Analysis of the Immune Response and Identification of Antibody Epitopes Against the Sigma C Protein of Avian Orthoreovirus Following Immunization with Live or Inactivated Vaccines.

Avian orthoreoviruses are causative agents of tenosynovitis and viral arthritis in both chickens and turkeys. Current commercial reovirus vaccines do not protect against disease caused by emerging variants. Custom-made inactivated reovirus vaccines are commonly utilized to help protect commercial poultry against disease. Antibody epitopes located on the viral attachment protein, σC, involved in virus neutralization, have not been clearly identified. In this study, the S1133 vaccine strain (Genetic Cluster 1 [GC1], a GC1 field isolate (117816), and a GC5 field isolate (94826) were determined to be genetically and serologically unrelated. In addition, chickens were vaccinated with either a commercial S1133 vaccine, 117816 GC1, or 94826 GC5, and sera were used in peptide microarrays to identify linear B-cell epitopes within the σC protein. Specific-pathogen-free (SPF) chickens were vaccinated twice with either: 1) live and live, 2) inactivated and inactivated, or 3) a combination of live and inactivated vaccines. Epitope mapping was performed on individual serum samples from birds in each group using S1133, 117816, and 94826 σC sequences translated into an overlapping peptides and spotted onto microarray chips. Vaccination with a combination of live and inactivated viruses resulted in a greater number of B-cell binding sites on the outer-capsid domains of σC for 117816 and 94826, but not for S1133. In contrast, the S1133-vaccinated birds demonstrated fewer epitopes, and those epitopes were located in the stalk region of the protein. However, within each of the vaccinated groups, the highest virus-neutralization titers were observed in the live/inactivated groups. This study demonstrates differences in antibody binding sites within σC between genetically and antigenically distinct reoviruses and provides initial antigenic characterization of avian orthoreoviruses and insight into the inability of vaccine-induced antibodies to provide adequate protection against variant reovirus-induced disease.

Análisis de la respuesta inmune e identificación de epítopos de anticuerpos contra la proteína Sigma C de Orthoreovirus aviar después de la inmunización con vacunas vivas o inactivadas. Los ortoreovirus aviares son agentes causantes de tenosinovitis y artritis viral tanto en pollos como en pavos. Las vacunas de reovirus comerciales actuales no protegen contra la enfermedad causada por variantes emergentes. Las vacunas de reovirus inactivadas hechas a medida se utilizan comúnmente para ayudar a proteger a las aves comerciales contra enfermedades. Los epítopos de anticuerpos ubicados en la proteína de unión viral, σC, involucrada en la neutralización del virus, no se han identificado claramente. En este estudio, se determinó que la cepa vacunal S1133 (Grupo genético 1 (GC1), un aislado de campo del grupo genético 1 (117816) y un aislado de campo del grupo genético 5 (GC5) (94826) no tenían ninguna relación genética ni serológica. Además, los pollos se vacunaron con una vacuna comercial S1133, 117816 GC1 o 94826 GC5, y se usaron sueros en micromatrices de péptidos para identificar epítopos de células B lineales dentro de la proteína σC. Pollos libres de patógenos específicos (SPF) fueron vacunados dos veces con: 1) vivo y vivo, 2) inactivado e inactivado, o 3) una combinación de vacunas vivas e inactivadas. El mapeo de epítopos se realizó en muestras de suero individuales de aves en cada grupo usando secuencias S1133, 117816 y 94826 σC colocadas en microchips. La vacunación con una combinación de virus vivos e inactivados dio como resultado un mayor número de sitios de unión de células B en los dominios de la cápside externa de σC para 117816 y 94826, pero no para S1133. Por el contrario, las aves vacunadas con S1133 demostraron menos epítopos y esos epítopos estaban ubicados en la región del tallo de la proteína. Sin embargo, dentro de cada uno de los grupos vacunados, los títulos de neutralización de virus más altos se observaron en los grupos vivos/inactivados. Este estudio demuestra diferencias en los sitios de unión de anticuerpos dentro de σC entre reovirus genética y antigénicamente distintos y proporciona una caracterización antigénica inicial de los ortoreovirus aviares y una idea de la incapacidad de los anticuerpos inducidos por la vacuna para proporcionar una protección adecuada contra la enfermedad inducida por reovirus variante.

Characterization of an Infectious Bronchitis Virus Isolated from Commercial Layers Suffering from False Layer Syndrome.

Infectious bronchitis virus (IBV) is a gammacoronavirus that primarily induces an upper respiratory disease in chickens, also affecting the urogenital tract and occasionally leading to a condition called false layer syndrome (FLS), where sexually mature hens ovulate normally but are unable to lay eggs. Here, we describe an outbreak of FLS in Arizona from which an IBV variant that is nearly 90% homologous to DMV/1639 using the Spike subunit 1 gene, named AZ/FLS/17, was isolated and used in challenge experiments. Three-day-old specific-pathogen-free chicks were challenged with AZ/FLS/17 or M41 in high and low doses, and the disease outcomes were compared. Overall, no differences in microscopic lesions or viral loads in the reproductive tract were detected between AZ/FLS/17- and M41-infected birds. To minimize the losses linked to FLS in the problematic flocks, an updated live-attenuated IBV vaccine protocol including the use of the Ma5 strain at the hatchery was implemented, resulting in a drastic reduction of false layers in the subsequent flocks. To monitor the circulation of wild-type and vaccine strains in this population, a molecular surveillance study was performed. Samples were collected at 1, 7, 14, and 21 days of age, and from laying hens at 30 and 36 wk. In older birds, the IBV strains detected were more diverse than at 1 and 7 days of age. Nevertheless, live vaccine combinations are still widely used to decrease the losses caused by FLS in commercial egg laying flocks worldwide.

Caracterización de un virus de la bronquitis infecciosa aislado de aves de postura comerciales que padecían el síndrome de la falsa ponedora. El virus de la bronquitis infecciosa (IBV) es un gammacoronavirus que induce principalmente una enfermedad de las vías respiratorias superiores en los pollos, que también afecta el tracto urogenital y ocasionalmente conduce a una condición llamada síndrome de la falsa ponedora (FLS), donde las gallinas sexualmente maduras ovulan normalmente pero no pueden producir huevos. En este estudio se describe un brote del síndrome de la falsa ponedora en Arizona a partir del cual se aisló una variante del virus de bronquitis que es casi 90% similar a la variante DMV/1639 usando el gene S1, la cual se denominó AZ/FLS/17, y se usó en experimentos de desafío. Pollos de tres días de edad libres de patógenos específicos (SPF) fueron desafiados con el virus AZ/FLS/17 o con el virus M41 en dosis altas y bajas, y se compararon los resultados de la enfermedad. En general, no se detectaron diferencias en las lesiones microscópicas o en las cargas virales en el tracto reproductivo entre las aves infectadas con el virus AZ/FLS/17 y el virus M41. Para minimizar las pérdidas relacionadas con el síndrome de la falsa ponedora en las parvadas problemáticas, se implementó un protocolo actualizado con vacuna viva atenuada contra la bronquitis infecciosa que incluía el uso de la cepa Ma5 en la incubadora, lo que resultó en una reducción drástica de las gallinas falsas ponedoras en las parvadas subsecuentes. Para monitorear la circulación de cepas de tipo silvestre y vacunales en esta población, se realizó un estudio de vigilancia molecular. Se recolectaron muestras a los 1, 7, 14 y 21 días de edad y de gallinas de postura a las 30 y 36 semanas. En aves mayores, las cepas del virus de bronquitis detectadas fueron más diversas que a los días 1 y 7 de edad. Sin embargo, las combinaciones de vacunas vivas todavía se utilizan ampliamente para disminuir las pérdidas causadas por el síndrome de la falsa ponedora en las parvadas de postura de huevo comercial en todo el mundo.

Detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa.

In situ hybridization and immunohistochemistry were utilized to identify tissues infected in ovo with infectious bronchitis virus (IBV). Chicken embryos were inoculated in ovo (chorioallantoic sac) with the Arkansas (Ark) serotype of IBV at 18 days of age. At 24, 48, 72, and 120 hr postinfection (HPI), bursa, lung, spleen, heart, and thymus were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. The digoxigenin-labeled antisense S1 riboprobe detected viral mRNA in the cytoplasm of respiratory epithelial cells in the primary bronchus at 24, 48, and 72 HPI. Viral mRNA was detected in bursa samples collected at 48 hr. Immunohistochemistry detected viral antigens in epithelial cells of the parabronchi and bursal tissues at 24 and 48 hr, respectively. No viral mRNA or antigen was detected by in situ hybridization or immunohistochemistry, respectively, in heart, thymus, or spleen at any time after inoculation. On the basis of these data, IBV apparently initially infects lung tissue, then migrates to and infects cells of the bursa. These results indicate that in situ hybridization can be useful in detection of IBV-infected chickens and in understanding the pathogenesis and virulence of IBV infection.

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates.

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization.

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.

Newcastle disease virus phosphoprotein gene analysis and transcriptional editing in avian cells.

Nucleotide sequence was determined for the phosphoprotein (P) gene from 23 Newcastle disease virus (NDV) isolates representing all defined pathotypes with different chronological and geographic origins. Sequence variation, with synonymous substitutions dominating, occurred throughout the P gene. An exception was a conserved central region containing the transcriptional editing site. Four G nucleotide additions were detected in NDV P gene mRNA potentially creating alternative open reading frames. However, only one in-frame stop codon exists with a single G addition among all isolates that would allow for a potential V protein. A second potential stop codon does not exist in the P gene consensus sequence among all isolates with more than one G nucleotide addition at the editing site. This precludes a possible W protein in these isolates. A second potential alternative in-frame start site exists among all isolates that could encode a predicted X protein for NDV. Comparison of the P gene editing sites among the Paramyxovirinae and predicted P gene usage demonstrates that NDV more closely resembles the respiroviruses and morbilliviruses. Phylogenetic analysis of P gene sequences among NDV isolates demonstrates there are two clades of these viruses. One group includes viruses isolated in the US prior to 1970, while a second cluster includes virulent viruses circulating worldwide.

The avian response to Newcastle disease virus.

Newcastle disease virus (NDV) is classified as a member of the superfamily Mononegavirales in the family Paramyxoviridae. This virus family is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. In 1993 the International Committee on the Taxonomy of Viruses rearranged the order of the Paramyxovirus genus and placed NDV within the Rubulavirus genus among the Paramyxovirinae. The enveloped virus has a negative sense single-stranded RNA genome of 15,186 kb which codes for an RNA directed RNA polymerase, hemagglutinin-neuraminidase protein, fusion protein, matrix protein, phosphoprotein and nucleoprotein in the 5' to 3' direction. The virus has a wide host range with most orders of birds reported to have been infected by NDV. Isolates are characterized by virulence in chickens and are categorized into three main pathotypes depending on severity of disease. Lentogenic isolates are of low virulence while viruses of intermediate virulence are termed mesogenic. Highly virulent viruses that cause high mortality in birds are termed neurotropic or viscerotropic velogenic. Velogenic NDV are List A pathogens that require reporting to the Office of International Epizootics and outbreaks result in strict trade embargoes. The primary molecular determinant for NDV pathogenicity is the fusion protein cleavage site amino acid sequence. Vaccination for NDV is primarily by mass application of live-virus vaccines among commercial poultry. Although protection is measured by presence of antibodies to NDV, vaccinated B-cell depleted chickens are resistant to disease. Consequently, immune protection involves responses that are presently incompletely defined.

Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates.

Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.

Antigenic and molecular characterization of three infectious bursal disease virus field isolates.

Three infectious bursal disease field viruses, identified as U28, 3212, and MISS and isolated in the early 1980s from the southeastern United States, were characterized both antigenically and genotypically. A panel of monoclonal antibodies (MAbs) was utilized in an antigen-capture enzyme-linked immunosorbent assay (ELISA) for antigenic characterization. The ELISA data indicated that U28 and the Delaware variants are antigenically related. The 3212 and the GLS variants were more closely related antigenically to each other than to other viruses analyzed. However, the MISS isolate reacted with MAbs that were specific for both classic and variant strains of infectious bursal disease virus (IBDV). Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify nucleotide sequences from the genome coding for the variable region of VP2 from IBDV field isolates U28, 3212, and MISS. Phylogenetic analysis of the deduced amino acid sequences revealed that U28 was most similar (98.3%) to the IBDV Delaware variants and that 3212 was most similar (97.1%) to the GLS variant. The MISS isolate was most similar (97.4%) to the classic 52/70 strain. Positive correlation occurred with the use of different methods to characterize IBDV isolates.