Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice.
Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.
Smith
Patrick M
PM
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA. psmith@lsuhsc.edu
Kahan
Shannon M
SM
Rorex
Colin B
CB
von Einem
Jens
J
Osterrieder
Nikolaus
N
O'Callaghan
Dennis J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D006566:Herpesviridae Infections / Q000473:pathology*; D004861:Herpesvirus 1, Equid / Q000472:pathogenicity; D007249:Inflammation / Q000473:pathology / Q000821:virology; D008168:Lung / Q000473:pathology* / Q000821:virology; D008171:Lung Diseases / Q000473:pathology / Q000821:virology*; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000235:genetics*; D014774:Virulence
D017931:DNA Primers; D004279:DNA, Viral; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; C077966:glycoprotein 300, Equid herpesvirus 1