Decreased Replication Capacity Research Articles

Virological characteristics of the SARS-CoV-2 BA.2.86 variant

In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both invitro and invivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.

Why is the SARS-CoV-2 Omicron variant milder?

Why is the SARS-CoV-2 Omicron variant milder?

Prevalence of the entecavir-resistance-inducing mutation rtA186T in a large cohort of Chinese hepatitis B virus patients

This study aimed to clarify whether rtA186T and rtI163V substitutions of hepatitis B virus (HBV) contributed to entecavir (ETV) resistance. A total of 22,009 Chinese patients with chronic HBV infection who received resistance testing at Beijing 302 Hospital from 2007 to 2016 were enrolled. Among them, 6170 patients had been treated with ETV. The HBV reverse transcriptase gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility. Classical ETV-resistance mutations rtT184/S202/M250substitution+rtM204V/I±L180M (LAM-r), rtA186T, and rtI163V were detected in 1252 (5.69%), 14 (0.06%), and 230 (1.05%) of the 22,009 patients, respectively. The rtA186T mutation always coexisted with LAM-r, but not with rtI163V. The 14 rtA186T-positive patients were all treated with LAM and ETV, and the emergence of the rtA186T+LAM-r was closely associated with virological breakthrough or inadequate virological response to ETV. By contrast, the emergence of rtI163V was not related to ETV treatment. Six rtA186T-positive patients were followed up longitudinally, showing that these patients all had received sequential adefovir and LAM monotherapies prior to ETV treatment. Compared to wild-type strain, two patient-derived mutants' rtL180M+A186T+M204V and rtL180M+T184S+A186T+M204V had 86.7% and 89.2% decreased replication capacity, 210- and 555-fold increased ETV resistance, respectively; and artificial elimination of rtA186T largely restored their ETV sensitivity. The rtA186T mutants remained sensitive to tenofovir. In conclusion, our study confirmed that rtA186T plus LAM-r is a novel ETV-resistance mutation pattern which conferred ETV resistance in multiple Chinese patients.

Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site.

Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.

Differential Impact of Resistance-Associated Mutations to Protease Inhibitors and Nonnucleoside Reverse Transcriptase Inhibitors on HIV-1 Replication Capacity

The effects of drug resistance on HIV-1 replication capacity have been studied, but data from clinical isolates are few. We accessed the patients with HIV-1 infection at the National Taiwan University Hospital who experienced virological failure. Genotypic susceptibility and replication capacity of clinical HIV-1 isolates were measured. There were 80 patients enrolled between September 2007 and August 2010. The HIV-1 replication capacity declined significantly with the increasing number of major resistance-associated mutations (RAMs) to protease inhibitors (PIs) (p<0.001); however, it did not decline significantly with the increasing RAMs to first-line nonnucleoside analogue reverse transcriptase inhibitors (NNRTIs) (p=0.098). Regarding the effects of resistance to antiretroviral drugs in salvage therapy, decreased replication capacity was noted with the increasing RAMs to darunavir/ritonavir (p<0.001) and specific RAMs (L100I, K101P, and Y181C/I/V) to etravirine (p<0.001). Although NNRTI-related RAMs have less remarkable effects, both PI- and NNRTI-related RAMs reduced replication capacity, especially RAMs to darunavir/ritonavir and etravirine, which are commonly used in salvage therapy for treatment of patients infected with highly resistant HIV. Thus, decreased viral fitness during the emergence of RAMs suggests the importance of continued optimal antiretroviral treatment even when virological failure was noted.

Hepatocytes polyploidization and cell cycle control in liver physiopathology.

Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels), oxidative stress, toxic insult, and chronic hepatitis etc.). Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

The YMDD and rtA194T Mutations Result in Decreased Replication Capacity in Wild-Type HBV as well as in HBV with Precore and Basal Core Promoter Mutations

A recent study indicated that addition of the hepatitis B e antigen (HBeAg) precore (PC) or basal core promoter (BCP) mutations to wild-type HBV offset the reduced replication of the HBV polymerase rtA194T±rtL180M+rtM204V mutations. rtA194T was reportedly associated with tenofovir resistance. We investigated these findings in genotype D HBV, where both PC and BCP naturally occur in vivo. A plasmid containing a wild-type 1.3 genome length genotype D HBV laboratory strain was used as a parent for PC, BCP, rtA194T±rtL180M+rtM204V, rtL180M+rtM204V and rtM204I mutants. Viral replication was evaluated by Southern blot analysis of intracellular HBV core DNA following transient transfection of HepG2 cells. Drug susceptibility was evaluated by quantitative PCR of intracellular HBV DNA. PC and BCP mutations each increased HBV DNA replication by approximately 200% over wild-type. rtA194T reduced replication by <40%, whereas rtL180M+rtM204V, rtL180M+rtA194T+rtM204V or rtM204I each reduced by >75% from their respective wild-type, PC or BCP genome backbone (P<0.05). The enhanced replication by PC or BCP offset the reduction by rtA194T; however, the other reverse transcriptase (RT) mutations in PC or BCP backbones remained significantly lower than wild-type (P<0.05). Regardless of the backbone, rtA194T±rtL180M+rtM204V remained susceptible to tenofovir in vitro. rtA194T alone remained susceptible to lamivudine, while rtL180M+rtM204V and rtL180M+rtA194T+rtM204V were resistant. PC or BCP mutations increased HBV DNA replication, offset the decreased replication by rtA194T alone, but they did not fully rescue the impaired replication conferred by other RT mutations as compared with wild-type. rtA194T±rtL180M+rtM204V did not confer tenofovir resistance.

Polyploidy and liver proliferation

Organisms containing an increase in DNA content by whole number multiples of the entire set of chromosomes are defined as polyploid. Cells that contain more than two sets of chromosomes were first observed in plants about a century ago, and it is now recognized that polyploid cells form in many eukaryotes under a wide variety of circumstances. Although it is less common in mammals, some tissues, including the liver, show a high percentage of polyploid cells. Thus, during post-natal growth, the liver parenchyma undergoes dramatic changes characterized by gradual polyploidization during which hepatocytes of several ploidy classes emerge as a result of modified cell-division cycles. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence and to both lead to a progressive loss of cell pluripotency and to a markedly decreased replication capacity. In adults, liver polyploidization is differentially regulated upon loss of liver mass and liver damage. Here we review the current state of understanding about how polyploidization is regulated during normal and pathological liver growth, and detail by which mechanisms hepatocytes become polyploid.

Reverse transcriptase mutation K65N confers a decreased replication capacity to HIV-1 in comparison to K65R due to a decreased RT processivity

In addition to K65R, the other mutation observed at HIV-1 RT codon 65 is K65N. While K65N appears to have a phenotypic effect similar to K65R, it is less frequent during clinical trials. We compared the relative impact of K → N with respect to K → R change on viral replication capacity (RC). Mutant viruses were created and replication kinetics assays were performed in PBM cells. Analysis of RCs revealed a significant loss in replication ( p = 0.004) for viruses containing K65N mutation in comparison to those with K65R mutation. RT processivity assays showed a significant decrease in the processivity of K65N RT in comparison to K65R RT. We demonstrated that the significant decrease in RC of K65N viruses is related to the impaired RT processivity of K65N RT in comparison to K65R, and that the selection of the K65R mutation may be favored in clinical use of antiretroviral drugs compared to K65N.

Reduced Replication Capacity of NL4-3 Recombinant Viruses Encoding Reverse Transcriptase–Integrase Sequences From HIV-1 Elite Controllers

Identifying viral and host determinants of HIV-1 elite control may help inform novel therapeutic and/or vaccination strategies. Previously, we observed decreased replication capacity in controller-derived viruses suggesting that fitness consequences of human leukocyte antigen (HLA) class I-associated escape mutations in Gag may contribute to this phenotype. This study examines whether similar functional defects occur in Pol proteins of elite controllers. Recombinant NL4-3 viruses encoding plasma RNA-derived reverse transcriptase-integrase sequences from 58 elite controllers and 50 untreated chronic progressors were constructed, and replication capacity measured in vitro using a green fluorescent protein (GFP) reporter T-cell assay. Sequences were analyzed for drug resistance and HLA-associated viral polymorphisms. Controller-derived viruses displayed significantly lower replication capacity compared with those from progressors (P < 0.0001). Among controllers, the most attenuated viruses were generated from individuals expressing HLA-B*57 or B*51. In viruses from B*57+ progressors (n = 8), a significant inverse correlation was observed between B*57-associated reverse transcriptase-integrase escape mutations and replication capacity (R = -0.89; P = 0.003); a similar trend was observed in B*57+ controller-derived viruses (n = 20, R = -0.36; P = 0.08). HIV-1 Pol function seemed to be compromised in elite controllers. As observed previously for Gag, HLA-associated immune pressure in Pol may contribute to viral attenuation and subsequent control of viremia.

Deletion of 1.8-kb mRNA of Marek's disease virus decreases its replication ability but not oncogenicity

BackgroundThe 1.8-kb mRNA was reported as one of the oncogenesis-related genes of Marek's disease virus (MDV). In this study, the bacterial artificial chromosome (BAC) clone of a MDV field strain GX0101 was used as the platform to generate mutant MDV to examine the functional roles of 1.8-kb mRNA.ResultsBased on the BAC clone of GX0101, the 1.8-kb mRNA deletion mutant GX0101Δ(A+C) was constructed. The present experiments indicated that GX0101Δ(A+C) retained a low level of oncogenicity, and it showed a decreased replication capacity in vitro and in vivo when compared with its parent virus, GX0101. Further studies in vitro demonstrated that deletion of 1.8-kb mRNA significantly decreased the transcriptional activity of the bi-directional promoter between 1.8-kb mRNA and pp38 genes of MDV.ConclusionThese results suggested that the 1.8-kb mRNA did not directly influence the oncogenesis but related to the replication ability of MDV.

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

BackgroundTwo HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.ResultsBiological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.ConclusionsThese preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.

Patients Infected with HIV Type 1 Subtype CRF01_AE and Failing First-Line Nevirapine- and Efavirenz-Based Regimens Demonstrate Considerable Cross-Resistance to Etravirine

AIDS Research and Human RetrovirusesVol. 26, No. 6 Letter to the EditorPatients Infected with HIV Type 1 Subtype CRF01_AE and Failing First-Line Nevirapine- and Efavirenz-Based Regimens Demonstrate Considerable Cross-Resistance to EtravirineWeerawat Manosuthi, David M. Butler, Wasun Chantratita, Chonlaphat Sukasem, Douglas D. Richman, and Davey M. SmithWeerawat ManosuthiBamrasnaradura Infectious Diseases Institute, Ministry of Public Health, Nonthaburi, Thailand.Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.Search for more papers by this author, David M. ButlerUniversity of California San Diego, La Jolla, California.Search for more papers by this author, Wasun ChantratitaFaculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.Search for more papers by this author, Chonlaphat SukasemFaculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.Search for more papers by this author, Douglas D. RichmanUniversity of California San Diego, La Jolla, California.Veterans Affairs San Diego Healthcare System, San Diego, California.Search for more papers by this author, and Davey M. SmithUniversity of California San Diego, La Jolla, California.Veterans Affairs San Diego Healthcare System, San Diego, California.Search for more papers by this authorPublished Online:20 Jun 2010https://doi.org/10.1089/aid.2009.0107AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited ByGlobal Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes3 November 2015 | Open Forum Infectious Diseases, Vol. 3, No. 2High prevalence of PI resistance in patients failing second-line ART in Vietnam11 December 2015 | Journal of Antimicrobial Chemotherapy, Vol. 71, No. 3A simian-human immunodeficiency virus carrying the rt gene from Chinese CRF01_AE strain of HIV is sensitive to nucleoside reverse transcriptase inhibitors and has a highly genetic stability in vivoMicrobes and Infection, Vol. 16, No. 6Extensive Drug Resistance in HIV-Infected Cambodian Children Who Are Undetected as Failing First-Line Antiretroviral Therapy by WHO 2010 Guidelines Mia Coetzer, Benjamin Westley, Allison DeLong, Chhraing Tray, Dim Sophearin, Eric Nerrienet, Leeann Schreier, and Rami Kantor13 June 2013 | AIDS Research and Human Retroviruses, Vol. 29, No. 7The Potential Cost and Benefits of Raltegravir in Simplified Second-Line Therapy among HIV Infected Patients in Nigeria and South Africa15 February 2013 | PLoS ONE, Vol. 8, No. 2High Plasma Efavirenz Concentration and CYP2B6 Polymorphisms in Thai HIV-1 InfectionsDrug Metabolism and Pharmacokinetics, Vol. 28, No. 5Unusual Insertion and Deletion at Codon 67 and 69 of HIV Type 1 Subtype C Reverse Transcriptase Among First-Line Highly Active Antiretroviral Treatment-Failing South Indian Patients: Association with Other Resistance Mutations Shanmugam Saravanan, Vidya Madhavan, Rami Kantor, Sathasivam Sivamalar, Selvamurthi Gomathi, Sunil S. Solomon, Nagalingeswaran Kumarasamy, Davey M. Smith, Robert T. Schooley, Suniti Solomon, and Pachamuthu Balakrishnan21 November 2012 | AIDS Research and Human Retroviruses, Vol. 28, No. 12Antiviral Drug Resistance and the Need for Development of New HIV-1 Reverse Transcriptase InhibitorsAntimicrobial Agents and Chemotherapy, Vol. 56, No. 10The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug ResistanceScientifica, Vol. 2012Short Communication Prevalence of Susceptibility to Etravirine by Genotype and Phenotype in Samples Received for Routine HIV Type 1 Resistance Testing in the United States Gaston Picchio, Johan Vingerhoets, Lotke Tambuyzer, Eoin Coakley, Mojgan Haddad, and James Witek29 November 2011 | AIDS Research and Human Retroviruses, Vol. 27, No. 12Genotypic Impact of Prolonged Detectable HIV Type 1 RNA Viral Load after HAART Failure in a CRF01_AE-Infected Cohort Maria Zolfo, Jonathan M. Schapiro, Vichet Phan, Olivier Koole, Sopheak Thai, Marc Vekemans, Katrien Fransen, and Lutgarde Lynen27 June 2011 | AIDS Research and Human Retroviruses, Vol. 27, No. 7Selection of nonnucleoside reverse transcriptase inhibitor-associated mutations in HIV-1 subtype C: evidence of etravirine cross-resistanceAIDS, Vol. 25, No. 8Reverse transcriptase mutation K65N confers a decreased replication capacity to HIV-1 in comparison to K65R due to a decreased RT processivityVirology, Vol. 414, No. 1Assessments of HIV Drug Resistance Mutations in Resource-Limited Settings15 April 2011 | Clinical Infectious Diseases, Vol. 52, No. 8Characterization of the E138K Resistance Mutation in HIV-1 Reverse Transcriptase Conferring Susceptibility to Etravirine in B and Non-B HIV-1 SubtypesAntimicrobial Agents and Chemotherapy, Vol. 55, No. 2 Volume 26Issue 6Jun 2010 InformationCopyright 2010, Mary Ann Liebert, Inc.To cite this article:Weerawat Manosuthi, David M. Butler, Wasun Chantratita, Chonlaphat Sukasem, Douglas D. Richman, and Davey M. Smith.Patients Infected with HIV Type 1 Subtype CRF01_AE and Failing First-Line Nevirapine- and Efavirenz-Based Regimens Demonstrate Considerable Cross-Resistance to Etravirine.AIDS Research and Human Retroviruses.Jun 2010.609-611.http://doi.org/10.1089/aid.2009.0107Published in Volume: 26 Issue 6: June 20, 2010Online Ahead of Print:May 27, 2010PDF download

RtE218G, a novel hepatitis B virus mutation with resistance to adefovir dipivoxil in patients with chronic hepatitis B

Antiviral therapy is a potentially successful approach for the treatment of patients with Hepatitis B virus (HBV) infection. One antiviral agent is the nucleoside analogue adefovir dipivoxil (ADV). Its efficiency is compromised by the emergence of drug-resistant HBV mutants. Although three major ADV-resistant mutations of HBV are known, rtA181T/V and rtN236T, HBV mutations associated with ADV resistance have not been fully identified. We analyzed DNA sequences that covered a 244 base pair region of the HBV polymerase gene from patients with clinical manifestations of ADV resistance. A novel pattern of amino acid substitutions in HBV polymerase was detected in 26 out of 86 patients. This mutant exhibited a substitution of glycine for glutamic acid at residue 218 (rtE218G). Transient transfection of the HBV replication-competent construct including the rtE218G mutation was performed in HepG2 cells in order to determine the relevance of this mutation to ADV resistance. Phenotypic analyses demonstrated that the rtE218G mutation could independently confer resistance to ADV in vitro, with a 50% inhibitory concentration (IC(50)) 5.5-fold higher than wild-type HBV. RtE218G-mutated HBV also showed a decreased replication capacity in vitro, equal to 87% of wild-type HBV. The present study showed that the rtE218G mutation may be a novel ADV-resistant mutation. Further work will focus on resistance surveillance and cross-resistance analyses, and the molecular mechanisms involved.

Successful Rescue Therapy with a Darunavir/Ritonavir and Etravirine Antiretroviral Regimen in a Child with Vertically Acquired Multidrug-Resistant HIV-1

An increasing prevalence of antiretroviral therapy (ART) resistance in ART-experienced and ART-naive pregnant women has been reported. Some studies suggest that antiretroviral drug-resistant viruses might have decreased replication capacity and transmissibility. However, cases of perinatal transmission of multidrug-resistant HIV type-1 (HIV-1) have been described. Here, we report the case of one child with vertically-acquired multidrug-resistant HIV-1 and the outcome of a rescue therapy with a darunavir/ritonavir- and etravirine-containing antiretroviral regimen. During the 15 months of therapy, the child showed clinical improvement, including no side effects, persistent suppression of viral replication and a great increase in CD4+ T-cell count. Paediatric HIV specialists should be prepared to manage a small, but increasing, number of babies with a 'nightmare' multidrug-resistant virus with no available treatment options. The use of experimental agents might become a compelling issue in vertically HIV-infected children born in the era of highly active ART.

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Alterations in the differentiation ability of mesenchymal stem cells in patients with nontraumatic osteonecrosis of the femoral head: Comparative analysis according to the risk factor

It has been suggested that decreased replication capacity of mesenchymal stem cells (MSCs) or decreased MSCs activity in the bone marrow is related to nontraumatic osteonecrosis (ON). However, little is known about differentiation ability of MSCs according to the risk factor of nontraumatic ON. We hypothesize that differentiation abnormalities in MSCs of the bone marrow of the proximal femurs might be related to nontraumatic ON of the femoral head. The purpose of this study was to investigate the osteogenic and adipogenic differentiation ability of MSCs in patients with nontraumatic ON of the femoral head. We examined the differentiation ability of MSCs in cultures derived from the bone marrow of the proximal femurs obtained from 10 patients with hip osteoarthritis (OA) and 37 patients with nontraumatic ON of the femoral head undergoing hip replacement surgery. We analyzed the osteogenic and adipogenic differentiation ability of MSCs according to the risk factor [alcohol-induced (15 patients), idiopathic (12 patients) and steroid-induced (10 patients)] of nontraumatic ON of the femoral head separately and compared it with patients with hip OA. The osteogenic activity was measured as the extracellular matrix calcification by alizarin red S staining and the alkaline phosphatase activity, and the adipogenic activity was measured as the accumulation of Oil red O-positive lipid vacuoles. The osteogenic differentiation ability of MSCs in patients with alcohol-induced and idiopathic ON was significantly reduced compared with that in patients with OA (p < 0.05 and p < 0.05, respectively). In patients with steroid-induced ON, the osteogenic differentiation ability was found to be increased, but the difference was not statistically significant. The adipogenic differentiation ability of MSCs was not significantly changed in patients with alcohol-induced, idiopathic, and steroid-induced ON compared to patients with OA. Our results indicate that altered osteogenic differentiation ability in MSCs is related to nontraumatic ON of the femoral head and the differentiation potential of MSCs in patients with nontraumatic ON differs according to its risk factor.

Replication capacity, biological phenotype, and drug resistance of HIV strains isolated from patients failing antiretroviral therapy.

The fitness of human immunodeficiency virus (HIV) in vivo depends on the interaction of a multitude of viral and host factors. The aim of this study was to analyze the biological phenotype and the intrinsic capacity of the HIV isolates with drug-resistance mutations to replicate efficiently in the absence of drugs. An open label multicenter cross-sectional study was undertaken on 28 HIV-infected patients failing antiretroviral treatment. The subjects were studied for CD4+ cell count, HIV viral load, syncytium-inducing phenotype, genotypic drug-resistance assay, and replication capacity of HIV isolates assessed by co-culture assay. All HIV isolates showed a decreased replication capacity compared with wild-type strains. The lowest replication capacity was detected in HIV strains with more than five drug-resistance mutations. The highest replication capacity was observed in strains carrying the K103N and Y181C primary mutations that emerged after treatment with non-nucleoside analogue inhibitors. Isolates with R5 biological phenotype had a higher number of resistant mutations than X4 isolates (P = 0.004). Particularly, the R5 phenotype was detected in all 6 isolates with more than 14 drug-resistance mutations. Patients with R5 strains had plasma viral load similar to patients with X4 strains, but marginally higher CD4+ cell counts, and their HIV isolates had significantly lower replication capacity of HIV isolates (P = 0.008). No patient carrying HIV with a maintained replication capacity had a viral load less than 30,000 copies/ml. In patients failing HAART, the detection of HIV isolates with the R5 biological phenotype correlates with CD4+ cell count, an impaired replication capacity, and a high number of drug-resistance mutations.

Polyploidy associated with oxidative injury attenuates proliferative potential of cells.

Polyploid cells are encountered ubiquitously but the biological significance of polyploidy is unclear. In view of their extensive capacity for regeneration, hepatocytes offer excellent systems for analyzing growth control mechanisms. We isolated hepatocytes from adult rats with and without two-third partial hepatectomy, which induces hepatic polyploidy. Polyploid hepatocytes showed evidence for oxidative injury with antioxidant depletion, lipid peroxidation and 8-hydroxy-adducts of guanine in nuclear DNA. Liver repopulation assays in intact animals showed markedly decreased replication capacity in polyploid hepatocytes. Recapitulation of polyploidy in cultured hepatocytes established that mitogenic stimulation in the presence of oxidative DNA injury was capable of inducing polyploidy. The findings provide novel frameworks in the context of polyploidy for understanding tissue development, regeneration and oncogenesis.