FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Front Immunol / Genome-wide CRISPR/Cas9 screening identifies host factors cr...
Frontiers in immunology2026; 17; 1764863; doi: 10.3389/fimmu.2026.1764863

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.
Get Full Text
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.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

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

Frontiers in immunology
ISSN: 1664-3224
NlmUniqueID: 101560960
Country: Switzerland
Language: English
Volume: 17
Pages: 1764863
PII: 1764863

Researcher Affiliations

Li, Ziqian
  • College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
Yu, Tingting
  • College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
Ge, Lijuan
  • College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
Lv, Shubing
  • College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang Uygur Autonomous Region, Urumqi, China.
Fu, Qiang
  • 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.
Shi, Huijun
  • 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.

References

This article includes 30 references
  1. Ma G, Azab W, Osterrieder N. Equine herpesviruses type 1 (EHV-1) and 4 (EHV-4)--masters of co-evolution and a constant threat to equids and beyond. Vet Microbiol. (2013) 167:123–34. doi:  10.1016/j.vetmic.2013.06.018, PMID:
    doi: 10.1016/j.vetmic.2013.06.018pubmed: 23890672google scholar: lookup
  2. Laval K, Poelaert KCK, Van Cleemput J, Zhao J, Vandekerckhove AP, Gryspeerdt AC, et al. The pathogenesis and immune evasive mechanisms of equine herpesvirus type 1. Front Microbiol. (2021) 12:662686. doi:  10.3389/fmicb.2021.662686, PMID:
    doi: 10.3389/fmicb.2021.662686pmc: PMC7970122pubmed: 33746936google scholar: lookup
  3. Oladunni FS, Horohov DW, Chambers TM. EHV-1: A constant threat to the horse industry. Front Microbiol. (2019) 10:2668. doi:  10.3389/fmicb.2019.02668, PMID:
    doi: 10.3389/fmicb.2019.02668pmc: PMC6901505pubmed: 31849857google scholar: lookup
  4. Huang T, Lehmann MJ, Said A, Ma G, Osterrieder N. Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis. J Virol. (2014) 88:12802–15. doi:  10.1128/jvi.02079-14, PMID:
    doi: 10.1128/jvi.02079-14pmc: PMC4248885pubmed: 25165105google scholar: lookup
  5. Hull MA, Pritchard SM, Nicola AV. Herpes simplex virus 1 envelope glycoprotein C shields glycoprotein D to protect virions from entry-blocking antibodies. J Virol. (2025) 99:e0009025. doi:  10.1128/jvi.00090-25, PMID:
    doi: 10.1128/jvi.00090-25pmc: PMC11998518pubmed: 40135897google scholar: lookup
  6. Myskiw C, Arsenio J, van Bruggen R, Deschambault Y, Cao J. Vaccinia virus E3 suppresses expression of diverse cytokines through inhibition of the PKR, NF-kappaB, and IRF3 pathways. J Virol. (2009) 83:6757–68. doi:  10.1128/jvi.02570-08, PMID:
    doi: 10.1128/jvi.02570-08pmc: PMC2698532pubmed: 19369349google scholar: lookup
  7. Visser LJ, Aloise C, Swatek KN, Medina GN, Olek KM, Rabouw HH, et al. Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression. PloS Pathog. (2020) 16:e1008702. doi:  10.1371/journal.ppat.1008702, PMID:
    doi: 10.1371/journal.ppat.1008702pmc: PMC7384677pubmed: 32667958google scholar: lookup
  8. Cheng KW, Bhave M, Markhard AL, Peng D, Bhatt KD, Travisano KA, et al. Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication. Nat Commun. (2025) 16:11028. doi:  10.1038/s41467-025-65979-3, PMID:
    doi: 10.1038/s41467-025-65979-3pmc: PMC12696002pubmed: 41372121google scholar: lookup
  9. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. (2014) 343:84–7. doi:  10.1126/science.1247005, PMID:
    doi: 10.1126/science.1247005pmc: PMC4089965pubmed: 24336571google scholar: lookup
  10. Hu S, Gan M, Wei Z, Shang P, Song L, Feng J, et al. Identification of host factors for livestock and poultry viruses: genome-wide screening technology based on the CRISPR system. Front Microbiol. (2024) 15:1498641. doi:  10.3389/fmicb.2024.1498641, PMID:
    doi: 10.3389/fmicb.2024.1498641pmc: PMC11619636pubmed: 39640855google scholar: lookup
  11. See WR, Yousefi M, Ooi YS. A review of virus host factor discovery using CRISPR screening. mBio. (2024) 15:e0320523. doi:  10.1128/mbio.03205-23, PMID:
    doi: 10.1128/mbio.03205-23pmc: PMC11559068pubmed: 39422472google scholar: lookup
  12. Suzuki T, Sato Y, Okuno Y, Goshima F, Mikami T, Umeda M, et al. Genome-wide CRISPR screen for HSV-1 host factors reveals PAPSS1 contributes to heparan sulfate synthesis. Commun Biol. (2022) 5:694. doi:  10.1038/s42003-022-03581-9, PMID:
    doi: 10.1038/s42003-022-03581-9pmc: PMC9296583pubmed: 35854076google scholar: lookup
  13. Han J, Perez JT, Chen C, Li Y, Benitez A, Kandasamy M, et al. Genome-wide CRISPR/cas9 screen identifies host factors essential for influenza virus replication. Cell Rep. (2018) 23:596–607. doi:  10.1016/j.celrep.2018.03.045, PMID:
    doi: 10.1016/j.celrep.2018.03.045pmc: PMC5939577pubmed: 29642015google scholar: lookup
  14. Shue B, Chiramel AI, Cerikan B, To TH, Frölich S, Pederson SM, et al. Genome-wide CRISPR screen identifies RACK1 as a critical host factor for flavivirus replication. J Virol. (2021) 95:e0059621. doi:  10.1128/jvi.00596-21, PMID:
    doi: 10.1128/jvi.00596-21pmc: PMC8610583pubmed: 34586867google scholar: lookup
  15. Stokes A, Allen GP, Pullen LA, Murray PK. A hamster model of equine herpesvirus type 1 (EHV-1) infection; passive protection by monoclonal antibodies to EHV-1 glycoproteins 13, 14 and 17/18. J Gen Virol. (1989) 70:1173–83. doi:  10.1099/0022-1317-70-5-1173, PMID:
    doi: 10.1099/0022-1317-70-5-1173pubmed: 2471805google scholar: lookup
  16. Trapp S, von Einem J, Hofmann H, Köstler J, Wild J, Wagner R, et al. Potential of equine herpesvirus 1 as a vector for immunization. J Virol. (2005) 79:5445–54. doi:  10.1128/jvi.79.9.5445-5454.2005, PMID:
    doi: 10.1128/jvi.79.9.5445-5454.2005pmc: PMC1082783pubmed: 15827159google scholar: lookup
  17. Wang SA. Adaptation of locally isolated equine herpesvirus (EHV-1) on different cell lines. J Vet Med Res. (2018) 18(1):52–6. doi:  10.47972/jvmr.2018.18.1.52
    doi: 10.47972/jvmr.2018.18.1.52google scholar: lookup
  18. Moezpoor MR, Stevenson M. Help or hinder: protein host factors that impact HIV-1 replication. Viruses. (2024) 16(8):1281. doi:  10.3390/v16081281, PMID:
    doi: 10.3390/v16081281pmc: PMC11360189pubmed: 39205255google scholar: lookup
  19. Finkel Y, Nachshon A, Aharon E, Arazi T, Simonovsky E, Dobešová M, et al. A virally encoded high-resolution screen of cytomegalovirus dependencies. Nature. (2024) 630:712–9. doi:  10.1038/s41586-024-07503-z, PMID:
    doi: 10.1038/s41586-024-07503-zpubmed: 38839957google scholar: lookup
  20. Liu LL, Yin YQ, Ma KX, Xing JC, Ren XX, Huang JY, et al. Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library. Vet Microbiol. (2024) 293:110099. doi:  10.1016/j.vetmic.2024.110099, PMID:
    doi: 10.1016/j.vetmic.2024.110099pubmed: 38677125google scholar: lookup
  21. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. (2014) 343:80–4. doi:  10.1126/science.1246981, PMID:
    doi: 10.1126/science.1246981pmc: PMC3972032pubmed: 24336569google scholar: lookup
  22. E X, Kowalik TF. A generally applicable CRISPR/cas9 screening technique to identify host genes required for virus infection as applied to human cytomegalovirus (HCMV) infection of epithelial cells. Methods Mol Biol. (2021) 2244:247–64. doi:  10.1007/978-1-0716-1111-1_13, PMID:
    doi: 10.1007/978-1-0716-1111-1_13pubmed: 33555591google scholar: lookup
  23. Slońska A, Cymerys J, Skwarska J, Golke A, Bańbura MW. Influence of importin alpha/beta and exportin 1 on equine herpesvirus type 1 (EHV-1) replication in primary murine neurons. Pol J Vet Sci. (2013) 16:749–51. doi:  10.2478/pjvs-2013-0106, PMID:
    doi: 10.2478/pjvs-2013-0106pubmed: 24597312google scholar: lookup
  24. Yang Q, Wu Y, Wang M, Chen S, Jia R, Yang Q, et al. The S-phase arrest of host cells caused by an alpha-herpesvirus genome replication facilitates viral recruitment of RNA polymerase II to transcribe viral genes. Cell Prolif. (2025) 58:e13811. doi:  10.1111/cpr.13811, PMID:
    doi: 10.1111/cpr.13811pmc: PMC12179542pubmed: 39870514google scholar: lookup
  25. Lai D, Sosicka P, Williams DJ, Bowyer ME, Ressler AK, Kohrt SE, et al. SLC35A2 loss-of-function variants affect glycomic signatures, neuronal fate and network dynamics. Brain. (2025) 148:4259–74. doi:  10.1093/brain/awaf198, PMID:
    doi: 10.1093/brain/awaf198pubmed: 40418734google scholar: lookup
  26. Huang W, Qian Z, Shi Y, Zhang Z, Hou R, Mei J, et al. PSMC2 is a novel prognostic biomarker and predicts immunotherapeutic responses: from pancreatic cancer to pan-cancer. Pharmgenomics Pers Med. (2023) 16:747–58. doi:  10.2147/pgpm.S418533, PMID:
    doi: 10.2147/pgpm.S418533pmc: PMC10423611pubmed: 37581119google scholar: lookup
  27. Schneider SM, Pritchard SM, Wudiri GA, Trammell CE, Nicola AV. Early steps in herpes simplex virus infection blocked by a proteasome inhibitor. mBio. (2019) 10(3):e00732-19. doi:  10.1128/mBio.00732-19, PMID:
    doi: 10.1128/mBio.00732-19pmc: PMC6520451pubmed: 31088925google scholar: lookup
  28. Amano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken). (2010) 67:545–54. doi:  10.1002/cm.20472, PMID:
    doi: 10.1002/cm.20472pmc: PMC3038199pubmed: 20803696google scholar: lookup
  29. Baldwin A, Popov IK, Keller R, Wallingford J, Chang C. The RhoGEF protein Plekhg5 regulates medioapical and junctional actomyosin dynamics of apical constriction during Xenopus gastrulation. Mol Biol Cell. (2023) 34:ar64. doi:  10.1091/mbc.E22-09-0411, PMID:
    doi: 10.1091/mbc.E22-09-0411pmc: PMC10295481pubmed: 37043306google scholar: lookup
  30. Słońska A, Cymerys J. Equid alphaherpesvirus 1 (EHV-1) infection of primary murine astrocytes: role of the cytoskeleton. Arch Virol. (2025) 171:19. doi:  10.1007/s00705-025-06494-0, PMID:
    doi: 10.1007/s00705-025-06494-0pmc: PMC12695969pubmed: 41372519google scholar: lookup

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,001 horse owners like you who receive our email updates on horse health and nutrition.