J. Michael Day

SUMMARY Recent metagenomic analyses of the enteric viromes in turkeys and chickens have revealed complex viral communities comprised of multiple viral families. Of particular significance are the novel avian picobirnaviruses (family Picobirnaviridae), multiple genera of tailed phages (family Siphoviridae), and undescribed avian enteric picornaviruses (family Picornaviridae). In addition to these largely undescribed—and therefore relatively poorly understood—poultry enteric viral families, these metagenomic analyses have also revealed the presence of well-known groups of enteric viruses such as the chicken and turkey astroviruses (family Astroviridae) and the avian rotaviruses and reoviruses (family Reoviridae). The order Picornavirales is a group of viruses in flux, particularly among the avian picornaviruses, since several new genera have been described recently based upon community analysis of enteric viromes from poultry and other avian species worldwide. Our previous investigation of the turkey enteri... RESUMEN Caracterización molecular de picornavirus entéricos de muestras archivadas de pavo y pollo de los Estados Unidos. Los análisis de metagenómica recientes de los viromas entéricos de pavos y pollos han revelado comunidades virales complejas compuestas de múltiples familias virales. De particular importancia son los nuevos picobirnavirus aviares (familia Picobirnaviridae), múltiples géneros de fagos con cola (familia Siphoviridae) y picornavirus entéricos aviares no descritos (familia Picornaviridae). Además de estas familias virales en aves comerciales en gran parte no descritas, y por lo tanto relativamente poco comprendidas, estos análisis metagenómicos también han revelado la presencia de grupos bien conocidos de virus entéricos tales como los astrovirus de pollo y pavo (familia Astroviridae) y los rotavirus aviares y reovirus (familia Reoviridae). El orden Picornavirales es un grupo cambiante de virus, en particular entre los picornavirus aviares, ya que varios nuevos géneros han sido descritos ...

There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds (“sentinels”) placed on farms revealed colonization by members of the Picornaviridae, Picobirnaviridae, Reoviridae, and Astroviridae that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus families and genera. Members of the avian enteric Reoviridae and Astroviridae have been well-characterized and have historically been implicated in poultry enteric disease; members of the Picobirnaviridae and Picornaviridae have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.

Despite the importance of the poultry gut, remarkably little is known about the complex gut microbial community. Enteric disease syndromes such as runting-stunting syndrome in broiler chickens and poult enteritis complex in young turkeys are difficult to characterize and reproduce in the laboratory. A great deal of work has been done to characterize the bacterial population in the poultry gut, leading to useful performance-based interventions such as direct-fed microbial preparations. Advances in the application of rapid molecular diagnostics and the advent of the next generation of nucleic acid sequencing have allowed researchers to begin to decipher the microbial community in complex environmental samples. Researchers have made great strides recently in placing names to some of the unknown and undescribed small viruses in the poultry gut such as parvoviruses, picornaviruses, picobirnavirus, and calicivirus. Investigation into the novel avian astroviruses continues, and recent progress has been made in the molecular characterization of the avian rotaviruses. This review will focus on the recent advances that have been made in the discovery, description, and characterization of the multitude of viruses that reside in the poultry gut.

The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.

Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses.

Turkey astrovirus type-2 (TAstV-2), turkey rotavirus (TRotV), and turkey reovirus (TReoV) have been implicated as possible causes of enteric diseases and poor production in turkeys; however, numerous studies with each individual virus have failed to reproduce the disease as observed in the field. Therefore, in this study we evaluated the pathogenesis of all possible combinations of one, two, or three viruses in comparison to sham inoculates in 3-day-old turkey poults. Body weights were recorded at 2, 4, 7, 10, and 14 days postinoculation (PI) and were decreased in virus-infected turkeys throughout the experiment as compared to sham inoculates. Although not significantly different from the other virus-exposed groups, the poults exposed to all three viruses had the lowest body weights throughout the experiment. Clinical signs, including huddling, diarrhea, and agitation, were only observed in groups exposed to TAstV-2 and/or TRotV. At 4 days PI, birds from each treatment group were necropsied, and pale intestines with watery contents and undigested feed were observed in the groups that were exposed to TRotV + TReoV or TRotV + TAstV-2 and the group exposed to all three viruses. Minimal microscopic lesions were observed in the intestines of turkeys infected with TAstV-2, TReoV, or a combination of both. In the turkeys infected with TRotV, either alone or in combination with other viruses, mild microscopic lesions were found in all sections of the small intestine and viral antigen was identified by immunohistochemical staining in mature enterocytes. No or very mild lesions were observed in other organs with the exception of the bursa of Fabricius, where mild to severe atrophy was observed in all virus-infected poults examined. Cloacal shedding of TAstV-2 and TRotV was evaluated by reverse-transcription PCR testing of cloacal swabs and minimal differences were observed among the treatment groups.

Recently, pathogenesis studies, using genetically distinct turkey-origin reoviruses (TRVs), revealed that poults infected with certain TRV isolates had moderate to severe bursal atrophy, suggesting virus-induced immune dysfunction. In order to characterize the effect of TRV infection on the turkey immune system, classical assays were undertaken to quantify the humoral and cell-mediated immune responses in small Beltsville and broad-breasted white poults infected with the TRV isolate NC/SEPR44/03. A marked effect on the cutaneous basophil hypersensitivity response, and on the antibody response to Newcastle disease virus (NDV) exposure, was noted in commercial and specific pathogen free (SPF) poults inoculated with NC/SEP-R44/03 at three days of age. Moderate to severe bursal atrophy, similar to that noted previously in SPF poults, occurred in commercial poults inoculated at three days of age. This immune dysfunction and bursal atrophy was not present in commercial poults inoculated at three weeks of age.