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 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).