Protection Against Infectious Bronchitis Virus Vaccine Recombinants and Chicken-Selected Vaccine Subpopulations.

Outbreaks of infectious bronchitis (IB) continue to occur from novel variants of IB virus (IBV) emerging from selection of vaccine subpopulations and/or naturally occurring recombination events. S1 sequencing of Arkansas (Ark) -type viruses obtained from clinical cases in Alabama broilers and backyard chickens shows both Ark Delmarva Poultry Industry (ArkDPI) vaccine subpopulations as well as Ark vaccine viruses showing recombination with other IB vaccine viruses. IB Ark-type isolates AL5, most similar to an ArkDPI vaccine subpopulation selected in chickens, AL4, showing a cluster of three nonsynonymous changes from ArkDPI subpopulations selected in chickens, and AL9, showing recombination with Massachusetts (Mass) -type IBV, were examined for pathogenicity and ability to break through immunity elicited by vaccination with a commercial ArkDPI vaccine. Analysis of predicted S1 protein structures indicated the changes were in regions previously shown to comprise neutralizing epitopes. Thus, they were expected to contribute to immune escape and possibly virulence. Based on clinical signs, viral load, and histopathology, all three isolates caused disease in naïve chickens, although AL9 and AL5 viral loads in trachea were statistically significantly higher (30- and 40-fold) than AL4. S1 gene sequencing confirmed the stability of the relevant changes in the inoculated viruses in the chickens, although virus in some individual chickens exhibited additional S1 changes. A single amino acid deletion in the S1 NTD was identified in some individual chickens. The location of this deletion in the predicted structure of S1 suggested the possibility that it was a compensatory change for the reduced ability of AL4 to replicate in the trachea of naïve chickens. Chickens vaccinated with a commercial ArkDPI vaccine at day of hatch and challenged at 21 days of age showed that vaccination provided incomplete protection against challenge with these viruses. Moreover, based on viral RNA copy numbers in trachea, differences were detected in the ability of the vaccine to protect against these IBV isolates, with the vaccine protecting the most poorly against AL4. These results provide additional evidence supporting that IBV attenuated vaccines, especially ArkDPI vaccines, contribute to perpetuating the problem of IB in commercial chickens.

Protección contra los virus de la bronquitis infecciosa vacunales recombinantes y las subpoblaciones de vacunas seleccionadas en pollos. Los brotes de la bronquitis infecciosa aviar continúan presentándose a partir de nuevas variantes de dicho virus, que surgen de la selección de subpoblaciones de vacunas y/o eventos de recombinación que ocurren naturalmente. La secuenciación del gene S1 de virus tipo Arkansas (Ark) obtenidos de casos clínicos en pollos de engorde y de traspatio de Alabama muestra que tanto las subpoblaciones de la cepa vacunal Arkansas Delmarva Poultry Industry (ArkDPI) así como los virus de la vacuna Arkansas muestran recombinación con otros virus vacunales de la bronquitis infecciosa. Los aislamientos del virus de la bronquitis infecciosa Arkansas tipo "AL5", más similares a una subpoblación de vacuna ArkDPI seleccionada en pollos, "AL4", que muestra un grupo de tres cambios no sinónimos de subpoblaciones de ArkDPI seleccionadas en pollos y el tipo "AL9", que muestra recombinación con el serotipo Massachusetts, se examinaron para determinar su patogenicidad y capacidad para traspasar la inmunidad generada por la vacunación con una vacuna comercial ArkDPI. El análisis de las estructuras predichas de la proteína S1 indicó que los cambios se produjeron en regiones que previamente se había demostrado comprendían epítopos neutralizantes. Por lo tanto, se esperaba que contribuyeran al escape inmunológico y posiblemente a la virulencia. Con base en los signos clínicos, la carga viral y la histopatología, los tres aislados causaron enfermedad en pollos sin exposición previa, aunque las cargas virales de AL9 y AL5 en la tráquea fueron estadísticamente significativamente mayores (30 y 40 veces) en comparación con AL4. La secuenciación del gene S1 confirmó la estabilidad de los cambios relevantes en los virus inoculados en los pollos, aunque el virus en algunos pollos individuales exhibió cambios adicionales en el gene S1. Se identificó una deleción de un solo aminoácido en el dominio terminal N del gene S1 (NTD S1) en algunos pollos individuales. La ubicación de esta eliminación en la estructura predicha del gene S1 sugirió la posibilidad de que se tratara de un cambio compensatorio por la capacidad reducida de AL4 para replicarse en la tráquea de pollos sin exposición previa. Los pollos vacunados con una vacuna comercial ArkDPI el día de la eclosión y desafiados a los 21 días de edad mostraron que la vacunación proporcionó una protección incompleta contra el desafío con estos virus. Además, basándose en el número de copias del ARN viral en la tráquea, se detectaron diferencias en la capacidad de la vacuna para proteger contra estos aislados del virus de la bronquitis infecciosa, siendo la vacuna con la protección más deficiente contra AL4. Estos resultados proporcionan evidencia adicional que respalda que las vacunas atenuadas contra el virus de la bronquitis infecciosa, especialmente las vacunas ArkDPI, contribuyen a perpetuar esta enfermedad en los pollos comerciales.

Changes in the Transcriptome Profile in Young Chickens after Infection with LaSota Newcastle Disease Virus.

Understanding gene expression changes in chicks after vaccination against Newcastle Disease (ND) can reveal vaccine biomarkers. There are limited data on chicks' early immune response after ND vaccination. Two trials focused on this knowledge gap. In experiment one, 42 13-day-old specific-pathogen-free (SPF) chicks were used. Harderian glands (Hgs) and tracheas (Tcs) from five birds per group were sampled at 12, 24, and 48 h post-vaccination (hpv) to evaluate the gene transcription levels by RNA sequencing (RNA-seq) and RT-qPCR. The results of RNA-seq were compared by glmFTest, while results of RT-qPCR were compared by t-test. With RNA-seq, a significant up-regulation of interferon-related genes along with JAK-STAT signaling pathway regulation was observed in the Hgs at 24 hpv. None of the differentially expressed genes (DEGs) identified by RNA-seq were positive for RT-qPCR. Experiment 2 used 112 SPF and commercial chickens that were 1 day old and 14 days old. Only the commercial birds had maternal antibodies for Newcastle Disease virus (NDV). By RNA-seq, 20 core DEGs associated with innate immunity and viral genome replication inhibition were identified. Genes previously unlinked to NDV response, such as USP41, were identified. This research present genes with potential as immunity biomarkers for vaccines, yet further investigation is needed to correlate the core gene expression with viral shedding post-vaccination.

Distribution of Infectious Bronchitis Virus Resistance in a Naïve Chicken Population.

Resistance to infectious bronchitis (IB) is a polygenic trait, but little is known about how resistance distributes in the host population. In this study, a relatively large number (n = 369) of specific-pathogen-free white leghorn chickens (Gallus gallus) were challenged with an Arkansas -type virulent IB virus (IBV), and resistance was evaluated 5 days after challenge by viral load (IBV RNA) in the trachea and cecal tonsils, as well as by tracheal histomorphometry (mucosal thickness and lymphocyte infiltration). Contrary to expectations, results showed a non-Gaussian distribution of resistance of the whole population against challenge. Indeed, most chickens accumulated toward higher resistance, i.e., lower viral loads and less tracheal damage. The current results also indicated limited differences in resistance to IBV between sexes. Tracheal viral load was significantly higher in males than that in females, but tracheal damage did not significantly differ between sexes. The difference in tracheal viral load found in males and females could have implications for viral spread in commercial chicken populations.

Nota de investigación­Distribución de la resistencia al virus de la bronquitis infecciosa en una población de pollos susceptibles. La resistencia a la bronquitis infecciosa es un rasgo poligénico, pero se sabe poco acerca de cómo se distribuye la resistencia en la población huésped. En este estudio, varios (n=369) pollos White Leghorn libres de patógenos específicos fueron desafiados con un virus de la bronquitis infecciosa virulento de tipo Arkansas y la resistencia se evaluó cinco días después del desafío mediante la carga viral en la tráquea y las tonsilas cecales, así como por histomorfometría traqueal (grosor de la mucosa e infiltración de linfocitos). Contrariamente a lo esperado, los resultados mostraron una distribución no gaussiana de la resistencia de toda la población frente al desafío. De hecho, la mayoría de los pollos se distribuyeron hacia una mayor resistencia, es decir, cargas virales más bajas y menos daño traqueal. Los resultados actuales también indicaron diferencias limitadas en la resistencia al virus de la bronquitis infecciosa entre sexos. La carga viral traqueal fue significativamente mayor en los machos en comparación con las hembras, pero el daño traqueal no fue significativamente diferente entre sexos. La diferencia en la carga viral traqueal encontrada en machos y hembras podría tener implicaciones para la propagación viral en las poblaciones de pollos comerciales.

Infectious bronchitis virus S2 expressed from recombinant virus confers broad protection against challenge.

We developed a recombinant Newcastle disease virus (NDV) LaSota (rLS) expressing the infectious bronchitis virus (IBV) S2 gene (rLS/IBV.S2). The recombinant virus showed somewhat-reduced pathogenicity compared to the parental lentogenic LaSota strain but effectively elicited hemagglutination inhibition antibodies against NDV and protected chickens against lethal challenge with virulent NDV/CA02. IBV heterotypic protection was assessed using a prime-boost approach with a commercially available attenuated IBV Massachusetts (Mass)-type vaccine. Specific-pathogen-free chickens primed ocularly with rLS/IBV.S2 at 4 days of age and boosted with Mass at 18 days of age were completely protected against challenge at 41 days of age with a virulent Ark-type strain. In a second experiment, we compared protection conferred by priming with rLS/IBV.S2 and boosting with Mass (rLS/IBV.S2+Mass) versus priming and boosting with Mass (Mass+Mass). We also modified the timing of vaccination to prime at 1 day of age and boost at 12 days of age. Challenge with virulent Ark was performed at 21 days of age. Based on clinical signs, both vaccinated groups appeared equally protected against challenge compared to unvaccinated challenged chickens. Viral loads in lachrymal fluids of birds receiving rLS/IBV.S2+Mass showed a clear tendency of improved protection compared to Mass+Mass; however, the difference did not achieve statistical significance. A significant difference (P < 0.05) was determined between these groups regarding incidence of detection of challenge IBV RNA in the trachea; viral RNA was detected in 50% of rLS/IBV.S2+Mass-vaccinated chickens while chickens vaccinated with Mass+Mass and unvaccinated challenged controls showed 84 and 90% incidence of IBV RNA detection in the trachea, respectively. These results demonstrate that overexposing the IBV S2 to the chicken immune system by means of a vectored vaccine, followed by boost with whole virus, protects chickens against IBV showing dissimilar S1.