions of different Regional Directorates of State Forests (RDSF) across Poland. Subtype identification of detected HEV strains was based on a phylogenetic analysis of the most conserved HEV ORF2 genome fragment. Out of 57 tested samples, consensus HEV ORF2 sequences of 348 bp were obtained for 45 strains. Nineteen strains were identified and belonged to the HEV gt 3a and 3i subtypes, whereas 26 were not assigned to any virus subtype. HEV gt 3i strains prevailed in the Polish wild boar population, 16 of such were identified, and they were significantly more often observed in the RDSF Katowice area (χ² = 28.6, p = 0.027 (<0.05)) compared to other regions of the country. Circulation of 3a strains was limited only to the RDSF Gdańsk territory (χ² = 48, p = 0.000 (<0.05)). The virus strains detected in the Polish population of wild boars representing previously identified HEV subtypes in wild boars, pigs, or humans in Europe are of epidemiological importance for public health. Full article

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting. Full article

To reduce global HIV-1 incidence, there is a need to understand and disentangle HIV-1 transmission dynamics and to determine the geographic areas and populations that act as hubs or drivers of HIV-1 spread. In Sub-Saharan Africa (sSA), the region with the highest HIV-1 burden, information about such transmission dynamics is sparse. Phylogenetic inference is a powerful method for the study of HIV-1 transmission networks and source attribution. In this review, we assessed available phylogenetic data on mixing between HIV-1 hotspots (geographic areas and populations with high HIV-1 incidence and prevalence) and areas or populations with lower HIV-1 burden in sSA. We searched PubMed and identified and reviewed 64 studies on HIV-1 transmission dynamics within and between risk groups and geographic locations in sSA (published 1995–2021). We describe HIV-1 transmission from both a geographic and a risk group perspective in sSA. Finally, we discuss the challenges facing phylogenetic inference in mixed epidemics in sSA and offer our perspectives and potential solutions to the identified challenges. Full article

Human papillomaviruses (HPV) cause a subset of head and neck cancers (HNSCC). HPV16 predominantly signs responsible for approximately 10% of all HNSCC and over 50% of tonsillar (T)SCCs. Prevalence rates depend on several factors, such as the geographical region where patients live, possibly due to different social and sexual habits. Smoking plays an important role, with non-smoking patients being mostly HPV-positive and smokers being mostly HPV-negative. This is of unparalleled clinical relevance, as the outcome of (non-smoking) HPV-positive patients is significantly better, albeit with standard and not with de-escalated therapies. The results of the first prospective de-escalation studies have dampened hopes that similar superior survival can be achieved with de-escalated therapy. In this context, it is important to note that the inclusion of p16^INK4A (a surrogate marker for HPV-positivity) in the 8th TMN-classification has only prognostic, not therapeutic, intent. To avoid misclassification, highest precision in determining HPV-status is of utmost importance. Whenever possible, PCR-based methods, still referred to as the "gold standard”, should be used. New diagnostic antibodies represent some hope, e.g., to detect primaries and recurrences early. Prophylactic HPV vaccination should lead to a decline in HPV-driven HNSCC as well. This review discusses the above aspects in detail. Full article

The sequence variability of the Epstein–Barr virus has been extensively studied throughout previous years in isolates from various geographic regions and consequent variations at both genetic and genomic levels have been described. However, isolates from South America were underrepresented in these studies. Here, we sequenced 15 complete EBV genomes that we analyzed together with publicly available raw NGS data for 199 EBV isolates from other parts of the globe by means of a custom-built bioinformatic pipeline. The phylogenetic relations of the genomes, the geographic structure and variability of the data set, and the evolution rates for the whole genome and each gene were assessed. The present work contributes to overcoming the scarcity of complete EBV genomes from South America and is the most comprehensive geography-related variability study, which involved determining the actual contribution of each EBV gene to the geographic segregation of the entire genome. Moreover, to the best of our knowledge, we established for the first time the evolution rate for the entire EBV genome based on a host–virus codivergence-independent assumption and assessed their evolution rates on a gene-by-gene basis, which were related to the encoded protein function. Considering the evolution of dsDNA viruses with a codivergence-independent approach may lay the basis for future research on EBV evolution. The exhaustive bioinformatic analysis performed on this new dataset allowed us to draw a novel set of conclusions regarding the genome evolution of EBV. Full article

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is genetically variable, allowing it to adapt to various hosts including humans. Indeed, SARS-CoV-2 has accumulated around two mutations per genome each month. The first relevant event in this context was the occurrence of the mutant D614G in the Spike gene. Moreover, several variants have emerged, including the well-characterized 20I/501Y.V1, 20H/501Y.V2, and 20J/501Y.V3 strains, in addition to those that have been detected within clusters, such as 19B/501Y or 20C/655Y in France. Mutants have also emerged in animals, including a variant transmitted to humans, namely, the Mink variant detected in Denmark. The emergence of these variants has affected the transmissibility of the virus (for example, 20I/501Y.V1, which was up to 82% more transmissible than other preexisting variants), its severity, and its ability to escape natural, adaptive, vaccine, and therapeutic immunity. In this respect, we review the literature on variants that have currently emerged, and their effect on vaccines and therapies, and, in particular, monoclonal antibodies (mAbs). The emergence of SARS-CoV-2 variants must be examined to allow effective preventive and curative control strategies to be developed. Full article

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4⁺ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4⁺ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4⁺ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing. Full article

Globally, chronic hepatitis B (CHB) infection is one of the leading causes of liver failure, decompensated cirrhosis, and hepatocellular carcinoma. Existing antiviral therapy can suppress viral replication but not fully eradicate the virus nor the risk of liver-related complications. Novel treatments targeting alternative steps of the viral cycle or to intensify/restore the host’s immunity are being developed. We discuss novel drugs that have already entered clinical phases of development. Agents that interfere with specific steps of HBV replication include RNA interference, core protein allosteric modulation, and inhibition of viral entry or viral protein excretion (NAPs and STOPS). Agents that target the host’s immunity include toll-like receptor agonists, therapeutic vaccines, immune checkpoint modulators, soluble T-cell receptors, and monoclonal antibodies. Most have demonstrated favorable results in suppression of viral proteins and genomic materials (i.e., HBV DNA and/or pre-genomic RNA), and/or evidence on host-immunity restoration including cytokine responses and T-cell activation. Given the abundant clinical experience and real-world safety data with the currently existing therapy, any novel agent for CHB should be accompanied by convincing safety data. Combination therapy of nucleos(t)ide analogue, a novel virus-directing agent, and/or an immunomodulatory agent will be the likely approach to optimize the chance of a functional cure in CHB. Full article

This article reviews the current knowledge on how viruses may utilize Extracellular Vesicle Assisted Inflammatory Load (EVAIL) to exert pathologic activities. Viruses are classically considered to exert their pathologic actions through acute or chronic infection followed by the host response. This host response causes the release of cytokines leading to vascular endothelial cell dysfunction and cardiovascular complications. However, viruses may employ an alternative pathway to soluble cytokine-induced pathologies—by initiating the release of extracellular vesicles (EVs), including exosomes. The best-understood example of this alternative pathway is human immunodeficiency virus (HIV)-elicited EVs and their propensity to harm vascular endothelial cells. Specifically, an HIV-encoded accessory protein called the “negative factor” (Nef) was demonstrated in EVs from the body fluids of HIV patients on successful combined antiretroviral therapy (ART); it was also demonstrated to be sufficient in inducing endothelial and cardiovascular dysfunction. This review will highlight HIV-Nef as an example of how HIV can produce EVs loaded with proinflammatory cargo to disseminate cardiovascular pathologies. It will further discuss whether EV production can explain SARS-CoV-2-mediated pulmonary and cardiovascular pathologies. Full article

The immunopathogenesis and molecular mechanisms involved during a hepatitis B virus (HBV) infection have made the approaches for research complex, especially concerning the patients’ responses in the course of the early acute stage. The study of molecular bases involved in the viral clearance or persistence of the infection is complicated due to the difficulty to detect patients at the most adequate points of the disease, especially in the time lapse between the onset of the infection and the viral emergence. Despite this, there is valuable data obtained from animal and in vitro models, which have helped to clarify some aspects of the early immune response against HBV infection. The diversity of the HBV (genotypes and variants) has been proven to be associated not only with the development and outcome of the disease but also with the response to treatments. That is why factors involved in the virus evolution need to be considered while studying hepatitis B infection. This review brings together some of the published data to try to explain the immunological and molecular mechanisms involved in the different stages of the infection, clinical outcomes, viral persistence, and the impact of the variants of HBV in these processes. Full article

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region. Full article

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families (Amalgaviridae, Caulimoviridae, Endornaviridae, Ophioviridae, Phenuiviridae, and Virgaviridae). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach. Full article

Viruses, far from being just parasites affecting hosts’ fitness, are major players in any microbial ecosystem. In spite of their broad abundance, viruses, in particular bacteriophages, remain largely unknown since only about 20% of sequences obtained from viral community DNA surveys could be annotated by comparison with public databases. In order to shed some light into this genetic dark matter we expanded the search of orthologous groups as potential markers to viral taxonomy from bacteriophages and included eukaryotic viruses, establishing a set of 31,150 ViPhOGs (Eukaryotic Viruses and Phages Orthologous Groups). To do this, we examine the non-redundant viral diversity stored in public databases, predict proteins in genomes lacking such information, and used all annotated and predicted proteins to identify potential protein domains. The clustering of domains and unannotated regions into orthologous groups was done using cogSoft. Finally, we employed a random forest implementation to classify genomes into their taxonomy and found that the presence or absence of ViPhOGs is significantly associated with their taxonomy. Furthermore, we established a set of 1457 ViPhOGs that given their importance for the classification could be considered as markers or signatures for the different taxonomic groups defined by the ICTV at the order, family, and genus levels. Full article

Infectious bronchitis viruses (IBVs) are evolving continuously via genetic drift and genetic recombination, making disease prevention and control difficult. In this study, we undertook genetic and pathogenic characterization of recombinant IBVs isolated from chickens in South Korea between 2003 and 2019. Phylogenetic analysis showed that 46 IBV isolates belonged to GI-19, which includes nephropathogenic IBVs. Ten isolates formed a new cluster, the genomic sequences of which were different from those of reference sequences. Recombination events in the S1 gene were identified, with putative parental strains identified as QX-like, KM91-like, and GI-15. Recombination detection methods identified three patterns (rGI-19-I, rGI-19-II, and rGI-19-III). To better understand the pathogenicity of recombinant IBVs, we compared the pathogenicity of GI-19 with that of the rGI-19s. The results suggest that rGI-19s may be more likely to cause trachea infections than GI-19, whereas rGI-19s were less pathogenic in the kidney. Additionally, the pathogenicity of rGI-19s varied according to the genotype of the major parent. These results indicate that genetic recombination between heterologous strains belonging to different genotypes has occurred, resulting in the emergence of new recombinant IBVs in South Korea. Full article

The possible role of viruses in feline liver disease has long remained neglected. However, in 2018, an analogue of human hepatitis B virus was identified in cats. Moreover, antibodies for human hepatitis E have been detected consistently at various prevalence rates in cats. Although the correlation between these viruses and the liver injury in cats must be clarified, hepatotropic viruses might represent an increasing risk for feline and public health. Full article

In recent years, major advances in research and experimental approaches have significantly increased our knowledge on the role of the HIV-1 capsid in the virus life cycle, from reverse transcription to integration and gene expression. This makes the capsid protein a good pharmacological target to inhibit HIV-1 replication. This review covers our current understanding of the role of the viral capsid in the HIV-1 life cycle and its interaction with different host factors that enable reverse transcription, trafficking towards the nucleus, nuclear import and integration into host chromosomes. It also describes different promising small molecules, some of them in clinical trials, as potential targets for HIV-1 therapy. Full article

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme. Full article

In European countries, autochthonous acute hepatitis E cases are caused by Hepatitis E Virus (HEV) genotype 3 and are usually observed as sporadic cases. In mid/late September 2019, a hepatitis E outbreak caused by HEV genotype 3 was recognized by detection of identical/highly similar HEV sequences in some hepatitis E cases from two Italian regions, Abruzzo and Lazio, with most cases from this latter region showing a link with Abruzzo. Overall, 47 cases of HEV infection were finally observed with onsets from 8 June 2019 to 6 December 2019; they represent a marked increase as compared with just a few cases in the same period of time in the past years and in the same areas. HEV sequencing was successful in 35 cases. The phylogenetic analysis of the viral sequences showed 30 of them grouped in three distinct molecular clusters, termed A, B, and C: strains in cluster A and B were of subtype 3e and strains in cluster C were of subtype 3f. No strains detected in Abruzzo in the past years clustered with the strains involved in the present outbreak. The outbreak curve showed partially overlapped temporal distribution of the three clusters. Analysis of collected epidemiological data identified pork products as the most likely source of the outbreak. Overall, the findings suggest that the outbreak might have been caused by newly and almost simultaneously introduced strains not previously circulating in this area, which are possibly harbored by pork products or live animals imported from outside Abruzzo. This possibility deserves further studies in this area in order to monitor the circulation of HEV in human cases as well as in pigs and wild boars. Full article

Despite advances in surgical resection and chemoradiation, high-grade brain tumors continue to be associated with significant morbidity/mortality. Novel therapeutic strategies and approaches are, therefore, desperately needed for patients and their families. Given the success experienced in treating multiple other forms of cancer, immunotherapy and, in particular, immunovirotherapy are at the forefront amongst novel therapeutic strategies that are currently under investigation for incurable brain tumors. Accordingly, herein, we provide a focused mini review of pertinent oncolytic herpes viruses (oHSV) that are being investigated in clinical trials. Full article

Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a β-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of β-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which β-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors. Full article

Mayaro virus (MAYV) hijacks the host’s cell machinery to effectively replicate. The mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK1/2 have emerged as crucial cellular factors implicated in different stages of the viral cycle. However, whether MAYV uses these MAPKs to competently replicate has not yet been determined. The aim of this study was to evaluate the impact of MAPK inhibition on MAYV replication using primary human dermal fibroblasts (HDFs) and HeLa cells. Viral yields in supernatants from MAYV-infected cells treated or untreated with inhibitors SB203580, SP600125, U0126, or Losmapimod were quantified using plaque assay. Additionally, viral protein expression was analyzed using immunoblot and immunofluorescence. Knockdown of p38⍺/p38β isoforms was performed in HDFs using the PROTACs molecule NR-7h. Our data demonstrated that HDFs are highly susceptible to MAYV infection. SB203580, a p38 inhibitor, reduced MAYV replication in a dose-dependent manner in both HDFs and HeLa cells. Additionally, SB203580 significantly decreased viral E1 protein expression. Similarly, knockdown or inhibition of p38⍺/p38β isoforms with NR-7h or Losmapimod, respectively, affected MAYV replication in a dose-dependent manner. Collectively, these findings suggest that p38 could play an important role in MAYV replication and could serve as a therapeutic target to control MAYV infection. Full article