Antonella Sarasini

Human Cytomegalovirus UL131-128 Genes Are Indispensable for Virus Growth in Endothelial Cells and Virus Transfer to Leukocytes

ABSTRACT Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

ABSTRACT Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.

Human Cytomegalovirus in Blood of Immunocompetent Persons during Primary Infection: Prognostic Implications for Pregnancy

Human cytomegalovirus (HCMV) was investigated in peripheral blood leukocytes (PBL) of 52 immunocompetent patients (40 pregnant women) with primary HCMV infection by quantitative determination of pp65 antigenemia, viremia, and leukoDNAemia. pp65 antigenemia was detected in 12 (57.1%) of 21, 4 (25%) of 16, and 0 of 10 patients examined 1, 2, and 3 months after onset, respectively. Viremia was detected in 5 (26.3%) of 19 patients during the first month only. LeukoDNAemia was detected in 20 of 20, 17 (89.5%) of 19, and 9 (47.3%) of 19 patients tested 1, 2, and 3 months after onset, respectively. Four (26.6%) of 15 patients were still DNAemia-positive at 4-6 months, whereas none were positive at >6 months. HCMV was not detected in PBL of 20 HCMV-immune donors or of 9 seropositive subjects with recurrent infection. Virus levels were low by all assays and did not correlate with clinical course of infection, intrauterine transmission, or severity of outcome. Invasive procedures in the presence of maternal leukoDNAemia did not seem to interfere with vertical transmission of HCMV infection.

Human cytomegalovirus serum neutralizing antibodies block virus infection of endothelial/epithelial cells, but not fibroblasts, early during primary infection

A panel of human sera exhibited a >or=128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10-15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity.

Diagnostic and prognostic value of human cytomegalovirus load and IgM antibody in blood of congenitally infected newborns

BACKGROUND Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. OBJECTIVES To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. STUDY DESIGN HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. RESULTS Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. CONCLUSIONS (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.

Fetal Human Cytomegalovirus Transmission Correlates with Delayed Maternal Antibodies to gH/gL/pUL128-130-131 Complex during Primary Infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.

Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13−UL128L+) or both RL13 and UL128L (RL13−UL128L−). RL13−UL128L− viruses produced greater yields of infectious progeny than RL13−UL128L+ viruses, and RL13−UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells

Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF. It was demonstrated that one of the four field isolates (VR6110) and its transfer-deficient variant were genetically related, but showed different curves of virus yield in HELF. In addition, neither the immediate-early (IE) mRNA nor the IE protein p72 were found to be transferred to PMNL before 72 h post-infection (late in infection) or in the presence of viral DNA replication inhibitors. These findings link EC and PMNL tropism and suggest that PMNL tropism is a late HCMV function.

Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients.

In the winter–spring seasons 2003–2004 and 2004–2005, 47 (5.7%) patients with acute respiratory infection associated with human coronavirus (hCoV) 229E‐, NL63‐, and OC43‐like strains were identified among 823 (597 immunocompetent and 226 immunocompromised) patients admitted to hospital with acute respiratory syndromes. Viral infections were diagnosed by either immunological (monoclonal antibodies) or molecular (RT‐PCR) methods. Each of two sets of primer pairs developed for detection of all CoVs (panCoV) failed to detect 15 of the 53 (28.3%) hCoV strains identified. On the other hand, all hCoV strains could be detected by using type‐specific primers targeting genes 1ab and N. The HuH‐7 cell line was found to be susceptible to isolation and identification of OC43‐ and 229E‐like strains. Overall, hCoV infection was caused by OC43‐like, 229E‐like, and NL63‐like strains in 25 (53.2%), 10 (21.3%), and 9 (19.1%) patients, respectively. In addition, three patients (6.4%) were infected by untypeable hCoV strains. NL63‐like strains were not found to circulate in 2003–2004, and 229E‐like strains did not circulate in 2004–2005, while OC43‐like strains were detected in both seasons. The monthly distribution reached a peak during January through March. Lower predominated over upper respiratory tract infections in each age group. In addition, hCoV infections interested only immunocompetent infants and young children during the first year of life, while all adults were immunocompromised patients. Coinfections of hCoVs and other respiratory viruses (mostly interesting the first year of life) were observed in 14 of the 47 (29.8%) patients and were associated with severe respiratory syndromes more frequently than hCoV single infections (P = 0.002). In conclusion, the use of multiple primer sets targeting different genes is recommended for diagnosis of all types of hCoV infection. In addition, the detection of still untypeable hCoV strains suggests that the number of hCoVs involved in human pathology might further increase. Finally, hCoVs should be screened routinely for in both infants and immunocompromised patients with acute respiratory infection. J. Med. Virol. 78:938–949, 2006.

Improved prenatal diagnosis of congenital human cytomegalovirus infection by a modified nested polymerase chain reaction

Two major variables may cause false‐negative results in prenatal diagnosis of congenital human cytomegalovirus (HCMV) infection: sensitivity of the technique(s) used; and time elapsed between maternal infection and antenatal testing. Previous results indicated that rapid HCMV isolation from amniotic fluid samples and viral DNA detection in amniotic fluid by nested polymerase chain reaction (nPCR) had comparable levels of sensitivity (69.2% and 76.9%, respectively). The nPCR protocol was reviewed following two additional false‐negative antenatal diagnosis in a twin pregnancy during which two procedures were performed at 18 and 23 weeks of gestation, respectively. In the new assay, multiple (instead of single) and 100 (instead of 20) μl amniotic fluid aliquots were individually amplified and tested by nPCR. By using this approach, low DNA levels (1–10 genome equivalents) were detected in 1–5/8 replicates of amniotic fluid samples taken from both twins during both procedures. In addition, viral DNA was detected in 5/6 replicates from two amniotic fluid samples still available from two previous false‐negative cases. However, nPCR on multiple amniotic fluid replicates did not anticipate positive prenatal results in a retrospective case, which required two procedures for correct diagnosis and, when prospectively employed, did not avoid one additional false‐negative prenatal diagnosis 8 weeks after maternal infection. Thus, delayed intrauterine transmission of the infection may be a potential cause of false‐negative results. However, the combination of a very sensitive technique with appropriate timing of prenatal testing can substantially increase the reliability of prenatal diagnosis results. J. Med. Virol. 56:99–103, 1998.