JVI Accepts, published online ahead of print on 28 October 2009

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J. Virol. doi:10.1128/JVI.01544-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

U_L47 Gene-Deleted Bovine Herpesvirus Type 1 Exhibits Impaired Growth in Cell Culture and Lack of Virulence in Cattle

Vladislav A. Lobanov, Sheryl L. Maher-Sturgess, Marlene G. Snider, Zoe Lawman, Lorne A. Babiuk, and Sylvia van Drunen Littel-van den Hurk^*

Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; and University of Alberta, 3-7 University Hall, Edmonton, Alberta, Canada

^* To whom correspondence should be addressed. Email: sylvia.vandenhurk{at}usask.ca.

Tegument protein VP8 encoded by the U_L47 gene of bovine herpesvirus 1 (BHV-1) is the most abundant constituent of mature virions. In the present report we describe the characterization of U_L47 gene-deleted BHV-1 in cultured cells and its natural host. The U_L47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease of intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U_L47 deletion mutant-virus infected cells, since bICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the infection course. Whereas VP16, glycoprotein (g) C, gB and VP5 were expressed to wild-type levels in the U_L47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U_L47 deletion mutant contained reduced amounts of gD, gB, gC and VP22, but similar amounts of VP16 compared to wild-type or revertant virus particles. In addition, the U_L47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U_L47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.