Characterization of strains of Anaplasma marginale from clinical cases in bovine using major surface protein 1a in Uruguay.

The major surface protein 1a (MSP1a) gene has been used to characterize Anaplasma marginale genetic diversity. This pathogen causes significant productivity and economic losses to the cattle industry. The objective of the present study was to report the first characterization of A. marginale genetic diversity in Uruguay based on MSP1a genotypes and their putative relationship with Rhipicephalus microplus. This cross-sectional study was conducted between 2016 and 2020. The study included whole blood samples from clinical cases of bovine anaplasmosis obtained from 30 outbreaks located in six Uruguay territorial departments. Diagnosis was performed using Giemsa-stained smears and confirmed by nested Polymerase Chance Reaction (nPCR) targeting the A. marginale major surface protein 5 gene. The genetic diversity of A. marginale strains was characterized by analyzing the microsatellite and tandem repeats of MSP1a. Based on the microsatellite structure, four genotypes were identified. Genotype E was the most prevalent. Analysis of MSP1a tandem repeats showed 28 different strains from the combination of 31 repeats, with τ-10-15 and α-ß-ß-ß-Γ being the most common. Repeats Γ, ß, α, and γ were associated with the absence of R. microplus with statistical significance (p < 0.05). Molecular observations showed that 46.7% of the strains identified in our samples lacked the ability to bind to tick cells; therefore, they were probably transmitted by other vectors. Strain genetic diversity provides valuable information for understanding the epidemiological behavior of A. marginale and could contribute to the development of effective vaccines for the control of this disease.

Bovine Polyomavirus-1 (Epsilonpolyomavirus bovis): An Emerging Fetal Pathogen of Cattle That Causes Renal Lesions Resembling Polyomavirus-Associated Nephropathy of Humans.

Bovine polyomavirus-1 (BoPyV-1, Epsilonpolyomavirus bovis) is widespread in cattle and has been detected in commercialized beef at supermarkets in the USA and Germany. BoPyV-1 has been questioned as a probable zoonotic agent with documented increase in seropositivity in people exposed to cattle. However, to date, BoPyV-1 has not been causally associated with pathology or disease in any animal species, including humans. Here we describe and illustrate pathological findings in an aborted bovine fetus naturally infected with BoPyV-1, providing evidence of its pathogenicity and probable abortigenic potential. Our results indicate that: (i) BoPyV-1 can cause severe kidney lesions in cattle, including tubulointerstitial nephritis with cytopathic changes and necrosis in tubular epithelial cells, tubular and interstitial inflammation, and interstitial fibroplasia; (ii) lesions are at least partly attributable to active viral replication in renal tubular epithelial cells, which have abundant intranuclear viral inclusions; (iii) BoPyV-1 large T (LT) antigen, resulting from early viral gene expression, can be detected in infected renal tubular epithelial cells using a monoclonal antibody raised against Simian Virus-40 polyomavirus LT antigen; and (iv) there is productive BoPyV-1 replication and virion assembly in the nuclei of renal tubular epithelial cells, as demonstrated by the ultrastructural observation of abundant arrays of viral particles with typical polyomavirus morphology. Altogether, these lesions resemble the "cytopathic-inflammatory pathology pattern" proposed in the pathogenesis of Human polyomavirus-1-associated nephropathy in immunocompromised people and kidney allograft recipients. Additionally, we sequenced the complete genome of the BoPyV-1 infecting the fetus, which represents the first whole genome of a BoPyV-1 from the Southern Hemisphere. Lastly, the BoPyV-1 strain infecting this fetus was isolated, causing a cytopathic effect in Madin-Darby bovine kidney cells. We conclude that BoPyV-1 is pathogenic to the bovine fetus under natural circumstances. Further insights into the epidemiology, biology, clinical relevance, and zoonotic potential of BoPyV-1 are needed.

Draft genome assembly for the colombian freshwater bocachico fish, Prochilodus magdalenae.

We report the first draft genome assembly for Prochilodus magdalenae, the leading representative species of the Prochilodontidae family in Colombia. This 1.2-Gb assembly, with a GC content of 42.0% and a repetitive content of around 31.0%, is in the range of previously reported characid species genomes. Annotation identified 34,725 nuclear genes, and BUSCO completeness value was 94.9%. Gene ontology and primary metabolic pathway annotations indicate similar gene profiles for P. magdalenae and the closest species with annotated genomes: blind cave fish (Astyanax mexicanus) and red piranha (Pygocentrus nattereri). A comparative analysis showed similar genome traits to other characid species. The fully sequenced and annotated mitochondrial genome reproduces the taxonomic classification of P. magdalenae and confirms the low mitochondrial genetic divergence inside the Prochilodus genus. Phylogenomic analysis, using nuclear single-copy orthologous genes, also confirmed the evolutionary position of the species. This genome assembly provides a high-resolution genetic resource for sustainable P. magdalenae management in Colombia and, as the first genome assembly for the Prochilodontidae family, will contribute to fish genomics throughout South America.

Recurrent Dissemination of SARS-CoV-2 Through the Uruguayan-Brazilian Border.

Uruguay is one of the few countries in the Americas that successfully contained the coronavirus disease 19 (COVID-19) epidemic during the first half of 2020. Nevertheless, the intensive human mobility across the dry border with Brazil is a major challenge for public health authorities. We aimed to investigate the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains detected in Uruguayan localities bordering Brazil as well as to measure the viral flux across this ∼1,100 km uninterrupted dry frontier. Using complete SARS-CoV-2 genomes from the Uruguayan-Brazilian bordering region and phylogeographic analyses, we inferred the virus dissemination frequency between Brazil and Uruguay and characterized local outbreak dynamics during the first months (May-July) of the pandemic. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 Brazilian lineages B.1.1.28 and B.1.1.33 into Uruguayan localities at the bordering region. The most probable sources of viral strains introduced to Uruguay were the Southeast Brazilian region and the state of Rio Grande do Sul. Some of the viral strains introduced in Uruguayan border localities between early May and mid-July were able to locally spread and originated the first outbreaks detected outside the metropolitan region. The viral lineages responsible for Uruguayan urban outbreaks were defined by a set of between four and 11 mutations (synonymous and non-synonymous) with respect to the ancestral B.1.1.28 and B.1.1.33 viruses that arose in Brazil, supporting the notion of a rapid genetic differentiation between SARS-CoV-2 subpopulations spreading in South America. Although Uruguayan borders have remained essentially closed to non-Uruguayan citizens, the inevitable flow of people across the dry border with Brazil allowed the repeated entry of the virus into Uruguay and the subsequent emergence of local outbreaks in Uruguayan border localities. Implementation of coordinated bi-national surveillance systems is crucial to achieve an efficient control of the SARS-CoV-2 spread across this kind of highly permeable borderland regions around the world.

Complete Genome Sequence of a Novel Recombinant Citrus Tristeza Virus, a Resistance-Breaking Isolate from Uruguay.

We report here the complete genome sequence of a Citrus tristeza virus (CTV) from Uruguay, sequenced by using Illumina and Sanger sequencing technology. This CTV DSST-17 genome clustered within genotype resistance breaking (RB) and presents two recombination events.

The Evolutionary History and Spatiotemporal Dynamics of the NC Lineage of Citrus Tristeza Virus.

Citrus tristeza virus (CTV) is a major pathogen affecting citrus trees worldwide. However, few studies have focused on CTV's evolutionary history and geographic behavior. CTV is locally dispersed by an aphid vector and long distance dispersion due to transportation of contaminated material. With the aim to delve deeper into the CTV-NC (New Clade) genotype evolution, we estimated an evolution rate of 1.19 × 10-3 subs/site/year and the most common recent ancestor in 1977. Furthermore, the place of origin of the genotype was in the United States, and a great expansion of the population was observed in Uruguay. This expansion phase could be a consequence of the increment in the number of naïve citrus trees in Uruguayan orchards encompassing citrus industry growth in the past years.

Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection.

Standard molecular methods for plant virus diagnosis require the purification of RNA or DNA extracts from a large number of samples, with sufficient concentration and quality for their use in PCR, RT-PCR, or qPCR analysis. Most methods are laborious and use either hazardous and/or costly chemicals. A previously published protocol for RNA isolation from several plant species yields high amounts of good quality RNA-DNA mixture in a simple, safe and inexpensive manner. In the present work, this method was tested to obtain RNA-DNA extracts from leaves of tomato, potato and three species of citrus, and was compared with two commercial kits. The results demonstrated that this protocol offers at least comparable nucleic acid quality, quantity and purity to those provided by commercial phenol-based or spin column systems and that are suitable to be used in PCR, RT-PCR and qPCR for virus and viroid detection. Because of its easy implementation and the use of safe and inexpensive reagents, it can be easily implemented to work in plant virus and viroid detection in different plant species.

Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage.

Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program.