Despite occasional reports of SARS-CoV-2 vertical transmission during pregnancy, the question of placental infection and its consequences for the newborn remain questionable. Here, we analyzed the placentas of 31 COVID-19-positive mothers by RT-PCR, immunohistochemistry and in situ hybridization. We only detected one case of placental infection, which was associated with intrauterine demise of the fetus. We then isolated and differentiated primary trophoblasts from non-pathological human placentas at term, and exposed them to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, we were not able to detect the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant four days after infection. As a mechanism of defense, we hypothesized that trophoblasts at term do not express ACE2 and TMPRSS, the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but the reported case raises concern about preterm infection.

ObjectiveATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs.MethodTwo recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments.ResultsTacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine.ConclusionABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response.

To improve insight in the drivers of local HIV-1 transmission in Belgium, phylogenetic, demographic, epidemiological and laboratory data from patients newly diagnosed between 2013 and 2015 were combined and analyzed. Characteristics of clustered patients, paired patients and patients on isolated branches in the phylogenetic tree were compared. The results revealed an overall high level of clustering despite the short time frame of sampling, with 47.6% of all patients having at least one close genetic counterpart and 36.6% belonging to a cluster of 3 or more individuals. Compared to patients on isolated branches, patients in clusters more frequently reported being infected in Belgium (95.1% vs. 47.6%; p < 0.001), were more frequently men having sex with men (MSM) (77.9% vs. 42.8%; p < 0.001), of Belgian origin (68.2% vs. 32.9%; p < 0.001), male gender (92.6% vs. 65.8%; p < 0.001), infected with subtype B or F (87.8% vs. 43.4%; p < 0.001) and diagnosed early after infection (55.4% vs. 29.0%; p < 0.001). Strikingly, Sub-Saharan Africans (SSA), overall representing 27.1% of the population were significantly less frequently found in clusters than on individual branches (6.0% vs. 41.8%; p < 0.001). Of the SSA that participated in clustered transmission, 66.7% were MSM and this contrasts sharply with the overall 12.0% of SSA reporting MSM. Transmission clusters with SSA were more frequently non-B clusters than transmission clusters without SSA (44.4% versus 18.2%). MSM-driven clusters with patients of mixed origin may account, at least in part, for the increasing spread of non-B subtypes to the native MSM population, a cross-over that has been particularly successful for subtype F and CRF02_AG. The main conclusions from this study are that clustered transmission in Belgium remains almost exclusively MSM-driven with very limited contribution of SSA. There were no indications for local ongoing clustered transmission of HIV-1 among SSA.

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