Search

The Varroa destructor mite's transmission of deformed wing virus (DWV) to honey bees is responsible for most winter mortalities of colonies worldwide. Four DWV genotypes (A, B, C and D) and numerous recombinants have been described. The most recent studies have reported the greater prevalence of DWV-B over DWV-A in several countries, including European ones, while C and D genotypes appear rare or extinct. However, no global evaluation of DWV-A and DWV-B distribution was available at the European level to date. In this study, we quantified both DWV genotypes by real-time PCR from pools or individual honey bees and from V. destructor mites sampled in 15 European countries between 2010 and 2017. These data and the sequencing of the viral RNA provide a first insight into DWV diversity, with a clear dominance of DWV-B and recombinants (A/B) in Europe. Chimeric sequencing reads were used to locate the recombinant junctions along the DWV genome. These were not randomly distributed, but mainly clustered in three genomic areas: the 5'UTR, leader peptide and helicase coding sequences. In our study, the DWV recombinant genomes shared at least the VP1-VP3 coding sequences with the DWV-B. Further studies are needed to explore the apicultural context explaining these differences in DWV genotype dominance. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) remains a leading cause of cancer-related deaths among women globally, highlighting the urgent need for more effective and targeted therapies. Traditional treatments, including surgery, chemotherapy, and radiation, face limitations such as drug resistance, metastasis, and severe side effects. Recent advancements in gene therapy, particularly CRISPR/Cas9 technology and Oncolytic Virotherapy (OVT), are transforming the BC treatment landscape. CRISPR/Cas9 enables precise gene editing to correct mutations in oncogenes like HER2 and MYC, directly addressing tumor growth and immune evasion. Simultaneously, OVT leverages genetically engineered viruses to selectively destroy cancer cells and stimulate robust antitumor immune responses. Despite their potential, gene therapies face challenges, including off-target effects, delivery issues, and ethical concerns. Innovations in delivery systems, combination strategies, and integrating gene therapy with existing treatments offer promising solutions to overcome these barriers. Personalized medicine, guided by genomic profiling, further enhances treatment precision by identifying patient-specific mutations, such as BRCA1 and BRCA2, allowing for more tailored and effective interventions. As research progresses, the constructive interaction between gene therapy, immunotherapy, and traditional approaches is paving the way for groundbreaking advancements in BC care. Continued collaboration between researchers and clinicians is essential to translate these innovations into clinical practice, ultimately improving BC patients' survival rates and quality of life. [ABSTRACT FROM AUTHOR]

This study investigates the crucial role of transfection methods in the manufacturability and potency of recombinant adeno-associated virus (rAAV) gene therapies. By employing a novel analytical approach, multiplex digital PCR (dPCR), we evaluated the impact of different transfection reagents and conditions on the scalability and quality of rAAV. Our research demonstrates that the selection of transfection approach significantly influences not only the yield and ease of scale-up but also the potency of the final product. Importantly, later changes to transfection parameters established in the early stages of development can be challenging, potentially compromising product quality and leading to comparability issues. Leveraging multiplex dPCR has proven instrumental in guiding these early-stage decisions, ensuring a reliable manufacturing process that consistently delivers high-quality therapeutic products. Our findings highlight the importance of optimizing transfection strategies early in development to guarantee the successful production of potent and cost-effective gene therapies, ultimately impacting patient accessibility. • Novel analytical approach: Multiplex dPCR reveals genome differences from different transfection reagents. • Optimal Plasmid Concentrations: FectoVIR excels at high plasmid levels; PEI at lower levels. • Purification Efficiency: FectoVIR yields higher vg titers and fewer partially filled capsids. • Biopotency: FectoVIR shows higher in vitro biopotency and longer vector genome fragments. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) and H9N2 avian influenza virus (AIV) represent significant pathogenic risks to the poultry industry, leading to considerable economic losses. Vaccination is a widely used preventive measure against these pathogens, yet the lack of a live bivalent vaccine targeting NDV and H9N2 AIV imposes a heavy vaccination burden. Previously, we constructed a genotype-matched chimeric NDV vector, LX-OAI4S, in which the genotype I NDV backbone was replaced with the ectodomain of haemagglutinin-neuraminidase (HN) and modified using the attenuated F gene from the genotype VII vaccine strain A-VII. Based on the LX-OAI4S vector, we successfully generated three H9N2 recombinant viruses: LX-OAI4S-NPU-HA, LX-OAI4S-MU-HA, and LX-OAI4S-HNU-HA. These recombinants incorporated the H9N2 HA gene, flanked by untranslated regions (UTRs) from the NP, M, or HN gene of the NDV LX strain, inserted between the P and M genes of LX-OAI4S. The vaccine candidate LX-OAI4S-NPU-HA induced a more robust immune response in chickens against H9N2 AIV and NDV than the other two recombinants. This response effectively protects against virus shedding and lethal virus challenge. Furthermore, spray vaccination with LX-OAI4S-NPU-HA showed protective efficacy against H9N2 AIV and NDV. This study offers a promising strategy for comprehensive protection in regions threatened by H9N2 AIV and NDV. [ABSTRACT FROM AUTHOR]

The H5N6 avian influenza virus (AIV) is constantly undergoing recombination and evolution with other subtypes of AIV, resulting in various types of recombinant H5N6 viruses. However, the risk to human public health of different recombinant types of H5N6 viruses remains unclear. Recently, two types of different recombinant H5N6 viruses were isolated from chickens. One of the viruses possessed six internal genes originating from H9N2, named A/Chicken/Hubei/112/2020 (H5N6) (abbreviated 112); the other virus possessed PB2, PB1, PA, and NP originating from H5N1, while the M and NS genes were derived from H9N2, named A/Chicken/Hubei/125/2020 (H5N6) (abbreviated 125). Here, we investigated the receptor binding properties, pathogenicity, and transmissibility of the two H5N6 AIVs. The results showed that 112 and 125 could bind α‐2,3‐linked sialic acid receptor (avian‐like receptor) and α‐2,6‐linked sialic acid receptor (human‐like receptor). However, 125 and 112 showed different pathogenicity in mice. Mice infected with 125 lost only a slight body weight and all survived, while mice infected with 112 lost weight rapidly and all died within a week of infection. Furthermore, in the transmission experiment, 125 could only transmit through direct contact, while 112 could transmit not only by direct contact but also by aerosol. The above results indicated that 112 exhibited enhanced pathogenicity and transmissibility compared to 125, suggesting that the H5N6 virus, whose internal genes were derived from H9N2, could pose a greater threat to human health. Therefore, continuous monitoring of different recombinant H5N6 viruses in poultry should be carried out to prevent their transmission to humans. [ABSTRACT FROM AUTHOR]

In herpesvirus, the terminase subunit pUL15 is involved in cleavage of the viral genome concatemers in the nucleus. Previous studies have shown that herpes simplex virus 1 (HSV-1) pUL15 can enter the nucleus without other viral proteins and help other terminase subunits enter the nucleus. However, this study revealed that duck plague virus (DPV) pUL15 cannot localize independently to the nucleus and can only be localized in the nucleus in the presence of pUL28 and pUL33. However, the data suggested the presence of a potential nuclear localization signal (NLS) in DPV pUL15, which is important for the localization of the terminase subunits. Subsequently, several single-point mutants were constructed to identify the vital amino acids within the NLS. The conserved amino acids K187, R188, and K190 are critical for the nuclear localization of pUL15, pUL28, and pUL33 but not for their interaction. Furthermore, corresponding recombinant viruses were constructed. The results revealed that the mutations rUL15K187Q, rUL15K188Q and rUL15K190Q had an obvious influence on concatemeric genome cleavage, but only K190Q significantly affected the production of progeny virions. These findings indicate that the NLS is important for the functions of DPV pUL15. Overall, a potential NLS and the key amino acids in DPV pUL15 were identified. Mutations in K187, K188 and K190 affected the cleavage of the concatemeric genome, but only mutations in K190 had an obvious effect on viral proliferation. [ABSTRACT FROM AUTHOR]

Background: The Epstein-Barr virus (EBV) is intricately linked to a range of human malignancies, with EBV latent membrane protein 2A (LMP2A) emerging as a potential target antigen for immunotherapeutic strategies in the treatment of nasopharyngeal carcinoma (NPC). Methods: The modified vaccinia virus Ankara (MVA) is universally used in vector vaccine research because of its excellent safety profile and highly efficient recombinant gene expression. Here, we constructed a novel MVA-LMP2A recombinant virus and investigated its specific immune response induction and oncolytic effect. Results: An immunization dose of 2 × 107 PFU induced the highest specific immune response, which was no longer increased by boost injections after four doses. Three weeks post-final immunization, the specific immune response reached its peak. The MVA-LMP2A vaccine-induced LMP2A-specific cytotoxic T lymphocytes (CTLs), which exhibited substantial efficacy against target cells and effectively inhibited tumor growth. Conclusions: Thus, the MVA-LMP2A recombinant virus effectively induces strong LMP2A-specific cellular and humoral immune responses and anti-tumor activity. This work provides a promising therapeutic strategy for developing NPC candidate vaccines, as well as a reference for the treatment of EBV LMP2-associated malignancies. [ABSTRACT FROM AUTHOR]

This study aimed to create a new recombinant virus by modifying the EV-A71 capsid protein, serving as a useful tool and model for studying human Enteroviruses. We developed a new screening method using EV-A71 pseudovirus particles to systematically identify suitable insertion sites and tag types in the VP1 capsid protein. The pseudovirus's infectivity and replication can be assessed by measuring postinfection luciferase signals. We reported that the site after the 100th amino acid within the VP1 BC loop of EV-A71 is particularly permissive for the insertion of various tags. Notably, the introduction of S and V5 tags at this position had minimal effect on the fitness of the tagged pseudovirus. Furthermore, recombinant infectious EV-A71 strains tagged with S and V5 epitopes were successfully rescued, and the stability of these tags was verified. Computational analysis suggested that viable insertions should be compatible with capsid assembly and receptor binding, whereas non-viable insertions could potentially disrupt the capsid's binding with heparan sulfate. We expect the tagged recombinant EV-A71 to be a useful tool for studying the various stages of the enterovirus life cycle and for virus purification, immunoprecipitation, and research in immunology and vaccine development. Furthermore, this study serves as a proof of principle and may help develop similar tags in enteroviruses, for which there are fewer available tools. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity. Therapeutic safety and efficacy depend on the correct encapsidation of the DNA in individual virus particles, which is often characterized by the single scalar value of the ratio of full capsids with complete viral genomes to the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of organizations. The analytical methods used were sedimentation velocity analytical ultracentrifugation (SV-AUC) with UV/Vis and/or Rayleigh interference optics, size exclusion chromatography (SEC) with multi-angle light scattering (MALS), and tandem UV/Vis and/or refractive index, cryogenic electron microscopy, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR–ELISA. The optical techniques (UV spectroscopy/SEC–MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever (ASF), a highly contagious disease caused by the ASF virus (ASFV), causes severe economic losses around domestic pigs and wild boars. In this study, we found that VSV-vectored ASFV vaccine effectively promoted the activation and maturation of BMDCs. The matured BMDCs secreted various inflammatory and chemotactic factors to stimulate the proliferation and activation of T cells and induce Th1- and Th17-type immune responses. Our research revealed the immune response mechanism of VSV-vectored ASFV vaccine induction in BMDCs, which provided more of a theoretical basis for VSV as a vaccine vector. The vesicular stomatitis virus (VSV)-vectored African swine fever virus (ASFV) vaccine can induce efficient immune response, but the potential mechanism remains unsolved. In order to investigate the efficacy of recombinant viruses (VSV-p35, VSV-p72)-mediated dendritic cells (DCs) maturation and the mechanism of inducing T-cell immune response, the functional effects of recombinant viruses on DC activation and target antigens presentation were explored in this study. The results showed that surface-marked molecules (CD80, CD86, CD40, and MHC-II) and secreted cytokines (IL-4, TNF-α, IFN-γ) were highly expressed in the recombinant virus-infected DCs. In addition, the co-culture results of recombinant virus-treated DCs with naive T cells showed that the Th1- and Th17-type responses were effectively activated. Taken together, the study indicated that the VSV-vectored ASFV vaccine activated the maturation of DCs and the Th1- and Th17-type immune response, which provided a theoretical basis for the development of novel ASF vaccines. [ABSTRACT FROM AUTHOR]

To prevent H9N2 avian influenza virus (AIV) and Avian metapneumonovirus/C (aMPV/C) infections, we constructed recombinant aMPV/C viruses expressing the HA protein of H9N2 AIV. In addition, EGFP was inserted into the intermediate non-coding region of P-M protein in the aMPV/C genome using a reverse genetic system. The conditions for rescuing the recombinant virus were enhanced followed by insertion of the H9N2 AIV HA gene into the same location in the aMPV/C. The constructed recombinant virus raMPV/C-HA expressed the H9N2 AIV HA protein and showed good stability. Immunization of chicks with raMPV/C-HA increased the generation of neutralizing antibodies against aMPV and H9N2 AIV for 21 days. In the late challenge experiment, raMPV/C-HA effectively inhibited the replication of the virus in vivo , decreased the incidence of infection and conferred protection effects. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) is an ideal model for exploring the mechanisms of the virus; it is also an optimal vector for developing vector vaccines and for cancer therapy. A reverse genetic system of NDV Mukteswar strain controlled by eukaryotic cellular RNA polymerase II promoter was established by reverse genetics technology. Based on the reverse genetic system, an open reading frame of the enhanced green fluorescent protein (EGFP) gene be inserted between the P and M genes of the viral genome and flanked with the gene start (GS) sequence and gene end (GE) sequence to form an independent transcription unit. The rescued virus was amplified in specific pathogen-free (SPF) chicken embryos for 10 generations, and the results showed that the recombinant virus could stably express the exogenous gene for at least 10 generations. Efficient expression of two exogenous genes synchronously is essential for the development of NDV-based multivalent vaccine candidates. Explore the possibility of simultaneous and efficient expression of two exogenous genes based on NDV vector. In the present study, a recombinant virus with co-expression of EGFP and cherry fluorescent protein (CFP) inserted between the intergenic regions of the P/M gene as two independent transcription units was successfully rescued. The results showed that the two exogenous genes could be expressed synchronously and efficiently. The results of biological analysis of the expression efficiency of exogenous genes showed that the EGFP in recombinant viruses with two exogenous genes was slightly lower than that of recombinant viruses with one exogenous gene, but the expression efficiency of CFP in recombinant viruses with two exogenous genes was higher than EGFP in both viruses. These recombinant viruses have similar growth kinetics but with a little attenuation in virulence compared with parental viruses. In conclusion, these data indicated that this study successfully established the reverse genetic system of the NDV Mukteswar strain and achieved the purpose of efficient expression of two exogenous genes synchronously in a novel approach, laying the foundation for the development of multivalent vaccines or tumor therapeutics using NDV as a vector. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common age-related disease that is correlated with a high number of senescent chondrocytes in joint tissues. Heterogeneous nuclear ribonucleoprotein D (HNRNPD) is an RNA-binding protein whose expression imbalance is associated with cell senescence, but the role of HNRNPD in the occurrence and development of OA has not been reported. In this study, HNRNPD was found to be associated with the chondrocyte senescence process. We determined the factors at the posttranscriptional level that regulated the expression of the genes that induce OA and found that HNRNPD was specifically highly expressed in OA-induced rat cartilage and in human OA cartilage. Recombinant adeno-associated virus (rAAV)-mediated HNRNPD gene overexpression alone did not significantly regulate the occurrence and development of OA in the physiological state of the joint. However, rAAV-HNRNPD significantly exacerbated experimental OA in rats subjected to destabilization of the medial meniscus. Overexpression of HNRNPD promoted mitochondrial dysfunction and the expression of FOXM1, which acts as a direct target. Furthermore, downregulation of FOXM1 in chondrocytes weakened the HNRNPD-mediated promotion of chondrocyte senescence and mitochondrial dysfunction. Our results suggest that the RNA-binding protein HNRNPD promotes chondrocyte senescence in the pathology of OA by upregulating FOXM1. RNA-binding protein HNRNPD promotes chondrocyte senescence in the pathology of OA by upregulating FOXM1, which lays a theoretical foundation for exploring new targets for the prevention and treatment of OA. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 5 (nsp5) is a cysteine protease involved in viral replication and suppression of the host immune system. The substrate-binding domain of nsp5 is important for its protease activity. However, the relationship between nsp5 protease activity and viral replication remains unclear. We confirmed the importance of amino acid T25 in the nsp5 substrate-binding domain for viral replication using a split luciferase assay. By generating recombinant viruses using bacterial artificial chromosomes, we found that the proliferation of viruses with the T25I mutation in nsp5 was cell-dependent in culture. Furthermore, mice infected with the T25I mutant recombinant virus with a mouse acclimation backbone showed weight loss and increased lung viral load, similar to the wild-type (WT) infected group, up to 3 days after infection. However, after day 4, the lung viral load was significantly reduced in the T25I-infected group compared to that in the WT-infected group. This suggests that nsp5 T25 is involved in the pathogenesis of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a lethal disease of domestic pigs that is currently challenging swine production in large areas of Eurasia. The causative agent, ASF virus (ASFV), is a large, double-stranded and structurally complex virus. The ASFV genome encodes for more than 160 proteins; however, the functions of most of these proteins are still in the process of being characterized. The ASF gene R298L, which has previously been characterized as able to encode a functional serine protein kinase, is expressed late in the virus infection cycle and may be part of the virus particle. There is no description of the importance of the R298L gene in basic virus functions such as replication or virulence in the natural host. Based on its evolution, it is proposed that there are four different phenotypes of R298L of ASFV in nature, which may have potential implications for R298L functionality. We report here that a recombinant virus lacking the R298L gene in the Georgia 2010 isolate, ASFV-G-∆R298L, does not exhibit significant changes in its replication in primary cultures of swine macrophages. In addition, when experimentally inoculated in pigs, ASFV-G-∆R298L induced a fatal form of the disease similar to that caused by the parental virulent ASFV-G. Therefore, deletion of R298L does not significantly affect virus replication and virulence in domestic pigs of the ASFV Georgia 2010 isolate. [ABSTRACT FROM AUTHOR]

Since the ban of neonicotinoid insecticides in the European Union, sugar beet production is threatened by outbreaks of virus yellows (VY) disease, caused by several aphid-transmitted viruses, including the polerovirus beet mild yellowing virus (BMYV). As the symptoms induced may vary depending on multiple infections and other stresses, there is an urgent need for fast screening tests to evaluate resistance/tolerance traits in sugar beet accessions. To address this issue, we exploited the virus-induced gene silencing (VIGS) system, by introducing a fragment of a Beta vulgaris gene involved in chlorophyll synthesis in the BMYV genome. This recombinant virus was able to generate early clear vein chlorosis symptoms in infected sugar beets, allowing easy and rapid visual discernment of infected plants across five sugar beet lines. The recombinant virus displayed similar infectivity as the wild-type, and the insert remained stable within the viral progeny. We demonstrated that the percentage of VIGS-symptomatic plants was representative of the infection rate of each evaluated line, and depending on the susceptibility of the line to BMYV infection, VIGS symptoms may last over months. Our work provides a polerovirus-based VIGS system adapted to sugar beet crop allowing visual and rapid large-scale screens for resistance or functional genomic studies. [ABSTRACT FROM AUTHOR]

Background/Objectives: African swine fever virus (ASFV) is a devastating disease affecting domestic and wild suids and causing significant economic losses in the global pig industry. Attenuated modified live virus (MLV) vaccines are the most promising approaches for vaccine development. This study aimed to evaluate the safety and efficacy of four recombinant ASFV genotype II strains, derived from the non-hemadsorbing (non-HAD) attenuated isolate Lv17/WB/Rie1, through the single or simultaneous deletion of virulence-associated genes. Methods: Recombinant viruses were engineered by deleting the UK, EP402R, and EP153R genes, either individually or in combination. Four recombinant strains were evaluated for safety and efficacy in domestic pigs vaccinated intramuscularly with 102 TCID₅₀. Clinical signs, viremia, virus shedding, and antibody responses were monitored. Protection efficacy was assessed by challenging vaccinated pigs with the virulent genotype II Armenia07 strain. Additionally, a reversion-to-virulence study involving an overdose of the vaccine candidate was conducted to evaluate its stability through serial immunizations. Results: Deletion of the UK gene alone increased virulence, whereas the double deletion of EP402R and EP153R (Lv17/WB/Rie1-ΔCD) significantly enhanced safety while maintaining full protective efficacy. Vaccinated pigs exhibited reduced viremia, no virus shedding, and robust virus-specific antibody responses, achieving complete protection against Armenia07. The reversion-to-virulence study revealed potential but limited pathogenicity after multiple passages, indicating areas for improvement in vaccine stability. Conclusions: The Lv17/WB/Rie1-ΔCD strain demonstrates excellent safety and efficacy, along with potential DIVA (differentiating infected from vaccinated animals) compatibility, positioning it as a strong candidate for an ASFV MLV vaccine. Further research is needed to refine the vaccine and address the potential risks of reversion to virulence. [ABSTRACT FROM AUTHOR]

Background: Duck adenovirus 3 (DAdV-3) is an emerging pathogen that has caused severe economic losses to the duck industry in China. Recently, the infection of ducks with serotype 4 fowl adenovirus (FAdV-4) has also been reported in China. Therefore, an efficient bivalent vaccine to control the diseases caused by DAdV-3 and FAdV-4 is extremely urgent. In our previous study, a recombinant FAdV-4 expressing Fiber-2 of DAdV-3 was generated and designated as rFAdV-4-Fiber-2/DAdV-3. Methods: Here, the recombinant virus rFAdV-4-Fiber-2/DAdV-3 was inactivated to serve as a bivalent vaccine, and its immunogenicity and protective efficacy against DAdV-3 were evaluated in Muscovy ducks. Results: The subcutaneous injection of rFAdV-4-Fiber-2/DAdV-3 could efficiently induce antibodies against Fiber-2 of DAdV-3 and neutralize antibodies against FAdV-4. After challenges with DAdV-3, in comparison with the non-immunized ducks, the immunized ducks did not show any bodyweight loss, gross lesions, or histopathologic change. Moreover, viral loads in livers and kidneys from immunized ducks were undetectable, whereas those in non-immunized ducks with challenge were significantly high. Conclusions: All these data demonstrate that the inactivated recombinant virus rFAdV-4-Fiber-2/DAdV-3 has the potential to be an efficient vaccine candidate against both FAdV-4 and DAdV-3, although efficacy for FAdV-4 needs to be confirmed experimentally. [ABSTRACT FROM AUTHOR]

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P [1] C73, G10P [11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV. • None of the described reverse genetics (RG) system have supported the recovery of bovine RVs (BRVs). • An optimized plasmid-based RG system for BRV culture-adapted strain and clinical isolates were developed. • We adopted the recombinant reporter BRV for high-throughput screening application. • Twelve candidates out of 1440 compounds have potential antiviral activities against rotavirus. • The BRV segment composition may influence the viral fitness. [ABSTRACT FROM AUTHOR]

Abstract Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome.

Introduction: Akabane virus (AKAV) is a worldwide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. Reporter-expressing recombinant virus represents a powerful tool to characterize the viral biology in vitro and in vivo. Methods: In this study, we have successfully established a reverse genetics system for AKAV. The recued virus possessed similar growth characteristics to the parental virus in vitro. Moreover, the recombinant AKAV reporter viruses expressing nanoluciferase (Nluc) or mWasabi were constructed by inserting into S segment, named rAKAV-Nluc and rAKAV-mWasabi, respectively. Results: We investigated the virological characteristics of rAKAV-Nluc and rAKAV-mWasabi and found that rAKAV-Nluc displayed similar growth kinetics as the parental virus and could stably produce the nano-luciferase even after 10 rounds of serial passages. rAKAV-mWasabi also exhibited comparable growth kinetics and genetic stability as the parental virus. We further used the two reporter viruses to test the susceptibility of different cell lines to AKAV and found that cell lines derived from various host species, including human, swine, cattle, and monkey enables AKAV replication efficiently, accelerating our understanding of the AKAV cell tropism range. Discussion: Taken together, our established reverse genetics system for AKAV provides more convenient screening tools and can be used to study AKAV virulence and tropism, and to elucidate the molecular biology of AKAV. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. Here we show that the host E3-ubiquitin ligase TRIM7 acts as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7-/- mice exhibit increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients reveal that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus shows reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology. TRIM7 acts as an antiviral factor during SARS-CoV-2 infection, by ubiquitinating the M protein on K14 and inhibiting caspase-6-dependent apoptosis. The natural K14 mutations in circulating strains support the physiological role of M ubiquitination. [ABSTRACT FROM AUTHOR]

Oncolytic viruses are either naturally occurring or genetically engineered viruses that can activate immune cells and selectively replicate in and destroy cancer cells without damaging healthy tissues. Oncolytic virus therapy (OVT) represents an emerging treatment approach for cancer. In this review, we outline the properties of oncolytic viruses and then offer an overview of the immune cells and tumor microenvironment (TME) across various OVTs. A thorough understanding of the immunological mechanisms involved in OVTs could lead to the identification of novel and more effective therapeutic targets for cancer treatment. [ABSTRACT FROM AUTHOR]

The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3′ untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90–115% recovery), and linearity (R2 > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products. [ABSTRACT FROM AUTHOR]

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time‐consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected. [ABSTRACT FROM AUTHOR]

Protein modification by the ubiquitin-like protein ISG15 (ISGylation) plays a crucial role in the immunological defense against viral infection. During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, innate immune signaling proteins are ISGylated, facilitating innate immunity. However, whether SARS-CoV-2 proteins are direct substrates for ISGylation remains unclear. In this study, we investigated whether SARS-CoV-2 proteins undergo ISGylation and whether ISGylation affects viral protein function. Co-transfection ISGylation analysis of SARS-CoV-2 proteins showed that the nucleocapsid (N) protein is ISGylated at several sites. Herc5 promoted N ISGylation and interacted with N, indicating that Herc5 acts as an E3 ligase for N ISGylation. Lys-261 (K261) within the oligomerization domain of N was identified as a potential ISGylation site that is necessary for efficient ISGylation of N. K261 is positioned at the center of the dimer interface in the crystal structure of the C-terminal domain dimer and the ISGylated form of N showed reduced protein dimerization in pull-down analysis. Importantly, a recombinant virus expressing K261R mutant N showed enhanced resistance to interferon-β treatment compared to its parental virus. We also found that viral PLpro removes conjugated ISG15 from N. Our findings demonstrate that ISGylation of SARS-CoV-2 N inhibits protein dimerization, resulting in viral growth more susceptible to type I interferon responses, and that viral PLpro counteracts this ISG15-mediated antiviral activity by removing conjugated ISG15 from N. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel betacoronavirus, is the causative agent of COVID-19, which has caused economic and social disruption worldwide. To date, many drugs and vaccines have been developed for the treatment and prevention of COVID-19 and have effectively controlled the global epidemic of SARS-CoV-2. However, SARS-CoV-2 is highly mutable, leading to the emergence of new variants that may counteract current therapeutic measures. Electron microscopy (EM) is a valuable technique for obtaining ultrastructural information about the intracellular process of virus replication. In particular, EM allows us to visualize the morphological and subcellular changes during virion formation, which would provide a promising avenue for the development of antiviral agents effective against new SARS-CoV-2 variants. In this review, we present our recent findings using transmission electron microscopy (TEM) combined with electron tomography (ET) to reveal the morphologically distinct types of SARS-CoV-2 particles, demonstrating that TEM and ET are valuable tools for visually understanding the maturation status of SARS-CoV-2 in infected cells. This review also discusses the application of EM analysis to the evaluation of genetically engineered RNA viruses. [ABSTRACT FROM AUTHOR]

The dynamic interplay between a multimeric phosphoprotein (P) and polymeric nucleoprotein (N) in complex with the viral RNA is at the heart of the functioning of the RNA-synthesizing machine of negative-sense RNA viruses of the order Mononegavirales. P multimerization and N phosphorylation are often cited as key factors in regulating these interactions, but a detailed understanding of the molecular mechanisms is not yet available. Working with recombinant rabies virus (RABV) N and P proteins and using mainly surface plasmon resonance, we measured the binding interactions of full-length P dimers and of two monomeric fragments of either circular or linear N-RNA complexes, and we analyzed the equilibrium binding isotherms using different models. We found that RABV P binds with nanomolar affinity to both circular and linear N-RNA complexes and that the dimerization of P protein enhances the binding affinity by 15–30-fold as compared to the monomeric fragments, but less than expected for a bivalent ligand, in which the binding domains are connected by a flexible linker. We also showed that the phosphorylation of N at Ser389 creates high-affinity sites on the polymeric N-RNA complex that enhance the binding affinity of P by a factor of about 360. [ABSTRACT FROM AUTHOR]

In an attempt to explore the RNA viromes of two German rivers, we searched the virus particle contents of one 50 L water sample each from the Teltow Canal and the Havel River for viruses assumed to infect invertebrates. More than 330 complete and partial virus genomes up to a length of 37 kb were identified, with noda-like and reo-like viruses being most abundant, followed by bunya-like and birna-like viruses. Viruses related to the Permutotetraviridae, Nidovirales, Flaviviridae, Rhabdoviridae and Chuviridae as well as the unclassified Jῑngmén virus and Negev virus groups were also present. The results indicate a broad extent of recombinant virus genomes, supporting the concept of the modularity of eukaryotic viruses. For example, novel combinations of genes encoding replicase and structural proteins with a jellyroll fold have been observed. Less than 35 viruses could be assigned to existing virus genera. These are (i) an avian deltacoronavirus which was represented by only one short contig, albeit with 98% similarity, (ii) a seadornavirus and a rotavirus, and (iii) some 30 nodaviruses. All remaining viruses are novel and too diverse for accommodation in existing genera. Many of the virus genomes exhibit ORFans encoding hypothetical proteins of up to 2000 amino acids without conserved protein domains. [ABSTRACT FROM AUTHOR]

Since 2011, the emergence of Pseudorabies virus (PRV) variants has led to significant vaccine failures, resulting in severe economic losses in China’s swine industry. Conventional PRV vaccines have shown limited efficacy against these emergent variants, underscoring the urgent need for novel immunization strategies. This study aimed to develop and evaluate a novel recombinant PRV vaccine candidate with improved safety and immunogenicity profiles. Utilizing the homology-directed repair (HDR)-CRISPR/Cas9 system, we generated a recombinant PRV strain, designated PRV SX-10ΔgI/gE/TK/UL24, with deletions in the gI, gE, TK, and UL24 genes. In vitro analyses demonstrated that the recombinant virus exhibited similar replication kinetics and growth curves comparable to the parental strain. The immunological properties of the recombinant PRV were assessed in murine and porcine models. All animals inoculated with PRV SX-10ΔgI/gE/TK/UL24 survived without exhibiting significant clinical signs or pathological alterations. Immunological assays revealed that PRV SX-10ΔgI/gE/TK/UL24 elicited significantly higher levels of gB-specific antibodies, neutralizing antibodies, and cytokines (including IFN-γ, IL-2, and IL-4) compared to both the Bartha-K61 and PRV SX-10ΔgI/gE/TK strains. Notably, both murine and porcine subjects immunized with PRV SX-10ΔgI/gE/TK/UL24 demonstrated enhanced protection against challenges with the variant PRV SX-10 strain, compared to other vaccine strains. These findings suggest that PRV SX10ΔgI/gE/TK/UL24 represents a promising PRV vaccine candidate strain, offering valuable insights for the prevention and control of PRV in clinical applications. [ABSTRACT FROM AUTHOR]

The highly pathogenic avian influenza 2.3.4.4b H5 viruses have been a cause for concern recently, as they have been responsible for continuous outbreaks since 2021. In China, the H5N6 subtype has been predominantly circulating in domestic poultry but has rarely been detected in wild birds over the past 3 years. In December 2023, novel reassortant 2.3.4.4b H5N6 viruses were resurgent in wild birds and domestic ducks in Eastern Asia. The viruses were reassorted with those of currently prevalent 2.3.4.4b H5N1 viruses of wild bird origin worldwide, as well as the H5N6 viruses that caused human infections in 2022 and low pathogenic avian influenza viruses, such as the H9N2 virus, which also contributed internal gene to the novel H5N6 viruses. Based on the phylogenetic analyses, we inferred that this recombination process occurred in migratory breeding sites in early 2023. Given the rapid transmission and high mutation capacity of currently circulating H5N1 viruses, as well as the strong pathogenicity of H5N6 viruses to humans, the novel recombinant viruses may continue to evolve and pose new threats to human health. Therefore, continuous surveillance of H5N6 viruses in wild birds and domestic poultry should be strengthened. [ABSTRACT FROM AUTHOR]

Engineered viral vectors designed to deliver genetic material to specific targets offer significant potential for disease treatment, safer vaccine development, and the creation of novel biochemical research tools. Viral tropism, the specificity of a virus for infecting a particular host, is often modified in recombinant viruses to achieve precise delivery, minimize off-target effects, enhance transduction efficiency, and improve safety. Key factors influencing tropism include surface protein interactions between the virus and host-cell, the availability of host-cell machinery for viral replication, and the host immune response. This review explores current strategies for modifying the tropism of recombinant viruses by altering their surface proteins. We provide an overview of recent advancements in targeting non-enveloped viruses (adenovirus and adeno-associated virus) and enveloped viruses (retro/lentivirus, Rabies, Vesicular Stomatitis Virus, and Herpesvirus) to specific cell types. Additionally, we discuss approaches, such as rational design, directed evolution, and in silico and machine learning-based methods, for generating novel AAV variants with the desired tropism and the use of chimeric envelope proteins for pseudotyping enveloped viruses. Finally, we highlight the applications of these advancements and discuss the challenges and future directions in engineering viral tropism. [ABSTRACT FROM AUTHOR]

Objective: High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. Methods: We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. Results: RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. Conclusion: The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) infection causes severe diarrhea and high mortality in neonatal piglets. Pseudorabies causes acute and often fatal infections in young piglets, respiratory disorders in growing pigs, and reproductive failure in sows. In late 2011, pseudorabies virus (PRV) variants occurred in Bartha-K61-vaccine-immunized swine herds, resulting in economic losses to the global pig industry. Therefore, it is essential to develop a safe and effective vaccine against both PEDV and PRV infections. In this study, we constructed a recombinant virus rPRV-PEDV S1 expressing the major neutralizing epitope region (COE, SS2, and SS6) of the PEDV S1 protein by homologous recombination technology and CRISPR/Cas9 gene editing technology, and then evaluated its biological characteristics in vitro and immunogenicity in pigs. The recombinant virus rPRV-PEDV S1 had similar growth kinetics in vitro to the parental rPRV NY-gE−/gI−/TK− strain, and was proven genetically stable in swine testicle (ST) cells and safe for piglets. PEDV S1-specific antibodies were detected in piglets immunized with rPRV-PEDV S1 on the 7th day post-immunization (dpi), and the antibody level increased rapidly at 14–21 dpi. Moreover, the immunized piglets receiving the recombinant virus exhibited alleviated clinical signs and reduced viral load compared to the unvaccinated group following a virulent PEDV HN2021 strain challenge. Also, piglets immunized with rPRV-PEDV S1 developed a PRV-specific humoral immune response and elicited complete protection against a lethal PRV NY challenge. These data indicate that the recombinant rPRV-PEDV S1 is a promising vaccine candidate strain for the prevention and control of PEDV and PRV infections. [ABSTRACT FROM AUTHOR]

The HIV-1 maturation inhibitor (MI) VH3739937 (VH-937) inhibits cleavage between capsid and spacer peptide 1 and exhibits an oral half-life in humans compatible with once-weekly dosing. Here, the antiviral properties of VH-937 are described. VH-937 exhibited potent antiviral activity against all HIV-1 laboratory strains, clinical isolates, and recombinant viruses examined, with half-maximal effective concentration (EC50) values ≤ 5.0 nM. In multiple-cycle assays, viruses less susceptible to other MIs, including A364V, were inhibited at EC50 values ≤ 8.0 nM and maximal percent inhibition (MPI) values ≥ 92%. However, VH-937 was less potent against A364V in single-cycle assays (EC50, 32.0 nM; MPI, 57%) and A364V emerged in one of four resistance selection cultures. Other substitutions were selected by VH-937, although re-engineered viruses with these sequences were non-functional in multiple-cycle assays. Measured dissociation rates from wild-type and A364V-containing VLPs help explain resistance to the A364V mutation. Overall, the in vitro antiviral activity of VH-937 supports its continued development as a treatment for HIV-1. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viruses (rAAVs) have been extensively studied for decades as carriers for delivering therapeutic genes. However, designing rAAV vectors with selective tropism for specific cell types and tissues has remained challenging. Here, we introduce a strategy for redirecting rAAV by attaching nanobodies with desired tropism at specific sites, effectively replacing the original tropism. To demonstrate this concept, we initially modified the genetic code of rAAV2 to introduce an azido-containing unnatural amino acid at a precise site within the capsid protein. Following a screening process, we identified a critical site (N587+1) where the introduction of unnatural amino acid eliminated the natural tropism of rAAV2. Subsequently, we successfully redirected rAAV2 by conjugating various nanobodies at the N587+1 site, using click and SpyTag-Spycatcher chemistries to form nanobody-AAV conjugates (NACs). By investigating the relationship between NACs quantity and effect and optimizing the linker between rAAV2 and the nanobody using a cathepsin B-susceptible valine-citrulline (VC) dipeptide, we significantly improved gene delivery efficiency both in vitro and in vivo. This enhancement can be attributed to the facilitated endosomal escape of rAAV2. Our method offers an exciting avenue for the rational modification of rAAV2 as a retargeting vehicle, providing a convenient platform for precisely engineering various rAAV2 vectors for both basic research and therapeutic applications. AAVs hold great promise in the treatment of genetic diseases, but their clinical use has been limited by off-target transduction and efficiency. Here, we report a strategy to construct NACs by conjugating a nanobody or scFv to an rAAV capsid site, specifically via biorthogonal click chemistry and a spy-spycatcher reaction. We explored the structure-effect and quantity-effect relationships of NACs and then optimized the transduction efficiency by introducing a valine-citrulline peptide linker. This approach provides a biocompatible method for rational modification of rAAV as a retargeting platform without structural disruption of the virus or alteration of the binding capacity of the nanobody, with potential utility across a broad spectrum of applications in targeted imaging and gene delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo. [ABSTRACT FROM AUTHOR]

Background: African swine fever virus (ASFV) continues to spread globally, causing severe economic losses to pig farmers. Vietnam licensed two live attenuated vaccines based on the ASFV strains ASFV-G-ΔI177L and ASFV-G-ΔMGF to control the ongoing ASF outbreaks. In 2023, newly emerging highly virulent recombinant ASF viruses (rASFV I/II) containing genetic elements from both p72 genotype I and II ASF viruses were reported from Northern Vietnam. Objective: This study evaluated whether the two vaccine strains were able to protect the pigs against the emerging rASFV I/II strain VNUA/rASFV/TN1/23. Results: Pigs vaccinated with ASFV-G-ΔMGF or ASFV-G-ΔI177L, when challenged with rASFV I/II, succumbed to the infection, or developed signs of chronic ASF. Conclusions: The findings from this study show that both vaccine strains that are licensed and used in Vietnam are unlikely to protect pigs from the emerging highly virulent rASFV I/II. This complicates the ongoing efforts to control ASF in Asia and globally and emphasizes the urgent need for a novel vaccine that can effectively protect pigs from the rASFV I/II. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viruses (rAAVs) have emerged as an attractive tool for gene delivery, and demonstrated tremendous promise in gene therapy and gene editing—therapeutic modalities with potential "one-and-done" treatment benefits compared to conventional drugs. Given their tropisms for the central nervous system (CNS) across various species including humans, rAAVs have been extensively investigated in both pre-clinical and clinical studies targeting neurodegenerative disease. However, major challenges remain in the application of rAAVs for CNS gene therapy, such as suboptimal vector design, low CNS transduction efficiency and specificity, and therapy-induced immunotoxicity. Therefore, continuing efforts are being made to optimize the rAAV vectors from their "core" genetic payloads to their "coat" or capsid structure. In this review, we describe current approaches for rAAV vector design tailored for transgene expression in the CNS, summarize the development of CNS-targeting AAV serotypes, and highlight recent advancements in AAV capsid engineering, aimed at generating a new generation of rAAVs with improved CNS tropism. Additionally, we discuss various administration routes for delivering rAAVs to the CNS and provide an overview of AAV-mediated gene therapies currently under investigation in clinical trials for the treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time‐consuming and expensive. Here, we developed a triplex real‐time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360‐14L genes to differentiate highly virulent genotype I recombinant viruses from low‐virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross‐reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low‐virulence genotype I virus, or genotype II virus. Thus, triplex qPCR provides an alternative tool for ASF surveillance in China. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10–40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs. • FectoVIR significantly increased rAAV9 potency compared to other reagents. • About 80 % of rAAV capsids are fully filled, with no difference due to reagent choice, as shown by AUC and mass photometry. • FectoVIR improves rAAV9 genome integrity, as shown by results from multiplex dPCR and Nanopore sequencing. • Preparation steps preceding Nanopore sequencing affect the distribution of fragments and the accuracy of titer measurements. • Employing a combination of mass- and sequence-based analytical methods is crucial for the thorough characterization of rAAV9s. [ABSTRACT FROM AUTHOR]

Motor learning (ML), which plays a fundamental role in growth and physical rehabilitation, involves different stages of learning and memory processes through different brain regions. However, the neural mechanisms that underlie ML are not sufficiently understood. Here, a previously unreported neuronal projection from the dorsal hippocampus (dHPC) to the zona incerta (ZI) involved in the regulation of ML behaviors is identified. Using recombinant adeno‐associated virus, the projections to the ZI are surprisingly identified as originating from the dorsal dentate gyrus (DG) and CA1 subregions of the dHPC. Furthermore, projection‐specific chemogenetic and optogenetic manipulation reveals that the projections from the dorsal CA1 to the ZI play key roles in the acquisition and consolidation of ML behaviors, whereas the projections from the dorsal DG to the ZI mediate the retrieval/retention of ML behaviors. The results reveal new projections from the dorsal DG and dorsal CA1 to the ZI involved in the regulation of ML and provide insight into the stages over which this regulation occurs. [ABSTRACT FROM AUTHOR]

Background/Objectives: α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. Methods: In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 1012 to 1.3 × 1013 vg/kg, bracketing those used in our clinical trials. Results: We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. Conclusions: Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 1013–1 × 1014 vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. Methods: In this study, the major antigen genes P1a of MP adhesion factor P1(3862‐4554 bases) and P30a of P30(49‐822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS‐P1a or NS‐P30a. The recombinant pHW2000 plasmids containing NS‐P1a or NS‐P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU‐P1a and rFLU‐P30a were rescued. RT‐PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU‐P1a and rFLU‐P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. Results: P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT‐PCR identification of the recombinant viruses rFLU‐P1a and rFLU‐P30a showed that P1a (693 bp), P30a (774 bp), NS‐P1a (1992bp) and NS‐P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. Conclusion: The recombinant viruses rFLU‐P1a and rFLU‐P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU‐P1a or rFLU‐P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is the causative agent of an often lethal disease in domestic pigs, African swine fever (ASF). ASF is currently a pandemic disease challenging pig production in Eurasia. While the ASFV genome encodes for over 160 proteins, the function of most of them are still not characterized. Among those ASF genes with unknown functions is the E111R gene. It has been recently reported that the deletion of the E111R gene from the genome of the virulent Chinese field isolate SY18 strain produced a reduction of virus virulence when pigs were inoculated at relatively low doses. Conversely, we report here that deletion of the ASFV gene E111R in the Georgia 2010 isolate does not alter the virulence of the parental virus in experimentally inoculated pigs. A recombinant virus lacking the E111R gene, ASFV-G-∆E111R was intramuscularly (IM) inoculated in domestic pigs at a dose of 102 HAD50 of ASFV-G-∆E111R and compared with animals that received a similar dose of virulent ASFV-G. Both, animals inoculated with either the recombinant ASFV-G-∆E111R or the parental virus developed a fatal form of the disease and were euthanized around the 6th–7th day post-inoculation (dpi). [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an acute infectious disease with a high mortality rate in both domestic and wild boars. Commercial vaccines or antiviral drugs for ASF were not available due to the complex diversity of the structure and genome of its pathogen African swine fever virus (ASFV). In recent years, there have been many reports on candidate strains of attenuated vaccines for ASFV. In this study, we obtained a recombinant virus named SY18ΔL60LΔCD2v by simultaneously deleting the L60L gene and CD2v gene from highly virulent strain SY18. In vitro, SY18ΔL60LΔCD2v displayed a decreased growth kinetic compared to that of parental SY18. In vivo, high doses (105 TCID50) of SY18ΔL60LΔCD2v can protect pigs (5/5) from attacks by the parental SY18 strain (102 TCID50). Low doses (102 TCID50) of SY18ΔL60LΔCD2v only protected 20% of pigs (1/5) from attacks by the parental SY18 strain (102 TCID50). The results indicated that the absence of these two genes in SY18 could induce protection against the homologous parental strain, and there were no obvious clinical symptoms or viremia. These results indicate that the SY18ΔL60LΔCD2v strain can serve as a new live attenuated vaccine candidate for the prevention and control of ASFV infection. [ABSTRACT FROM AUTHOR]

Objective: Emotional memory has been associated with many psychiatric diseases. Understanding emotional memory could be beneficial in comprehending and discovering new therapies for diseases related to emotional memory, such as depression and post-traumatic stress disorder (PTSD). Our previous study revealed that Dnmt3a expression in the dentate gyrus (DG) contributes to fear memory. However, is there a correlation between Dnmt3a expression in the DG and reward memory? This study aims to explore the relationship between Dnmt3a expression and reward memory. Methods: We induced fear memory (Fear group) or reward memory (Reward group) using fear conditioning and social interaction in females, respectively. We then measured the expression levels of Dnmt3a and c-fos after the retrieval of different types of memory. Additionally, we used a recombinant Adeno-Associated Virus (rAAV) to overexpress Dnmt3a in the DG and conducted conditioned place preference (CPP) tests to assess changes in reward memory. Results: We observed a significant increase in Dnmt3a and c-fos expression in the Fear group compared with the Reward group. Overexpression of Dnmt3a in the DG led to an increase in time spent in the white box during CPP tests. Conclusion: Dnmt3a expression levels varied after the retrieval of fear or reward memory, and overexpression of Dnmt3a in the DG enhanced reward memory. These findings suggest that Dnmt3a expression in the DG plays a role in reward memory. [ABSTRACT FROM AUTHOR]

The chicken infectious anemia virus (CIAV) has been reported in major poultry-producing countries and poses a significant threat to the poultry industry worldwide. In this study, two Marek's disease virus (MDV) recombinants, rMDV-CIAV-1 and rMDV-CIAV-2, were generated by inserting the CIAV VP1 and VP2 genes into the MDV vaccine strain 814 at the US2 site using the fosmid-based rescue system. For rMDV-CIAV-1, an internal ribosome entry site was inserted between VP1 and VP2, so that both proteins were produced from a single open reading frame. In rMDV-CIAV-2, VP1 and VP2 were cloned into different open reading frames and inserted into the MDV genome. The recombinant viruses simultaneously expressed VP1 and VP2 in infected chicken embryo fibroblasts and exhibited growth kinetics similar to those of the parent MDV. The two recombinant viruses induced antibodies against CIAV in chickens. A single dose of the recombinant viruses provided strong protection against CIAV-induced anemia in chickens. These recombinant VP1- and VP2-expressing MDVs are potential vaccines against CIAV in chickens. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains. [ABSTRACT FROM AUTHOR]