Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome

Abstract

Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a DynamicIn VitroModel

“…All viruses used in the study were recovered from infectious bacterial artificial chromosome (BAC) clones. Those were BACs of EHV-1 strain Ab4 (33) and EHV-4 strain TH20p (34), as well as modified BACs EHV-1_gB4, EHV-4_gB1, revertant EHV-1_gB1r (20), EHV1_gD4, EHV-4_gD1 (35), EHV-1⌬US3, and EHV-1 that contained US3 of EHV-4 (EHV-1_US3_4) in lieu of authentic US3, fully rescuing the parental EHV-1 phenotype and functioning as revertant for this study (A. Proft and W. Azab, unpublished data). monomeric red fluorescent protein (mRFP1)-labeled EHV-1 was previously constructed by inserting mRFP1 into VP26 of EHV-1 strain RacL11 (36).…”

Glycoproteins D of Equine Herpesvirus Type 1 (EHV-1) and EHV-4 Determine Cellular Tropism Independently of Integrins

“…EHV-1 strain L11⌬gp2 (59) was reconstituted after transfection of 2 g of bacterial artificial chromosome (BAC) DNA into rabbit kidney (RK13) cells, using Lipofectamine 2000 (Invitrogen). Recombinant WA79 was derived from an EHV-4 infectious BAC clone that was generated by the insertion of a loxP-flanked BAC vector into the intergenic region between genes 58 and 59 (7). Cells.…”

“…EHV-1 strain RacL11 cloned as a BAC (pL11) contains the enhanced green fluorescent protein (EGFP) gene instead of the nonessential gene 71 (59). The EHV-4 BAC clone pYO03 was generated by the insertion of mini F plasmid sequences flanked by loxP sites into the intergenic region between genes 58 and 59 (7). Both pL11 and pYO03 BACs were maintained in Escherichia coli GS1783 cells (a kind gift from Greg Smith, Northwestern University, Chicago, IL).…”

“…The EHV-1 mutants and revertants, EHV-1gD4 and EHV-1R, were reconstituted after transfection of 2 g of purified BAC DNA into RK13 cells using Lipofectamine 2000 (Invitrogen) as described previously (59). For EHV-4 mutants and revertants, EHV-4gD1, EHV-4 152D , and EHV-4R, the viruses were reconstituted by transfection of purified DNA into 293 cells as described earlier (7,8). Three days later, the supernatant and cells were collected and used to infect confluent NBL-6 cells.…”

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin

“…transfecting BAC DNA into human embryonic kidney (293T) cells, as described previously (Azab et al, 2009(Azab et al, , 2010aRudolph et al, 2002). Supernatants and infected cells were collected 48 h after transfection and high titre stocks of each virus were produced by passaging the transfection product on equine dermal (ED) cells.…”