Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Women in the European Virus Bioinformatics Center.

Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.

Covidex: An ultrafast and accurate tool for SARS-CoV-2 subtyping.

The epidemiological surveillance of SARS-CoV-2 by means of whole-genome sequencing has revealed the emergence and co-existence of multiple viral lineages or subtypes throughout the world. Moreover, it has been shown that several subtypes of this virus display particular phenotypes, such as increased transmissibility or reduced susceptibility to neutralizing antibodies, leading to the denomination of Variants of Interest (VOI) or Variants of Concern (VOC). Thus, subtyping of SARS-CoV-2 is a crucial step for the surveillance of this pathogen. Here, we present Covidex, an open-source, alignment-free machine learning subtyping tool. It is a shiny web app that allows an ultra-fast and accurate classification of SARS-CoV-2 genome sequences into the three most used nomenclature systems (GISAID, Nextstrain, Pango lineages). It also categorizes input sequences as VOI or VOC, according to current definitions. The program is cross-platform compatible and it is available via Source-Forge https://sourceforge.net/projects/covidex or via the web application http://covidex.unlu.edu.ar.

Isolation of endophytic bacteria from the medicinal, forestal and ornamental tree Handroanthus impetiginosus.

Plant interactions with endophytic bacteria produce mutual benefits and contribute to environmental sustainability. Handroanthus impetiginosus (Mart. ex DC.) Mattos 'pink lapacho' (syn. Tabebuia impetiginosa, Bignoniaceae) is a medicinal, ornamental and forestal native tree from South and Mesoamerica. Plant growth promoting bacteria (PGPB) isolated from pink lapacho are scarcely described. The aim of this work was to isolate and characterize native endophytic bacteria from pink lapacho. Ten bacterial strains were isolated from leaves and six from roots of naturally growing trees in Luján (Central-Eastern region of Argentina). Endophytes were identified as Bacillus, Paenibacillus, Pseudomonas, Rhizobium, Rummeliibacillus and Methylobacterium genera, according to 16S rRNA gene sequencing and phylogenetic analysis. In the present study, a strain of the Rummelibacillus genus (L14) has been first ever reported as endophyte. This strain was capable of growing in Nfb medium and exhibited zinc solubilization ability. A high percentage of strains showed PGPB traits; namely 88% fixed nitrogen, 63% solubilized zinc, 69% solubilized phosphate and 63% produced indole compounds such as IAA. Most strains were salt tolerant that confer them a potential competitive advantage to survive in saline conditions. To the best of our knowledge, this is the first study reporting an approach to assess the diversity of cultivable endophytic bacteria of H. impetiginosus tree and its plant growth promoting capacity. The knowledge about this kind of associations could contribute to environmental sustainability by developing effective biofertilizers that minimize the use of chemical fertilizers and pesticides.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

Baculovirus AcMNPV induces type I interferons and NK/NKT cells-mediated protection against foot-and-mouth disease virus in mice.

Foot-and-mouth disease is a viral illness that affects cloven-hoofed animals causing serious economic losses. Inactivated vaccines against its causative agent, foot-and-mouth disease virus (FMDV), require approximately seven days to induce protection. Therefore, antiviral strategies are needed to provide earlier protection and to stop the spread of this highly contagious virus during outbreak situations. In this way, our group has previously demonstrated that the baculovirus (BV) Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an insect virus with immunostimulant effects, induces a nonspecific antiviral status that protects C57BL/6 mice against a lethal challenge with FMDV A/Arg/01 at 3 hours or 3 days post inoculation. In this work, we studied the immunological mechanisms involved in this protection. Firstly, we compared the protection elicited by AcMNPV in wild type mice and in knock-out mice lacking the subunit IFNAR1 of the receptor for type I interferons (IFNs). Our results showed that type I IFNs are key to prevent the death of the animals after the FMDV challenge. On the other hand, we evaluated the role of NK and NKT cells by depleting these cell subsets with anti-NK1.1 monoclonal antibody. These cells proved to be necessary for the induction of IFN-γ by AcMNPV and to prevent the onset of a severe disease after the FMDV challenge. We propose BV as a novel tool for the development of antiviral strategies because of the high levels of IFNs induced and the NK/NKT cells-mediated immune response elicited.

A beginner's guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing.

Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workflow proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.

Differential replication of Foot-and-mouth disease viruses in mice determine lethality.

Adult C57BL/6J mice have been used to study Foot-and-mouth disease virus (FMDV) biology. In this work, two variants of an FMDV A/Arg/01 strain exhibiting differential pathogenicity in adult mice were identified and characterized: a non-lethal virus (A01NL) caused mild signs of disease, whereas a lethal virus (A01L) caused death within 24-48h independently of the dose used. Both viruses caused a systemic infection with pathological changes in the exocrine pancreas. Virus A01L reached higher viral loads in plasma and organs of inoculated mice as well as increased replication in an ovine kidney cell line. Complete consensus sequences revealed 6 non-synonymous changes between A01L and A10NL genomes that might be linked to replication differences, as suggested by in silico prediction studies. Our results highlight the biological significance of discrete genomic variations and reinforce the usefulness of this animal model to study viral determinants of lethality.

Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in biological processes such as development. MiRNAs silence target mRNAs by binding to complementary sequences in the 3'untranslated regions (3'UTRs). The parasitic helminths of the genus Echinococcus are the causative agents of echinococcosis, a zoonotic neglected disease. In previous work, we performed a comprehensive identification and characterization of Echinococcus miRNAs. However, current knowledge about their targets is limited. Since target prediction algorithms rely on complementarity between 3'UTRs and miRNA sequences, a major limitation is the lack of accurate sequence information of 3'UTR for most species including parasitic helminths. We performed RNA-seq and developed a pipeline that integrates the transcriptomic data with available genomic data of this parasite in order to identify 3'UTRs of Echinococcus canadensis. The high confidence set of 3'UTRs obtained allowed the prediction of miRNA targets in Echinococcus through a bioinformatic approach. We performed for the first time a comparative analysis of miRNA targets in Echinococcus and Taenia. We found that many evolutionarily conserved target sites in Echinococcus and Taenia may be functional and under selective pressure. Signaling pathways such as MAPK and Wnt were among the most represented pathways indicating miRNA roles in parasite growth and development. Genome-wide identification and characterization of miRNA target genes in Echinococcus provide valuable information to guide experimental studies in order to understand miRNA functions in the parasites biology. miRNAs involved in essential functions, especially those being absent in the host or showing sequence divergence with respect to host orthologs, might be considered as novel therapeutic targets for echinococcosis control.

Artificial microRNAs as antiviral strategy to FMDV: structural implications of target selection.

RNA interference (RNAi) appears as a promising strategy to control virus replication. While the antiviral power of short-hairpin RNAs or small-interfering RNAs against FMDV has been demonstrated widely, safer RNAi effectors such as artificial microRNAs (amiRs) have not been evaluated extensively. In this work, transgenic monoclonal cell lines constitutively expressing different amiRs targeting FMDV 3D-coding region or 3'UTR were established. Certain cell lines showed an effective, sequence-specific amiR-mediated silencing activity that was accomplished by degradation of the target mRNA, as demonstrated in co-transfection experiments of reporter genes fused to FMDV target sequences. However, FMDV replication in these amiR-expressing cells was affected barely. Experiments aimed at elucidating the cause of RNAi failure demonstrated limited accessibility of the targeted region in the molecular environment of the viral RNA. Since RNAi is mediated by large-dimension silencing complexes containing the siRNA and not simply by a linear oligonucleotide, we propose that target selection should consider not only the local RNA structure but also the global conformation of target RNA.

Enhanced IRES activity by the 3'UTR element determines the virulence of FMDV isolates.

A reverse genetics approach was used to identify viral genetic determinants of the differential virulence displayed by two field foot-and-mouth disease virus (FMDV) strains (A/Arg/00 and A/Arg/01) isolated in Argentina during the 2000-2001 epidemics. A molecular clone of A/Arg/01 strain and viral chimeras containing the S-fragment or the internal ribosome entry site (IRES) of A/Arg/00 in the A/Arg/01 backbone were constructed and characterized. The IRES appeared as a determining factor of the lower level of A/Arg/00 replication in cell culture. High-throughput RNA probing revealed structural differences between both IRESs. Translation experiments using either synthetic viral RNAs (in vitro) or bicistronic plasmids (in vivo) showed that these IRESs' activities differ when the viral 3' untranslated region (UTR) is present, suggesting that their function is differentially modulated by this region. This work provides experimental evidence supporting the role of the IRES-3'UTR modulation in determining the level of FMDV replication in field strains.

Dynamic changes in viral population structure and compartmentalization during chronic hepatitis C virus infection in children.

Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2-9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability. Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure.

Phylogenetic analysis of previously nontypeable hepatitis C virus isolates from Argentina.

Phylogenetic analysis of hepatitis C virus isolates from Argentina that were previously nontypeable by restriction fragment length polymorphism (RFLP) analysis revealed that they belong to genotype 1a. A substitution at position 107 (G-->A), which is the landmark of these strains, was shown to be distributed among isolates worldwide. The RFLP patterns obtained for these isolates should be added to the ones reported for genotype 1 isolates.

Hepatitis C virus infection in infants and children from Argentina.

Hepatitis C virus (HCV) infection is uncommon in children, and its natural history is still unknown. Our aim was to analyze exposure to HCV in 48 infants and children in Argentina and to evaluate consecutive samples in 26 of them to study the outcome of HCV infection in early stages. HCV viremia, as determined by reverse transcription-PCR (RT-PCR) from the 5' untranslated region, showed continuously positive, occasionally positive, and negative patterns during follow-up. Restriction fragment length polymorphism was performed on RT-PCR-positive samples to evaluate HCV genotype. Genotype 1 turned out to be predominant, and no patient displayed a genotype shift during the observation period. Perinatal HCV infection was predominantly observed in patients born to mothers coinfected with HCV and human immunodeficiency virus. HCV viral load was detected by means of the AMPLICOR MONITOR, version 2.0, kit. No correlation was observed between HCV viral load and alanine aminotransferase and aspartate aminotransferase levels, although we detected a trend towards higher levels among patients displaying consecutive positive HCV RT-PCR results. Our results demonstrate that pediatric HCV infection is characterized by high viral loads and diverse HCV viremia patterns, independent of both age and route of transmission in the population under study. Further research is necessary to determine whether the high rate of HCV replication is related to virus variability or to host immune response.

Hepatitis C virus isolates from Argentina disclose a novel genotype 1-associated restriction pattern.

Hepatitis C virus isolates which disclosed a novel genotype 1-associated restriction pattern by restriction fragment length polymorphism analysis were characterized. Except for a mother and child pair, the patients were unrelated. Sequence analysis showed a G-->A substitution leading to a new RsaI recognition site. Phylogenetic analysis revealed that these isolates constitute a novel genetic lineage within the main cluster of genotype 1 strains.

Epstein Barr virus associated pediatric nasopharyngeal carcinoma: its correlation with p53 and bcl-2 expression.

BACKGROUND: Pediatric nasopharyngeal carcinoma (NPC) is relatively rare. The Epstein Barr virus (EBV) association with the oncogenesis of NPC is well established. Apoptosis-related proteins, p53 and bcl-2, have also been described in adult NPC pathogenesis. PROCEDURE: From 1988 to 1998, 16 patients with NPC were treated at R. Gutierrez Children's Hospital and the National J.P. Garrahan Pediatric Hospital. Their median age was 12 years (range 8-20), 2 females and 14 males. The presence of p53, bcl-2 and latent membrane protein-1 (LMP-1) of EBV expression was studied by immunohistochemistry and Epstein Barr encoded RNAs (EBERs) by in situ hybridization in tissue sections from formalin-fixed, paraffin-embedded NPC biopsies RESULTS: EBV presence and LMP-1 expression in epithelial tumor cells were detected in all the biopsies studied. p53 was expressed in 13/16 NPCs, but the frequency of positive malignant cells differed from case to case, ranging from less than 25 to 100% with heterogeneous staining intensity. Bcl-2 positive staining in tumor epithelial cells was detected in 2/16; whereas 10/16 cases showed bcl-2 positivity in infiltrating lymphocytes. CONCLUSIONS: Although our series is small, we conclude that the pathogenesis of pediatric NPC as a multistep process may well involve EBV infection. This leads to LMP-1 expression and p53 overexpression in epithelial tumor cells, whereas bc-2 seems unrelated to the development of this disorder.

Caracterización genotípica de la infección por el virus de hepatitis C en niños / Genotype characterization of hepatitis C virus infection in children

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5'untranslated region (5'UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36 per cent) were always positive for HCV RNA, in 5 (20 per cent) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36 per cent) showed alternating RT-nested PCR results and in 2 (8 per cent) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the host's response in the initial stages of it.