Robust heavy-traffic approximations for service systems facing overdispersed demand.

Arrival processes to service systems often display fluctuations that are larger than anticipated under the Poisson assumption, a phenomenon that is referred to as overdispersion. Motivated by this, we analyze a class of discrete-time stochastic models for which we derive heavy-traffic approximations that are scalable in the system size. Subsequently, we show how this leads to novel capacity sizing rules that acknowledge the presence of overdispersion. This, in turn, leads to robust approximations for performance characteristics of systems that are of moderate size and/or may not operate in heavy traffic.

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector.

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4(+) and CD8(+) T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4(+) and CD8(+) T cell subsets.

Lymphotoxins and cytomegalovirus cooperatively induce interferon-beta, establishing host-virus détente.

Tumor necrosis factor (TNF)-related cytokines regulate cell death and survival and provide strong selective pressures for viruses, such as cytomegalovirus (CMV), to evolve counterstrategies in order to persist in immune-competent hosts. Signaling by the lymphotoxin (LT)-beta receptor or TNF receptor-1, but not Fas or TRAIL receptors, inhibits the cytopathicity and replication of human CMV by a nonapoptotic, reversible process that requires nuclear factor kappa B (NF-kappa B)-dependent induction of interferon-beta (IFN-beta). Efficient induction of IFN-beta requires virus infection and LT signaling, demonstrating the need for both host and viral factors in the curtailment of viral replication without cellular elimination. LT alpha-deficient mice and LT beta R-Fc transgenic mice were profoundly susceptible to murine CMV infection. Together, these results reveal an essential and conserved role for LTs in establishing host defense to CMV.

Modification of maternal and congenital cytomegalovirus infection by anti-glycoprotein b antibody transfer in guinea pigs.

Prepregnancy human and guinea pig cytomegalovirus immunity reduces rates of congenital infection in subsequent pregnancies. Inbred JY-9 strain guinea pigs were used to study the role of hyperimmune anti-glycoprotein B (gB) serum in modification of congenital infection in early pregnancy. Significantly shorter duration of primary maternal viremia and fewer pregnancy losses occurred in passively immunized dams, compared with nonimmune dams. Placentas from recipients of negative control serum were smaller and had marked mononuclear cell infiltrates and focal necrosis and more viral foci than did those from recipients of anti-gB hyperimmune serum. Significantly higher intrauterine growth retardation occurred in pups of negative control serum recipients than in pups of passively immunized dams. Significantly higher proportions of pups and placentas from recipients of negative control serum were positive on viral culture than from passively immunized dams. Thus, anti-gB passive immunization decreased fetal infection and intrauterine growth retardation, shortened maternal viremia, and reduced pregnancy losses and placental inflammation and infection.

Intrauterine transmission of cytomegalovirus to infants of women with preconceptional immunity.

BACKGROUND: Preconceptional immunity against cytomegalovirus (CMV) provides only partial protection against intrauterine transmission of the virus. Whether congenital CMV infection in the offspring of women who are seropositive for CMV can occur after maternal reinfection with a different strain of CMV is unknown. METHODS: Serum specimens from 46 women with preconceptional immunity against CMV that were obtained during the previous pregnancy and the current pregnancy were analyzed for antibodies against the strain-specific epitopes of CMV glycoprotein H. Virus-neutralizing activity in maternal serum samples was measured against the AD169 laboratory strain of CMV and the CMV isolates available from seven infected infants. In addition, the nucleotide sequences of the glycoprotein H gene from the seven CMV isolates were determined. RESULTS: Eleven of the 16 mothers with infected infants (69 percent) had antibodies against the glycoprotein H epitopes present on two laboratory strains of CMV, AD169 and Towne. Ten of the 16 mothers with infected children (62 percent) acquired new antibody specificities against glycoprotein H, as compared with only 4 of the 30 mothers of uninfected infants (13 percent, P<0.001). The samples obtained at the time of the current delivery from four of the seven mothers contained at least twice as many neutralizing antibodies against the CMV isolated from their infants as were present in the samples obtained at the previous delivery. The specificity of the newly acquired maternal antibodies reflected the amino acid sequence of the glycoprotein H epitope of CMV from these four infants. CONCLUSIONS: In women who are seropositive for CMV, reinfection with a different strain of CMV can lead to intrauterine transmission and symptomatic congenital infection.

B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses.

Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus.

This investigation consisted of a longitudinal study of the effects of congenital cytomegalovirus (CMV) infection on hearing sensitivity in 860 children with documented asymptomatic or symptomatic congenital CMV infection. Of the 651 children with asymptomatic CMV infection, 48 (7.4%) developed sensorineural hearing loss (SNHL), compared to 85 (40.7%) of the children with symptomatic CMV infection. Children in both groups experienced latent effects consisting of delayed onset of loss, threshold fluctuations, and/or progressive loss of hearing. It can be concluded that congenital CMV infection is a leading cause of SNHL in children. The late onset and progression of loss necessitates continued monitoring of hearing sensitivity in this population.

Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment.

Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for alpha- and beta-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [(3)H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.

Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly.

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

Symptomatic congenital cytomegalovirus infection in infants born to mothers with preexisting immunity to cytomegalovirus.

OBJECTIVES: To determine the frequency of symptomatic congenital cytomegalovirus (CMV) infection in the offspring of women with a recurrent maternal CMV infection and to characterize the demographic and newborn findings. METHODS: Study subjects consisted of infants with symptomatic congenital CMV infection identified by a newborn virologic screening program at the University of Alabama Hospital between 1991 and 1997 and were enrolled in a long-term follow-up study. Maternal infections were categorized by an analysis of archival serum specimens collected before pregnancy and at the time of delivery. Demographic data and clinical findings at birth were collected from maternal and newborn hospital records and from parents at the time of initial evaluation. RESULTS: Of the 47 infants with symptomatic congenital CMV infection identified during the study period, 8 were born to mothers with a confirmed nonprimary or recurrent CMV infection. The type of maternal infection could be ascertained in only approximately 43% (20/47) of the children with symptomatic congenital CMV infection born at the University of Alabama Hospital during the study period. There were no significant differences in demographic characteristics of the recurrent infection group and the infants who were born to mothers with either primary CMV infection during pregnancy or unclassified maternal infection. Similarly, the range of severity of clinical abnormalities during the newborn period did not differ in the two groups of children. Furthermore, there were no significant differences in the incidence of sequelae at long-term follow-up in the two groups of children. CONCLUSIONS: Symptomatic congenital CMV infection can occur after a nonprimary or recurrent maternal infection. However, the exact incidence of symptomatic congenital CMV infection among children born to women with preexisting immunity remains to be defined.

Human cytomegalovirus infection of caco-2 cells occurs at the basolateral membrane and is differentiation state dependent.

Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts.

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta).

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.

Ganciclovir treatment of symptomatic congenital cytomegalovirus infection: results of a phase II study. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group.

Congenital cytomegalovirus (CMV) infection occurs in approximately 1% of newborns in the United States. A phase II evaluation was done of ganciclovir for the treatment of symptomatic congenital CMV infection. Daily doses of 8 or 12 mg/kg were administered in divided doses at 12-h intervals for 6 weeks. Clinical and laboratory evaluations sought evidence of toxicity, quantitative virologic responses in urine, plasma drug concentrations, and clinical outcome. A total of 14 and 28 babies received 8 and 12 mg/kg/day, respectively. Five additional babies received ganciclovir on a compassionate plea basis. Significant laboratory abnormalities included thrombocytopenia (< or = 50,000/mm3) in 37 babies and absolute neutropenia (< or = 500 mm3) in 29 babies. Quantitative excretion of CMV in the urine decreased; however, after cessation of therapy, viruria returned to near pretreatment levels. Hearing improvement or stabilization occurred in 5 (16%) of 30 babies at 6 months or later, indicating efficacy.

Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex.

Previous studies have described three disulfide-bonded glycoprotein complexes within the envelope of human cytomegalovirus (HCMV). These have been designated gCI, gCII, and gCIII. Although gCI has been identified as homodimeric glycoprotein B (gB, gpUL55), the compositions of gCII and gCIII remain incompletely defined. Earlier studies suggested that gCIII was composed of glycoprotein H (gH, gpUL75) complexed with a second glycoprotein, the gL homolog of HCMV. We characterized the gCIII complex of HCMV using recombinant vaccinia virus-expressed gH and gL. Our results indicated that authentic gCIII was not reconstituted by coexpression of gH and gL. The presence of a third, structurally and antigenically unique glycoprotein with an estimated molecular mass of 125,000 Da in virion-derived gCIII complexes suggested that at least three proteins were necessary for formation of this envelope glycoprotein complex. This third glycoprotein, gp125, contained both simple and complex N-linked carbohydrates and had an estimated deglycosylated mass of 64,000 Da. Furthermore, we demonstrated that mature gH existed as both a covalently complexed and noncovalently associated component of the gCIII complex within the envelope of infectious extracellular virions. These findings provide further evidence for the structural complexity of the envelope of HCMV and emphasize the uncertainties associated with the previous assignment of specific functions to envelope proteins of HCMV.

Progressive and fluctuating sensorineural hearing loss in children with asymptomatic congenital cytomegalovirus infection.

OBJECTIVE: To determine the prevalence and temporal changes of sensorineural hearing loss (SNHL) among children with clinically inapparent (asymptomatic) congenital cytomegalovirus (CMV) infection identified from a cohort of newborn infants screened for congenital CMV infection. METHODS: The study population consisted of 307 children with documented asymptomatic congenital CMV infection, 76 uninfected siblings of children with asymptomatic congenital CMV infection, and 201 children whose neonatal screen for congenital CMV infection showed negative results. Audiologic evaluations were completed for all children to determine their hearing status. RESULTS: SNHL occurred only in children with congenital CMV infection. Of the children with asymptomatic congenital CMV infection, 22 (7.2%; 95% confidence interval, 4.5% to 10.6%) had SNHL. Among the children with hearing loss, further deterioration of hearing occurred in 50.0%, with the median age at first progression at 18 months (range, 2 to 70 months). Delayed-onset SNHL was observed in 18.2% of the children, with the median age of detection at 27 months (range, 25 to 62 months). Fluctuating SNHL was documented in 22.7% of the children with hearing loss. CONCLUSIONS: Asymptomatic congenital CMV infection is likely a leading cause of SNHL in young children. The continued deterioration of hearing and delayed onset of SNHL in these children emphasizes the need for continued monitoring of their hearing status.

Neuroradiographic findings in the newborn period and long-term outcome in children with symptomatic congenital cytomegalovirus infection.

OBJECTIVE: To determine whether newborn cranial computed tomographic (CT) scan abnormalities predict an adverse neurodevelopmental outcome in children with symptomatic congenital cytomegalovirus (CMV) infection and to examine the association between clinical findings at birth and imaging abnormalities. METHODS: The data from 56 children with symptomatic congenital CMV infection who underwent cranial CT scans as newborns and were enrolled in a long-term follow-up study were analyzed. The incidence of sequelae was compared between the groups of children with normal and abnormal imaging studies. The relationship between CT scan results and other newborn findings was also examined. RESULTS: Abnormal CT scans were noted in 70% of subjects; intracerebral calcification was the most frequent finding. Most of the children with an abnormal newborn CT scan (90%) developed at least one sequela, compared with 29% of those with a normal study. Only 1 child with a normal CT scan had an IQ < 70, in contrast to 59% of those with imaging abnormalities. In addition, almost half of the children with CT abnormalities had an IQ < 50 compared with none of those with a normal CT scan. Newborn CT abnormalities were also associated with an abnormal hearing screen at birth and hearing loss on follow-up. None of the neonatal neurologic findings were predictive of an abnormal CT scan. CONCLUSION: In neonates with symptomatic congenital CMV infection, a cranial CT scan is a good predictor of an adverse neurodevelopmental outcome. In addition, newborn clinical and laboratory findings did not predict neuroradiographic abnormalities in neonates with symptomatic congenital CMV infection.

Detection of antibodies to respiratory syncytial virus attachment and nucleocapsid proteins with recombinant baculovirus-expressed antigens.

The ability to measure antibodies against individual respiratory syncytial virus (RSV) proteins is important in the analysis of immune responses to RSV. We expressed the nucleocapsid (N) protein and the group A and B RSV attachment (G) proteins from recombinant baculoviruses. The three recombinant RSV proteins were used individually in an enzyme-linked immunosorbent assay (ELISA; bac-ELISA for results from assays of all three proteins). The bac-ELISA results were compared to the results obtained by a whole-virus ELISA (RS-ELISA for results from assays of both group A and B viruses). Antibody samples from 113 children were tested. The determination of seronegative or seropositive status by the bac-ELISA was compared to the same determination by the RS-ELISA; the sensitivity of bac-ELISA was 87% (95% confidence interval [CI], 78 to 93%), the specificity was 82% (CI, 59 to 94%), and the positive and negative predictive values were 95% (CI, 86 to 98%) and 60% (CI, 41 to 77%), respectively. The group specificity of the G-protein ELISA was confirmed by testing antibodies from experimentally immunized animals. Thus, the bac-ELISA was shown to be comparable to the whole-virus ELISA in detecting antibody responses to RSV, while it offered the advantage of measuring specific antibody responses to individual RSV proteins.

Expression, purification, and characterization of a soluble form of human cytomegalovirus glycoprotein B.

The human cytomegalovirus glycoprotein B gene (gB; gpUL55) was truncated at amino acid 692 and recombined into Autographa californica nuclear polyhedrosis virus (baculovirus). Infection of insect cells with the recombinant baculovirus resulted in high-level expression and secretion of the truncated gB protein (gB-S) into the culture medium. Purification of gB-S by monoclonal antibody affinity chromatography yielded a protein of ca. 200 kDa. Characterization of the 200-kDa purification product indicated that the recombinant gB protein retained many structural and functional features of the viral gB. Comparison of electrophoretic migration patterns in reduced versus nonreduced protein samples and immune blotting analysis with antibodies specific for the amino or carboxy-terminus of gB demonstrated that the recombinant protein was composed of disulfide linked 69 kDa amino terminal and 35-kDa carboxy-terminal fragments. In addition, recognition of the 200-kDa gB-S by a conformational-dependent, oligomer-specific monoclonal antibody suggested that gB-S was properly folded and dimeric. Like the viral gB, gB-S had heparin binding ability. One heparin binding site was found to reside within the 35-kDa carboxy-terminal fragment (aa 492-692). Heparin binding was abolished when gB-S was denatured. These data suggest that gB contains a novel heparin binding motif that is at least partially conformational dependent.