24 research outputs found
Identificazione del corecettore utilizzato da HIV per l'ingresso nella cellula: confronto tra saggio fenotipico in vitro e fenotipo virtuale
No full textOBIETTIVI: L’introduzione nella pratica clinica degli antagonisti del CCR5 come nuova classe di inibitori dell’entry ha determinato la necessità di conoscere il coreceptor usage del virus prima di iniziare la terapia. Molti clinici ottengono questa informazione utilizzando il test Trofile nell’ ultima versione ultrasensibile (ESTA).Il largo utilizzo di questo saggio è limitato da alti costi e da lunghi tempi di attesa per la risposta. Sono perciò stati fatti numerosi sforzi per ottenere risultati in minor tempo possibile e con bassi costi migliorando i test genotipici che prevedono l’utilizzo di algoritmi interpretativi.
A causa dell’elevata variabilità del gene env i saggi genotipici sono tuttavia inaffidabili mentre i saggi biologici sono quasi sempre in grado di definire il tropismo attuale. A tal fine abbiamo messo a punto un vettore di espressione fluorescente che si caratterizza per consentire il clonaggio delle sequenze V3 amplificate dal paziente di interesse. Questo plasmide è dotato di un gene per la proteina fluorescente GFP che viene espressa quando il virus replica nelle cellule, è pertanto possibile conoscere sia il fenotipo conferito da quella specifica sequenza V3 che la capacità replicativa della variante V3 attraverso l’analisi fluorimetrica delle cellule dopo un saggio di transfezionne-infezione.
Obiettivo di questo lavoro è confrontare il saggio fenotipico home-brew con il saggio commerciale Trofile nella sua forma più sensibile l’Enhanced Trofile (ESTA) e conseguire una valutazione comparativa tra i diversi saggi fenotipici e tra i saggi fenotipici e saggi virtuali nella definizione del coreceptor usage.
MATERIALI E METODI: E’ stata utilizzata una strategia molecolare ricombinante che ha permesso di clonare in pNLmodΔV3GFP la sequenza V3 amplificata dal virus di 38 pazienti analizzati generando chimere virali replicative.
La regione V3 delle varianti virali è stata sequenziata e analizzata al geno2pheno, al fine di ottenere la predizione del fenotipo.
E’ importante determinare il livello di significatività, cioè di selezionare quanto conservativa è la determinazione del virus X4, questo livello di significatività è determinato dal tasso di falsa positività per la classificazione di un virus X4. Questo tasso detto FPR (False Positive Rate) è un cut off, una soglia al di sopra della quale i virus sono definiti R5 e al di sotto X4. Attualmente l’FPR utilizzato dalla comunità scientifica è del 10%; questo valore sembra raggiungere un buon compromesso tra sensibilità e specificità.
RISULTATI: sono stati ottenuti 117 cloni dal plasma di 38 pazienti HIV-1 positivi, il saggio ricombinante home brew ha identificato 90 cloni con fenotipo R5 corrispondenti a 28 pazienti, 20 cloni con fenotipo D/M corrispondenti a 9 pazienti e tutte le chimere virali ottenute da 1 paziente sono risultate essere non replicative (risultato DM con ESTA).
Il saggio ESTA ha identificato 25 pazienti con fenotipo R5, 9 pazienti con fenotipo D/M e 4 NR (due di questi sono risultai D/M e uno R5 con il nostro saggio).
CONCLUSIONI: il saggio fenotipico home brew descritto si mostra più sensibile del saggio ESTA ed inoltre offre la possibilità di analizzare la quasi specie virale del paziente consentendo di studiare l’influenza che ogni residuo aminoacidico della regione V3 ha sul fenotipo.
I nostri risultati mostrano che il 33% delle sequenze V3 clonate identificate come X4 dall’algoritmo geno2pheno quando inserite nel backbone di NL 4-3 conferiscono alla chimera virale un fenotipo R5. Il livello di FPR dovrebbe pertanto essere scelto con molta attenzione
Analysis of the functional relationship between V3 loop and gp120 context with regard to human immunodeficiency virus coreceptor usage using naturally selected sequences and different viral backbones
No full textAbstractThe human immunodeficiency virus type 1 (HIV-1) gp120 V3 loop plays a predominant role in chemokine receptor usage; however, other linear and nonlinear gp120 domains are involved in this step of the HIV-1 replication cycle. At present, the functional relationship between V3 and these domains with regard to coreceptor usage is unclear. To gain insights into the nature of this relationship in naturally selected viral variants, we developed a recombinant strategy based on two different gp120 backbones derived from CXCR4 (X4)- and CCR5 (R5)-tropic viral strains, respectively. Using this recombinant model system, we evaluated the phenotype patterns conferred to chimeric viruses by exogenous V3 loops from reference molecular clones and samples from infected subjects. In 13 of 17 recombinants (76%), a comparable phenotype was observed independently of the gp120 backbone, whereas in a minority of the recombinant viruses (4/17, 24%) viral infectivity depended on the gp120 context. No case of differential tropism using identical V3 sequence in the two gp120 contexts was observed. Site-directed mutagenesis experiments were performed to evaluate the phenotypic impact of specific V3 motifs. The data indicate that while the interaction of HIV-1 with chemokine receptors is driven by V3 loop and influenced by its evolutionary potential, the gp120 context plays a role in influencing the replication competence of the variants, suggesting that compensatory mutations occurring at sites other than V3 are necessary in some cases
Distribution and molecular analysis of mef(A)-containing elements in tetracycline-susceptible and -resistant Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance.
No full textOBJECTIVES:
To analyse the distribution and molecular features of mef(A)-containing elements in a large collection of different Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance. To further characterize a tet(O)-mef(A) element.
METHODS:
Gene detection was carried out by PCR using primers designed from established sequences or from sequences in this study. From a tet(O)-mef(A) element (approximately 60 kb), an 11 972 bp region including the tet(O) and mef(A) genes was sequenced.
RESULTS:
In the tetracycline-susceptible isolates (n =28), the mef(A) gene was contained in a regular Tn1207.1 transposon (7.2 kb), which was inserted into one of two previously described elements, Tn1207.3 (approximately 52 kb) or a 58.8 kb chimeric element, both flanked by the comEC gene. In the tetracycline-resistant isolates (n =61), all of which carried the tet(O) gene, the mef(A) gene was part of a variable Tn1207.1-related transposon inserted into unique elements which contained the tet(O) gene approximately 2.3 to 5.5 kb upstream of the mef(A) gene and were not flanked by the comEC gene. In the Tn1207.1-like transposon of these tet(O)-mef(A) elements, only msr(D) (orf5) and a modified orf6, in addition to mef(A), were detected by PCR in all isolates tested; while orf1 and orf2 were always undetectable, orf3, orf7 and orf8 were found in variable percentages. In an orf3-positive element, sequencing identified four new open reading frames downstream of the tet(O) gene, followed by three short sequences with homology to sequences of the pneumococcal mega element.
CONCLUSIONS:
The mef(A) gene is carried on different chromosomal genetic elements depending on whether the isolates are susceptible or resistant to tetracycline
Identification of six putative novel human papillomaviruses (HPV) and characterization of type 87 (candHPV87)
No full textResistance and replicative capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treatment.
No full textGenotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term treatment.
No full textThe human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced
into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral
agents. In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost
evolution of HIV-1. The genotype and phenotype patterns and the relative replication capacity (rRC) of
enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders
and 4 responders) who received the compound for more than 1 year in combination with different regimens.
Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp41 N-terminal
heptad repeat was observed in samples from the seven virological nonresponders but not in those from
responders. In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected
within 3 months. Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to
enfuvirtide, with the 50% inhibitory concentrations (IC50s) ranging from 0.6 to 12.8 g/ml, whereas the IC50
for isolates with baseline sequences was 0.013 0.010 g/ml. Interestingly, long-term monitoring of resistant
variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes. A limited
drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing
mutations associated with resistance. Overall, the data indicate that the different genotype patterns associated
with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized
by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance,
and limited influence on the viral rRC
Genotype and phenotype patterns of human immunodeficiency virus type 1 to enfuvirtide during long-term treatments
No full textThe human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral agents. In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost evolution of HIV-1. The genotype and phenotype patterns and the relative replication capacity (rRC) of enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders and 4 responders) who received the compound for more than 1 year in combination with different regimens. Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp4l N-terminal heptad repeat was observed in samples from the seven virological nonresponders but not in those from responders. In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected within 3 months. Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to enfuvirtide, with the 50% inhibitory concentrations (IC(50)s)ranging from 0.6 to 12.8 mug/ml, whereas the IC50 for isolates with baseline sequences was 0.013 +/- 0.010 mug/ml. Interestingly, long-term monitoring of resistant variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes. A limited drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing mutations associated with resistance. Overall, the data indicate that the different genotype patterns associated with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance, and limited influence on the viral rRC
Identification of six putative novel human papillomaviruses (HPV) and characterization of candidate HPV type 87.
No full textMolecular analysis of hepatitis B virus (HBV) in an HIV co-infected patient with reactivation of occult HBV infection following discontinuation of lamivudine-including antiretroviral therapy
No full textAbstract Background Occult hepatitis B virus (HBV) infection (OBI) is characterized by HBV DNA persistence even though the pattern of serological markers indicates an otherwise resolved HBV infection. Although OBI is usually clinically silent, immunocompromised patients may experience reactivation of the liver disease. Case presentation We report the case of an individual with human immunodeficiency virus (HIV) infection and anti-HBV core antibody positivity, who experienced severe HBV reactivation after discontinuation of lamivudine-including antiretroviral therapy (ART). HBV sequencing analysis showed a hepatitis B surface antigen escape mutant whose presence in an earlier sample excluded reinfection. Molecular sequencing showed some differences between two isolates collected at a 9-year interval, indicating HBV evolution. Resumption of ART containing an emtricitabine/tenofovir combination allowed control of plasma HBV DNA, which fell to undetectable levels. Conclusion This case stresses the ability of HBV to evolve continuously, even during occult infection, and the effectiveness of ART in controlling OBI reactivation in HIV-infected individuals.</p
