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 < 0.001) and hypoxia (P < 0.015). When the presence of RSV alone and the RSV-hMPV co-infections were considered, RSV mono-infected patients resulted to have longer hospitalization and higher hypoxia (P < 0.001). The data highlight that (i) RSV has a central role as a respiratory pathogen of infants, (ii) the wide circulation of recently identified viruses does not reduce the clinical and epidemiological importance of RSV, and that (iii) recently identified agents (hMPVs, hBoVs, and hCoVs) act as primary pathogens or co-pathogens.

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

During HIV pathogenesis, infected macrophages behave as “viral reservoirs” that accumulate and retain virions within dedicated internal Virus-Containing Compartments (VCCs). The nature of VCCs remains ill characterized and controversial. Using wild-type HIV-1 and a replication-competent HIV-1 carrying GFP internal to the Gag precursor, we analyzed the biogenesis and evolution of VCCs in primary human macrophages. VCCs appear roughly 14 hours after viral protein synthesis is detected, initially contain few motile viral particles, and then mature to fill up with virions that become packed and immobile. The amount of intracellular Gag, the proportion of dense VCCs, and the density of viral particles in their lumen increased with time post-infection. In contrast, the secretion of virions, their infectivity and their transmission to T cells decreased overtime, suggesting that HIV-infected macrophages tend to pack and retain newly formed virions into dense compartments. A minor proportion of VCCs remains connected to the plasma membrane overtime. Surprisingly, live cell imaging combined with correlative light and electron microscopy revealed that such connections can be transient, highlighting their dynamic nature. Together, our results shed light on the late phases of the HIV-1 cycle and reveal some of its macrophage specific features.