Isolation and genomic characterization of a novel avian orthoreovirus strain in Korea, 2014.

In this study, we isolated a novel avian reovirus (ARV) strain, K738/14, from a broiler chicken with viral arthritis in South Korea. Genome sequence comparisons showed relatively low nucleotide identity with previously identified ARV strains. Phylogenetic analyses suggested multiple reassortment events between reovirus strain S1133 and reoviruses of Hungarian, Chinese, and US origin had occurred. In addition, recombination analyses showed evidence of intra-segmental recombination in the M2 and S2 genes. Based on our genetic analyses, multiple reassortment events, intra-segmental recombination, and accumulation of point mutations have possibly contributed to the emergence of this novel genotype of ARV, identified in Korea.

Investigating Turkey Enteric Picornavirus and Its Association with Enteric Disease in Poults.

Previous research into the viral community in the poultry gastrointestinal tract has revealed a number of novel and partially described enteric viruses. It is evident that the poultry gut viral community remains minimally characterized and incompletely understood. Investigations into the microbiome of the poultry gut have provided some insight into the geographical distribution and the rapidly evolving taxonomy of the avian enteric picornaviruses. The present investigation was undertaken to produce a comparative metagenomic analysis of the gut virome from a healthy turkey flock versus a flock placed in the field. This investigation revealed a number of enteric picornavirus sequences that were present in the commercial birds in the field that were completely absent in the healthy flock. A novel molecular diagnostic assay was used to track the shedding of field strains of turkey enteric picornavirus in commercial poults inoculated with picornavirus-positive intestinal homogenates prepared from turkeys that were experiencing moderate enteric disease. The propagation of this novel enteric picornavirus in commercial poults resulted in significant reduction in weight gain, and suggests that this common inhabitant of the turkey gut may result in performance problems or enteric disease in the field.

Host Specificity and Phylogenetic Relationships of Chicken and Turkey Parvoviruses.

Inoculation of specific-pathogen-free chickens and turkeys with five chicken parvoviruses (ChPV) and five turkey parvoviruses (TuPV) resulted in productive virus replication only in the homologous host species. A phylogenetic tree based on nucleotide sequences of the VP1 gene segment revealed a host-specific clustering of the virus strains. These results suggest that the VP1 gene plays an essential role in host specificity of ChPV and TuPV strains and could be a relevant target sequence for strain classification.

Molecular and phylogenetic analysis of a novel turkey-origin picobirnavirus.

A previous metagenomic analysis of the turkey gut RNA virus community identified novel enteric viruses that may play roles in poultry enteric diseases or in performance problems noted in the field. As part of the molecular characterization of these novel enteric viruses, a reverse transcriptase-PCR diagnostic assay was developed, targeting a novel turkey-origin picobirnavirus (PBV) initially identified in a pooled intestinal sample from turkey poults in North Carolina. Little detailed molecular information exists regarding the family Picobirnaviridae, particularly for the PBVs that have been described in avian species. This diagnostic assay targets the turkey PBV RNA-dependent RNA polymerase gene and produces an 1135-bp amplicon. This assay was validated using in vitro transcribed RNA and was tested using archived enteric samples collected from turkey flocks in the southeastern United States. Further, a phylogenetic analysis suggests the turkey PBV is unique because it does not group closely with the recognized PBV genogroups circulating in mammalian hosts.

Investigating turkey enteric coronavirus circulating in the Southeastern United States and Arkansas during 2012 and 2013.

Periodic monitoring of poultry flocks in the United States via molecular diagnostic methods has revealed a number of potential enteric viral pathogens in continuous circulation in turkeys and chickens. Recently turkey integrators in the Southeastern United States and Arkansas experienced an outbreak of moderate to severe enteritis associated with turkey enteric coronavirus (TCoV), and numerous enteric samples collected from turkey flocks in these areas tested positive for TCoV via real-time reverse-transcriptase PCR (RRT-PCR). This report details the subsequent sequence and phylogenetic analysis of the TCoV spike glycoprotein and the comparison of outbreak-associated isolates to sequences in the public database. TCoVs investigated during the present outbreak grouped geographically based upon state of origin, and the RRT-PCR assay was a good indicator of subsequent seroconversion by TCoV-positive turkey flocks.

Recent progress in the characterization of avian enteric viruses.

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.

Chicken parvovirus-induced runting-stunting syndrome in young broilers.

Previously we identified a novel parvovirus from enteric contents of chickens that were affected by enteric diseases. Comparative sequence analysis showed that the chicken parvovirus (ChPV) represented a new member in the Parvoviridae family. Here, we describe some of the pathogenic characteristics of ChPV in young broilers. Following experimental infection, 2-day-old broiler chickens showed characteristic signs of enteric disease. Runting-stunting syndrome (RSS) was observed in four of five experimental groups with significant growth retardation between 7 and 28 days postinoculation (DPI). Viral growth in small intestine and shedding was detected at early times postinoculation, which was followed by viremia and generalization of infection. ChPV could be detected in most of the major tissues for 3 to 4 wk postinoculation. Immunohistochemistry staining revealed parvovirus-positive cells in the duodenum of inoculated birds at 7 and 14 DPI. Our data indicate that ChPV alone induces RSS in broilers and is important determinant in the complex etiology of enteric diseases of poultry.

The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system.

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.

Structural analysis of kasugamycin inhibition of translation.

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).

Pathogenic evaluation of a turkey coronavirus isolate (TCoV NC1743) in turkey poults for establishing a TCoV disease model.

Turkey coronavirus (TCoV) can cause a highly contagious enteric disease in turkeys with severe economic losses in the global turkey industry. To date, no commercial vaccines are available for control of the disease. In the present study, we isolated a field strain (NC1743) of TCoV and evaluated its pathogenicity in specific-pathogen-free (SPF) turkey poults to establish a TCoV disease model. The results showed that the TCoV NC1743 isolate was pathogenic to turkey poults with a minimal infectious dose at 106 EID50/bird. About 50 % of one-day-old SPF turkeys infected with the virus's minimal infectious dose exhibited typical enteric disease signs and lesions from 6 days post-infection (dpi) to the end of the experiment (21 dpi). In contrast, fewer than 20 % of older turkeys (1- or 2-week-old) infected with the same amount of TCoV displayed enteric disease signs, which disappeared after 15-18 dpi. Although all infected turkeys, regardless of age, shed TCoV, the older turkeys shed less virus than the younger birds, and 50 % of the 2-week-old birds even cleared the virus at 21 dpi. Furthermore, the viral infection caused day-old turkeys more body-weight-gain reduction than older birds. The overall data demonstrated that the TCoV NC1743 isolate is a highly pathogenic strain and younger turkeys are more susceptible to TCoV infection than older birds. Thus, one-day-old turkeys infected with the minimal infectious dose of TCoV NC1743 could be used as a TCoV disease model to study the disease pathogenesis, and the TCoV NC1743 strain could be used as a challenge virus to evaluate a vaccine protective efficacy.

Metagenomic analysis of the turkey gut RNA virus community.

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.

Astrovirus, reovirus, and rotavirus concomitant infection causes decreased weight gain in broad-breasted white poults.

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.

Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses.

Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A polymerase chain reaction (PCR) assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detecting parvoviruses in experimentally infected chickens. In a nationwide survey, a total of 138 field enteric samples from poultry flocks were tested by PCR for parvovirus presence. Of the tested chicken samples that were collected in 54 farms, 77% showed the presence of parvovirus, while 78% of the turkey samples that were received from 29 farms were parvovirus positive. For the first time, our data clearly demonstrate that parvoviruses are widely distributed in commercial poultry flocks in the United States. The high prevalence of parvovirus infection in birds from enteric disease-affected flocks suggests a potential role of these viruses in the etiology of enteric disease of poultry. Phylogenetic analyses comparing NS gene segments showed that most of the chicken and turkey parvovirus isolates formed separate phylogenetic groups. These findings suggest that the chicken and turkey parvoviruses might have diverged from a common ancestor and have subsequently undergone host-specific adaptation.

Pathogenesis of type 2 turkey astroviruses with variant capsid genes in 2-day-old specific pathogen free poults.

The pathogenicity of three different type 2 turkey astroviruses (TAstV-2) was studied in specific pathogen free turkeys. These viruses differ based on sequence analysis of the capsid gene. Poults were inoculated at 2 days of age and examined during 14 days for clinical signs and virus shedding. All inoculated poults presented signs of enteric disease including diarrhoea and growth depression. Virus presence and shedding was detected by real-time reverse transcriptase-polymerase chain reaction from intestinal contents and cloacal swabs collected at 3, 7 and 14 days post-inoculation. Viraemia was also confirmed by this method. Common lesions observed at necropsy were dehydration; distended intestines filled with watery contents and undigested feed, and dilated caeca with foamy contents. Microscopic lesions present in the intestines consisted of mild crypt hyperplasia, villous atrophy and lymphocytic infiltration, and were most common in the jejunum. Presence of the viruses was demonstrated by immunohistochemistry and by in situ hybridization in both villi and crypt enterocytes in the jejunum and, less frequently, the duodenum, ileum and caeca. Mild lesions consisting mainly of lymphocytic infiltration were also observed in other organs including the pancreas, liver, spleen and kidneys. Mild to moderate bursal atrophy occurred in all TAstV-2-infected poults examined; however, no specific viral staining was observed in this organ or any other tissues examined apart from the intestines. In conclusion, TAstV-2 viruses with variant capsids produce a similar enteric disease in young turkeys and may also affect the immune system of the birds by causing bursal lymphoid depletion.

Turkey origin reovirus-induced immune dysfunction in specific pathogen free and commercial turkey poults.

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/SEP-R44/03. 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.

Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006.

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.

A multiplex RT-PCR test for the differential identification of turkey astrovirus type 1, turkey astrovirus type 2, chicken astrovirus, avian nephritis virus, and avian rotavirus.

Recent studies have revealed the presence ofastroviruses and rotavirus in numerous poorly performing and healthy chicken and turkey flocks in the United States. The phylogenetic analysis of the sequence data produced during these studies has identified four groups of avian astroviruses circulating in the United States: turkey astrovirus types 1 and 2 (TAstV-1 and TAstV-2), avian nephritis virus (ANV), and a chicken-origin astrovirus (CAstV). As the molecular epidemiology of poultry enteric disease is poorly understood, the development of updated diagnostic assays is crucial to the continued surveillance and management of enteric disease in affected as well as healthy flocks. This report details the development of a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for astroviruses and avian rotavirus in turkey-origin and chicken-origin samples. The assay consists of two multiplex tests, one for turkey-origin samples and one for chicken-origin samples. The turkey sample test differentially identifies TAstV-1, TAstV-2, ANV, and avian rotavirus. The test for chicken-origin samples differentially identifies CAstV, ANV, and avian rotavirus. Assay sensitivity varied by target sequence between approximately 10 copies for avian rotavirus alone and approximately 2 x 10(6) copies for TAstV-2 in the presence of a heterologous competitor RNA sequence. Each test was shown to be specific for the intended target by testing for cross-reaction with other common avian enteric viruses. The specificity was further shown by testing 109 chicken specimens and 32 turkey specimens from commercial flocks with the appropriate test and sequencing the RT-PCR amplicons to confirm amplification of the correct target.

Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms.

A longitudinal survey to detect enteric viruses in intestinal contents collected from turkeys in eight commercial operations and one research facility was performed using molecular detection methods. Intestinal contents were collected from turkeys prior to placement, with each flock resampled at 2, 4, 6, 8, 10, and 12 wk of age. The samples were screened for astrovirus, rotavirus, reovirus, and turkey coronavirus (TCoV) by a reverse transcriptase and polymerase chain reaction (RT-PCR), and for groups 1 and 2 adenovirus by PCR. Rotavirus was the only virus detected prior to placement (7 of 16 samples examined). All of the commercial flocks were positive for rotavirus and astrovirus from 2 until 6 wk of age, and most were intermittently positive until 12 wk of age, when the birds were processed. Of the 96 samples collected from birds on the farms, 89.5% were positive for astrovirus, and 67.7% were positive for rotavirus. All flocks were negative for TCoV, reovirus, and group 1 adenovirus at all time points, and positive for group 2 adenovirus (hemorrhagic enteritis virus) at 6 wk of age. All the flocks monitored were considered healthy or normal by field personnel. Turkeys placed on research facilities that had been empty for months and thoroughly cleaned had higher body weights and lower feed conversion rates at 5 wk of age when compared to turkeys placed on commercial farms. Intestinal samples collected at 1, 2, and 3 wk of age from these turkeys were free of enteric viruses. This report demonstrates that astroviruses and rotaviruses may be present within a turkey flock through the life of the flock. Comparison of infected birds with one group of turkeys that were negative for enteric viruses by the methods used here suggests that astrovirus and/or rotavirus may affect production. The full impact on flock performance needs to be further determined.

Molecular Characterization of Enteric Picornaviruses in Archived Turkey and Chicken Samples from the United States.

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 enteric picornaviruses suggests the avian enteric picornaviruses may contribute to the enteric disease syndromes and performance problems often observed in turkeys in the Southeastern United States. This report describes our recent phylogenetic analysis of turkey and chicken enteric samples archived at the Southeast Poultry Research Laboratory from 2004 to present and is a first step in placing these novel avian picornaviruses within the larger Picornaviridae family.

A diarrheic chicken simultaneously co-infected with multiple picornaviruses: Complete genome analysis of avian picornaviruses representing up to six genera.

In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1, KT880670) were identified by viral metagenomic and RT-PCR methods from a single specimen of a diarrheic chicken suffering from a total of eight picornavirus co-infections, in Hungary. The complete genomes of six picornaviruses were determined and their genomic and phylogenetic characteristics and UTR RNA structural models analyzed in details. Picornaviruses belonged to genera Sicinivirus (the first complete genome), Gallivirus, Tremovirus, Avisivirus and "Orivirus" (two potential genotypes). In addition, the unassigned phacoviruses were also detected in multiple samples of chickens in the USA. Multiple co-infections promote and facilitate the recombination and evolution of picornaviruses and eventually could contribute to the severity of the diarrhea in chicken, in one of the most important food sources of humans.