Genetic and antigenic heterogeneity of infectious bronchitis virus in South America: implications for control programmes.

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.

Phylodynamic analysis of avian infectious bronchitis virus in South America.

Infectious bronchitis virus (IBV) is a coronavirus of chickens that causes great economic losses to the global poultry industry. The present study focuses on South American IBVs and their genetic relationships with global strains. We obtained full-length sequences of the S1 coding region and N gene of IBV field isolates from Uruguay and Argentina, and performed Phylodynamic analysis to characterize the strains and estimate the time of the most recent common ancestor. We identified two major South American genotypes, which were here denoted South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive South American lineage that emerged in the 1960s. The A/SAII genotype may have emerged in Asia in approximately 1995 before being introduced into South America. Both SAI and A/SAII genotype strains clearly differ from the Massachusetts strains that are included in the vaccine formulations being used in most South American countries.

Comparative histopathological and immunological study of two field strains of chicken anemia virus.

Infection of poultry with chicken anemia virus (CAV) is implicated in several field problems in broiler flocks due to the immunosuppression generated and, consequently, the increased susceptibility to secondary infections. Recently, we have reported an increased occurrence of clinical cases caused by CAV strains distantly related to those commonly used for vaccination. In order to understand the behavior of two Argentinean CAV strains (CAV-10 and CAV-18) in two-week-old chickens, an immune and histopathological study was performed. Neither mortality nor clinical signs were observed in the infected or control groups. Thymus lobes from chickens infected with both CAV viruses were smaller compared to the negative control group. At 14 days post-infection (dpi), only chickens inoculated with CAV-10 show a severe depletion of lymphocytes in the thymus cortex and in follicles from the bursa of Fabricius. Also thymopoiesis disorders, such as reduction in the percentage of total DP (CD4 + CD8α+) thymocytes and alteration in the percentages of DP subpopulations, were more important in animals inoculated with the CAV-10 than the CAV-18 strain. In addition, only animals infected with CAV-10 show a decrease in CD8αß splenocytes. Altogether our results show that, although both Argentinean CAV strains produce subclinical infections in chickens causing immunosuppression at 14 dpi, they might differ in their in vivo pathogenicity.

Study of coinfection with local strains of infectious bursal disease virus and infectious bronchitis virus in specific pathogen-free chickens.

Immunosuppressive diseases cause great losses in the poultry industry, increasing the susceptibility to infections by other pathogens and promoting a suboptimal response to vaccination. Among them, infectious bursal disease virus (IBDV) arises as one of the most important around the world. IBDV infects immature B lymphocytes, affecting the immune status of birds and facilitating infections by other pathogens such as avian infectious bronchitis virus (IBV). Although it has been reported that the interaction between these viruses increases IBV clinical signs, there are no actual studies about the interaction between regional circulating isolates that validate this statement. In this context, the objective of our work was to evaluate the effect of the interaction between local isolates of IBDV (belonging to genogroup 4) and IBV (lineage GI-16) in chickens. Thus, specific pathogen-free chickens were orally inoculated with IBDV genogroup (G) 4 or with PBS at 5 d of age. At 14-days postinoculation (dpi) the animals were intratracheally inoculated with a GI-16 IBV or with PBS. At multiple time points, groups of birds were euthanized and different parameters such as histological damage, viral load, lymphocyte populations and specific antibodies were evaluated. The success of IBDV infection was confirmed by the severity of bursal atrophy, viral detection, and presence of anti-IBDV antibodies. In IBV-infected animals, the presence of viral genome was detected in both kidney and bursa. The coinfected animals showed higher degree of lymphocyte infiltration in kidney, higher rate of animals with IBV viral genome in bursa at 28 dpi, and a clear decrease in antibody response against IBV at 28, 35, and 40 dpi. The results indicate that the infection with the local isolate of IBDV affects the immune status of the chickens, causing major severe damage, in response to IBV infection, which could consequently severely affect the local poultry industry.

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters.

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.

Evaluation of the Efficiency of Commercial Vaccines Against Infectious Bronchitis Virus (IBV) Belonging to the GI-16 Lineage Isolated in an Argentinean Outbreak.

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.

Nota de investigación­Evaluación de la eficacia de vacunas comerciales contra el virus de la bronquitis infecciosa (IBV) perteneciente al linaje GI-16 aislado durante un brote argentino. En este estudio se evaluó la efectividad de agregar la vacuna del serotipo 793B a un programa de inmunización para controlar al virus de la bronquitis infecciosa (con las siglas en inglés IBV) linaje GI-16. Por tanto, se realizaron dos experimentos diferentes. Primeramente, se llevó a cabo una prueba de neutralización cruzada de virus, que indicó que ni los serotipos Massachusetts (Mass) ni 793B están antigénicamente relacionados con el aislado de campo A13 (GI-16). También se realizó una prueba de desafío para evaluar si la combinación Massachussets/793B era más eficiente que Massachussets/Connecticut (Conn) para proteger a los pollos contra la variante argentina A13. De esta forma, 40 pollos se organizaron en cuatro grupos. Los pollos del Grupo A se vacunaron al día de edad con el serotipo Massachussets y luego a los 14 días con los serotipos Massachussets más Connecticut. Los pollos del Grupo B recibieron los serotipos Massachussets y 793B a los 1 y 14 días de edad, respectivamente. Los grupos C y D permanecieron sin vacunar. A los 28 días de edad, los Grupos A, B y C fueron desafiados con el aislado A13, mientras que el Grupo D permaneció como control negativo. El análisis estadístico de la evaluación de la ciliostasis, realizada a los 7 días después del desafío (dpch), indicó que la diferencia entre el tratamiento Massachussets/793B y Massachussets/Connecticut no fue significativa (P> 0.05). Sin embargo, la comparación con el control negativo mostró que solo el Grupo A fue significativamente diferente, lo que sugiere un desempeño ligeramente mejor en el bloqueo de la ciliostasis para la combinación Massachussets/793B. Por otro lado, no se observaron diferencias significativas (P> 0.05) en la carga viral, cuantificada mediante transcripción reversa y PCR cuantitativa en tiempo real de hisopos traqueales y riñones (a 3 y 7 días después del desafío, respectivamente) entre los grupos vacunados. Además, se encontraron algunas cantidades del genoma viral en ambos grupos vacunados que podrían indicar que ni las combinaciones Massachussets/793B ni Massachussets/Connecticut inhibieron totalmente la replicación viral. Esta replicación viral en pollos vacunados debe tenerse muy en cuenta porque podría promover la selección de nuevas variantes en el futuro.

Upper alimentary tract papillomas in calves related to papillomavirus infection.

This study reports 3 cases of spontaneous papillomavirus infection in 1-week-old calves. Thickening of the omasum and abomasum wall, with acute inflammation, necrosis, ulceration, and neoplastic changes were seen in 1 calf. In the other 2, small papillomas were observed in the omasal mucosa, exhibiting proliferation of the parakeratinized epithelium. Papillomavirus antigens were detected by immunohistochemistry and virus-like particles were seen through electron microscopy.

Origin and global spreading of an ancestral lineage of the infectious bursal disease virus.

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.

Molecular characterization of avian infectious bronchitis virus strains from outbreaks in Argentina (2001-2008).

Twenty infectious bronchitis virus isolates were recovered from broilers and layers in different outbreaks amongst commercial poultry flocks in different geographic regions of Argentina from 2001 to 2008. The viruses were isolated from the tracheas, lungs, and caecal tonsils of birds that were showing respiratory signs. Further analysis based on their nucleotide and amino acid sequences in hypervariable region (HVR) 1 and the intervening sequence including HVRs 1 and 2 (HVR1/2) of the S1 gene was done to determine the genetic relationships among them and reference strains. Five isolates were highly related to the Massachusetts or Connecticut serotypes, indicating the probability of the detection and isolation of vaccine strains. The other Argentinean isolates formed three separate clusters (A, B and C), distant from the vaccine serotypes, with no correlation between the generated clusters and a geographic pattern. These observations could explain the failure of the Massachusetts serotype vaccination programmes to control IBV in these flocks. In addition, the utilization of HVR1/2 and HVR1 sequences resulted in trees with similar topology but the phylogenetic relationships using HVR1/2 nucleotide sequences were better supported by higher bootstrap values. Therefore, the sequences of the HVR1/2 region are recommended for phylogenetic studies.

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5.

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype "BoHV-5b", were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays. Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5.

Genetically diverse herpesviruses in South American Atlantic coast seabirds.

Different herpesviruses have been associated with respiratory and enteric disease and mortality among seabirds and waterfowl. In 2011, a respiratory disease outbreak affected 58.3% (98/168) of the Magellanic penguins undergoing rehabilitation due to an oil spill off the southern Brazilian coast. Etiology was attributed to a novel herpesvirus identified by histopathology, immunohistochemistry, electron microscopy and molecular studies with partial DNA sequencing. Since migration, rehabilitation and translocation may facilitate the spread of pathogens between populations and trigger the onset of clinical disease in animals with latent infections, investigation of herpesvirus occurrence in asymptomatic seabirds was performed. Samples from free-ranging seabirds were collected in Argentinian Patagonia (Magellanic penguins) and the Abrolhos Archipelago in Brazil (Brown boobies, Masked boobies, Red-billed tropicbirds, White-tailed tropicbirds and South American tern). Furthermore, asymptomatic seabirds housed at the facility where the outbreak occurred were also sampled. In total, 354 samples from eight seabird species were analyzed by PCR for herpesvirus. Four different sequences of herpesviruses were identified, one in Yellow-nosed Albatross, one in Boobies and Tropicbirds and two in Magellanic penguins. Magellanic penguin herpesvirus 1 was identified during the penguin outbreak at the rehabilitation facility in Brazil, while Magellanic penguin herpesvirus 2 was recovered from free-ranging penguins at four reproduction sites in Argentina. Phylogenic analysis of the herpesviruses sequences tentatively identified suggested that the one found in Suliformes and the one associated with the outbreak are related to sequences of viruses that have previously caused seabird die-offs. These findings reinforce the necessity for seabird disease surveillance programs overall, and particularly highlight the importance of quarantine, good hygiene, stress management and pre-release health exams in seabirds undergoing rehabilitation.

Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus.

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.

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.

Genetic characterization of porcine circovirus type 2 from pigs with porcine circovirus associated diseases in Argentina.

Porcine circovirus type 2 (PCV-2) has been associated with syndromes grouped by the term porcine circovirus associated diseases (PCVAD). The PCV-2 isolates have been grouped into two major groups or genotypes according to their nucleotide sequence of whole genomes and/or ORF-2: PCV-2b, which have, in turn, been subdivided into three clusters (1A-1C), and PCV-2a, which has been subdivided into five clusters (2A-2E). In the present study, we obtained 16 sequences of PCV-2 from different farms from 2003 to 2008, from animals with confirmatory diagnosis of PCVAD. Since results showed an identity of 99.8% among them, they were grouped within a common cluster 1A-B. This preliminary study suggests a stable circulation of PCV-2b among the Argentinean pig population.