Engineering of recombinant porcine deltacoronavirus stably expressing foreign gene.

Knockout of nAChR subunits in spider mites and their phytoseiid predators confers spinosyn cross-resistance and reveals a conserved mode of action in mites.

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for Escherichia coli transformation and cloning of RABV full-genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.IMPORTANCEReverse genetics systems allow researchers to generate recombinant viruses with precise genetic modifications, advancing studies of viral replication, pathogenicity, and vaccine development. However, constructing a full-length viral genome expressing plasmids is often time-consuming and technically demanding. To bypass the cloning process, a simple, cloning-free reverse genetics platform based on the circular polymerase extension reaction (CPER) has been applied for positive-sense RNA viruses. In this study, we applied the CPER-based reverse genetics system for rabies virus (RABV), a mononegavirus, enabling rapid and flexible generation of recombinant RABVs, including mutant, reporter-expressing, and chimeric clones. Our approach greatly facilitates genetic engineering of RABV and provides a versatile framework that can be extended to other mononegaviruses, thereby accelerating both basic and applied virology research.

De novo recovery of Ghana virus, an African bat Henipavirus, reveals differential tropism and attenuated pathogenicity compared to Nipah virus.

The Simbu serogroup, part of the Peribunyaviridae family, includes arboviruses associated with febrile illnesses in humans and fetal congenital malformations due to viral neurotropism in ruminants. These viruses possess a tripartite, negative-sense RNA genome lacking the poly(A) tail. Notably, the 5’ untranslated region (UTR) of the small (S) genomic segment contains conserved RNA elements, including a stem-loop (SL) structure and a sequence-based motif (GC signal) flanking the messenger RNA (mRNA) termination site. Although their functions remain unclear, their conservation and specific location suggest a potential role in mRNA transcription termination and translation initiation. A reverse genetics system for Schmallenberg virus (SBV) was used to create a viral recombinant library bearing deliberate mutations in both motifs. Replication kinetics, S segment transcription termination, and Nucleocapsid protein (N) abundance of rescued virus mutants were evaluated in mammalian and insect cell culture. Virulence was assessed in an immunocompetent mouse model. Characterization of the mutant viruses indicated that the SL structure is essential for viral production, with the stem length as a key feature; more than three complementary base pairs between the stem arms are necessary for replication. A shorter stem length impaired replicative fitness, N protein abundance and altered the mRNA to genomic RNA ratio. Point mutations in the GC signal disrupted proper mRNA termination, thereby limiting viral N protein synthesis and, thus, virion assembly. In vivo, attenuated viruses resulted in lower viral loads, reduced dissemination in mice brains, and improved survival rates compared to wild-type SBV. The GC signal mutants exhibited strong attenuation while still maintaining active transcription. Overall, these findings indicate that the SL and GC signal serve as cis-regulatory elements and are indirect determinants of SBV virulence, regulating viral replication and influencing neuropathogenesis.

Coral reefs are one of the most biodiverse and productive ecosystems on Earth. However, corals are currently under threat from increasing ocean temperatures driven by climate change. Despite the known importance of these fragile ecosystems, our understanding of the molecular mechanisms driving ecologically important traits has been constrained by a lack of genetic tools for functional characterization. To address this limitation, we have developed straightforward and efficient methods to genetically modify corals and study gene function throughout various life history stages using CRISPR-Cas9-based mutagenesis. In this protocol, we first describe how to spawn and collect gametes from the coral Acropora millepora during seasonal spawning events. Next, we describe a method for microinjection of one-cell coral zygotes with CRISPR-Cas9 reagents. We include considerations about effective single-guide RNA design, methods for identifying successfully injected animals, strategies for rearing mutant larvae and juveniles, and methods for the detection and quantification of genomic modifications. This protocol is currently the only way to perform gene editing in corals and takes ~2-4 weeks to complete and has been successfully applied to study genes controlling heat tolerance in coral larvae and skeleton formation in coral juveniles. These technical advances set the foundation for a new field using reverse genetics to study ecologically important traits in corals, such as the establishment of symbiosis and its breakdown upon heat stress.

Development of an inactivated H9N2 subtype avian influenza serological DIVA vaccine using the chimeric A/B NA epitope approach.

The advancement of genomics has reshaped the process of gene discovery, integrating both forward and reverse genetic strategies to elucidate gene function and regulation. Forward genetics progresses from phenotype to genotype, identifying genes responsible for specific traits through mutagenesis, phenotypic screening, and mapping techniques such as QTL analysis and positional cloning. In contrast, reverse genetics begins with a known gene sequence and determines its function through targeted alterations using approaches like TILLING, EcoTILLING, RNA interference (RNAi), virus-induced gene silencing (VIGS), and homologous recombination. Recent developments in genome-editing technologies, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas system, have enabled precise, efficient, and cost-effective genetic modifications. Each approach offers distinct advantages and limitations, depending on species, research objectives, and available resources. The convergence of forward and reverse genetics, coupled with next-generation sequencing and bioinformatics, provides an integrated framework for functional genomics, enabling rapid identification, validation, and manipulation of genes associated with desirable agronomic traits, thereby accelerating plant breeding and crop improvement.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and older adults. Currently approved vaccines target only the viral fusion protein, but live-attenuated vaccines - e.g. through inactivation of nonstructural protein 1 (NS1) - likely induce a broader immune response. NS1 inhibits the immune response by repressing interferon production, but this has mostly been shown in immortalized cell lines, which do not necessarily represent the in vivo situation. Here, we assessed the effect of NS1 mutations on replication and host responses in physiologically relevant differentiated primary human nasal epithelial cells (HNEC) cultured at air-liquid interface (ALI). Using yeast-based reverse genetics, NS1-inactivating mutations were introduced. In differentiated HNECs, NS1 mutants showed delayed replication compared to wild-type virus. Bulk RNA sequencing early after infection revealed stronger antiviral signatures in HNEC infected with NS1 mutants compared to wild-type, characterized by upregulation of interferons and chemokines. Cytokine analysis confirmed these results. Finally, an indirect immune cell migration assay revealed that both WT and NS1-mutant viruses induce migration of mainly neutrophils. In conclusion, this study shows that RSV NS1 supports viral replication not only via inhibition of the production of interferons, but also by reducing early chemokine production and secretion by epithelial cells. Together, our data highlight the suitability of the ALI transwell model for preclinical assessment of live-attenuated vaccine candidates.

IntroductionIron is an essential microelement for animals, humans, and plants. Notably, approximately one-third of the world’s soils are alkaline, leading to iron deficiency. Therefore, understanding the mechanism of iron absorption and transport in plants is crucial for improving iron bioavailability in crops. MethodsIn this research, reverse genetics was used to identify the transcription factor MYB30 as a positive regulator of the plant response to iron deficiency.Results and DiscussionPhenotype analysis demonstrated that MYB30 mutant plants were sensitive to iron deficiency, exhibiting reduced root length, lower chlorophyll content, and elevated lipid peroxidation, whereas MYB30 overexpression lines showed enhanced tolerance. Metabolomic analysis of myb30 plant roots by mass spectrometry indicated decreased antioxidant activity and detoxification capacity under iron-deficient conditions. Interestingly, 22 metabolic pathways were altered in the myb30 plant under iron deficiency. This metabolic reprogramming likely compromises plant growth. Furthermore, MYB30 reduced reactive oxygen species accumulation under iron deficiency stress by activating related genes and enhancing antioxidant enzyme activity. In summary, metabolite analysis provides detailed molecular insights into plant iron deficiency stress and supports molecular genetic breeding efforts to improve mineral nutrition in crops.

The rich information encoded in cis-regulatory DNA sequences has not been fully exploited for gene function prediction in reverse genetics. Here we show that orthologous cis-regulatory sequences that diverged approximately 160 million years ago share little sequence similarity, yet remarkably retain semantic similarity that can be effectively captured by a deep learning model, PhytoBabel. Although trained solely on orthologous cis-regulatory sequence pairs from 15 angiosperms, PhytoBabel implicitly learned spatio-temporal gene expression patterns, conserved noncoding sequences, semantically similar fragments and phylogenetic relationships among species. Furthermore, PhytoBabel enables the discovery of evolutionarily unrelated but semantically similar cis-regulatory sequences, facilitating the identification of novel genes with functions of interest. As a proof of concept, we identified somatic embryogenesis-related morphogenic regulators in maize that exhibit semantic similarity to known Arabidopsis morphogenic regulators. By bridging the gap in the cis-regulatory sequence → semantics → gene function information chain, PhytoBabel provides a valuable tool for gene function prediction in reverse genetics.

Antagonism of the host responses that limits viral replication is critical to the success of infection. Recently, we identified that the hypervariable region (HVR) of SARS-CoV-2 NSP3 binds to FXR1 and disrupts stress granule formation during the early stages of infection. Despite variation across the rest of the HVR, a 20-amino acid region, highly conserved in the Sarbecovirus family, is required for NSP3-FXR1 binding, but the critical residues remained unresolved. In this study, we explore the individual residues in NSP3 driving FXR1 binding and determine their impact on viral replication, pathogenesis, and stress granule formation. Our results indicate that the tyrosine at position 138 (Y138) and a phenylalanine at position 145 (F145) are required for FXR1 binding and affinity. Using reverse genetics, we showed that mutating NSP3 Y138A/F145A (YF mutant) reduced viral replication in vitro and in vivo. Importantly, we demonstrate that attenuation is not due to differential type I interferon responses but rather loss of stress granule control by the NSP3 mutant as compared to wild type. Together, our findings demonstrate the importance of Y138 and F145 within the NSP3-HVR in regulating stress granule formation at the early times post-infection.IMPORTANCEStress granules play a key role in host-antiviral defenses, and viruses have developed strategies to antagonize their activity. For SARS-CoV-2, the virus has two proteins that antagonize stress granules, with NSP3 acting early and nucleocapsid acting at late times. Here, we show that key NSP3 residues Y138 and F145, conserved across the Sarbecovirus family, are necessary to bind FXR1 and disrupt its activity in stress granule formation. Mutating these residues results in attenuation of SARS-CoV-2 replication and induces stress granule formation at early times post-infection. These results show the importance of these NSP3 residues in disrupting stress granule formation early and highlight multiple approaches SARS-CoV-2 uses to antagonize stress granule activation.

Encephalomyocarditis virus (EMCV) is a rodent-borne picornavirus that has repeatedly caused severe outbreaks resulting in the deaths of zoo mammals - most notably elephants - for decades. However, within North America, little is known regarding the diversity of EMCV that exists in nature, the reservoir or amplifying hosts important for maintaining the virus, and the epidemiology of zoo-associated outbreaks. This lack of knowledge of the EMCV strains that circulate in North America has impeded a more thorough understanding of how genetic and antigenic variation may affect pathogenicity or potentially vaccine-induced protection from disease. Herein, following a zoological outbreak of EMCV in Florida, we conducted the most comprehensive comparative phylogenomic analysis of virus isolates from fatal zoo animal cases and local rodent species to date, identifying both non-native, invasive rodents and native mice and rat species as potentially important in precipitating and/or maintaining outbreaks, with multiple transmissions to zoo animals. After development of an autogenous vaccine, we investigated the duration and magnitude of neutralizing antibody responses in elephants monthly for multiple years, providing a unique fine-scale, long-term profile of responses to vaccination. To better understand how antigenic variation may affect vaccine-induced protection, we constructed a reverse genetics system to determine the level of cross-protection afforded by autogenous vaccination against capsids derived from various divergent EMCV strains and serotypes. These results provide new advancements in understanding EMCV transmission and antigenicity in North America, which can be used as a foundation to ultimately enable zoos to better protect animals from this important pathogen.

Rotavirus is a leading cause of severe, dehydrating diarrhea in infants and young animals, causing significant global morbidity and mortality. For decades, research was hindered by challenges in establishing reverse genetics systems due to the virus's complex segmented genome and poor cell culture adaptation. The first helper virus-dependent system (2006) was limited by low efficiency. A 2017 breakthrough established the first fully plasmid-based system, which eliminated helper viruses and revolutionized the field. Subsequent optimizations, such as codon modification and CRISPR/Cas9 integration, have significantly enhanced efficiency, enabling viable systems for diverse human and animal strains. This narrative review summarizes the evolution and technological milestones of rotavirus reverse genetics. We discuss critical applications in analyzing viral gene function, developing novel vaccines, screening antiviral drugs, and investigating cross-species transmission. Finally, we provide an outlook on the future prospects of this transformative technology.

Plant viral vectors are powerful tools for transient gene overexpression and silencing, enabling rapid functional analysis without the need for genetic transformation. Rice stripe mosaic virus (RSMV) is an emerging plant rhabdovirus transmitted propagatively by the leafhopper Recilia dorsalis. Leveraging its cross-kingdom replication ability, here we report the development of RSMV as a versatile vector for regulatable foreign gene expression and virus-induced gene silencing (VIGS) in rice and its insect vector. We first established an efficient reverse genetics system for RSMV using Nicotiana benthamiana as a model host. Recombinant virus particles recovered from N. benthamiana leaves were infectious to R. dorsalis and efficiently transmitted to rice. RSMV-based vectors stably accommodated at least two foreign genes, totaling up to 3.7 kb, and maintained stable expression across multiple passages. As proof-of-concept, the RSMV-VIGS vector achieved >90% knockdown of a target gene in R. dorsalis, producing near-knockout phenotypes that persisted throughout adulthood, and also induced efficient gene silencing in infected rice plants. Our work enables genetic manipulation of RSMV for molecular studies and provides a robust tool for functional genomics in both rice and insect hosts.

Many plant pathogenic fungi penetrate host surfaces mechanically, using turgor pressure generated by specialized infection cells called appressoria. These appressoria develop semipermeable cell walls and accumulate osmolytes internally to create turgor by osmosis. Although melanin is known to be important for turgor generation, the mechanism underlying wall semipermeability remains unclear. By using reverse genetics, we identified that the enzymes PKS2 and PBG13 are required for forming the semipermeable barrier in fungi causing anthracnose and rice blast diseases. These enzymes synthesize 3,5-dihydroxyhexanoic acid polymers that are essential for pathogenicity. These polymers reduce cell wall permeability and generate turgor, independently of melanization. Our findings uncover a mechanism of fungal turgor generation, linking enzyme function to pathogen penetration and disease potential, presenting new targets for disease control.

The increasing challenges posed by climate change, resource degradation, and the demand for sustainable agriculture necessitate advanced yet accessible tools for crop improvement. TILLING and its variant, Eco-TILLING, are non-transgenic reverse genetics approaches that enable the identification of induced and naturally occurring genetic variation across diverse crop species. TILLING combines chemical mutagenesis with high-throughput mutation detection to identify functional alleles in specific genes, while Eco-TILLING facilitates the discovery of natural polymorphisms in germplasm collections and wild relatives. These techniques effectively bridge functional genomics and applied plant breeding, particularly in polyploid, orphan, and vegetatively propagated crops. However, limitations include the low frequency of true knockout mutations, the predominance of silent or weak-effect alleles, the need for extensive population screening, and the chimeric nature of M1 plants that complicates early-generation selection. Despite these constraints, TILLING-based approaches remain valuable tools for modern breeding programs, with strong regulatory acceptance and adaptability across agro-ecological conditions.

A reverse genetics system for the feline panleukopenia virus (FPV) strain JL2280 was successfully established in this study. By optimizing the cloning strategy for the terminal hairpin structures, a full-length infectious clone, pFPV, harboring a KpnI genetic marker was constructed. The recombinant virus (rFPV) was successfully rescued in CRFK cells and exhibited replication kinetics, pathogenicity, and morphological characteristics comparable to those of the wild-type virus.To evaluate the potential of FPV as a live vector, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated rFPV-EGFP, was generated by inserting a P2A-EGFP cassette downstream of the NS1 gene. The rFPV-EGFP virus mediated efficient EGFP expression in infected cells; however, the fluorescence intensity gradually diminished with serial passages.The reverse genetics platform developed herein provides a valuable tool for investigating the genomic functions, pathogenic mechanisms, and evolution of FPV. Furthermore, the successful rescue of rFPV-EGFP demonstrates the preliminary feasibility of FPV as a live vector for foreign gene expression. Nevertheless, strategies such as optimizing insertion sites or modifying the viral backbone are required to enhance the stability of exogenous protein expression. This study lays the groundwork for the development of novel FPV-based genetically engineered vaccines and antiviral therapeutics.

Research progress and applications of reverse genetics systems for infectious bronchitis virus.

Experimental and computational approaches to adaptive viral evolution: Linking molecular variation to phenotypic outcomes.