This review comprehensively evaluates the rational design of classical animal herpesvirus vectors, including pseudorabies virus, herpesvirus of turkeys, and feline herpesvirus type 1, providing perspectives on how continuous biotechnological innovations will empower herpesvirus vectors to serve as formidable prophylactic tools against emerging and re-emerging infectious diseases.
Abstract
Recombinant herpesvirus vectors have emerged as highly potent platforms for next-generation vaccine development, distinguished by their genomic capacity and their unique ability to elicit durable, long-lasting immunity through persistent latent infections. In this review, we trace the evolutionary trajectory of herpesvirus vector engineering—transitioning from traditional homologous recombination to advanced bacterial artificial chromosome (BAC) systems and precise, scarless CRISPR/Cas9 gene editing. We comprehensively evaluate the rational design of classical animal herpesvirus vectors, including pseudorabies virus (PRV), herpesvirus of turkeys (HVT), and feline herpesvirus type 1 (FHV-1). Specifically, we highlight their distinct advantages in molecular attenuation, the optimization of non-essential insertion loci, and the application of tissue-specific promoters for multiplexed antigen presentation. Furthermore, we discuss the strategic deployment of multivalent herpesvirus vaccines within the “One Health” framework, emphasizing their critical role in simplifying immunization protocols and interrupting the transmission chains of zoonotic diseases. Finally, we address the prevailing translational bottlenecks, including scalable manufacturing challenges and stringent regulatory frameworks regarding environmental release, providing perspectives on how continuous biotechnological innovations will empower herpesvirus vectors to serve as formidable prophylactic tools against emerging and re-emerging infectious diseases.
This review provides a comprehensive overview of the current status of viral and non-viral vector systems for in vivo and ex vivo applications, and key comparisons are made across safety, efficacy, scalability, and immune responses.
ABSTRACT A non-replicating Tiantan strain-based vaccinia virus (NTV) holds significant application prospects for vaccination and gene therapy and has recently entered clinical trials as a novel and safer vaccine candidate against monkeypox. However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-ΔF1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-ΔF1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (103 PFU) or a single dose (10⁵ PFU) of NTV-ΔF1L-C7L, comparable to that conferred by VTT. These findings demonstrate that NTV-ΔF1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak. IMPORTANCE A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox. A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.
Jiao Ren, Shiyuan Liu, Hang Yuan et al.· Journal of Virology· 0 citations
This review examines the critical challenge of evolving animal virus resistance to vaccines, a phenomenon threatening veterinary medicine, global food security, and public health. While effective for controlling bacterial diseases, vaccination against rapidly mutating RNA viruses often imposes strong selective pressures, driving the emergence of antigenic variants that evade host immunity. These vaccines escape mutants, evidenced in viruses like canine parvovirus (CPV), avian influenza (AIV), and foot-and-mouth disease (FMD), lead to outbreaks in vaccinated populations and complicate disease management. The paper elucidates the mechanisms behind this resistance, primarily genetic mutations in antigenic sites and sophisticated immune evasion strategies, which enable viruses to circumvent neutralization by vaccine-induced antibodies. It critically evaluates the limitations of current vaccine technologies, including inactivated, live-attenuated, and subunit vaccines, noting that imperfect immunity can inadvertently promote the selection of resistant strains. To address this ongoing evolutionary arms race, the authors advocate for a paradigm shift in vaccine design. They propose leveraging advanced technologies such as viral vector platforms, structural biology, and predictive modeling to develop next-generation vaccines targeting conserved epitopes. Furthermore, the review emphasizes the necessity of global surveillance programs to monitor viral evolution in real-time and calls for a collaborative, interdisciplinary approach to create more robust and future-proof vaccination strategies, thereby mitigating the risk of vaccine-driven viral and ensuring long term efficacy.
Z. Al-Talabani, Anmar Ayoub Al-Obaidi, Shahad Abdul Majeed Aswad· SAR Journal of Pathology and...· 0 citations
Viral reverse genetics enables the rescue of infectious virions from cloned cDNA and serves as a core technique for mapping viral genotype–phenotype relationships, dissecting RNA viral life cycles, and developing antiviral countermeasures. This review systematically summarizes the evolution, technical framework and optimization strategies of RNA virus reverse genetics, with three prototype viruses covering all major RNA genome types: SARS-CoV-2 (+ssRNA), non-segmented negative-sense Newcastle disease virus (NDV), and segmented negative-sense influenza A virus. Core modules including infectious clone construction, diverse promoter systems, hammerhead/HDV ribozymes, and solutions for large unstable genomes such as BAC and ISA are elaborated. We summarize its irreplaceable applications in vaccine development, pathogenesis research, virus-host interaction analysis and high-throughput antiviral screening. Current bottlenecks include low rescue efficiency, cDNA genetic instability and biosafety hazards. We also introduce non-infectious surrogate platforms and establish a three-tier antiviral screening pipeline. Future advances will integrate CRISPR/Cas editing, standardized modular tools, biosafety engineering, AI and big data. Distinct from previous reviews focusing on single viral genera, this work conducts cross-type horizontal comparisons and summarizes universal technical obstacles and tailored optimizations, offering comprehensive references for basic virology, accelerated vaccine innovation and precise antiviral design.
Yu Guo, Ting Xue, Jianhua Wang et al.· Frontiers in Virology· 0 citations
Recombinant measles virus (rMeV) vectors are promising platforms for vaccine development against emerging infectious diseases due to their safety, stability, and potent immunogenicity. However, conventional rMeV rescue systems frequently exhibit low efficiency, thereby constraining their scalability and throughput. In this study, we developed a modular, helper-virus-free and high-efficiency rescue platform based on an orthogonal transcription system utilizing orthogonal promoters and engineered RNA polymerases fused to an mRNA capping enzyme. This innovative system facilitated robust cytoplasmic manufacture of both genomic and auxiliary components, eliminating the need for helper virus co-infection (such as modified vaccinia virus) and enhancing rescue efficiency by more than 50-fold relative to traditional rescue approaches. Utilizing this technology, we demonstrated the versatility of the platform by successfully generating six rMeV-based vaccine antigen candidates from influenza virus, Pseudomonas aeruginosa, and Brucella spp. All rescued vaccine candidates exhibited stable transgene expression, sustained replication, and strong antigen production. Immunization studies in golden Syrian hamsters verified that the vaccine candidates elicited high titers of neutralizing and antigen-specific antibodies without any observable adverse effects. These results demonstrate that our orthogonal transcription-based platform facilitates the efficient and safe production of rMeV vectors and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.
Zihan Ma, Weijun Wang, Qiuli Lou et al.· Synthetic and Systems Biotec...· 0 citations
Porcine epidemic diarrhea virus (PEDV) G2c variants have recently emerged, posing significant challenges to swine health management. As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus–host interactions.
Fan Zhang, He-Lu Liu, Linlong Ji et al.· Viruses· 0 citations
