Emilia Genini

Human Cytomegalovirus Replicates Abortively in Polymorphonuclear Leukocytes after Transfer from Infected Endothelial Cells via Transient Microfusion Events

ABSTRACT Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

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.

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.

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients

Abstract Background Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. Objective To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. Study design Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. Results Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January–May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. Conclusion HKU1 hCoV strains circulated in northern Italy during the winter–spring season 2005–2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter–spring season

Abstract Background Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. Objectives To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. Study design Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter–spring season 2003–2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. Results RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1–A2 and B1–B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. Conclusion (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2–3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.

Serum antibody response to the gH/gL/pUL128-131 five-protein complex of human cytomegalovirus (HCMV) in primary and reactivated HCMV infections

BACKGROUND Recently, a new human cytomegalovirus (HCMV) glycoprotein complex has been identified and potentially proposed as a vaccine. OBJECTIVE The aim of this study was to determine whether the HCMV gH/gL/pUL128-pUL130-pUL131 (gH/gL/pUL128-131) 5-protein (pentameric) complex (which has been recently found to be indispensable for the infection of endothelial and epithelial cells) is able to elicit a consistent antibody response in both primary and reactivated HCMV infections. STUDY DESIGN The antibody response was determined by both indirect immunofluorescence (IFA) and ELISA, using fixed (IFA) or lysed (ELISA) epithelial (ARPE-19) cells infected with one or more adenoviral vectors, each carrying one HCMV gene and, in parallel, with a control adenovirus vector. RESULTS The specificity of results was determined by the reactivity of human neutralizing mAbs recognizing two, three, or four proteins of the complex. In 14 cases of primary infection, an IgG antibody seroconversion to the UL128-131 gene products was consistently detected within 2-4 weeks after onset of infection, while antibodies persisted for at least 12 months. The IgG antibody response to UL128-131 gene products was generally superior to the response to gH and appeared to follow the neutralizing antibody response (as determined in epithelial cells). In reactivated infections, the antibody response showed a trend reminiscent of a booster response. IgG antibodies were detected in HCMV-seropositive healthy adult controls, but not in HCMV-seronegative individuals. CONCLUSIONS The IgG antibody response to the pentameric complex could be a major target for the evaluation of the antibody response to a pentamer-based vaccine.

Comparative evaluation of eight commercial human cytomegalovirus IgG avidity assays

BACKGROUND The interpretation of a positive IgM antibody result to human cytomegalovirus (HCMV) in a pregnant woman is of major importance for the correct management of the pregnancy. Determination of HCMV-specific IgG avidity is considered an useful approach for distinguishing IgM antibody due to primary HCMV infection from IgM antibody elicited during non-primary infection. OBJECTIVE Comparative evaluation of eight commercial HCMV IgG avidity assays currently available in Europe. STUDY DESIGN A panel of 198 sequential samples collected from 65 pregnant women at 0-90, 91-180, and >180 days after the onset of primary HCMV infection was retrospectively tested by Abbott, BioMérieux, Bio-Rad, DiaSorin, Diesse, Euroimmun, Radim, and Technogenetics HCMV IgG avidity assays according to the manufacturers instructions. RESULTS None of the 198 samples tested yielded identical scores by the kits under evaluation. The Euroimmun and Radim assays showed the best correlation with expected results in terms of low (0-90 days), intermediate (90-180 days) and high (>180 days) avidity results, respectively. The best accuracy in diagnosing a recent (<90 days after the onset) or non-recent (>180 days after the onset) primary HCMV infection was shown by Radim followed by Euroimmun and Diesse. The best correlation with a well established in-house developed HCMV IgG avidity assay was shown by Radim. CONCLUSIONS HCMV IgG avidity kits need to be improved and standardized. In the meantime, highly specific IgM assays are preferable for screening purposes in pregnant women.

Lymphoproliferative Response in Primary Human Cytomegalovirus (HCMV) Infection Is Delayed in HCMV Transmitter Mothers

BACKGROUND The T cell-mediated immune response to human cytomegalovirus (HCMV) after primary infection, as well as the determinants of intrauterine transmission, are poorly understood. METHODS Sequential peripheral blood leukocyte samples from 74 pregnant women and 29 nonpregnant individuals with primary infection were examined for HCMV-specific CD4+ T cells by cytokine flow cytometry (CFC) and lymphoproliferative response (LPR) analysis. Immunological results for 19 transmitter and 21 nontransmitter mothers were compared. RESULTS Comparison of CFC and LPR analysis results showed that (1) there was no difference between pregnant and nonpregnant individuals; (2) HCMV-specific CD4+ T cells were detected by CFC, in the absence of an LPR to HCMV, in the great majority or the totality (according to different intervals) of samples collected from both pregnant and nonpregnant individuals during follow-up; and (3) LPR to HCMV was significantly (P<.001) lowered or delayed in transmitter mothers, compared with that in nontransmitter mothers. CONCLUSIONS Pregnancy does not influence the HCMV-specific immune response. A dissociation between CFC response and LPR is commonly observed in patients with primary infections, and ad hoc studies aimed at understanding the mechanism(s) of the reduced LPR in transmitter mothers are warranted.

Declining levels of rescued lymphoproliferative response to human cytomegalovirus (HCMV) in AIDS patients with or without HCMV disease following long-term HAART.

Objective: To investigate the lymphoproliferative response (LPR) to human cytomegalovirus (HCMV) in two groups of AIDS patients undergoing long‐term highly active antiretroviral therapy (HAART): group 1 (n = 22) with nadir CD4+ T cell count <50/&mgr;l and no HCMV disease; group 2 (n = 16) with <50/&mgr;l CD4+ T‐cell count and HCMV disease. All patients had previously undergone antiretroviral monotherapy or dual therapy before initiating HAART. Study Design and Methods: The two groups of patients were tested prospectively for CD4+ T cell count, HIV RNA load, HCMV viremia, and LPR to HCMV at baseline, and then after 3 and 4 years of HAART. A control group of 13 recently diagnosed treatment‐naive AIDS patients with CD4+ T‐cell counts <100/&mgr;l was also investigated. Results: No LPR to HCMV was found in any of the treatment‐naive patients nor in any patient of the two groups examined at baseline, when HCMV viremia was 13.6% in the patient group without disease and 87.5% in the group with disease (p < .0001). After 3 years of HAART, the frequency of patients who recovered an LPR to HCMV was not significantly different (81.8% in the group without HCMV disease, and 68.7% in the group with HCMV disease), whereas, compared with baseline, the HIV load decreased and the CD4+ T‐cell count increased significantly and to a comparable extent in the two groups of patients. In addition, the frequency of patients with HCMV viremia, although reduced, became comparable in both groups. After 4 years of HAART, the frequency of responders to HCMV without and with HCMV disease dropped to comparable levels (50.0 vs. 56.3%, respectively) in association with high median CD4+ T‐cell counts and low median HIV RNA plasma levels. In parallel, the frequency of patients with HCMV viremia did not change significantly. In addition, after between 3 and 4 years of HAART, although the frequency of stable responders and nonresponders remained unchanged (50%) in both groups, most of the remaining patients showed declining levels of responsiveness to HCMV. Although some patients from both groups were found to have CD4+ T‐cell counts >150/&mgr;l in the absence of LPR to HCMV, thus suggesting dissociation of specific and nonspecific immune reconstitution, a significant correlation was found between CD4+ T‐cell count and LPR to HCMV (r = 0.44; p < .001). From a clinical standpoint, anti‐HCMV therapy could be safely discontinued in 8 patients with HCMV retinitis showing CD4+ T‐cell counts >150/&mgr;l, recovery of HCMV LPR, and no HCMV viremia. Conclusions: Declining levels of the previously recovered LPR to HCMV are often observed after long‐term HAART. However, because the role of LPR in the evolution of HCMV infection and disease during HAART remains to be defined, the clinical impact of the declining LPR to HCMV must still be clarified in long‐term prospective studies.

Monitoring of human cytomegalovirus (HCMV)-specific CD4+ T cell frequency by cytokine flow cytometry as a possible indicator for discontinuation of HCMV secondary prophylaxis in HAART-treated AIDS patients

OBJECTIVE Absolute CD4+ T cell count and human cytomegalovirus (HCMV)-specific CD4+ T cell frequency (as determined by cytokine flow cytometry, CFC) were compared for their ability to predict HCMV disease and safe discontinuation of HCMV secondary prophylaxis. STUDY DESIGN Three groups of AIDS patients with previous nadir CD4+ T cell count <100/microl were studied. Group A included 48 HAART-treated patients with no HCMV disease. Group B included 11 HAART-treated patients with previous HCMV disease who discontinued HCMV prophylaxis. Group C included 23 HAART-treated (n = 16) or -naive (n = 7) patients with previous HCMV disease either continuing or starting HCMV prophylaxis. Patients underwent follow-up for detection of HCMV viremia or disease (groups A and B) and for discontinuation of HCMV secondary prophylaxis on the basis of either HCMV-specific or absolute CD4+ T cell count (group C). RESULTS During follow-up, while some patients showed a stable HCMV-specific CD4+ T cell response, others had a fluctuating response (unstable responders) or showed no response at all. In detail, 13/48 group A patients were either HCMV non-responders or unstable responders and 2 of them developed HCMV viremia; 3/11 group B patients were unstable responders, none developing either HCMV viremia or disease; finally, 9 group C patients discontinued HCMV prophylaxis based on absolute CD4+ T cell count > 150 cells/microl, but in 2 of them lacking HCMV-specific response HCMV retinitis relapsed. None of the seven group C patients discontinuing HCMV prophylaxis on the basis of CFC showed HCMV disease relapse. CONCLUSIONS CFC may support absolute CD4+ T cell count for both guiding HCMV prophylaxis discontinuation and better monitoring HCMV infection in AIDS patients with no previous HCMV disease or having discontinued HCMV prophylaxis.