In vitroandin vivocharacterization of a recombinant rhesus cytomegalovirus containing a complete genome
Authors: Taher Husam , Mahyari Eisa , Kreklywich Craig , Uebelhoer Luke S. , McArdle Matthew R. , Moström Matilda J. , Bhusari Amruta , Nekorchuk Michael , Whitmer Travis , Scheef Elizabeth A. , Sprehe Lesli M. , Roberts Dawn , Hughes Colette M. , Jackson Kerianne A. , Selseth Andrea N. , Ventura Abigail B. , Yue Yujuan , Schmidt Kimberli A. , Shao Jason , Edlefsen Paul T. , Smedley Jeremy , Stanton Richard J. , Axthelm Michael K. , Estes Jacob D. , Hansen Scott G. , Kaur Amitinder , Barry Peter A. , Bimber Benjamin N. , Picker Louis J. , Streblow Daniel N. , Früh Klaus , Malouli Daniel 
Published: June 2020

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence,in vivoexamination 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 commonly used 68-1 strain of RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptation that cause reduced viremia in RhCMV-naïve animals and limited shedding compared to low passage isolates. Using sequence information from primary RhCMV isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant replication in the blood similar to primary isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post infection. In contrast, viral dissemination and viremia 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 bothin vitroandin vivoconsistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displaysin vitroandin vivocharacteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Author Summary

Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) andMycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68-1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.