Francesco Saladini

A novel methodology for large-scale phylogeny partition

Phylogenetic analysis is used to identify transmission chains, but no software is available for the automated partition of large phylogenies. Prosperiet al. apply a new search algorithm to identify transmission clusters within the phylogeny of HIV-1gene sequences linking molecular and epidemiological data. Supplementary information The online version of this article (doi:10.1038/ncomms1325) contains supplementary material, which is available to authorized users.

Changing patterns in HIV-1 non-B clade prevalence and diversity in Italy over three decades*

HIV‐1 non‐B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non‐B clades and their association with demographic factors, over the entire course of the HIV‐1 epidemic, have not been fully investigated in Italy.

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Significance Human DEAD-box polypeptide 3 (DDX3) is an ATPase/RNA helicase involved in the replication of many viral pathogens. We reported herein the first inhibitor, to our knowledge, of the helicase binding site of DDX3 endowed with a broad spectrum antiviral activity [HIV-1 WT, HIV drug-resistant strains, Hepatitis C virus (HCV), Dengue virus (DENV), and West Nile virus (WNV)]. The good toxicity profile suggests that the DDX3 activity, although essential for viruses, could be dispensable to the cells, validating DDX3 as a pharmaceutical target. Our results clearly showed that DDX3 inhibitors could be exploited to treat HIV/HCV coinfections, emerging infectious diseases (such as DENV and WNV), and HIV-1 patients carrying drug-resistant strains. Each of these three medical conditions currently represents a major challenge for clinical treatment. Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors

Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I.

Use of Peripheral Blood DNA for Genotype Antiretroviral Resistance Testing in Drug-Naive HIV-Infected Subjects

Parallel analysis of peripheral blood mononuclear cell DNA and plasma RNA from 169 drug-naive human immunodeficiency virus-infected subjects revealed that evaluation of both compartments increases the sensitivity of detection of drug resistance-related mutations, compared with examination of either source alone. Peripheral blood mononuclear cell DNA may play a role in the surveillance of transmitted antiretroviral resistance.

Identification of a possible ancestor of the subtype A1 HIV Type 1 variant circulating in the former Soviet Union.

The HIV-1 subsubtype A1 epidemic of the former Soviet Union (FSU) was caused by an A1 variant apparently derived from a single introduction event. We identified an A1 variant highly related to the A1 lineage circulating in the FSU in a patient from Conakry, Republic of Guinea, who was diagnosed with HIV-1 when he moved to Italy in 2002. The most probable route of infection was two blood transfusions received in his country of origin in 1998. Full-length (9781 bp) molecular characterization revealed that this strain was evolutionarily parental to, but distinct from, the A1 lineage circulating in the FSU. Similar results were obtained analyzing partial genome sequences. A full-length sequence similarity plot and bootscanning analysis supported this evidence and excluded any potential recombination events with other HIV-1 variants. This viral strain represents the first evidence of an African patient infected by an A1 subtype related to the A1 variants spreading in FSU countries. The identification of this distinct A1 variant may support the origin of the Russian A epidemic from West Africa.

Prevalence of HIV-1 integrase mutations related to resistance to dolutegravir in raltegravir naïve and pretreated patients.

The prevalence of HIV-1 integrase mutations related to resistance to the next-generation integrase inhibitor (INI), dolutegravir (DTG), was assessed in 440 INI-naïve subjects and in 120 patients failing a raltegravir (RTG)-containing regimen. Of the mutations selected by DTG in vitro, S153FY was not detected in any isolate while L101I and T124A were highly prevalent in both groups and significantly associated with non-B subtype. RTG-selected double and triple mutants, mostly the G140S/Q148H variant, were detected in only 32 (26.7%) RTG-treated patients. As L101I and T124A do not appear to exert any major effect in vivo and double and triple mutants resistant to DTG are infrequently selected by RTG, DTG can be effectively used in INI-naïve patients and may retain activity in many patients failing RTG.

Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms

Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six different RNA-folding algorithms on the URF parental strains. These findings suggest that such hairpins may play a role in the higher recombination rates detected at these positions.

Agreement between an in-house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV-1 protease, reverse transcriptase, and integrase inhibitors

Although clinical management of drug resistance is routinely based on genotypic methods, phenotypic assays remain necessary for the characterization of novel HIV‐1 inhibitors, particularly against common drug‐resistant variants. We describe the development and assessment of the performance of a recombinant virus assay for measuring HIV‐1 susceptibility to protease (PR), reverse transcriptase (RT), and integrase (IN) inhibitors.

Natural NS5A inhibitor resistance associated substitutions in hepatitis C virus genotype 1 infected patients from Italy

OBJECTIVES Genetic variability in NS5A is associated with different levels of resistance to the currently licensed NS5A inhibitors. The aim of this study was to detect NS5A inhibitor resistance associated substitutions (RASs) in hepatitis C virus (HCV) genotype 1 (GT1) patients who are naive to direct-acting HCV antivirals. METHODS Amplification, Sanger sequencing and phylogenetic analysis of the HCV NS5A region were performed on plasma obtained from 122 consecutive patients with HCV chronic infection attending four different clinics in Italy. RESULTS NS5A inhibitor RASs were detected in 14/61 (23.0%) HCV GT1b and 3/61 (4.9%) HCV GT1a infected patients (p 0.007). The pan-genotypic RAS Y93H was detected in 1 (1.6%) GT1a and 4 (6.6%) GT1b patients. GT1a sequences clustered into two different clades with RASs detected in 1/34 (2.9%) clade I and 2/27 (7.4%) clade II sequences. CONCLUSIONS Although the impact of naturally occurring NS5A RASs might be limited with upcoming pan-genotypic treatment regimens, this information is still useful to map naturally occurring HCV variants in different geographic areas in the context of current HCV therapy.