The mealworm (Tenebrio molitor) is one of the most commonly mass-reared insects for food and feed. Monitoring the health status of commercially reared mealworm populations is of great importance for the early detection of entomopathogens and for preventing pathogen outbreaks. Metagenomic screening is a suitable and commonly used method for detecting entomopathogens. The approach used here previously enabled the discovery of the Tenebrio molitor densovirus (TmDV) (family Parvoviridae, subfamily Densovirinae) in symptomatic larvae. In the present study, the search for TmDV was extended to larvae, pupae and adults of T. molitor, including 19 symptomatic and asymptomatic samples obtained from a commercial mealworm mass-rearing facility. The presence of TmDV in all life stages of T. molitor was demonstrated, and its relative abundance was quantified using Nanopore sequencing. The infectivity of TmDV to T. molitor was demonstrated by isolating viral particles from sample LD2 and feeding them to mealworms. The experiment confirmed T. molitor as a susceptible host but showed a rather asymptomatic course of the infection with little effect on larval growth during 56 days of observation. It is hypothesized that this largely covert infection may explain the lack of reports of TmDV in mealworms or other insects, despite its detection in metagenomics surveillance studies of various insectivorous vertebrates. The complete genomes of 15 different TmDV genotypes present in various ratios in the different life stages of T. molitor could be reconstructed. Including these genotype sequences in phylogenetic analyses allowed us to re-evaluate the relationship and diversity of previously reported TmDV and related isolates, all belonging to the species Blattambidensovirus incertum1. Our findings suggest that T. molitor and possibly other insects are susceptible hosts of viruses of Blattambidensovirus incertum1, while its occasional detection in metagenomic datasets of insectivorous vertebrates may not represent true densovirus host associations.
Atkinsonella hypoxylon virus (AhV) is a fungi-infecting betapartitivirus and the typical member of the Partitiviridae, a family of persistent viruses that infect a broad range of organisms. Partitiviruses have been largely overlooked following their designation as cryptic viruses. However, evidence is accumulating that they play an important role in the ecology of their hosts. Since the capsid proteins of partitiviruses have been implicated in virus-host interactions, exploring their structural biology may give clues into the evolution, horizontal transmission and host adaptation of partitiviruses. The capsid of AhV shares the same organization of other partitiviruses with 60 dimeric capsid protein protomers arranged with T=1 icosahedral symmetry. The structure, determined by cryo-electron microscopy to 2.4 Å, shows that AhV has a unique iteration on the protrusion domain with an extensive network of hydrophobic interactions among equivalent interdigitating loops at the dimerization interface. AhV also shares a conserved helical core in the shell domain, which we extend to all genera of the recognized partitiviruses using protein structure prediction. The helical core appears to be a conserved element of the picobirnavirus lineage of capsid protein folds and provides a template onto which various elaborations of the protrusion domain have evolved. The involvement of the protrusion in virus-host interactions has previously been proposed, and our findings provide evidence of a structural device enabling capsid protein diversification during the evolution of the Partitiviridae.
Coronaviruses evolve rapidly, with recombination and mutation fostering the emergence of variant strains. The avian coronavirus infectious bronchitis virus (IBV) is an important poultry pathogen and a valuable natural model for studying coronaviruses. Australian strains have evolved independently of those infecting chickens elsewhere in the world, so understanding the biology and evolution of these strains can further our understanding of factors driving the emergence of novel coronaviruses. We infected groups of specific pathogen-free Leghorn chickens with six Australian IBVs (from five distinct genotypes) isolated between 1962 and 2013. All six affected the respiratory tract, but only one was nephropathogenic (N1/62). All six induced significant lesions and actively replicated in the upper respiratory tract, but they had lower levels of replication and induced less severe lesions in the middle and lower trachea. There were significant differences between the six strains in the severity of the lesions they induced and in their tissue tropism and effect on tracheal ciliary motility. Strains N1/62 (strain T) and N1/03 caused the most severe tracheal ciliostasis and replicated to the highest levels in tissues. Strain N1/03 caused the most severe lesions at 9 days post-infection. Only strain N1/03 caused lesions in the lower trachea. Overall, strains N1/03 and N1/62 were the most virulent. This study is the first to characterize the histological changes induced by the recently isolated Australian IBVs and compare them directly with older strains. Recombination between field and vaccine strains of IBV has yielded emergent IBVs in Australia that appear to have enhanced virulence for the respiratory tract.
Hepatitis B virus (HBV) infects human populations worldwide. HBV strains are classified into 10 genotypes, of which the HBV genotype D (HBV/D) infection is particularly prevalent in several countries. The HBV core promoter regulates viral replication and transcription, and the naturally occurring A1762T/G1764A double mutation (CP1) in the core promoter accelerates HBV replication. Previous clinical studies showed that a new core-promoter mutation, G1764T/C1766G (CP2), is frequently observed in genomes containing the G1757A substitution, which is unique to HBV/D; however, CP2 is not observed in genomes containing the 1762T/1764A double mutation. In this study, we found that the CP2 mutation dramatically increased viral replication and transcription efficiency in two cell lines; the degree of stimulation was comparable to that induced by CP1. Introduction of the 1757A substitution reduced the increase in viral replication induced by the CP1 mutation. By contrast, the addition of the 1757A substitution significantly increased the effect of the CP2 mutation. The transcriptional activity of CP1 was decreased by the 1757A substitution, due to a reduction in HNF1 binding affinity, suggesting that 1757G is an important component of the HNF1 binding consensus sequence. The HBV/D-specific CP2 mutation creates a binding site for the transcription factor HNF3, thereby increasing its transcriptional activity. HBX proteins containing substitutions reflecting the two types of core-promoter mutations did not affect the efficiency of viral replication. Therefore, we hypothesize that the introduction of the CP2 mutation represents a survival strategy for HBV/D, allowing it to escape the effect of the 1757A substitution.
Hepatitis C virus (HCV) is often associated with chronic liver diseases and significant alterations in host cellular signalling. However, the molecular mechanisms underlying HCV-related liver pathogenesis remain to be elucidated. The Hippo signalling pathway, a key regulator of cell proliferation and survival, plays a critical role in maintaining liver homeostasis. Here, we investigated the role of the Hippo pathway in HCV-related pathogenesis. We demonstrated that HCV infection induces degradation of LATS1, a key regulator of the Hippo pathway. Degradation of LATS1 protein was restored by a proteasomal inhibitor, but not a lysosome inhibitor, indicating that HCV promotes proteasomal degradation of LATS1 protein. HCV-induced degradation of LATS1 protein was suppressed in si-Itch-transfected Huh-7.5 cells. These results suggest that Itch ubiquitin ligase is involved in ubiquitin-dependent degradation of LATS1 protein. Cell fractionation assays and immunofluorescence staining revealed that HCV infection promoted nuclear translocation of YAP1 protein, suggesting that HCV infection suppresses the Hippo pathway. Furthermore, the transcription of YAP1 target genes, CYR61 and CTGF, that are involved in tissue remodelling and proliferation, was upregulated in HCV-infected Huh-7.5 cells and in HCV-infected patients. Taken together, we propose that HCV promotes the ubiquitin-dependent proteasomal degradation of LATS1 protein, leading to suppression of the Hippo pathway, thereby upregulating transcription of CYR61 and CTGF genes, which may contribute to HCV-related pathogenesis.
Curly top disease (CTD) affects sugar beet, tomato and other crops, resulting in stunted plants with severely curled leaves and reduced yields. The disease occurs worldwide and is caused by geographically associated monopartite geminiviruses transmitted mostly by leafhoppers. However, the aetiology of CTD in South America remains unknown because the disease disappeared in the 1950s. Here, we describe how the chance finding of tomato plants with CTD-like symptoms in Brazil in 2016 and high-throughput sequencing helped identify a novel ~2.6 kb geminivirus DNA associated with curly top disease in Mato Grosso (GV-CTD-MT) that induced CTD symptoms in agroinoculated tomato and Nicotiana benthamiana Domin. plants and produced geminivirus-like virions in infected plants. Evidence GV-CTD-MT may be the genomic DNA of the historic Brazilian curly top virus (BraCTV) includes (i) occurring in the same geographic location (Brazil); (ii) inducing nearly identical CTD symptoms in tobacco (Nicotiana tabacum L.) and tomato plants to those described in the 1940s for the BraCTV; (iii) inducing CTD in a similar broad host range of plant species as previously reported for BraCTV based on leafhopper transmission experiments; (iv) the co-phylogenetic analysis predicting the vector of GV-CTD-MT is Agallia sp. leafhoppers; and, importantly, (v) transmission experiments showing Agallia albidula, the known vector of BraCTV, is a competent vector of GV-CTD-MT. Sequence and phylogenetic analyses revealed that this novel CTD-inducing geminivirus has a chimeric genome, a long evolutionary history, and is closely related to monopartite geminiviruses recently identified in South America. These viruses were placed in the new genus Topilevirus, the fifth genus whose members induce CTD. Thus, our results suggest that BraCTD, which disappeared over 70 years ago, and possibly historic CTDs of sugar beet in South America were caused by topileviruses transmitted by indigenous leafhoppers, thereby solving the conundrum left by first-generation researchers.
Virus-like particles (VLPs) based on hepatitis B core antigen (HBc) represent an immunogenic and modular platform for epitope presentation. In this study, the VLPs formed by the modified recombinant hepatitis B core antigen from genotype G (HBc/G) were used as carriers for the presentation of a receptor-binding motif (RBM) of the Delta variant of Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). The RBM was inserted at the C-terminus of the modified HBc/G extended by the addition of a second, specifically modified C-terminal domain of HBc/G. All arginine residues in the extra domain were replaced with glycine, resulting in a 'two-tailed' HBc/G-Gly vector. The resulting HBc/G-Gly-RBM construct successfully formed regular VLPs in Escherichia coli and elicited specific antibody responses in mice. Despite the moderate immunogenicity of the RBM insert compared with the HBc carrier, sera from RBM-VLP-immunized animals exhibited neutralizing activity against MLV particles pseudotyped with the SARS-CoV-2 Delta spike and showed cross-reactivity with receptor-binding domains from the Wuhan and Omicron variants. To enhance the immune response, a replication-deficient Semliki Forest virus (SFV) vector expressing IL-12 was evaluated alone and in combination with the squalene-based adjuvant AddaVax. The co-administration of SFV-IL12 and AddaVax modestly improved virus neutralization rates and promoted a Th1 response, characterized by increased IgG2a production and IFN-γ secretion. These findings demonstrate the feasibility of combining classical and genetic adjuvants with the HBc-based VLP platform and provide preliminary insights for further optimization toward more potent and protective SARS-CoV-2 vaccine candidates.
Diagnostic inaccuracies are a major yet often overlooked threat to global health, leading to delayed treatment, preventable harm and systemic gaps in disease control. Among the most affected domains are Orthoflavivirus infections, which pose ongoing diagnostic challenges due to antigenic cross-reactivity, overlapping clinical symptoms and the narrow temporal sensitivity of standard tools such as serology and reverse transcription polymerase chain reaction. These constraints have led to widespread misdiagnoses and underreporting, ultimately hampering both effective clinical management and public health response. Recent advances in metagenomic and metatranscriptomic sequencing offer a transformative solution by enabling unbiased, simultaneous pathogen detection and real-time profiling of viral and host transcriptomics. In this review, we assess the diagnostic performance and translational value of these approaches in resolving Orthoflavivirus infections, with case examples from clinical settings in countries like the USA, UK, China and Germany which have already implemented these approaches into routine diagnosis in some settings. We examine key methodological considerations, including optimal sample timing, sample types and processing, sequencing strategy selection and the diagnostic performance of various platforms. We highlight the growing use of metatranscriptomics for detecting active infections, profiling viral and host responses, identifying coinfections and supporting real-time surveillance. We also discuss the key challenges such as technical expertise, lack of standardization, cost, turnaround time and regulatory approval that currently limit global implementation. Finally, we highlight emerging international efforts to integrate sequencing-based diagnostics into routine hospital workflows. Together, these innovations mark a critical shift toward precision diagnostics for Orthoflavivirus infections, with broad implications for clinical settings.
Senecavirus A (SVA) is a picornavirus that was first isolated in the USA in 2002; however, there is evidence that the virus was circulating in swine herds since 1988. Despite frequent reports of vesicular disease outbreaks caused by SVA infection in swine in Brazil since 2014, there is limited data on the genetic diversity and evolution of the virus in the country. SVA was isolated from swine exhibiting vesicular lesions, with samples originating from farms or slaughterhouses across 57 municipalities in 8 Brazilian states between 2018 and 2022. We obtained 501 SVA genomes through Sanger and Oxford Nanopore sequencing. Phylogenetic analysis revealed that Brazilian SVA sequences are genetically distinct from sequences from other countries, including China, USA and Canada, and form a monophyletic cluster, indicating a common ancestor for the viruses currently circulating in Brazil. Furthermore, there are two main clusters with sequences from the Midwest and Southern regions, suggesting that SVA is evolving independently in the swine population of the country. Pairwise sequence comparisons allowed us to identify seven unique mutations with high frequency in the Brazilian SVA sequences. Notably, mutations were identified in specific regions of the capsid proteins that interact with the host cell receptor (ANTRX1) and in surface-exposed residues, suggesting potential evolutionary changes due to receptor interaction or immune pressure. Recombination analysis provided evidence of at least five recombination events among the Brazilian strains. These findings offer new insights into the evolution of SVA circulating in Brazil and into the global epidemiology/evolutionary dynamics of the virus.
Orthohantaviruses are emerging zoonotic pathogens that can cause life-threatening diseases in humans. Their tripartite, negative-sense RNA genome is encapsidated by the viral nucleoprotein, but the subcellular localization and dynamics of these viral RNAs and proteins remain poorly characterized. Here, we present a comprehensive microscopy-based analysis of Puumala virus, the most prevalent orthohantavirus in northern and western Europe. Using fluorescence in situ hybridization (FISH) and Multiple Sequential FISH, we mapped the distribution of viral mRNAs, viral genomic RNAs (vRNAs), nucleoproteins and associated host cell factors, quantifying their intracellular abundance, co-localization and subcellular positioning. We observed distinct clustering of vRNAs with varying degrees of nucleoprotein association, a progressive increase in nucleoprotein expression levels during infection and a concomitant rise in the abundance of P-bodies. Moreover, we report a marked spatial reorganization of actin, microtubules and P-bodies, indicating substantial structural remodelling of host cells during orthohantavirus infections. Using a novel end-specific FISH assay, we observed a preferential 5'-end degradation of vRNAs in P-bodies, shedding new light on orthohantavirus RNA turnover within host RNA-processing compartments. Finally, co-localization analyses revealed the formation of potential 'viral factories' composed of nucleoprotein, vRNAs and viral mRNAs, indicating an intricate assembly hierarchy. Collectively, these findings improve our understanding of orthohantavirus replication and highlight the dynamic interplay between virus and host cell components.
Ovine gammaherpesviruses 2 (OvGHV2), a member of the Macavirus genus within the Orthoherpesviridae family, causes lymphoproliferative diseases in susceptible species, most notably sheep-associated malignant catarrhal fever. Research on OvGHV2 has been hindered by the absence of a permissive cell culture system, limiting investigations into viral replication, entry mechanisms and cell tropism. This challenge constrains progress towards understanding OvGHV2 pathogenesis and developing effective vaccines or therapeutics. We investigated the molecular mechanisms underlying OvGHV2 infection, focusing on the nuclear trafficking pathways of the latency-associated nuclear antigen (LANA), encoded by ORF73. In other gammaherpesviruses, LANA is known to mediate viral episome maintenance, chromatin tethering and latency through its nuclear localization. We identified and functionally characterized a novel bipartite nuclear localization signal (NLS) within the C-terminal region of OvGHV2 LANA and elucidated its interactions with host nuclear import receptors. Using high-resolution crystallography and quantitative binding assays, we mapped the key residues responsible for binding to importin alpha (IMPα) and demonstrated isoform-specific variations in binding affinity. Confocal microscopy revealed that the OvGHV2 LANA predominantly localizes to the nucleus through an IMPα/β1-dependent pathway, as mutation or inhibition of the NLS significantly reduced nuclear accumulation. Interestingly, partial nuclear localization under these conditions suggests an additional IMPα/β1-independent nuclear import mechanism. Collectively, our biochemical and structural analyses confirm that the identified NLS is essential for IMPα-mediated nuclear import of OvGHV2 LANA. These findings provide new insights into OvGHV2 host interactions and establish a molecular basis for developing targeted antiviral strategies against ovine gammaherpesvirus 2 infection.
Cervical squamous cell carcinoma (SCC) is classified by the World Health Organization based on its association with human papillomavirus (HPV) into HPV-associated (HPVA) and HPV-independent (HPVI) categories. HPVI SCCs can be p53 wild-type or p53 abnormal, the latter harboring a driver alteration in TP53 and portending worse survival. Recent developments have expanded the spectrum of squamous intraepithelial lesions (SILs) in the cervix. In the HPVA category, seborrheic keratosis-like lesions are now established as having an aetiological association with HPV42, a common low-risk HPV type. Papillary immature metaplasia, a further low-risk HPVA lesion, also falls into this category. More recently, potential HPVI squamous precursor lesions have been described; these form a wide morphologic spectrum and are difficult to diagnose, with the use of p16, p53, and HPV testing mandatory. When these ancillary tests are used, the HPVI SILs, similar to their invasive counterparts, stratify into p53 abnormal and p53 wild-type categories. Different genomic alterations are seen within the two groups, supporting their neoplastic nature. In this review, we provide a historical perspective and comprehensive description of all cervical SILs, with a focus on emerging entities and premalignant lesions, and appraise the terminology used over past years and that recently proposed for new entities. Key clinical, colposcopic, and morphologic features, differential diagnosis, treatment, follow-up, and prognosis are discussed. Special attention is paid to ancillary studies that assist in resolving differential diagnoses and their interpretation in light of recent scientific publications and international guidelines. We also discuss the clinical impact of a pathologic diagnosis of HPVA versus HPVI SILs.
Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that causes severe neurological diseases in humans. The assembly of flaviviruses is initiated by the interaction between capsid (C) proteins and viral genomic RNA, yet the molecular determinants that govern RNA encapsidation remain unclear. In this study, we established a TBEV virus-like particle (VLP) system to analyse viral factors that influence viral RNA incorporation independently of productive infection. Using a reporter-containing TBEV minigenome, we investigated the contribution of UTRs to the incorporation of RNA into extracellular particles. Truncation of the 5' UTR, 3' UTR or both did not significantly affect the levels of minigenome RNA detected in pelleted extracellular fractions, indicating that RNA incorporation occurs largely in a non-specific manner. We further examined the role of C protein dimerization by introducing alanine substitutions into residues that form the α2-α2' and α4-α4' dimer interfaces. Paradoxically, these substitutions increased the levels of minigenome RNA detected in pelleted extracellular particles without altering intracellular RNA expression, indicating a complex relationship between the integrity of the C protein dimer interface and levels of extracellular viral RNA. Finally, we showed that TBEV proteins and minigenome RNA can be detected in extracellular vesicle (EV)-associated fractions under VLP-producing conditions. Using immunoaffinity purification, we demonstrate the presence of viral components in EVs, underscoring EV-associated release as a factor that complicates the analysis of flaviviral particle assembly.
There is an urgent need for alternative solutions to combat multidrug-resistant (MDR) E. coli infections. In recent years, there has been an increase in MDR strains causing urinary tract infections (UTIs), which has resulted in more challenging treatment options, increased healthcare costs and prolonged hospital stays. The utilization of bacteriophages as a prospective modality for the management of bacterial infections has garnered significant attention. The objective of this study was to isolate and describe a phage capable of infecting MDR E. coli strains isolated from the urine of patients affected with UTI. The phage EcoPhCCP1 was isolated using the plaque assay from the influent of a wastewater treatment plant. The phage was characterized by phenotypic and genomic features. Morphological characteristics such as shape and size were determined using electron microscopy, and its host range was determined against multiple MDR strains. The complete genome of the phage was subjected to whole-genome sequencing and then assembled and annotated to search for virulence or antimicrobial resistance gene (ARG). VIRIDIC was employed to compare the closest phage genomes, while VICTOR and taxMyPhage were used to construct its phylogeny. EcoPhCCP1 is a tailed phage capable of infecting and propagating in multiple MDR E. coli strains recovered from UTI. The phage genome is 44,482 bp in length, with a GC content of 50.7 mol%, and encodes 87 ORFs, 33 of which have been previously functionally annotated. Phage EcoPhCCP1 is a Kagunavirus, in the recently created Sarkviridae family. Notably, phage EcoPhCCP1 does not harbour ARGs or virulence genes, thus rendering it a promising candidate for phage therapy against clinically significant MDR E. coli strains. Moreover, phage EcoPhCCP1 possesses putative anti-CRISPR proteins.
The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small-animal model for congenital cytomegalovirus, a leading cause of cognitive impairment and hearing loss in newborns. GPCMV encodes human cytomegalovirus (HCMV) homologues of viral entry glycoprotein complexes, which are neutralizing-antibody vaccine targets. As with HCMV, GPCMV has two pathways of cell entry (direct and endocytic). Specific viral gH/gL-based complexes are necessary for receptor interaction and cell entry: gH/gL/gO trimer (direct) and pentamer complex (PC) (endocytic). Both pathways also require gB as the fusogenic protein. Direct GPCMV cell entry requires platelet-derived growth factor receptor alpha (PDGFRA), but an endocytic PC receptor remains unknown. We hypothesized that cellular knockout of direct and endocytic receptors would completely block infection, which cannot be achieved by gB-based antibodies. Candidate receptors including neuropilin proteins (NRP1, NRP2) and CD147 present on all established guinea pig cell lines were selected based on importance as common virus receptors or in fetal development. Results demonstrated that NRP2 interacted with PC, unlike NRP1 or CD147, in immunoprecipitation assays and eliminated NRP1/NRP2 heterodimer receptor interaction. The viral trimer only interacted with PDGFRA. Double knockout of PDGFRA/NRP2 completely blocked GPCMV infection. In contrast, the CD147/PDGFRA double knockout had limited GPCMV inhibition, and the single knockout of CD147 had no impact. Knockout of the various receptors had no effect on control HSV-1 infection. Ectopic expression of guinea pig cell receptors restored GPCMV infection but not human NRP2/PDGFRA, indicating a basis for the species-specific barrier for GPCMV and HCMV infection. Overall, results increase the translational relevance of GPCMV for the development of CMV intervention strategies.
Cyvirus cyprinidallo2 (CyHV-2) is an alloherpesvirus and the causative agent of herpesviral haematopoietic necrosis in goldfish. Whole-genome sequence comparison of the developed live-attenuated vaccine P7-P8 with virulent CyHV-2 strains revealed seven single-nucleotide polymorphisms, five deletions and one inversion in the ORFs, which may be involved in attenuation. A start codon loss in ORF 113, a putative apoptosis-inhibition gene, was observed in the mutations. In vitro assays indicated that apoptosis-related genes were upregulated in cells inoculated with the vaccine or virulent virus compared to uninfected cells. However, the vaccine group showed increased phosphatidylserine externalization and DNA damage, suggesting the apoptosis-inducing properties of P7-P8. In the in vivo experiment, histopathology demonstrated that vaccinated goldfish exhibited immune responses, such as leucocyte aggregation and melanomacrophage centre formation, without marked degeneration. Gene expression analysis showed upregulation of proinflammatory and granzyme B genes in vaccinated fish. In addition, the vaccine strain triggered apoptosis of the infected cells during the early stage of infection, potentially promoting virus clearance and preventing excessive virus replication. The results show that the P7-P8 potentially induces apoptosis and immune responses, contributing to low virus propagation without tissue damage. This study provides insights into CyHV-2 pathogenesis, suggesting that apoptosis-related genes can be the targets for vaccine development against alloherpesviruses in aquaculture species.
Despite their great agronomic interest and widespread occurrence in germplasm resources, the quantitative resistance and tolerance of plants to their parasites have rarely been studied in terms of durability potential. Using experimental evolution under controlled conditions for 9 months, we compared the evolution of potato virus Y (PVY) (Potyvirus yituberosi) virulence, measured by the effect of viral infection on plant fresh weight, and replicative fitness, measured by systemic viral load, in five pepper (Capsicum annuum) lines contrasting in their levels of quantitative resistance and tolerance. PVY evolutionary trajectories differed between pepper lines. Three lines revealed either an increase in PVY replicative fitness or an increase or decrease in PVY virulence. Two other lines did not reveal any significant change in PVY replicative fitness or virulence. The tolerance level of three pepper lines also differed significantly when measured with initial and evolved PVY populations, often associated with changes in PVY virulence. PVY evolutionary trajectories were partly explained by parameters linked to plant resistance operating at different stages of infection (inoculation, colonization of inoculated leaves and systemic infection). This study provides information on the durability potential of quantitative resistance and tolerance to PVY in pepper.
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac16 (BV/ODV-E26) is a multifunctional protein found exclusively in group I NPV genomes. Here, we report a novel role for Ac16 in nucleolar localization. Using protein truncation and subcellular localization analysis, we characterized that the residues 78-113 of Ac16 contain nuclear (NLS) and nucleolar (NoLS) localization signals. Further multiple point mutation analysis within this region demonstrated that two basic-amino-acid-rich clusters, 78HKKKLRH84 and 108KKTTHR113, function as NLSs, and together, they constitute a functional NoLS. However, Ac16 itself was not observed to localize to nucleoli, while it displayed an overlapping distribution with IE1 during AcMNPV infection. Co-expression assay revealed that IE1, an Ac16-interacting protein, alters the subcellular localization of Ac16, and this effect is independent of the Ac16 NoLS. By yeast two-hybrid library screening, vacuolar (H+)-ATPase subunit D (SfVhaD) was identified as a candidate interaction partner of Ac16, which was further confirmed by co-immunoprecipitation. Ac16 affected the localization of SfVhaD; both proteins predominantly colocalized in nucleoli in transient co-expression assays, while they primarily colocalized within the virogenic stroma during AcMNPV infection. Together, these data suggest that Ac16 contains a functional NoLS and may facilitate the transport of its interaction partners from nucleoli to viral replication centres during AcMNPV infection.
Understanding the genomic diversity of the Mpox (formerly known as 'monkeypox') virus (MPXV) is important for monitoring viral evolution and dissemination. We encountered three clinical cases in our hospital during the outbreak period of Mpox in 2023 in Japan. The present study aims to report the largest genomic rearrangement observed to date in one of the clinical isolates and to demonstrate its viability in cell culture. Whole-genome sequencing and digital PCR were used to characterize the viral genomes. Viral isolation, microscopic observation and growth kinetics in Vero cells were performed to confirm viral replication. All three patients were men living with human immunodeficiency virus (HIV) and presented typical Mpox symptoms, such as rash, fever and pustules on the body surfaces, including near the genitals. Virus isolation was successful in all three cases. All viral strains belonged to clade IIb, lineage C.1. Notably, one strain exhibited a large-scale genomic rearrangement: a 5.5 kb deletion at the left variable region replaced by a 30.5 kb inverted sequence, the largest reported in clinical isolates. Despite this extensive genomic change, the strain maintained robust replication capacity and marked fusogenicity in vitro. We report, for the first time in clinical isolates, a massive genomic rearrangement in MPXV that does not impair viral replicability. This finding represents an example of genomic plasticity and provides a rare but noteworthy resource for future studies. Taken together, when performing genomic analyses of MPXV, aberrant genomic rearrangements should also be carefully considered alongside single-base substitutions.
Cytomegalovirus (CMV) is a beta herpesvirus that persists quiescently in healthy individuals but causes severe disease in immunocompromised patients and congenitally infected newborns. Side effects posed by the currently available antivirals necessitate the development of new antivirals with improved safety profiles. This study aims to optimize the potency of an anti-CMV peptide (P10) mimicking conserved region 2 of tegument pp150, fused with an elastin-like polypeptide (ELP). Our previous study has demonstrated that ELP-P10 inhibits murine cytomegalovirus (MCMV) growth in vitro and in vivo with enhanced pharmacokinetic properties relative to the free peptide. To enhance the potency of ELP-P10 at a lower concentration, this study utilizes a cell-penetrating peptide, SynB1, to facilitate the delivery of ELP-P10 into the cells. SynB1 was added to the N terminus of ELP-P10 to generate SynB1-ELP-P10. Antiviral efficacy and cytotoxic effects of ELP-P10 and SynB1-ELP-P10 were studied in cell culture. Pharmacokinetics, biodistribution and antiviral efficacy were studied in a mouse model of CMV infection. While ELP-P10 maintained significant antiviral activity against human cytomegalovirus (HCMV) in cell culture at a higher concentration, SynB1-ELP-P10 shows potency against HCMV and MCMV at a threefold lower concentration compared to ELP-P10. SynB1-ELP-P10 had similar bioavailability after subcutaneous administration as ELP-P10, and SynB1 conjugation to ELP-P10 significantly enhanced its accumulation in the kidneys. Moreover, in an in vivo model of CMV infection, ELP-P10 and SynB1-ELP-P10 treatment led to a significant reduction in the viral titre compared to controls. In conclusion, the strategic modification of ELP-P10 with SynB1 potentiated CMV inhibition, allowing for the use of lower therapeutic doses and mitigating potential side effects.