High Replication Rate Research Articles

Low-pH Stability of Influenza A Virus Sialidase Contributing to Virus Replication and Pandemic.

The spike glycoprotein neuraminidase (NA) of influenza A virus (IAV) has sialidase activity that cleaves the terminal sialic acids (viral receptors) from oligosaccharide chains of glycoconjugates. A new antigenicity of viral surface glycoproteins for humans has pandemic potential. We found "low-pH stability of sialidase activity" in NA. The low-pH stability can maintain sialidase activity under acidic conditions of pH 4-5. For human IAVs, NAs of all pandemic viruses were low-pH-stable, whereas those of almost all human seasonal viruses were not. The low-pH stability was dependent on amino acid residues near the active site, the calcium ion-binding site, and the subunit interfaces of the NA homotetramer, suggesting effects of the active site and the homotetramer on structural stability. IAVs with the low-pH-stable NA showed much higher virus replication rates than those of IAVs with low-pH-unstable NA, which was correlated with maintenance of sialidase activity under an endocytic pathway of the viral cell entry mechanism, indicating contribution of low-pH stability to high replication rates of pandemic viruses. The low-pH-stable NA of the 1968 H3N2 pandemic virus was derived from the low-pH-stable NA of H2N2 human seasonal virus, one of two types classified by both low-pH stability in N2 NA and a phylogenetic tree of N2 NA genes. The 2009 H1N1 pandemic virus acquired low-pH-stable NA by two amino acid substitutions at the early stage of the 2009 pandemic. It is thought that low-pH stability contributes to infection spread in a pandemic through enhancement of virus replication.

ViQuaS: an improved reconstruction pipeline for viral quasispecies spectra generated by next-generation sequencing.

The combined effect of a high replication rate and the low fidelity of the viral polymerase in most RNA viruses and some DNA viruses results in the formation of a viral quasispecies. Uncovering information about quasispecies populations significantly benefits the study of disease progression, antiviral drug design, vaccine design and viral pathogenesis. We present a new analysis pipeline called ViQuaS for viral quasispecies spectrum reconstruction using short next-generation sequencing reads. ViQuaS is based on a novel reference-assisted de novo assembly algorithm for constructing local haplotypes. A significantly extended version of an existing global strain reconstruction algorithm is also used. Benchmarking results showed that ViQuaS outperformed three other previously published methods named ShoRAH, QuRe and PredictHaplo, with improvements of at least 3.1-53.9% in recall, 0-12.1% in precision and 0-38.2% in F-score in terms of strain sequence assembly and improvements of at least 0.006-0.143 in KL-divergence and 0.001-0.035 in root mean-squared error in terms of strain frequency estimation, over the next-best algorithm under various simulation settings. We also applied ViQuaS on a real read set derived from an in vitro human immunodeficiency virus (HIV)-1 population, two independent datasets of foot-and-mouth-disease virus derived from the same biological sample and a real HIV-1 dataset and demonstrated better results than other methods available.

Current and Future Targets of Antiviral Therapy in the Hepatitis C Virus Life Cycle

ABSTRACT Advances in our understanding of the hepatitis C virus (HCV) life cycle have enabled the development of numerous clinically advanced direct-acting antivirals. Indeed, the recent approval of first-generation direct-acting antivirals that target the viral NS3–4A protease and NS5B RNA-dependent RNA polymerase brings closer the possibility of universally efficacious and well-tolerated antiviral therapies for this insidious infection. However, the complexities of comorbidities, unforeseen side effects or drug–drug interactions, viral diversity, the high mutation rate of HCV RNA replication and the elegant and constantly evolving mechanisms employed by HCV to evade host and therapeutically implemented antiviral strategies remain as significant obstacles to this goal. Here, we review advances in our understanding of the HCV life cycle and associated opportunities for antiviral therapy.

Hepatitis C virus treatment revolution: need for close monitoring

Hepatitis C virus (HCV) infection is a relevant clinical and public health problem. The virus infects approximately 3% of the global population, and is one of the leading causes of end-stage liver diseases and hepatocellular carcinoma, causing >350 000 deaths yearly [1]. Unlike from other agents causing chronic infections (e.g. human immunodeficiency virus and hepatitis B virus), HCV cannot establish a permanent intracellular genomic archive, and its persistence relies on continuous replication. Hence, HCV can, theoretically, be eradicated from the host, provided that new replication cycles are prevented [2]. For >20 years, the combination of pegylated interferon (Peg-IFN) and ribavirin was the pillar of anti-HCV therapy, often being referred to as the standard of care (SOC). However, the success rate of the SOC is only approximately 50% overall. Furthermore, patients in whom there is a failure to clear HCV with SOC have little (if any) chance of subsequent treatment with the same combination being successful, and, perhaps more importantly, patients with comorbidities are ineligible to receive Peg-IFN at all [2]. Recently, compounds with direct antiviral activity (DAAs) have been discovered. The first-generation protease inhibitors (PIs) telaprevir and boceprevir were approved by the FDA on May 2011 for HCV genotype 1 in combination with the SOC. These combinations significantly improved viral clearance, but with a significant increase in adverse events. Moreover, the issue of subjects ineligible to receive Peg-IFN remained unsolved [3,4]. Subsequently, two new drugs, sofosbuvir (NS5B polymerase inhibitor) and simeprevir (second-generation PI), have been approved. Many other compounds, with different viral targets, will enter clinical practice soon, including three NS5A inhibitors (ledipasvir, daclatasvir, and ombitasvir), two-second generation PIs (asunaprevir and ABT-450), and one NS5B inhibitor (dasabuvir). Combinations of these compounds promise all-oral, once-daily, ultra-short (8–12 weeks), interferon-free treatment with outstanding efficacy, an improved safety profile, and no comorbidity contraindications. It is common opinion that, owing to these new options, HCV will not represent a significant health issue in the near future [3–5]. However, this optimism hides several critical issues. Among them are: prohibitive cost of the therapy; the best drug combination(s); when to start treatment and how long to treat; and transmission by asymptomatic carriers. Furthermore, it is our firm opinion that another topical issue that is not sufficiently addressed by current guidelines is therapeutic response monitoring, which is focused on the measurement of HCV RNA in the blood, and is aimed at optimizing therapy duration, to prompt early discontinuation to prevent potential side effects, and to reduce unnecessary costs. Currently, it is assumed that the best chance of HCV eradication in a patient occurs when viral replication has been halted for a sufficient time to allow its disappearance from every infected cell. However, even in cases when HCV RNA is not detected with the most sensitive assay, it is still possible that minute, undetected amounts of virus are present. Previous experience with the SOC has indicated that assays with higher sensitivity perform better in predicting sustained virological response in patients who have reached the end of therapy response. In this respect, the diagnostic armamentarium for monitoring HCV therapy has improved greatly, and the sensitivity of current real-time PCR-based assays has substantially improved as compared with the first-generation assays. It is not clear whether the sensitivity of current assays could be further improved, and whether such improvements would offer any advantage in monitoring DAA-based therapy. There is a general consensus that the rate of decay of HCV RNA is predictive of response. Clear stopping rules have been established for the SOC, to allow treatment duration to be adapted on the basis of early decay of viral load, and even for treatment to be stopped in cases of insufficient or absent decline. However, for the DAA-containing regimens, therapy tailoring indications and stopping rules have been formulated for a very limited number of compounds; in addition, there is a great degree of variability in the cut-off levels to adopt, even for DAAs with similar mechanisms of action and administration schedules. In fact, negative and positive predictive values of different cut-off levels have been established with respect to different endpoints and criteria, so that a uniform decision pathway is lacking [3]. Another highly debated issue in monitoring strategies is resistance to antivirals. Owing to the high replication rate of HCV, and to the lack of proofreading activity of viral polymerase, HCV is highly variable, and each possible mutation, and even each combination of mutations, arises every day within each infected individual, representing the basis for selection of resistant variants by DAAs [6]. Despite reduced

Key parameters and optimal design of a single-layered induction coil for external rapid mold surface heating

Setting high mold temperatures for injection-molding plastics facilitates favorable flow conditions for filling cavities with melted materials and provides an esthetically pleasing surface as well as a high replication rate of high-quality products; however, the cooling times are typically prolonged. Electromagnetic induction heating incorporating surface heating instead of conventional volume heating for mold-heating processes is advantageous because it provides a rapid heating time and a reduced cooling time, is environmentally friendly, and saves energy; therefore, it has been adopted in various variotherm injection-molding systems. Although previous studies have discussed how induction heating is influenced by major factors, such as the number of coil turns, working frequency, and heating distance, few studies have investigated other crucial factors, such as the thickness of the heated target and the position of the induction coil. In this study, the effects of the thickness of a heated target, pitch of coil turns, heating distance, position of the induction coil, working frequency, and waiting time on the heating rate and temperature uniformity of induction heating on a mold surface by using a single-layered coil were analyzed. In addition, the Taguchi method and principal component analysis were applied to determine the optimal combination of control factors for achieving a high heating rate and low temperature deviation. Both simulation and experimental results indicated that the thickness of a heated target plays a crucial role in affecting the heating rate; specifically, a thicker workpiece slows the heating process and generates rapid heat dissipation after induction heating. Moreover, the position of the induction coil exerts the most notable effect on heating uniformity.

Emergence and transmission of arbovirus evolutionary intermediates with epidemic potential.

The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapitulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary trajectories can be predicted and studied in the short term before new variants displace currently circulating strains.

Mutations in HIV-1 Reverse Transcriptase Affect the Errors Made in a Single Cycle of Viral Replication

The genetic variation in HIV-1 in patients is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Some of the mutations in reverse transcriptase (RT) that alter the deoxynucleoside triphosphate (dNTP)-binding pocket, including those that confer resistance to nucleoside/nucleotide analogs, affect dNTP selection during replication. The effects of mutations in RT on the spectrum (nature, position, and frequency) of errors made in vivo are poorly understood. We previously determined the mutation rate and the frequency of different types of mutations and identified hot spots for mutations in a lacZα (the α complementing region of lacZ) reporter gene carried by an HIV-1 vector that replicates using wild-type RT. We show here that four mutations (Y115F, M184V, M184I, and Q151M) in the dNTP-binding pocket of RT that had relatively small effects on the overall HIV-1 mutation rate (less than 3-fold compared to the wild type) significantly increased mutations at some specific positions in the lacZα reporter gene. We also show that changes in a sequence that flanks the reporter gene can affect the mutations that arise in the reporter. These data show that changes either in HIV-1 RT or in the sequence of the nucleic acid template can affect the spectrum of mutations made during viral replication. This could, by implication, affect the generation of drug-resistant mutants and immunological-escape mutants in patients. RT is the viral enzyme that converts the RNA genome of HIV into DNA. Errors made during replication allow the virus to escape from the host's immune system and to develop resistance to the available anti-HIV drugs. We show that four different mutations in RT which are known to be associated with resistance to anti-RT drugs modestly increased the overall frequency of errors made during viral replication. However, the increased errors were not uniformly distributed; the additional errors occurred at a small number of positions (hot spots). Moreover, some of the RT mutations preferentially affected the nature of the errors that were made (some RT mutations caused an increase in insertion and deletion errors; others caused an increase in substitution errors). We also show that sequence changes in a region adjacent to a target gene can affect the errors made within the target gene.

Resistance mutations are rare among protease inhibitor treatment-naive hepatitis C genotype-1 patients with or without HIV coinfection.

HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. This diversity may lead to emergence of resistant strains in direct-acting antiviral therapy. The frequency of naturally occurring HCV protease inhibitor (PI) mutations has been addressed in many countries, but there are few data on the prevalence of these mutations in Brazilian patients. We evaluated the sequence of HCV NS3 protease gene in 247 patients (135 HCV-monoinfected and 112 HIV-HCV-coinfected patients). HCV RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. HIV-HCV-coinfected patients were more likely to be older than 40 years and have an HCV subtype-1a infection. Overall, 21.9% of patients had at least one amino acid substitution in the NS3 region; 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R) and the Q80K mutation was not found in our case series. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Baseline HCV NS3 amino acid substitutions identified herein are considered mostly natural polymorphisms with no clinical impact on PI-based therapy. The identified resistance mutations may be associated with low-level resistance to PIs in vitro. Q80K substitution seems to be a rare event in Brazil. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample.

Identification of Virulence Factors in Leishmania infantum Strains by a Proteomic Approach

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.

A false occult hepatitis B virus infection developed in a patient with psoriatic arthritis under infliximab and methotrexate therapy

Despite lacking of international guidelines about the management of patients with occult hepatitis B virus infection (OBI) starting TNF-α blockers, there is some evidence from real life settings that these drugs are safe in OBI patients with rheumatic diseases. On the contrary, the management of the so-called false OBI patients appears still undefined. We describe a case of acute hepatitis B virus (HBV) infection occurred in an anti-HBs and anti- HBc positive patient affected by psoriatic arthritis, who had been treated for five years with infliximab. Baseline HBV-DNA analysis had not been performed. Although HBs Ag was still negative and the transaminases in the normal range, HBV-DNA serum analysis surprisingly showed high replication rate. Entecavir was added, and three months later HBV-DNA was no longer detectable. Even if HBs Ag is persistently negative, the assessment of HBV-DNA should be recommended at least at baseline in order to rule out hidden active necro-inflammatory liver disease.

Identification of a new HIV-1 BC circulating recombinant form (CRF60_BC) in Italian young men having sex with men

HIV-1 recombination, reverse transcriptase (RT) low fidelity and high replication rate are the drivers of variability and evolution on the global scale. Only few of these HIV-1 chimeric forms have been characterized in Europe, despite 20% of infections are due to unique or circulating recombinant forms worldwide. An outbreak of BC recombinants has been recently described in a southern region of Italy, Apulia, in men having sex with men (MSM) seeking sexual partners on-line.We analyzed the full length genome of HIV-1 BC recombinants harbored by three recently infected subjects, two MSM and a heterosexual woman, with no evidence of epidemiological link. The recombination analysis showed a unique recombination pattern of a subtype C genome with 3 subtype B fragments corresponding to HXB2 positions: [1–463] in the 5′LTR , [2804–3037] in RT and [8662–9548] corresponding to the C-terminal segment of gp41, nef and most of 3′LTR.Phylogenetic analysis revealed the South American origin of the C subtype parental strain. A research conducted in an Italian nationwide database provided six additional similar sequences from other Italian regions with identical recombination pattern in pol gene; a further BLAST search retrieved one full length genome isolated in France with the same mosaic pattern, except an additional B subtype short fragment in the integrase region. These recombinant isolates, designated CRF60_BC, led to the identification of the first Italian circulating recombinant form, which gave rise to an epidemic burst mainly involving MSM.

Microbiological characterization of Göttingen minipigs

Xenotransplantation using pig cells, tissues or organs may be associated with transmission of porcine microorganisms which in the worst case scenario may induce zoonoses. To prevent this, a detailed analysis of the viruses, bacteria, fungi and protozoa in the donor pigs should be performed. This includes characterization of the herd as well as of each individual donor animal. Furthermore, the final transplant, e.g., the islet cell preparation, should be analyzed.At present, the Auckland island pigs are the best characterized animals and the presence of numerous bacteria, viruses and protozoa is analyzed regularly in the animals and in islet cell preparations [1]. Preclinical [2] and clinical [3] trials transplanting islet cells from these animals did not result in transmission of porcine microorganisms including the porcine endogenous retroviruses (PERVs). PERV‐A and PERV‐B are present in the genome of all pigs, and they may infect human cells (for review see [4]). PERV‐C are not present in all pigs, they infect only pig cells, however recombinant PERV‐A/C infect human cells and are characterized by high replication rates. Previously it was shown that the expression of PERVs was different among breeds including German landrace, Duroc, Schwäbisch‐Hällisch, Large White, as well as (multi)transgenic crossbreds generated for xenotransplantation [5]. The highest expression was found in Yucatan minipigs [6]. Although PERV expression was well studied in these breeds, the prevalence of other microorganisms remains unclear.In addition to the Auckland island pigs the Göttingen minipigs, which are held at Ellegaard, Denmark, represent another well‐characterized pig breed. The animals are well‐defined concerning their physiologic parameters, health status, and genetics since this herd was established with the purpose to supply minipigs for preclinical pharmacology and toxicology [7]. Health monitoring includes testing for a broad spectrum of viruses, bacteria, fungi and protozoa and health reports are regularly published at Ellegaard website [8]. When the prevalence and the expression of PERVs were analyzed in these animals, it was demonstrated that although PERV‐A, ‐B and ‐C proviruses were found in all animals, their expression was low [9]. Unfortunately the health monitoring of the herd does not include xenozoonotic infectious agents such as cytomegalovirus, gamma‐lymphotropic herpesvirus and hepatitis E [10]. Approaches to detect these viruses are under development and additional investigations are required to assess the suitability of Göttingen minipigs and other animals for xenotransplantation in terms of microbiological safety.

Differential impact of APOBEC3-driven mutagenesis on HIV evolution in diverse anatomical compartments

Previous studies on HIV quasispecies have revealed HIV compartmentalization in various tissues within an infected individual. Such HIV variation is a result of a combination of factors including high replication and mutation rates, recombination, and APOBEC3-host selective pressure. To evaluate the differential impact of APOBEC3 editing in HIV-1 compartments, we analyzed the level of G-to-A hypermutation in HIV-1 protease and reverse transcriptase sequences among 30 HAART-treated patients for whom peripheral blood mononuclear cells and body tissues or fluids [cerebral spinal fluid (CSF), rectal tissue, or renal tissue] were collected on the same day. APOBEC3-mediated hypermutation was identified in 36% (11/30) of participants in at least one viral reservoir. HIV hypermutated sequences were often observed in viral sanctuaries (total n = 10; CSF, n = 6; renal tissue, n = 1; rectal tissue n = 3) compared with peripheral blood (total n = 4). Accordingly, APOBEC3 editing generated more G-to-A drug resistance mutations in sanctuaries: three patients' CSF (i.e. G73S in protease; M184I, M230I in reverse transcriptase) and two other patients' rectal tissues (M184I, M230I in reverse transcriptase) while such mutations were absent from paired peripheral blood mononuclear cells. APOBEC3-induced mutations observed in peripheral blood underestimate the overall proportion of hypermutated viruses in anatomical compartments. The resulting mutations may favor escape to antiretrovirals in these compartments in conjunction with a lower penetration of drugs in some sanctuaries. On the other side, because hypermutated sequences often harbor inactivating mutations, our results suggest that accumulation of defective viruses may be more dominant in sanctuaries than in peripheral blood of patients on effective HAART.

Molecular and Genetic Characterization of Natural HIV-1 Tat Exon-1 Variants from North India and Their Functional Implications

BackgroundDesigning an ideal vaccine against HIV-1 has been difficult due to enormous genetic variability as a result of high replication rate and lack of proofreading activity of reverse transcriptase leading to emergence of genetic variants and recombinants. Tat transactivates HIV-1 LTR, resulting in a remarkable increase in viral gene expression, and plays a vital role in pathogenesis. The aim of this study was to characterize the genetic variations of Tat exon-1 from HIV-1 infected patients from North India.MethodsGenomic DNA was isolated from PBMCs and Tat exon-1 was PCR amplified with specific primers followed by cloning, sequencing and sequence analyses using bioinformatic tools for predicting HIV-1 subtypes, recombination events, conservation of domains and phosphorylation sites, and LTR transactivation by luciferase assay.ResultsPhylogenetic analysis of Tat exon-1 variants (n = 120) revealed sequence similarity with South African Tat C sequences and distinct geographical relationships were observed for B/C recombinants. Bootscan analysis of our variants showed 90% homology to Tat C and 10% to B/C recombinants with a precise breakpoint. Natural substitutions were observed with high allelic frequencies which may be beneficial for virus. High amino acid conservation was observed in Tat among Anti Retroviral Therapy (ART) recipients. Barring few changes, most of the functional domains, predicted motifs and phosphorylation sites were well conserved in most of Tat variants. dN/dS analysis revealed purifying selection, implying the importance of functional conservation of Tat exon-1. Our Indian Tat C variants and B/C recombinants showed differential LTR transactivation.ConclusionsThe possible role of Tat exon-1 variants in shaping the current HIV-1 epidemic in North India was highlighted. Natural substitutions across conserved functional domains were observed and provided evidence for the emergence of B/C recombinants within the ORF of Tat exon-1. These events are likely to have implications for viral pathogenesis and vaccine formulations.

Hepatitis B virus genetic mutations and evolution in liver diseases.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length. Owing to a lack of proofreading capacity during reverse transcription and a high replication rate, HBV exhibits as quasispecies. To detect the genetic mutations of HBV, many methods with different sensitivities and throughputs were developed. According to documentary records, HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome. In this review, we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression. Since some of the results were controversial from different laboratories, it remains to be seen whether functional analyses will confirm their role in modifying the course of infection.

A continuous phenotype space model of RNA virus evolution within a host

Due to their very high replication and mutation rates, RNA viruses can serve as an excellent testing model for verifying hypothesis and addressing questions in evolutionary biology. In this paper, we suggest a simple deterministic mathematical model of the within-host viral dynamics, where a possibility for random mutations incorporates. This model assumes a continuous distribution of viral strains in a one-dimensional phenotype space where random mutations are modelled by Brownian motion (that is, by diffusion). Numerical simulations show that random mutations combined with competition for a resource result in evolution towards higher Darwinian fitness: a stable pulse traveling wave of evolution, moving towards higher levels of fitness, is formed in the phenotype space. The advantage of this model, compared with the previously constructed models, is that this model is mechanistic and is based on commonly accepted model of virus dynamics within a host, and thus it allows an incorporation of features of the real-life host-virus system such as immune response, antiviral therapy, etc.

Boosting the Power of Schizophrenia Genetics by Leveraging New Statistical Tools

Genome-wide association studies (GWAS) have identified a large number of gene variants associated with schizophrenia, but these variants explain only a small portion of the heritability. It is becoming increasingly clear that schizophrenia is influenced by many genes, most of which have effects too small to be identified using traditional GWAS statistical methods. By applying recently developed Empirical Bayes statistical approaches, we have demonstrated that functional genic elements show differential contribution to phenotypic variance, with some elements (regulatory regions and exons) showing strong enrichment for association with schizophrenia. Applying related methods, we also showed abundant genetic overlap (pleiotropy) between schizophrenia and other phenotypes, including bipolar disorder, cardiovascular disease risk factors, and multiple sclerosis. We estimated the number of gene variants with effects in schizophrenia and bipolar disorder to be approximately 1.2%. By applying our novel statistical framework, we dramatically improved gene discovery and detected a large number of new gene loci associated with schizophrenia that have not yet been identified with standard GWAS methods. Utilizing independent schizophrenia substudies, we showed that these new loci have high replication rates in de novo samples, indicating that they likely represent true schizophrenia risk genes. The new statistical tools provide a powerful approach for uncovering more of the missing heritability of schizophrenia and other complex disorders. In conclusion, the highly polygenic architecture of schizophrenia strongly suggests the utility of research approaches that recognize schizophrenia neuropathology as a complex dynamic system, with many small gene effects integrated in functional networks.

HIV-1 Drug-Resistant Minority Variants: Sweating the Small Stuff

Within each human immunodeficiency virus (HIV)–infected individual, there exists a remarkably heterogeneous collection of viruses. Over the course of a typical HIV infection, a starting population of relatively homogeneous virus may undergo substantial diversification, such that mutations may be detected at >10% of nucleotide positions [1]. This diversity is driven both by the error-prone nature of HIV reverse transcription and the high rate of viral replication. The HIV reverse transcriptase enzyme lacks a proofreading mechanism, and it is estimated that up to 5 mutations may be introduced during each replication cycle [2, 3]. In addition, the virus is thought to undergo 10–100 million rounds of replication daily, leading to the production of more than a billion new virions [4, 5].

Accelerated Self‐Replication under Non‐Equilibrium, Periodic Energy Delivery

Self-replication is a remarkable phenomenon in nature that has fascinated scientists for decades. In a self-replicating system, the original units are attracted to a template, which induce their binding. In equilibrium, the energy required to disassemble the newly assembled copy from the mother template is supplied by thermal energy. The possibility of optimizing self-replication was explored by controlling the frequency at which energy is supplied to the system. A model system inspired by a class of light-switchable colloids was considered where light is used to control the interactions. Conditions under which self-replication can be significantly more effective under non-equilibrium, cyclic energy delivery than under equilibrium constant energy conditions were identified. Optimal self-replication does not require constant energy expenditure. Instead, the proper timing at which energy is delivered to the system is an essential controllable parameter to induce high replication rates.

Resistencia a telaprevir

Telaprevir is an extremely potent antiviral drug. However, as with other direct-acting antiviral agents against the hepatitis C virus (HCV), this potency can be compromised by the rapid emergence of resistance mutations. This phenomenon is favored by the high rate of HCV replication, the lack of corrective activity to HCV polymerase errors, a low genetic barrier to resistance, and poor treatment adherence. Like other HCV protease inhibitors, telaprevir has a low genetic barrier to resistance. Phase II/III clinical trials (ADVANCE, ILLUMINATE and REALIZE) have characterized resistance to telaprevir in combination with pegylated interferon and ribavirin both genotypically and phenotypically. Mutations are selected mainly in positions 36, 54, 55, 155 and 156. The resistance profile depends on the genetic subtype of HCV, with selection of mutations in positions 36 and 155 for subtype 1a and in positions 36, 54, 55 and 156 for subtype 1b. The V36M+R155K and A156F/T/V variants confer a high degree of resistance (>25-fold increase), while the impact on resistance of other mutations such as V36A/G/M, T54A/S, R155G/K/M/T and A156S is lower (3-25- fold increase). The presence of polymorphisms or mutations associated with telaprevir resistance is very low (< 1%), especially for mutations with a higher impact on resistance. There is a high degree of cross resistance among protease inhibitors. However, the rapid reversion of telaprevir resistance mutations after the end of treatment could allow the use of “recycling” strategies with protease inhibitors.