The epidemiology and pathogenesis of CMV infections among pregnant women have been intensely studied over the last three decades. This paper highlights recent developments that make either universal or limited serologic screening for CMV during pregnancy potentially attractive. The developments include an understanding of the pathogenesis of CMV infections, a knowledge of high-risk women, the availability of accurate methods for the serologic diagnosis of a primary CMV infection using either single or serial blood samples, accurate methods for the diagnosis of fetal infection via amniotic fluid, sensitive fetal and placental indicators for neonatal outcomes, and the availability of potentially effective interventions.
We observed previously that African American adolescents in Richmond reporting infrequent sexual activity had cytomegalovirus (CMV) seroprevalence rates one half that of their adult mothers and caregivers. We therefore sought to determine if sexually active African American adolescents have higher rates of CMV infection than sexually inactive African American adolescents.
Cases (aged 13–18 years) sought care for a sexually transmitted infection (STI) or pregnancy. Controls were sexually inactive and matched to cases for age, race, and gender and enrolled at the same clinic as cases and sought medical treatment unrelated to an STI. Subjects completed a questionnaire, provided saliva for antibody testing, and were interviewed for determination of sexual activity.
Two groups of sexually active cases were enrolled. The first group had a diagnosis of an STI. In this group, both cases and matched controls were seropositive at a rate of 32% (7/22 for cases and 7/22 for controls). In the second group, cases self-reported an STI but objective evidence was lacking. In this group, cases were seropositive at a rate of 38% (six of 16) compared with matched controls among whom 6.3% were seropositive (one in 16). The overall rate of seropositivity in all 38 cases was 34% compared with a rate of 21% for all controls (P=0.3, odds ratio 2.0, 95% confidence interval 0.6–7.5).
Our results confirm lower rates of CMV infection among the current generation of African American adolescents compared with African American adults in Richmond, and suggest that this is not associated with sexual activity.
cytomegalovirus; sexually transmitted disease; adolescents; African Americans; seroprevalence
Over 90% of the world's population acquires a cytomegalovirus (CMV) infection. This infection, although asymptomatic or self-limiting, is a major burden to the immune system. For this reason, and because CMV immunization is possible, determining whether CMV can cause reduced longevity, particularly among those with coronary artery disease, is important and previous reports have been conflicting. Thus our objective was to assess the association between CMV infection as defined serologically and antibody levels against CMV and long-term survival (18 years). We completed a prospective observational cohort study of 915 consecutive patients (mean age 58 years) undergoing coronary angiography. CMV immunoglobulin levels were measured at baseline using either a whole cell CMV antigen or a purified protein antigen (gB). After adjustment for potentially confounding variables (age, race, gender, body mass index, the presence or absence of coronary artery disease, the number of diseased vessels, diabetes, renal disease, hypertension, dialysis, congestive heart failure, and the maximum percent reduction in luminal diameter), Cox's proportional hazards models showed no association between CMV seropositivity or levels of antibodies against CMV by either assay and longevity for both patients with or without coronary artery disease (CAD) nor for those under or over 70 years of age at baseline. Our observations suggest that universal immunization against CMV may not improve longevity.
cytomegalovirus; coronary artery disease; mortality.
Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease.
Cytomegalovirus; Antibodies; Spread inhibition; Epithelial cells
Acute and recurrent acute urticaria are often associated with multiple factors including infections and recent data suggest a role for herpesviruses.
To test the null hypothesis, that is, there is no association of herpesvirus infections with urticaria.
Thirty-seven patients between one month and 15 years of age were age matched to 37 controls who were healthy or had mild acute respiratory infections but without urticaria. Patients and controls were followed for 1 to 6 years. Diagnostic studies included DNA detection by real-time PCR for herpes simplex virus (HSV) types 1 and 2, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6). Tests for other infections included adenovirus, parvovirus B 19, respiratory syncytial virus, influenza A, Group A streptococci, rotavirus, and parasites.
Specific infections were diagnosed in 26 of 37 cases and among 9 of 37 control children (P=0.0002). Single or concomitant herpesvirus infections occurred in 24 cases and in 4 controls (65% vs 11 %, p=0.0003). Cases had 10 HHV-6 infections, 8 CMV infections, 5 EBV infections, and 4 HSV-1 infections.
Herpesvirus infections are associated with acute or recurrent acute urticaria.
Congenital cytomegalovirus (CMV) disease is the leading cause of permanent disability in neonates in the United States. Neutralizing antibodies in saliva may protect against maternal CMV infection by blocking viral entry into oral epithelial cells, but the antibody response to CMV in the saliva following natural infection is not well characterized. Saliva specimens from naturally infected individuals were tested for CMV-neutralizing activity using epithelial and fibroblast cells. Saliva from seronegative adults had no inherent anti-CMV activity. Neutralizing activity of saliva from naturally infected adults was not detectable using fibroblast cells, and saliva from young children, adolescents, and Towne vaccine recipients did not have activity using either cell type. However, when using epithelial cells, neutralizing activity was present in saliva from 50% of seropositive adults, correlated with serum-neutralizing activity, and was more prevalent in mothers of children in day care than in non-day care-associated adults. Three day care mothers with high salivary neutralizing activities (>1:20) had exceptionally high serum-neutralizing titers (3- to 8-fold higher than typical seropositives) and were immunoblot positive for serum antibodies to the epithelial entry mediator UL130. These results suggest that salivary neutralizing activities are attainable by induction of high serum IgG levels and could be utilized to evaluate candidate cytomegalovirus vaccines.
Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.
Cytomegalovirus infections are an important cause of disease for which no licensed vaccine exists. Recent studies have focused on the gH/gL/UL128-131 complex as antibodies to gH/gL/UL128-131 neutralize viral entry into epithelial cells. Prior studies have used cells from the retinal pigment epithelium, while to prevent transmission, vaccine-induced antibodies may need to block viral infection of epithelial cells of the oral or genital mucosa. We found that gH/gL/UL128-131 is necessary for efficient viral entry into epithelial cells derived from oral and genital mucosa, that short peptides from UL130 and UL131 elicit high titer neutralizing antibodies in rabbits, and that such antibodies neutralize viral entry into epithelial cells derived from these relevant tissues. These results suggest that single subunits or peptides may be sufficient to elicit potent epithelial entry neutralizing responses and that secretory antibodies to such neutralizing epitopes have the potential to provide sterilizing immunity by blocking initial mucosal infection.
Cytomegalovirus; Vaccine; Mucosal infection; Neutralizing antibody
To determine the frequency of pregnancy and exposure to CMV among mothers contemplating a possible additional pregnancy and with a child less than 2 years of age in group day care.
We performed a prospective observational study which included a demographic questionnaire and serologic and virologic monitoring of mothers and their children in day care.
Of 60 women, 62% were seronegative and 20% had a child shedding CMV. Of the 60 women, 23 women or 38% (95% CI = 0.27, 0.51) became pregnant on average 10 months after enrollment. During pregnancy, eight or 35% (95% CI = 0.19, 0.55) of these pregnant women had a child in day care who shed CMV
These results illustrate the potential magnitude of the public problem associated with exposure to a silent viral infection during pregnancy. Our data, when extrapolated to the U.S. population, estimate that every two years between 31,000 and 168,000 susceptible pregnant women will be exposed to CMV by an infected child.
Cytomegalovirus; pregnancy; birth defects; day care
Antibodies that neutralize cytomegalovirus (CMV) entry into fibroblasts are predominantly directed against epitopes within virion glycoproteins that are required for attachment and entry. However, the mechanism of CMV entry into epithelial and endothelial cells differs from fibroblast entry. Using assays that simultaneously measured neutralizing activities against CMV entry into fibroblasts and epithelial cells, we found that human immune sera and CMV-hyperimmuneglobulins have on average 48-fold higher neutralizing activities against epithelial cell entry compared to fibroblast entry, suggesting that natural CMV infections elicit neutralizing antibodies that are epithelial entry-specific. This activity could not be adsorbed with recombinant gB. The Towne vaccine and the gB/MF59 subunit vaccine induced epithelial entry-specific neutralizing activities that were on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following natural infection. These results suggest that CMV vaccine efficacy may be enhanced by induction of epithelial entry-specific neutralizing antibodies.
cytomegalovirus; vaccines; neutralizing antibody
Human cytomegalovirus (HCMV) viral chemokine, UL146, and TNF α- like receptor UL144 genes show a high degree of hypervariability in clinical isolates. These proteins are predicted to be immune modulators and may contribute to the pathogenesis of HCMV infections. We analyzed the UL146 and UL144 genetic variation of 51 HCMV isolates from congenitally infected children and 13 isolates from children in childcare. There was no statistically significant correlation between UL146 and UL144 genotypes and HCMV disease and/or sequelae. However, there were some groups that had a relatively large proportion of asymptomatic outcomes. These included UL146 group 8 (7/8 asymptomatic) and UL146 group 10 (3/3 asymptomatic). UL144 group B had 11/15 (73%) asymptomatic. UL146 and UL144 genes remained stable in serial isolates from children in daycare for intervals up to three years. These results indicate that most UL146 and UL144 genotypes do not predict clinical sequelae following congenital HCMV infections.
cytomegalovirus; cytokine; UL144; UL146; UL147; chemokine; congenital; sequelae; variability
Since African-Americans have twice the prevalence of cytomegalovirus (CMV) infections as age-matched Caucasians we sought to determine the ages and possible sources of infection of African-American children.
Subjects were 157 African-American healthy children and adolescents and their 113 household adults in Richmond VA. Families completed a questionnaire, provided saliva for antibody testing, and adolescents were interviewed regarding sexual activity.
Regardless of age CMV seropositivity was not associated with gender, breast feeding, health insurance, sexual activity, or household income, education, or size. In the final regression model, prior CMV infection in adults was over two-fold higher than in children (chi-square = 18.8, p < 0.0001). At one year of age the CMV seropositivity rate was 11% (95%CI = 4% – 24%) and increased 1.8% each year until age 13 years. Between ages 13 and 20 years the CMV seropositivity rate remained between 22% and 33%. For adults, the CMV seropositivity rate was 84% in 21 year olds (95%CI = 69%–.92%). There was no association between CMV infections of the children and their mothers but CMV infections among siblings were associated.
We observed that African-American children had CMV seroprevalence rates by age 20 years at less than one-half of that of their adult mothers and caregivers. Sibling-to-sibling transmission was a likely source of CMV infections for the children. The next generation of African-American women may be highly susceptible to a primary CMV infection during pregnancy and may benefit from a CMV vaccine.
Background. Congenital cytomegalovirus (CMV) infection is a leading cause of disabilities in children, yet the general public appears to have little awareness of CMV. Methods. Women were surveyed about newborn infections at 7
different geographic locations. Results. Of the 643 women surveyed, 142 (22%) had heard of congenital CMV. Awareness increased with increasing levels of education (P < .0001). Women who had worked as a healthcare professional had a higher
prevalence of awareness of CMV than had other women (56% versus 16%, P < .0001). Women who were aware of CMV were most likely to have heard about it from a healthcare
provider (54%), but most could not correctly identify modes of
CMV transmission or prevention. Among common causes of birth defects and
childhood illnesses, women's awareness of CMV ranked last. Conclusion. Despite its large public health burden, few women had heard of congenital CMV, and even fewer were aware of prevention strategies.
To confirm an association between cytomegalovirus (CMV) infection and the presence of antibodies to Smith (Sm), to ribonucleoprotein (RNP), and to a component of the U1 ribonucleoproteins (U1-70 kD), we measured antibodies to these protein antigens using an enzyme immunoassay and an immunoblot. The antibodies were measured in the sera of 80 healthy subjects, one-half of whom were naturally CMV seropositive and one-half were CMV seronegative, and in eight subjects immunized with a live attenuated strain of CMV. None of the vaccinees developed antibodies to Sm, to RNP, or to U1-70 kD at either 4 or 12 months after immunization. Additionally, there was no statistically significant association between levels of antibodies to Sm or to RNP and between sera obtained from vaccinees, natural CMV seropositive individuals, and CMV seronegative individuals. One CMV seropositive serum and one CMV seronegative serum tested positive for antibodies to U1-70 kD. These data indicate that neither wild-type infection nor the live-attenuated Towne vaccine frequently induce autoantibody production.
autoantibodies; cytomegalovirus; RNP antigen; Sm antigen; U1-70kD
Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.
The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.