Rapid spread of the novel respiratory syncytial virus A ON1 genotype, central Italy, 2011 to 2013.

Respiratory infections positive for human respiratory syncytial virus (RSV) subtype A were characterised in children admitted to hospitals in Rome and Ancona (Italy) over the last three epidemic seasons. Different strains of the novel RSV-A genotype ON1, first identified in Ontario (Canada) in December 2010, were detected for the first time in Italy in the following 2011/12 epidemic season. They bear an insertion of 24 amino acids in the G glycoprotein as well as amino acid changes likely to change antigenicity. By early 2013, ON1 strains had spread so efficiently that they had nearly replaced other RSV-A strains. Notably, the RSV peak in the 2012/13 epidemic season occurred earlier and, compared with the previous two seasons, influenza-like illnesses diagnoses were more frequent in younger children; bronchiolitis cases had a less severe clinical course. Nonetheless, the ON1-associated intensive care unit admission rate was similar, if not greater, than that attributable to other RSV-A strains. Improving RSV surveillance would allow timely understanding of the epidemiological and clinicopathological features of the novel RSV-A genotype.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010.

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.

Human case of autochthonous West Nile virus lineage 2 infection in Italy, September 2011.

On 10 September 2011, a patient in his 50s was admitted to hospital in Ancona, Italy, after six days of high fever and no response to antibiotics. West Nile virus (WNV) infection was suspected after tests to determine the aetiology of the fever were inconclusive. On 20 September, WNV-specific IgM and IgG antibodies were detected in the patient's serum. Genomic sequencing of the viral isolate showed that the virus belonged to WNV lineage 2.

Molecular surveillance of pandemic influenza A(H1N1) viruses circulating in Italy from May 2009 to February 2010: association between haemagglutinin mutations and clinical outcome.

Haemagglutinin sequences of pandemic influenza A(H1N1) viruses circulating in Italy were examined, focusing on amino acid changes at position 222 because of its suggested pathogenic relevance. Among 169 patients, the D222G substitution was detected in three of 52 (5.8%) severe cases and in one of 117 (0.9%) mild cases, whereas the D222E mutation was more frequent and evenly distributed in mild (31.6%) and severe cases (38.4%). A cluster of D222E viruses among school children confirms reported human-to-human transmission of viruses mutated at amino acid position 222.

Intra-host evolution of human immunodeficiency virus type 1 and viral fitness.

RNA viruses are frequently tolerant to high levels of mutagenesis. In contrast, DNA viruses are less errorprone and coevolve along with their specific hosts over long time periods. Although both strategies have been successful, the "RNA-strategy" (directly linked to the pathogenic potential of these agents) most often generates novelty (new variants, new strains, and even new viral pathogens). For several decades, intra-host virus evolution has been considered to be a speculative field, far from the main issues of clinical virology. This concept is now changed, due to the evidence that RNA virus evolution is intimately linked to failures in viral disease control and prevention. Antiviral strategies using single and fixed elements (i.e. treatments using one antiviral compound, immunizations using a single recombinant protein) have been unable to control highly dynamic quasispecies, such as human immunodeficiency virus type I (HIV-1) and hepatitis C virus (HCV). The development of combinatorial treatments in HIV-1 infection and the recognition that vaccines should be multivalent are important steps in adapting disease control strategies to the complexity of viral populations. The present report summarizes the strategies adopted to address HIV-1 evolution and its phenotypic consequences, including changes in susceptibility to antiviral compounds, viral fitness, and pathogenic potential. In particular, it is highlighted that sequence-function analyses of the intra-host HIV-I evolution, including studies of viral fitness, have opened up new perspectives not only to studying the pathogenic mechanisms and the virus-host relationships, but also to designing new strategies for monitoring antiviral therapies.

Resistance and replicative capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treatment.

Enfuvirtide is the prototype member of a new class of anti HIV-1 agents, the fusion inhibitors (FI). In recent clinical trials, the compound has shown its efficacy in combination with other antiretroviral agents in vivo. However mutant strains resistant to the action of the drug arise quite rapidly in vitro and in vivo. To analyze the process of selection and evolution of HIV-1 strains resistant to enfuvirtide in vivo and to evaluate the impact of resistance on viral fitness, 12 HIV-1 infected subjects treated with T20 (enfuvirtide) for at least one year were included in the study. Gp41-coding sequences were amplified from plasma samples of these subjects at baseline and at different time points during treatment. Seven of the 12 subjects showed selection of gp41 mutations under the selective pressure of enfuvirtide. In particular, these mutations clustered in two distinct regions: (i) a mutational hot-spot localized, as previously described, in the first residues of the N-HR domain, with position number 38 as the most heavily mutated, but including also a G36V, a N42D/T, a N43D, a L44M and a L45M; (ii) other mutations were localized further downstream, within N-HR/C-HR junction and in the C-HR. A recombinant assay specifically designed for the determination of HIV-1 phenotype to FI was developed and validated. Using this assay, we observed that all of the 7 mutated clones displayed substantially reduced susceptibility to T20, IC50 ranging from 0.6 to12.8 microg/ml (>100 fold change). The residues whose mutation was associated with a potent reduction in susceptibility were V38, N42, and N43, other positions such as G36, N44 and L45 playing a minor role. None of the mutant HIV isolates showed cross-resistance to T-1249. By the same method, the HIV-1 replicative capacity of the recombinant clones was tested in the absence of drugs, and for each subject, pre-therapy clones were compared to post-therapy ones. In 3/7 subjects a significant decrease in replicative capacity of the recombinant clones was observed. The phenotypic data from this study suggest that the secondary additional mutations, could be associated with improved resistance or recovery of replicative capacity (compensatory mutations).

Immune reconstitution in HIV-1 infected children with discordant response to antiretroviral therapies: patterns of HIV-1 env gene evolution driven by restoration of thymic function.

The evolution of human immunodeficiency virus type 1 (HIV-1) env gp120 region was addressed in HIV-1 infected children showing virological failure to antiretroviral therapy (ART). Sequence analysis of the replicating plasma virus at baseline and after one year of therapy documented evolution of gp120 in all subjects but one. Analysis of the host's selective pressure showed that the values of Ka/Ks ratios were higher in the V3 sequence than in the whole C2-V5 region in 4 of 5 children with improvement of thymic output. Moreover, in 2 of the 4 chidren, the V3 evolution paralleled with a reverse shift of the viral phenotype (from CXCR4-tropic to CCR5-tropic). These results suggest that, under ART, the V3 evolution towards less pathogenic viral variants may be driven by the host's increased selective pressure following restoration of thymic function and immune reconstitution.

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a multicenter cohort of HIV-1-infected Italian patients with virologic failure.

We evaluated the prevalence of both Q151M and 6-bp insert at position 69 of RT region responsible for multiple dideoxynucleoside analogue-resistant (MddNR) HIV-1 variants in 177 patients who failed to respond to combination therapy. Patients had received protease inhibitors (PI) and/or nonnucleoside reverse transcriptase inhibitors (NNRTIs) after a long-term experience with nucleoside reverse transcriptase inhibitors (NRTIs) (including zidovudine monotherapy). Two of 177 patients (1.1%) showed the specific complex of Q151M mutation, while 4 (2.3%) had the 69 6-bp insert. Mutations that belong to the 151 set in the absence of the pivotal Q151M substitution were detected in as many as 3.9% of the patients. One patient exhibited a 69S [VG] insert that has not been previously phenotypically characterized. This HIV-1 isolate had high levels of resistance to all NRTIs except stavudine. MddNR is an emerging problem after sequential therapy with this class of compounds among HIV-1-infected patients. Either didanosine (ddI) or zidovudine (ZDV) monotherapy allowed the emergence of MddNR variants containing Q151M complex. Monotherapy with ZDV and ddI or subsequent treatments with various NRTI combinations were the common background in the patients with the 69 insert. The overall prevalence of MddNR (3.4%) in Italy is comparable with that observed in several other European countries (3.4%-6.5%). These data suggest that patients failed by NRTI regimens should be analyzed for the presence of both patterns of MddNR.

Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy.

OBJECTIVE: To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). DESIGN AND METHODS: A non-replicative HIV-1 molecular vector (designated pdelta prodelta env) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pdelta prodelta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). RESULTS: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P = 0.0038 and P = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P = 0.022; ritonavir, P = 0.0466) were observed. CONCLUSION: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.

Complexity and dynamics of HIV-1 quasispecies.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediate virus entry into target cells by binding receptors of the cell membrane and fusing viral and cellular structures. In particular, recent crystallographic studies have clarified the complex role of the glycoprotein gp120 in the early phase of the infection. In this context the inter- and intra-host variability of the HIV-1 gp120 poses a major problem for the development of effective methods of immunization against this virus. In the present report, the relevant aspects emerging from the study of HIV-1 variability are addressed and several methodological approaches to evaluate HIV-1 diversity discussed.

Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection.

Deletion of the nef gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived clones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants.

Identification of two distinct subsets of long-term nonprogressors with divergent viral activity by stromal-derived factor 1 chemokine gene polymorphism analysis.

Stromal-derived factor (SDF)-1, the natural ligand for CXCR4, is present in a common polymorphic variant defined by a G-->A transition in the 3' untranslated region of the gene. In persons infected with human immunodeficiency virus type 1 (HIV-1), the homozygous genotype (SDF1-3'A/3'A) has been postulated to interfere with the appearance of T-tropic syncytium-inducing strains. The polymorphism of SDF1 was correlated with HIV-1 phenotype, plasma viremia, and unspliced and multiply spliced specific transcripts in 158 virologically characterized HIV-1-infected patients (39 recent seroconverters, 75 typical progressors, and 44 AIDS patients) and in 42 HIV-1-infected long-term nonprogressors (LTNPs). Analysis of SDF1 allele distribution revealed that SDF1-3'A/3'A status is associated with low CD4 cell count (P=.0449) but not with a specific HIV-1 phenotype. In LTNPs, SDF1-+/+ condition defined a subset of persons with lower HIV-1 replication than in heterozygous subjects. The low viral activity in SDF1-+/+ LTNPs suggests that other factors play a major role in vivo in determining the course of HIV-1 infection.

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution.

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.

A combination of nucleoside analogues and a protease inhibitor reduces HIV-1 RNA levels in semen: implications for sexual transmission of HIV infection.

Direct contact with semen is the major route of sexual acquisition of human immunodeficiency virus (HIV) in homosexual and heterosexual partners of seropositive men. In this study, we show that concentrations of HIV-1 RNA molecules in plasma and semen of seropositive patients are related to the duration and type of antiretroviral agents used in treatment. In patients treated with zidovudine alone, 1, 3 and 6 months after the start of therapy, the mean HIV-1 load in plasma was reduced by 0.57, 0.38 and 0.21 log10 and in semen by 0.66, 0.50 and 0.15 log10, respectively. In patients treated with zidovudine plus didanosine at months 1, 3 and 6, the mean decrease in plasma HIV-1 RNA was 1.40, 1.25 and 1.12 log10 and in semen 1.10, 1.41 and 1.32 log10, respectively. In patients treated with a combination of a protease inhibitor and two nucleoside analogues the mean log10 decrease was 1.77, 1.83, 1.71 and 2.38 log10 in plasma and 1.17, 1.74, 2.19 and 3.02 log10 in semen at 1, 2, 3 and 4 months, respectively. Treatment with a combination of a protease inhibitor and two nucleoside analogues caused a dramatic decrease in cell-free HIV-1 RNA in semen, which is a reliable measure of viral load. These findings could have implications for the sexual transmission of HIV-1.

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors.

OBJECTIVE: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. DESIGN AND METHODS: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. RESULTS: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. CONCLUSIONS: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Early increase of CD4+ CD45RA+ and CD4+ CD95- cells with conserved repertoire induced by anti-retroviral therapy in HIV-infected patients.

Administration of anti-retroviral drugs induces a decrease of viral load associated with increase of CD4+ cell count in most HIV-infected patients. To investigate the early changes in CD4+ cell phenotype induced by anti-retroviral therapy, six patients with CD4+ cell count > 100/mm3 and never treated with anti-HIV therapy were enrolled and blood samples collected several times within 14 days from the initiation of therapy with Zidovudine plus Didanosine. CD4+ cell count and HIV viraemia were investigated at each time point, as well as the expression of CD45RA, CD45RO and CD95/Fas molecules on CD4+ cells, and the T cell receptor (TCR) Vbeta repertoire of CD4+ cells. All patients showed a rapid and dramatic decrease in viral load with a corresponding increase of CD4+ cell count. The main remodelling of CD4+ cell subpopulations took place in the first 14 days of therapy, and consisted of: (i) increased CD4+CD45RA+/CD4+CD45RO+ ratio; (ii) decrease of CD95/Fas expression. The rise in absolute number of CD4+CD45RA+ cells was paralleled by an increase of CD4+CD95/Fas- cells and accounted for most of the early increment of CD4+ cell count. The TCR Vbeta repertoire of CD4+ cells was conserved after anti-HIV therapy, with the exception of two patients with expanded CD4+Vbeta12+ cells, which also tested CD45RA+ and CD95/Fas-. These experiments show that newcomer CD4+ lymphocytes are CD45RA+CD95/Fas- cells, suggesting that blocking HIV replication causes an early and antigen-independent proliferation of possibly 'naive' cells unprimed for CD95/Fas-mediated apoptosis. These cells expressed a conserved and widespread TCR repertoire, suggesting that their capability for antigenic recognition is intact.