Development of an indirect enzyme-linked immunosorbent assay test for detecting antibodies to chicken astrovirus in chicken sera.

The development of an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of Group B chicken astrovirus (CAstV) infections is described. The test was based on the use of an affinity-purified capsid antigen, specific to CAstV isolate 11672, produced as a glutathione-S-transferase N-terminal fusion protein by a recombinant baculovirus. Strongly positive ELISA signals were elicited against experimentally produced antisera raised to CAstVs from Group B (subgroups i and ii) but were negative for antisera raised to a Group A CAstV. Using a panel of 240 selected serum samples, 99% agreement was observed when the results obtained by ELISA were compared to those from an indirect immunofluorescence test for CAstV 11672. The ELISA test was applied to 68 serum sets comprising 1864 samples, which were obtained from parent and grandparent flocks originating mainly in the UK. Of the 52 sets containing ELISA-positive samples, 24 sets had >75% samples positive and nine sets had <25% samples positive and were regarded as having high and low seropositivities, respectively. Of the 1864 serum samples tested 1090 (58.5%) were ELISA positive and of these, 234 sera (21.5%) produced strongly positive signals, whereas moderately positive and weakly positive signals were produced by 562 (51.5%) and 294 (27%) sera. When used for flock screening purposes, this ELISA test can be used to (i) investigate the occurrence of first-time CAstV infections of parent flocks during lay and the possible adverse effects caused by vertically transmitted CAstV infections on broiler hatchability and performance and (ii) diagnose Group B CAstV infections within specific pathogen free flocks.

Chicken astrovirus capsid proteins produced by recombinant baculoviruses: potential use for diagnosis and vaccination.

Chicken astroviruses (CAstVs) have been characterized recently. Due to their relatively poor growth in cell culture, virus-specific antigens are not readily available for the development of diagnostic reagents and vaccines. For this purpose two capsid protein antigens, specified by the 11672 isolate of CAstV, were produced in insect cells following infection with recombinant baculoviruses. The GST-11672 capsid protein, a fusion protein comprising the capsid protein and glutathione-S-transferase (GST) as an N-terminal affinity tag, and the 11672 capsid protein alone were detected by western blotting as proteins of ~100 and 70 kDa, respectively. Immunization with the affinity-purified GST-11672 capsid protein produced a polyclonal rabbit antiserum, which reacted by indirect immunofluorescence with Group B CAstVs but which showed no reactivity with the Group A CAstV isolate, 612. When used as part of an immunoperoxidase-based immunohistochemical procedure, this rabbit antiserum facilitated the detection of CAstV antigen in formalin-fixed, paraffin-embedded kidney tissue at the sites of histopathology characteristic of nephritis. Although further evaluation with sera from commercial chickens is required, a prototype indirect antibody-detecting enzyme-linked immunosorbent assay (ELISA) based on affinity-purified GST-11672 capsid protein as coating antigen demonstrated considerable potential with low ELISA absorbance values being generated with sera from specific pathogen free (SPF) chickens, and high absorbance values being generated with serum samples from experimentally infected chickens. Immunization experiments of SPF chickens showed that, when administered as mixtures with oil adjuvant, crude cell lysates containing the GST-11672 capsid protein or the 11672 capsid protein elicited virus-specific antibody responses that were detectable by indirect immunofluorescence and by virus neutralization assays.

Capsid protein sequence diversity of chicken astrovirus.

The complete capsid gene sequences of 24 chicken astroviruses (CAstVs), collected in the UK, Germany, the Netherlands and South Africa from the 1980s to 2008, were determined and compared with that of a US CAstV (UGA-2006). Pairwise comparisons and phylogenetic analysis demonstrated the existence of two major capsid groups, designated A and B, which shared 38 to 40% amino acid identity. CAstVs from groups A and B shared capsid protein identities ranging from 26 to 38% with other avian astroviruses. The group A CAstVs comprised three subgroups, which displayed inter-subgroup identities ranging from 77 to 82%, while group B comprised two clearly separated subgroups, Bi and Bii, which displayed intra-subgroup identities of 97 to 99% and 94 to 99%, respectively, and shared inter-subgroup identities of 84 to 85%. Phylogenetic analyses performed with contiguous open reading frame 1b (polymerase) and open reading frame 2 (capsid) CAstV sequences showed that CAstVs from capsid subgroup Bi had polymerase genes that differed from those possessed by CAstVs belonging to group A and subgroup Bii. The N-terminal capsid regions (residues 1 to 415) were more conserved than the C-terminal regions, with the C-terminal regions of the subgroup Bi and Bii CAstVs sharing 76 to 78% amino acid identity, while the C-terminal regions of the A subgroups displayed identities less than 75%. CAstVs representative of both capsid groups and more than one subgroup were detected within the same broiler flock. The high level of capsid sequence diversity observed in this study has important implications for both the control and diagnosis of CAstV infections.

Capsid protein sequence diversity of avian nephritis virus.

The capsid gene sequences of 25 avian nephritis viruses (ANVs), collected in the UK, Germany and Belgium from the 1980s to 2008, were determined and compared with those of serotype 1 (ANV-1) and serotype 2 (ANV-2) ANV isolates. Amino acid identities as low as 51% were determined. Pairwise comparisons supported by phylogenetic analysis identified six ANVs, including ANV-1 and ANV-2, which shared<80% amino acid identities with one another, and which were selected to be representative of six groups. The ANVs were not distributed according to geographical location or year of sampling, and the detection of ANVs from five different groups in 11 samples sourced from six flocks belonging to the same UK organization within a 4-month period indicated that sequence-diverse ANVs were co-circulating. Amino acid alignments demonstrated the existence of variable regions throughout the capsid protein, nine of which were selected for detailed comparisons. With most ANVs, the variable region sequences were similar to those of one of the six representative ANVs, but some ANV capsids displayed novel variable region profiles, in which variable regions that were characteristic of more than one representative ANV were present. Phylogenetic analysis based on C-terminal sequences of approximately 260 amino acids and SimPlot analysis provided evidence that RNA recombination events located in the 1250 to 1350 nucleotide region resulted in new combinations of the N-terminal and C-terminal capsid regions. The high level of capsid sequence diversity observed in the present study has important implications for both the control and diagnosis of ANV infections.

Development and evaluation of real-time TaqMan® RT-PCR assays for the detection of avian nephritis virus and chicken astrovirus in chickens.

The development and preliminary evaluations of two TaqMan®-based, real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays for the quantitative detection of avian nephritis virus (ANV) and chicken astrovirus (CAstV) RNAs are described. The assays used amplicons generated from the 3' untranslated region of the ANV genome and a conserved region of CAstV open reading frame 1b including its junction with open reading frame 2. High virus RNA levels (>10(5.99) viral copies) were detected for ANV and CAstV in 81% and 67% gut content samples from growth-retarded broiler flocks. Results from longitudinal surveys of two broiler flocks showed that ANV and CAstV RNAs were detected in most gut content and kidney samples collected at all time points from day 0 to day 35, with RNA levels of both astroviruses being higher in the gut contents than in the kidneys, and with the ANV RNA levels being greater than those of CAstV especially at early (days 7 and 14) time points. When the results obtained for the days 4/5 time-point samples from four broiler flocks with varying growth performances were compared, the two better-performing flocks had 100-fold to 1000-fold less ANV viral copies than the flocks that performed least well. Application of the rRT-PCR tests to samples collected from broiler chicks, which were experimentally infected with a crude gut content inoculum, demonstrated that ANV RNA could be detected in gut content and kidney samples at levels similar to those found at corresponding time points in longitudinal survey samples, whereas CAstV RNA was detected at lower levels than in the longitudinal survey samples, especially in kidney samples.

Development and application of an RT-PCR test for detecting avian nephritis virus.

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting avian nephritis virus (ANV) is described. Primers, which amplified a fragment of 182 base pairs (bp), were located in the conserved 3' untranslated region (UTR) of the genome. The limit of detection of the test was estimated to be approximately 18 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative ANV samples, some of which were not detected by previously described RT-PCR tests for detecting ANV, but other avian astroviruses including chicken astrovirus isolates and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples from UK, German and US broiler flocks with enteritis/growth problems, ANVs were detected by RT-PCR in 82/82 (100%) samples. ANVs were also detected in 80/96 (83%) pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performance. Whereas all samples collected on day 0 from the surveys were negative for ANV, all samples collected at days 4/5, 7, 10, 14, 21 and 28 tested positive. Sequence determinations performed with amplicons produced with 14 field samples confirmed the ANV specificity of the test, while comparative and phylogenetic analyses based on 109-nucleotide 3'-UTR sequences demonstrated that the majority of ANVs investigated were more closely related to the serotype 2 ANV (accession number AB 046864) than to the serotype 1 ANV (accession number NC 003790).

Detection of chicken astrovirus by reverse transcriptase-polymerase chain reaction.

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting chicken astroviruses (CAstV) is described. Primers, which amplified a fragment of 510 base pairs, were located in conserved regions within the ORF 1b (RNA polymerase) gene. The limit of detection of the test was estimated to be approximately 60 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative CAstV samples, some of which were not detected by a previously described RT-PCR test for detecting CAstV, but other avian astroviruses including avian nephritis virus and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples and swabs from UK and German broiler flocks with growth problems, CAstVs were detected by RT-PCR in 50/52 (96%) samples. CAstVs were detected in between 30% and 72.5% pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performances. Whereas all day 0 samples were CAstV-negative, high detection rates were observed when the surveyed birds were aged 4, 5 and 7 days. Based on partial ORF 1b sequences, a phylogenetic analysis of 20 CAstVs indicated the existence of two groups. One group comprised four CAstV isolates, including FP3 and 11672, and two field CAstVs, which shared >94% nucleotide identity. The remaining 14 CAstVs, comprising the first characterized CAstV and 612 isolates and 12 field CAstVs, shared 85% to 99% nucleotide identity and displayed 76% to 79% nucleotide identity with the 11672-like and FP3-like CAstVs.

Avian encephalomyelitis virus in reared pheasants: a case study.

An outbreak of neurological disease occurred in pheasant chicks on a game farm in 2007. The disease was first seen in the 10th hatching of chicks on the farm. Affected chicks showed trembling and incoordination from the time of hatching, and subsequently blindness and cataract formation was seen in some of the affected chicks at 3 weeks of age. The peak mortality and culling figure was 21.0% in the worst affected hatch, compared with a maximum of 11.7% in the first nine hatches. No further cases were evident by 7.5 weeks of age. Histopathological examination showed a moderate acute encephalomyelitis in some, but not all, of the chicks with neurological signs. The clinical presentation and histopathological findings were typical of vertically transmitted avian encephalomyelitis as seen in chickens, although avian encephalomyelitis virus could not be detected in inoculated embryonated chicken eggs. However, serological testing by enzyme-linked immunosorbent assay for antibodies to the virus was positive in four of five affected 3-week-old birds and in 23 out of 29 adult breeding birds, and reverse transcriptase-polymerase chain reaction testing of RNA extracted from brain and pancreas tissue of affected chicks yielded nucleotide sequences aligned 82% and 83% with three avian encephalomyelitis sequences in a sequence database. The evidence suggested that the neurological disease was attributable to infection with a strain of avian encephalomyelitis virus that appeared to have entered the flock at the start of the breeding season, and was possibly introduced by carrier pheasants brought on to the farm early in the season.

Identification of chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses.

Earlier work identified and biologically characterized antigenically distinct enterovirus-like viruses (ELVs) of chickens. Three of these ELVs can now be identified as astroviruses. Characterization involved the use of a hitherto undescribed, degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) to amplify astrovirus open reading frame (ORF) 1b-specific cDNA fragments followed by nucleotide sequence determination and analysis of the amplified fragments. ELV-1 was confirmed as an isolate of the astrovirus avian nephritis virus (ANV). ELV-4 (isolate 612) and ELV-3 (isolates FP3 and 11672) were antigenically and genetically related to the second characterized astrovirus of chickens, namely chicken astrovirus (CAstV). Using indirect immunofluorescence, the FP3 and 11672 ELV-3 isolates were very closely related to one another, and less closely related to ELV-4 and the previously described CAstV (P22 18.8.00 reference isolate). Comparative analyses based on the ORF 1b amplicon sequences showed that the FP3 and 11672 ELV-3 isolates shared high nucleotide (95%) and amino acid (98%) identities with one another, and lower nucleotide (76% to 79%) and amino acid (84% to 85%) identity levels with ELV-4 and the reference CAstV P22 18.8.00 isolates. The combined degenerate primer RT-PCR and sequencing methods also provided a nucleotide sequence specific to duck hepatitis virus type 2 (DHV-2) (renamed duck astrovirus) and duck hepatitis virus type 3 (DHV-3), which, for the first time, can also be identified as an astrovirus. Phylogenetic analyses based on the amplified ORF 1b sequences showed that ANV was the most distantly related avian astrovirus, with DHV-3 being more closely related to turkey astrovirus type 2 than DHV-2.