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.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56−CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16− NK cells from HSPC. These CD56−CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.

Approximately 1 in 200 infants is born with congenital cytomegalovirus (CMV), making it the most common congenital infection. About 1 in 5 congenitally-infected babies will suffer long-term sequelae, including sensorineural deafness, intellectual disability, and epilepsy. CMV infection is highly species-dependent, and the Rhesus CMV (RhCMV) infection of rhesus monkey fetuses is the only animal model that replicates essential features of congenital CMV infection in humans, including placental transmission, fetal disease, and fetal loss. To better understand the determinants and dynamics of congenital CMV transmission, we developed a mathematical model for placental transmission, comprising of maternal, placental, and fetal compartments using parameters from literature and experimental data from RhCMV seronegative rhesus macaques inoculated with RhCMV at 7.7-9.0 weeks of pregnancy. The model was then used to study the effect of the timing of inoculation, maternal immune suppression, and hyper-immune globulin infusion on the risk of placental transmission in the context of primary and reactivated chronic maternal CMV infection.

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-...