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

Finite-temperature relativistic Landau problem and the relativistic quantum Hall effect

This paper presents a study of the free energy and particle density of the relativistic Landau problem, and their relevance to the quantum Hall effect. We study first the zero temperature Casimir energy and fermion number for Dirac fields in a 2+1-dimensional Minkowski space-time, in the presence of a uniform magnetic field perpendicular to the spatial manifold. Then, we go to the finite-temperature problem, with a chemical potential, introduced as a uniform zero component of the gauge potential. By performing a Lorentz boost, we obtain Hall's conductivity in the case of crossed electric and magnetic fields.Comment: Final version, to appear in Journal of Physics A: Mathematical and Genera

Multiple changes in gene expression in chronic human Achilles tendinopathy

Atlas™ cDNA cell interaction arrays (CLONTECH) were used to examine degenerate tissue from four patients with Achilles tendon disorders, in order to identify changes in expression of genes important in cell–cell and cell–matrix interactions. The greatest difference between normal (post-mortem) and degenerate tissue samples was in the level of MMP-3 (stromelysin) mRNA, which was down-regulated in all the degenerate samples. Quantitative RT-PCR assay of RNA extracted from paired ‘normal’ and degenerate Achilles tendon tissue samples taken from tendons during surgery mirrored the results of the arrays. Levels of MMP-3 mRNA were lower, whereas levels of type-I and type-III collagen mRNAs were significantly higher, in the degenerate compared to the ‘normal’ samples. Immunoblotting of proteins extracted from the same tendon samples showed that three of four normal tissue samples taken from individuals without apparent tendon disorder had much higher levels of MMP-3 protein than ‘normal’ or degenerate samples from patients with tendinosis. We suggest that MMP-3 may play an important role in the regulation of tendon extracellular matrix degradation and tissue remodelling

Effects of feeding omega-3 fatty acids and vitamin E on the chemical composition and microbial population of broiler meat

Thesis (MScAgric) -- University of Stellenbosch, 2000.ENGLISH ABSTRACT: Lipids remain one of the most important nutrients required by broilers. The growing awareness that some Western societies have too high a dietary ratio of n-6/n-3 polyunsaturated fatty acids is of direct relevance to broiler nutrition and lipid metabolism. Meaningful quantities of n-3 polyunsaturates have been incorporated into major poultry tissues, so that the production of broiler meat with high n-3 polyunsaturates becomes advantageous for the broiler industry as they are perceived as having a 'healthier' lipid profile. Unfortunately, such broiler meat is rather susceptible to oxidative deterioration, and oxidation often determines shelf life of poultry meat products. The addition of a-tocopherol (vitamin E) to broiler diets is an effective means of improving the oxidative stability of broiler meat. Elevated a-tocopherol levels in broiler feeds increase tissue concentrations thereof resulting in improved stability of membranal structures which may be expected to increase the oxidative stability of broiler meat and meat products. Three investigations were done at Mariendahl Poultry Research Station in Stellenbosch. The broilers were kept in 1 x 0.4 x 0.5 m cages in a broiler rearing house. All the trials started with day-old chicks, except experiment 1 where 3-week old broilers were used. At the end of trials 2 and 3 the 6-week old broilers were slaughtered and the carcasses prepared for chemical analysis. Experiment 1: Metabolisabie energy of Canola acid oil and Famarol acid oil for broiler chickens. In trials with 21-day-old male broilers the true metabolisabie energy value, corrected for nitrogen retention (TMEn) was determined by the balance method for Canala acid oil (CAO) and Famarol acid oil (FAO). The trials were duplicated, each time using different samples of the two oils from the same source (experiment 1 and 2). Each of the two oils were blended in two ratios with a basal diet to form the test diets, viz. 100% Basal; 96% Basal: 4% Oil; 92% Basal: 8% Oil. In experiment 3, 50 % bran was added to the maize to form the basal diet. The balance trials lasted for 3 days after an adaptation period of 4 days. The TMEn values determined by regression for the broilers of CAO did not differ significantly (P>0.05) between experiments 1 and 2. However, the value for experiment 3 was significantly (P<0.05) higher than those for experiments 1 and 2. The TMEn values of FAO also did not differ significantly (P>0.05) between experiments 1 and 2, although the value for experiment 3 was significantly higher than that of experiment 1. The addition of 50 % bran to the basal diet in experiment 3 could have stimulated the digestive breakdown process and hence increase the secretion of digestive enzymes. This could lead to an increase in the utilisation of the test lipid and therefore an increase in the TMEn value. The TMEn values of CAO differed significantly (P<0.05) from those of FAO for all three the experiments (exp. 1:30.6 ± 0.399 MJ/kg for CAO vs. 25.9 ± 0.441 MJ/kg for FAO; expo 2: 31.0 ± 0.633 MJ/kg for CAO vs. 26.1 ± 0.668 MJ/kg for FAO: expo 3: 32.1 ± 0.867 MJ/kg for CAO vs. 27.1 MJ/kg for FAO).Experiment 2: of broilers. The dietary effects of various combinations of Canala acid oil (CAO, a high level of C18:3n-3 and MUFA) and Famarol Effects of various dietary n-6/n-3 fatty acid ratios on the perfonnance and body composition acid oil (FAO, a high level of 18:2n-6 and SFA) on tissue fatty acid composition were studied in broiler carcasses and abdominal fat pads. From day-old to six weeks, chicks were fed one of six diets containing 100% FAO, 80% FAO-20% CAO, 60% FAO-40% CAO, 40% FAO-60% CAO, 20% FAO-80% CAO, 100% CAO. There were no statistical differences (P>0.05) in average daily gain (1.71 ± 0.059 g) or feed conversion ratios (1.97 ± 0.051) among dietary groups. No statistical differences (P>0.05) were found in the chemical proximate composition of the carcasses for the moisture (66.20 ± 0.112 %), protein (17.63 ± 0.484 %), lipid (15.92 ± 1.507 %) and ash (0.95 ± 0.115 %) content among dietary groups. No statistical differences (P>0.05) were found in the chemical proximate composition of the abdominal fat pads for the moisture (28.77 ± 0.112 %), protein (3.03 ± 0.484 %), lipid (63.32 ± 9.789 %) and ash (0.45 ± 0.135 %) content among dietary groups. With the increase in dietary CAO levels, the percentages of C18:2n-6 and C20:4n-6 in the carcasses decreased respectively with 1.78 % from 20.88 % and 0.35 % from 1.05 %, whilst C18:3n-3 and longer chain n-3 fatty acids such as C20:5n-3 and C22:6n-3 increased respectively with 2.25 % from 1 %, 0.1 % from 0.1 % and 0.67 % from 0.2 %. The same tendency was seen in the abdominal fat pads where C18:2n-6 and C20:4n-6 decreased respectively with 1.55 % from 20.75 % and 0.98 % from 1.2 % with an increase in dietary CAO, whilst C18:3n-3, C20:5n-3 and C22:6n-3 increased respectively with 2.13 % from 1.15 %, 0.45 % from 0.03 % and 0.95 % from 0.05 %. The n-3/n-6 ratio in the carcasses and abdominal fat pads increased respectively with 0.16 % from 0.06 % and 0.19 % from 0.06 % with an increase in dietary CAO. These results clearly indicate that dietary CAO enriched with a-linolenic acid lower saturated fatty acids respectively in broiler carcasses and abdominal fat pads with 4.88 % from 31.6 % and 10.63% from 31.1 %, whilst increasing monounsaturated fatty acids with 3.87 % from 44.95 % and 7.25 % from 46.7 % respectively and polyunsaturated fatty acids with 1.02 % from 23.45 % and 2.38 % from 23.2 % respectively. Experiment 3: Effect of dietary vitamin E on the performance of broilers and oxidative stability, colour, microbiological stability, fatty acid composition and pH of broiler meat during refrigerated and frozen storage. Experiment 1 was carried out with 220 one-day-old broiler chicks to evaluate the effect of eleven concentrations of vitamin E (0, 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 mg a-tocopheryl acetate 1 kg diet) on their production performance and the oxidative stability of their frozen broiler carcasses. The diets with vitamin E levels 0 to 100 mg were fed from day-old to 42 days of age while the diets with vitamin E levels 120 to 200 mg were fed from 21 to 42 days of age. The oxidative stability, evaluated by thiobarbituric acid reactive substances (TBARS) values, was determined after 30, 90, 120 and 150 days of storage at -20°C. There were no statistical differences (P>0.05) in average daily gain (1.85 ± 0.111 g) or feed conversion ratios (2.29 ± 0.397) among dietary groups. TBARS values increased significantly (P<0.05) with increasing time of storage (basal diet: day 30 = 1.71 ± 0.51; day 150 = 4.89 ± 0.51), but decreased significantly (P<0.05) with increasing vitamin E levels (day 150: basal = 4.89 ± 0.51; 100 mg / kg = 1.09 ± 0.27). Experiment 2 was carried out with day-old broiler chicks to evaluate the effect of five concentrations of vitamin E (0, 40, 80, 120 and 160 mg atocopheryl acetate / diet) on their performance and the oxidative stability of their refrigerated carcasses. The experimental diets were fed from day-old to 42 days of age. The oxidative stability, evaluated by TBARS values, colour deterioration and microbiological stability were determined after 0, 4, 8, 10 and 12 days of storage at 4°C. Fatty acid analysis was done on the samples of days 0 and 12. There were no statistical differences (P>0.05) in average daily gain (1.88 ± 0.117 g) or feed conversion ratios (2.37 ± 0.467) among dietary groups. TBARS values increased significantly (P<0.05) with increasing time of storage, but decreased significantly (P<0.05) with increasing vitamin E levels. There were no statistical differences (P>0.05) in colour measurements for L* (44.97 ± 0.662), a* (5.23 ± 0.315) or b* (12.76 ± 0.321) values between treatments. Microbiological counts increased significantly (P<0.05) over time with vitamin E concentration showing no effect. There were no statistical differences (P>0.05) for any of the fatty acid groups measured (SFA: Day 0 = 26.1 ± 1.13%, Day 12 = 26.1 ± 1.17%; MUFA: Day 0 = 41.4 ± 1.46%, Day 12 = 40.2 ± 2.28%; PUFA: Day 0 = 32.4 ± 1.95%, Day 12 = 33.8 ± 2.52%) among dietary groups. Similarly, none of the fatty acids showed statistical significant (P>0.05) concentration changes over time. There were no statistical differences (P>0.05) in pH (6.01 ± 0.206) among dietary groups.AFRIKAANSE OPSOMMING: Lipiede is steeds een van die mees belangrike voedingstowwe wat deur braakuikens benodig word. Die groeiende bewuswording dat sekere Westerse gemeenskappe 'n te hoë verhouding van n-6/n-3 poli-onversadigde vetsure in hul dieet het, is direk relevant vir braaikuikenvoeding en lipiedmetabolisme. Betekenisvolle hoeveelhede n-3 polionversadigde vetsure is geïnkorporeer in die belangrikste hoendersnitte, met die gevolg dat die produksie van braaikuikenvleis met hoë n-3 poli-onversadigde vetsure voordelig is vir die braaikuikenindustrie en geag word 'n meer "gesonde" beeld te hê. Ongelukkig is sodanige braaikuikenvleis redelik vatbaar vir oksidatiewe bederf, en oksidasie bepaal dikwels die rakleeftyd van hoendervleisprodukte. Die byvoeging van a-tokoferol (vitamine E) by braaikuikendiëte is 'n effektiewe manier om die oksidatiewe stabiliteit van braaikuikenvleis te verbeter. Verhoogde a-tokoferol vlakke in braakuikenvoere verhoog die weefselkonsentrasie wat verhoogde stabiliteit van die membraanstrukture en derhalwe moontlike verhoogde oksidatiewe stabiliteit van braakuikenvleis en -produkte tot gevolg het. Drie ondersoeke is onderneem by Mariendahl Pluimvee Navorsingstasie te Stellenbosch. Die braakuikens is aangehou in 1 x 0.4 x 0.5m hokke in braaikuikenhuise. In al die proewe is dagoud kuikens gebruik, behalwe eksperiment 1 waar drieweek oue kuikens gebruik is. Aan die einde van proewe 2 en 3 is die ses-week oue braaikuikens geslag en die karkasse voorberei vir analise. Eksperiment 1: braaikuikens. Die ware metaboliseerbare energie waarde van Canola voergraadolie (CAO) en Famarol voergraadolie (FAO), Metaboliseerbare energie van Canola voergraadolie en Famarol voergraadolie vir gekorregeer vir stikstof retensie (WMEn), is by wyse van proewe op 21 dae oue braaikuikenhaantjies bepaal deur van die balansrnetode gebruik te maak. Die proewe is tweemaal herhaal vir verhoogde akkuraatheid, met die gebruik van verskillende monsters van die twee olies vanaf dieselfde bron. Die olies is in twee verhoudings met 'n basale diëet gemeng om die proef dieet te vorm, nl. 100% Basaal; 96% Basaal: 4% Olie en 92% Basaal: 8% Olie. Die balans proewe het 3 dae geduur na afloop van 'n aanpassingsperiode van 4 dae. Die WMEn waardes van CAO, bepaal deur middel van regressie analise, het nie betekenisvol verskil (P>0.05) tussen eksperimente 1 en 2 nie. Die waarde van eksperiment 3 was betekenisvol hoër (P<0.05) as die van die eerste twee eksperimente. Die WMEn waardes van FAO het ook nie betekenisvol verskil (P>0.05) tussen eksperimente 1 en 2 nie, maar die waarde vir eksperiment 3 was betekenisvol hoër as dié van eksperiment 1. Die WMEn waardes van COA het betekenisvol verskil (P< 0.05) van dié van FAO vir al die eksperimente (exp. 1: 30.6 ± 0.399 MJ/kg vir CAO vs. 25.9 ± 0.441 MJ/kg vir FAO; expo 2: 31.0 ± 0.633 MJ/kg vir CAO vs. 26.1 ± 0.668 MJ/kg vir FAO: expo 3: 32.1 ± 0.867 MJ/kg vir CAO vs. 27.1 MJ/kg vir FAO). Eksperiment 2: Die invloed van verskeie rantsoen n-6/n3 vetsuurverhoudings op die produksie en liggaamsamestelling van braaikuikens. Die rantsoeneffek van verskeie kombinasies Canola voergraadolie (CAO, 'n hoë vlak van C18:3n-3 en monoonversadigde vetsure) en Famarol voergraadolie (FAO, 'n hoë vlak van 18:2n-6 en versadigde vetsure) op die weefselvetsuursamestelling is bestudeer in braaikuikenkarkasse en abdominale vetneerlegging. Die kuikens is van dagoud to op ses-weke ouderdom een van ses diëte gevoer met die volgende samestellings: 100% FAO, 80% FAO-20% CAO, 60% FAO - 40%CAO, 40% FAO - 60% CAO, 20% FAO - 80% CAO, 100% CAO. Daar was geen statistiese verskil tussen die rantsoengroepe (P>0.05) in die gemiddelde daaglikse toename (1.71 ± 0.059 g) of die voeromsetverhoudings (1.97 ± 0.051) nie. Geen statistiese verskil (P>0.05) is gevind in die chemiese samestelling van die karkasse vir vog (66.20 ± 0.112 %), proteïn (17.63 ± 0.484 %), lipied (15.92 ± 1.507 %) en as (0.95 ± 0.115 %) inhoud tussen die rantsoen groepe nie. Geen statistiese verskille (P>0.05) is gevind in die chemiese samestelling van die abdominale vetneerlegging vir vog (28.77 ± 0.112 %), proteien (3.03 ± 0.484 %), lipied (63.32 ± 9.789 %) en as (0.45 ± 0.135 %) inhoud onder die rantsoengroepe nie. Met die verhoging in die rantsoen CAO vlakke het die persentasie van C18:2n-6 en C20:4n-6 in die karkasse verminder met 1.78 % en 0.35 % respektiewelik, terwyl C18:3n-3 en langer ketting n-3 vetsure soos C20:5n-3 en C22:6n-3 respektiewelik met 2.25 %, 0.1 % en 0.67 % verhoog het. Dieselfde tendens is opgemerk in die abdominale vetneerlegging waar C18:2n-6 en C20:4n-6 afgeneem het met 1.55 % en 0.98 % respektiewelik met die verhoging van rantsoen CAO, terwyl C18:3n-3, C20:5n-3 en C22:6n-3 verhoog het met 2.13 %, 0.45 % en 0.95 % respektiewelik. Die n- 3/n-6 verhouding in die karkasse en abdominale vetneerlegging het verhoog met 0.16 % en 0.19 % respektiewelik met die verhoging van die rantsoen CAO. Die resultate toon onomwonde aan dat rantsoen CAO verryk met c-Iinoletensuur, verlaag versadigde vetsure in braaikuikenkarkasse en -adbdominale vetneerleggings met 4.88 % en 10.63% respektiewelik, terwyl die mono-onversadigde vetsure met 3.87 % en 7.25 % respektiewelik verhoog word en polionversadigde vetsure met 1.02 % en 2.38 % respektiewelik verhoog word. Eksperiment 3: Die invloed van vitamine E op die produksie van braaikuikens en die oksidatiewe stabiliteit, kleur, mikrobiologiese stabilitiet, vetsuursamestelling en pH van braaikuikenvleis gedurende verkoelde en bevrore berging. Eksperiment 1 is uitgevoer met 220 dagoud braaikuikens ten einde die effek van elf konsentrasies van vitamine E (0, 20, 40, 60, 80, 100, 120, 140, 160, 180 en 200 mg a-tokoferyl acetaat / kg voer) op hul produksieprestasie en die oksidatiewe stabiliteit van hul gevriesde braakuikenkarkasse te evalueer. Die diëte met vitamine E vlakke 0 tot 100 mg is vanaf dagoud tot 42-dae-ouderdom gevoer, terwyl die diëte met vitamine E vlakke van 120 tot 200mg gevoer is vanaf 21 tot 42- dae-ouderdom. Die oksidatiewe stabiliteit, soos geëvalueer deur tiobarbituriese suur reaktiewe stowwe (TBARS) waardes, is bepaal na 30, 90, 120 en 150 dae van berging teen -20°C. Daar was geen statistiese verskille (P>0.05) in die gemiddelde daaglikse toename (1.85 ± 0.111 g) of voeromsetverhoudings (2.29 ± 0.397) tussen die rantsoengroepe nie. TBARS waardes het betekenisvol toegeneem (P<0.05) met die verhoging in bergingsperiode, maar het betekenisvol afgeneem (P<0.05) met verhoogde vitamine E vlakke. Eksperiment 2 is uitgevoer met dagoud braaikuikens ten einde die effek van vyf konsentrasies van vitamine E (0, 40, 80, 120 and 160 mg a-tokoferyl acetaat / kg voer) op hul prestasie en die oksidatiewe stabiliteit van hul verkoelde karkasse te evalueer. Die eksperimentele diëte is gevoer vanaf dagoud tot 42- dae-ouderdom. Die oksidatiewe stabiliteit, geëvalueer deur middel van TBARS waardes, kleur afname en mikrobiologiese stabiliteit is bepaal na 0, 4, 8, 10 en 12 dae van berging teen 4°C. Vetsuuranalises is gedoen op die monsters van dae 0 en 12. Daar was geen statistiese verskille (P>0.05) in die gemiddelde daaglikse toename (1.88 ± 0.117 g) of voeromsetverhoudings (2.37 ± 0.467) tussen die rantsoengroepe nie. TBARS waardes het betekenisvol verhoog (P<0.05) met die verlengde bergingsperiode, maar het betekenisvol afgeneem (P<0.05) met verhoogde viatmine E vlakke. Daar was geen statistiese verskille (P>0.05) in kleur metings vir L* (44.97 ± 0.662), a* (5.23 ± 0.315) of b* (12.76 ± 0.321) waardes tussen behandelings nie. Mikrobiologiese tellings het betekenisvol verhoog (P0.05) vir enige van die vetsuurgroepe tussen die behandelings nie. Soortgelyks het geen van die vetsure statisties betekenisvolle (P>0.05) konsentrasieveranderings oor tyd aangetoon nie. Daar was geen statistiese verskil (P>0.05) in die pH (6.01 ± 0.206) tussen die rantsoengroepe nie

Human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in adult allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection

Background Human cytomegalovirus infection is the most frequent viral complication in patients undergoing hematopoietic stem cell transplantation. We investigated the development of human cytomegalovirus-specific T cells in adult recipients of hematopoietic stem cell transplants. Design and Methods From May 2003 through October 2006 a total of 45 patients were monitored for human cytomegalovirus-specific T-cell reconstitution. Human cytomegalovirus-infected autologous dendritic cells were used as a stimulus to detect interferon-γ-producing human cytomegalovirus-specific CD8+ and CD4+ T cells during the first year after transplantation. Interleukin-2 production by specific T cells was also determined. ![Figure 1.][1] Figure 1. Probability of HCMV infection development and HCMV-specific CD4+ and CD8+ T-cell immunity reconstitution. A: cumulative incidence curves of HCMV infection according to donor (D) and recipient (R) HCMV-serostatus. B: cumulative incidence curves of HCMV infection and HCMV-specific CD8+ and CD4+ T-cell reconstitution (i.e. corresponding to a specific T-cell number greater than 0.4 cells/μL blood). C: cumulative incidence curves of HCMV-specific CD8+ T-cell reconstitution according to D/R HCMV-serostatus. D: cumulative incidence curves of HCMV-specific CD4+ T-cell reconstitution according to D/R HCMV-serostatus. Results Human cytomegalovirus infection was detected in the blood of 39/45 patients at a median of 29 days after transplantation. Human cytomegalovirus-specific T-cell reconstitution followed reactivation of latent human cytomegalovirus infection at a median time of about 2 months after transplantation. Only donor human cytomegalovirus-seronegativity and bone marrow as a stem cell source were found to delay specific T-cell reconstitution significantly. Levels of three CD8+ and one CD4+ human cytomegalovirus-specific T-cells/μL blood had a positive predictive value of around 80% for identifying patients able to control human cytomegalovirus infection spontaneously. Five patients who received high doses of steroids for treatment of graft-versus-host disease developed human cytomegalovirus infection requiring pre-emptive treatment despite high levels of interferon-γ-producing T cells in response to human cytomegalovirus. Specific interleukin-2 production was not detected in patients with human cytomegalovirus infection requiring treatment, while 90% of patients who spontaneously controlled human cytomegalovirus infection had T cells that produced interleukin-2 and interferon-γ. Conclusions Pre-transplant human cytomegalovirus infection of the recipient is a major factor driving human cytomegalovirus-specific immune reconstitution. Control of human cytomegalovirus infection likely requires the presence of both interferon-γ and interleukin-2 producing T cells. Corticosteroid treatment may favor active viral replication even in patients with specific T cells. [1]: pending:ye

Comparison of Quantitative Cytomegalovirus Real-time PCR in Whole Blood and pp65 Antigenemia Assay: Clinical Utility of CMV Real-time PCR in Hematopoietic Stem Cell Transplant Recipients

Successful preemptive therapy for cytomegalovirus (CMV) infection in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infection. Although the pp65 antigenemia assay has been widely used for this purpose, real-time quantification of CMV DNA has recently been recognized as an alternative diagnostic approach. However, the guidelines for antiviral therapy based on real-time quantitative polymerase chain reaction (RQ-PCR) have yet to be established. From November 2004 to March 2005, a total of 555 whole blood samples from 131 hematopoietic stem cell transplant (HSCT) recipients were prospectively collected. RQ-PCR was conducted using an Artus® CMV LC PCR kit (QIAGEN). Both qualitative and quantitative correlations were drawn between the two methods. Exposure to the antiviral agent influenced the results of the two assays. Additionally, the discrepancy was observed at low levels of antigenemia and CMV DNA load. Via ROC curve analysis, the tentative cutoff value for preemptive therapy was determined to be approximately 2×104 copies/mL (sensitivity, 80.0%; specificity, 50.0%) in the high risk patients, and approximately 3×104 copies/mL (sensitivity, 90.0%; specificity, 70.0%) in the patients at low risk for CMV disease. Further study to validate the optimal cutoff value for the initiation of preemptive therapy is currently underway

Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trial

Impact of sequence variation in the ul128 locus on production of human cytomegalovirus in fibroblast and epithelial cells

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism

Reconstitution of Human Cytomegalovirus-Specific CD4+ T Cells is Critical for Control of Virus Reactivation in Hematopoietic Stem Cell Transplant Recipients but Does Not Prevent Organ Infection

The relative contribution of human cytomegalovirus (HMCV)-specific CD4(+) and CD8(+) T cells to the control of HCMV infection in hematopoietic stem cell transplant (HSCT) recipients is still controversial. HCMV reactivation and HCMV-specific CD4(+) and CD8(+) T cell reconstitution were monitored for 1 year in 63 HCMV-seropositive patients receiving HSCT. HCMV reactivation was detected in all but 2 patients. In 20 of 63 (31.7%) patients (group 1) HCMV infection resolved spontaneously, whereas 32 of 63 (50.8%) patients (group 2) controlled the infection after a single short-course of pre-emptive therapy and the remaining 9 (14.3%) patients (group 3) suffered from relapsing episodes of HCMV infection, requiring multiple courses of antiviral therapy. The kinetics and magnitude of HCMV-specific CD8(+) T cell reconstitution were comparable among the 3 groups, but HCMV-specific CD4(+) T cells were lower in number in patients requiring antiviral treatment. HCMV-seronegative donors, as well as unrelated donors (receiving antithymocyte globulin) and acute graft-versus-host disease (GVHD) were associated with both delayed HCMV-specific CD4(+) T cell reconstitution and severity of infection. Conversely, these risk factors had no impact on HCMV-specific CD8(+) T cells. Eight patients with previous GVHD suffered from HCMV gastrointestinal disease, although in the presence of HCMV-specific CD4(+) and CD8(+) systemic immunity and undetectable HCMV DNA in blood. Reconstitution of systemic HCMV-specific CD4(+) T cell immunity is required for control of HCMV reactivation in adult HSCT recipients, but it may not be sufficient to prevent late-onset organ localization in patients with GVHD. HCMV-specific CD8(+) T cells contribute to control of HCMV infection, but only after HCMV-specific CD4(+) T cell reconstitution