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During a series of pathology surveys in four production complexes of a U.S. broiler integrator, the technical services veterinarians of an animal health company noted a high incidence of severe gizzard erosions and ulcerations (GEU), prompting further clinical investigation and a battery trial. No growth-promoting antibiotics or ionophore coccidiostats were used during the period of these surveys. All used tribasic copper chloride (TBCC) at ≤120 ppm added copper in broiler rations. Clostridium perfringens was isolated from 83% and 67% of gizzard lesions cultured in two complexes, and cecal C. perfringens most probable number determinations were higher in severely affected than in mildly affected or unaffected birds. Histopathology revealed both acellular koilin fusion defects characteristic of copper toxicity, as well as inflammatory cell infiltrates. Intralesional bacilli suggestive of C. perfringens were noted in 78% of affected flocks examined. Species E Aviadenovirus was isolated from one bird in one complex, and that bird had a single intranuclear inclusion body; no other flocks had Adenoviruses isolated or detected on PCR, nor any inclusion bodies. Other viruses detected were thought to be incidental. A pilot study using feed with supplemental copper from TBCC or copper sulfate and challenge with one of the isolated C. perfringens strains reproduced the lesions. A battery study was conducted with an unchallenged negative control group fed a diet with 16 ppm added copper, a group fed the control diet and orally challenged with 10 8 organisms of a field strain of C. perfringens at 21 and 22 days, and a group treated with the same diet containing 250 ppm added copper from TBCC and orally challenged with C. perfringens . Birds were necropsied at 23 and 28 days. All challenged groups developed lesions, with those receiving both TBCC and C. perfringens having significantly higher gross and histopathological lesion scores than the unchallenged negative controls. Lesions were qualitatively similar to those in the field and contained suspected C. perfringens bacilli. Because the levels of TBCC used in the commercial birds and in the battery trial generally have been considered safe, and because C. perfringens is usually regarded as a pathogen of the lower GI tract, the possible association of these two agents with GEU is a novel observation and warrants further investigation.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.

Prevention of tenosynovitis/viral arthritis caused by variant avian reoviruses within commercial broiler production has become increasingly more challenging because of the lack of protection afforded by the current commercially available vaccines. Avian reoviruses isolated from clinical cases of tenosynovitis/viral arthritis in recent years are antigenically distinct from nearly all of the commercially licensed modified live and inactivated biologics available in the United States. The emergence of new variants is likely shaped by a lack of homologous protection coupled with selection pressure influences and results in antigenically diverse populations of avian reoviruses. One tool available to the poultry industry is the use of autogenous (custom) vaccines. Although these can be effective, isolation, characterization, and screening of isolates from clinical cases is paramount for the selection of isolates to include in these vaccines. With no treatment options, control can only be attained via prevention of infection. To achieve this goal, commercially licensed products with antigenic applicability and broadly cross-protective vaccine strains are needed.

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

The Reoviridae family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus-single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5' and 3' ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses.

Reoviral-induced tenosynovitis/viral arthritis is an economically significant disease of poultry. Affected birds present with lameness, unilateral or bilateral swollen hock joints or shanks, and/or reluctance to move. In severe cases, rupture of the gastrocnemius or digital flexor tendons may occur, and significant culling may be necessary. Historically, vaccination with a combination of modified live and inactivated vaccines has successfully controlled disease. Proper vaccination reduced vertical transmission and provided maternal-derived antibodies to progeny to protect against disease, at an age when they were most susceptible. Starting in 2011-2012, an increased incidence of tenosynovitis/viral arthritis was observed in chickens and turkeys. In chickens, progeny from reovirus-vaccinated breeders were affected, suggesting commercial vaccines did not provide adequate protection against disease. In turkeys, clinical disease was primarily in males, although females can also be affected. The most significant signs were observed around 14-16 wks of age and include reluctance to move, lameness, and limping on one or both legs. The incidence of tenosynovitis/viral arthritis presently remains high. Reoviruses isolated from clinical cases are genetically and antigenically characterized as variants, meaning they are different from vaccine strains. Characterization of the field isolates reveals multiple new genotypes and serotypes that are significantly different from commercial vaccines and each other. In 2012, a single prevalent virus was isolated from a majority of the cases submitted to the Poultry Diagnostic and Research Center at the University of Georgia. Genetic characterization of the σC protein revealed the early isolates belonged to genetic cluster (GC) 5. Soon after the initial identification of the GC5 variant reovirus, many broiler companies incorporated these isolates from their farms into their autogenous vaccines and continue to do so today. The incidence of GC5 field isolates has decreased significantly, likely because of the widespread use of the isolates in autogenous vaccines. Unfortunately, variant reoviruses belonging to multiple GCs have emerged, despite inclusion of these isolates in autogenous vaccines. In this review, an overview of nomenclature, sample collection, and diagnostic testing will be covered, and a summary of variant reoviruses isolated from clinical cases of tenosynovitis/viral arthritis over the past 10 yrs will be provided.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

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

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1 -targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Multiple oncogenic viruses, including lymphoproliferative disease virus (LPDV) and reticuloendotheliosis virus (REV), have been detected in wild turkeys ( Meleagris gallopavo ). The prevalence of infection with these viruses appears to be more common than overt disease; thus, data on the manifestation of associated disease in wild turkeys are scarce. Diagnostic records from wild turkeys submitted to the Southeastern Cooperative Wildlife Disease Study from 1980 to 2017 were reviewed to identify cases of neoplasia. Neoplasia was reported in 59 of 851 (6.9%) wild turkeys submitted. Of the cases of neoplasia tested by polymerase chain reaction, LPDV was detected in 34 of 58 (59%), REV in 10 of 39 (26%), both viruses in 3 of 39 (8%), and no retroviruses detected in 5 of 39 (13%) turkeys. The most common gross lesions observed among turkeys with neoplasms were emaciation (30/40; 75%); nodules in the skin (26/59; 44%), liver (17/59; 29%), or spleen (9/59; 15%); and splenomegaly (14/59; 24%). Microscopically, nodules were composed of pleomorphic round cells with large eccentric nuclei and prominent nucleoli resembling lymphocytes or lymphoblasts (57/59; 97%) except for 2 cases, one of myeloid cell origin and the other with primarily spindloid cells. This study indicates the need to characterize the pathogenesis and potential health threat posed by REV and LPDV to wild turkeys. Experimental infection studies and the development of additional diagnostic tests to confirm the role of retroviruses in lymphoproliferative disease are warranted.

Despite descriptions of runting-stunting syndrome (RSS) in broiler chickens dating back over 40 years, the aetiology has not yet been described. A novel chicken astrovirus (CkAstV) was isolated in an LMH liver cell line from the intestines of chickens affected with RSS. Clinical RSS is characterized by retarded growth and cystic crypt lesions in the small intestine. In 1-day-old broiler chickens infected with the CkAstV isolate, virus was only detected in the intestinal epithelial cells during the first few days after infection. Notably, the preferred host cells are the crypt epithelial cells following initial replication in the villous epithelial cells, thus implying viral preference for immature intestinal cells. Nevertheless, the CkAstV isolate did not induce remarkable pathological changes, despite the presence of the virus in situ. Serial chicken-to-chicken passages of the virus induced increased virulence, as displayed by decreased weight gain and the presence of cystic lesions in the small intestine reproducing clinical RSS in chickens. The analysis of the full-length genome sequences from the isolated CkAstV and the CkAstV from the bird-to-bird passages showed >99 % similarity. The data obtained in this study suggest that the CkAstV isolate is capable of inducing RSS following serial bird-to-bird passages in broilers and is as an aetiological agent of the disease.

Avian reoviruses are the causative agent of viral arthritis/tenosynovitis in chickens and turkeys. Clinical signs of disease include swelling of the hock joints accompanied by lesions in the gastrocnemius and digital flexor tendons causing lameness in addition to hydropericardium. The economic impact is significant as it results in poor weight gain, increased feed conversion ratios and condemnations at the processing plant. Vaccination with both live attenuated and inactivated oil emulsion vaccines have been used successfully for decades to control the disease. Current commercial vaccine strains belong to the same serotype and are antigenically and serologically distinct from circulating variant field viruses isolated from clinical cases of tenosynovitis. Since 2012, there has been a dramatic increase in the number of clinical cases of tenosynovitis in commercial poultry and commercial vaccines are unable to provide adequate levels of protection against disease. Producers have elected to use custom inactivated vaccines in the absence of any commercially available homologous vaccines. Identification and selection of field isolates for use in autogenous vaccines can be difficult especially when multiple reoviruses are co-circulating among flocks. In addition, field data suggests that in some cases the custom vaccines are providing adequate protection against disease but as new genetic variants emerge, new vaccines are needed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Two broiler chicken houses containing 17,500 chicks each experienced an extreme elevation in chick mortality beginning on day 3 after placement. Clinical signs observed upon farm visit included numerous small chicks for their age; depressed, lethargic, and comatose chicks; and chicks huddling near feed pans and under heaters. Necropsied chicks were markedly pale and had atrophy of the thymus and bursa, swollen and edematous proventriculus, erosions in the koilin and in the proventricular-ventricular junction, pale kidneys, and yellowish to brownish-orange liver often with linear pale areas. The chicks had watery blood and hematocrits measured from 9.5% to 18%. Chicken infectious anemia was initially suspected based on the clinical signs and gross lesions. Histopathology revealed multifocal acute hepatic degeneration and necrosis with golden-brown pigment in the cytoplasm of hepatocytes and Kupffer cells, moderate to severe koilin degeneration and fragmentation, multifocal mild to moderate proventricular necrosis, mild to moderate necrosis and loss of enterocytes, blunting of small intestinal villi, lymphoid depletion in the thymus and bursa, erythrophagocytosis in the liver and spleen, and acute renal tubular degeneration and necrosis. Special stains revealed mild to abundant accumulation of copper pigment in the cytoplasm of hepatocytes and iron pigment in the cytoplasm of Kupffer cells. Feed analysis revealed 2140 to 2393 parts per million of copper in the starter ration, and heavy metal analysis detected markedly elevated copper levels in formalin-fixed samples of the liver. Excessive amounts of tribasic copper chloride in the starter ration caused copper toxicosis in these chicks. Similar clinical signs and lesions were reproduced when the suspect feed was used in an experimental pen trial.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus causing infectious bursal disease in chickens. IBDV undergoes antigenic drift, so characterizing the antigenicity of IBDV plays an important role for identification and selection of vaccine candidates. In this study, an in vivo experimental model was developed to differentiate a new antigenic variant of IBDV. To this end, a hyper-immune serum to IBDV E/Del-type virus was generated in specific pathogen-free chickens and a standard volume of the hyper-immune serum was serially diluted and injected in specific pathogen-free birds via intravenous, subcutaneous, or intramuscular routes. The chickens were bled at different time points in order to evaluate the dynamics of virus neutralization titres. Based on the results, chickens were injected with different serum dilutions by the subcutaneous route. Twenty-four hours later, chickens were bled and then challenged with 100 median chicken infectious doses of the E/Del virus and a new IBDV variant. Chickens were euthanized at 7 days post infection and the bursa of Fabricius was removed for microscopic evaluation to determine the bursal lesion score. The determined virus neutralization titre along with the bursal lesion score was used to determine the breakthrough titre in the in vivo chicken model. Based on the data obtained, an antigenic subtype of IBDV was identified and determined to be different from E/Del. This model is a sensitive model for determination of IBDV antigenicity of non-tissue culture adapted IBDV.

The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hcl, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J-induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J-infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.

The genomic RNA of a novel chicken astrovirus was determined. The full length sequence is 7520 nucleotides and encodes three open reading frames (1a, 1b, 2) for three proteins. The genomic organization was similar to other astroviruses with two exceptions. The open reading frame of the RNA-dependent RNA polymerase contains its own start codon which is different from other astroviruses described to date, providing evidence for a replication mechanism different than what has previously been described for astroviruses. Furthermore, the stem-loop structure located at the potential ribosomal frameshift signal described for other astroviruses has been shown to be a hairpin structure for the novel chicken astrovirus. Phylogenic analysis of the full length sequence revealed that this chicken astrovirus formed a branch independent from other astroviruses, indicating that this astrovirus is significantly different from astroviruses described to date.

Currently, the aetiology of runting and stunting syndrome (RSS) in chickens is unknown. The impact of RSS on weight gain and microscopic lesions in immunological organs and the duodenum, was investigated in 1-day-old commercial broilers at 12 days following exposure to RSS-contaminated litter. Furthermore, the presence of the viral nucleic acids of three astroviruses and one parvovirus was analysed by in situ hybridization from days 1 through 5 post exposure. A 70% decrease in weight was observed in the RSS-exposed group at the end of the experiments when compared with the unexposed controls. Lesions in the bursa of Fabricius and thymus were present in both groups but were significantly higher at the end of the study in the RSS-exposed group. In contrast, no significant difference in Harderian gland lesions was observed between the groups. Histological lesions in the duodenum were already present 24 h after exposure in the RSS-exposed group only, peaked at day 4 and declined until the end of the study. Results of the in situ hybridization studies clearly indicate replication of three astroviruses (chicken astrovirus, avian nephritis virus [ANV]-1, ANV-2) in the duodenum but not in other organs evaluated. Chicken astrovirus nucleic acids were detected on days 1 and 2 post exposure, while ANV-1 and ANV-2 nucleic acids were observed on several days during the period investigated. Surprisingly, no viral nucleic acid specific for the chicken parvovirus was observed. The results indicate that astroviruses probably play an important role during RSS due to the concurrence of viral RNA detection and lesions in the duodenum.

The antigenic profiles of over 300 infectious bursal disease virus (IBDV) isolates were analyzed using a panel of monoclonal antibodies in a reverse genetics system. In addition, the sequences of a large portion of the neutralizing-antibody-inducing VP2 of IBDV were determined. Phylogenetic analysis of nucleotide and amino acid sequences in combination with the antigenic profiles obtained using the monoclonal antibody panel, revealed a lack of correlation between antigenicity and isolate's placement within the phylogenetic tree. In-depth analysis of amino acid exchanges revealed that changes within a certain region of the VP2 molecule resulted in differences in the antigenicity of the virus. This comprehensive analysis of VP2 sequences indicated a high selective pressure in the field that was likely due to vaccination programs, which increase the rate of evolution of the virus.

The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.

Captive-reared Whooping Cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997-98 and 2001-02 release seasons. Exposure to infectious bursal disease virus (IBDV) serotype 2 as evidenced by seroconversion was suspected to be the factor that precipitated these mortality events. Very little is known about the incidence of IBD in wild bird populations. Before this study, natural exposure had not been documented in wild birds of North America having no contact with captive-reared cranes, and the prevalence and transmission mechanisms of the virus in wild birds were unknown. Sentinel chickens (Gallus gallus) monitored on two Whooping Crane release sites in central Florida, USA, during the 2003-04 and 2004-05 release seasons seroconverted, demonstrating natural exposure to IBDV serotype 2. Blood samples collected from Wild Turkeys (Meleagris gallopavo) and Sandhill Cranes (Grus canadensis) in eight of 21 counties in Florida, USA, and one of two counties in southern Georgia, USA, were antibody-positive for IBDV serotype 2, indicating that exposure from wild birds sharing habitat with Whooping Cranes is possible. The presence of this virus in wild birds in these areas is a concern for the resident flock of Whooping Cranes because they nest and raise their chicks in Florida, USA. However, passively transferred antibodies may protect them at this otherwise vulnerable period in their lives.

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).

Infectious bursal disease virus (IBDV) is an immunosuppressive virus which primarily infects IgM B-cells in the bursa of Fabricius. Flow cytometric analysis was used to phenotype B-cell populations in the bursa and spleen following IBDV infection. In the bursa, two IgM B-cell subpopulations, designated as A and B, were identified based on cell size and granularity. While both subpopulations differentially expressed IgM and Bu-1b surface markers, both groups displayed major histocompatibility complex class II surface antigens at equal levels. Following IBDV challenge of nonvaccinated birds, the B subpopulation was significantly reduced between 7 and 21 days postchallenge compared to either nonchallenged birds or vaccinated-challenged birds. However, the reduction of subpopulation B in the bursa, following IBDV exposure, did not reduce the levels of total serum IgA, IgG, and IgM, nor did it affect IgG and IgA B-cells in the spleen. Phenotypic analysis of the subpopulations identified differential expression of Lewis(x), IgM, Bu-1b, and MUI78 surface antigens between the subpopulations. Overall, these are the first studies to identify two distinct IgM B-cell subpopulations in the chicken bursa, and the first to describe the decrease in the IgM B-cell population relative to IgA and IgG B-cells following IBDV infection.

Infectious bursal disease virus (IBDV) serotype 1 is the causative agent of a highly contagious immunosuppressive disease of young chickens. In the past, a number of antigenic, as well as pathogenic, subtypes have been described. The determination of the antigenic makeup of circulating strains is of vital interest to the poultry industry because changes in the antigenicity of circulating field strains have an impact on the use of vaccines. To obtain a more comprehensive overview of the relationship between the nucleotide and amino acid sequence and the antigenic makeup of field isolates, a system based on reverse genetics of IBDV was established. Using this approach, a database for field isolates from three different states in the United States (Georgia, Alabama, and Louisiana), consisting of nucleotide sequence, amino acid sequence, and a reaction pattern based on a panel of monoclonal antibodies, was established. The obtained results showed that phylogenic analysis, which is based on the similarity of sequences, would lead to false conclusions regarding a possible antigenic makeup of the particular isolate. Sequences of field samples were divided into three groups: 1) those that grouped with variant strain E/Del sequences but were antigenically different, 2) those that did not group with sequences of E/Del but were similar in their antigenic makeup, and 3) those that did not group with E/Del sequences and were antigenically different. In addition, using the reverse-genetics approach, a number of field isolates showed no reactivity with any of the used monoclonal antibodies, indicating that an unknown, antigenic subtype of IBDV serotype 1 is circulating in the field.

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.

A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of two enteric viruses of poultry: turkey enteric coronavirus (TCV) and turkey astrovirus (TAstV). PCR primers were designed to conserved regions within the nucleocapsid gene of TCV and to the polymerase gene of TAstV-2. The primer pairs were successfully used in a multiplex RT-PCR to detect nucleic acid of TAstV-2 and TCV. The test was optimized for use with intestines/feces from naturally infected turkeys. The primers were specific and did not amplify other common RNA or DNA avian viruses. The detection limit was determined to be 10 ng of RNA used as starting template. The use of this specific test allows the rapid and early diagnosis of two financially costly viruses affecting the commercial turkey industry.

The open reading frame of the S3 segment encoding the sigma2 protein of four turkey reovirus field isolates was analyzed for sequence heterogeneity. The turkey reoviruses we present here have a 97% amino acid identity to turkey NC 98. The S3 nucleotide and amino acid sequence similarity was < or =61% and 78%-80%, respectively, when compared to the chicken reovirus isolates. Comparison of amino acid sequences from chickens and turkeys with that of a duck isolate revealed a 53% and 55% similarity, respectively. Phylogenetic analyses, based on both nucleotide and amino acid sequence, resulted in three major groups among the avian reoviruses; these groups were clearly separated by species. The results of this study provide further evidence, based on the deduced sigma2 sequence, that turkey reoviruses form a distinct, separate group relative to chicken and duck isolates. In addition, as a result of the limited sequence identity with their avian counterparts, turkey reoviruses could potentially be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

Nucleotide sequence was determined for the RNA-dependent RNA polymerase (L) gene of 16 Newcastle disease virus (NDV) isolates from diverse geographic and chronological origins. The observed consensus amino acid sequence conformed to the six domains previously identified among paramyxovirus L proteins, and the putative 749QGDNQ753 active site was strictly conserved among all isolates. Analysis of predicted amino acid sequences allowed us to identify a sequencing error in the previously reported L genes for NDV. The correct sequences reported herein provided a more accurate alignment with predicted l-amino acid sequences of other paramyxoviruses. Comparison of L gene coding sequences among isolates revealed that synonymous substitutions dominated non-synonymous substitutions, as observed previously with other NDV genes. However, the overall substitution rate was lower than other genes examined making the L gene the most conserved of the NDV genome. Phylogenetic analysis of L genes among NDV isolates was consistent with previous results that suggested the existence of two major lineages. One group contained strains isolated in North America prior to 1970 and included virulent and vaccine strains, while the second group included virulent viruses isolated worldwide. A comparison of the NDV L coding sequences to other Paramyxoviridae illustrated the unique clustering of the avian-specific paramyxoviruses, further justifying the newly created Avulavirus genus.

During 2001, a mild infectious laryngotracheitis virus (ILTV) infection occurred in broiler flocks in the southeastern United States. Clinical signs included mild tracheitis, swollen sinuses, and conjunctivitis, with no increased mortality and minimal serologic response. Infrequent intranuclear inclusion bodies with or without syncytial cell formation were observed in eyelid, trachea, and larynx and in the chorioallantoic membrane of infected embryos. Immunohistochemistry and a nested infectious laryngotracheitis polymerase chain reaction (ILT PCR) were utilized to confirm the presence of ILTV nucleic acid in fixed tissues. In addition, 2-wk-old specific-pathogen-free (SPF) birds inoculated with field material exhibited the mild signs observed in broilers in the field. Tracheal swabs and tissues taken from these SPF birds were also positive by nested ILT PCR. Restriction fragment length polymorphism analysis of ILT PCR products indicated that ILT virus associated with mild respiratory disease in the southeast is related to the chicken embryo origin vaccine type strains.

In this study we compared protection by DNA vaccination with the F (pCMV-F) or N (pCMV-N) gene from avian metapneumovirus (aMPV) in turkeys. One-week-old turkey poults received two intramuscular injections 2 wk apart. Birds were challenged with a turkey-embryo-adapted aMPV at 5 wk of age. Birds vaccinated with pCMV-F had decreased clinical signs of disease as well as significantly reduced virus load in tracheal swabs compared with birds vaccinated with pCMV-N or unvaccinated control birds. Serum neutralizing antibodies were significantly higher in birds receiving pCMV-F compared with all other groups. These results indicate that DNA vaccination with the F, but not N, gene of aMPV can induce significant protection against aMPV infection.

We have constructed a DNA vaccine (pDKArkS1-DPI) expressing the S1 glycoprotein (Arkansas DPI) of infectious bronchitis virus (IBV) to examine protective immunity after in ovo and intramuscular DNA immunization. Birds receiving in ovo DNA followed by live virus vaccination at 2 wk of age were 100% protected from clinical disease. Birds receiving only live virus vaccine or only in ovo DNA vaccination were < or = 80% protected. IBV was detected up to 10 days postchallenge in unvaccinated control groups, whereas birds receiving in ovo DNA and live virus vaccination cleared IBV from tracheal samples before day 5 postchallenge. Transcription of the S1 gene was confirmed in lung tissue after in ovo vaccination by an antisense riboprobe, and the S1 protein was detected by immunohistology in the heart and bursa. In a separate experiment, birds were injected intramuscularly with either 50, 100, or 150 microg of the DNA vaccine at 1 day of age and then again with either 100, 200, or 300 microg of the DNA vaccine, respectively, at 14 days of age. At 10 days postchallenge, no clinical signs were observed and no challenge virus was reisolated from the birds vaccinated with 150 microg and 300 microg of DNA. Between DNA-vaccinated birds and nonvaccinated control birds, no statistical differences were observed for IBV-specific serum antibodies as detected by enzyme-linked immunosorbent assay or the virus neutralization test. These data indicate that DNA vaccination with the S1 gene either in ovo or intramuscularly can provide birds with some protection against clinical disease after homologous IBV challenge.

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

The nucleocapsid (N) protein genes from 24 Newcastle disease virus (NDV) isolates representing various pathotypes with different geographical and chronological origins were cloned and sequenced. The N-terminal region of the N protein to residue 401 was highly conserved among isolates with several conservative substitutions occurring that correlated with phylogenetic relationships. Variability of the N protein was detected in the C-terminal portion similar to what has been reported for other members of the Paramyxovirinae. Amino acids previously identified as invariant or highly conserved in N proteins of other paramyxoviruses were also present in the NDV protein. Phylogenetic analysis of N gene coding sequences among NDV isolates again demonstrated the existence of two major groups. One clade contained viruses that included vaccine and virulent strains isolated in the USA prior to 1970 while a second clade included vaccine and virulent viruses isolated worldwide. Comparison of N protein amino acid sequences among members of the Paramyxoviridae resulted in NDV and avian paramyxovirus 6 separating as a cluster distinct from the Rubulavirus genus. This provides further support for avian paramyxoviruses being considered for their own genus among the Paramyxovirinae.

The deduced sigma-2 protein sequence from the S3 gene segment of a novel turkey reovirus, designated NC98, isolated from the bursa of birds exhibiting poult enteritis and mortality syndrome was determined. The isolate, serologically distinct from other avian reoviruses, was isolated in turkey embryo kidney cells and RNA was purified for cDNA synthesis. Oligonucleotide primers were designed based on conserved avian S3 nucleotide sequence data. The NC98 S3 open reading frame comprised 1,101 base pairs and encoded 366 amino acids with a predicated molecular mass of 40.5 kDa. Although the S3 nucleotide sequence from several chicken isolates share at least 86% identity, they share only 64% with the NC98 turkey isolate. Interestingly, the S3 nucleotide sequence from a muscovy duck reovirus shares 55% identity with NC98 and 53% identity with chicken isolates. As observed in other avian reovirus sigma2 protein sequences, a zinc-binding motif and double-stranded RNA binding domain were found within the predicted amino acid sequence of NC98. Phylogenetic analysis of the deduced sigma2 sequence demonstrated that NC98 separated as a distinct virus relative to other avian strains. The results of this study indicate that NC98 is a novel turkey reovirus that shares limited genomic sequence identity to isolates of chicken and duck origin and should be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

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

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

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

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

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

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