Mixed Infections Unravel Novel HCV Inter-Genotypic Recombinant Forms within the Conserved IRES Region.

Hepatitis C virus (HCV) remains a significant global health challenge, affecting millions of people worldwide, with chronic infection a persistent threat. Despite the advent of direct-acting antivirals (DAAs), challenges in diagnosis and treatment remain, compounded by the lack of an effective vaccine. The HCV genome, characterized by high genetic variability, consists of eight distinct genotypes and over ninety subtypes, underscoring the complex dynamics of the virus within infected individuals. This study delves into the intriguing realm of HCV genetic diversity, specifically exploring the phenomenon of mixed infections and the subsequent detection of recombinant forms within the conserved internal ribosome entry site (IRES) region. Previous studies have identified recombination as a rare event in HCV. However, our findings challenge this notion by providing the first evidence of 1a/3a (and vice versa) inter-genotypic recombination within the conserved IRES region. Utilizing advanced sequencing methods, such as deep sequencing and molecular cloning, our study reveals mixed infections involving genotypes 1a and 3a. This comprehensive approach not only confirmed the presence of mixed infections, but also identified the existence of recombinant forms not previously seen in the IRES region. The recombinant sequences, although present as low-frequency variants, open new avenues for understanding HCV evolution and adaptation.

The SIRAH force field: A suite for simulations of complex biological systems at the coarse-grained and multiscale levels.

The different combinations of molecular dynamics simulations with coarse-grained representations have acquired considerable popularity among the scientific community. Especially in biocomputing, the significant speedup granted by simplified molecular models opened the possibility of increasing the diversity and complexity of macromolecular systems, providing realistic insights on large assemblies for more extended time windows. However, a holistic view of biological ensembles' structural and dynamic features requires a self-consistent force field, namely, a set of equations and parameters that describe the intra and intermolecular interactions among moieties of diverse chemical nature (i.e., nucleic and amino acids, lipids, solvent, ions, etc.). Nevertheless, examples of such force fields are scarce in the literature at the fully atomistic and coarse-grained levels. Moreover, the number of force fields capable of handling simultaneously different scales is restricted to a handful. Among those, the SIRAH force field, developed in our group, furnishes a set of topologies and tools that facilitate the setting up and running of molecular dynamics simulations at the coarse-grained and multiscale levels. SIRAH uses the same classical pairwise Hamiltonian function implemented in the most popular molecular dynamics software. In particular, it runs natively in AMBER and Gromacs engines, and porting it to other simulation packages is straightforward. This review describes the underlying philosophy behind the development of SIRAH over the years and across families of biological molecules, discussing current limitations and future implementations.

Genome Sequences of SARS-CoV-2 Strains from the Republic of Moldova.

The whole-genome sequences of 15 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains from nasopharyngeal swab samples collected in the Republic of Moldova in June 2020 to September 2021 were determined. Little variability was observed in the early stages, when mostly clade 19A was circulating, followed by clade 20B. Later, multiple introductions of SARS-CoV-2 lineages B.1.1., B.1.1.7, and B.1.1.525 were detected. The B.1.1.7 lineage became predominant between December 2020 and June 2021, followed by the Delta variant.

The stressed life of a lipid in the Zika virus membrane.

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, these kinds of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we use multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between both membrane layers. These findings lead to the idea that the Flaviviral membrane may store a significant amount of elastic energy, playing an active role in the membrane fusion process.

Resistance-associated substitutions and response to treatment in a chronic hepatitis C virus infected-patient: an unusual virological response case report.

BACKGROUND: Direct-Acting agents (DAAs) target and inhibit essential viral replication proteins. They have revolutionized the treatment of Hepatitis C virus (HCV) infection reaching high levels of sustained virologic response. However, the detection of basal resistance-associated substitutions (RASs) to DAAs in naïve patients could be important in predicting the treatment outcome in some patients exhibiting failures to DAA-based therapies. Therefore, the aim of this work was to evaluate the presence of RASs as minority variants within intra-host viral populations, and assess their relationship to response to therapy on a multiple times relapser patient infected chronically with HCV. CASE PRESENTATION: A male HCV infected-patient with a genotype 1a strain was evaluated. He had previously not responded to dual therapy (pegylated interferon-α plus ribavirin) and was going to start a direct-acting agent-based therapy (DAAs). He showed no significant liver fibrosis (F0). Viral RNA was extracted from serum samples taken prior and after therapy with DAAs (sofosbubir/ledipasvir/ribavirin). NS5A and NS5B genomic regions were PCR-amplified and the amplicons were sequenced using Sanger and next-generation sequencing (NGS) approaches. RASs were searched in in-silico translated sequences for all DAAs available and their frequencies were determined for those detected by NGS technology. Sanger sequencing did not reveal the presence of RASs in the consensus sequence neither before nor after the DAA treatment. However, several RASs were found at low frequencies, both before as well as after DAA treatment. RASs found as minority variants (particularly substitutions in position 93 within NS5A region) seem to have increased their frequency after DAA pressure. Nevertheless, these RASs did not become dominant and the patient still relapsed, despite perfect adherence to treatment and having no other complications beyond the infection (no significant fibrosis, no drug abuse). CONCLUSIONS: This report shows that some patients might relapse after a DAA-based therapy even when RASs (pre- and post-treatment) are detected in very low frequencies (< 1%) within intra-host viral populations. Increased awareness of this association may improve detection and guide towards a personalized HCV treatment, directly improving the outcome in hard-to-treat patients.

Wrapping Up Viruses at Multiscale Resolution: Optimizing PACKMOL and SIRAH Execution for Simulating the Zika Virus.

Simulating huge biomolecular complexes of million atoms at relevant biological time scales is becoming accessible to the broad scientific community. That proves to be crucial for urgent responses against emergent diseases in real time. Yet, there are still issues to sort regarding the system setup so that molecular dynamics (MD) simulations can be run in a simple and standard way. Here, we introduce an optimized pipeline for building and simulating enveloped virus-like particles (VLP). First, the membrane packing problem is tackled with new features and optimized options in PACKMOL. This allows preparing accurate membrane models of thousands of lipids in the context of a VLP within a few hours using a single CPU. Then, the assembly of the VLP system is done within the multiscale framework of the coarse-grained SIRAH force field. Finally, the equilibration protocol provides a system ready for production MD simulations within a few days on broadly accessible GPU resources. The pipeline is applied to study the Zika virus as a test case for large biomolecular systems. The VLP stabilizes at approximately 0.5 µs of MD simulation, reproducing correlations greater than 0.90 against experimental density maps from cryo-electron microscopy. Detailed structural analysis of the protein envelope also shows very good agreement in root-mean-square deviations and B-factors with the experimental data. The level of details attained shows for the first time a possible role for anionic phospholipids in stabilizing the envelope. Combining an efficient and reliable setup procedure with an accurate coarse-grained force field provides a valuable pipeline for simulating arbitrary viral systems or subcellular compartments, paving the way toward whole-cell simulations.

Prevalence and molecular epidemiology of bovine leukemia virus in Colombian cattle.

Bovine leukemia virus (BLV) is one of the five agents considered most significant for cattle. It is important to determine the prevalence and molecular epidemiology of BLV throughout the country in order to gain a more thorough understanding of the current situation of BLV and to reveal the possibility of masked genotypes that the primers used by OIE are unable to identify. Blood samples were collected at random from 289 cows distributed in 75 farms across the country. PCR amplification of env, gag and tax gene segments was performed. The obtained amplicons were sequenced and then subjected to phylogenetic analyses. A total of 62% of the cows present at 92% of the farms were BLV-positive for gag fragment. Genotype 1 was exclusively detected by env gene segment when analyzed using previously reported primers. However, tax gene analysis revealed circulation of genotype 6 variants, which were also detected based on env gene analysis with newly designed primers. These results indicate that current genotyping approaches based on partial env sequencing may bias BLV genetic variability approaches and underestimate the diversity of the detected BLV genotypes. This report is one of the first molecular and epidemiological studies of BLV conducted in Colombia, which contributes to the global epidemiology of the virus; it also highlights the substantial impact of BLV on the country's livestock and thus is a useful resource for farmers and government entities.

The SIRAH 2.0 Force Field: Altius, Fortius, Citius.

A new version of the coarse-grained (CG) SIRAH force field for proteins has been developed. Modifications to bonded and non-bonded interactions on the existing molecular topologies significantly ameliorate the structural description and flexibility of a non-redundant set of proteins. The SIRAH 2.0 force field has also been ported to the popular simulation package AMBER, which along with the former implementation in GROMACS expands significantly the potential range of users and performance of this CG force field on CPU/GPU codes. As a non-trivial example of its application, we undertook the structural and dynamical analysis of the most abundant and conserved calcium-binding protein, calmodulin (CaM). CaM is composed of two calcium-binding motifs called EF-hands, which in the presence of calcium specifically recognize a cognate peptide by embracing it. CG simulations of CaM bound to four calcium ions in the presence or absence of a binding peptide (holo and apo forms, respectively) resulted in good and stable ion coordination. The simulation of the holo form starting from an experimental structure sampled near-native conformations, retrieving quasi-atomistic precision. Removing the binding peptide enabled the EF-hands to perform large reciprocal movements, comparable to those observed in NMR structures. On the other hand, the isolated peptide starting from the helical conformation experienced spontaneous unfolding, in agreement with previous experimental data. However, repositioning the peptide in the neighborhood of one EF-hand not only prevented the peptide from unfolding but also drove CaM to a fully bound conformation, with both EF-hands embracing the cognate peptide, resembling the experimental holo structure. Therefore, SIRAH 2.0 shows the capacity to handle a number of structurally and dynamically challenging situations, including metal ion coordination, unbiased conformational sampling, and specific protein-peptide recognition.

Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients.

Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.

Evidence of increasing diversification of Zika virus strains isolated in the American continent.

Zika virus (ZIKV) is a member of the family Flaviviridae. ZIKV emerged in Brazil in 2015, causing an unprecedented epidemic and since then the virus has rapidly spread throughout the Americas. These facts highlight the need of detailed phylogenetic studies to understand the emergence, spread, and evolution of ZIKV populations. For these reasons, a Bayesian coalescent Markov Chain Monte Carlo analysis of complete genome sequences of ZIKV strains recently isolated in the American continent was performed. The results of these studies revealed an increasing diversification of ZIKV strains in different genetic lineages and co-circulation of distinct genetic lineages in several countries in the region. The time of the most recent common ancestor (tMRCA) was established to be around February 20, 2014 for ZIKV strains circulating in the American region. A mean rate of evolution of 1.55 × 10-3 substitutions/site/year was obtained for ZIKV strains included in this study. A Bayesian skyline plot indicate a sharp increase in population size from February 2014 to July 2015 and a decline during 2016. These results are discussed in terms of the emergence and evolution of ZIKV populations in the American continent.

Host influence in the genomic composition of flaviviruses: A multivariate approach.

Flaviviruses present substantial differences in their host range and transmissibility. We studied the evolution of base composition, dinucleotide biases, codon usage and amino acid frequencies in the genus Flavivirus within a phylogenetic framework by principal components analysis. There is a mutual interplay between the evolutionary history of flaviviruses and their respective vectors and/or hosts. Hosts associated to distinct phylogenetic groups may be driving flaviviruses at different pace and through various sequence landscapes, as can be seen for viruses associated with Aedes or Culex spp., although phylogenetic inertia cannot be ruled out. In some cases, viruses face even opposite forces. For instance, in tick-borne flaviviruses, while vertebrate hosts exert pressure to deplete their CpG, tick vectors drive them to exhibit GC-rich codons. Within a vertebrate environment, natural selection appears to be acting on the viral genome to overcome the immune system. On the other side, within an arthropod environment, mutational biases seem to be the dominant forces.

Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries.

Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130-150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients' management and outcome prediction in developing countries.

A detailed comparative analysis of codon usage bias in Zika virus.

Zika virus (ZIKV) is a member of the family Flaviviridae and its genome consists of a single-stranded positive sense RNA molecule with 10,794 nucleotides. Clinical manifestations of disease caused by ZIKV infection range from asymptomatic cases to an influenza-like syndrome. There is an increasing concern about the possible relation among microcephaly and ZIKV infection. To get insight into the relation of codon usage among viruses and their hosts is extremely important to understand virus survival, fitness, evasion from host's immune system and evolution. In this study, we performed a comprehensive analysis of codon usage and composition of ZIKV. The overall codon usage among ZIKV strains is similar and slightly biased. Different codon preferences in ZIKV genes in relation to codon usage of human, Aedes aegypti and Aedes albopictus genes were found. Most of the highly frequent codons are A-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. G+C compositional constraint as well as dinucleotide composition also influence the codon usage of ZIKV. The results of these studies suggest that the emergence of ZIKV outside Africa, in the Pacific and the Americas may also be reflected in ZIKV codon usage. No significant differences were found in codon usage among strains isolated from microcephaly cases and the rest of strains from the Asian cluster enrolled in these studies.

Bayesian coalescent inference reveals high evolutionary rates and diversification of Zika virus populations.

Zika virus (ZIKV) is a member of the family Flaviviridae. In 2015, ZIKV triggered an epidemic in Brazil and spread across Latin America. By May of 2016, the World Health Organization warns over spread of ZIKV beyond this region. Detailed studies on the mode of evolution of ZIKV strains are extremely important for our understanding of the emergence and spread of ZIKV populations. In order to gain insight into these matters, a Bayesian coalescent Markov Chain Monte Carlo analysis of complete genome sequences of recently isolated ZIKV strains was performed. The results of these studies revealed a mean rate of evolution of 1.20 × 10(-3) nucleotide substitutions per site per year (s/s/y) for ZIKV strains enrolled in this study. Several variants isolated in China are grouped together with all strains isolated in Latin America. Another genetic group composed exclusively by Chinese strains were also observed, suggesting the co-circulation of different genetic lineages in China. These findings indicate a high level of diversification of ZIKV populations. Strains isolated from microcephaly cases do not share amino acid substitutions, suggesting that other factors besides viral genetic differences may play a role for the proposed pathogenesis caused by ZIKV infection. J. Med. Virol. 88:1672-1676, 2016. © 2016 Wiley Periodicals, Inc.

An evolutionary insight into Newcastle disease viruses isolated in Antarctica.

The disease caused by Newcastle disease virus (NDV) is a severe threat to the poultry industry worldwide. Recently, NDV has been isolated in the Antarctic region. Detailed studies on the mode of evolution of NDV strains isolated worldwide are relevant for our understanding of the evolutionary history of NDV. For this reason, we have performed Bayesian coalescent analysis of NDV strains isolated in Antarctica to study evolutionary rates, population dynamics, and patterns of evolution. Analysis of F protein cleavage-site sequences of NDV isolates from Antarctica suggested that these strains are lentogenic. Strains isolated in Antarctica and genotype I reference strain Ulster/67 diverged from ancestors that existed around 1958. The time of the most recent common ancestor (MRCA) was established to be around 1883 for all class II viruses. A mean rate of evolution of 1.78 × 10(-3) substitutions per site per year (s/s/y) was obtained for the F gene sequences of NDV strains examined in this study. A Bayesian skyline plot indicated a decline in NDV population size in the last 25 years. The results are discussed in terms of the possible role of Antarctica in emerging or re-emerging viruses and the evolution of NDV populations worldwide.

Phylogenetic analysis of the neuraminidase gene of pandemic H1N1 influenza A virus circulating in the South American region.

Molecular characterization of circulating influenza A viruses (IAV) in all regions of the world is essential to detect mutations potentially involved in increased virulence, anti-viral resistance and immune escape. In order to gain insight into these matters, a phylogenetic analysis of the neuraminidase (NA) gene of 146 pandemic H1N1 (H1N1pdm) influenza A virus strains isolated in Argentina, Brazil, Chile, Paraguay, Peru and Uruguay from 2009 to 2013 was performed. Comparison of vaccine strain A/California/7/2009 included in the influenza vaccine recommended for the Southern hemisphere from 2010 through 2013 influenza seasons and strains isolated in South America revealed several amino acid substitutions. Mapping of these substitutions revealed that most of them are located at the surface of the protein and do not interfere with the active site. 3.4% of the strains enrolled in these studies carried the H275Y substitution that confers resistance to oseltamivir. Strains isolated in South America differ from vaccine in two predicted B-cell epitope regions present at positions 102-103 and 351-352 of the NA protein. Moreover, vaccine and strains isolated in Paraguay differ also in an epitope present at position 229. These differences among strains isolated in South America and vaccine strain suggests that these epitopes may not be present in strains isolated in this region. A potential new N-linked glycosylation site was observed in the NA protein of an H1N1pdm IAV strain isolated in Brazil. The results of these studies revealed several genetic and antigenic differences in the NA of H1N1pdm IAV among vaccine and strains circulating in South America. All these findings contribute to our understanding of the course of genetic and antigenic evolution of H1N1pdm IAV populations circulating in the South American region and, consequently, contribute to the study and selection of future and more appropriate vaccines and anti-viral drugs.

Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development.

BACKGROUND: Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity. The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 IAV strain (H1N1pdm) in Mexico and USA. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. A comprehensive study to investigate the effect of selection pressure imposed by the human host on the codon usage of an emerging, pandemic IAV strain and the trends in viral codon usage involved over the pandemic time period is much needed. RESULTS: We performed a comprehensive codon usage analysis of 310 IAV strains from the pandemic of 2009. Highly biased codon usage for Ala, Arg, Pro, Thr and Ser were found. Codon usage is strongly influenced by underlying biases in base composition. When correspondence analysis (COA) on relative synonymous codon usage (RSCU) is applied, the distribution of IAV ORFs in the plane defined by the first two major dimensional factors showed that different strains are located at different places, suggesting that IAV codon usage also reflects an evolutionary process. CONCLUSIONS: A general association between codon usage bias, base composition and poor adaptation of the virus to the respective host tRNA pool, suggests that mutational pressure is the main force shaping H1N1 pdm IAV codon usage. A dynamic process is observed in the variation of codon usage of the strains enrolled in these studies. These results suggest a balance of mutational bias and natural selection, which allow the virus to explore and re-adapt its codon usage to different environments. Recoding of IAV taking into account codon bias, base composition and adaptation to host tRNA may provide important clues to develop new and appropriate vaccines.

Bayesian coalescent analysis of pandemic H1N1 influenza A virus circulating in the South American region.

The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 influenza A virus strain (H1N1pdm). Understanding the evolution of H1N1pdm populations within the South American region is essential for studying global diversification, emergence, resistance and vaccine efficacy. In order to gain insight into these matters, we have performed a Bayesian coalescent Markov Chain Monte Carlo analysis of hemagglutinin (HA) and neuraminidase (NA) gene sequences of all available and comparable HA and NA sequences obtained from H1N1pdm IAV circulating in the South American region. High evolutionary rates and fast population growths characterize the population dynamics of H1N1pdm strains in this region of the world. A significant contribution of first codon position to the mean evolutionary rate was found for both genes studied, revealing a high contribution of non-synonymous substitutions to the mean substitution rate. In the 178days period covered by these studies, substitutions in all HA epitope regions can be observed. HA substitutions D239G/N and Q310H have been observed only in Brazilian patients. While substitution D239G/N is not particularly associated to a specific genetic lineage, all strains bearing substitution Q310H were assigned to clade 6, suggesting a founder effect. None of the substitutions found in the NA proteins of H1N1pdm strains isolated in South America appears sufficiently close to affect the drug binding pocket for the three NA inhibitor antivirals tested. A more detailed analysis of NA proteins revealed epitope differences among 2010 vaccine and H1N1pdm IAV strains circulating in the South American region.