Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells

Abstract

Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (<1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine.IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We studied CMV acquisition in infants and found that infections are usually brief and self-limited and are successfully established relatively rarely. Thus, although most people eventually acquire CMV infection, it usually requires numerous exposures. Our analyses indicate that this is because the virus is surprisingly inefficient, barely replicating well enough to spread to neighboring cells in the mouth. Greater knowledge of why CMV infection usually fails may provide insight into how to prevent it from succeeding.

Keywords: Epstein-Barr virus; basic reproduction number; cytomegalovirus; founder population; herpes simplex virus; human herpesviruses; mathematical modeling; oral shedding; transient infection.

FIG 1

Transient oral CMV infections and shedding in Ugandan infants. (A and B) Examples of CMV DNA concentrations measured in two infants without primary infection, showing 6 and 9 transient CMV infections (A), and two infants with primary infection (gray), showing 2 and 3 preceding transient CMV infections (B). (C) Durations of transient CMV infections (n = 107) are generally less than 2 weeks. The duration was categorized based on total consecutive positive swabs to account for sampling frequency. Overlap occurred because the exact day the infection ended was not observed. (D) Comparison of maximum CMV load distributions in infants during transient CMV infections and primary infections. (E) CMV DNA is present more frequently than that of other human herpesviruses in infant oral swabs prior to primary infection. (F) Viral DNA is detected at similar frequencies in infants who do and do not go on to develop primary infection. The boxplots represent the IQRs, the whiskers represent the ranges of values within 1.5 IQR of the nearest quartile, and the dots represent data points outside the ranges.

FIG 2

Transient CMV infections are common prior to primary CMV infection in Ugandan infants. Shown is the estimated cumulative distribution of primary infection incidence by total observed transient infections among all of the infants and restricted to infants with primary infection only. For example, among all of the infants, 50% experienced primary infection after 4 transient infections were observed. The distributions were estimated using Kaplan-Meier analysis to incorporate infants with transient infections but no observed primary infection during the study (indicated by ×). The probability that an infection was transient was estimated from these distributions ranged from 76% (primary infected infants only) to 88% based on estimates from the distributions (Table 1).

FIG 3

In mathematical model simulations of CMV infection, a low basic reproductive number (R₀) and a low initial number of infected cells (I₀) increase the probability that observed CMV infections are transient. (A) Fifty example simulations with either 1 or 10 initially infected cells and with an R₀ value of either 1.05 or 1.5. Prolonged low-viral-load transient infections were observed most commonly at low R₀ and high I₀. (B) Probabilities that observed infections were transient (color map and contours) across ranges of R₀ (1 to 1.5) and I₀ (1 to 100) based on 10,000 simulations of CMV infection. Compared with probabilities estimated from the cohort (Table 1), a 76% probability of an observed transient CMV infection occurred at an R₀ approximately equal to 1.1 for an I₀ equal to 1 (dotted line) and an 88% probability occurred at an R₀ approximately equal to 1.05 for an I₀ equal to 1.

FIG 4

Model simulations recapitulate observed CMV transient-infection viral loads and durations at low I₀ and low R₀. (A) Distributions of CMV loads based on simulations of observable transient infections for varying R₀ and I₀ values. The solid red line denotes the median CMV DNA concentration observed from transient infections in the Ugandan infant cohort, and the dashed red lines are the boundaries for the IQR. The boxplots represent IQRs, the whiskers represent the ranges of values within 1.5 IQR of the nearest quartile, and the dots represent data points outside the ranges. (B) Bar plots displaying distributions of observed consecutive positive swabs based on the simulated transient infections for varying R₀ and I₀ values. The count of consecutive positive swabs is an estimate of duration that accounts for the granularity and variation of the sampling frequency in the cohort. Consecutive positive swabs observed from transient infections in the Ugandan infant cohort (presented unstacked in Fig. 1B) (Data) are repeated in each panel for comparison. Prolonged transient CMV episodes (>4 consecutive positive swabs) were not observed in the data. (C) Deviance scores summarizing agreement between the distributions of transient-infection duration (categorized by the total consecutive positive swabs shown in panel B) between the infant cohort data and the model for different I₀ and R₀ values. The deviance scores were calculated using the G test statistic (51); lower values correspond to higher agreement between the model and the data.