ABSTRACT
EUL47 is a major component of the tegument of equine herpesvirus 1 (EHV-1). To determine its function, we used Red/ET cloning to delete its gene (gene 13) from EHV-1 strain Ab4p inserted into a bacterial artificial chromosome (BAC), yielding Ab4pattBΔ13. We also examined the reverted virus (Ab4pattB13R). Ab4pattBΔ13 replicated in rabbit kidney (RK)-13 cells, indicating that ORF13 is dispensable for virus replication in cell culture. Its intracellular and extracellular titers were about 10- and 100-fold lower than those of the revertant and parent strains, respectively. In addition, the plaque size was half the plaque sizes of the other two strains. The particle-to-plaque forming unit ratio of Ab4pattBΔ13 was 21-fold greater than the ratios of the revertant and parent strains. No enveloped virions were detected in the cytoplasm of Ab4pattBΔ13-infected cells by transmission electron microscopy. In hamster, Ab4pattBΔ13 caused clinical signs and weight loss after only 1 day, but induced less severe neurological signs than did the revertant and parent strains. These results indicate that EUL47 is structurally required for normal virus replication, viral morphogenesis and viral infectivity, and that loss of EUL47 moderately attenuates the neuropathogenicity of EHV-1 in the hamster model.
Subject(s)
Herpesvirus 1, Equid/physiology , Herpesvirus 1, Equid/ultrastructure , Viral Structural Proteins/metabolism , Virus Replication , Animals , Cell Line , Cricetinae , Disease Models, Animal , Gene Deletion , Herpesviridae Infections/pathology , Herpesviridae Infections/virology , Herpesvirus 1, Equid/growth & development , Herpesvirus 1, Equid/pathogenicity , Mesocricetus , Microscopy, Electron, Transmission , Rabbits , Viral Load , Viral Plaque Assay , Viral Structural Proteins/genetics , Virion/ultrastructure , VirulenceABSTRACT
Equine herpesvirus 1 (EHV-1) bacterial artificial chromosome clone (Ab4p BAC) was established based on neuropathogenic strain Ab4p. ORF37 encoding UL24 was replaced with a selection cassette, rpsL-neo gene, to produce an ORF37 deletion mutant, Ab4pORF37. Transfection of RK-13 cells with Ab4pORF37 genome DNA produced infectious virus, indicating that ORF37 is not essential for EHV-1 replication in cell culture. Deletion of ORF37 had no effect on the transcript expression of neighboring genes, ORF36 and ORF38, and the growth activity in MDBK cells. Ab4pDeltaORF37 lost neuropathogenicity in CBA/N1 mice as indicated by the absence of any neurological disorders and death. The growth of Ab4pDeltaORF37 in cultivated neural cells was one order of magnitude lower than that of parental and revertant viruses. These results indicated that the ORF37 is a neuropathogenicity determinant of EHV-1 in the mouse encephalitis model.
Subject(s)
Encephalitis, Viral/virology , Herpesvirus 1, Equid/pathogenicity , Viral Proteins/physiology , Virulence Factors/physiology , Animals , Body Weight , Brain/pathology , Brain/virology , Cattle , Cell Line , Encephalitis, Viral/pathology , Gene Knockout Techniques , Herpesvirus 1, Equid/genetics , Herpesvirus 1, Equid/growth & development , Lung/virology , Male , Mice , Mice, Inbred CBA , Rabbits , Viral Load , Viral Plaque Assay , Viral Proteins/genetics , Virulence Factors/geneticsABSTRACT
The pathogenicity of equine herpesvirus 1 (EHV-1) isolates of Japan were evaluated by using the CBA mouse model. CBA mice were inoculated with eight Japanese EHV-1 strains (89c1, 90c16, 90c18, 97c11, 98c12, 00c19, 01c1 and HH-1) and one British strain (Ab4p). 89c1 caused slight body weight loss and nervous signs in mice at 8 days post infection (dpi). Severe weight loss and nervous signs were observed in mice inoculated with Ab4p at 6 dpi. The other strains did not cause apparent clinical signs. Infectious viruses were recovered from the lungs of all groups at 2 dpi. Histopathological analysis revealed interstitial pneumonia in the lungs of all mice inoculated with EHV-1. Encephalitis or meningoencephalitis was observed in the brains of mice inoculated with 89c1, 90c18, 97c11, 98c12, 01c1 and Ab4p. Japanese EHV-1 strains showed low pathogenicity in CBA mice, whereas the sequential affects of infection are similar to those of the highly pathogenic strain Ab4p. These results suggest that field isolates of EHV-1 have varying degrees of pathogenicity in CBA mice.
