Changes in adenoviral chromatin organization precede early gene activation upon infection.

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

FEZ1 Plays Dual Roles in Early HIV-1 Infection by Independently Regulating Capsid Transport and Host Interferon-Stimulated Gene Expression.

Fasciculation and elongation factor zeta 1 (FEZ1), a multifunctional kinesin-1 adaptor, binds human immunodeficiency virus type 1 (HIV-1) capsids and is required for efficient translocation of virus particles to the nucleus to initiate infection. However, we recently found that FEZ1 also acts as a negative regulator of interferon (IFN) production and interferon-stimulated gene (ISG) expression in primary fibroblasts and human immortalized microglial cell line clone 3 (CHME3) microglia, a natural target cell type for HIV-1 infection. This raises the question of whether depleting FEZ1 negatively affects early HIV-1 infection through effects on virus trafficking or IFN induction or both. Here, we address this by comparing the effects of FEZ1 depletion or IFN-ß treatment on early stages of HIV-1 infection in different cell systems with various IFN-ß responsiveness. In either CHME3 microglia or HEK293A cells, depletion of FEZ1 reduced the accumulation of fused HIV-1 particles around the nucleus and suppressed infection. In contrast, various doses of IFN-ß had little to no effect on HIV-1 fusion or the translocation of fused viral particles to the nucleus in either cell type. Moreover, the potency of IFN-ß's effects on infection in each cell type reflected the level of induction of MxB, an ISG that blocks subsequent stages of HIV-1 nuclear import. Collectively, our findings demonstrate that loss of FEZ1 function impacts infection through its roles in two independent processes, as a direct regulator of HIV-1 particle transport and as a regulator of ISG expression. IMPORTANCE As a hub protein, fasciculation and elongation factor zeta 1 (FEZ1) interacts with a range of other proteins involved in various biological processes, acting as an adaptor for the microtubule (MT) motor kinesin-1 to mediate outward transport of intracellular cargoes, including viruses. Indeed, incoming HIV-1 capsids bind to FEZ1 to regulate the balance of inward/outward motor activity to ensure net forward movement toward the nucleus to initiate infection. However, we recently showed that FEZ1 depletion also induces interferon (IFN) production and interferon-stimulated gene (ISG) expression. As such, it remains unknown whether modulating FEZ1 activity affects HIV-1 infection through its ability to regulate ISG expression or whether FEZ1 functions directly, or both. Using distinct cell systems that separate the effects of IFN and FEZ1 depletion, here we demonstrate that the kinesin adaptor FEZ1 regulates HIV-1 translocation to the nucleus independently of its effects on IFN production and ISG expression.

Rapid identification of early infections in febrile patients after CD19 target CAR-T cell therapy for B-cell malignancies.

BACKGROUND: CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy stands out as a revolutionary intervention, exhibiting remarkable remission rates in patients with refractory/relapsed (R/R) B-cell malignancies. However, the potential side effects of therapy, particularly cytokine release syndrome (CRS) and infections, pose significant challenges due to their overlapping clinical features. Promptly distinguishing between CRS and infection post CD19 target CAR-T cell infusion (CTI) remains a clinical dilemma. Our study aimed to analyze the incidence of infections and identify key indicators for early infection detection in febrile patients within 30 days post-CTI for B-cell malignancies. METHODS: In this retrospective cohort study, a cohort of 104 consecutive patients with R/R B-cell malignancies who underwent CAR-T therapy was reviewed. Clinical data including age, gender, CRS, ICANS, treatment history, infection incidence, and treatment responses were collected. Serum biomarkers procalcitonin (PCT), interleukin-6 (IL-6), and C-reactive protein (CRP) levels were analyzed using chemiluminescent assays. Statistical analyses employed Pearson's Chi-square test, t-test, Mann-Whitney U-test, Kaplan-Meier survival analysis, Cox proportional hazards regression model, Spearman rank correlation, and receiver operating characteristic (ROC) curve analysis to evaluate diagnostic accuracy and develop predictive models through multivariate logistic regression. RESULTS: In this study, 38 patients (36.5%) experienced infections (30 bacterial, 5 fungal, and 3 viral) within the first 30 days of CAR T-cell infusion. In general, bacterial, fungal, and viral infections were detected at a median of 7, 8, and 9 days, respectively, after CAR T-cell infusion. Prior allogeneic hematopoietic cell transplantation (HCT) was an independent risk factor for infection (Hazard Ratio [HR]: 4.432 [1.262-15.565], P = 0.020). Furthermore, CRS was an independent risk factor for both infection ((HR: 2.903 [1.577-5.345], P < 0.001) and severe infection (9.040 [2.256-36.232], P < 0.001). Serum PCT, IL-6, and CRP were valuable in early infection prediction post-CAR-T therapy, particularly PCT with the highest area under the ROC curve (AUC) of 0.897. A diagnostic model incorporating PCT and CRP demonstrated an AUC of 0.903 with sensitivity and specificity above 83%. For severe infections, a model including CRS severity and PCT showed an exceptional AUC of 0.991 with perfect sensitivity and high specificity. Based on the aforementioned analysis, we proposed a workflow for the rapid identification of early infection during CAR-T cell therapy. CONCLUSIONS: CRS and prior allogeneic HCT are independent infection risk factors post-CTI in febrile B-cell malignancy patients. Our identification of novel models using PCT and CRP for predicting infection, and PCT and CRS for predicting severe infection, offers potential to guide therapeutic decisions and enhance the efficacy of CAR-T cell therapy in the future.

Natural IgG protects against early dissemination of vesicular stomatitis virus.

Neonatal Fc receptor (FcRn) recycles immunoglobulin G, and inhibition of FcRn is used clinically for treatment of autoimmune diseases. In this work, using the vesicular stomatitis virus (VSV) mouse infection model system, we determined the role of FcRn during virus infection. While induction of neutralizing antibodies and long-term protection of these antibodies was hardly affected in FcRn deficient mice, FcRn deficiency limited the amount of natural IgG (VSV-specific) antibodies. Lack of natural antibodies (nAbs) limited early control of VSV in macrophages, accelerated propagation of virus in several organs, led to the spread of VSV to the neural tissue resulting in fatal outcomes. Adoptive transfer of natural IgG into FcRn deficient mice limited early propagation of VSV in FcRn deficient mice and enhanced survival of FcRn knockout mice. In line with this, vaccination of FcRn mice with very low dose of VSV prior to infection similarly prevented death after infection. In conclusion we determined the importance of nAbs during VSV infection. Lack of FcRn limited nAbs and thereby enhanced the susceptibility to virus infection.

Highly sensitive SERS platform for pathogen analysis by cyclic DNA nanostructure@AuNP tags and cascade primer exchange reaction.

The capacity to identify small amounts of pathogens in real samples is extremely useful. Herein, we proposed a sensitive platform for detecting pathogens using cyclic DNA nanostructure@AuNP tags (CDNA) and a cascade primer exchange reaction (cPER). This platform employs wheat germ agglutinin-modified Fe3O4@Au magnetic nanoparticles (WMRs) to bind the E. coli O157:H7, and then triggers the cPER to generate branched DNA products for CDNA tag hybridization with high stability and amplified SERS signals. It can identify target pathogens as low as 1.91 CFU/mL and discriminate E. coli O157:H7 in complex samples such as water, milk, and serum, demonstrating comparable or greater sensitivity and accuracy than traditional qPCR. Moreover, the developed platform can detect low levels of E. coli O157:H7 in mouse serum, allowing the discrimination of mice with early-stage infection. Thus, this platform holds promise for food analysis and early infection diagnosis.

Multi-omic profiling reveals early immunological indicators for identifying COVID-19 Progressors.

We sought to better understand the immune response during the immediate post-diagnosis phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14-CD16+ and intermediate CD14+CD16+ monocytes. In lymphocytes, the CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. These early stage observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19. BACKGROUND: Much of the literature on immune response post-SARS-CoV-2 infection has been in the acute and post-acute phases of infection. TRANSLATIONAL SIGNIFICANCE: We found differences at early time points of infection in approximately 160 participants. We compared multi-omic signatures in immune cells between individuals progressing to needing more significant medical intervention and non-progressors. We observed widespread evidence of a state of increased inflammation associated with progression, supported by a range of epigenomic, transcriptomic, and proteomic signatures. The signatures we identified support other findings at later time points and serve as the basis for prognostic biomarker development or to inform interventional strategies.

Liver transplantation for autoimmune hepatitis: Pre-transplant does not predict the early post-transplant outcome.

BACKGROUND AND AIMS: Autoimmune hepatitis (AIH) is a rare indication (<5%) for liver transplantation (LT). The aim of this study was to describe the early outcome after LT for AIH. METHODS: A multicenter retrospective nationwide study including all patients aged ≥16 transplanted for AIH in France was conducted. Occurrences of biliary and vascular complications, rejection, sepsis, retransplantation and death were collected during the first year after LT. RESULTS: A total of 344 patients (78.8% of women, 17.0% of (sub)fulminant hepatitis and 19.2% of chronic liver diseases transplanted in the context of acute-on-chronic liver failure [ACLF]) were included, with a median age at LT of 43.6 years. Acute rejection, sepsis, biliary and vascular complications occurred in respectively 23.5%, 44.2%, 25.3% and 17.4% of patients during the first year after LT. One-year graft and patient survivals were 84.3% and 88.0% respectively. The main cause of early death was sepsis. Pre-LT immunosuppression was not associated with an increased risk for early infections or surgical complications. Significant risk factors for septic events were LT in the context of (sub)fulminant hepatitis or ACLF, acute kidney injury at the time of LT (AKI) and occurrence of biliary complications after LT. AKI was the only independent factor associated with graft (HR = 2.5; 95% CI: 1.1-5.4; p = .02) and patient survivals (HR = 2.6; 95% CI: 1.0-6.5; p = .04). CONCLUSION: Early prognosis is good after LT for AIH and is not impacted by pre-LT immunosuppression but by the presence of AKI at the time of LT.

Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato.

Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt in host plants, a particularly serious problem in potato cultivation. Several pathogenicity-related proteins play important roles in the host infection process, hence, identifying such proteins, especially those with unknown functions, will surely aid in understanding the mechanism responsible for the pathogenesis of the fungus. Here, tandem mass tag (TMT) was used to quantitatively analyze the differentially expressed proteins in V. dahliae during the infection of the susceptible potato cultivar "Favorita". Potato seedlings were infected with V. dahliae and incubated for 36 h, after which 181 proteins were found to be significantly upregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that most of these proteins were involved in early growth and cell wall degradation. The hypothetical, secretory protein with an unknown function, VDAG_07742, was significantly upregulated during infection. The functional analysis with knockout and complementation mutants revealed that the associated gene was not involved in mycelial growth, conidial production, or germination; however, the penetration ability and pathogenicity of VDAG_07742 deletion mutants were significantly reduced. Therefore, our results strongly indicate that VDAG_07742 is essential in the early stage of potato infection by V. dahliae.

[Report on COVID-19 infection prevention and control training sessions for care homes provided by Kawaguchi city and comparison with those provided by the local governments in Japan].

Objectives　We compared COVID-19 prevention and control information provided to care homes (CHs) by the Kawaguchi City public health center (PHC), which utilizes our precedent advice on nfection, with the information from several local governments (LGs) in Japan. This study aimed to highlight the role of LG-associated doctors in providing information to CHs, utilizing their precedent advice on infection control in CHs and medical facilities. This study analyzed the sector and type of information the LGs should provide to CHs to prevent and control COVID-19.Methods　We compared training sessions on COVID-19 prevention and control information provided to CHs by the Kawaguchi City PHC with training sessions offered by several other LGs in Japan that are available on their websites.Results　The Kawaguchi City PHC has been providing COVID-19 information to CHs when needed, including prevention and control information, through their doctors, utilizing our precedent advice on infection control, management of health conditions of staff and residents, and early detection of COVID-19. In contrast, 68 LGs announced that they have provided training sessions to CHs for the prevention and control of COVID-19 through their official homepages from March to September 2022. These training sessions involved information dissemination by infection control specialist nurses (42.6%), clinic or hospital doctors (32.4%), infection control specialist doctors (11.8%), and staff from LG headquarters, PHC, or LG-associated doctors (51.5%). Among the 68 LGs, 41 provided information that included hand hygiene (95.1%), personal protective equipment (92.7%), proper ventilation (51.2%), and management of staff (90.2%) and resident (58.5%) health conditions. Furthermore, Kawaguchi City PHC and several LGs provided information for the early detection of COVID-19.Conclusion　We suggest that LGs provide COVID-19 training sessions conducted by LG doctors that include management of staff and resident health conditions, provision of early detection information, and utilization of precedent advice on infection in CHs and medical facilities.

Long-acting granulocyte colony-stimulating factor in primary prophylaxis of early infection in patients with newly diagnosed multiple myeloma.

PURPOSE: This study sought to compare the efficacy of prophylactic long-acting and standard granulocyte colony-stimulating factor (G-CSF) on febrile neutropenia, early infections, and treatment delay in patients with newly diagnosed multiple myeloma (MM) receiving the therapeutic regimen of bortezomib, lenalidomide, and dexamethasone (VRd). METHODS: A prospective study with 68 consecutive patients with MM was conducted in three regional hospitals. Participants were randomly treated with the VRd regimen in combination with prophylactic long-acting G-CSF (treatment group) or prophylactic standard G-CSF (control group). The primary endpoints were the incidence rates of febrile neutropenia, early infection, and treatment delays. The secondary endpoint was clinical outcomes. RESULTS: Thirty-three patients were assigned to the treatment group, and thirty-five patients were assigned to the control group. The incidence of febrile neutropenia was 6.1% and 17.1% in the treatment and control groups, respectively (p = 0.297). However, the rates of early infection and treatment delay were markedly lower in the treatment group than in the control group (6.1% vs. 25.7% and 9.1% vs. 31.4%; p < 0.05). Notably, all early infections occurred during the first four cycles of VRd therapy, and the most common type of infection was pneumonia. No significant difference in clinical efficacy was found between the two groups. All participants achieved at least partial remission. CONCLUSIONS: Prophylactic administration of domestic long-acting G-CSF markedly reduced the rates of early infection and treatment delay as compared with standard G-CSF in patients newly diagnosed with MM. Notably, all early infections occurred during the first four cycles of VRd therapy. As such, it seems appropriate to administer long-acting G-CSF with the aim of primary prophylaxis of early infection in the setting of newly diagnosed MM.

Significance of initiating antiretroviral therapy in the early stage of HIV infection.

A growing number of guidelines now recommend that human immunodeficiency virus (HIV) infected patients should be given early antiretroviral therapy (ART), especially in acute HIV infection. ART during early infection can limit viral reservoirs and improve immune cell function. From a societal prospect, early-infected individuals who achieve a state of viral suppression through ART can reduce the chance of HIV transmission and reduce the acquired immunodeficiency syndrome (AIDS)-related disease burden. However, there are many problems in the early diagnosis and treatment of HIV infection, including personal and social factors, which hinder the implementation and development of early treatment. It is recommended that initiating ART in the early stage of HIV infection, combined with other treatment strategies, so as to achieve functional cure.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

Impact of Antiretroviral Therapy Duration on HIV-1 Infection of T Cells within Anatomic Sites.

Understanding the impact of antiretroviral therapy (ART) duration on HIV-infected cells is critical for developing successful curative strategies. To address this issue, we conducted a cross-sectional/inter-participant genetic characterization of HIV-1 RNA from pre- and on-therapy plasmas and HIV-1 DNA from CD4+ T cell subsets derived from peripheral blood (PB), lymph node (LN), and gut tissues of 26 participants after 3 to 17.8 years of ART. Our studies revealed in four acute/early participants who had paired PB and LN samples a substantial reduction in the proportion of HIV-infected cells per year on therapy within the LN. Extrapolation to all 12 acute/early participants estimated a much smaller reduction in the proportion of HIV-1-infected cells within LNs per year on therapy that was similar to that in the participants treated during chronic infection. LN-derived effector memory T (TEM) cells contained HIV-1 DNA that was genetically identical to viral sequences derived from pre- and on-therapy plasma samples. The proportion of identical HIV-1 DNA sequences increased within PB-derived TEM cells. However, the infection frequency of TEM cells in PB was stable, indicating that cellular proliferation that compensates for T cell loss over time contributes to HIV-1 persistence. This study suggests that ART reduces HIV-infected T cells and that clonal expansion of HIV-infected cells maintains viral persistence. Importantly, LN-derived TEM cells are a probable source of HIV-1 genomes capable of producing infectious HIV-1 and should be targeted by future curative strategies.IMPORTANCE HIV-1 persists as an integrated genome in CD4+ memory T cells during effective therapy, and cessation of current treatments results in resumption of viral replication. To date, the impact of antiretroviral therapy duration on HIV-infected CD4+ T cells and the mechanisms of viral persistence in different anatomic sites is not clearly elucidated. In the current study, we found that treatment duration was associated with a reduction in HIV-infected T cells. Our genetic analyses revealed that CD4+ effector memory T (TEM) cells derived from the lymph node appeared to contain provirus that was genetically identical to plasma-derived virions. Moreover, we found that cellular proliferation counterbalanced the decay of HIV-infected cells throughout therapy. The contribution of cellular proliferation to viral persistence is particularly significant in TEM cells. Our study emphasizes the importance of HIV-1 intervention and provides new insights into the location of memory T cells infected with HIV-1 DNA, which is capable of contributing to viremia.

HSP/HSC70 activity is required for Bombyx mori nucleopolyhedrovirus replication at the early infectious phase.

Bombyx mori nucleopolyhedrovirus caused large amounts of silk loss annually, although it also could be used as silkworm bioreactor expression vector effectively and efficiently. Many heat shock (cognate) proteins 70 (HSP/HSC70) were induced by baculovirus and found existence in viral structure assembly. However, the concrete mechanism still need further elucidation for understanding host and virus interaction. In this study, the application of HSP/HSC70 inhibitor VER155008 is virus infectious phase-dependent for figuring out the role of intact molecular chaperone HSP/HSC70 activity in different stages of BmNPV proliferation progress. All the data had shown that HSP/HSC70 played a vital role in viral genome replication, virus protein abundance, BmNPV proliferation and budded virus production at the early infectious phase. This finding may provide new insights to unravel the interaction between baculovirus and silkworm in the initial infectious stage.

Brucella abortus-infected platelets modulate the activation of neutrophils.

Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Platelets (PLTs) have been widely involved in the modulation of the immune response. We have previously reported the modulation of Brucella abortus-mediated infection of monocytes. As a result, PLTs cooperate with monocytes and increase their inflammatory capacity, promoting the resolution of the infection. Extending these results, in this study we demonstrate that patients with brucellosis present slightly elevated levels of complexes between PLTs and both monocytes and neutrophils. We then assessed whether PLTs were capable of modulating functional aspects of neutrophils. The presence of PLTs throughout neutrophil infection increased the production of interleukin-8, CD11b surface expression and reactive oxygen species formation, whereas it decreased the expression of CD62L, indicating an activated status of these cells. We next analyzed whether this modulation was mediated by released factors. To discriminate between these options, neutrophils were treated with supernatants collected from B. abortus-infected PLTs. Our results show that CD11b expression was induced by soluble factors of PLTs but direct contact between cell populations was needed to enhance the respiratory burst. Additionally, B. abortus-infected PLTs recruit polymorphonuclear (PMN) cells to the site of infection. Finally, the presence of PLTs did not modify the initial invasion of PMN cells by B. abortus but improved the control of the infection at extended times. Altogether, our results demonstrate that PLTs interact with neutrophils and promote a proinflammatory phenotype which could also contribute to the resolution of the infection.

Outcome of COVID-19 in liver transplant recipients: A preliminary report from Northwestern Italy.

Covid-19 pandemic is deeply affecting transplant activity worldwide. It is unclear whether solid organ transplant recipients are at increased risk of developing severe complications and how they should be managed, also concerning immunosuppression. This is a report about the course and management of SARS-CoV-2 infection in liver transplant recipients from a single center in Northwestern Italy in the period March-April 2020. Three patients who were treated at our institution are reported in detail, whereas summary data are provided for those managed at peripheral Hospitals. Presentation varied from asymptomatic to rapidly progressive respiratory failure due to bilateral interstitial pneumonia. Accordingly, treatment and changes to immunosuppression were adapted to the severity of the disease. Overall mortality was 20%, whereas Covid-related mortality was 10%. Two cases of prolonged (>2 months) viral carriage were observed in two asymptomatic patients who contracted the infection in the early course after transplant. Besides depicting Covid-19 course and possible treatment scenarios in liver transplant patients, these cases are discussed in relation to the changes in our practice prompted by Covid-19 epidemic, with potential implications for other transplant programs.

Ledipasvir/Sofosbuvir for 8 Weeks to Treat Acute Hepatitis C Virus Infections in Men With Human Immunodeficiency Virus Infections: Sofosbuvir-Containing Regimens Without Interferon for Treatment of Acute HCV in HIV-1 Infected Individuals.

BACKGROUND: Current guidelines for the management of hepatitis C virus (HCV) infections provide varying recommendations for the optimal treatment of acute HCV infections. There are limited data from small cohort studies to provide guidance on the best approach to treatment of this important patient population. METHODS: Sofosbuvir-Containing Regimens Without Interferon for Treatment of Acute HCV in HIV-1 Infected Individuals is an open-label, 2-cohort, Phase 1 clinical trial in which the second cohort assessed the safety and efficacy of 8 weeks of ledipasvir/sofosbuvir for the treatment of acute HCV infections in participants with chronic human immunodeficiency virus (HIV)-1 infections. This final analysis of the second cohort had a planned accrual of 27 participants, based on non-inferiority criteria, compared to the study-defined, historical, sustained virologic response (SVR) of 60% with pegylated-interferon/ribavirin. RESULTS: We enrolled 27 men (9 Hispanic; 11 White, non-Hispanic; 5 Black, non-Hispanic; 2 Asian or Pacific Islander; median age 46 years). Most (96%) had HCV genotype-1 infection and 59% had the favorable interleukin 28B CC genotype. The median baseline HCV RNA load was 6.17 log10 IU/mL (interquartile range 4.51 - 6.55). All participants (100%) achieved the primary outcome of a sustained virologic response 12 weeks after the date of the last dose of study treatment (90% confidence interval 90-100%), achieving non-inferiority versus the 60% historic benchmark. No treatment discontinuations occurred. CONCLUSIONS: This multicenter clinical trial, investigating 8 weeks of ledipasvir/sofosbuvir for acute HCV infections in men with HIV infections, reports a 100% SVR. This study provides the rationale for larger studies of shortened courses of direct-acting antiviral therapies in persons with HIV infections, including those with high baseline HCV RNA loads. CLINICAL TRIALS REGISTRATION: NCT02128217.

Central Nervous System Inflammation and Infection during Early, Nonaccelerated Simian-Human Immunodeficiency Virus Infection in Rhesus Macaques.

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neurological AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this nonaccelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4+ T cell counts mirrored early human immunodeficiency virus (HIV) infection in humans. At 12 weeks postinfection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+ CD4- cellular infiltrate in the brain parenchyma without a concomitant increase in CD68/CD163+ monocytes, macrophages, and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAScope in situ hybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF are predominantly mediated by T cell-mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late-stage disease utilizing SIV. In the era of antiretroviral therapy, manifestations of late-stage HIV are less common. Furthermore, new interventions, such as monoclonal antibodies and therapeutic vaccinations, target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with SHIV expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans and that early neurologic inflammation is characterized by predominantly T cell-mediated inflammation accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end-stage disease.

Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection.

Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1-/- and SUN2-/- cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection.IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis.

Unveiling the early events of Pseudomonas aeruginosa adaptation in cystic fibrosis airway environment using a long-term in vitro maintenance.

Pseudomonas aeruginosa chronic infections are the major cause of high morbidity and mortality in cystic fibrosis (CF) patients due to the use of sophisticated mechanisms of adaptation, including clonal diversification into specialized CF-adapted phenotypes. In contrast to chronic infections, very little is known about what occurs after CF lungs colonization and at early infection stages. This study aims to investigate the early events of P. aeruginosa adaptation to CF environment, in particular, to inspect the occurrence of clonal diversification at early stages of infection development and its impact on antibiotherapy effectiveness. To mimic CF early infections, three P. aeruginosa strains were long-term grown in artificial sputum (ASM) over 10 days and phenotypic diversity verified through colony morphology characterization. Biofilm sub- and inhibitory concentrations of ciprofloxacin were applied to non- and diversified populations to evaluate antibiotic effectiveness on P. aeruginosa eradication. Our results demonstrated that clonal diversification might occur after ASM colonization and growth. However, this phenotypic diversification did not compromise ciprofloxacin efficacy in P. aeruginosa eradication since a biofilm minimal inhibitory dosage would be applied. The expected absence of mutators in P. aeruginosa populations led us to speculate that clonal diversification in the absence of ciprofloxacin treatments could be driven by niche specialization. Yet, biofilm sub-inhibitory concentrations of ciprofloxacin seemed to overlap niche specialization as "fitter" variants emerged, such as mucoid, small colony and pinpoint variants, known to be highly resistant to antibiotics. The pathogenic potential of all emergent colony morphotypes-associated bacteria, distinct from the wild-morphotypes, revealed that P. aeruginosa evolved to a non-swimming phenotype. Impaired swimming motility seemed to be one of the first evolutionary steps of P. aeruginosa in CF lungs that could pave the way for further adaptation steps including biofilm formation and progress to chronic infection. Based on our findings, impaired swimming motility seemed to be a candidate to disease marker of P. aeruginosa infection development. Despite our in vitro CF model represents a step forward towards in vivo scenario simulation and provided valuable insights about the early events, more and distinct P. aeruginosa strains should be studied to strengthen our results.