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.

Genetic characterization of bovine herpesvirus 4 (BoHV-4) isolates from Argentine cattle suggests a complex evolutionary scenario.

Bovine herpevsirus 4 (BoHV-4) is a gammaherpesvirus that has been associated with different clinical conditions in cattle. In Argentina, BoHV-4 was detected in diverse bovine samples. The aim of this study was to analyze the genetic relationship of 48 field BoHV-4 strains isolated from cattle in Argentina. According to thymidine kinase (tk) gene sequences, BoHV-4 isolates belong to genotypes 1, 2 and 3. Phylogenetic analyses confirmed the presence of the three previously described viral genotypes. However, some of the studied isolates presented conflicting phylogenetic signals between the studied markers. This suggests a complex evolutionary background, that is a history of recombination, incomplete lineage sorting (deep coalescence) or a combination of these, which requires further study. These potential events make difficult the diagnosis of BoHV-4 from clinical samples of cattle and may pose a significant problem for the control of the virus in the herds.

Analysis of the anti-apoptotic v-Bcl2 and v-Flip genes and effect on in vitro programmed cell death of Argentinean isolates of bovine gammaherpesvirus 4 (BoHV-4).

Some viruses encode inhibitory factors of apoptosis during infection to prolong cell viability and then to achieve a higher production of viral progeny or facilitate persistent infections. There is evidence that some gammaherpesviruses, including BoHV-4, carry genes that can both inhibit or induce apoptosis. BoHV-4 possesses two genes (ORF16 and ORF71) that code for proteins with anti-apoptotic functions, such as v-Bcl2 and v-Flip, respectively. Thus, it is relevant to study BoHV-4 in relation to the modulation of apoptosis in infected cells as a strategy for persistence in the host. The objective of this work was to analyze whether variations in v-Flip and v- Bcl2 of six phylogenetically divergent Argentinean isolates of BoHV-4 can influence the capacity of these strains to induce apoptosis in cell cultures. In this study, variations were mainly detected in the v-Flip gene and protein of the BoHV-4 strains belonging to genotype 3. Thus, it is possible to infer that sequence variations could be associated with some BoHV-4 genotype. Induction of apoptosis was not a significant event for any of the genetically distinct local isolates of BoHV-4 and there was not an evident relationship between the variability of both genes with the apoptotic effect of the phylogenetically distinct strains.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Influence of mismatch of Env sequences on vaccine protection by live attenuated simian immunodeficiency virus.

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.

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.

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.

Drug resistance mutations in HIV pol sequences from Argentinean patients under antiretroviral treatment: subtype, gender, and age issues.

We studied drug resistance mutations (DRMs) in 2623 pol sequences. Out of 94,828 amino acid substitutions that were detected, 8749 corresponded to nucleoside reverse transcriptase inhibitor (NRTI), 3765 to nonnucleoside reverse transcriptase inhibitor (NNRTI), and 7141 to protease inhibitor (PI) resistance-associated mutations. The most common DRMs were L10I, I54V, L90M, V82A, A71V, L10V, M46I, M184V, M41L, T215Y, D67N, L210W, K70R, N348I, V118I, K103N, Y181C, G190A, K101E, V108I, L100I, V90I, K101Q, and A98G. As expected, DRMs frequencies depended on viral genotype. The amounts of NRTI and PI resistance mutations among B and BF sequences from children were higher than among sequences from adults. The frequencies of PI and NRTI resistance mutations among B and BF sequences from adult men were higher than among sequences from women. Some of these observations can be explained in light of the available epidemiological information, but some cannot, indicating that further studies are needed to understand the antiretroviral resistance epidemics in Argentina.

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

Glycoprotein gene sequence variation in rhesus monkey rhadinovirus.

Gene sequences for seven glycoproteins from 20 independent isolates of rhesus monkey rhadinovirus (RRV) and of the corresponding seven glycoprotein genes from nine strains of the Kaposi's sarcoma-associated herpesvirus (KSHV) were obtained and analyzed. Phylogenetic analysis revealed two discrete groupings of RRV gH sequences, two discrete groupings of RRV gL sequences and two discrete groupings of RRV gB sequences. We called these phylogenetic groupings gH(a), gH(b), gL(a), gL(b), gB(a) and gB(b). gH(a) was always paired with gL(a) and gH(b) was always paired with gL(b) for any individual RRV isolate. Since gH and gL are known to be interacting partners, these results suggest the need of matching sequence types for function of these cooperating proteins. gB phylogenetic grouping was not associated with gH/gL phylogenetic grouping. Our results demonstrate two distinct, distantly-related phylogenetic groupings of gH and gL of RRV despite a remarkable degree of sequence conservation within each individual phylogenetic group.

In-depth analysis of the origins of HIV type 1 subtype C in South America.

The South American HIV-1 epidemic is characterized by the co-circulation of subtype B and BF recombinant variants. Together with the B and BF genotypes, HIV-1 subtype C (HIV-1C), F1, and several other recombinants have been reported. The epidemiological significance and immune correlates of these "non-B-non-BF" strains circulating in South America are still uncertain and therefore are increasingly attracting the interest of the scientific community. In this study, the South American HIV-1C epidemic was studied using new technologies for the phylogenetic analysis of large datasets. Our results indicate that there is a major clade encompassing most of the South American HIV-1C strains. These analyses also agreed that some strains do not group inside this major clade, suggesting that there could be HIV-1C sequences of different origins circulating in South America. Others have proposed different hypotheses about the origins of HIV-1C strains from South America. This study shows that an exact single origin cannot be determined, a fact that could be attributed to sampling problems, phylogenetic uncertainty, and the shortage of historical and epidemiological data. Currently, the reported data indicate that HIV-1C strains were introduced in Brazil and afterward spread to other regions of South America. By using character optimization on the obtained phylogenetic trees, we observed that Argentina could also be a point in which the HIV-1C epidemic entered South America.