Wildlife population die-offs are frequently driven by pathogen introductions. In El Palmar National Park (northeastern Argentina), crab-eating foxes (Cerdocyon thous) and Pampas foxes (Lycalopex gymnocercus) experienced increased mortality likely associated with canine distemper virus (CDV) during 2006–2007. In 2017, the incidental detection of a crab-eating fox with neurological signs of CDV-compatible paresis, together with an abrupt decline in fox abundance, raised concerns for a renewed outbreak. Questionnaire surveys of park staff and affiliated hunters documented recurrent discoveries of fox carcasses and foxes manifesting clinical signs characteristic of CDV. CDV RNA was later confirmed in a dying crab-eating fox with paresis using RT-PCR; the same individual tested negative for rabies by direct immunofluorescence, cell culture, and RT-PCR. Phylogenetic analysis of whole-genome sequences revealed that the 2018 fox CDV lineage clustered with contemporaneous lineage from domestic dogs in the region, and diverged from vaccinal lineages. Spotlight road counts indicated exponential growth of crab-eating foxes during 2006–2015 followed by collapse, whereas Pampas foxes remained stable at lower levels. Indices of relative abundance of both species declined sharply during 2017–2018 —concordantly across spotlight surveys, camera traps, and hunter sightings—, indicating a mass mortality event that transiently altered the fox species ratio. Population recovery was slow. Multiple, convergent lines of evidence implicate CDV —most plausibly of canine origin— as the proximate driver of the fox population collapse. These findings underscore the need to increase dog vaccination coverage and to promote responsible ownership within the surrounding watershed.

Carnivore protoparvovirus 1 (CPPV-1) is a significant pathogen of domestic carnivores, but its occurrence and genomic diversity in South American wildlife remain poorly characterized. Here we report the first detection and complete-genome characterization of an FPV-like CPPV-1 strain in a Procyon cancrivorus from Northeastern Argentina. Viral DNA was sequenced using tiling-tailed amplicons and NGS (95.4% genomic coverage, mean depth >1700×). Phylogenetic analysis of global genomes indicated that the P. cancrivorus strain clustered robustly within the FPV-like clade and was closely related to FPV variants circulating in domestic cats in Argentina. The CPPV-1 positive individual was clinically healthy, suggesting subclinical spillover at the wildlife-domestic interface. These findings extend the recognized host range of FPV-like parvoviruses in South America and underscore the need for genomic surveillance to monitor cross-species transmission at the wildlife-domestic interface.

Canine morbillivirus, also known as canine distemper virus (CDV), is a multi-host pathogen infecting both carnivorous and non-carnivorous species worldwide. However, the limited availability of complete CDV genomes hinders detailed phylogenetic and epidemiological research. Here, we developed a multiplex PCR-based genomic panel optimized for Illumina next-generation sequencing to obtain full-length CDV genomes from clinical samples directly. Designed from 173 global sequences, it was validated using a commercial vaccine strain and 15 field isolates from five Latin American countries. The method consistently achieved over 97% genome coverage, with average depths exceeding 1200×, even in samples with high Ct values. Whole-genome phylogenetic analysis of the obtained sequences resolved three lineages with higher resolution than H gene trees, revealing previously undetected sub-lineage structure. This approach also detected intra-host variants, offering insights into the natural diversity of CDV. This cost-effective, scalable method allows high-quality genome recovery without virus isolation, broadening genomic surveillance and enhancing the molecular epidemiology of CDV.

Honey bees (Apis mellifera) play a crucial role in global food production through pollination services. However, their populations are threatened by various stressors, like the ectoparasitic mite Varroa destructor and associated viral pathogens. In this study, we aimed to characterise and compare the viral communities (viromes) in V. destructor-resistant and susceptible colonies using high-throughput sequencing. Our findings revealed differences in virome composition associated with the season and not with the resistance or susceptibility to V. destructor. Furthermore, we detected Apis mellifera filamentous virus (AmFV) and Lake Sinai virus (LSV) for the first time in Uruguay, and obtained the complete or partial genomes of both viruses, along with those of other previously described viruses, such as Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Deformed wing virus (DWV), and Sacbrood virus (SBV). This study contributes to a deeper understanding of the virome dynamics in honey bees. It highlights the importance of this type of study for the early detection of new viral pathogens, which could help to understand the tripartite network involving V. destructor, honey bees, and viruses.

Human respiratory and enteric viruses are responsible for substantial morbidity and mortality worldwide. Wastewater-based epidemiology utilizing next-generation sequencing serves as an effective tool for monitoring viral circulation dynamics at the community level. However, these complex environmental samples are often laden with other microorganisms and host genomic material, which can hinder the sensitivity of viral detection. To address this limitation, targeted enrichment sequencing is emerging as a preferred strategy, facilitating the acquisition of a more comprehensive understanding of specific pathogens. In this study, we evaluated the performance of a targeted enrichment sequencing panel for 42 excreted respiratory viruses (including Picornaviridae, Adenoviridae, Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Orthoherpesviridae, Pneumoviridae, and Parvoviridae families), known as the Respiratory Pathogen ID/AMR enrichment panel (RPIP), coupled with Explify bioinformatics analysis in 3 sewage samples from Uruguay. RPIP panel successfully identified sequences from frequently circulating viruses, along with some that had not been documented previously. We identified and characterized various viruses, including human Enterovirus (Coxsackievirus A1 and A19), Influenza A-H1N1, and full-length sequences of SARS-CoV-2. Additionally, several other viral pathogens were detected, such as human Bocavirus, human Parechovirus, Enterovirus A71, and Enterovirus D68; however, for these viruses further analysis was limited due to the small genomic regions or low-read coverage obtained. While the RPIP panel necessitates substantial sequencing depth and may introduce bias towards the more predominant strains present in the samples, this approach suggests its viability as a genomic epidemiological tool for assessing respiratory and enteric viruses in wastewater.

Canine parvovirus is a highly contagious pathogen affecting domestic dogs and other carnivores globally. Monitoring CPV through continuous genomic surveillance is crucial for mapping variability and developing effective control measures. Here, we developed a method using multiplex-PCR-next-generation sequencing to obtain full-length CPV genomes directly from clinical samples. This approach utilizes tiling and tailed amplicons to amplify overlapping fragments of roughly 250 base pairs. This enables the creation of Illumina libraries by conducting two PCR reaction runs. We tested the assay in 10 fecal samples from dogs diagnosed with CPV and one CPV-2 vaccine strain. Furthermore, we applied it to a feline sample previously diagnosed with the feline panleukopenia virus. The assay provided 100 % genome coverage and high sequencing depth across all 12 samples. It successfully provided the sequence of the coding regions and the left and right non-translated regions, including tandem and terminal repeats. The assay effectively amplified viral variants from divergent evolutionary groups, including the antigenic variants (2a, 2b, and 2c) and the ancestral CPV-2 strain included in vaccine formulations. Moreover, it successfully amplified the entire genome of the feline panleukopenia virus found in cat feces. This method is cost-effective, time-efficient, and does not require lab expertise in Illumina library preparation. The multiplex-PCR-next-generation methodology facilitates large-scale genomic sequencing, expanding the limited number of complete genomes currently available in databases and enabling real-time genomic surveillance. Furthermore, the method helps identify and track emerging CPV viral variants, facilitating molecular epidemiology and control. Adopting this approach can enhance our understanding of the evolution and genetic diversity of Protoparvovirus carnivoran1.

The Western equine encephalitis virus is transmitted between mosquitoes and wild birds. Mosquitoes spread the virus to horses and human populations, causing severe encephalitis and death. The most recent large outbreak occurred in South America from November 2023 to April 2024. We identified and genetically characterized Uruguayan strains during this outbreak to understand their evolutionary trends and rapid expansion. We obtained genomes from 15 strains using a novel multiplex PCR assay combined with next-generation Illumina sequencing. The phylogenetic analysis revealed that viruses from Uruguay and Brazil collected during the outbreak and an Argentine strain from 1958 share a common evolutionary origin and are distinct from North American strains. Phylogenetic and epidemiological data suggest that the outbreak originated in Argentina and spread to Uruguay and Brazil, likely by movements of infected birds. Genomic analysis also revealed mispairing in real-time PCR primers and probes that may affect official diagnostic protocols.

The microbiota composition of the bovine female reproductive tract influences reproductive efficiency, susceptibility to genital pathogens, and the health of newborn calves. However, knowledge about cervico-vaginal microbiota during gestation is scarce. Therefore, the present study aimed to analyze the taxonomic profile of the cervico-vaginal bovine microbiota throughout pregnancy and after calving using high-throughput sequencing of a fragment of the 16S ribosomal RNA gene.Healthy nulliparous Holstein heifers (n = 13) with similar age and body conditional score were selected to collect samples from the cervico-vaginal area with a sterile swab at 5 timepoints. We sequenced the V1-V2 region of the 16S ribosomal RNA gene and analyzed data using the DADA2, phyloseq and vegan R Studio packages.No differences were observed in alpha and beta diversity across sampling points, accounting for the stability of the microbiota throughout pregnancy. The most abundant phyla are Firmicutes, Bacteroidota, Proteobacteria and Actinobacteria, and are present as the main taxa in all five sampling points. Also, several of the least abundant taxa can be observed to change with time.Our comprehensive study of the cervico-vaginal bacterial microbiota during the gestation period contributes to the knowledge of microbiota dynamics on the bovine reproductive tract during and after pregnancy and can serve as a baseline for future research and the development of potential therapeutic interventions.

Canine parvovirus (CPV) is a significant pathogen in domestic dogs worldwide, causing a severe and often fatal disease. CPV comprises three antigenic variants (2a, 2b, and 2c) distributed unevenly among several phylogenetic groups. The present study compared genetic variability and evolutionary patterns in South American CPV populations. We collected samples from puppies suspected of CPV infection in the neighboring Argentina and Uruguay. Antigenic variants were preliminarily characterized using PCR-RFLP and partial vp2 sequencing. Samples collected in Argentina during 2008-2018 were mainly of the 2c variant. In the Uruguayan strains (2012-2019), the 2a variant wholly replaced the 2c from 2014. Full-length coding genome and vp2 sequences were compared with global strains. The 2c and 2a strains fell by phylogenetic analysis into two phylogroups (Europe I and Asia I). The 2c strains from Argentina and Uruguay clustered in the Europe I group, with strains from America, Europe, Asia, and Oceania. Europe I is widely distributed in South America in the dog population and is also being detected in the wildlife population. The 2a strains from Uruguay formed the distinct Asia I group with strains from Asia, Africa, America, and Oceania. This Asia I group is increasing its distribution in South America and worldwide. Our research reveals high genetic variability in adjacent synchronic samples and different evolutionary patterns in South American CPV. We also highlight the importance of ancestral migrations and local diversification in the evolution of global CPV strains.

The infectious bursal disease virus (IBDV) causes a severe immunosuppressive disorder in young chickens. IBDV evolution resulted in the emergence of strains with divergent genetic, antigenic, and pathogenic characteristics. Genetic classification is typically performed by sequencing the coding region of the most immunogenic region of the viral protein 2 (VP2). Sequencing both double-stranded RNA genome segments is essential to achieve a more comprehensive IBDV classification that can detect recombinants and reassortments. Here, we report the development and standardization of a tiled PCR amplicon protocol for the direct and cost-effective genome sequencing of global IBDV strains using next-generation technology. Primers for tiled PCR were designed with adapters to bypass expensive and time-consuming library preparation steps. Sequencing was performed on Illumina MiniSeq equipment, and fourteen complete genomes of field strains were assembled using reference sequences. The PCR-enrichment step was used to obtain genomes from low-titer biological samples that were difficult to amplify using traditional sequencing. Phylogenetic analyses of the obtained genomes confirmed previous strain classification. By combining the enrichment methodology with massive sequencing, it is possible to obtain IBDV genomic sequences in a fast and affordable manner. This procedure can be a valuable tool to better understand virus epidemiology.

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.

Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.

Canine distemper virus (CDV) is a Morbillivirus (Canine morbillivirus) that greatly impacts domestic and wildlife carnivores worldwide. The CDV RNA genome has high genetic variability, evidenced by several lineages that follow a global geographic pattern. The evolutionary trajectories and population dynamics of CDV lineages are still unclear and debatable, particularly in South America, where relatively few sequences are available. We performed phylogenetic and Bayesian analyses using an updated dataset of the highly variable hemagglutinin (H) gene, including seven South American countries. The time to the most recent common ancestor (tMRCA) of the current CDV lineages was dated to the early 1900s in North America. Maximum likelihood and Bayesian maximum clade credibility phylogenies showed similar topologies with two main branches (L1 and L2) corresponding to the NA1 lineage (L1) and the remaining lineages worldwide (L2). The four circulating lineages in South America (EU1/SA1, SA2, SA3, NA4/SA4) arose from independent migration events from North America and Europe. North American strains colonized most northern South American countries via Ecuador and then Colombia and Peru, originating the SA3 and NA4/SA4 lineages during their spread. The entry and expansion in the southern part of South America (Argentina, Brazil, Chile, and Uruguay) occurred through three independent migration events and gave rise to the EU1/SA1 and SA2 lineages. South American lineages have specific combinations of amino acids under positive selection that constitute signatures of taxonomic and evolutionary relevance. Our findings provide a comprehensive scenario for the origin and migration routes of Canine morbillivirus in South America and highlight the importance of phylodynamics in understanding the geographic patterns of modern genetic variability.

The genetic variability of SARS-CoV-2 (genus Betacoronavirus, family Coronaviridae) has been scrutinized since its first detection in December 2019. Although the role of structural variants, particularly deletions, in virus evolution is little explored, these genome changes are extremely frequent. They are associated with relevant processes, including immune escape and attenuation. Deletions commonly occur in accessory ORFs and might even lead to the complete loss of one or more ORFs. This scenario poses an interesting question about the origin and spreading of extreme structural rearrangements that persist without compromising virus viability. Here, we analyze the genome of SARS-CoV-2 in late 2021 in Uruguay and identify a Delta lineage (AY.20) that experienced a large deletion (872 nucleotides according to the reference Wuhan strain) that removes the 7a, 7b, and 8 ORFs. Deleted viruses coexist with wild-type (without deletion) AY.20 and AY.43 strains. The Uruguayan deletion is like those identified in Delta strains from Poland and Japan but occurs in a different Delta clade. Besides providing proof of the circulation of this large deletion in America, we infer that the 872-deletion arises by the consecutive occurrence of a 6-nucleotide deletion, characteristic of delta strains, and an 866-nucleotide deletion that arose independently in the AY.20 Uruguayan lineage. The largest deletion occurs adjacent to transcription regulatory sequences needed to synthesize the nested set of subgenomic mRNAs that serve as templates for transcription. Our findings support the role of transcription sequences as a hotspot for copy-choice recombination and highlight the remarkable dynamic of SARS-CoV-2 genomes.

BACKGROUND:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in domestic animals have occurred from the beginning of the pandemic to the present time. Therefore, from the perspective of One Health, investigating this topic is of global scientific and public interest.OBJECTIVES:The present study aimed to determine the presence of SARS-CoV-2 in domestic animals whose owners had coronavirus disease 2019 (COVID-19).METHODS:Nasopharyngeal and faecal samples were collected in Uruguay. Using quantitative polymerase chain reaction (qPCR), we analysed the presence of the SARS-CoV-2 genome. Complete genomes were obtained using ARTIC enrichment and Illumina sequencing. Sera samples were used for virus neutralisation assays.FINDINGS:SARS-CoV-2 was detected in an asymptomatic dog and a cat. Viral genomes were identical and belonged to the P.6 Uruguayan SARS-CoV-2 lineage. Only antiserum from the infected cat contained neutralising antibodies against the ancestral SARS-CoV-2 strain and showed cross-reactivity against the Delta but not against the B.A.1 Omicron variant.MAIN CONCLUSIONS:Domestic animals and the human SARS-CoV-2 P.6 variant comparison evidence a close relationship and gene flow between them. Different SARS-CoV-2 lineages infect dogs and cats, and no specific variants are adapted to domestic animals. This first record of SARS-CoV-2 in domestic animals from Uruguay supports regular surveillance of animals close to human hosts.

The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.

BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels’ importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

Chicken anaemia virus (CAV) is a widespread pathogen that causes immunosuppression in chickens. The virus-induced immunosuppression often results in secondary infections and a sub-optimal response to vaccinations, leading to high mortality rates and significant economic losses in the poultry industry. The small circular ssDNA genome (2.3 kb) has three partially overlapping genes: vp1, vp2 and vp3. VP1 capsid protein is highly variable and contains the neutralizing epitopes. Here, we analysed CAV strains from Uruguay using the full-length vp1 gene and performed a global comparative analysis to provide new evidence about the origin, dispersion and genetic variability of the virus. The phylogenetic analysis classified CAV in three or four major clades. Two clades (II and III) grouped most of the strains circulating worldwide including the Uruguayan strains. The phylodynamic analyses indicated that CAV emerged in the early 1900s and diverged to originate clade II and III. This early period of viral emergence was characterised by local diversification promoted by the extremely high substitution rate inferred for the virus (3.8 x 10(-4) substitutions/site/year). Later, the virus underwent a global spreading by intra- and inter-continental migrations that correlates with a significant rise in the effective population size. In South America, CAV was introduced in three different migratory events and spread across the continent. Our findings suggest that the current CAV distribution is the consequence of its continuous expansion capability that homogenizes the populations and prevents the detection of clear temporal and geographic patterns of evolution in most strains.

Two severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants.

Canine parvovirus type 2 (CPV2) is one of the most important intestinal pathogens in dogs and puppies. CPV2 has been evolved into three genetic and antigenic variants (2a, 2b, and 2c), which are distributed worldwide. We reported the first study of genetic diversity of CPV2 in Chile. Sixty-five samples were collected from puppies presenting with severe gastroenteritis and different vaccination statuses. PCR, restriction fragment length polymorphism (RFLP), and partial sequencing of the coding region of the structural viral protein VP2 was performed. Thirty of a total of 65 samples tested positive by PCR out of which 19 were further classified as CPV2c and one as CPV2a using RFLP and Sanger sequencing. The phylogeny was in concordance with the RFLP analysis. This is the first report of the genetic characterization of CPV2 in Chile and reveals a high occurrence of CPV2c.