Rare unclassified 16S rRNA operational taxonomic units from the uncharted Engaño Bay (Argentinean Patagonia).

Rare microbes make up most of the diversity of marine microbiomes, and recent works have highlighted their importance for microbial community dynamics and in fragmented habitats. Rare taxa have been infrequently studied in comparison with abundant groups, and rare unclassified sequences are common in culture-independent studies. Here, we describe a detailed analysis of nonclassifiable sequences from the Chubut river estuary at the Argentinean Patagonia. Standard taxonomic assignments of environmental 16S rRNA sequences resulted in about 13% unclassified operational taxonomic units (OTUs). The potential affiliations of these OTUs could be narrowed by mapping the classification software assignments on a phylogeny obtained directly from our environmental sequence data. Customized BLAST analyses were remarkably consistent with these phylogenetic assignments, especially when the unclassified OTUs were blasted against sequences from cultured and type microorganisms. In addition, our BLAST analyses revealed significant similarities between several unclassified OTUs and a plethora of unclassified sequences from around the world. Further phylogenetic comparisons with 6194 carefully selected reference sequences showed that these unclassified sequences may correspond to 5 unnamed groups, possibly encompassing ranks from subclass to family inside the Alphaproteobacteria, and to an unknown Gracilibacteria lineage. Overall, these results demonstrate the value of straight phylogenetic analysis, customized BLAST searches, and comparisons with sequences from type material, for the systematic study of rare unclassified sequences.

Are ocean currents too slow to counteract SAR11 evolution? A next-generation sequencing, phylogeographic analysis.

This work set out to shed light on the phylogeography of the SAR11 clade of Alphaproteobacteria, which is probably the most abundant group of heterotrophic bacteria on Earth. In particular, we assessed the degree to which empirical evidence (environmental DNA sequences) supports the concept that SAR11 lineages evolve faster than they are dispersed thus generating vicariant distributions, as predicted by recent simulation efforts. We generated 16S rRNA gene sequences from surface seawater collected at the South West Atlantic Ocean and combined these data with previously published sequences from similar environments from elsewhere. Altogether, these data consisted in about 1e6 reads, from which we generated 355,306 high quality sequences of which 95,318 corresponded to SAR11. Quantitative phylogeographic analyses supported the existence of a spatially explicit distribution of SAR11 species and provided evidence in favor of the idea that dispersal limitations significantly contribute to SAR11 radiation throughout the world's oceans. Likewise, pairwise phylogenetic distances between the communities studied here were significantly correlated with the genetic divergences predicted by a previously proposed neutral model. As discussed in the paper, these findings are compatible with the concept that the ocean surface constitutes a homogeneous environment for SAR11, in agreement with previous experimental data. We discuss the implications of this hypothesis in a global change scenario. This is the first study combining high throughput sequencing and phylogenic analysis to study bacterial phylogeography and reporting a distance decay pattern of phylogenetic distances for bacteria.

Virus evolution during chronic hepatitis B virus infection as revealed by ultradeep sequencing data.

Despite chronic hepatitis B virus (HBV) infection (CHB) being a leading cause of liver cirrhosis and cancer, HBV evolution during CHB is not fully understood. Recent studies have indicated that virus diversity progressively increases along the course of CHB and that some virus mutations correlate with severe liver conditions such as chronic hepatitis, cirrhosis and hepatocellular carcinoma. Using ultradeep sequencing (UDS) data from an intrafamilial case, we detected such mutations at low frequencies among three immunotolerant patients and at high frequencies in an inactive carrier. Furthermore, our analyses indicated that the HBV population from the seroconverter patient underwent many genetic changes in response to virus clearance. Together, these data indicate a potential use of UDS for developing non-invasive biomarkers for monitoring disease changes over time or in response to specific therapies. In addition, our analyses revealed that virus clearance seemed not to require the virus effective population size to decline. A detailed genetic analysis of the viral lineages arising during and after the clearance suggested that mutations at or close to critical elements of the core promoter (enhancer II, epsilon encapsidation signal, TA2, TA3 and direct repeat 1-hormone response element) might be responsible for a sustained replication. This hypothesis requires the decline in virus load to be explained by constant clearance of virus-producing hepatocytes, consistent with the sustained progress towards serious liver conditions experienced by many CHB patients.

Hepatitis B virus resistance substitutions: long-term analysis by next-generation sequencing.

HBV phylogenetics and resistance-associated mutations (RAMs) were surveyed by next-generation sequencing of 21 longitudinal samples from seven patients entering antiviral therapy. The virus populations were dominated by a few abundant lineages that coexisted with substantial numbers of low-frequency variants. A few low-frequency RAMs were observed before treatment, but new ones emerged, and their frequencies increased during therapy. Together, these results support the idea that chronic HBV infection is dominated by a few virus lineages and that an accompanying plethora of diverse, low-frequency variants may function as a reservoir that potentially contribute to viral genetic plasticity, potentially affecting patient outcome.

Characterization and phylogeny of Isaria spp. strains (Ascomycota: Hypocreales) using ITS1-5.8S-ITS2 and elongation factor 1-alpha sequences.

The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

A clustering phenomenon among HCV-1a strains among patients coinfected with HIV from Buenos Aires, Argentina.

The human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same transmission routes which lead to high coinfection rates. Among HIV-infected individuals such rates reached 21% in Argentina, being HCV-1a the most predominant subtype. In this work, 25 HCV subtype 1a (HCV-1a) strains from Argentinean patients coinfected with HIV were studied based on E2 and NS5A sequences. Phylogenetic analyses indicated that 12 strains were highly related to each other, constituting a highly supported (posterior probability = 0.95) monophyletic group that we called "M." The remaining HCV strains (group dispersed or "D") were interspersed along the phylogenetic trees. When comparing both groups of HCV-1a, 10 amino acid differences were located in functional domains of E2 and NS5A proteins that appeared to affect eventually the peptides binding to MHC-I molecules thus favoring immune escape and contributing to the divergence of HCV genotypes. Bayesian coalescent analyses for HCV-1a cluster M isolates indicated that the time to the most recent common ancestor (tMRCA) overlaps with the age estimated recently for the HIV-BF epidemic in Argentina. Furthermore, the genomic characterization based on pol gene analysis from HIV viremic patients showed that most HIV isolates from patients coinfected with HCV-1a cluster M were BF recombinants with identical recombination patterns. In conclusion, these results suggest the presence of an HCV-1a monophyletic cluster with a potential HIV co-transmission by phylogenetic analyses.

Phylogenetic analysis of Ostreococcus virus sequences from the Patagonian Coast.

A phylogenetic analysis of new Ostreococcus virus (OV) sequences from the Patagonian Coast, Argentina, and homologous sequences from public databases was performed. This analysis showed that the Patagonian sequences represented a divergent viral clade and that the rest of OV sequences analyzed here were clustered into six additional phylogenetic groups. Analyses of 18S gene libraries supported a close relationship of the Patagonian Ostreococcus host with clade A sequences described elsewhere, corroborating previous studies indicating that clade A strains are ubiquitous. Besides the Patagonian OV sequences, several phylogenetic groupings were linked to particular geographic locations, suggesting a role for allopatric cladogenesis in viral diversification. However, and in agreement with previous observations, other viral lineages included sequences with diverse geographic origins. These findings, together with analyses of ancestral trait trajectories performed here, are consistent with an evolutionary dynamics in which geographical isolation has a role in OV diversification but can be followed by rapid dispersion to remote places.

Stable human T-cell lymphotropic virus type 1 (HTLV-1) subtype a/subgroup a endemicity in Amerindians from Northwest Argentina: a health problem to be resolved.

Jujuy province, in Northwest Argentina, is known to be endemic for HTLV-1 infection. Moreover, foci of HTLV-1 associated pathologies have also been described in this region. To gain an insight into the current situation of HTLV-1/2 in this endemic area, a seroprevalence and phylogenetic study was performed among a Kolla community from Abra Pampa city and surroundings. Out of 112 individuals, 11 (9.8%) were confirmed as HTLV-1 positive and no HTLV-2 infection was detected. The phylogenetic analysis of the LTR region showed that all the HTLV-1 sequences belonged to the Cosmopolitan subtype a/transcontinental subgroup A, and were closely related to reference sequences from Peru, Argentina, and the South of Brazil (P = 0.82). Considering the cultural and historical features of this community and in spite of the mandatory detection of anti-HTLV-1/2 antibodies in blood banks since 2005, it would be important to implement new public health measures focused on decreasing HTLV-1 transmission in this endemic area.

The human fungal pathogen Paracoccidioides brasiliensis (Onygenales: Ajellomycetaceae) is a complex of two species: phylogenetic evidence from five mitochondrial markers.

Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. In order to study the diversity of P. brasiliensis mitochondrial genes, to evaluate previous taxonomic proposals, and to explore the hypothesis that the previously described "divergent isolate" B30 (also called Pb01) could represent a new P. brasiliensis species, we undertook a molecular phylogenetic analysis based on five mitochondrial markers. Mitochondrial sequences of 59 P. brasiliensis isolates obtained from clinical and environmental samples, and the orthologous genes from outgroup species, are reported and analysed using parsimony and Bayesian methods. The combined data set comprised 2364 characters, of which 426 were informative. One of the studied strains presented a 376-nt insertion at the apocytochrome b (cob) gene. The corresponding sequence had a high similarity (79%) with an intron found in the Neurospora crassa cob gene. Interestingly, this intron is absent in the previously published sequence of the P. brasiliensis mitochondrial genome. Our trees were moderately congruent with the previous P. brasiliensis taxonomic proposals. Furthermore, we identified a new monophyletic group of strains within P. brasiliensis. Nevertheless, the phylogenetic species recognition (PSR) analyses described here suggested that these groups of strains could represent geographical variants rather than different Paracoccidioides cryptic species. In addition, and as previously proposed by other authors, these analyses supported the existence of a new specie of Paracoccidioides, which includes the previously described, divergent isolate B30/Pb01. This is the first report providing evidence, independent of nuclear markers, for the split of this important human pathogen into two species. We support the formal description of the B30/Pb01 as new specie. ©The Willi Hennig Society 2010.

Quantitative phylogenomic evidence reveals a spatially structured SARS-CoV-2 diversity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent RNA virus that spread around the planet in about 4 months. The consequences of this rapid dispersion are under investigation. In this work, we analyzed thousands of genomes and protein sequences from Africa, America, Asia, Europe, and Oceania. We provide statistically significant evidence that SARS-CoV-2 phylogeny is spatially structured. Remarkably, the virus phylogeographic patterns were correlated with ancestral amino acidic substitutions, suggesting that such mutations emerged along colonization events. We hypothesize that geographic structuring is the result of founder effects occurring as a consequence of, and local evolution occurring after, long-distance dispersion. Based on previous studies, the possibility that this could significantly affect the virus biology is not remote.

Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades.

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave unique names to the identified groups and no study has yet integrated all the available sequences. Thus, we hypothesized that many of the different groups previously identified actually correspond to a limited group of genotypes that are unevenly distributed worldwide. To examine this hypothesis, we sequenced the env gene from 28 BLV field strains and compared these sequences to 46 env sequences that represent all the genetic groupings already identified. By using phylogenetic analyses, we recovered six clades, or genotypes, that we have called genotypes 1, 2, 3, 4, 5 and 6. Genotypes 1-5 have counterparts among the sequence groupings identified previously. One env sequence did not cluster with any of the others and was highly divergent when compared with the six genotypes identified here. Thus, an extra genotype, which we named 7, may exist. Similarity comparisons were highly congruent with phylogenetic analyses. Furthermore, our analyses confirmed the existence of geographical clusters.

Genetic analysis of the invasive alga Didymosphenia geminata in Southern Argentina: Evidence of a Pleistocene origin of local lineages.

The diatom Didymosphenia geminata has gained notoriety due to the massive growths which have occurred in recent decades in temperate regions. Different explanations have been proposed for this phenomenon, including the emergence of new invasive strains, human dispersion and climate change. Despite the fact in Argentina nuisance growths began in about 2010, historical records suggest that the alga was already present before that date. In addition, preliminary genetic data revealed too high a diversity to be explained by a recent invasion. Here, we estimate the divergence times of strains from southern Argentina. We integrate new genetic data and secondary, fossil and geological calibrations into a Penalized Likelihood model used to infer 18,630 plausible chronograms. These indicate that radiation of the lineages in Argentina began during or before the Pleistocene, which is hard to reconcile with the hypothesis that a new variant is responsible for the local mass growths. Instead, this suggests that important features of present distribution could be the result of multiple recent colonizations or the expansion of formerly rare populations. The text explains how these two possibilities are compatible with the hypothesis that recent nuisance blooms may be a consequence of climate change.

Phylogenetic Diversity in Core Region of Hepatitis C Virus Genotype 1a as a Factor Associated with Fibrosis Severity in HIV-1-Coinfected Patients.

High hepatitis C virus (HCV) genetic diversity impacts infectivity/pathogenicity, influencing chronic liver disease progression associated with fibrosis degrees and hepatocellular carcinoma. HCV core protein is crucial in cell-growth regulation and host-gene expression. Liver fibrosis is accelerated by unknown mechanisms in human immunodeficiency virus-1- (HIV-1-) coinfected individuals. We aimed to study whether well-defined HCV-1a core polymorphisms and genetic heterogeneity are related to fibrosis in a highly homogeneous group of interferon-treated HIV-HCV-coinfected patients. Genetic heterogeneity was weighed by Faith's phylogenetic diversity (PD), which has been little studied in HCV. Eighteen HCV/HIV-coinfected patients presenting different liver fibrosis stages before anti-HCV treatment-initiation were recruited. Sampling at baseline and during and after treatment was performed up to 72 weeks. At inter/intrahost level, HCV-1a populations were studied using molecular cloning and Sanger sequencing. Over 400 complete HCV-1a core sequences encompassing 573 positions of C were obtained. Amino acid substitutions found previously at positions 70 and 91 of HCV-1b core region were not observed. However, HCV genetic heterogeneity was higher in mild than in severe fibrosis cases. These results suggest a potential utility of PD as a virus-related factor associated with chronic hepatitis C progression. These observations should be reassessed in larger cohorts to corroborate our findings and assess other potential covariates.

Genomic characterization and molecular evolution analysis of subtype B and BF recombinant HIV-1 strains among Argentinean men who have sex with men reveal a complex scenario.

Currently, data on HIV-1 circulating strains among men who have sex with men (MSM) in Argentina is scarce. In South America, the distribution and the prevalence of BF recombinants are dissimilar and exhibit an underappreciated heterogeneity of recombinant structures. Here, we studied for the first time the genetic diversity of HIV-1 BF recombinants and their evolution over time through in-depth phylogenetic analysis and multiple recombination detection methods involving 337 HIV-1 nucleotide sequences (25 near full-length (NFL) and 312 partial pol gene) obtained from Argentinean MSM. The recombination profiles were studied using multiple in silico tools to characterize the genetic mosaicism, and phylogenetic approaches to infer their relationships. The evolutionary history of BF recombinants and subtype B sequences was reconstructed by a Bayesian coalescent-based method. By phylogenetic inference, 81/312 pol sequences clustered within BF clade. Of them, 46 sequences showed a genetic mosaic with CRF12_BF-like patterns, including plausible second-generation recombinants. Other CRFs_BF like (CRF17, 28, 29, 39, 42, 44, 47) and probable URFs_BF were less frequently found. Phylogenetic and recombination analyses on NFL sequences allowed a meticulous definition of new BF mosaics of genomic patterns. The Bayesian analyses pointed out quite consistent onset dates for the CRFs_BF clade based on B and F gene datasets (~1986 and ~1991 respectively). These results indicate that the CRFs_BF variants have been circulating among Argentinean MSM for about 30 years. This study reveals, through growing evidence showing the importance of MSM in the dynamics of the HIV-1 epidemic in Argentina, the coexistence of CRF12_BF-like and high diversity of strains exhibiting several BF mosaic patterns, including non-reported URFs that may reflect active clusters as potential intervention targets to hinder HIV-1 transmission.

A long distance RT-PCR able to amplify the Pestivirus genome.

A method to amplify long genomic regions (up to approximately 12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer_Arg reference strain obtained using this method is presented and characterized.

Evolution of hepatitis C virus in HIV coinfected patients under antiretroviral therapy.

Five patients (P) were followed-up for an average of 7.73years after highly active antiretroviral therapy (HAART) initiation. Patients' immune and virological status were determined by periodical CD4+T-cell counts and HIV and HCV viral load. HCV populations were studied using longitudinal high throughput sequence data obtained in parallel by virological and immunological parameters. Two patients (P7, P28) with sub-optimal responses to HAART presented HCV viral loads significantly higher than those recorded for two patients (P1, P18) that achieved good responses to HAART. Interestingly, HCV populations from P7 and P28 displayed a stable phylogenetic structure, whereas HCV populations from P1 and P18showeda significant increase in their phylogenetic structure, followed by a decrease after achieving acceptable CD4+T-cell counts (>500 cell/µl). The fifth patient (P25) presented high HCV viral loads, preserved CD4+T-cell counts from baseline and all along the follow-up, and displayed a constant viral phylogenetic structure. These results strongly suggest that HAART-induced immune recovery induces a decrease in HCV viral load and an increase in the HCV population phylogenetic structure likely reflecting the virus diversification in response to the afresh immune response. The relatively low HCV viral load observed in the HAART responder patients suggests that once HCV is adapted it reaches a maximum number of haplotypes higher than that achieved during the initial stages of the immune response as inferred from the two recovering patients. Future studies using larger number of patients are needed to corroborate these hypotheses.

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.

HIV-1 tropism dynamics and phylogenetic analysis from longitudinal ultra-deep sequencing data of CCR5- and CXCR4-using variants.

OBJECTIVE: Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships. METHODS: Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained. RESULTS: Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence. CONCLUSIONS: CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.

Effects of UV radiation on the taxonomic composition of natural bacterioplankton communities from Bahía Engaño (Patagonia, Argentina).

In order to gain insights into the effects of solar ultraviolet radiation (UVR, 280-400 nm) on the composition of marine bacterioplankton communities from South Atlantic waters - Bahía Engaño (Patagonia, Argentina), we performed microcosms experiments during the Austral summer of 2010. Water samples were exposed to three solar radiation treatments in 25 L microcosms during 8 days: PAR+UV-A+UV-B (280-700 nm; PAB treatment), PAR+UV-A (320-700 nm; PA treatment), and PAR only (400-700 nm; P treatment). The taxonomic composition of the bacterial communities, at the beginning and at the end of the experiment, were studied by the analyses of 16S rDNA gene libraries. Multivariate and phylogenetic analyses demonstrated substantial differences in the community composition so that the samples exposed to PAR and PAR+UV-A presented more similar taxa assemblages among them than compared to the PAR+UV-A+UV-B exposed one. Our results indicate that overall, exposure to different radiation treatments can shape the taxonomic composition of marine bacterial populations, grown in microcosms, from this Patagonian area.