This review systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens.
Abstract
Viral infectious diseases in livestock and poultry cause substantial economic losses worldwide and pose persistent zoonotic threats to public health. Elucidating virus-host interactions is essential for understanding viral pathogenesis and developing effective control strategies. In recent years, genome-wide CRISPR/Cas9 functional screening has emerged as a powerful and unbiased high-throughput approach for identifying host dependency and restriction factors. Enabled by the development of species-specific sgRNA libraries, this technology has significantly advanced research in veterinary virology. In this review, we systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens. We highlight key host factors involved in viral entry, replication, and egress, and integrate these findings to delineate conserved cross-viral dependency networks, such as sialic acid biosynthesis, endosomal–lysosomal trafficking, double-membrane vesicle (DMV) formation, and interferon signaling pathways. Furthermore, we discuss the translational potential of these genomic discoveries for practical agricultural applications, particularly in the development of host-targeted broad-spectrum antivirals and the generation of disease-resistant livestock (e.g., receptor-edited pigs and chickens) through precise genome editing. Finally, we outline future perspectives, including the integration of single-cell transcriptomics and in vivo validation, thereby providing a comprehensive framework for advancing disease control and sustainable breeding in animal agriculture.
Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized.
In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection.
We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN’s trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry.
Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.
Jinyan Zhang, Hailong Liu, Jian Du et al.· Frontiers in Cell and Develo...· 0 citations
This review provides an overview of recent advances in the development of genetically engineered pigs with enhanced resistance to major viral diseases, focusing on targeted gene modifications using knockout, knock-in, and overexpression approaches.
Z. Namula, T. Otoi, Aya Nakai et al.· Wetchasan sattawaphaet = The...· 0 citations
Highly pathogenic RNA viruses, such as Ebola, SARS-CoV-2, and influenza, cause severe disease in humans. High mutation rates, which enable RNA viruses to evade immunity and escape antivirals, and their ability to spread from animals to humans and cause pandemics and outbreaks, make RNA viruses significant threats to public health. Diseases caused by Ebola, SARS-CoV-2, and influenza are prevented and treated with only a limited number of approved antiviral drugs, the effectiveness of which is limited by mutations in the viral targets. It is crucial to understand the structural determinants, molecular mechanisms, and host interactions of pathogenic RNA viruses to develop effective antiviral strategies. In this review, we discuss selected RNA viruses, focusing on the structure of their RNA polymerases and interactions with host factors during the different stages of the viral lifecycle, as well as the traditional antivirals targeting these structures and pathways. Furthermore, emerging concepts such as liquid–liquid phase separation and biomolecular condensates, and novel promising antiviral strategies are discussed. Understanding shared and distinct structures, molecular mechanisms, and host interactions across highly pathogenic RNA viruses enables the discovery of new and more effective antiviral strategies, ultimately improving clinical outcomes against evolving RNA viruses.
Hana Krnjić, Adna Hrapović, Aiša Galijatović et al.· Viruses· 0 citations
The efficiency of cell culture-based vaccine production is fundamentally constrained by the antiviral defenses of host cells, creating a major bottleneck for rapid and large-scale vaccine manufacturing. Key antiviral proteins-such as RNase L, PKR, and JAK1 act as intrinsic brakes on viral replication, limiting efficient propagation of many clinically relevant viruses. To overcome this challenge, we generated HEK293T, Vero, and MDCK cell lines with targeted knockouts of multiple antiviral genes. Notably, these engineered cells maintained normal growth and viability while supporting markedly increased viral yields. Multi-gene deletions enhance the replication of both enveloped viruses, including influenza A virus, pseudotyped lentivirus, and porcine epidemic diarrhea virus (PEDV), and non-enveloped viruses such as coxsackievirus. The magnitude of enhancement scaled proportionally with the number of genes disrupted. By systematically removing host restriction factors, this platform provides a versatile and powerful strategy for accelerating viral propagation, offering a strong foundation for more efficient development and large-scale production of cell culture-based anti-viral vaccines.
Zhengmei Xu, Jaemyeong Jeon, Jinsoo Oh et al.· Frontiers in Bioengineering...· 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
BACKGROUND
Bovine viral diarrhea virus (BVDV) is a viral pathogen that may cause reproductive disorders, health, and performance. BVDV serotypes have undergone dynamic changes due to sustained genetic diversity, particularly in BVDV type 1 (BVDV1), which has compromised the specificity and sensitivity of diagnostic assays. Furthermore, BVDV1 is prevalent worldwide, causing significant losses to the livestock industry. Therefore, in order to further enhance the control and elimination of BVDV, it is necessary to detect and prevent the spread of BVDV. Therefore, establishing a faster and more accurate on-site detection method is particularly important.
METHODS
Traditional PCR and ELISA methods are limited by cost, sensitivity, and equipment dependence. In this study, we designed primers based on the conserved 5'Untranslated Region (5'UTR) of BVDV1 and developed a method that combines one-step reverse transcription recombinase assisted amplification (RT-RAA) and CRISPR/Cas12a based fluorescent substrates in a mixture to quickly screen and accurately distinguish BVDV1 for the diagnosis and differentiation of common bovine viruses such as Bovine Coronavirus (BCoV) and Bovine Herpesvirus type 1 (BoHV) et al. The established RT-RAA-CRISPR/Cas12a scheme can achieve the entire inspection process in a portable metal bath at 40℃. We further optimized the reaction conditions of RT-RAA-CRISPR/Cas12a to detect its sensitivity, specificity, and repeatability. The established RT-RAA-CRISPR/Cas12a and conventional PCR were used to detect 300 clinical samples of cattle, verifying the actual detection ability.
RESULTS
This method did not show any cross-reactivity with the other nine common bovine viruses, demonstrating excellent specificity. The minimum template concentration required to trigger significant trans cleavage activity without RT-RAA amplification is 10 copies/µL of BVDV template. However, combined with RT‑RAA amplification, the detection limit was significantly decreased to one copy of the BVDV template per reaction. In simulated sample testing, this method achieved 100% accuracy, and the entire detection process can be completed within 30 min. In clinical sample testing, RT-RAA-CRISPR/Cas12a showed a higher sensitivity rate for BVDV1, as the positive detection rate was 1.85 times higher than that of conventional PCR.
CONCLUSION
In summary, the BVDV-1 RT-RAA-CRISPR/Cas12a detection method established in this study offers several advantages, including fast operation, high sensitivity, strong targeting, and good repeatability. Moreover, the same test tube reaction system makes it widely applicable in resource-limited environments, making it of great value in large-scale screening during BVDV outbreaks. This method not only expands the diagnostic toolbox of BVDV but also provides an up-and-coming solution for controlling the spread of BVDV in cattle herds.
