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Genome-wide CRISPR/Cas9 screening identifies host factors critical for antiviral defense against equine herpesvirus type 1.
Abstract: Equine herpesvirus type 1 (EHV-1) is a major veterinary pathogen causing significant economic losses in the livestock industry. Despite its impact, effective vaccines and targeted antiviral strategies remain limited, largely due to an incomplete understanding of host factors regulating viral replication and pathogenesis. Unassigned: To systematically identify host genes essential for EHV-1 infection, we established a BHK-21 cell line stably expressing Cas9 and performed a genome-wide CRISPR/Cas9 knockout screen using a pooled lentiviral single-guide RNA library. Significantly enriched candidate genes from positive selection were validated by generating knockout cell lines. Viral replication and protein expression were assessed using quantitative polymerase chain reaction and Western blot analysis. Pathway enrichment and protein interaction network analyses were subsequently conducted. Unassigned: Genome-wide CRISPR/Cas9 screening identified multiple host factors critical for EHV-1 replication. Pathway enrichment analysis revealed that these genes were involved in key cellular signaling and regulatory networks associated with viral infection. Functional validation demonstrated that knockout of selected host genes significantly suppressed EHV-1 replication and viral protein synthesis. Unassigned: These findings highlight essential host determinants required for EHV-1 replication and suggest that targeting host factors may represent a promising strategy for antiviral intervention. This study provides a foundation for the development of host-directed immunotherapeutic and antiviral approaches against EHV-1 infection.
Copyright © 2026 Li, Yu, Ge, Lv, Fu and Shi.
Publication Date: 2026-02-02 PubMed ID: 41705235PubMed Central: PMC12907326DOI: 10.3389/fimmu.2026.1764863Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study used a genome-wide CRISPR/Cas9 knockout screen to identify host genes crucial for the replication and infection of equine herpesvirus type 1 (EHV-1), a virus causing economic losses in livestock.
- The research highlights specific host factors necessary for viral replication and suggests that targeting these host genes could lead to new antiviral strategies.
Background and Objectives
- Equine herpesvirus type 1 (EHV-1) is a significant pathogen in horses, leading to respiratory diseases, abortions, and neurological disorders, impacting livestock health and economic stability.
- Current vaccines and antiviral treatments are limited, partly due to a lack of comprehensive understanding of the host cellular factors that facilitate or restrict EHV-1 infection.
- The primary objective was to systematically identify host genes essential for EHV-1 infection by conducting a genome-wide knockout screen using the CRISPR/Cas9 technology in BHK-21 cells (a cell line derived from baby hamster kidney cells) engineered to stably express Cas9.
Methodology
- Established a BHK-21 cell line that stably expresses the Cas9 nuclease, enabling efficient editing of genomic DNA.
- Utilized a pooled lentiviral single-guide RNA (sgRNA) library covering the genome to perform a knockout screen, thereby inactivating different host genes across the cell population.
- Exposed these edited cells to EHV-1 to identify those with altered susceptibility based on gene knockouts that either promoted survival or increased virus resistance.
- Identified significantly enriched candidate genes by positive selection — genes whose knockout led to reduced viral replication or cell death resistance.
- Validated top candidate genes by individually knocking them out and assessing their effects on viral replication using quantitative polymerase chain reaction (qPCR) to quantify viral genome copies and Western blot to measure viral protein levels.
- Conducted pathway enrichment analysis and protein interaction network studies to understand the biological functions and interactions of the identified genes involved in viral infection pathways.
Key Findings
- Multiple host genes were identified as critical for the replication of EHV-1.
- These genes clustered in specific cellular signaling and regulatory networks, which are key to viral entry, replication, or evasion of host immunity.
- Functional experiments showed that knocking out these genes significantly suppressed EHV-1 replication and reduced the production of viral proteins, confirming their role as facilitators of infection.
- Pathway analyses suggested that these host factors are involved in signaling pathways that viruses typically exploit to establish infection or evade host immune responses.
Implications and Future Directions
- The identification of essential host determinants for EHV-1 replication opens new avenues for antiviral intervention focused on targeting host factors rather than the virus itself.
- Host-directed therapies may reduce the risk of viral resistance compared to direct-acting antivirals since viruses are less able to adapt to changes in host cellular machinery.
- This research provides a foundational framework for developing immunotherapeutic strategies or antiviral drugs that inhibit key host proteins or pathways critical for EHV-1 survival.
- Further studies could explore the detailed mechanisms by which these identified genes influence EHV-1 infection and whether similar host factors are involved in infections by related herpesviruses.
- The validated host genes also present potential biomarkers for EHV-1 susceptibility or targets for genetic modification aimed at improving disease resistance in horses.
Cite This Article
APA
Li Z, Yu T, Ge L, Lv S, Fu Q, Shi H.
(2026).
Genome-wide CRISPR/Cas9 screening identifies host factors critical for antiviral defense against equine herpesvirus type 1.
Front Immunol, 17, 1764863.
https://doi.org/10.3389/fimmu.2026.1764863 Publication
Researcher Affiliations
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University Key Laboratory of New Drug Research and Innovation for Herbivorous Animals of Xinjiang, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
- College of Veterinary Medicine, Xinjiang Agricultural University Key Laboratory of New Drug Research and Innovation for Herbivorous Animals of Xinjiang, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
MeSH Terms
- Animals
- CRISPR-Cas Systems
- Herpesvirus 1, Equid / physiology
- Herpesvirus 1, Equid / immunology
- Virus Replication / genetics
- Cell Line
- Host-Pathogen Interactions / genetics
- Host-Pathogen Interactions / immunology
- Herpesviridae Infections / immunology
- Herpesviridae Infections / virology
- Herpesviridae Infections / genetics
- Herpesviridae Infections / veterinary
- Horses
- Gene Knockout Techniques
- Horse Diseases / virology
- Horse Diseases / immunology
- Horse Diseases / genetics
- Genome-Wide Association Study
Conflict of Interest Statement
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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