To determine the effect of the human cytomegalovirus (CMV) major immediate-early (MIE) enhancer or promoter on the efficiency of viral replication in permissive human cells, we constructed recombinant viruses with their human MIE promoter, enhancer, and promoter plus enhancer replaced with the murine CMV components. After a low multiplicity of infection (MOI) (0.01 PFU/cell), recombinant human CMV with the murine CMV promoter replicated like the wild type but recombinant virus with the murine enhancer replicated less efficiently. Immediate-early (IE) viral protein pIE72 (UL123), early viral protein (UL44), and viral DNA synthesis were significantly decreased. The effect of the human CMV enhancer substitution with the murine CMV enhancer was also demonstrated in different cell types by using recombinant virus with the UL127 promoter, driving the expression of green fluorescent protein (GFP). After an MOI of 1, GFP expression was high with the human CMV enhancer and significantly lower with the murine CMV enhancer. Even though at a high MOI (10 PFU/cell), the murine CMV enhancer was as efficient as the human CMV enhancer for the transcription of IE genes in human foreskin fibroblast cells, at lower MOIs, the murine CMV enhancer was less efficient. Proximal and distal chimeras of the human and murine enhancers also replicated less efficiently at a low MOI and expressed lower levels of GFP from the UL127 promoter. These experiments demonstrate that the entire human CMV enhancer has evolved for the efficient expression of the viral IE and early genes in human cells. Possible functions of the human CMV enhancer and promoter at a low MOI are discussed.
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The detection of human cytomegalovirus (CMV) DNA in the plasma of immunocompromised hosts has been reported (1, 10, 15, 17, 21). This observation is of interest because it suggests that (i) there may be an alternate mechanism for dissemination of infectious virus within the host in addition to cell-to-cell transmission and (ii) plasma may be a convenient source of patient material for detecting a disseminated CMV infection. Although the presence of CMV DNA in peripheral leukocytes (WBC) is a sensitive marker for virus dissemination (9, 11, 13, 16, 22), detection of CMV DNA in plasma may be useful when counts of circulating WBC are low, such as in immunocompromised hosts. A positive correlation between detection of infectious CMV in WBC and CMV DNA in plasma has been reported (1, 21). Since the highest levels of CMV DNA are usually seen in patients with symptomatic disease, most often detected as retinitis (19), it is possible that the presence of CMV DNA in plasma is reflecting the quantity in WBC. The purpose of this study was to compare quantities of CMV DNA in WBC and in plasma and to see how they relate to symptomatic CMV disease in human immunodefi- ciency virus (HIV)-infected patients.
Infection with Cytomegalovirus (CMV) continues to be a major threat in organ transplant recipients and conge- nitally-infected children [1,2]. Although existing sys- temic therapies are effective in suppressing virus replication, serious side effects and the emergence of resistant viral strains pose significant treatment dilem- mas . The need to identify and develop new anti- CMV compounds coincides with the advancement of rapid, sensitive, and high-throughput methods for screening of lead compounds. While the plaque reduc- tion assay remains the gold-standard for screening of anti-viral compounds, new techniques based on recom- binant DNA technology and highly sensitive molecular assays have recently been suggested as alternatives [4-6]. Real-time PCR, the standard of care in the management of CMV disease in high- risk patient populations, may also provide a sensitive tool for drug screening [7-12]
Between August 2006 and September 2008, a total of 558 consecutive blood samples were received by our Labora- tory for the simultaneous determination of pp65 antigen- emia, nested-PCR and real-time PCR in leukocytes. The samples came from 30 adult allogeneic HSCT recipients (17 males and 13 females; median age: 40.5, range: 16-56) with human leukocyte antigen (HLA) identical sibling donors at risk for CMV infection (CMV seropositive donor and/or recipient) at the Bone Marrow Transplant Unit, Hemocenter, University of Campinas - Unicamp, SP, Brazil (table 1). The median number of samples per patient was 20 (range: 5-23). These patients were pro- spectively monitored for active CMV infection at weekly intervals from D+0 to D+150 post-transplant. The proto- col was designed in accordance with the requirements for research involving human subjects in Brazil, was approved by the Institutional Ethics Committee and an informed written consent was received from each patient.
The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclu- sion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant ( ⌬ UL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with ⌬ UL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented ⌬ UL32-BAC replication but murine CMV M32 failed to complement, consistent with evolu- tionary divergence of rodent and primate cytomegaloviruses. Infection with ⌬ UL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.
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Human cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality among transplant recipients. Molecular assays have been developed for the detection and quantification of CMV nucleic acid. In evaluating the clinical utility of these assays, correlations with clinical outcome are essential. The Amplicor CMV Monitor and NucliSens CMV pp67 tests were compared to the CMV antigenemia assay for 45 transplant recipients and 1 patient with Wegener’s granulomatosis. Twenty-three patients remained anti- genemia negative throughout the monitoring period, none of whom developed CMV disease. In this patient group, both the Amplicor and NucliSens assays showed very high specificity; only 1 of the 324 specimens assayed by NucliSens and none of the 303 specimens assayed by Amplicor were positive. Twenty-three patients were antigenemia positive during the monitoring period, 12 of whom developed 13 episodes of symptomatic CMV disease. In this patient group, the NucliSens assay was positive at or before the development of symptoms in 12 of the 13 episodes of CMV disease. All eight patients with symptomatic CMV disease who were tested by the Amplicor assay were positive at or before the development of disease. For the 11 asymptomatic patients, the NucliSens assay was positive less frequently than the antigenemia or Amplicor assays. The NucliSens assay was more likely to be positive at higher antigenemia or viral load levels. Both the NucliSens and Amplicor assays appear to have clinical utility in monitoring patients for CMV disease.
sites and frozen at 2 20 8 C to 2 80 8 C until shipped on dry ice to the central repository. Specimens were then stored at 2 80 8 C until pro- cessed. CMV DNA was measured from 10 m l of plasma using a CMV DNA PCR procedure (14, 15). Specimens were first evaluated by qualitative assay as to the presence of CMV DNA in plasma using methods previously described except CMV DNA was extracted using a silica extraction method (17). Briefly, 100 m l of each plasma was treated with 900 m l lysis buffer and 40 m l silica suspension for 10 min at room temperature in a 2 ml microtube. The lysis buffer contained 4.23M guanidinium thiocyanate, 20 mM disodium-EDTA, and 1.0% Triton X-100 in 0.1M Tris-HCl (pH 6.4). The nucleic acid-silica com- plex was spun down for 15 s at 12,000 g in a microcentrifuge. The pel- let was washed twice with wash buffer containing 4.23 M guanidinium thiocyanate in 0.1M Tris-HCl (pH 6.4), followed by two washes with 70% ethanol and a wash with acetone. The pellet was dried at 56 8 C for 10 min in a heat block, resuspended with 200 m l sterile Millipore water, and incubated for 10 min at 56 8 C. The nucleic acid supernatant was recovered after centrifugation for 2 min at 12,000 g in a microcen- trifuge. This supernatant was then clarified by one or more centrifu- gations to be completely void of silica particles and stored at 2 20 8 C until used in PCR (14, 15). The sensitivity of the qualitative PCR as- say was 500 copies per ml. Those identified as DNA positive were quantitated for CMV DNA using a competitive plasma DNA PCR (QC-PCR) technique (15, 18). The assay could quantitate the pres- ence of $ 25 copies per 10 m l ( $ 2,500 copies per milliliter) CMV DNA in plasma. Plasma specimens positive for CMV DNA were cat- egorized as either positive but below 2,500 copies per milliliter or quantitated when above 2,500 copies per milliliter.
Local treatment of diabetic ulcer with AMPs had at least an equipotent anti-inﬂ ammatory eﬀ ect compared to antibiotics . In recent years, there have been at least 20 diﬀ erent peptides in several steps of development scheduled for anti-infective trials . Two studies on human lactoferrin 1-11 peptide (hLF1-11) were aborted at Phase I and II stages. Th e purpose of these studies was to establish tolerance to treatment with hLF1-11 adminis tered intravenously as a single daily dose for 10 consecutive days. Th e target population was patients with bacteremia due to Staphylococcus epidermidis (clinicaltrials.gov-identiﬁ er NCT00509847) or patients with proven candidemia (clinicaltrials.gov-identiﬁ er NCT00509834). Th e reasons given for withdrawal were that recruitment was not feasible within the timeframe (bacteremia) or the patient population was not available (candidemia).
Previously, we demonstrated that cationic liposomes are ef- fective for gene transfer in the kidney (8–10). Cationic lipo- somes, composed of a cationic lipid and a neutral lipid, have been used as vehicles for gene transfer both in vitro and in vivo. Since the initial report of lipofection in cultured cells by Felgner et al. in 1987 (11), the efficacy and safety of the in vivo use of liposomes have been demonstrated in studies of experi- mental animals (12, 13) and human gene therapy clinical trials (14, 15). Cationic liposomes are useful for gene delivery in the liver (16), lung (17), and cancers (13). When a mixture of Lipo- fectin (GIBCO BRL, Gaithersburg, MD), a cationic liposome preparation, and the cytomegalovirus (CMV)/ b -galactosidase expression plasmid was injected into the renal artery or retro- gradely infused via the renal pelvis, b -galactosidase was ex- pressed only in renal tubular cells, but not in glomerular, vas- cular, or interstitial compartments of the kidney (8–10). In this study, we further evaluated the efficacy and safety of intrare- nal–pelvic liposomal delivery of the CAII gene on correction of renal tubular acidosis in the CAII-deficient mice model.
Transfection into 293T cells revealed enhanced splicing when transcription was directed via the CMV (Fig. 3B, lanes 1 and 2). Whereas the parental MLV clone showed an equal ratio of unspliced and spliced RNAs, the CMV-driven construct showed an almost twofold enhancement of splicing (Fig. 3C). To evaluate this effect on the protein level, we probed cell lysates with anticapsid or anti-green fluorescent protein (GFP) antibodies (Fig. 2D), since GFP is encoded within the Env ORF (Fig. 3A). CMOV11GFP displayed less Gag expression and enhanced Env expression in comparison to the wild-type MLV (Fig. 3D) in agreement with the Northern blot data (Fig. 3B). This effect can also be measured via flow cytometry de- tecting the GFP. Here, a fourfold increase in the mean fluo- rescence intensity in the case of CMOV11GFP also indicates enhanced splicing, leading to more of the GFP Env fusion protein (Fig. 3E). To determine whether this change in gene expression has any impact on viral titer and therefore more biological relevance, we took supernatants of transient trans- fections and determined their titers on murine fibroblasts by use of the GFP as a reporter. The enhanced Env expression and reduced Gag expression led to a significant drop in titer, showing that a proper balance of Gag and Env indeed deter- mines infectivity (Fig. 3F). It will be interesting to look for
with LMV. Our patient has been re-challenged with the compound after a first short time exposure of 8 days. Thus, the question arose whether the selection of LMV-resistant viruses has already occurred during the first period of administration, because at that time virus loads were very high. On the other hand, re-administration of the drug resulted in successful suppression of virus replication for at least 30 days. Then, however, the resistant CMV subpopulation ap- parently prevailed. Unfortunately, due to the lack of appropriate patient specimens the question concern- ing the exact time point of emergence of LMV resist- ance cannot be answered unequivocally. Additional studies are needed to decide whether the re-exposure to letermovir during repeated periods of CMV reacti- vation poses a particular risk for the development of antiviral drug resistance.
relatively poor: only about 50% of CMV IgM-positive individuals have primary infection . Profound and long-term immunodeficiency and immunological switching which follows HSCT procedure is serious especially now with the development of haploidentical family donors. The current therapy for CMV disease with either ganciclovir or valganciclovir is associated with significant toxicity, therefore it shows unacceptable side effect after HSCT for preventive use: myelotoxicity. In clinical practice a fast and accurate diagnostic test before the decision for preemptive therapy is precious: the above mentioned CMV- or ganciclovir- induced suppression of myelopoiesis appears to be crucial for hematological recovery dilemma between two courses of medical action, sometimes both undesirable. Therefore, assessing immune parameter, especially specific response to cytomegalovirus, represents an appealing strategy for identifying immunodeficient patients and transplant recipients at risk of CMV opportunistic infection. Contrary to IgM and disadvantages of pentamer analysis (significant decrease below detection limit was observed) CMV-specific IgG level, CMV- quantiferon analysis and significant decrease of such specific immunity may be used in clinical practice. The intensive and significant decrease of CMV-quantiferon and specific IgG precedes virus reactivation. During severe lymphopenia, before the second wave of immunoreconstitution, the specific IgG is a “big player”. In the first phase after HSCT, after fast myeloid cell line recovery IgG acts by:
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established by evaluation of 20 human sera. Ten negative con- trol sera had no detectable virus-specific antibodies in WN virus IgM capture and IgG ELISAs. The mean microsphere MFI for these sera was 247 ⫾ 74, establishing an MFI of ⱖ988 (P/N ratio of ⱖ4.0) as a cutoff for a positive result. The 10 sera from WN viral encephalitis patients all tested positive for an- tibodies to WN virus E protein. The mean MFI for the patient sera was 7,626 ⫾ 4,312 (P ⬍ 0.001; range, 2,763 to 17,188), corresponding to a mean P/N ratio of 30.8 ⫾ 17.4 (range, 11.2 to 69.4). MIA results from repeated experiments were com- pared to determine interoperator reproducibility. For intra- assay imprecision studies, 10 aliquots of a WN virus encepha- litis patient serum pool and 10 aliquots of a negative control serum pool were tested separately in the rWNV-E MIA. The intra-assay imprecision of the positive pool provided a coeffi- cient of variation (CV) of 7%. The intra-assay imprecision of the negative serum pool provided a CV of 11%. These same negative and positive serum pools were analyzed on several days for estimates of interassay imprecision. The interassay CVs for the positive and negative serum pools were 17 and 32%, respectively. MIA results for 91 positive and negative sera independently analyzed by two individuals demonstrated interoperator assay reproducibility (kappa ⫽ 0.85; P/N r 2 ⫽
Tissue sections were retrospectively analyzed for EBV and other HHV (CMV, HHV-6, − 7, − 8) by quantitative real-time PCR assays . HPV detection and genotyping was performed using a multiplex real-time PCRs based on hybridization-fluorescence detection of amplified products (PANA RealTyperTM HPV assay – Panagene; DBA Italia, Milano, Italy). The assay was performed for detection and differentiation of 20 high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, and 82), two low-risk genotypes (6 and 11) from clinical speci- mens. Eighteen genotypes (30, 32, 34, 40, 42, 43, 44, 54, 55, 61, 62, 67, 74, 81, 83, 84, 87, and 90) are detected, but not typed.
occurs in Africa independent of Human immunodefi- ciency virus (HIV) infection. In children progression is fulminant and outcome fatal whereas in adults presen- tation is similar to classic KS. Immunosuppression- or transplantation-associated KS occurs in organ-transplant recipients and patients receiving immunosuppressive therapy for other medical conditions. This type tends to be aggressive, involving lymph nodes, mucosa and vis- ceral organs, sometimes in the absence of skin lesions. Epidemic or acquired immune deficiency syndrome (AIDS) associated Kaposi sarcoma is the best known variant and is commonly known to be an AIDS defining
CMV antigenemia has been previously shown to predict CMV disease in lung transplant recipients (10). In our patient population, which received intensive ganciclovir and CMVIG prophylaxis when CMV disease was anticipated, antigenemia testing had a sensitivity, specificity, PPV, and NPV of 48, 99, 85, and 98%, respectively. These results are in contrast with those reported by Egan et al. (10), who studied nine lung transplant patients, among others, and found that antigenemia testing had a 100% sensitivity and 93.7% specificity. The dif- ferences between the two studies with respect to the number of patients (41 versus 9), posttransplantation antiviral manage- ment (intense prophylaxis against CMV versus no prophylaxis at all), and duration of the observation period (12 months versus 8 months) might have contributed to these differences. In our study, CMV disease occurred in association with low and high numbers of positive cells, which implies that preemp- tive anti-CMV therapy needs to be entertained with any pos- itive antigenemia test result. On treatment, the number of positive cells rapidly decreased and the antigenemia test result became negative after 1 month of therapy for all patients, including those who later experienced a recurrence of the disease. Unlike PCR, antigenemia testing did not identify the patients at high risk of recurrent CMV.
Effect of receptor expression on phosphatidylinositol turn- over. A classical way of studying constitutive signaling of re- ceptors is to perform gene dose experiments, where cells are transfected with increasing amounts of a plasmid coding for the receptor and the relevant second messenger is measured in the cells in the absence of an added receptor ligand (3, 34). Figure 2 shows that of the four tested CMV proteins expressed in COS-7 cells, only US28 induced a clear increase in phos- phatidylinositol turnover in a gene-dose-dependent manner above the control. M33 and UL33 each showed a marginal degree of constitutive activity, whereas US27 did not display any constitutive activity above the baseline (pTEJ8). This lack of signaling cannot be accounted for by a failure of receptor expression, as this was verified by fluorescence detection of GFP-tagged receptors (see below).
Human CMV infection represents a major infectious com- plication in immunocompromised patients. Methods have been developed to detect and/or quantitate the virus or its components in blood, for example, blood culture, pp65 Ag, PBL, and plasma DNAemia and mRNAemia assays. The avail- ability of more sensitive tests means that it is possible to have positive laboratory assay results far before the onset of clinical symptoms. Identification of this asymptomatic stage is impor- tant because initiation of preemptive therapy at this time might prove beneficial. In addition, some investigators have found CMV DNA levels in plasma or PBLs to be reliable markers of replication in disseminated human CMV infections (4, 14, 32, 35). However, earlier protocols were poorly standardized, and only now are test results becoming comparable because of commercially available assays. In our study, we compared two conventional assays (the CMV viremia and pp65 Ag assays) and new molecular CMV assays with both PBLs (bDNA CMV 2.0 [Chiron]) and plasma specimens (P-AMP and CMM [Roche]).
Two analyses were then conducted. The first analysis used nonparametric receiver operating characteristic (ROC) curve comparisons (9) to determine the most predictive test of CMV retinitis. The test determined as most predictive in this step was then entered into a multivariate generalized estimating equation (GEE) model (26) to determine significant factors associated with CMV retinitis, as described below. To assess the validity of the different tests (antigenemia, Digene, and Amplicor), appropriate cutoff points for those tests were deter- mined; to determine which test is most appropriate, ROC curves were computed, plotted, and compared. Computations of all sensitivities and specificities were made using SPSS 9.0 for Windows statistical software. The sensitivites and
We performed additional multivariable analyses by using generalized linear mixed models to assess the effect of CMV reactivation on the time course of each individual biomarker. In the mixed model analyses, we assessed whether baseline biomarker levels were comparable be- tween matched groups (that is, coefficient for CMV reacti- vation) as well as the effect of CMV on the course of the biomarker levels over time (that is, coefficient for inter- action term between time and CMV reactivation). A priori we chose to model the established immune markers IL-6 and IL-10. Based on the observed divergence in the sym- metric percentage differences over time between groups, we conducted the multivariable analyses also for the pro-inflammatory chemokine IP-10 and the anti-inflamma- tory cytokine IL-1RA. Since not all CMV reactivation pa- tients were included in both comparisons, we performed separate mixed model analyses for the primary and second- ary comparisons. Thus, in total, eight models were built (for each of the four biomarkers in each of the two compar- isons). For each model, SOFA score at t = 0 (that is, the day of reactivation) and age were included as confounders since we used a range (instead of an exact value) as match- ing criteria for these co-variables. For the fixed part of th emodels a polynomial term for time was evaluted (that is, quadratic time effect). Furthermore, a random intercept and a rondom slowe were evaluted for each model. Re- stricted maximum likelihood estimation (REML) was used to generate unbiased variance estimates for the final models . Different ways to model the time course for each host response marker were compared by using the likelihood ratio test and Akaike ’ s information criterion.