In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

by Husam Taher, Eisa Mahyari, Craig Kreklywich, Luke S. Uebelhoer, Matthew R. McArdle, Matilda J. Moström, Amruta Bhusari, Michael Nekorchuk, Xiaofei E, Travis Whitmer, Elizabeth A. Scheef, Lesli M. Sprehe, Dawn L. Roberts, Colette M. Hughes, Kerianne A. Jackson, Andrea N. Selseth, Abigail B. Ventura, Hillary C. Cleveland-Rubeor, Yujuan Yue, Kimberli A. Schmidt, Jason Shao, Paul T. Edlefsen, Jeremy Smedley, Timothy F. Kowalik, Richard J. Stanton, Michael K. Axthelm, Jacob D. Estes, Scott G. Hansen, Amitinder Kaur, Peter A. Barry, Benjamin N. Bimber, Louis J. Picker, Daniel N. Streblow, Klaus Früh, Daniel Malouli

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

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