Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

The number and pattern of viral genomic reassortments are not necessarily identifiable from segment trees.

Reassortment is an evolutionary process common in viruses with segmented genomes. These viruses can swap whole genomic segments during cellular co-infection, giving rise to novel progeny formed from the mixture of parental segments. Because large-scale genome rearrangements have the potential to generate new phenotypes, reassortment is important to both evolutionary biology and public health research. However, statistical inference of the pattern of reassortment events from phylogenetic data is exceptionally difficult, potentially involving inference of general graphs in which individual segment trees are embedded. In this paper, we argue that, in general, the number and pattern of reassortment events are not identifiable from segment trees alone, even with theoretically ideal data. We call this fact the fundamental problem of reassortment, which we illustrate using the concept of the `first-infection tree', a typically but not always counterfactual genealogy that would have been observed in the segment trees had no reassortment occurred. Further, we illustrate four additional problems that can arise logically in the inference of reassortment events and show, using simulated data, that these problems are not rare and can potentially distort our perception of reassortment even in small data sets. Finally, we discuss how existing methods can be augmented or adapted to account for not only the fundamental problem of reassortment but also the four additional situations that can complicate the inference of reassortment.

A story of viral co-infection, co-transmission and co-feeding in ticks: how to compute an invasion reproduction number.

With a single circulating vector-borne virus, the basic reproduction number incorporates contributions from tick-to-tick (co-feeding), tick-to-host and host-to-tick transmission routes. With two different circulating vector-borne viral strains, resident and invasive, and under the assumption that co-feeding is the only transmission route in a tick population, the invasion reproduction number depends on whether the model system of ordinary differential equations possesses the property of neutrality. We show that a simple model, with two populations of ticks infected with one strain, resident or invasive, and one population of co-infected ticks, does not have Alizon's neutrality property. We present model alternatives that are capable of representing the invasion potential of a novel strain by including populations of ticks dually infected with the same strain. The invasion reproduction number is analysed with the next-generation method and via numerical simulations.

Salt-rich versus salt-poor structural scenarios in the central Northern Calcareous Alps: implications for the Hallstatt facies and early Alpine tectonic evolution (Eastern Alps, Austria).

One of the most remarkable features of the central Northern Calcareous Alps (Eastern Alps, Austria) is the widespread presence of Upper Triassic deep-water carbonates (the Hallstatt facies) and Permo-Triassic evaporites resting on deep-water Middle Jurassic strata and their underlying Upper Triassic shallow-water carbonate platform successions. The Hallstatt facies and accompanying evaporites have been classically interpreted to originate either from a location south of the time-equivalent carbonate platforms, or to have been deposited in deeper water seaways within the broad platform domain. To date, this dispute has been addressed mostly through the analysis of Triassic and Jurassic facies distribution in map view, which, however, is subject to some degree of ambiguity and subjectivity. In this contribution we present, for the first time, sequentially restored regional cross-sections through the central Northern Calcareous Alps to understand the implications of the contrasting paleogeographic models. We present (a) an interpretation based on a highly allochthonous origin of the Triassic deep-water units and (b) an interpretation based on their relative autochthony in which we incorporate the potential influence of salt tectonics in the central NCA. The restored cross-sections provide a framework within which the alternative scenarios and their paleogeographic implications can be better understood. Through this analysis we propose that salt tectonics in the central NCA can provide a valid explanation for apparent inconsistencies in the relative autochthony scenario and thus constitutes a reasonable alternative to the currently accepted allochthony scenario.

Eddy Current Position Measurement in Harsh Environments: A Temperature Compensation and Calibration Approach.

Eddy current displacement sensors (ECDSs) are widely used for the noncontact position measurement of small displacements (lift-offs). Challenges arise with larger displacements as the sensitivity of the ECDSs decreases. This leads to a more pronounced impact of temperature variations on the inductance and, consequently, an increased position error. Design solutions often rely on multiple coils, suitable coil carrier materials, and compensation measures to address the challenges. This study presents a single-coil ECDS for large displacement ranges in environments with high temperatures and temperature variations. The analysis is based on a sensor model derived from an equivalent circuit model (ECM). We propose design measures for both the sensing coil and the target, focusing on material selection to handle the impact of temperature variations. A key part of improving performance under varying temperatures includes model-based temperature compensation for the inductance of the sensing coil. We introduce a method to calibrate the sensor for large displacements, using a modified coupling coefficient based on field simulation data. Our analysis shows that this single-coil ECDS design maintains a position error of less than 0.2% full-scale for a temperature variation of 100 K for the sensing coil and 110 K for the target.

Inferring Viral Transmission Time from Phylogenies for Known Transmission Pairs.

When the time of an HIV transmission event is unknown, methods to identify it from virus genetic data can reveal the circumstances that enable transmission. We developed a single-parameter Markov model to infer transmission time from an HIV phylogeny constructed of multiple virus sequences from people in a transmission pair. Our method finds the statistical support for transmission occurring in different possible time slices. We compared our time-slice model results to previously described methods: a tree-based logical transmission interval, a simple parsimony-like rules-based method, and a more complex coalescent model. Across simulations with multiple transmitted lineages, different transmission times relative to the source's infection, and different sampling times relative to transmission, we found that overall our time-slice model provided accurate and narrower estimates of the time of transmission. We also identified situations when transmission time or direction was difficult to estimate by any method, particularly when transmission occurred long after the source was infected and when sampling occurred long after transmission. Applying our model to real HIV transmission pairs showed some agreement with facts known from the case investigations. We also found, however, that uncertainty on the inferred transmission time was driven more by uncertainty from time calibration of the phylogeny than from the model inference itself. Encouragingly, comparable performance of the Markov time-slice model and the coalescent model-which make use of different information within a tree-suggests that a new method remains to be described that will make full use of the topology and node times for improved transmission time inference.

Predicting Impacts of Contact Tracing on Epidemiological Inference from Phylogenetic Data.

Robust sampling methods are foundational to many inference problems in the phylodynamic field, yet the impact of using contact tracing, a type of non-uniform sampling used in public health applications, is not well understood. To investigate and quantify how this non-uniform sampling method influences recovered phylogenetic tree structure, we developed a new simulation tool called SEEPS (Sequence Evolution and Epidemiological Process Simulator) that allows for the simulation of contact tracing and the resulting transmission tree, pathogen phylogeny, and corresponding virus genetic sequences. Importantly, SEEPS takes within-host evolution into account when generating pathogen phylogenies and sequences from transmission histories. Using SEEPS, we demonstrate that contact tracing can significantly impact the structure of the resulting tree as described by popular tree statistics. Contact tracing generates phylogenies that are less balanced than the underlying transmission process, less representative of the larger epidemiological process, and affects the internal/external branch length ratios that characterize specific epidemiological scenarios. We also examine a 2007-2008 Swedish HIV-1 outbreak and the broader 1998-2010 European HIV-1 epidemic to highlight the differences in contact tracing and expected phylogenies. Aided by SEEPS, we show that the Swedish outbreak was strongly influenced by contact tracing even after downsampling, while the broader European Union epidemic showed little evidence of universal contact tracing, agreeing with the known epidemiological information about sampling and spread. SEEPS is available at github.com/MolEvolEpid/SEEPS.

A novel candidate hepatitis C virus genotype 4 subtype identified by next generation sequencing full-genome characterization in a patient from Saudi Arabia.

Background and aim: Hepatitis C virus (HCV) infection is a major global public health concern, being a leading cause of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The virus is classified into 8 genotypes and 93 subtypes, each displaying distinct geographic distributions. Genotype 4 is the most predominant in the Middle East and Eastern Mediterranean and is associated with high rates of hepatitis C infection worldwide. This study used next-generation sequencing to fully characterize the HCV genome and identify a novel subtype within genotype 4 isolated from a 64-year-old Saudi man diagnosed with hepatitis C. Methods: We analyzed the complete genome of the 141-HCV isolate using whole-genome sequencing. Results: Our phylogenetic reconstructions, based on the entire genome of HCV-4 strains, revealed that the 141-HCV isolate formed a distinct group within the genotype 4 classification, providing valuable new insights into the variability of HCV. Conclusion: This discovery of a previously unclassified HCV subtype within genotype 4 sheds light on the ongoing evolution and diversity of the virus. Such knowledge has significant implications for diagnostic and therapeutic approaches, as different subtypes may exhibit varying drug sensitivities and resistance profiles.

Last in first out: SIV proviruses seeded later in infection are harbored in short-lived CD4+ T cells.

HIV can persist in a latent form as integrated DNA (provirus) in resting CD4+ T cells of infected individuals and as such is unaffected by antiretroviral therapy (ART). Despite being a major obstacle for eradication efforts, the genetic variation and timing of formation of this latent reservoir remains poorly understood. Previous studies on when virus is deposited in the latent reservoir have come to contradictory conclusions. To reexamine the genetic variation of HIV in CD4+ T cells during ART, we determined the divergence in envelope sequences collected from 10 SIV infected rhesus macaques. We found that the macaques displayed a biphasic decline of the viral divergence over time, where the first phase lasted for an average of 11.6 weeks (range 4-28 weeks). Motivated by recent observations that the HIV-infected CD4+ T cell population is composed of short- and long-lived subsets, we developed a model to study the divergence dynamics. We found that SIV in short-lived cells was on average more diverged, while long-lived cells harbored less diverged virus. This suggests that the long-lived cells harbor virus deposited starting earlier in infection and continuing throughout infection, while short-lived cells predominantly harbor more recent virus. As these cell populations decayed, the overall proviral divergence decline matched that observed in the empirical data. This model explains previous seemingly contradictory results on the timing of virus deposition into the latent reservoir, and should provide guidance for future eradication efforts.

Modeling the evolution of segment trees reveals deficiencies in current inferential methods for genomic reassortment.

Reassortment is an evolutionary process common in viruses with segmented genomes. These viruses can swap whole genomic segments during cellular co-infection, giving rise to new viral variants. Large-scale genome rearrangements, such as reassortment, have the potential to quickly generate new phenotypes, making the understanding of viral reassortment important to both evolutionary biology and public health research. In this paper, we argue that reassortment cannot be reliably inferred from incongruities between segment phylogenies using the established remove-and-rejoin or coalescent approaches. We instead show that reassortment must be considered in the context of a broader population process that includes the dynamics of the infected hosts. Using illustrative examples and simulation we identify four types of evolutionary events that are difficult or impossible to reconstruct with incongruence-based methods. Further, we show that these specific situations are very common and will likely occur even in small samples. Finally, we argue that existing methods can be augmented or modified to account for all the problematic situations that we identify in this paper. Robust assessment of the role of reassortment in viral evolution is difficult, and we hope to provide conceptual clarity on some important methodological issues that can arise in the development of the next generation of tools for studying reassortment.

Inferring viral transmission time from phylogenies for known transmission pairs.

When the time of an HIV transmission event is unknown, methods to identify it from virus genetic data can reveal the circumstances that enable transmission. We developed a single-parameter Markov model to infer transmission time from an HIV phylogeny constructed of multiple virus sequences from people in a transmission pair. Our method finds the statistical support for transmission occurring in different possible time slices. We compared our time-slice model results to previously-described methods: a tree-based logical transmission interval, a simple parsimony-like rules-based method, and a more complex coalescent model. Across simulations with multiple transmitted lineages, different transmission times relative to the source's infection, and different sampling times relative to transmission, we found that overall our time-slice model provided accurate and narrower estimates of the time of transmission. We also identified situations when transmission time or direction was difficult to estimate by any method, particularly when transmission occurred long after the source was infected and when sampling occurred long after transmission. Applying our model to real HIV transmission pairs showed some agreement with facts known from the case investigations. We also found, however, that uncertainty on the inferred transmission time was driven more by uncertainty from time-calibration of the phylogeny than from the model inference itself. Encouragingly, comparable performance of the Markov time-slice model and the coalescent model-which make use of different information within a tree-suggests that a new method remains to be described that will make full use of the topology and node times for improved transmission time inference.

Recombination smooths the time signal disrupted by latency in within-host HIV phylogenies.

Within-host Human immunodeficiency virus (HIV) evolution involves several features that may disrupt standard phylogenetic reconstruction. One important feature is reactivation of latently integrated provirus, which has the potential to disrupt the temporal signal, leading to variation in the branch lengths and apparent evolutionary rates in a tree. Yet, real within-host HIV phylogenies tend to show clear, ladder-like trees structured by the time of sampling. Another important feature is recombination, which violates the fundamental assumption that evolutionary history can be represented by a single bifurcating tree. Thus, recombination complicates the within-host HIV dynamic by mixing genomes and creating evolutionary loop structures that cannot be represented in a bifurcating tree. In this paper, we develop a coalescent-based simulator of within-host HIV evolution that includes latency, recombination, and effective population size dynamics that allows us to study the relationship between the true, complex genealogy of within-host HIV evolution, encoded as an ancestral recombination graph (ARG), and the observed phylogenetic tree. To compare our ARG results to the familiar phylogeny format, we calculate the expected bifurcating tree after decomposing the ARG into all unique site trees, their combined distance matrix, and the overall corresponding bifurcating tree. While latency and recombination separately disrupt the phylogenetic signal, remarkably, we find that recombination recovers the temporal signal of within-host HIV evolution caused by latency by mixing fragments of old, latent genomes into the contemporary population. In effect, recombination averages over extant heterogeneity, whether it stems from mixed time signals or population bottlenecks. Furthermore, we establish that the signals of latency and recombination can be observed in phylogenetic trees despite being an incorrect representation of the true evolutionary history. Using an approximate Bayesian computation method, we develop a set of statistical probes to tune our simulation model to nine longitudinally sampled within-host HIV phylogenies. Because ARGs are exceedingly difficult to infer from real HIV data, our simulation system allows investigating effects of latency, recombination, and population size bottlenecks by matching decomposed ARGs to real data as observed in standard phylogenies.

Molecular and Genetic Characterization of Hepatitis B Virus (HBV) among Saudi Chronically HBV-Infected Individuals.

The study aimed to characterize the genotype and subgenotypes of HBV circulating in Saudi Arabia, the presence of clinically relevant mutations possibly associated with resistance to antivirals or immune escape phenomena, and the possible impact of mutations in the structural characteristics of HBV polymerase. Plasma samples from 12 Saudi Arabian HBV-infected patients were analyzed using an in-house PCR method and direct sequencing. Saudi patients were infected with mainly subgenotype D1. A number of mutations in the RT gene (correlated to antiviral resistance) and within and outside the major hydrophilic region of the S gene (claimed to influence immunogenicity and be related to immune escape) were observed in almost all patients. Furthermore, the presence of mutations in the S region caused a change in the tertiary structure of the protein compared with the consensus region. Clinical manifestations of HBV infection may change dramatically as a result of viral and host factors: the study of mutations and protein-associated cofactors might define possible aspects relevant for the natural and therapeutic history of HBV infection.

Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ∼1 year after infection. Intact SIV genomes in circulating CD4+T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6-2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals.

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4+ T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4+ T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

A deep learning approach to real-time HIV outbreak detection using genetic data.

Pathogen genomic sequence data are increasingly made available for epidemiological monitoring. A main interest is to identify and assess the potential of infectious disease outbreaks. While popular methods to analyze sequence data often involve phylogenetic tree inference, they are vulnerable to errors from recombination and impose a high computational cost, making it difficult to obtain real-time results when the number of sequences is in or above the thousands. Here, we propose an alternative strategy to outbreak detection using genomic data based on deep learning methods developed for image classification. The key idea is to use a pairwise genetic distance matrix calculated from viral sequences as an image, and develop convolutional neutral network (CNN) models to classify areas of the images that show signatures of active outbreak, leading to identification of subsets of sequences taken from an active outbreak. We showed that our method is efficient in finding HIV-1 outbreaks with R0 ≥ 2.5, and overall a specificity exceeding 98% and sensitivity better than 92%. We validated our approach using data from HIV-1 CRF01 in Europe, containing both endemic sequences and a well-known dual outbreak in intravenous drug users. Our model accurately identified known outbreak sequences in the background of slower spreading HIV. Importantly, we detected both outbreaks early on, before they were over, implying that had this method been applied in real-time as data became available, one would have been able to intervene and possibly prevent the extent of these outbreaks. This approach is scalable to processing hundreds of thousands of sequences, making it useful for current and future real-time epidemiological investigations, including public health monitoring using large databases and especially for rapid outbreak identification.

Combining biomarker and virus phylogenetic models improves HIV-1 epidemiological source identification.

To identify and stop active HIV transmission chains new epidemiological techniques are needed. Here, we describe the development of a multi-biomarker augmentation to phylogenetic inference of the underlying transmission history in a local population. HIV biomarkers are measurable biological quantities that have some relationship to the amount of time someone has been infected with HIV. To train our model, we used five biomarkers based on real data from serological assays, HIV sequence data, and target cell counts in longitudinally followed, untreated patients with known infection times. The biomarkers were modeled with a mixed effects framework to allow for patient specific variation and general trends, and fit to patient data using Markov Chain Monte Carlo (MCMC) methods. Subsequently, the density of the unobserved infection time conditional on observed biomarkers were obtained by integrating out the random effects from the model fit. This probabilistic information about infection times was incorporated into the likelihood function for the transmission history and phylogenetic tree reconstruction, informed by the HIV sequence data. To critically test our methodology, we developed a coalescent-based simulation framework that generates phylogenies and biomarkers given a specific or general transmission history. Testing on many epidemiological scenarios showed that biomarker augmented phylogenetics can reach 90% accuracy under idealized situations. Under realistic within-host HIV-1 evolution, involving substantial within-host diversification and frequent transmission of multiple lineages, the average accuracy was at about 50% in transmission clusters involving 5-50 hosts. Realistic biomarker data added on average 16 percentage points over using the phylogeny alone. Using more biomarkers improved the performance. Shorter temporal spacing between transmission events and increased transmission heterogeneity reduced reconstruction accuracy, but larger clusters were not harder to get right. More sequence data per infected host also improved accuracy. We show that the method is robust to incomplete sampling and that adding biomarkers improves reconstructions of real HIV-1 transmission histories. The technology presented here could allow for better prevention programs by providing data for locally informed and tailored strategies.

Large Evolutionary Rate Heterogeneity among and within HIV-1 Subtypes and CRFs.

HIV-1 is a fast-evolving, genetically diverse virus presently classified into several groups and subtypes. The virus evolves rapidly because of an error-prone polymerase, high rates of recombination, and selection in response to the host immune system and clinical management of the infection. The rate of evolution is also influenced by the rate of virus spread in a population and nature of the outbreak, among other factors. HIV-1 evolution is thus driven by a range of complex genetic, social, and epidemiological factors that complicates disease management and prevention. Here, we quantify the evolutionary (substitution) rate heterogeneity among major HIV-1 subtypes and recombinants by analyzing the largest collection of HIV-1 genetic data spanning the widest possible geographical (100 countries) and temporal (1981-2019) spread. We show that HIV-1 substitution rates vary substantially, sometimes by several folds, both across the virus genome and between major subtypes and recombinants, but also within a subtype. Across subtypes, rates ranged 3.5-fold from 1.34 × 10-3 to 4.72 × 10-3 in env and 2.3-fold from 0.95 × 10-3 to 2.18 × 10-3 substitutions site-1 year-1 in pol. Within the subtype, 3-fold rate variation was observed in env in different human populations. It is possible that HIV-1 lineages in different parts of the world are operating under different selection pressures leading to substantial rate heterogeneity within and between subtypes. We further highlight how such rate heterogeneity can complicate HIV-1 phylodynamic studies, specifically, inferences on epidemiological linkage of transmission clusters based on genetic distance or phylogenetic data, and can mislead estimates about the timing of HIV-1 lineages.

Genetic Subtypes and Natural Resistance Mutations in HCV Genotype 4 Infected Saudi Arabian Patients.

This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.

Reuse of Ti6Al4V Powder and Its Impact on Surface Tension, Melt Pool Behavior and Mechanical Properties of Additively Manufactured Components.

The quality and characteristics of a powder in powder bed fusion processes play a vital role in the quality of additively manufactured components. Its characteristics may influence the process in various ways. This paper presents an investigation highlighting the influence of powder deterioration on the stability of a molten pool in a laser beam powder bed fusion (LB-PBF, selective laser melting) process and its consequences to the physical properties of the alloy, porosity of 3D-printed components and their mechanical properties. The intention in this was to understand powder reuse as a factor playing a role in the formation of porosity in 3D-printed components. Ti6Al4V (15 µm-45 µm) was used as a base material in the form of a fresh powder and a degraded one (reused 12 times). Alloy degradation is described by possible changes in the shape of particles, particle size distribution, chemical composition, surface tension, density and viscosity of the melt. An approach of 3D printing singular lines was applied in order to study the behavior of a molten pool at varying powder bed depths. Single-track cross-sections (STCSs) were described with shape parameters and compared. Furthermore, the influence of the molten pool stability on the final density and mechanical properties of a material was discussed. Electromagnetic levitation (EML) was used to measure surface tension and the density of the melt using pieces of printed samples. It was found that the powder degradation influences the mechanical properties of a printed material by destabilizing the pool of molten metal during printing operation by facilitating the axial flow on the melt along the melt track axis. Additionally, the observed axial flow was found to facilitate a localized lack of fusion between concurrent layers. It was also found that the surface tension and density of the melt are only impacted marginally or not at all by increased oxygen content, yet a difference in the temperature dependence of the surface tension was observed.