Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys

Abstract

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.

Fig. 1.

Genetic organization of the UL/b′ region of three strains of RhCMV. ORF homologous to HCMV ORF are designated by “RhUL” (48, 61). ORF without homologues in HCMV are designated with the naming scheme of Hansen et al. (indicated by “rh”) (34). Hatched boxes, three genes coding for proteins involved in epithelial tropism (RhUL128, RhUL130, and RhUL131). An inversion (segments B and C) and deletion (segment A) in the UL/b′ region of 68-1 resulted in the loss of UL128 and one exon of RhUL130, leaving only the RhUL131 ORF intact. Shaded boxes, six alpha-chemokine-like ORF. A deletion of segment E in 68-1 resulted in the loss of three of these ORF (RhUL146a, RhUL146b, and rh161.1) and a premature truncation of a fourth (rh161.2, indicated by the asterisk).

Fig. 2.

Longitudinal analysis of RhCMV coinfection in two rhesus macaques (RM). Real-time PCR detection of gB copies in plasma (A), saliva (B), and urine (C). This assay did not discriminate between individual RhCMV strains. The arrow represents the limit of detection (200 copies of RhCMV/ml).

Fig. 3.

Differential PCR detection of RhCMV strains UCD52, UCD59, and 68-1. (A) Demonstration of the specificity of amplification of RhCMV strains with corresponding primer pairs (see Materials and Methods for details). The presence of UCD59 was determined by detection of an amplicon with Pr 59 and the absence of an amplicon with Pr 68-1. (B) Strain-specific detection of RhCMV DNA by differential PCR of saliva and urine samples from coinfected RM at different time points (in weeks). +, detection of appropriately sized amplicons; −, absence of detectable amplicon.

Fig. 4.

Longitudinal analysis of RhCMV single-strain infection. Real-time PCR detection of gB copies in plasma (A), saliva (B), and urine (C). For plasma, only weeks 0 to 10 are presented since no RhCMV DNA was detected in plasma after 10 weeks. Urine samples were not available for weeks 2, 3, 10, 12, and 14. The arrows represent the limit of detection (200 copies of RhCMV/ml).

Fig. 5.

Horizontal transmission of RhCMV to seronegative macaques. A total of 15 macaques that were RhCMV seronegative were relocated to housing together with a single RhCMV-infected monkey. The observed cumulative numbers of cohoused macaques that contained detectable IgG antibodies to RhCMV antigens are presented relative to the time (in weeks) following relocation. A theoretical seroconversion rate in which an RhCMV-infected monkey transmits RhCMV to another monkey every 7 weeks is shown for comparison.

Fig. 6.

Longitudinal detection of RhCMV in the saliva of naturally infected juvenile rhesus macaques at the CNPRC over 11 consecutive weeks. The dashed line and arrow represent the limit of detection (200 copies of gB/ml).