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OBJECTIVES To investigate the value of tropism (determined by genotypic testing) to predict CD4 depletion in HIV-infected antiretroviral-naive patients with high CD4 counts. METHODS Viral tropism was determined by geno2pheno (false positive rate = 10%) in 223 HIV-infected subjects naive to antiretrovirals with CD4 count ≥350 cells/μL and HIV-RNA >500 copies/mL enrolled in the ICONA Foundation Study for whom a stored plasma sample (baseline) was retrospectively tested. We monitored CD4 cell count and identified predictors of decline before antiretroviral therapy initiation, applying a mixed linear model with covariates (age, gender, tropism, HIV risk factor, calendar year of HIV infection, months from HIV diagnosis to baseline, hepatitis C virus status, CD4 and HIV-RNA at sample collection and duration of follow-up). RESULTS Two hundred and twenty-three subjects met the eligibility criteria; 137 (61%) were male and the median age was 35 (31-40) years. Median follow-up was 16.4 (3.2-37.2) months. Median CD4 decrease during follow-up was -157 (-278 to -13) cells/μL. At baseline, 192 (86%) subjects were defined as harbouring R5 virus and 31 (14%) non-R5. Median CD4 count was 571 (458-729) cells/μL and median HIV-RNA was 4.08 (3.57-4.55) log(10) copies/mL. At multivariable analysis, a greater mean CD4 decrease was associated with non-R5 viral tropism (-159.9 ± 12.22, P = 0.0002) at baseline. Other significant covariates were female gender, older age, intravenous drug use, longer duration of follow-up, and higher CD4 cell count and higher HIV-RNA at sample collection. CONCLUSIONS In patients with CD4 counts ≥350 cells/μL, non-R5 viral tropism by geno2pheno is predictive of CD4 decrease independent of their viral set point and CD4 counts.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co‐infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients. J. Med. Virol. 82:153–156, 2010. © 2009 Wiley‐Liss, Inc.

To understand the dynamic viral evolution observed during failure on raltegravir-containing regimens, we studied the genotypic and phenotypic patterns of resistance to raltegravir and the residual replication capacity (rRC) of HIV-1 variants selected in vivo.Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4-24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients' plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells.In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC(50)) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants.Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

During the recent severe acute respiratory syndrome (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M isolate. By using both real-time reverse transcription-polymerase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 mg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and bioloaic features.

Human immunodeficiency virus (HIV) -infected patients with HIV RNA loads of < 50 copies/mL were followed-up for a median (interquartile range) of 30.8 (11.7–32.9) months to study the effect of residual viraemia (RV) on virological rebound (VR). At baseline, 446 (60.3%) patients had undetectable HIV RNA (group A) and 293 (39.7%) had RV (1–49 HIV RNA copies/mL, group B) by kinetic PCR. VR occurred in 4 (0.9%) patients in group A and in 12 (4.1%) patients in group B (p 0.007). Time to VR was shorter among patients of group B (Log-rank test: p 0.003). However, the proportion of VR was extremely low also among patients with RV.

Objectives: It is currently debated whether patients with residual viraemia are at higher risk of virological failure than those attaining ,1 HIV RNA copy/mL. We therefore investigated the effect of residual viraemia on virological rebound. Methods: We used a prospective, non-interventional, single-centre, study. This analysis was based on HIVinfected patients with two consecutive HIV RNA viral loads (VLs) of ,50 copies/mL as tested by Versant bDNA, followed by two HIV RNA VLs of ,50 copies/mL as tested using the Versant kinetic PCR molecular system (kPCR; limit of quantification ¼ 1 copy/mL). Virological rebound was defined as two consecutive HIV RNA values of .50 copies/mL after baseline, and the time to virological rebound was calculated using the Kaplan ‐ Meier method. Results: There were 739 eligible patients; 446 (60.4%) had HIV RNA ,1 copy/mL (group A) and 293 (39.6%) had residual viraemia (1 ‐49 HIV RNA copies/mL; group B). After a follow-up (median 48.9 weeks), virological rebound occurred in four patients in group A (0.9%) and six patients in group B (2%); the time to virological rebound was similar in the two groups (log-rank test P ¼ 0.231). CD4+ cell recovery (slope) was significantly less in the patients with residual viraemia; +14.3 (27.7, 43.9) cells/mm 3 per year versus +21.2 (22.5, 53.2) cells/mm 3 per year; P ¼0.036. Conclusions: Residual viraemia assessed by kPCR was not associated with virological rebound during 1 year of follow-up. However, the patients attaining ,1 HIV RNA copy/mL showed a small but statistically significant improvement in CD4+ cell recovery.

Objective : Evaluate HIV-1 subtype B integrase gene evolution in patients failing raltegravir (RAL)-based savage regimens by clonal analysis of the replicating viral quasispecies. Design : Seven triple class failure HIV-1 (subtype B)-infected patients, followed at San Raffaele Hospital and enrolled in the RAL Expanded Access Program (MK0518-023), were evaluated. Patients were followed up for 24-48 weeks and due to the absence of other active drugs, RAL was maintained in their regimens even if resistance mutations were detected. Methods : Immunologic and virologic parameters were recorded every 4 weeks, and amplification and clonal analysis of viral populations were performed at baseline and every 4-12 weeks in all patients. Results : Resistance to RAL appeared initially associated with selection of single variants (Y143R, Q148R N155H) in the majority of patients; however, in three patients, complex patterns of viral mutations were observed. The clonal analysis of viral quasispecies allowed to describe the evolution of each viral population and the progressive accumulation of RAL resistance-associated mutations and polymorphisms associated with therapy failure. Conclusion : The complex patterns of resistance mutations observed, including novel variants evolved under continuous RAL pressure, suggesting that they are the result of the equilibrium between drug resistance and enzyme function. Despite the efficacy of this compound, our data discourage its use in a functional monotherapy and maintaining RAL even in presence of RAL resistance-associated mutations may lead to the progressive formation of viral reservoirs with multiple integrase inhibitor-resistant variants that may limit the future efficacy of other integrase inhibitors due to cross-resistance.

The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.

A prospective 2‐year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co‐pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV‐OC43, 35.7% hCoV‐NL63, 21.4% hCoV‐HKU1, and 3.6% hCoV‐229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co‐infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P

Sequential nasopharyngeal aspirates from patients without respiratory symptoms undergoing hematopoietic stem cell transplantation (HSCT) were tested for genomic RNA of human metapneumovirus (hMPV). Persistent hMPV infection was documented in most of them and confirmed by virus isolation. hMPV infection etiology was also evaluated during the same period in samples from pediatric patients with acute respiratory diseases (ARDs). Sequence analysis of hMPV in HSCT recipients documented infection by hMPV genotype A and strong interhost similarity; this pattern differs from that observed in pediatric patients with ARDs. The data indicate that HSCT recipients may frequently develop symptomless hMPV infection.

To understand the evolutionary characteristics of HIV- 1 variants resistant to protease inhibitors ( PI), the replicating plasma virus was analyzed in three patients shifted to a PI- sparing regimen after virological failure. The dynamic features of carryover of mutations associated with PI resistance in the absence of selective pressure on the protease gene indicate that viral variants resistant to reverse transcriptase inhibitors and bearing mutations of the protease sequence can maintain efficient replication capacity in vivo.

In this single-centre, retrospective study, we analyzed data of 194 patients receiving antiretroviral therapy with

The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4–24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4 + T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process.