Isolation and evolutionary analyses of gout-associated goose astrovirus causing disease in experimentally infected chickens

Abstract

Astroviruses are a common cause of gastroenteritis in humans and animals. They are also associated with extraintestinal infections, including hepatitis in ducklings, nephritis in chickens, as well as fatal meningitis and encephalitis in humans and other mammals. Since 2014, outbreaks of disease characterized by visceral gout and swelling of kidneys have been reported in goslings and ducklings in China, with the causative agent revealed to be a novel avian astrovirus designated goose astrovirus (GoAstV). In the present study, this novel gout-associated GoAstV was identified in diseased goslings from 2 farms in Hunan province, China. Three genomes were successfully sequenced and analyzed and were shown to have high identities of 99.7 to 99.8% between each other, with some specific amino acid alterations revealed in open reading frame 2 when compared with other gout-associated GoAstVs. Two strains were further efficiently isolated in the DF-1 chicken fibroblast cell line with high virus titers of 10¹¹ viral genomic copies per mL of culture media. A pilot virus challenge study using GoAstV in chickens demonstrated that this virus can cause clinical visceral gout in chickens, indicating its ability to cross the species barrier. Based on the phylogenetic analyses of capsid sequences, the identified GoAstVs were proposed to be classified into 2 genotypes, GoAstV1 and GoAstV2, and the novel gout-associated GoAstVs were all clustered in GoAstV2. Further Bayesian inference analyses indicated a nucleotide substitution rate of 1.46 × 10^-3 substitutions/site/year for avian astrovirus based on open reading frame 2 sequences, and the time to the most recent common ancestor of GoAstVs was estimated to be around 2011. This is the first report to confirm GoAstV can infect chickens while also providing an estimation of the evolutionary rates of Avastroviruses.

Keywords: chicken; evolution; goose astrovirus; infection; time to the most recent common ancestor (TMRCA).

Figure 1

Indirect immunofluorescence assay demonstrating DF-1 cells infected with GoAstV isolated in the present study. (A) GoAstV-infected cells immunostained with mouse serum against partial capsid cp1 (green). (B) GoAstV-infected cells immunostained with DAPI (blue). (C) Merge of (A) and (B). (D) GoAstV-infected cells immunostained with mouse serum against partial capsid cp2 (green). (E) GoAstV-infected cells immunostained with DAPI. (F) Merge of (D) and (E). (G–I) Mock infection. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GoAstV, goose astrovirus.

Figure 2

Phylogenetic analyses based on genomes (A) or the complete amino acid sequences of ORF2 (B) of the present 3 GoAstV strains and other representative AAstV strains obtained from GenBank. The tree was constructed by using the Neighbor-Joining method based on the p-distance model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (only >70% are shown). The tree is drawn to scale, with branch lengths measured in numbers of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 5,600 and 549 positions in the final dataset for (A) and (B), respectively. Evolutionary analyses were conducted in MEGA7. The strains of AAstV species with a solid triangle in (B) were previously defined by ICTV and others (Bosch et al., 2011; Guix et al., 2013). The sequences indicated with solid circles are GoAstV2 strains obtained in the present study. Abbreviations: avian astrovirus, AAstV; ICTV, International Committee on Taxonomy of Viruses; GoAstV, goose astrovirus; ORF2, open reading frame 2.

Figure 3

Bayesian MCC tree based on the ORF2 sequences of GoAstVs and other reference AAstVs. The branches are colored according to the most probable ancestral location country of their descendent nodes. The branch length of each taxon is shown above the branches. Abbreviations: Avian astroviruses, AAstVs; GoAstV, goose astrovirus; maximum clade credibility, MCC; ORF2, open reading frame 2.

Figure 4

Clinical signs and gross lesions indicated by white arrows in chickens experimentally infected with GoAstV. (A) Diarrhea with brown-white feces. (B) Mild swollen leg joint. (C) Mild swellings of kidneys and urate deposition on the renal surface and air sacs.