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.Fil: Jones, Leandro Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales y Ciencias de la Salud - Sede Trelew. Laboratorio de Virología y Genética Molecular; ArgentinaFil: Manrique, Julieta Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales y Ciencias de la Salud - Sede Trelew. Laboratorio de Virología y Genética Molecular; Argentin

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.Fil: Manrique, Julieta Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; ArgentinaFil: Calvo, Andrea Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; ArgentinaFil: Jones, Leandro Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentin

An optimized DNA extraction protocol for benthic Didymosphenia geminata

Didymosphenia geminata mats display few cells in relation to extracellular material and contain polysaccharides and heavy metals that interfere with molecular studies. We describe an optimized DNA extraction protocol that help to overcome these difficulties. Our protocol outperformed five previously described DNA extraction techniques.Fil: Uyua, Noelia Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Manrique, Julieta Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentin

Linear polyethylenimine-decorated gold nanoparticles: One-step electrodeposition and studies of interaction with viral and animal proteins

Polyethylenimine (PEI)-decorated gold nanoparticles (AuNP) were electrodeposited on conductive surfaces in one-step procedure. Solution-state NMR evidenced that chloride from PEI.HCl was partially exchanged by AuCl 4 − prior to AuNP formation. XPS studies indicated the presence of Au° together with the interaction between AuNP with nitrogen atoms of the PEI polymer. The particle size by DLS was 49 nm. The electrochemical behavior of bare electrode and of glassy carbon electrode modified with PEI-decorated AuNP was compared using [Fe(CN) 6 ] 3-/4- redox probe, to determine the potential role of the nanomaterial and of the polymer in the detection of proteins. The most relevant experimental variables from cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) were used for the characterization of protein uptake. The adsorption by electrostatic interaction between the biomolecules and the positively charged polymer (PEIH + ) affected negatively the current response (Ip) of the probe, especially when the negatively charged protein was involved. This platform resulted adequate to immobilize proteins and to characterize this process, for further applications as a tool in bioanalysis or biotechnology.Fil: Lazaro Martinez, Juan Manuel. Universidad de Málaga; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Byrne, Agustin Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Rodríguez Castellón, Enrique. Universidad de Málaga; EspañaFil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales y Ciencias de la Salud - Sede Trelew. Laboratorio de Virología y Genética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales y Ciencias de la Salud - Sede Trelew. Laboratorio de Virología y Genética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campodallorto, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentin

Genomic analysis of bovine herpesvirus type 4 (BoHV-4) from Argentina: High genetic variability and novel phylogenetic groups

Bovine herpesvirus 4 (BoHV-4) is a γ-herpesvirus that has been isolated both from apparently healthy animals and from cattle with a variety of clinical signs, including post-partum endometritis and abortion. BoHV-4 causes either a persistent or a latent infection in cells of the monocyte/macrophage lineage. Two groups of BoVH-4 strains have been defined based on their restriction patterns: the Movar-like strains (European prototype) and the DN 599-like strains (American prototype). The purpose of the present study was to genetically characterize wild type BoHV-4 strains isolated from vaginal discharges of aborted cows in Argentina. The virus was identified by isolation and nested PCR in all vaginal discharge samples from aborted cows, either as a sole agent or in association with other pathogens. Restriction enzyme profiling and phylogenetic analysis demonstrated that there is a high genetic variability among the studied field isolates. The existence of three groups of strains, which were designated as genotypes 1, 2 and 3, is described. Genotypes 1 and 2 possibly correspond to the Movar-like and DN 599-like groups, respectively, whereas Genotype 3 corresponds to a novel group. Two viral strains did not cluster into any of these three groups, indicating that other genotypes could be circulating in Argentina. These results suggest a complex epidemiological background for the Argentinean BoHV-4 strains, probably influenced by independent events of genetic drift. This hypothesis cannot be rejected based on the available data. However, there is no direct evidence supporting this possibility. Thus, it seems speculative to suggest that interspecific jumps are responsible for the observed phylogenetic grouping. On the other hand, our analyses suggest a geographical structure for the observed viral genotypes, since genotypes 1 and 2 included the European (Movar-like) and American (DN599-like) reference strains, respectively. Geographic dispersion is known to be a driver of herpes viruses diversification, and independent evolution in geographical isolated places ensures the emergence of particular mutations in each location due to genetic drift (Compans, 2007; Zong et al., 1999). Therefore, at this point, the genetic drift hypothesis is the one that requires less ad-hoc considerations and thus, to our understanding, is the one that fits to the findings from this study. The involvement of this genetic variability in the detection and pathogenesis of BoHV-4 remains to be investigated.Fil: Verna, Andrea Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Manrique, Julieta Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; ArgentinaFil: Pérez, Sandra Elizabeth. Universidad Nacional del Centro de la Provincia de Buenos Aires; ArgentinaFil: Leunda, M. R.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Pereyra, Susana Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Jones, Leandro Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; ArgentinaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; Argentin

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.EEA BalcarceFil: Gonzalez Altamiranda, Erika Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Sandra. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Verna, Andrea Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Influence of glyceryl guaiacolate ether on anesthetics in tilapia compared to benzocaine and eugenol

ABSTRACT Objective. The study aimed to investigate the effectiveness of glyceryl guaiacolate ether (GGE) and compare the times of induction, recovery, hematological changes, total protein and glycaemia among anesthetics in Nile tilapia, Oreochromis niloticus. Materials and methods. A total of 60 tilapia distributed in 3 aquariums (N=20) were used, which formed the group benzocaine (100 mg/L), eugenol (50 mg/L) and guaiacol glyceryl ether (9.000 mg/L). After the induction of anesthesia fish blood samples were collected to determine the complete hemogram and glycemia. Then the animals were placed in aquariums with running water for assessing the anesthesia recovery. Results. It was verified that GGE showed longer induction and recovery times as well a significant increase (p0.05). An increase in the number of monocytes in the group treated with benzocaine (p <0.05) was observed in the analysis of the hematological parameters with no difference between groups for other variables. Conclusions. Eugenol and benzocaine allow rapid induction and recovery in Nile tilapia, without evidence of stress during handling and GGE showed high induction and recovery times, being inadequate for anesthetic use in Nile tilapia

Genetic data generated from virus host complexes obtained by membrane co-immobilization are equivalent to data obtained from tangential filtrate virus concentrates and virus cultures

The sieving and immobilization of virus–host complexes using impact filtration (aka membrane co-immobilization or MCI) is a novel approach to the study of plankton viruses. One of the most interesting characteristics of the method is the possibility of generating data on potential viral hosts without the need of culturing hosts cells. MCI has demonstrated to be useful for studying viruses of picoalgae, but studies comparing data generated by MCI to data obtained by other techniques are lacking. In this work, Ostreococcus virus (OV) and Ostreococcus sp. sequences generated from virus–host complexes obtained by MCI were compared to sequences obtained from tangential filtration (TF) concentrates and virus cultures (VC). Statistical parsimony, phylogenetic analyses, pairwise distance comparisons, and analysis of molecular variance showed that the viral and host sequences obtained by the three methods were highly related to each other, indicating that MCI, TF, and VC produce equivalent results. Minor differences were observed among viral sequences obtained from VC and TF concentrates as well as among host sequences generated from VC and MCI. As discussed in the body of the paper, the divergence observed for cultured cells could be due to selective pressures exerted by culture conditions, whereas the correlate observed for the corresponding viral sequences could obey to a hitchhiking effect.Fil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jones, Leandro Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia ; Argentin

Are ocean currents to 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.Fil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The peril of PCR inhibitors in environmental samples: the case of Didymosphenia geminata

Since the introduction of polymerase chain reaction (PCR) biodiversity study has been significantly influenced by the chance of generating unprecedented amounts of molecular data. Although it is a robust technique, those applications requiring high sensitivity and reproducibility, that is PCR detection and quantitative PCR, can be negatively affected by PCR inhibition. This is particularly challenging for diverse kinds of samples included the environmental ones, which usually contain complex mixtures of a variety of inhibitory substances. The problem of PCR inhibition can be overcome, or ameliorated, by implementing adequate protocols of nucleic acids purification, internal controls and modern analytical approaches focused on PCR kinetics. Herein, we remark these procedures and describe the general techniques that can be used to optimize DNA extraction protocols for PCR applications. In addition, we show that PCR inhibition might have negative consequences in molecular studies of Didymosphenia geminata, an invasive microalga that have recently developed massive blooms in temperate regions worldwide, and provide general guidelines for dealing with this problem.Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Uyua, Noelia Mariel. Universidad Nacional de la Patagonia ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin