Search Results (224)

Mosquitoes are recognized as the arthropod group with the greatest vectorial capacity, and the viruses they transmit constitute a significant concern in the context of global One Health. In addition, these insects act as hosts for a wide diversity of insect-specific viruses (ISVs), which exclusively infect arthropods. Expanding knowledge of ISVs is particularly relevant, given their potential influence on arbovirus replication and their role in elucidating the evolutionary processes that shape virus–vector interactions. In this study, we report the isolation and molecular analysis of three negeviruses associated with different mosquito species of the genera Culex, Coquillettidia, Mansonia, and Ochlerotatus, collected in Belém, Pará State, in the Brazilian Amazon: Loreto virus, Wallerfield virus, and a putative new species, designated Terra firme virus. Eleven pools exhibited cellular alterations consistent with cytopathic effects in invertebrate C6/36 cells but showed no evidence of replication in vertebrate Vero cells. Notably, simultaneous infections by two or three negeviruses were detected in some mosquito pools, indicating the occurrence of multiple viral infections within individual samples. Genomic analyses revealed that the isolated strains share conserved domains with previously described isolates from other countries. Phylogenetic inferences demonstrated that the investigated strains are classified within the clades Nelorpivirus and Sandewavirus. Taken together, these findings expand the currently known diversity of the negevirus group and contribute to a more comprehensive understanding of its host range and geographic distribution. Full article

Viral internal ribosome entry sites (IRESs) are specialized RNA structures that facilitate cap-independent translation as a strategy to usurp the host translational machinery. The Type 6 IRESs are the most streamlined mechanism to date, as they adopt a three pseudoknot RNA structure to initiate factorless translation initiation by directly recruiting the ribosome and drive translation. The Halastavi árva virus (HalV) IRES represents the most minimalistic subclass identified to date, whereby the IRES lacks specific pseudoknot domains that bind to the 40S subunit but instead recruits pre-assembled 80S ribosomes via a mechanism that is not fully understood. Here, we examined cellular conditions that can support HalV IRES translation. We demonstrated that the HalV IRES is translationally active in insect Sf21 lysates and Drosophila S2 cells, but inactive in mammalian RRL and wheat germ extract. Cells treated with heat shock or serum starvation suppressed HalV IRES activity, whereas virus infection robustly enhanced HalV IRES-mediated translation. Finally, the HalV IRES can support viral translation and replication using a heterologous viral replicon. These findings highlight the context-specific cellular conditions that allow ribosome assembly and translation by a factorless minimalist IRES. Full article

The single von Willebrand factor C (SVWC) domain-containing protein family represents a crucial class of immune molecules recently identified in insects and crustaceans. Initially regarded as functional analogs of vertebrate interferons (IFNs) due to their virus-induced expression and activation of the Janus kinase-signal transducer and activator of the transcription (JAK-STAT) pathway, recent studies have revealed that SVWC proteins possess far more complex functions. Many SVWC members are themselves a novel class of pattern recognition receptors (PRRs) that can directly bind to viruses and bacteria. Importantly, SVWCs are not a single entity but a highly diverse family—multiple subtypes exist in Drosophila, Bombyx mori, and shrimp—a gene expansion that implies functional differentiation. This review systematically examines the multifunctionality of SVWC proteins in insects and crustaceans, with a particular focus on the functional specialization driven by subtype diversity. We delve into the complex regulatory networks governing SVWC expression, including the differential activation by nuclear factor kappa B (NF-κB) pathways (Dorsal, Rel-2, Relish) and interferon regulatory factor (IRF) pathways. We detail the unique signaling mechanism by which SVWCs activate the JAK-STAT pathway via integrins, rather than the canonical Domeless receptor. Furthermore, we extend the discussion to the emerging roles of SVWCs as PRRs in humoral immunity (activating Toll/IMD pathways to induce antimicrobial peptides) and cellular immunity (mediating hemocyte phagocytosis). Based on current evidence, We propose that diverse SVWC subtypes may recognize distinct pathogens, bind to different integrin receptors, and activate specific STAT variants via disparate upstream induction pathways, thereby establishing a systematic and hierarchical immunoregulatory network. This understanding positions the SVWC protein family as a central hub in the insect immune network and offers a novel perspective on the complexity and evolution of invertebrate immunity. Full article

Aedes aegypti is a primary vector for globally significant arboviruses such as dengue virus (DENV). The mosquito’s metavirome, particularly its insect-specific virus (ISV) component, is recognized as a key modulator of arboviral transmission. However, the natural ecology of these interactions in populations remains poorly understood. This study presents the first comparative analysis of the metavirome in wild-caught A. aegypti from Vietnam based on natural DENV infection status. Metaviromic analysis was performed on 69 DENV-positive pools from six central provinces. The results obtained were compared with previously obtained metaviromic data from 7 DENV-negative pools (from the same region). Analysis suggests the presence of a stable ‘core metavirome’ of 11 ISVs present in both groups. Interestingly, six ISVs were detected only in DENV-negative mosquitoes, which may suggest potential antagonistic interactions requiring further investigation. Conversely, five ISVs were found only in DENV-positive pools, including Aedes partiti-like virus 1 and Aedes anphevirus. The latter may suggest possible synergistic relationships that facilitate arboviral replication. Phylogenetic analysis of prevalent ISVs, such as Phasi Charoen-like phasivirus (PCLV) and Chaq-like virus, revealed patterns of both local circulation and genetic diversity. The findings describe distinct ISV profiles associated with DENV infection in a natural setting, providing a data-driven foundation for hypothesizing specific virus–virus interactions. The data underscores the complexity of the mosquito metavirome. Here, we identified several candidate ISVs for future experimental studies aimed at understanding potential functional impact on arboviral vector competence. Full article

The management of Culex pipiens (Diptera: Culicidae), key vectors of arboviruses like West Nile virus, necessitates sustainable alternatives to chemical insecticides. This study screened indigenous entomopathogenic fungi (EPF) from forest soils in Achaia, Greece, for their larvicidal efficacy against Cx. pipiens biotype molestus. Fifteen fungal isolates were obtained via insect baiting and identified as Beauveria and Metarhizium species. A comprehensive bioassay at 1 × 10⁸ conidia mL⁻¹ revealed significant variation in pathogenicity after 72 h. Two isolates, Beauveria bassiana (BB) (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (K3(1)) (Hypocreales: Clavicipitaceae), exhibited the highest virulence among the tested isolates, each causing 60% mortality with a rapid median lethal time (LT₅₀) of ~18.5 h. Survival analysis, Cox modeling, and non-linear kinetic modeling (Gompertz/Richards) classified three distinct virulence clusters: high/rapid, moderate/consistent, and low/delayed. A pathogenicity network analysis and a composite virulence index further validated BB and K3(1) as the most effective candidates. These results demonstrate the high isolate specificity of fungal efficacy and underscore the importance of screening local fungal diversity. The identified high-virulence isolates represent promising, environmentally sound candidates for the development of targeted biopesticides. Future research should focus on formulation for aquatic environments and integration into resistance-resilient integrated vector management programs. Full article

The overuse of chemical insecticides highlights the urgent need for novel vector control strategies. Insect-specific viruses (ISVs), such as the cell-fusing agent virus (CFAV), have shown potential to block arbovirus transmission by inhibiting viral replication in mosquitoes. However, the effects of CFAV beyond its natural host, Aedes aegypti, remain largely unexplored. In this study, we established a CFAV infection model in Aedes albopictus, a major vector for Zika virus (ZIKV), via intrathoracic injection. Stable infection was achieved, with viral loads reaching up to 10⁷ copies per mosquito by day 10 post-injection. Nevertheless, high post-injection mortality (median survival: 3 days) was observed, which we attribute primarily to mechanical injury. No evidence of vertical transmission of CFAV was detected in Ae. albopictus. Co-injection of CFAV and ZIKV did not significantly affect ZIKV replication in this species. In contrast, in Ae. aegypti pre-infected with CFAV followed by oral ZIKV challenge, CFAV significantly reduced ZIKV infection rates in the ovaries at day 4 and viral loads in salivary glands at day 10. These findings demonstrate that while CFAV can productively infect Ae. albopictus, it does not undergo vertical transmission in this species, and has no inhibitory effect on ZIKV under the co-infection conditions tested. This study underscores challenges associated with using single ISVs such as CFAV for arbovirus control and highlights the complex, bidirectional role of multiple ISV co-infections. While exploring multi-ISV combinations may offer a potential strategy to enhance antiviral efficacy, their net effect—whether suppression or enhancement of arboviruses—warrants careful investigation. Full article

Background/Objectives: Epidemiological studies have proven that coxsackievirus B3 (CVB3) is the major virus that causes acute and chronic myocarditis and pancreatitis. Currently, there are no antiviral therapeutic drugs or vaccines that are available for use as clinical therapeutics or vaccines. Subunit polypeptides-based vaccines, especially when combined with adjuvants, represent safe and effective vaccine platforms because they are considered to be better immunogens. The viral capsid protein VP1 of CVB3 is the most immunogenic viral polypeptide, providing opportunities for its use in designing subunit polypeptide vaccines. In the present study, we designed and produced a CVB3 vaccine candidate based on the recombinant expression of the major immunogenic viral protein VP1 of a wild-type CVB3 strain. Methods: We assessed its induced humoral and cellular immune responses and then evaluated its protective immunity against pathogenic CVB3 strain challenges in a Balb/c mouse model. Neutralizing specific antibodies and cytokine interferon gamma (INF-γ) production were determined in the sera of both prime- and prime-boost-immunized mice with the vaccine candidate. Results: Our results demonstrate that the recombinant rVP1 expressed in a eukaryotic insect cell baculovirus vector system elicited cellular and humoral immune responses, protecting Balb/c mice from lethal challenges. Conclusions: Hence, the vaccine produced based on the recombinant expression of VP1 is a promising and potential candidate against natural CVB3 infections. Full article

The family Flaviviridae has been expanded to include the highly divergent flavi-like viruses into three new families, Flaviviridae, Pestiviridae, and Hepaciviridae, in the order Amarillovirales. Classical flavivirids are small, enveloped viruses with positive-sense ssRNA genomes lacking a 3′ poly(A) tail and ~9.0–13.0 kb in length, with a single open reading frame (ORF) encoding structural proteins at the N-terminus and nonstructural proteins at the C-terminus. Members infect a wide range of mammals, birds, and insects, and many are host-specific and pathogenic. Although the RNA-directed RNA polymerase (RdRP) gene sequences of the flavi-like viruses group phylogenetically with those of classical flavivirids, flavi-like viruses often encode larger polyproteins and possess substantially longer genomes of up to ~40 kb, and some have a 3′ poly(A) tail. Their host range extends across the whole animal kingdom and angiosperm plants. This review describes the reported flavi-like viruses of aquatic animals, providing a meaningful update on all three new families in Amarillovirales that have been discovered using metagenomics in fish, crustaceans, mollusks, and echinoderms. These amarilloviruses include pathogenic viruses of aquatic animals, such as Cyclopterus lumpus virus (CLuV) detected in moribund lumpfish, and infectious precocity virus (IPV) found in iron prawn syndrome (IPS)-affected farmed giant freshwater prawns. Full article

Cryo-electron microscopy (cryo-EM) has revolutionized structural virology, enabling routine structure determination at 2–4 Å resolution, with exceptional cases reaching 1.56 Å. The structural diversity of viruses across vertebrate, plant, and insect hosts provides fundamental insights into infection mechanisms, host–pathogen coevolution, and therapeutic target identification. However, analysis of Electron Microscopy Data Bank entries reveals notable disparities in structural coverage: among 11,717 eukaryotic virus structures (excluding bacteriophages), vertebrate viruses constitute 97.6% (n = 11,432) of deposited entries, while plant viruses (1.0%; n = 117) and insect viruses (1.4%; n = 168) remain significantly underrepresented. This bias stems from distinct technical barriers including size limitations for giant viruses exceeding 200 nm, the loss of asymmetric information during symmetry-imposed processing, and the morphological complexity of filamentous and pleomorphic viruses. Each barrier has driven the development of specialized methodological solutions: block-based local refinement overcomes through-focus variations in giant viruses, cryo-electron tomography (cryo-ET) validates and reveals asymmetric features lost in symmetrized reconstructions, and subtomogram averaging enables structural analysis of pleomorphic assemblies. This review synthesizes recent methodological advances, critically evaluates their capacity to address specific technical barriers, and proposes strategies for expanding structural investigations across underrepresented host systems to achieve comprehensive understanding of viral structural biology. Full article

Advances in diagnostic techniques, along with environmental changes driven by human activity, have intensified the surveillance and monitoring of virus and arbovirus circulation on the Amazon. These efforts have increased the detection of insect-specific viruses in field-collected hematophagous arthropods. This study reports the first isolation of the Aguas Brancas virus from mosquitoes collected in the Brazilian Amazon and in a rural area of Brasília, Federal District, Brazil. Arthropods of the family Culicidae, genus Limatus durhamii, were collected at ground level in forest fragments. Sample BEAR812610 originated from Ananindeua, Pará, within the Evandro Chagas Institute’s grounds, and sample BEAR839941 from a forest fragment in Brasília (Ceilândia—Núcleo Rural Boa Esperança, Site B4). Specimens were identified to the species/genus level, macerated, and the supernatant inoculated into C6/36 and Vero cell cultures for viral isolation. The presence of arboviruses was determined by indirect immunofluorescence using antibodies against major arbovirus groups. Positive samples were sequenced for nucleotide and amino acid identification, and phylogenetic analysis confirmed the virus as belonging to the genus Orthoflavivirus. This represents the first report of the isolation and characterization of the insect-specific Aguas Brancas virus. Full article

The Orthoflavivirus genus includes a variety of human-pathogenic, mosquito-borne flaviviruses (MBFs) including dengue, Zika, and West Nile viruses, which pose significant global public health threats. Insect-specific flaviviruses (ISFs) are another group within the genus that exclusively replicate in mosquitoes and are incapable of infecting vertebrates. ISFs have recently attracted growing research interest due to their potential applications in vaccine development. In addition, multiple studies have demonstrated that prior infection with ISFs such as Palm Creek virus and Binjari virus can suppress subsequent infection with human-pathogenic MBFs. This phenomenon, known as superinfection exclusion (SIE), opens the avenue for the potential applications of ISFs in MBF transmission control. This prompted a growing number of studies into ISFs and their interactions with MBFs in mosquito hosts. In this review, we provide an overview on ISFs, with a particular emphasis on the capacity of different ISFs to cause SIE, the current insights into the mechanisms of this phenomenon, and the potential use of ISFs in the SIE-based biocontrol strategies. Full article

Background: Pathogenic flaviviruses pose a serious threat to human health, and vaccines are an effective means of prevention and control. Although related vaccines have achieved significant progress, safety and efficacy limitations still exist, urgently requiring the development of novel vaccine platforms. The insect-specific flavivirus Chaoyang virus (CYV), with a structure similar to pathogenic flaviviruses and limited to insect cell replication, has potential as a safe vaccine vector. Methods: To systematically evaluate CYV’s potential as a universal flavivirus vaccine backbone and provide a vaccine candidate for type I Japanese encephalitis virus (JEV) prevention, this study constructed a chimeric JEV genotype I (GI) prME protein vaccine candidate CYV-JEV using CPER technology, systematically assessing its safety and immunoprotective effects. Results: Using the CPER method, CYV-JEV was successfully rescued, showing efficient replication in mosquito cells but defective replication in mammalian cells. As a vaccine backbone, CYV did not induce inflammatory responses or immune cell subset imbalances in IFNAR^−/− mice. CYV-JEV exhibited no pathogenicity in adult and suckling IFNAR^−/− mice. Immunisation of IFNAR^−/− mice with 10⁶ FFU twice provided complete protection against lethal challenge (100%) and effectively reduced paralysis rates (62.5%). Single-cell sequencing further revealed extensive T- and B-cell activation in the immune spleen. Conclusions: The results demonstrate that the CYV-based CYV-JEV candidate vaccine demonstrates both safety and efficacy, representing a promising alternative to attenuated JEV vaccines, with CYV showing potential as a safe and effective universal flavivirus vaccine backbone. Full article

The production and quality of wheat and maize grain can be significantly affected by various pests and pathogens, with phytoplasmas posing a particular threat due to their rapid spread and potential to cause severe damage to cultivated crops. The objective of this investigation was to evaluate the risk associated with these wall-less bacteria in wheat and maize crops. To achieve this, a survey was conducted in commercial fields located in southwestern Poland. Samples of winter wheat and fodder maize were collected at two distinct developmental stages, including both symptomatic and asymptomatic plants. Symptoms observed in wheat included yellowing, stunting, and excessive tillering, while maize plants showed yellow leaf striping, red discoloration, and stunted growth. Polymerase chain reaction (PCR) assays using phytoplasma-specific primers, followed by Sanger sequencing and sequence analysis, confirmed phytoplasma infections in 2% of wheat and 1.5% of maize samples. Virtual restriction fragment length polymorphism (RFLP) analysis identified the wheat-infecting phytoplasmas as belonging to subgroup 16SrI-C (‘Candidatus Phytoplasma tritici’-related strain)—a pathogen of major concern for wheat, while maize-infecting phytoplasmas were classified into subgroups 16SrI-B and 16SrV-C. Additionally, wheat plants collected during the early elongation phase were tested for Mastrevirus hordei (former wheat dwarf virus, WDV) using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), which confirmed the presence of WDV in all tested samples. Preliminary screening of field-collected leafhoppers revealed that 7.5% of Psammotettix alienus, the predominant species in wheat fields, carried 16SrI-C phytoplasmas. In maize fields, Zyginidia scutellaris was the most prevalent species, with 1.7% of individuals carrying 16SrV-C phytoplasma. These findings suggest that these insect species may contribute to the transmission of phytoplasmas in wheat and maize. This study provides the first documented evidence of 16SrI-C phytoplasma infecting wheat in Poland, and of 16SrV-C and 16SrI-B phytoplasmas infecting maize, expanding the known host range of these subgroups in the country and highlighting their potential phytosanitary importance. Full article

Piscine orthoreovirus genotype 1 (PRV-1) is an emerging viral pathogen in salmon aquaculture that causes Heart and Skeletal Muscle Inflammation (HSMI), with high prevalence in salmon-producing countries such as Chile. A significant obstacle in PRV-1 vaccine development is the inability to culture the virus in vitro, which limits the scalability and production of traditional inactivated or DNA-based vaccine strategies. This study describes the development of a novel virus-like particle (VLP)-based vaccine against PRV-1. Recombinant VLP were produced by co-expressing the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) using a baculovirus-based expression system in insect cells. In addition, to enable differentiating infected from vaccinated animals (DIVA) strategies, the σ1 protein was modified by adding of a cmyc epitope tag. The results demonstrated that the native VLP vaccine (VLP6n) significantly reduced viral loads in Atlantic salmon challenged with PRV-1. Moreover, in rainbow trout, the cmyc-tagged VLP-like vaccine (VLP6c) elicited a specific antibody response against the cmyc epitope, allowing differentiation between vaccinated and naturally infected fish. Overall, this VLP-based vaccine platform represents a promising strategy for controlling PRV-1 prevalence in salmon-producing counties, supporting the implementation of serological surveillance programs. Full article

Background: Seasonal influenza vaccines must be reformulated annually due to the high genetic variability and antigenic drift of circulating influenza viruses. The annual update, guided by World Health Organization (WHO) recommendations, results in significant challenges, including compressed production time periods, elevated manufacturing costs, and global distribution pressures. Moreover, mismatches between vaccine strains and circulating viruses can severely reduce protective efficacy, underscoring the urgent need for broadly protective and long-lasting influenza vaccines. Methods: In this study, we developed an adjuvanted trivalent recombinant influenza virus-like particle vaccine (a-RIV) using the baculovirus–insect cell expression system and formulated it with an AS01-like adjuvant. The vaccine comprises full-length hemagglutinin (HA) proteins from WHO-recommended seasonal influenza strains: A/H1N1 (AH1), A/H3N2 (AH3), and B/Victoria (B/vic) lineages. The purified HA proteins were subsequently formulated with a liposomal adjuvant to enhance the immunogenicity. Results: In mouse immunization studies, the a-RIV vaccine elicited significantly stronger humoral and cellular immune responses than the licensed recombinant vaccine Flublok and the conventional inactivated influenza vaccine (IIV). High levels of functional anti-HA antibodies and antigen-specific T cell responses persisted for at least six months post-vaccination. Moreover, a-RIV induced broadly reactive antibodies capable of cross-binding to heterologous AH1 and AH3 influenza strains. Conclusions: Our data demonstrate that the a-RIV elicits enhanced, durable, and broadly cross-reactive immune responses against multiple influenza subtypes. These findings support the potential of adjuvanted recombinant HA-based vaccine as a promising candidate for the development of next-generation influenza vaccine. Full article