In vitroandin vivocharacterization of a recombinant rhesus cytomegalovirus containing a complete genome

Abstract

In vitro and in vivo characterization of a recombinant1 rhesus cytomegalovirus containing a complete genome 2 3 Abstract (300 words) 42 Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species 43 specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models 44 using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has 45 been established as a representative model for infection of humans with HCMV due to the close 46 evolutionary relationships of both host and virus. However, the commonly used 68-1 strain of 47 RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due 48 to tissue culture adaptation that cause reduced viremia in RhCMV-naïve animals and limited 49 shedding compared to low passage isolates. Using sequence information from primary RhCMV 50 isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-51 1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve 52 RM with the reconstituted virus resulted in significant replication in the blood similar to primary 53 isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post 54 infection. In contrast, viral dissemination and viremia was greatly reduced upon deletion of genes 55 also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-56 like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and 57 in vivo consistent with an important immunomodulatory function of the respective proteins. We 58 conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while 59 being amenable to genetic modifications through BAC recombineering techniques.60 61 4 Author Summary (150-200 word non-technical summary) 62 Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy 63 immunocompetent individuals, but HCMV can cause serious disease after congenital infection and 64 in individuals with immunocompromised immune systems. Since HCMV is highly species specific 65 and cannot productively infect immunocompetent laboratory animals, experimental infection of 66 rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related 67 animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting 68 cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines 69 against chronic and recurring infections with pathogens such as the human immunodeficiency 70 virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted 71 with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in 72 tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a 73 full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve 74 RM with FL-...

Cytomegaloviral determinants of CD8+T cell programming and RhCMV/SIV vaccine efficacy

“…We next asked the question of whether abrogating both the PRC and non-PRC-related immune programming activities of Rh157.5 and Rh157.4 was sufficient to convert a wildtype-like RhCMV, with conventional MHC-Ia-restricted CD8 + T cell targeting, to a 68-1-like vector with exclusively MHC-IIand MHC-E-restricted CD8 + T cell responses. Using sequence information from primary RhCMV isolates, including the original 68-1 isolate 17, we reconstructed a full length RhCMV clone (RhCMV FL) in which all suspected deletions, inversions, frameshifts and premature termination codons affecting viral open reading frames were repaired and the Rh13.1 open reading frame (ORF) was replaced by an SIVgag insert (18). RhCMV FL has robust in vivo spread (18), and as expected for a wildtype genetic configuration (9), elicits MHC-Ia-restricted, CD8 + T cell responses ( Fig.…”

“…Using sequence information from primary RhCMV isolates, including the original 68-1 isolate 17, we reconstructed a full length RhCMV clone (RhCMV FL) in which all suspected deletions, inversions, frameshifts and premature termination codons affecting viral open reading frames were repaired and the Rh13.1 open reading frame (ORF) was replaced by an SIVgag insert (18). RhCMV FL has robust in vivo spread (18), and as expected for a wildtype genetic configuration (9), elicits MHC-Ia-restricted, CD8 + T cell responses ( Fig. 2A).…”

Engineering, Decoding and Systems-Level Characterization of Chimpanzee Cytomegalovirus

“…Second, and more important, the large and complex CMV genomes become available for prokaryotic recombination techniques, allowing for efficient and reliable gene targeting. In addition to multiple HCMV strains cloned as BACs (Wilkinson et al, 2015), animal CMV BACs are utilized as cloning platforms for in vitro and in vivo models and thereby provide the framework for a profound understanding of CMV infection and pathogenesis (Roark et al, 2020;Taher et al, 2020). In particular, the rhesus CMV (RhCMV) BAC platform has become a popular model system for HCMV due to many shared features in infection, replication and immune control (Itell et al, 2017;Powers and Früh, 2008).…”