Occupational HIV Infection in a Research Laboratory With Unknown Mode of Transmission: A Case Report.

A laboratory worker was infected with human immunodeficiency virus (HIV) type 1 in a biosafety level 2 containment facility, without any apparent breach. Through full-genome sequencing and phylogenetic analyses, we could identify the source of infection in a replication-competent clone that unknowingly contaminated a safe experiment. Mode of transmission remains unclear. Caution is warranted when handling HIV-derived constructs.

Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage.

Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression.

Next-generation sequencing provides an added value in determining drug resistance and viral tropism in Cameroonian HIV-1 vertically infected children.

With limited and low-genetic barrier drugs used for the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa, vertically transmitted HIV-1 drug-resistance (HIVDR) is concerning and might prompt optimal pediatric strategies.The aim of this study was to ascertain HIVDR and viral-tropism in majority and minority populations among Cameroonian vertically infected children.A comparative analysis among 18 HIV-infected children (7 from PMTCT-exposed mothers and 11 from mothers without PMTCT-exposure) was performed. HIVDR and HIV-1 co-receptor usage was evaluated by analyzing sequences obtained by both Sanger sequencing and ultra-deep 454-pyrosequencing (UDPS), set at 1% threshold.Overall, median (interquartile range) age, viremia, and CD4 count were 6 (4-10) years, 5.5 (4.9-6.0) log10 copies/mL, and 526 (282-645) cells/mm, respectively. All children had wild-type viruses through both Sanger sequencing and UDPS, except for 1 PMTCT-exposed infant harboring minority K103N (8.31%), born to a mother exposed to AZT+3TC+NVP. X4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS). Rate of X4-tropic viruses was 0% (0/6) below 5 years (also as minority species), and became relatively high above 5 years (55.6% [5/9], P = .040. X4-tropic viruses were higher with CD4 ≤15% (4/9 [44.4%]) versus CD4 >15% (1/6 [16.7%], P = .580); similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200 (2/11 [18.2%] cells/mm, P = .077.NGS has the ability of excluding NRTI- and NNRTI-mutations as minority species in all but 1 children, thus supporting the safe use of these drug-classes in those without such mutations, henceforth sparing ritonavir-boosted protease inhibitors or integrase inhibitors for the few remaining cases. In children under five years, X4-tropic variants would be rare, suggesting vertical-transmission with CCR5-tropic viruses and possible maraviroc usage at younger ages.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

BACKGROUND: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. METHODS: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. RESULTS: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

The HIV-1 reverse transcriptase polymorphism A98S improves the response to tenofovir disoproxil fumarate+emtricitabine-containing HAART both in vivo and in vitro.

The impact of baseline HIV-1 reverse transcriptase (RT) polymorphisms on response to first-line modern HAART containing tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) was evaluated. The impact of each RT polymorphism on virological success (VS) was evaluated in 604 HIV-1 subtype B-infected patients starting TDF+FTC-containing HAART. TDF and FTC antiviral activity was also tested in PBMCs infected by mutagenised HIV. Structural analysis based on docking simulations was performed. A98S was the only mutation significantly correlated with an increased proportion of patients achieving VS at 24 weeks (94.0% vs. 84.3%; P=0.03). Multivariate regression and Cox model analyses confirmed this result. At concentrations close to the minimal concentration achieved in patient plasma, TDF and FTC exhibited higher potency in the presence of A98S-mutated virus compared with wild-type (IC90,TDF, 8.6±1.1 vs. 19.3±3.5nM; and IC90,FTC, 12.4±7.7 vs. 16.8±9.8nM, respectively). The efficacy of FTC, abrogated by M184V, was partially restored by A98S (IC90,FTC, 5169±5931nM for A98S+M184V vs. 18477±12478nM for M184V alone). Docking analysis showed the higher potency of TDF and FTC in the presence of A98S-mutated virus was mainly due to higher binding affinity between drugs and mutated RT compared with wild-type. In the presence of FTC, A98S also partially restored the RT binding affinity impaired by M184V alone. A98S polymorphism improves virological response to TDF+FTC-containing HAART. This may help clinicians in the choice of the optimal NRTI backbone aimed at achieving maximal virological inhibition.

Recent Transmission Clustering of HIV-1 C and CRF17_BF Strains Characterized by NNRTI-Related Mutations among Newly Diagnosed Men in Central Italy.

BACKGROUND: Increased evidence of relevant HIV-1 epidemic transmission in European countries is being reported, with an increased circulation of non-B-subtypes. Here, we present two recent HIV-1 non-B transmission clusters characterized by NNRTI-related amino-acidic mutations among newly diagnosed HIV-1 infected men, living in Rome (Central-Italy). METHODS: Pol and V3 sequences were available at the time of diagnosis for all individuals. Maximum-Likelihood and Bayesian phylogenetic-trees with bootstrap and Bayesian-probability supports defined transmission-clusters. HIV-1 drug-resistance and V3-tropism were also evaluated. RESULTS: Among 534 new HIV-1 non-B cases, diagnosed from 2011 to 2014, in Central-Italy, 35 carried virus gathering in two distinct clusters, including 27 HIV-1 C and 8 CRF17_BF subtypes, respectively. Both clusters were centralized in Rome, and their origin was estimated to have been after 2007. All individuals within both clusters were males and 37.1% of them had been recently-infected. While C-cluster was entirely composed by Italian men-who-have-sex-with-men, with a median-age of 34 years (IQR:30-39), individuals in CRF17_BF-cluster were older, with a median-age of 51 years (IQR:48-59) and almost all reported sexual-contacts with men and women. All carried R5-tropic viruses, with evidence of atypical or resistance amino-acidic mutations related to NNRTI-drugs (K103Q in C-cluster, and K101E+E138K in CRF17_BF-cluster). CONCLUSIONS: These two epidemiological clusters provided evidence of a strong and recent circulation of C and CRF17_BF strains in central Italy, characterized by NNRTI-related mutations among men engaging in high-risk behaviours. These findings underline the role of molecular epidemiology in identifying groups at increased risk of HIV-1 transmission, and in enhancing additional prevention efforts.