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Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log 10 dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%-98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

Infectious bronchitis virus (IBV) is a highly infectious and transmissible gammacoronavirus that is nearly impossible to control through biosecurity. Coronaviruses are RNA viruses with an enormous capacity for rapid replication and high rates of mutation, leading to a tremendous amount of genetic diversity. Viral evolution occurs when selection working on genetic diversity leads to new mutations being fixed in the population over time. For IBV, the emergence of variant viruses is likely due to a combination of selection acting on existing genetic diversity, as well as on newly created mutations as the virus replicates, or genetic drift. Immunity against IBV creates a strong selection pressure; however, immunity can also reduce the viral load, decreasing replication and the development of new mutations. Examining the balance between immunity reducing infection, replication, and genetic diversity, and immune pressure selecting for new variants, is extremely difficult at best. Nonetheless, vaccination and immunity do play a role in the emergence of new antigenic variants of IBV. To complicate the situation even more, coronaviruses can undergo recombination, and several studies in the literature report recombination between IBV vaccines and field viruses. However, to our knowledge, unlike genetic drift, recombination alone has not been shown to result in a new antigenic and pathogenic IBV type emerging to cause widespread disease in poultry. Vaccines against IBV that result in an immune population can reduce transmission (basic reproductive number R 0 less than 1), making vaccines for IBV the best control strategy available. However, IBV control remains extremely challenging because of the high number of antigenic variants causing disease in poultry and a limited number of vaccines that mostly provide only partial protection against infection and replication of those variants. Currently, there is one major variant IBV circulating in all sectors of US commercial poultry production: DMV/1639/11. This virus was initially detected in 2011, but only began causing significant disease in 2014/2015. Since then, it has affected all three sectors of poultry production (layers, breeders, broilers) and continues to predominate in certain regions of the United States. Additionally, a previously classified variant IBV, which is no longer considered a variant virus, GA08, is highly prevalent. This is attributed to heavy GA08-type IBV vaccine usage because disease caused by the GA08-type virus is rare. Interestingly, the major IBV detected in poultry for several decades, ArkDPI, is no longer among the most detected viruses in the United States. This change corresponds to the shift away from ArkDPI vaccine usage in the broiler sector as GA08 vaccine usage has increased and highlights the role IBV vaccines play in influencing viral populations in commercial chickens.

Two or more different live attenuated infectious bronchitis virus (IBV) vaccine types are often given to broilers to induce homologous protection as well as to broaden protection against other IBV types in the field. However, the ability of broilers to respond to three or four different antigenic types of IBV vaccine has not been examined experimentally. In this study, we vaccinated one-day-old broiler chicks by eyedrop with three or four different IBV vaccine types simultaneously. The presence and relative amount of each vaccine was examined in all of the birds by IBV type-specific real-time RT-PCR at 5 days post-vaccination and each vaccine was detected in all of the birds given that vaccine. The birds were challenged at 28 days of age and protection was measured by clinical signs, virus detection and by ciliostasis. Birds vaccinated with three different IBV types (Ark, Mass and GA98) were protected against challenge with each of those IBV types and were partially protected against challenge with the GA08 virus. Birds vaccinated with four different IBV types (Ark, Mass, GA98 and GA08) were protected against challenge with each of those IBV types with the exception of Mass challenged birds which clearly had 3/11 birds not protected based on individual ciliostasis scores, but had an average ciliostasis score of >50% which is considered protected. The results are important for the control of IBV because they indicate that simultaneous vaccination with up to four different IBV vaccine types can provide adequate protection against challenge for each type.

Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log 10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Commercial broilers are commonly exposed to gaseous ammonia (NH 3 ) originating from degradation of nitrogen-containing excreta in the litter during the grow-out period. Ammonia concentrations in the air are higher in poorly ventilated houses and appear to coincide with the elevated incidence of respiratory disease occurring during the winter months. This study examined the effect of NH 3 on the immune response to infectious bronchitis virus (IBV) vaccination and protection against homologous serotype challenge in commercial broiler chickens. One-day-old chicks were administered IBV vaccine and exposed to 30-60 ppm of NH 3 . At 28 DOA, birds were challenged oculonasally with a pathogenic homologous IBV, and protection was measured by viral detection, clinical signs, ciliostasis, and presence of airsacculitis. IBV-specific serum IgG and lacrimal fluid IgA titers, as well as Harderian gland (HG) immune cell phenotypes, were evaluated. Ammonia exposure was associated with an increased incidence of airsacculitis among non-vaccinated, challenged birds. Vaccinated, NH 3 -exposed birds were completely protected from IBV challenge. Ammonia had subtle effects on cilia morphology and function but did not affect vaccine or challenge virus replication and clearance, clinical signs, ciliostasis, tracheal histopathology scores, or immune responses. In the HG of vaccinated birds, the percent of leukocytes, MHC I + /MHC II hi expression, IgM + expression, and CD8 + expression was increased, while mucosal IgA and serum IgG titers were nominal. Non-vaccinated, IBV-challenged birds exhibited an increased percent of leukocytes, MHC I + /MHC II hi expression, and IgM + expression in the HG at 5 dpc, followed by increased mucosal IgA and serum IgG titers and CD8 + expression at 10-14 dpc. In contrast, vaccinated, IBV-challenged birds had a minimal increase in MHC I + /MHC II hi expression, and serum IgG antibody titers in vaccinated birds increased rapidly. The results indicate that commercial broilers exposed to moderate levels of ambient NH 3 are equally protected against IBV challenge if appropriately vaccinated, and the absence of robust immune activation in vaccinated, challenged birds suggests that the challenge virus was efficiently neutralized before establishing infection. In contrast, ambient NH 3 exposure was associated with a higher incidence of airsacculitis in non-vaccinated, challenged birds, despite the apparent lack of differences in the immune response between birds in the NH 3 -exposed and NH 3 control groups., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

During summer and early fall of 2012, the US experienced the largest outbreak of hemorrhagic disease (HD) on record; deer (both Odocoileus virginianus and Odocoileus hemionus) in 35 states were affected, including many northern states where HD typically does not occur. Epizootic hemorrhagic disease virus (EHDV) was the predominant virus isolated, with serotype 2 (EHDV-2) representing 66% (135/205) of all isolated viruses. Viruses within the EHDV serogroup are genetically similar, but we hypothesized that subtle genetic distinctions between viruses would exist across the geographic range of the outbreak if multiple EHDV-2 strains were responsible. We examined viral relatedness and molecular epidemiology of the outbreak by sequencing the mammalian binding protein (VP2) gene and the insect vector binding protein (VP7) gene of 34 EHDV-2 isolates from 2012 across 21 states. Nucleotide sequences of VP2 had 99.0% pairwise identity; VP7 nucleotide sequences had 99.1% pairwise identity. Very few changes were observed in either protein at the amino acid level. Despite the high genetic similarity between isolates, subtle nucleotide differences existed. Both VP2 and VP7 gene sequences separated into two distinct clades based on patterns of single-nucleotide polymorphisms after phylogenetic analysis. The clades were divided geographically into eastern and western clades, although those divisions were not identical between VP2 and VP7. There was also an association between percent sequence identity and geographic distance between isolates. We concluded that multiple EHDV-2 strains contributed to this outbreak.

Avian respiratory disease causes significant economic losses in commercial poultry. Because of the need to protect long-lived poultry against respiratory tract pathogens from an early age, vaccination programs for pullets typically involve serial administration of a variety of vaccines, including infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and infectious laryngotracheitis virus (ILTV). Often the interval between vaccinations is only a matter of weeks, yet it is unknown whether the development of immunity and protection against challenge when vaccines are given in short succession occurs in these birds, something known as viral interference. Our objective was to determine whether serially administered, live attenuated vaccines against IBV, NDV, and ILTV influence the development and longevity of immunity and protection against challenge in long-lived birds. Based on a typical pullet vaccination program, specific-pathogen-free white leghorns were administered multiple live attenuated vaccines against IBV, NDV, and ILTV until 16 weeks of age (WOA), after which certain groups were challenged with IBV, NDV, or ILTV at 20, 24, 28, 32, and 36 WOA. Five days post-challenge, viral load, clinical signs, ciliostasis, tracheal histopathology, and antibody titers in serum and tears were evaluated. We demonstrate that pullets serially administered live attenuated vaccines against IBV, NDV, and ILTV were protected against homologous challenge with IBV, NDV, or ILTV for at least 36 weeks, and conclude that the interval between vaccinations used in this study (at least 2 weeks) did not interfere with protection. This information is important because it shows that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent.

Almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (IBV) using live attenuated vaccines mass administered by spray at day of hatch. Although many different types of IBV vaccines are used successfully, the ArkDPI serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. In this study, we examined a different Ark vaccine strain (Ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. Further attenuation of the Ark99 vaccine was achieved by passage in embryonated eggs but ArkGA P1, P20, and P40 (designated ArkGA after P1) were still too reactive to be suitable vaccine candidates. However, ArkGA P60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. In addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. The full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the Illumina platform and the data showed single nucleotide polymorphisms (SNPs) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. ArkGA P60 accumulated the most SNPs in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. These results indicate that the ArkGA P60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic Ark-type virus., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To determine the genetic and epidemiological relationship of infectious bronchitis virus (IBV) isolates from commercial poultry to attenuated live IBV vaccines we conducted a phylogenetic network analysis on the full-length S1 sequence for Arkansas (Ark), Massachusetts (Mass) and Delmarva/1639 (DMV/1639) type viruses isolated in 2015 from clinical cases by 3 different diagnostic laboratories. Phylogenetic network analysis of Ark isolates showed two predominant groups linked by 2 mutations, consistent with subpopulations found in commercial vaccines for this IBV type. In addition, a number of satellite groups surrounding the two predominant populations were observed for the Ark type virus, which is likely due to mutations associated with the nature of this vaccine to persist in flocks. The phylogenetic network analysis of Mass-type viruses shows two groupings corresponding to different manufacturers vaccine sequences. No satellite groups were observed for Mass-type viruses, which is consistent with no persistence of this vaccine type in the field. At the time of collection, no vaccine was being used for the DMV/1639 type viruses and phylogenetic network analysis showed a dispersed network suggesting no clear change in genetic distribution. Selection pressure analysis showed that the DMV/1639 and Mass-type strains were evolving under negative selection, whereas the Ark type viruses had evolved under positive selection. This data supports the hypothesis that live attenuated vaccine usage does play a role in the genetic profile of similar IB viruses in the field and phylogenetic network analysis can be used to identify vaccine and vaccine origin isolates, which is important for our understanding of the role live vaccines play in the evolutionary trajectory of those viruses.

The Arkansas Delmarva Poultry Industry (ArkDPI) infectious bronchitis virus (IBV) vaccine is effective when administered by eye drop, where the vaccine virus is able to infect and replicate well in birds and is able to induce protection against homologous challenge. However, accumulating evidence indicates that the ArkDPI vaccine is ineffective when applied by hatchery spray cabinet using the same manufacturer-recommended dose per bird. For this study, we aimed to determine the minimum infectious dose for the spray-administered ArkDPI vaccine, which we designate as the dose that achieves the same level of infection and replication as the eye drop-administered ArkDPI vaccine. To this end, we used increasing doses of commercial ArkDPI vaccine to vaccinate 100 commercial broiler chicks at day of hatch, using a commercial hatchery spray cabinet. The choanal cleft of each bird was swabbed at 7 and 10 days postvaccination, and real-time reverse-transcriptase PCR was performed. We observed that the level of infection and replication with spray vaccination matches with that of eye drop vaccination when chicks received 100 times the standard dose for the commercial ArkDPI vaccine. We further examined the S1 spike gene sequence from a subset of reisolated ArkDPI vaccine virus samples and observed that certain nucleotide changes arise in vaccine viruses reisolated from chicks, as previously reported. This suggests that the ArkDPI vaccine has a certain virus subpopulation that, while successful at infecting and replicating in chicks, represents only a minor virus subpopulation in the original vaccine. Thus, the minimum infectious dose for the ArkDPI vaccine using a hatchery spray cabinet appears to be dependent on the amount of this minor subpopulation reaching the chicks.

Infectious bronchitis virus is an important respiratory pathogen in chickens. The IBV S1 spike is a viral structural protein that is responsible for attachment to host receptors and is a major target for neutralizing antibodies. To date, there is no experimentally determined structure for the IBV S1 spike. In this study, we sought to find a predicted tertiary structure for IBV S1 using I-TASSER, which is an automated homology modeling platform. We found that the predicted structures obtained were robust and consistent with experimental data. For instance, we observed that all four residues (38, 43, 63, and 68) that have been shown to be critical for binding to host tissues, were found at the surface of the predicted structure of Massachusetts (Mass) S1 spike. Together with antigenicity index analysis, we were also able to show that Ma5 vaccine has higher antigenicity indices at residues close to the receptor-binding region than M41 vaccine, thereby providing a possible mechanism on how Ma5 achieves better protection against challenge. Examination of the predicted structure of the Arkansas IBV S1 spike also gave insights on the effect of polymorphisms at position 43 on the surface availability of receptor binding residues. This study showcases advancements in protein structure prediction and contributes useful, inexpensive tools to provide insights into the biology of IBV., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Infectious bronchitis virus (IBV) is the causative agent of a highly contagious disease that results in severe economic losses to the global poultry industry. The virus exists in a wide variety of genetically distinct viral types, and both phylogenetic analysis and measures of pairwise similarity among nucleotide or amino acid sequences have been used to classify IBV strains. However, there is currently no consensus on the method by which IBV sequences should be compared, and heterogeneous genetic group designations that are inconsistent with phylogenetic history have been adopted, leading to the confusing coexistence of multiple genotyping schemes. Herein, we propose a simple and repeatable phylogeny-based classification system combined with an unambiguous and rationale lineage nomenclature for the assignment of IBV strains. By using complete nucleotide sequences of the S1 gene we determined the phylogenetic structure of IBV, which in turn allowed us to define 6 genotypes that together comprise 32 distinct viral lineages and a number of inter-lineage recombinants. Because of extensive rate variation among IBVs, we suggest that the inference of phylogenetic relationships alone represents a more appropriate criterion for sequence classification than pairwise sequence comparisons. The adoption of an internationally accepted viral nomenclature is crucial for future studies of IBV epidemiology and evolution, and the classification scheme presented here can be updated and revised novel S1 sequences should become available., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, we examined the association among clinical signs, ciliostasis, virus detection, and histopathology for evaluating protection of vaccinated chickens against homologous and heterologous infectious bronchitis virus (IBV) challenge. At 5 days following challenge with IBV, we found a good correlation among clinical signs, ciliostasis in the trachea, challenge virus detection, and microscopic lesions in the trachea, with all four criteria being negative in fully protected birds and positive in fully susceptible birds. In partially protected birds we observed clinical signs and detected challenge virus; however, the ciliated epithelium was intact. In a second experiment, we challenged fully protected, partially protected, and fully susceptible birds with IBV, and then at 5 days postchallenge we gave the birds an opportunistic bacterium intranasally. Twenty Bordetella avium colonies were recovered from one of five fully protected birds, and only five colonies were isolated from two of five partially protected birds without ciliostasis, whereas in birds with ciliostasis, numerous colonies were isolated. Obviously, decreasing IBV infection and replication in the upper respiratory tract will decrease transmission and mutations, leading to variant viruses, and herein we demonstrate that protection of the cilia will decrease secondary bacterial infections, which have been shown to lead to condemnations and increased mortality. Thus, it appears that examining both criteria would be important when evaluating IBV vaccine efficacy.

studies in our laboratory showed that the Arkansas-Delmarva Poultry Industry (Ark-DPI) vaccine given to 1-day-old chickens by hatchery spray cabinet replicated poorly and failed to adequately protect broilers against homologous virus challenge, whereas the same vaccine given by eye-drop did replicate and the birds were protected following homologous virus challenge. To determine if mechanical damage following spray application plays a role in failure of the Ark-DPI vaccine, we examined the morphology of three Ark-DPI vaccines from different manufacturers using an electron microscope and included a Massachusetts (Mass) vaccine as control. One of the Ark-DPI vaccines (vaccine A) and the Mass vaccine had significantly (P < 0.005) fewer spikes than the other two Ark-DPI vaccines. We also found that the Ark-DPI and Mass vaccines had significantly (P < 0.005) fewer spike proteins per virus particle when compared to their respective challenge viruses. This observation is interesting and may provide some insight into the mechanism behind infectious bronchitis virus attenuation. No obvious differences were observed in virus morphology and no consistent trend in the number of spikes per virion was found in before- and after-spray samples. We also determined the vaccine titer before and after spray in embryonated eggs and found that both Ark-DPI and Mass vaccines had a similar drop in titer, 0.40 logi and 0.310 logi, respec10ively. Based on these data, it appears that mechanical damage to the Ark-DPI vaccine is not occurring when delivered by a hatchery spray cabinet, suggesting that some other factor is contributing to the failure of that vaccine when given by that method.

Coronaviruses (CoVs) are worldwide in distribution, highly infectious, and difficult to control because of their extensive genetic diversity, short generation time, and high mutation rates. Genetically diverse CoVs have been reported from wild aquatic birds that may represent a potential reservoir for avian CoVs as well as hosts for mutations and recombination events leading to new serotypes or genera. We tested 133 pooled samples representing 700 first-passage (in eggs) and 303 direct cloacal swab transport media samples from wild aquatic birds in the US that were avian influenza-negative. We isolated RNA from frozen samples and performed reverse transcriptase-PCR using a published universal CoV primer set. Of the samples tested, one from a Ruddy Turnstone (Arenaria interpres) was positive for CoV, showing nucleotide sequence similarity to a duck coronavirus (DK/CH/HN/ZZ2004). These data indicate a possible low prevalence of CoVs circulating in wild aquatic birds in the eastern half of the US.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Feline infectious peritonitis virus (FIPV), an alpha Coronavirus, is the causative agent of a fatal immune mediated disease in cats. It is currently unclear if this virus circulates in the field or develops in felines that are infected with Feline enteric coronavirus. To better understand the genomic changes associated with viral adaptation, we sequenced the complete genomes of FIPV WSU 79-1146 at different tissue passage levels: passage 1, passage 8, and passage 50 tissue culture. Twenty-one amino acid differences were observed in the polyprotein 1a/ab between the different passages. Only one residue change was observed in the spike glycoprotein, which reverted back on subsequent passages, four changes were observed in the 3c protein, and one change was observed in each 3a, small membrane, nucleocapsid and 7a proteins. The mutation rate was calculated to be 5.08-6.3 × 10(-6) nucleotides/site/passage in tissue culture suggesting a relatively stable virus. Our data show that FIPV has a low mutation rate as it is passed in cell culture but has the capacity for change specifically in nsp 2, 3c, and 7b as it is passed in cell culture.

Avian coronavirus (commonly known as Infectious bronchitis virus [IBV]) is of major economic importance to commercial chicken producers worldwide. Due to the existence of multiple serotypes and variants of the virus that do not cross-protect, it is important to diagnose circulating serotypes and choose the right vaccine type for successful protection. In an effort to improve conventional diagnostic tests, a microsphere-based assay was developed and evaluated for simultaneous detection of the most common IBV vaccine serotypes in the United States: Arkansas (Ark), Connecticut (Conn), Massachusetts (Mass), Delaware (DE072), and Georgia 98 (GA98). The analytical specificity and sensitivity, and diagnostic specificity and sensitivity, were evaluated. The microsphere-based assay was highly specific to designated serotypes and generated reproducible data. Comparing the microsphere-based assay to nucleotide sequencing, the 2 methods agreed more than 93% (kappa value > .77). In addition, the microsphere-based assay could detect coinfections in clinical samples. The results demonstrate the utility of the microsphere-based assay as a rapid and accurate diagnostic tool with the potential for high throughput diagnosis.

Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Infectious bronchitis virus (IBV) is a gamma coronavirus that causes a highly contagious disease in chickens. The virus can affect the upper respiratory tract and the reproductive tract, and some strains can cause a nephritis. Different serotypes and genetic types of the virus have been identified worldwide and for the most part do not cross-protect. In addition, new types of the virus continue to arise due to mutations and recombination events in the viral genome, making this virus difficult to identify and extremely difficult to control. Surveillance and identification of IBV types is extremely important for control of the disease and the advancement of molecular methods have aided in this pursuit. Genetic typing of IBV, which involves reverse transcription-PCR amplification and sequence analysis of the S1 glycoprotein gene, has revolutionized diagnosis and identification of this virus by making it possible to type and compare the relatedness of a large number of virus isolates in a short period of time. The purpose of this review is to give an update on the strains of IBV currently circulating in commercial chickens worldwide and hopefully to present a clear picture of the relationship between many of these viruses. The information on IBV types presented herein is from published manuscripts, submissions to GenBank, our own unpublished data, and personal communications with scientists and diagnosticians working with IBV worldwide.

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.

Coronaviruses, which are single stranded, positive sense RNA viruses, are responsible for a wide variety of existing and emerging diseases in humans and other animals. The gammacoronaviruses primarily infect avian hosts. Within this genus of coronaviruses, the avian coronavirus infectious bronchitis virus (IBV) causes a highly infectious upper-respiratory tract disease in commercial poultry. IBV shows rapid evolution in chickens, frequently producing new antigenic types, which adds to the multiple serotypes of the virus that do not cross protect. Rapid evolution in IBV is facilitated by strong selection, large population sizes and high genetic diversity within hosts, and transmission bottlenecks between hosts. Genetic diversity within a host arises primarily by mutation, which includes substitutions, insertions and deletions. Mutations are caused both by the high error rate, and limited proof reading capability, of the viral RNA-dependent RNA-polymerase, and by recombination. Recombination also generates new haplotype diversity by recombining existing variants. Rapid evolution of avian coronavirus IBV makes this virus extremely difficult to diagnose and control, but also makes it an excellent model system to study viral genetic diversity and the mechanisms behind the emergence of coronaviruses in their natural host., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n = 6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anasplatyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 x 10(4.7) median embryo infectious dose (EID50)/ml (range: 1 x 10(4.3) to 1 x 10(5.4) EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

Infectious bronchitis virus (IBV) Cal99 variant was isolated from the kidneys of seven 2-5-mo-old game chickens with nephritis and respiratory disease. IBV Cal99 variant is usually associated with respiratory disease in broiler chickens in California. Macroscopically, the majority of the birds had moderately to severely enlarged and mottled pale kidneys, with increased urates in the ureters. Microscopically, most of the birds had acute nephrosis and interstitial nephritis. The birds also had sinusitis, tracheitis, bronchopneumonia, airsacculitis, salivary gland adenitis, and lymphoid depletion in the thymus and bursa of Fabricius. Immunohistochemistry was strongly positive for IBV antigen in the cytoplasm of tubular epithelial cells in the kidneys and also in the epithelium of the respiratory tract, salivary glands, proventriculus, intestine, and bursa of Fabricius. Infectious bronchitis virus was isolated from the trachea, lungs, kidneys, and cecal tonsils. Sequencing of the hypervariable region of the S1 gene of the kidney IBV isolate, designated IBV/CA99variant/07, revealed that the virus was 98% homologous to the Cal99 serotype of IBV.

The full-length genomes of 11 infectious bronchitis virus (IBV) field isolates from three different types of the virus; Massachusetts (Mass), Connecticut (Conn) and California (CAL) isolated over a 41, 25 and 8 year period respectively, were sequenced and analyzed to determine the mutation rates and level of polymorphisms across the genome. Positive selection was not detected and mutation rates ranged from 10(-4) to 10(-6)substitutions/site/year for Mass and Conn IBV types where attenuated live vaccines are routinely used to control the disease. In contrast, for CAL type viruses, for which no vaccine exists, positive selection was detected and mutation rates were 10 fold higher ranging from 10(-2) to 10(-3)substitutions/site/year. Lower levels of genetic diversity among the Mass and Conn viruses as well as sequence similarities with vaccine virus genomes suggest that the origin of the Mass and all but one of the Conn viruses was likely vaccine virus that had been circulating in the field for an unknown but apparently short period of time. The genetic data also identified a recombinant IBV isolate with 7 breakpoints distributed across the entire genome suggesting that viruses within the same serotype can have a high degree of genetic variability outside of the spike gene. These data are important because inaccurate measures of genetic diversity and mutation rates could lead to underestimates of the ability of IBV to change and potentially emerge to cause disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The main aims of the present study were to characterize NS1 protein from H9N2 avian influenza viruses (AIVs) isolated in Israel and to investigate the possibility to use NS1-based indirect ELISA. To achieve these purposes, the non-structural gene (NS1) of 79 AIVs of the H9N2 subtype isolated in Israel in 2000-2009 was sequenced and genetically analyzed. The phylogenetic analysis demonstrated that four distinct introductions of H9N2 occurred in Israel during this period. Analysis of the inferred amino acid sequences of the NS1 proteins showed high, about 10%, differences between viruses of the 3rd and 4th introductions. Antibodies against NS1 protein in immune sera were tested by means of indirect ELISA using recombinant NS1 as antigen. Immune sera were obtained from experimentally H9N2-infected chicken after infection on 4, 7, 10, 14, and 21 days. All sera from chickens experimentally infected with 3rd- or 4th-introduction AIV contained anti-NS1 antibodies that were detected by enzyme-linked immunosorbent assay (NS1-ELISA) even though the recombinant NS1 used as antigen for NS1-ELISA differed significantly in its amino acid sequences from the NS1 protein of AIV that caused infection in experimental birds. These findings indicate that the sites of the NS1 protein by which viruses belonging to 3rd and 4th introduction are out of antigenic epitope positions were responsible for the results of NS1-based iELISA.

In the present study we describe the rapid development of an attenuated live vaccine for GA08, a new serotype of infectious bronchitis virus, using a heat-treatment method. Incubation of the GA08 strain of IBV at 56 degrees C and passage in embryonated eggs was used as a method to fast track the attenuation process. The virus was incubated in a 56 degrees C water bath and aliquots were removed every 5 min for up to 1 h, and then each aliquot was inoculated into 10-day-old embryonated eggs. Virus with the longest incubation time that produced lesions in the embryos was harvested, again incubated at 56 degrees C as described and passaged in embryonated eggs. Attenuation of the virus, designated GA08/GA08HSp16/08, was verified in 1-day-old specific pathogen free chicks. A 10x dose of the vaccine was found to be safe for 2-week-old broiler chicks of commercial origin. The efficacy of the heat-treated attenuated virus was determined by vaccinating broiler chicks of commercial origin at 1 and 14 days of age intraocularly/intranasally. Vaccinated birds that were challenged with 10(4.5) median embryo infectious doses of pathogenic GA08 virus/bird at 28 days of age were protected from the disease, and challenge virus was only detected in the trachea of one of 21 birds by real-time reverse transcriptase-polymerase chain reaction at 5 days post challenge. The attenuation process took 10 weeks to complete, which is a substantially shorter time than required to attenuate infectious bronchitis virus by serial passage in embryonated eggs without heat treatment (38 weeks or more).

Anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated QR448(a), was examined in vitro and in vivo. Treatment of avian infectious bronchitis virus (IBV) with QR448(a) reduced the virus titer as measured in two laboratory host systems, Vero E6 cells and embryonating eggs. The effect of QR448(a) on IBV in chickens was also investigated. Administering QR448(a) to chickens at a 1:20 dilution by spray, 2h before challenge with IBV was determined to be the most effective treatment. Treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral RNA in the trachea. Treatment with QR448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of IBV over a 14-day period. Anti-IBV activity of QR448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. In addition, QR448(a) had activity against both Massachusetts and Arkansas type IB viruses, indicating that it can be expected to be effective against IBV regardless of serotype. To our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus IBV., (Copyright 2010 Elsevier B.V. All rights reserved.)

Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses., (Copyright 2009 Elsevier Inc. All rights reserved.)

Low pathogenic avian influenza H6N2 viruses were biologically characterized by infecting chickens and ducks in order to compare adaptation of these viruses in these species. We examined the clinical signs, virus shedding, and immune response to infection in 4-wk-old white leghorn chickens and in 2-wk-old Pekin ducks. Five H6N2 viruses isolated between 2000 and 2004 from chickens in California, and one H6N2 virus isolated from chickens in New York in 1998, were given intrachoanally at a dose of 1 x 10(6) 50% embryo infectious dose per bird. Oral-pharyngeal and cloacal swabs were taken at 2, 4, and 7 days postinoculation (PI) and tested by real-time reverse-transcriptase polymerase chain reaction for presence of virus. Serum was collected at 7, 14, and 21 days PI and examined for avian influenza virus antibodies by commercial enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) testing. Virus shedding for all of the viruses was detected in the oral-pharyngeal swabs from chickens at 2 and 4 days PI, but only three of the five viruses were detected at 7 days PI. Only two viruses were detected in the cloacal swabs from the chickens. Virus shedding for four of the five viruses was detected in the oral-pharyngeal cavity of the ducks, and fecal shedding was detected for three of the viruses (including the virus not shed by the oral-pharyngeal route) in ducks at 4 and 7 days PI. All other fecal swabs from the ducks were negative. Fewer ducks shed virus compared to chickens. Both the chickens and the ducks developed antibodies, as evidenced by HI and ELISA titers. The data indicate that the H6N2 viruses can infect both chickens and ducks, but based on the number of birds shedding virus and on histopathology, the viruses appear to be more adapted to chickens. Virus shedding, which could go unnoticed in the absence of clinical signs in commercial chickens, can lead to transmission of the virus among poultry. However, the viruses isolated in 2004 did not appear to replicate or cause more disease than earlier virus isolates.

The pathogenesis, virus shedding, and serologic response in specific-pathogen-free (SPF) chickens and commercial turkeys against H4, H6, and H9 type low pathogenic avian influenza viruses (LPAI) from wild birds was examined. Four-week-old chickens and three-week-old turkeys were given 1 x 10(6) EID50 of LPAI per bird, intrachoanally, and examined for clinical signs for 3 wk. Oropharyngeal and cloacal swabs, and fecal samples, were collected at 2, 4, and 7 days postinoculation (PI) for virus detection by real-time RT-PCR. Serum was collected at 7, 14, and 21 days PI and examined for antibodies against avian influenza virus (AIV) by the enzyme-linked immunosorbant assay (ELISA) and hemagglutination inhibition tests. Tissue samples for histopathology were collected from three birds per group at 3 days PI. The hemagglutinin genes of the viruses were sequenced, and phylogenetic analysis was conducted. Clinical signs ranged from no clinical signs to moderate depression, decreased activity, and decreased food and water consumption. Based on virus detection results, SPF chickens were generally found to be shedding more virus from both the oropharynx and cloaca than were commercial turkeys. Microscopic lesion results in both species showed the predominance of lesions in the respiratory and gastrointestinal tract, which is consistent with the fact that these viruses are of low pathogenicity. In chickens and turkeys, oropharyngeal shedding strongly correlated with the lesions found in the upper respiratory tract. Turkeys had fewer lesions in the respiratory tract and more lesions in the gastrointestinal tract compared to chickens. Thirteen LPAI viruses caused seroconversion in commercial turkeys, whereas only 6 LPAI viruses caused seroconversion in SPF chickens. Phylogenetic analysis of the HA genes showed that the H4, H6, and H9 viruses evaluated here represented the full genetic diversity of North American AIVs of their respective subtypes. This data is important for surveillance and control because some of the LPAI viruses (of wild bird origin and examined in this study) that can infect and be shed by chickens and turkeys would be difficult to detect in commercial poultry. Specifically, detection is difficult because these viruses did not cause overt clinical disease or mortality, but only induced mild microscopic lesions and exhibited poor seroconversion.

In this study, the sequence of the H5 and PB1 genes of the low-pathogenic avian influenza virus (LPAI) A/Black Duck/NC/674-964/06 isolate were determined for replikin peptides and used to design and chemically synthesize a vaccine. The vaccine was used to immunize specific-pathogen-free (SPF) leghorn chickens held in Horsfall isolation units, by the upper respiratory route, at 1, 7, and 14 days of age. The birds were challenged at 28 days of age with 1 x 10(6) 50% embryo infective dose of the LPAI Black Duck/NC/674-964/06 H5N1 virus per bird. Oropharyngeal and cloacal swabs were collected at 2, 4, and 7 days postinoculation (PI) for virus detection by real-time RT-PCR. Serum was collected at 7, 14, and 21 days PI and examined for antibodies against avian influenza virus by the enzyme-linked immunosorbent assay and hemagglutination inhibition (HI) tests. Tissue samples for histopathology were collected from three birds per group at 3 days PI. The experimental design consisted of a negative control group (not vaccinated and not challenged) and a vaccinated group, a vaccinated and challenged group, and a positive control group (challenged only). None of the nonchallenged birds, the vaccinated birds, or the vaccinated and challenged birds showed overt clinical signs of disease during the study. A slight depression was observed in the nonvaccinated challenged birds on day 2 postchallenge. Although the numbers of birds per group are small, no shedding of the challenge virus was detected in the vaccinated and challenged birds, whereas oropharyngeal and cloacal shedding was detected in the nonvaccinated and challenged birds. HI antibodies were detected in the vaccinated and nonchallenged group as well as in the vaccinated and challenged group, but rising antibody titers, indicating infection with the LPAI challenge virus, were not detected. Rising HI titers were observed in the nonvaccinated and challenged group. In addition, no antibodies were detected in the nonchallenged birds. Noteworthy microscopic lesions were not observed in the vaccinated and challenged birds, whereas nonvaccinated-challenged birds had microscopic lesions consistent with infection with LPAI viruses. Taken together, these data indicate that a replikin peptide vaccine, specifically made against the H5N1 Black Duck/NC/674-964/06 isolate, and administered three times to the upper respiratory tract, was capable of protecting chickens from infection and from shedding of the homologous virus, which is extremely important because reduced virus shedding and transmission decreases the potential for H5 LPAI viruses to become HPAI viruses. The study is also important because it shows that the vaccine can be effectively mass-delivered to the upper respiratory tract.

To determine the coverage of infectious bronchitis virus (IBV) vaccine field boost in commercial broilers, estimate the relative amount of vaccine virus in the trachea, and follow the clearance of the vaccine, we collected approximately 100 tracheal swabs at various times postvaccination from 10 different flocks and used real-time reverse transcriptase-PCR (RT-PCR) to detect the virus. This allowed us to detect vaccine virus in as few as 3% of the birds in a flock of 20,000 birds with a 95% confidence level. We found that the number of birds positive for IBV vaccine following vaccination in the field resembled a parabolic-shaped curve that peaked around 14 days postvaccination, or it resembled a sinusoidal-type wave with a frequency of about 2 wk. The patterns did not appear to correlate with water or spray vaccination methods, nor did they correlate with the type of backpack sprayer used. The highest number of positive birds in a flock ranged from 66% to 100%. The viral genome copies in the tracheal swabs, as determined by real-time RT-PCR, ranged from 1 x 10(2.6)/ml to 1 x 10(5.2)/ml and, in most studies, had a positive correlation with the number of birds positive for vaccine virus in the flock. On the last sample day of each study, 21, 28, or 35 days postvaccination, from 12% to 66% of the birds were still positive for vaccine virus, and although different IBV vaccine types were used in each study, only Arkansas vaccine virus was identified in selected samples on those days. Arkansas vaccine virus was also the only virus identified in selected samples at 1, 3, and 5 days postvaccination, clearly indicating that Arkansas vaccine virus is persisting in the birds. Protection studies conducted on birds vaccinated with Arkansas- and Delaware-type vaccines and removed from the field at 21 days postvaccination showed complete protection against challenge with Delaware (except for one bird), whereas protection against Arkansas challenge was between 37.5% and 62.5%. Our findings show that introduction of IBV vaccines into a commercial broiler flock do not necessarily follow a seemingly logical pattern of a high number of birds infected followed by clearance from the trachea, but resembled either a parabolic curve or a sinusoidal-type wave. In addition, Arkansas vaccine viruses are clearly persisting in commercial broilers, which may be because of incomplete protection observed for that IBV type.

Intestinal samples collected from 43 commercial broiler and 33 commercial turkey flocks from all regions of the United States during 2005 and 2006 were examined for the presence of astrovirus, rotavirus, reovirus, and coronavirus by reverse transcription-polymerase chain reaction (PCR), and for the presence of groups 1 and 2 adenovirus by PCR. Phylogenetic analysis was performed to further characterize the viruses and to evaluate species association and geographic patterns. Astroviruses were identified in samples from 86% of the chicken flocks and from 100% of the turkey flocks. Both chicken astrovirus and avian nephritis virus (ANV) were identified in chicken samples, and often both viruses were detected in the same flock. Turkey astrovirus type-2 and turkey astrovirus type-1 were found in 100% and 15.4% of the turkey flocks, respectively. In addition, 12.5% of turkey flocks were positive for ANV. Rotaviruses were present in 46.5% of the chicken flocks tested and in 69.7% of the turkey flocks tested. Based upon the rotavirus NSP4 gene sequence, the chicken and turkey origin rotaviruses assorted in a species-specific manner. The turkey origin rotaviruses also assorted based upon geographical location. Reoviruses were identified in 62.8% and 45.5% of chicken and turkey flocks, respectively. Based on the reovirus S4 gene segment, the chicken and turkey origin viruses assorted separately, and they were distinct from all previously reported avian reoviruses. Coronaviruses were detected in the intestinal contents of chickens, but not turkeys. Adenoviruses were not detected in any chicken or turkeys flocks. Of the 76 total chicken and turkey flocks tested, only three chicken flocks were negative for all viruses targeted by this study. Most flocks were positive for two or more of the viruses, and overall no clear pattern of virus geographic distribution was evident. This study provides updated enteric virus prevalence data for the United States using molecular methods, and it reinforces that enteric viruses are widespread in poultry throughout the United States, although the clinical importance of most of these viruses remains unclear.

In this study, we were interested in determining if high titered egg adapted modified live infectious bronchitis virus (IBV) vaccines contain spike gene related quasispecies that undergo selection in chickens, following vaccination. We sequenced the spike glycoprotein of 12 IBV vaccines (5 different serotypes from 3 different manufacturers) directly from the vaccine vial, then compared that sequence with reisolated viruses from vaccinated and contact-exposed birds over time. We found differences in the S1 sequence within the same vaccine serotype from different manufacturers, differences in S1 sequence between different vaccine serials from the same manufacturer, and intra-vaccine differences or quasispecies. Comparing the sequence data of the reisolated viruses with the original vaccine virus, we were able to identify in vivo selection of viral subpopulations as well as mutations. To our knowledge, this is the first report showing selection of a more fit virus subpopulation as well as mutations associated with replication of modified live IBV vaccine viruses in chickens. This information is important for our understanding of how attenuated virus vaccines, including potential vaccines against the SARS-CoV, can ensure long-term survival of the virus and can lead to changes in pathogenesis and emergence of new viral pathogens. This information is also valuable for the development of safer modified live coronavirus vaccines.

Avian leukosis subgroup J (ALV-J) causes a variety of tumors and mortality in meat-type chickens. Since its discovery in the late 1980s, ALV-J has spread to breeding stock produced by most primary breeding companies of North America, the European Union, and Asia. ALV-J seems to have been eradicated from elite breeding stock produced by most primary breeders, albeit ALV-J still circulates in some commercial poultry. This study was undertaken to examine the molecular epidemiology and evolution of ALV-J detected in breeding stock and broiler chickens representing eight primary breeding companies over a period of approximately 20 yr (1988-2007). The redundant transmembrane region of the envelope gene has been deleted in some isolates, suggesting that this region is dispensable for viral fitness. Within the 3' untranslated region (3' UTR), the direct repeat 1 was present in 100% of the ALV-J isolates studied. In contrast, the E element has undergone substantial deletions in >50% of the ALV-J proviruses studied. Overall, the unique region 3 was the least conserved within the 3' long terminal repeat (LTR), albeit the transcriptional regulatory elements typical of avian retroviruses (CAAT, CArG, PRE, TATA, and Y boxes) were highly conserved. The direct repeat region of the LTR was identical in all of the proviruses, and the 3' unique region 5 was relatively well conserved. Thus, the 3' UTR of ALV-J has evolved rapidly, reflecting significant instability of this region. Some of the mutations in the 3' UTR have resulted in the emergence of moderately distinct genetic lineages representing each primary breeding company from which ALV-J was isolated.

In this study, we characterized three variant infectious bronchitis virus (IBV) strains isolated in 2003 and 2004 from broiler chickens in California and compared them to previously isolated California variant viruses and to common vaccine serotypes used in the United States. We conducted genetic, serologic, and pathogenicity studies on all three isolates, then tested different vaccines against one of the viruses. Genetically the three variant IBV strains, designated CA557/03, CA706/03, and CA1737/04, were not related to each other. GenBank BLAST database search and phylogenetic analysis of the hypervariable region of the S1 subunit of the spike gene to determine the most closely related viruses to the three variants showed the CA557/03 variant to be 81.8% similar to the CAV/CA56b/91 whereas the CA706/03 and CA1737/04 variant viruses were only distantly related to Dutch/D1466/81 (72.2%), a vaccine strain used in Europe, and Korea/K142/02 (72.7%), a Korean field isolate, respectively. Cross virus-neutralization testing showed that none of the 2003-04 California IBV variant viruses were serologically related to each other or to Ark, Conn, or Mass vaccine strains. In addition the CA1737/04 isolate was also tested against DE072 and found not to be serologically related. All three variant viruses were pathogenic in 1-wk-old broilers and vaccination with Mass/Conn followed by Holland/Conn provided 80% protection against the CA1737/04 virus. The 2003-04 California variant viruses were not compared with variants isolated in California during 1970s and 1980s because, to our knowledge, no genetic information is available and those viruses are no longer obtainable. This study shows that the CA557/03 virus was distantly related to the CAV-type viruses isolated in California in the early 1990s, but that none of the 2003-04 viruses were similar genetically or serologically to the CAL99-type viruses, indicating that new IBV variants continue to emerge and cause disease in commercial chickens in California.

Because sequence data on H9 avian influenza virus (AIV) from wild birds are currently limited, we set out to determine the sequence of the hemagglutinin (HA) gene of H9 viruses circulating in North American waterfowl and shorebirds. In this study, we examined the HA gene from H9 AIV isolated from mallards (Anas platyrhynchos) sampled during 1998 and 1999 in Minnesota and ruddy turnstones (Arenaria interpres) sampled during 2003 in Delaware and New Jersey. At these sites, the H9N2 subtype represented 12% and 4% of the avian influenza isolates from mallards in 1998 and 1999, respectively, and 8% of the AIVs isolated from shorebirds between 2000 and 2002. Sequences from these viruses were compared with sequences from H9 AIV isolated from commercial poultry and aquatic birds from North America, Europe, Asia, and the Middle East: four previously reported and three new clades were observed. Sequence data from the HA gene of North American waterfowl and shorebird isolates generated in this study most closely group with the Eurasian H9 viruses in the Y439 clade. In addition, the HA cleavage site (AASNR/G) and receptor binding site was identical to the representative virus of that group (DK/Hong Kong/Y439/97). Viruses in that clade are commonly found in ducks and chickens in Hong Kong and Korea. Positive evolutionary selection (dNonsynonymous > dSynonymous) was observed for the HA gene among the North American waterfowl and shorebird H9N2 viruses, indicating that the H9N2-type viruses are changing in their natural hosts.

It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

In February 2003, a severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in humans in Guangdong Province, China, and caused an epidemic that had severe impact on public health, travel, and economic trade. Coronaviruses are worldwide in distribution, highly infectious, and extremely difficult to control because they have extensive genetic diversity, a short generation time, and a high mutation rate. They can cause respiratory, enteric, and in some cases hepatic and neurological diseases in a wide variety of animals and humans. An enormous, previously unrecognized reservoir of coronaviruses exists among animals. Because coronaviruses have been shown, both experimentally and in nature, to undergo genetic mutations and recombination at a rate similar to that of influenza viruses, it is not surprising that zoonosis and host switching that leads to epidemic diseases have occurred among coronaviruses. Analysis of coronavirus genomic sequence data indicates that SARS-CoV emerged from an animal reservoir. Scientists examining coronavirus isolates from a variety of animals in and around Guangdong Province reported that SARS-CoV has similarities with many different coronaviruses including avian coronaviruses and SARS-CoV-like viruses from a variety of mammals found in live-animal markets. Although a SARS-like coronavirus isolated from a bat is thought to be the progenitor of SARS-CoV, a lack of genomic sequences for the animal coronaviruses has prevented elucidation of the true origin of SARS-CoV. Sequence analysis of SARS-CoV shows that the 5' polymerase gene has a mammalian ancestry; whereas the 3' end structural genes (excluding the spike glycoprotein) have an avian origin. Spike glycoprotein, the host cell attachment viral surface protein, was shown to be a mosaic of feline coronavirus and avian coronavirus sequences resulting from a recombination event. Based on phylogenetic analysis designed to elucidate evolutionary links among viruses, SARS-CoV is believed to have branched from the modern Group 2 coronaviruses, suggesting that it evolved relatively rapidly. This is significant because SARS-CoV is likely still circulating in an animal reservoir (or reservoirs) and has the potential to quickly emerge and cause a new epidemic.

The objective of this study was to compare the presence of the Arkansas (Ark) and Massachusetts (Mass) serotypes of infectious bronchitis virus (IBV) in the tracheas and cecal tonsils of commercial broilers after vaccination at 1 day of age by coarse spray. When given as a single serotype vaccine, the Mass strain was detected by reverse transcriptase-polymerase chain reaction (RT-PCR)-restriction fragment length polymorphism (RFLP) only in the tracheas, whereas the Ark strain was detected in both the tracheas and cecal tonsils. By in situ hybridization, the Mass and Ark nucleocapsid (Nc) genes were detected only at 7 days in the tracheas. When both strains were given in the mixed vaccine, the Mass strain was more consistently detected by RT-PCR-RFLP in the tracheas and cecal tonsils at early stages of infection (up to 14 days) and the Arkansas strain was more consistently detected at late stages of infection (21 and 28 days). By in situ hybridization, the IBV Nc gene was more consistently detected in the trachea at early stages of infection (7, 14, and 21 days) and in the cecal tonsils at late stages of infection (21, 28, and 35 days). In general, the Mass strain was more frequently recovered from the tracheal and cecal tonsil tissues at earlier stages of infection and the Ark strain was recovered at later stages of infection.

Sixteen infectious bronchitis virus (IBV) isolates were recovered from broilers and layers from five geographic poultry regions in Colombia. The viruses were isolated from tracheas, lungs, and cecal tonsils of birds, previously vaccinated with the Massachusetts strain, that were showing respiratory signs. Further analysis of the IBV isolates was achieved by phylogenetic analysis comparing their deduced amino acid sequences in the hypervariable region 1 of the S1 gene with reference strains. Four unique genotype clusters containing isolates with indigenous genotypes were observed. One isolate was found to be the Connecticut genotype and three isolates were found to be the Massachusetts genotype.

In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.

Hammerhead ribozymes are catalytic RNA molecules that specifically cleave a target RNA molecule. Herein, we report the design, synthesis, and in vitro analysis of a hammerhead ribozyme targeted to the infectious bronchitis virus (IBV) nucleocapsid mRNA. At a concentration of 0.5 or 10 microM, the ribozyme, designated IBV-N-Rz, effectively cleaved target RNAs in trans (37 C, 10 mM MgCl2, 50 mM Tris). Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and relative quantitative reverse transcription-polymerase chain reaction. The amount of target RNA continually declined over a 5-hr period, indicating that the ribozyme was truly catalytic. Although stability and delivery problems must be overcome, a hammerhead ribozyme targeted to the IBV nucleocapsid mRNA most likely has antiviral activity and may be an effective therapeutic/prophylactic reagent in the future.