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. 2021 Feb 3;13(2):235.
doi: 10.3390/v13020235.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3

Paula Lagan Tregaskis  1 , Ryan Devaney  1 , Victoria J Smyth  1
Affiliations

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3

Paula Lagan Tregaskis et al. Viruses. .

Abstract

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5' and 3' regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

Keywords: astrovirus; avian nephritis virus; genotyping and recombination; molecular characterisation; phylogenetics.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic representation of ANV genotype 3 whole genome and ribosomal shift mechanism. (a) Schematic representation of ANV genotype 3 whole genome structural organisation comprising a partially overlapping open reading frame 1a (ORF1(a) and ORF1b and separate ORF2. Motifs and secondary structures located in ORF1a nuclear localisation signal NLS) bipartite KKKGKTK and TEEEY, ORF1b RdRp motifs DWTRFD, GNPSG, YGDD, and FGMWVK. (b) Schematic representation of the partially overlapping and –1 (signified by an arrow) ORF1a and 1b ribosomal frameshift signal with mapped pseudoknot, heptameric slippery sequence, and stemloop. The lysine amino acid (K) in frame 3 where translation recommences after the ribosomal frameshifting signal (RFS) is marked as *.
Figure 2
Figure 2
Astroviridae ribosomal frameshift mechanism ORF1a and ORF1b. Alignment of the Avastroviridae whole genome heptameric slippery sequence and stemloop region including ANV-3 strains and MT585643 VF14-92-A2 and MT585644 VF16-03-164b. Red box is ANV VF14-92-A2 and VF16-03-64b, blue box and * identify the methionine/putative start codons, and the black box marks the heptameric slippery sequence.
Figure 3
Figure 3
(a,b) RNAfold secondary structures of pseudoknot, heptameric slippery sequence, and stemloop with a minimum free energy (MFE) of -16.8 kcal/mol for MT585643 VF14-92-A2 and 164b -17 kcal/mol MFE for MT585644 VF16-03-164b.
Figure 4
Figure 4
Genome coverage of ANV-3. (a) The whole genome coverage validation of ANV-3 reads from MT585643 VF14-92-A2 with MT585644 VF16-03-164b used as a reference sequence. The Geneious read mapping algorithm, set at 5 iterations, resulted in whole genome coverage with 9241 reads. The ORF1a 3′ region had the highest coverage at a 1042 read depth. (b) The BWA-MEM read aligner/Tablet visualisation showed a similar profile to Geneious and whole genome breadth and depth of coverage.
Figure 5
Figure 5
Astroviridae whole genome sequence (WGS) phylogeny. The Avastroviridae whole genome nucleotides of 78 sequences, starting from ORF1a phylogenetic analysis in MEGA X from MUSCLE-aligned sequences. Evolutionary history was inferred by maximum likelihood with bootstrapping of 1000 replicates. The tree was annotated into clades and ANV genotypes coloured blue with sequences from this study, with MT585643, VF14-92-A2, and MT585644 VF16-03-164b positioned in Avastrovirus genotype 3. Clade abbreviations; CAstV = Chicken astrovirus, DAstV = Duck astrovirus, GAstV = Goose astrovirus, ANV = Avian nephritis virus, and TAstV = turkey astrovirus.
Figure 6
Figure 6
Astroviridae capsid ORF2 phylogeny. The Avastroviridae capsid ORF2 amino acids of 145 sequences phylogenetic analysis in MEGA X from MUSCLE-aligned sequences. Evolutionary history was inferred by maximum likelihood with bootstrapping of 1000 replicates. The tree is annotated in clades and ANV genotypes coloured blue with sequences from this study (in bold), with MT585643, VF14-92-A2, and MT585644 VF16-03-164b positioned in avian nephritis genotype 3. Clade abbreviations; CAstV = Chicken astrovirus, DAstV = Duck astrovirus, GAstV = Goose astrovirus, ANV = Avian nephritis virus, and TAstV = turkey astrovirus.
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