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.

Intestinal Tropism of an Infectious Bronchitis Virus Isolate Not Explained by Spike Protein Binding Specificity.

An infectious bronchitis virus (IBV) with an unusual enteric tropism (CalEnt) was isolated from a California broiler flock exhibiting runting-stunting syndrome. IBV was detected in the small intestine, but not in the respiratory tract or kidney. During virus isolation in embryos, it did not replicate in chorioallantoic membrane (CAM) but could be recovered from intestines. Its S1 protein showed 93% amino acid sequence identity to a California variant isolated in 1999 (Cal99). Intestinal lesions were reproduced following ocular/nasal inoculation of specific-pathogen-free chickens, but respiratory signs and lesions were also present. The virus was detected in both respiratory and intestinal tissues. To determine whether the novel tropism of IBV CalEnt was due to an increased ability of its S1 protein to bind to the intestinal epithelium, we compared the binding of soluble trimeric recombinant S1 proteins derived from CalEnt and Cal99 to chicken tissues. Contrary to expectations, the CalEnt S1 protein did not bind to small intestine and, unlike Cal99 S1, did not bind to the respiratory epithelium or CAM. Using only the CalEnt S1 N-terminal domain or including the S2 ectodomain (lacking membrane and cytoplasmic domains), which have been shown to improve ArkDPI S1 protein binding, did not lead to detectable binding at the standard protein concentration to any tissue tested. Our results indicate no/poor binding of the CalEnt spike protein to both respiratory and intestinal tissues and thus do not support better attachment to intestinal epithelial cells as a reason for CalEnt's extended tropism. These results might reflect shortcomings of the assay, including that it does not detect potential contributions of the S1 C-terminal domain to attachment. We used bioinformatic approaches to explore the possibility that the unique tropism of CalEnt might be a result of functions of the S protein in cell-entry steps subsequent to attachment. These analyses suggest that CalEnt's S2 coding region was acquired through a recombination event and encodes a unique amino acid sequence at the putative recognition site for the protease that activates the S protein for fusion. Thus, S2 activation by tissue-specific proteases might facilitate CalEnt entry into intestinal epithelial cells and compensate for poor binding by its S1 protein.

Tropismo intestinal de un aislamiento del virus de la bronquitis infecciosa con una especificidad de unión a la proteína espícula inusual. Se aisló un virus de la bronquitis infecciosa (IBV) con un tropismo entérico inusual (CalEnt) de una parvada de pollos de engorde de California que presentaba síndrome de retraso en el crecimiento. Se detectó al virus de bronquitis en el intestino delgado, pero no en el tracto respiratorio o en el riñón. Durante el aislamiento del virus en huevos embrionados de pollo, no se replicó en la membrana corioalantoidea (CAM), pero pudo recuperarse de los intestinos. Su proteína S1 mostró una identidad de secuencia de aminoácidos del 93% con una variante de California aislada en el año 1999 (Cal99). Las lesiones intestinales se reprodujeron después de la inoculación ocular/nasal de pollos libres de patógenos específicos, pero también hubo signos y lesiones respiratorias. El virus se detectó en los tejidos respiratorios e intestinales. Para determinar si el nuevo tropismo de este virus de la bronquitis infecciosa CalEnt se ocasionaba por una mayor capacidad de su proteína S1 para unirse al epitelio intestinal, se comparó la unión a los tejidos de pollo de las proteínas S1 recombinantes triméricas solubles derivadas de los virus CalEnt y Cal99. Contrariamente a lo esperado, la proteína CalEnt S1 no se unió al intestino delgado y a diferencia del virus Cal99 S1, no se unió al epitelio respiratorio o CAM. Mediante el uso de solo el dominio N-terminal de la proteína S1 del virus CalEnt o por la inclusión del ectodominio S2 (que carece de dominios de membrana y citoplasmáticos), que se ha demostrado mejora la unión de la proteína S1 del serotipo Arkansas DPI, no se observó una unión detectable a ningún tejido analizado a la concentración de proteína estándar. Estos resultados indican una unión nula o deficiente de la proteína de la espícula del virus CalEnt a los tejidos respiratorios e intestinales y por lo tanto, no respaldan la preferencia de la unión a las células epiteliales intestinales como una razón para el tropismo extendido del virus CalEnt. Estos resultados pueden reflejar las deficiencias del ensayo, incluyendo el hecho de que no detecta posibles contribuciones del dominio C-terminal de la proteína S1 en la unión. Se utilizaron enfoques bioinformáticos para explorar la posibilidad de que el tropismo único del virus CalEnt podría ser el resultado de las funciones de la proteína S en los pasos de entrada a las células posteriores a la unión. Estos análisis sugieren que la región de codificación S2 del virus CalEnt se adquirió a través de un evento de recombinación y codifica una secuencia de aminoácidos única en el supuesto sitio de reconocimiento de la proteasa que activa la proteína S para la fusión. Por lo tanto, la activación de S2 por proteasas específicas de tejido podría facilitar la entrada del virus CalEnt en las células epiteliales intestinales y compensar la unión deficiente por su proteína S1.

Correction to: Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs.

The Figure 3 in the original version of this article was incorrectly published. In this article the top panel of Figure 3 that describes the amino acid sequence alignment is now added. The original article has been corrected.

Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs.

The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 restricts the antigen specific CTL immune response.

Protection against infectious bronchitis virus by spike ectodomain subunit vaccine.

The avian coronavirus infectious bronchitis virus (IBV) S1 subunit of the spike (S) glycoprotein mediates viral attachment to host cells and the S2 subunit is responsible for membrane fusion. Using IBV Arkansas-type (Ark) S protein histochemistry, we show that extension of S1 with the S2 ectodomain improves binding to chicken tissues. Although the S1 subunit is the major inducer of neutralizing antibodies, vaccination with S1 protein has been shown to confer inadequate protection against challenge. The demonstrated contribution of S2 ectodomain to binding to chicken tissues suggests that vaccination with the ectodomain might improve protection compared to vaccination with S1 alone. Therefore, we immunized chickens with recombinant trimeric soluble IBV Ark-type S1 or S-ectodomain protein produced from codon-optimized constructs in mammalian cells. Chickens were primed at 12days of age with water-in-oil emulsified S1 or S-ectodomain proteins, and then boosted 21days later. Challenge was performed with virulent Ark IBV 21days after boost. Chickens immunized with recombinant S-ectodomain protein showed statistically significantly (P<0.05) reduced viral loads 5days post-challenge in both tears and tracheas compared to chickens immunized with recombinant S1 protein. Consistent with viral loads, significantly reduced (P<0.05) tracheal mucosal thickness and tracheal lesion scores revealed that recombinant S-ectodomain protein provided improved protection of tracheal integrity compared to S1 protein. These results indicate that the S2 domain has an important role in inducing protective immunity. Thus, including the S2 domain with S1 might be promising for better viral vectored and/or subunit vaccine strategies.

Infectious Bronchitis Virus S2 of 4/91 Expressed from Recombinant Virus Does Not Protect Against Ark-Type Challenge.

We previously demonstrated that chickens primed with a recombinant Newcastle disease virus LaSota (rLS) expressing the S2 gene of infectious bronchitis virus (IBV) and boosted with an attenuated IBV Massachusetts (Mass)-type vaccine were protected against IBV Arkansas (Ark)-type virulent challenge. A possible basis for the reported ability of IBV 4/91 (serotype 793/B) vaccine to protect against divergent IBV strains (e.g., QX, Q1, and D1466) in a prime-boost approach with an IBV Mass vaccine is that an immune response against the S2 protein of IBV 4/91 is cross-protective. Therefore, we evaluated the protective capabilities of the S2 protein of IBV 4/91 expressed from rLS. The level of S2 amino acid sequence identity between 4/91 and the Ark challenge strain used in this study (90.7%) is within the range of S2 amino acid sequence identities between 4/91 and Q1 (91%-94%) and QX (89%-94%) strains. Chickens primed with attenuated Mass IBV at 1 day of age and boosted with rLS/IBV.S2-4/91 at 14 days of age were challenged with a virulent Ark IBV strain at 28 days of age. Protection (reduction of clinical signs and viral loads) assessed 5 days postchallenge showed nonsignificant differences between chickens primed with Mass vaccine and boosted with rLS/IBV.S2-4/91 and chickens vaccinated with Mass only. Thus, the observed level of protection is attributable only to the effect of the Mass vaccine, indicating that the S2 of IBV 4/91 does not induce broad cross-protective immunity.

Combined infectious bronchitis virus Arkansas and Massachusetts serotype vaccination suppresses replication of Arkansas vaccine virus.

Polyvalent infectious bronchitis virus vaccination is common worldwide. The possibility of vaccine interference after simultaneous combined vaccination with Arkansas (Ark) and Massachusetts (Mass)-type vaccines was evaluated in an effort to explain the high prevalence of Ark-type infectious bronchitis virus in vaccinated chickens. Chickens ocularly vaccinated with combinations of Ark and Mass showed predominance of Mass vaccine virus before 9 days post-vaccination (DPV) in tears. Even when Mass and Ark vaccines were inoculated into separate eyes, Mass vaccine virus was able to outcompete Ark vaccine virus. Although Mass vaccine virus apparently had a replication advantage over Ark vaccine in ocular tissues, Ark vaccine virus appeared to have an advantage in spreading to and/or replicating in the trachea. When chickens vaccinated with Ark or Mass vaccine were housed together, Mass vaccine virus was able to spread to Ark-vaccinated chickens, but the Ark vaccine was not detected in Mass-vaccinated chickens. Only Mass vaccine was detected in tears of sentinel birds introduced into groups receiving both vaccines. Furthermore, Ark vaccine virus RNA was not detectable until 10 DPV in most tear samples from chickens vaccinated with both Ark and Mass vaccines at varying Ark vaccine doses, while high concentrations of Mass virus RNA were detectable at 3-7 DPV. In contrast, Ark vaccine virus replicated effectively early after vaccination in chickens vaccinated with Ark vaccine alone. The different replication dynamics of Ark and Mass viruses in chickens vaccinated with combined vaccines did not result in reduced protection against Ark challenge at 21 DPV. Further studies are needed to clarify if the viral interference detected determines differences in protection against challenge at other time points after vaccination.

Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies.

Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs) was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry.

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.

Comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different Arkansas Delmarva Poultry Industry -derived infectious bronchitis virus vaccines.

Factors responsible for the persistence of Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis vaccines in commercial flocks and the high frequency of isolation of ArkDPI-type infectious bronchitis viruses in respiratory cases are still unclear. We compared dynamics of vaccine viral subpopulations, viral loads, persistence in trachea and cloaca, and the magnitude of infectious bronchitis virus (1BV)-specific antibody induction after vaccination with two commercial ArkDPI-derived Arkansas (Ark) serotype vaccines. One of the vaccines (coded vaccine B) produced significantly higher vaccine virus heterogeneity in vaccinated chickens than the other vaccine (coded A). Chickens vaccinated with vaccine B had significantly higher viral loads in tears at 5 days postvaccination (DPV) than those vaccinated with vaccine A. Vaccine B also induced a significantly higher lachrymal immunoglobulin M response at 11 DPV, an earlier peak of IBV-specific lachrymal immunoglobulin A, and higher serum antibodies than vaccine A. In addition, a significantly higher proportion of birds vaccinated with vaccine B had vaccine virus detected in the trachea at 20 DPV than those vaccinated with vaccine A. Furthermore, the virus detected at 20 DPV in most of the chickens vaccinated with vaccine B was a single specific subpopulation (subpopulation 4) selected from multiple vaccine subpopulations detected earlier at 5 and 7 DPV in the same chickens. On the other hand, a higher proportion of chickens vaccinated with vaccine A had virus detected in cloacal swabs at 20 DPV. Thus we found differences in mucosal antibody induction and selection and persistence of vaccine viruses between two ArkDPI-derived vaccines from different manufacturers. The higher vaccine virus heterogeneity observed in chickens vaccinated with vaccine B compared with those vaccinated with vaccine A may be responsible for these differences. Thus the high frequency of Ark IBV viruses in the field may be due to the inherent ability of some ArkDPI-derived vaccine viruses to be selected and persist in vaccinated chickens. Vaccine virus persistence may offer genetic material for recombination or may undergo mutations with the potential to result in increased virulence.

Efficient heterologous antigen gene delivery and expression by a replication-attenuated BoHV-4-based vaccine vector.

Bovine Herpesvirus 4 (BoHV-4) is a gammaherpesvirus belonging to the Rhadinovirus genus and due to its biological characteristics has been proposed as a vaccine vector for veterinary vaccines. Because viral vector-associated risk is a major concern for viral vector applications, attenuation is a desirable feature. Therefore, efforts are directed toward the development of highly attenuated viral vectors. BoHV-4 naturally exhibits limited pathogenicity and a further attenuation, in terms of replication, was obtained by disrupting the late gene encoding the 1.7-kb polyadenylated RNA (L1.7). An L1.7 deleted mutant BoHV-4 (BoHV-4-A-KanaGalKΔL1.7), as well as its revertant (BoHV-4-A-Rev), was generated by homologous recombination from the genome of a BoHV-4 isolate (BoHV-4-A) cloned as a bacterial artificial chromosome (BAC). BoHV-4-A-KanaGalKΔL1.7 showed attenuation in terms of competence to reconstitute infectious virus, viral replication, and plaque size when compared to BoHV-4-A, BoHV-4-A-Rev, and BoHV-4-A-KanaGalKΔTK, a recombinant control virus where the KanaGalK selectable marker was inserted into the thymidine kinase open reading frame. The capability of BoHV-4-A-KanaGalKΔL1.7 to deliver and express a heterologous antigen was investigated by replacing the KanaGalK cassette with a vesicular stomatitis virus glycoprotein (VSVg) expression cassette to generate BoHV-4-A-EF1αVSVgΔL1.7. BoHV-4-A-EF1αVSVgΔL1.7 infected cells robustly expressed VSVg, thus confirming that the replication deficiency resulting from L1.7 disruption did not prevent heterologous gene delivery and expression. Although further work is needed to identify the specific function of the BoHV-4 L1.7 gene, the L1.7 gene may represent an ideal targeting locus for the integration of a heterologous antigen expression cassette, resulting in attenuation of the viral vector.

Genetic diversity and selection regulates evolution of infectious bronchitis virus.

Conventional and molecular epidemiologic studies have confirmed the ability of infectious bronchitis virus (IBV) to rapidly evolve and successfully circumvent extensive vaccination programs implemented since the early 1950s. IBV evolution has often been explained as variation in gene frequencies as if evolution were driven by genetic drift alone. However, the mechanisms regulating the evolution of IBV include both the generation of genetic diversity and the selection process. IBV's generation of genetic diversity has been extensively investigated and ultimately involves mutations and recombination events occurring during viral replication. The relevance of the selection process has been further understood more recently by identifying genetic and phenotypic differences between IBV populations prior to, and during, replication in the natural host. Accumulating evidence suggests that multiple environmental forces within the host, including immune responses (or lack thereof) and affinity for cell receptors, as well as physical and biochemical conditions, are responsible for the selection process. Some scientists have used or adopted the related quasispecies frame to explain IBV evolution. The quasispecies frame, while providing a distinct explanation of the dynamics of populations in which mutation is a frequent event, exhibits relevant limitations which are discussed herein. Instead, it seems that IBV populations evolving by the generation of genetic variability and selection on replicons follow the evolutionary mechanisms originally proposed by Darwin. Understanding the mechanisms underlying the evolution of IBV is of basic relevance and, without doubt, essential to appropriately control and prevent the disease.

Infectious bronchitis virus subpopulations in vaccinated chickens after challenge.

Infectious bronchitis coronavirus (IBV) shows extensive genotypic and phenotypic variability. The evolutionary process involves generation of genetic diversity by mutations and recombination followed by replication of those phenotypes favored by selection. In the current study, we examined changes occurring in a wild Arkansas (Ark) challenge strain in chickens that were vaccinated either ocularly with commercially available attenuated ArkDPI-derived vaccines or in ovo with a replication-defective recombinant adenovirus expressing a codon-optimized IBV Ark S1 gene (AdArkIBV.S1(ck)). Commercial IBV Ark vaccines A, B, and C provided slightly differing levels of protection against homologous challenge. Most importantly for the current study, chickens vaccinated with the different vaccines displayed significant differences in specific B-lymphocyte responses in the Harderian gland (i.e., the challenge virus encountered differing immune selective pressure during invasion among host groups). Based on S1 sequences, five predominant populations were found in different individual vaccinated/challenged chickens. Chickens with the strongest immune response (vaccine A) were able to successfully impede replication of the challenge virus in most chickens, and only the population predominant in the challenge strain was detected in a few IBV-positive birds. In contrast, in chickens showing less than optimal specific immune responses (vaccines B and C) IBV was detected in most chickens, and populations different from the predominant one in the challenge strain were selected and became predominant. These results provide scientific evidence for the assumption that poor vaccination contributes to the emergence of new IBV strains via mutation and/or selection. In ovo vaccination with a low dose of AdArkIBV.S1(ck) resulted in a mild increase of systemic antibody and reduced viral shedding but no protection against IBV signs and lesions. Under these conditions we detected only virus populations identical to the challenge virus. Possible explanations are discussed. From a broad perspective, these results indicate that selection is an important force driving IBV evolution.

Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift.

We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV+IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV+IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.

The proportion of specific viral subpopulations in attenuated Arkansas Delmarva poultry industry infectious bronchitis vaccines influences vaccination outcome.

We investigated the significance of differing proportions of specific subpopulations among commercial Arkansas (Ark) Delmarva poultry industry (DPI) vaccines with regard to vaccination outcome. Two ArkDPI-derived vaccines that contain a higher proportion of viruses with S1 genes that become selected during replication in chickens exhibited more rapid establishment of those selected subpopulations in chickens, produced significantly higher viral loads in tears, and induced higher antibody responses compared with two other ArkDPI vaccines with lower proportions of viruses that become selected in chickens. The presence of higher proportions of selected subpopulations was also associated with a significantly higher incidence of respiratory signs early after vaccination and in some cases more severe tracheal lesions. However, one of the ArkDPI-derived vaccines with a lower proportion of selected subpopulations, despite producing a lower viral load in tears, also induced a higher incidence of respiratory signs later after vaccination and more severe tracheal lesions. Furthermore, one of the ArkDPI-derived vaccines with a higher proportion of selected subpopulations, despite producing a higher viral loads in tears, resulted in less severe tracheal damage. These discrepancies suggest that infectious bronchitis virus (IBV) load in tears may not always predict degree of tracheal damage and that phenotypic characteristics other than S1 may also be involved in severity of vaccine reactions following ArkDPI vaccine administration. We observed lower antibody responses to the vaccines that produced lower viral loads, which might contribute to the persistence of Ark serotype IBV vaccines observed in commercial flocks.

Infectious bronchitis virus in testicles and venereal transmission.

Even though males represent only 8%-12% of the birds of a breeder flock, their role in infectious bronchitis virus (IBV) dissemination is largely unknown. We first assessed the effect of IBV replication in the chicken testes. Ten-week-old males were inoculated with Arkansas (Ark) or Massachusetts (Mass) IBV virulent strains. Seven days postinoculation (DPI) IBV RNA was detected in testicles of 100% of M41- and in 96% of Ark-infected males. Marginal nonsynonymous variation was detected in spike (S) gene of the predominant population of IBV replicating in the testes compared to the S gene of the predominant population of viruses prior to inoculation. IBV M41 and Ark were detected in spermatogonia and Sertoli cells of testicles of infected roosters by immunofluorescence, without evident histopathological changes. We next assessed venereal transmission of IBV by artificially inseminating 54-wk-old hens either with semen from IBV-infected roosters or with IBV suspended in naïve semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.

Bovine herpesvirus 4 immediate early 2 (Rta) gene is an essential gene and is duplicated in bovine herpesvirus 4 isolate U.

The ORF50/Rta gene has been shown to be an essential gene for many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U.

Integration of bovine herpesvirus 4 genome into cultured persistently infected host cell genome.

Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play a secondary causal role, along with bacterial infection, in bovine post-partum metritis. Mechanisms of maintenance of BoHV-4 persistent infection are not understood. We previously generated in vitro models of BoHV-4 persistent infection in human rhadomyosarcoma and bovine macrophage cell lines by drug selection of cells infected with BoHV-4 carrying a drug-resistance marker, and demonstrated circular episomal BoHV-4 genomes. In the present study, we used fluorescent in situ hybridization (FISH) to demonstrate BoHV-4 genomes also integrated into the genomes of these persistently infected cells.

Host intraspatial selection of infectious bronchitis virus populations.

Arkansas (Ark)-type infectious bronchitis virus (IBV) subpopulations with an S gene sequence distinct from the vaccine predominant consensus were previously found in the upper respiratory tract of chickens within 3 days after inoculation. This finding indicated that a distinct virus subpopulation was rapidly positively selected by the chicken upper respiratory tract. We hypothesized that during host invasion, the replicating IBV population further changes as it confronts the distinct environments of different tissues, leading to selection of the most fit population. We inoculated 15-day-old chickens with 10(4) 50% embryo infective doses of an Ark-type IBV commercial vaccine via the ocular and nasal routes and characterized the sequences of the S1 gene of IBV contained in tear fluid, trachea, and reproductive tract of individual chickens at different times postinoculation. The predominant IBV phenotype contained in the vaccine (before inoculation) became a minor or nondetectable population at all times in all tissues after replication in the majority of the chickens, corroborating our previous findings. Five new predominant populations designated component (C) 1 through C5, showing distinct nonsynonymous changes, i.e., nucleotide changes resulting in different amino acids encoded and thus in a phenotypic change of the predominant virus population, were detected in the tissues or fluids of individual vaccinated chickens. Due to the different biochemical properties of some amino acids that changed in the S1 glycoprotein, we anticipate that phenotypic shift occurred during the invasion process. Significant differences were detected in the incidence of some distinct IBV predominant populations in tissues and fluids; e.g., phenotype C1 showed the highest incidence in the reproductive tract of the chickens, achieving a significant difference versus its incidence in the trachea (P < 0.05). These results indicate for the first time that IBV undergoes intraspatial variation during host invasion, i.e., the dominant genotype/phenotype further changes during host invasion as the microenvironment of distinct tissues exerts selective pressure on the replicating virus population.

Rapid selection in chickens of subpopulations within ArkDPI-derived infectious bronchitis virus vaccines.

We examined spike (S) gene sequences of the virus populations of four different commercial ArkDPI-derived infectious bronchitis coronavirus vaccines before and during a single passage in specific pathogen free chickens. We found different degrees of genetic heterogeneity among the four vaccines before passage in chickens, ranging from no apparent heterogeneity to heterogeneity in 20 positions in the S gene. In all except one position, nucleotide differences were non-synonymous. The majority of amino acid differences were in the S1 subunit of the protein. For three of the four ArkDPI-derived vaccines, a single subpopulation with an S gene sequence distinct from the vaccine majority consensus at 5 to 11 codons was selected in chickens within 3 days after ocular vaccination. In contrast, we obtained no evidence for selection of specific subpopulations of the fourth ArkDPI-derived vaccine or Massachusetts or DE072 serotype vaccines. The virus subpopulations within each vaccine selected by chickens are similar in their S1 gene sequences, but distinct in the 3' portion of the S2 subunit gene for each of the three vaccines. In the S1 gene, the selected subpopulations are more similar to the virulent parental ArkDPI isolate than to the predominant vaccine population. The different proportions of distinct subpopulations in Ark vaccines apparently more fit for replication in the respiratory tract of chickens might cause different degrees of damage to respiratory epithelium and/or immune responses in vaccinated chickens. Sequence comparisons provided no evidence to support that ArkDPI-like field isolates were derived directly from host-selected vaccine virus subpopulations.