Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes one of the most significant infectious diseasesof dogs. Although the virus dispersed over long distances in the past, current populations are considered to be spatiallyconfined and with only a few instances of migration between specific localities. It is unclear whether these dynamicsoccur in South America where global studies have not been performed. The aim of this study is to analyze the patterns ofgenetic variability in South American CPV populations and explore their evolutionary relationships with global strains.Genomic sequences of sixty-three strains from South America and Europe were generated and analyzed using a phylodynamicapproach. All the obtained strains belong to the CPV-2a lineage and associate with global strains in four monophyleticgroups or clades. European and South American strains from all the countries here analyzed are representative of awidely distributed clade (Eur-I) that emerged in Southern Europe during 1990?98 to later spread to South America in theearly 2000s. The emergence and spread of the Eur-I clade were correlated with a significant rise in the CPV effective populationsize in Europe and South America. The Asia-I clade includes strains from Asia and Uruguay. This clade originated in Asia during the late 1980s and evolved locally before spreading to South America during 2009?10. The third clade (Eur-II)comprises strains from Italy, Brazil, and Ecuador. This clade appears in South America as a consequence of an early introductionfrom Italy to Ecuador in the middle 1980s and has experienced extensive local genetic differentiation. Some strainsfrom Argentina, Uruguay, and Brazil constitute an exclusive South American clade (SA-I) that emerged in Argentina in the1990s. These results indicate that the current epidemiological scenario is a consequence of inter- and intracontinentalmigrations of strains with different geographic and temporal origins that set the conditions for competition and local differentiationof CPV populations. The coexistence and interaction of highly divergent strains are the main responsible for thedrastic epidemiological changes observed in South America in the last two decades. This highlights the threat of invasionfrom external sources and the importance of whole-genome resolution to robustly infer the origin and spread of new CPVvariants. From a taxonomic standpoint, the findings herein show that the classification system that uses a single aminoacid to identify variants (2a, 2b, and 2c) within the CPV-2a lineage does not reflect phylogenetic relationships and is not suitableto analyze CPV evolution. In this regard, the identification of clades or sublineages within circulating CPV strains is thefirst step towards a genetic and evolutionary classification of the virus.Fil: Grecco, Sofia. Universidad de la República; UruguayFil: Iraola, Gregorio. Universidad de la República; UruguayFil: Decaro, Nicola. Università degli Studi di Bari; ItaliaFil: Alfieri, Alice. Universidade Estadual de Londrina; BrasilFil: Alfieri, Amauri. Universidade Estadual de Londrina; BrasilFil: Gallo Calderon, Marina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: da Silva, Ana Paula. Universidade Estadual de Londrina; BrasilFil: Name, Daniela. Universidad de la República. Facultad de Ciencias; UruguayFil: Aldaz, Jaime. Universidad Estatal de Bolivar; EcuadorFil: Calleros, Lucia. Universidad de la República. Facultad de Ciencias; UruguayFil: Marandino, Ana. Universidad de la República. Facultad de Ciencias; UruguayFil: Gonzalo, Tomas. Universidad de la República. Facultad de Ciencias; Urugua

A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences

Background: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. Results: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. Conclusions: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species.EEA BalcarceFil: Iraola, Gregorio. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay. Institut Pasteur Montevideo. Unidad de Bioinformática; UruguayFil: Pérez, Ruben. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; UruguayFil: Betancor, Laura. Universidad de la República. Facultad de Medicina. Instituto de Higiene. Departamento de Bacteriología y Virología; UruguayFil: Marandino, Ana. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; UruguayFil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Mendez, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Piccirillo, Alessandra. Università degli Studi di Padova. Dipartimento di Biomedicina Comparata e Alimentazione; ItaliaFil: Tomás, Gonzalo. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; UruguayFil: Velilla, Alejandra Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Calleros, Lucía. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Urugua

A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences

Background: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. Results: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. Conclusions: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus

The infectious bursal disease virus (IBDV) is a major health threat to the world’s poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDVnegative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 103 and 108 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen

Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes one of the most significant infectious diseases of dogs. Although the virus dispersed over long distances in the past, current populations are considered to be spatially confined and with only a few instances of migration between specific localities. It is unclear whether these dynamics occur in South America where global studies have not been performed. The aim of this study is to analyze the patterns of genetic variability in South American CPV populations and explore their evolutionary relationships with global strains. Genomic sequences of sixty-three strains from South America and Europe were generated and analyzed using a phylodynamic approach. All the obtained strains belong to the CPV-2a lineage and associate with global strains in four monophyletic groups or clades. European and South American strains from all the countries here analyzed are representative of a widely distributed clade (Eur-I) that emerged in Southern Europe during 1990–98 to later spread to South America in the early 2000s. The emergence and spread of the Eur-I clade were correlated with a significant rise in the CPV effective population size in Europe and South America. The Asia-I clade includes strains from Asia and Uruguay. This clade originated in Asia during the late 1980s and evolved locally before spreading to South America during 2009–10. The third clade (Eur-II) comprises strains from Italy, Brazil, and Ecuador. This clade appears in South America as a consequence of an early introduction from Italy to Ecuador in the middle 1980s and has experienced extensive local genetic differentiation. Some strains from Argentina, Uruguay, and Brazil constitute an exclusive South American clade (SA-I) that emerged in Argentina in the 1990s. These results indicate that the current epidemiological scenario is a consequence of inter- and intracontinental migrations of strains with different geographic and temporal origins that set the conditions for competition and local differentiation of CPV populations. The coexistence and interaction of highly divergent strains are the main responsible for the drastic epidemiological changes observed in South America in the last two decades. This highlights the threat of invasion from external sources and the importance of whole-genome resolution to robustly infer the origin and spread of new CPV variants. From a taxonomic standpoint, the findings herein show that the classification system that uses a single amino acid to identify variants (2a, 2b, and 2c) within the CPV-2a lineage does not reflect phylogenetic relationships and is not suitable to analyze CPV evolution. In this regard, the identification of clades or sublineages within circulating CPV strains is the first step towards a genetic and evolutionary classification of the virus

A deletion in SARS-CoV- 2 ORF7 identified in COVID-19 outbreak in Uruguay

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

Genome sequences of SARS-CoV-2 P.1 (Variant of Concern) and P.2 (Variant of Interest) identified in Uruguay

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

Research Note: high genetic diversity of infectious bronchitis virus from Mexico

The avian infectious bronchitis virus (IBV) is a highly mutable coronavirus that causes an acute and highly contagious disease responsible for economic losses to the poultry industry worldwide. Preventing and controlling bronchitis disease is difficulted by the numerous IBV circulating types with limited antigenic cross rotection that hamper the prevention and control by heterologous vaccines. The coding region of the variable spike S1 receptor-attachment domain is used to classify IBV in 7 genotypes (GI–GVII) comprising 35 viral lineages (1–35). Knowledge of the circulating IBV types causing outbreaks in a specific geographic region is beneficial to select better the appropriate vaccine(s) and contribute to disease control. In the study, 17 avian infectious bronchitis virus strains were obtained from chickens showing signs of illness in Mexico from 2007 to 2021. We detected 4 lineages within genotype I, three already known (GI-3, GI-9, GI-13) and one newly described (GI-30). In addition, we identified 2 divergent monophyletic groups that are tentatively described as lineages of new genotypes (GVIII-1 and GIX-1). Our findings revealed that Mexico's high genetic IBV diversity results from the co-circulation of divergent lineages belonging to different genotypes. Mexican IBV lineages differ significantly from Massachusetts and Connecticut vaccine strains, indicating that the currently used vaccines may need to be updated

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion

BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in nonstructural, 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

Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes

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 transmem- brane 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