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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.
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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.
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Doenças do Cão , Genômica , Infecções por Parvoviridae , Parvovirus Canino , Filogenia , Parvovirus Canino/genética , Parvovirus Canino/classificação , Animais , Cães , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/epidemiologia , Doenças do Cão/virologia , Doenças do Cão/epidemiologia , Genômica/métodos , Variação Genética , América do Sul/epidemiologia , Genoma Viral , Uruguai/epidemiologia , Argentina/epidemiologiaRESUMO
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.
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Doenças do Cão , Infecções por Parvoviridae , Parvovirus Canino , Vacinas , Gatos , Animais , Cães , Parvovirus Canino/genética , Infecções por Parvoviridae/diagnóstico , Vírus da Panleucopenia Felina/genética , Variação Antigênica , Doenças do Cão/diagnóstico , FilogeniaRESUMO
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.
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Vírus da Doença Infecciosa da Bursa , Animais , Vírus da Doença Infecciosa da Bursa/genética , Filogenia , Galinhas , Reação em Cadeia da Polimerase , Sequência de BasesRESUMO
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.
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COVID-19 , Coinfecção , Vírus da Influenza A , Influenza Humana , Vírus de RNA , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Criança , Adulto , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias , RNA , Teste para COVID-19 , Coinfecção/diagnóstico , Coinfecção/epidemiologia , SARS-CoV-2/genética , Vírus de RNA/genética , Vírus Sincicial Respiratório Humano/genética , Vírus da Influenza A/genética , Sequenciamento de Nucleotídeos em Larga Escala , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Influenza Humana/epidemiologiaRESUMO
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.
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COVID-19 , Doenças do Gato , Doenças do Cão , Gatos , Animais , Humanos , Cães , SARS-CoV-2/genética , Uruguai , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Animais DomésticosRESUMO
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.
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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.
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COVID-19/virologia , Linhagem da Célula , Deleção de Genes , Genoma Viral , Fases de Leitura Aberta/genética , SARS-CoV-2/genética , Proteínas Virais/genética , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/genética , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Uruguai/epidemiologiaRESUMO
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.
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COVID-19 , Fases de Leitura Aberta , SARS-CoV-2 , COVID-19/epidemiologia , COVID-19/virologia , Genoma Viral , Humanos , SARS-CoV-2/genética , Deleção de Sequência , Uruguai/epidemiologiaRESUMO
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.
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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.
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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 (2.3â 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 × 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.RESEARCH HIGHLIGHTS Current strains of chicken anaemia virus emerged in Asia in the early 1900s.Chicken anaemia virus has a high substitution rate.The phylogenetic analysis classified chicken anaemia virus in four major clades.Evolution in South America was characterized by long migration and local spreading.
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Vírus da Anemia da Galinha , Animais , Vírus da Anemia da Galinha/genética , Galinhas , Filogenia , América do Sul/epidemiologiaRESUMO
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.
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COVID-19 , SARS-CoV-2 , Deleção de Sequência , COVID-19/virologia , Surtos de Doenças , Genoma Viral , Humanos , Filogenia , SARS-CoV-2/genética , Uruguai/epidemiologiaRESUMO
Carnivore protoparvovirus 1 is one of the most important pathogens affecting both wild and domestic carnivores. Here, we reported the genetic characterization of canine parvovirus (CPV-2) strains from a rescued guiña (Leopardus guigna) and domestic dogs from Chile. Guiña strain was classified as CPV-2c, and phylogenetic analysis of the complete coding genome showed that the guiña CPV-2c strain shares a recent common ancestor with Chilean domestic dogs' strains. These viruses showed >99% identity and exhibited three changes in the NS1 protein (V596A, E661K and L582F). This is the first detection and genetic characterization of CPV-2c infection in guiña worldwide, and one of the few comparative studies that show the source of infection was domestic dogs. The current findings highlight the fact that guiña is a susceptible species to protoparvovirus infection and that domestic dogs represent an important threat to its conservation. The CPV-2 cross-species transmission between domestic dogs and guiña should be taken into account for protection programmes of this endangered species.
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Doenças do Cão/transmissão , Felidae , Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Animais , Chile , Doenças do Cão/virologia , Cães , Infecções por Parvoviridae/virologiaRESUMO
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.
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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.
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Infectious bursal disease virus (IBDV) is an economically relevant and widespread pathogen that produces immunosuppression in young chickens. IBDV is genetically classified into seven genogroups (G1-G7), where the traditional classic, variant and very virulent strains correspond to G1, G2 and G3, respectively. The G4 strains, also known as 'distinct' (dIBDV), have recently acquired increased relevance because of their prevalence and notorious impair to the poultry industry in South America. Here, worldwide dIBDV strains were studied using phylogenetic and phylodynamic approaches. The phylogenetic analyses performed using partial and complete sequences of both viral segments (A and B) consistently clustered the dIBDV strains in a monophyletic group. The analyses of the VP5, polyprotein and VP1 coding regions identified amino acid residues that act as markers for the identification of the entire dIBDV group or different sub-populations. The phylodynamic analyses performed using the hypervariable region of VP2 indicated that the dIBDV strains emerged in the early 1930s in Eastern Europe, shortly after the emergence of classic strains (1927) and before variant (1949) and very virulent strains (1967). The analysis of the migration routes indicated that after its emergence, the dIBDV strains spread to Eastern Asia around 1959, to Brazil around 1963, and to Argentina around 1990. These inter-continental migrations resulted in three sub-populations that are currently represented by strains from (a) Brazil, (b) Eastern Asia and Canada, and (c) Eastern Europe, Argentina and Uruguay. Taken together, our results highlight the complex evolutionary history of IBDV and the importance of new phylodynamic data to unravel and nearly follow the different evolutionary pathways taken by this important poultry pathogen.
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Evolução Biológica , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/fisiologia , Filogenia , Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/classificação , Vírus da Doença Infecciosa da Bursa/genética , Proteínas Virais/análiseRESUMO
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.
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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, IBDV-negative 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.
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Infecções por Birnaviridae/veterinária , Galinhas/virologia , Vírus da Doença Infecciosa da Bursa/classificação , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Proteínas Estruturais Virais/genética , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Bolsa de Fabricius/virologia , Primers do DNA/genética , Sondas de DNA , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Doenças das Aves Domésticas/virologia , RNA de Cadeia Dupla/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus (27.6kb) that causes one of the most persistent respiratory disease in poultry. The virus is classified in genotypes with different epidemiological relevance and clinical implications. The present study reports the development and validation of specific RT-qPCR assays for the detection of two major IBV genotypes: South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive and widespread South American lineage while the A/SAII genotype is distributed in Asia, Europe and South America. Both identification assays employ TaqMan probes that hybridize with unique sequences in the spike glycoprotein gene. The assays successfully detected all the assessed strains belonging to both genotypes, showing high specificity and absence of cross-reactivity. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable determination coefficients, PCR efficiencies and relatively small intra- and inter-assay variability. The assays demonstrated a wide dynamic range between 10(1)-10(7) and 10(2)-10(7) RNA copies/reaction for SAI and A/SAII strains, respectively. The possibility to characterize a large number of samples in a rapid, sensitive and reproducible way makes these techniques suitable tools for routine testing, IBV control, and epidemiological research in poultry.