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Vaccines2026; 14(7); 634; doi: 10.3390/vaccines14070634

Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage Florida Clade 2).

Abstract: Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing the hemagglutinin (HA) gene of H3N8 EIV (sub-lineage: Florida clade 2) was generated and characterized in vitro. Methods: The HA gene of EIV (Florida clade 2) was used for antigen gene cloning. The expression cassette for the HA gene was commercially synthesized and inserted into the backbone of EHV1∆IR6 BAC using an mutagenesis strategy. Recombinant clones were selected using antibiotic selection, PCR, and RFLP. Further, the recombinant virus was regenerated in RK-13 cells via transfection and characterized in vitro for plaque size, growth kinetics and immunofluorescence antibody test (IFAT). Results: PCR and RFLP confirmed the successful insertion of the HA gene into pEHV1∆IR6/gE BAC. The recombinant virus, vEHV1∆IR6/gE-HA(FC2), was successfully rescued in RK13 cells and demonstrated expression of the EIV haemagglutinin proteins by immunofluorescence assay. Although plaque size was reduced in the generated mutant virus in comparison to parental virus, the growth kinetics of the recombinant viruses were comparable to those of vEHV1∆IR6/gE. Conclusions: These findings demonstrate the successful expression of immunodominant hemagglutinin protein of EIV by recombinant EHV-1 and indicate the potential suitability of EHV-1 BAC as a vector platform for foreign gene expression.
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Publication Date: 2026-07-20 PubMed ID: 42506671PubMed Central: PMC13416966DOI: 10.3390/vaccines14070634Google Scholar: Lookup
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  • 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 research focuses on developing a recombinant equine herpesvirus type 1 (EHV-1) that expresses the key hemagglutinin protein from the equine influenza virus (EIV), aiming to create a novel live-attenuated vaccine vector to protect horses against both viruses.

Background

  • Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are significant causes of respiratory disease in horses, leading to economic losses in the equine industry.
  • Bacterial Artificial Chromosome (BAC) technology allows precise genetic manipulation of viral genomes, facilitating the development of genetically modified, live-attenuated viruses for vaccine purposes.
  • The authors previously created an attenuated EHV-1 vaccine by deleting certain genes associated with virulence using BAC technology, providing a foundation for using this mutant EHV-1 as a vector for expressing foreign genes.

Objective

  • The study aimed to engineer a recombinant EHV-1 virus that expresses the hemagglutinin (HA) protein from the EIV subtype H3N8, specifically the Florida clade 2 sub-lineage, which is an important antigen for inducing immunity against EIV.
  • The goal was to characterize this recombinant virus in vitro to assess its growth properties and expression of the foreign HA protein.

Methods

  • The HA gene from the Florida clade 2 subtype of EIV was selected as the foreign antigen gene.
  • A commercially synthesized expression cassette containing the HA gene was inserted into the genome of the previously developed attenuated EHV-1 mutant (pEHV1∆IR6/gE BAC) using mutagenesis techniques facilitated by BAC technology.
  • Recombinant viral clones were identified and selected using:
    • Antibiotic selection
    • Polymerase chain reaction (PCR) to verify the presence of the inserted gene
    • Restriction fragment length polymorphism (RFLP) analysis to confirm correct genetic insertion
  • Recombinant virus was recovered by transfecting the modified BAC DNA into RK-13 cells (rabbit kidney cell line).
  • The recombinant virus was characterized using:
    • Plaque size assays to evaluate viral replication and spread
    • Growth kinetics studies to compare replication rates with parent virus
    • Immunofluorescence antibody test (IFAT) to confirm expression of the hemagglutinin protein

Results

  • PCR and RFLP analyses confirmed successful insertion of the HA gene into the EHV-1 BAC genome.
  • The recombinant virus, named vEHV1∆IR6/gE-HA(FC2), was successfully rescued in RK-13 cells.
  • Immunofluorescence assays demonstrated that the recombinant virus expressed the hemagglutinin protein from EIV effectively, indicating successful foreign gene expression.
  • Although the recombinant virus produced smaller plaques compared to the parent virus—suggesting some attenuation of spread—the overall viral growth kinetics were similar, indicating robust viral replication capability.

Conclusions and Implications

  • This study successfully demonstrated that the immunodominant hemagglutinin protein of equine influenza virus can be expressed by a recombinant, attenuated EHV-1 vector.
  • The recombinant virus maintains comparable replication properties to its parent, despite reduced plaque size, which may be advantageous for vaccine safety and attenuation.
  • These findings support the potential of EHV-1 BAC-based vectors as a platform for developing multivalent vaccines that could protect horses against multiple respiratory pathogens simultaneously.
  • This approach could enhance vaccine efficacy and disease control strategies in the equine industry by combining protections into a single live-attenuated viral vector vaccine.

Cite This Article

APA
Bera BC, Bernela M, Madhwal A, Pradhan SS, Balena V, Anand T, Kandasamy S, Pavulraj S, Ahlawat W, Kandpal D, Mor P, Bishnoi G, Vasdev N, Tripathi BN, Bhattacharya TK, Virmani N. (2026). Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage Florida Clade 2). Vaccines (Basel), 14(7), 634. https://doi.org/10.3390/vaccines14070634

Publication

Vaccines
ISSN: 2076-393X
NlmUniqueID: 101629355
Country: Switzerland
Language: English
Volume: 14
Issue: 7
PII: 634

Researcher Affiliations

Bera, Bidhan Chandra
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Bernela, Manju
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Madhwal, Aashwina
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
  • Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India.
Pradhan, Stephanie S
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
  • Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India.
Balena, Venkataramireddy
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
  • Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India.
Anand, Taruna
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Kandasamy, Supriya
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
  • Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India.
Pavulraj, Selvaraj
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Ahlawat, Wandit
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Kandpal, Diksha
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Mor, Priya
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Bishnoi, Gurmesh
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Vasdev, Nishant
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Tripathi, Bhupendra Nath
  • Indian Council of Agricultural Research, Krishi Bhawan, New Delhi 110012, Delhi, India.
Bhattacharya, Tarun Kumar
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.
Virmani, Nitin
  • ICAR-National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India.

Grant Funding

  • BGAM 9005 / ICAR National Science Agriculture Fund

Conflict of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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