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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.

Infectious bronchitis virus (IBV) is a persistent sanitary problem for the South American poultry industry despite extensive vaccination. The IBV single-stranded RNA genome has high rates of mutation and recombination that generate a notorious virus variability. Since most IBV vaccines are type-specific, there is a need for constant surveillance of the circulating lineages and knowledge about their genetic and antigenic properties. Here we present an integrative analysis that provides the pattern of genetic variation of the South American IBV strains and information about their antigenic characteristics. The genetic analysis was performed using the S1 complete coding sequences of all available South American strains, including newly obtained Argentine and Uruguayan field samples. Our phylogenetic and phylodynamic analyses evidence that three main lineages (GI-1, GI-11 and GI-16) are extensively circulating in South American flocks. Strains of the GI-1 lineage (Massachusetts-type) were detected in Argentina, Brazil, Chile and Colombia. The GI-11 lineage is an exclusively South American lineage that emerged in the 1950s, and is the predominant lineage in Brazil and Uruguay at present. The GI-16 lineage emerged around 1979, and is currently circulating in most South American territories (Argentina, Chile, Uruguay, Colombia and Peru). The virus cross-neutralization test performed here reveals very low antigenic relatedness between GI-11 and GI-16 lineages (i.e. they are different serotypes). The results of this study extend our knowledge about the present and past IBV variability in South America and provide relevant elements to improve the control programmes by considering the genetic and antigenic attributes of IBV.

In this study we evaluated the effectiveness of adding serotype 793B vaccine to an immunization program in order to control the infectious bronchitis virus (IBV) GI-16 lineage. Therefore, two different experiments were performed. First, a virus cross-neutralization test was carried out, which indicated that neither the Massachusetts (Mass) nor 793B serotypes are antigenically related to the field isolate A13 (GI-16). We also performed a challenge trial to evaluate if the Mass/793B combination is more efficient than Mass/Connecticut (Conn) to protect chickens against the Argentinian variant A13. Thus, 40 chickens were organized in four groups. Chickens in Group A were vaccinated at 1 day of age with Mass serotype and then at 14 days old with Mass plus Conn serotypes. Chickens in Group B received Mass and 793B serotypes at 1 and 14 days old, respectively. Groups C and D remained unvaccinated. At 28 days of age, Groups A, B, and C were challenged with the A13 isolate, while Group D remained as the negative control. The statistical analysis of the ciliostasis evaluation, performed at 7 days postchallenge (dpch), indicated that the difference between Mass/793B and Mass/Conn was not significant (p > 0.05). However, the comparison against the negative control showed that only Group A was significantly different, suggesting a slightly better performance on blocking ciliostasis for the Mass/793B combination. On the other hand, no significant differences were observed in the viral load, quantified by reverse-transcription quantitative real-time PCR (RT-qPCR) in tracheal swabs and kidneys (at 3 and 7 dpch, respectively) between vaccinated groups. Furthermore, some amounts of the viral genome were found in both vaccinated groups that could indicate that neither the Mass/793B nor the Mass/Conn combinations totally inhibited the viral replication. Such viral replication in vaccinated chickens should seriously be taken into consideration because it could promote the selection of new variants in the future.

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.

Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) cause relevant immunosuppressive diseases in poultry. Clinical diagnosis of these viruses is challenging given the different disease presentations and the frequent occurrence of co-infections with other pathogens. Here, we standardized and validated simplex and duplex RT-qPCR assays for the straightforward detection of IBDV and CAV. The qPCR assays are based on primers and hydrolysis probes that target highly conserved regions of IBDV and CAV genomes. Analytical sensitivity tests on 10-fold serial dilutions containing 100-108 viral genomes indicated that the simplex assays have good determination coefficients and efficiency and detect a wide range of virus doses (102 to 108 molecules copies/reactions). The relatively small values of intra- and inter-assay variability ensure the repeatability and support its reproducibility in different diagnostic and research facilities. The assays are also efficient tools for absolute quantification as indicated by the analytical performance analysis. The assays have an excellent specificity and absence of cross-reactivity with negative samples, or with other common avian viruses. The simplex IBDV and CAV assays use probes labelled with different dyes (FAM and HEX) and can be multiplexed for the simultaneous detection of both viruses. The determination coefficients, PCR efficiencies, and relatively small intra- and inter-assay variability were comparable to the simplex assays. This duplex assay is the first to simultaneously detect IBDV and CAV using the same RNA extraction from the bursa of Fabricius in a single and straightforward step. Therefore, this method is time saving, provides quantitative results for both targets without any cross-reaction, and reduces the risk of carrying-over contaminations. The qPCR assays here developed can be used in simplex and duplex formats for detection and quantification of large number of samples with reliable sensitivity and specificity. These tools are expected to improve surveillance and control of these ubiquitous viruses.

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.

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described “distinct IBDV” (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage. Research HighlightsA South American strain of the dIBDV lineage was phenotypically characterized.The strain produced subclinical infections with a marked bursal atrophy.Infected chickens were severely immunosuppressed.The dIBDV strains are antigenically divergent from other IBDV lineages. A South American strain of the dIBDV lineage was phenotypically characterized. The strain produced subclinical infections with a marked bursal atrophy. Infected chickens were severely immunosuppressed. The dIBDV strains are antigenically divergent from other IBDV lineages.

Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus that causes one of the most persistent respiratory diseases in poultry. The virus is classified in genotypes and lineages with different epidemiological relevance. Two lineages of the GI genotype (11 and 16) have been widely circulating for decades in South America. GI-11 is an exclusive South American lineage while the GI-16 lineage is distributed in Asia, Europe and South America. Here, we obtained the whole genome of two Uruguayan strains of the GI-11 and GI-16 lineages using Illumina high-throughput sequencing. The strains here sequenced are the first obtained in South America for the infectious bronchitis virus and provide new insights into the origin, spreading and evolution of viral variants. The complete genome of the GI-11 and GI-16 strains have 27,621 and 27,638 nucleotides, respectively, and possess the same genomic organization. Phylogenetic incongruence analysis reveals that both strains have a mosaic genome that arose by recombination between Euro Asiatic strains of the GI-16 lineage and ancestral South American GI-11 viruses. The recombination occurred in South America and produced two viral variants that have retained the full-length S1 sequences of the parental lineages but are extremely similar in the rest of their genomes. These recombinant virus have been extraordinary successful, persisting in the continent for several years with a notorious wide geographic distribution. Our findings reveal a singular viral dynamics and emphasize the importance of complete genomic characterization to understand the emergence and evolutionary history of viral variants., Highlights • Genomic analysis was performed in two main lineages of Infectious bronchitis virus. • Lineages differ in their S1 sequences but are similar in the rest of the genome. • Genomic similarity between both lineages arise by inter-lineage recombination. • Inter-lineage recombination occurred in South America between European/Asiatic and local strain. • Recombinant forms have persisted in the continent for several years with wide geographic distribution.

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.

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.

Infectious bursal disease virus is a relevant avian pathogen that affects poultry production. Here, we report the full-length coding sequence of the Uruguayan strain dIBDV/UY/2014/2202, isolated from a commercial broiler flock. The strain belongs to the distinct IBDV lineage that is widely distributed in South America.

Infectious Bursal Disease Virus (IBDV) is one of the most concerning sanitary problems to the world poultry production. IBDV comprises four well-defined evolutionary lineages known as classic (cIBDV), classic virulent (cvIBDV), variant (vaIBDV) and hypervirulent (vvIBDV) strains. In the present study, we characterized IBDV samples in Argentina and Uruguay by sequencing the coding region of the hypervariable domain of the capsid protein VP2. Samples belonging to three strains (cIBDV, vvIBDV, and cvIBDV) were unambiguously classified by the presence of molecular markers and phylogenetic analysis. Notably, a high proportion of samples (60 %) could not be accurately assign to any of the previously described strains, and were then denoted as novel IBDV (nIBDV). These Uruguayan and Argentine nIBDVs constitute an independent evolutionary lineage that also includes viruses from others countries of America and Asia. The hypervariable VP2 sequence of these nIBDVs shares amino acids markers with other IBDV strains, but has a unique and conserved molecular signature (272T, 289P, 290I and 296F) that may be considered a diagnostic character for classification. A discriminant analysis of principal components (DAPC) also identified the nIBDVs as a cluster of genetically related viruses separated from the typical IBDV strains. DAPC and genetic distance estimation indicated that the nIBDV is one of the most genetically divergent lineages of IBDV. Together, the present study suggests that the highly divergent nIBDV lineage is a previously undescribed IBDV group that is widely circulating in the world poultry production. Further studies using antigenic, pathogenic, epidemiologic, and additional genetic studies are needed to completely characterize this lineage and determine if it should be considered alongside conventional IBDV strains. Fil: Hernández, Martín. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Gonzalo, Tomas. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Marandino, Ana. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Iraola, Gregorio. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Maya, Leticia. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Mattion, Nora Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología ; Argentina Fil: Hernandez, Diego. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Villegas, Pedro. University of Georgia. College of Veterinary Medicine. Poultry Diagnostic and Research Center; Estados Unidos Fil: Banda, Alejandro. Mississippi State University. College of Veterinary Medicine. Poultry Research and Diagnostic Laboratory; Estados Unidos Fil: Panzera, Yanina. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay Fil: Perez, Ruben. Universidad de la República. Facultad de Ciencias. Instituto de Biología. Sección Genética Evolutiva, Departamento de Biología Animal; Uruguay