Chicken Astrovirus (CAstV) Molecular Studies Reveal Evidence of Multiple Past Recombination Events in Sequences Originated from Clinical Samples of White Chick Syndrome (WCS) in Western Canada

Abstract

In this study, we aimed to molecularly characterize 14 whole genome sequences of chicken astrovirus (CAstV) isolated from samples obtained from white chick syndrome (WCS) outbreaks in Western Canada during the period of 2014-2019. Genome sequence comparisons showed all these sequences correspond to the novel Biv group from which no confirmed representatives were published in GenBank. Molecular recombination analyses using recombination detection software (i.e., RDP5 and SimPlot) and phylogenetic analyses suggest multiple past recombination events in open reading frame (ORF)1a, ORF1b, and ORF2. Our findings suggest that recombination events and the accumulation of point mutations may have contributed to the substantial genetic variation observed in CAstV and evidenced by the current seven antigenic sub-clusters hitherto described. This is the first paper that describes recombination events in CAstV following analysis of complete CAstV sequences originated in Canada.

Keywords: chicken astrovirus; molecular epidemiology; recombination; runting-stunting syndrome; white chick syndrome; whole genome sequencing.

Figure 1

Hatching of normal (yellow) and affected (white) chicks. Progenitor broiler breeders had a drop in production, and low hatchability (case 14-1235). Apparently normal chick on the left and an affected “white chick” on the right in (a). Chick box after quality check containing apparently normal chicks on the left, and affected chicks on the right (b).

Figure 2

Post-mortem examination on dead-in-shell embryos and culls of case 15-1262a, and histopathology of cases 14-1235a; and 15-1262a, respectively. Dead-in-shell embryo with enlarged firm green livers in (a). Day-old culled chick with white plumage showing enlarged firm green liver and unabsorbed yolk-sac contents with visible green discoloration in (b). CAstV microphotographs of histopathological liver lesions on white chick syndrome (WCS) clinical cases. Case 14-1235a shows in 20× (c), proliferating bile ducts in black arrows, bile in canalicular lumens in blue arrows, and heterophils and macrophages in the portal vain in the green arrow. Case 15-1262a (d) shows in 40× one small foci of acute hepatic necrosis (black arrow).

Figure 3

Nucleotide ML phylogenetic tree of complete CAstV sequences. Different colors indicate different genotypes according to ORF2 analysis described in Smyth et al. 2017 (i.e., Aiii, Bi, Bii, Biii, and Biv in red) [5]. The included sequences are described in Supplementary Table S1. Canadian sequences are in bold.

Figure 4

Amino acid ML phylogenetic tree of 52 ORF2 CAstV sequences. Different colors indicate different genotypes according to ORF2 analysis described by Smyth 2017 [5]. The included sequences are described in Supplementary Table S1.

Figure 5

Bootscan analysis of recombinant CAstV sequences for confirming recombination was performed using Simplot program v3.5.1. Each analysis considers different parent sequences (different colors) plotted in a graph considering in the vertical-axis Percentage of permuted trees, and on the horizontal axis, position on the genome of the query sequence. Recombinant CAstV query sequences are 19-0981/CA-SK/19 (Supplementary Figure S3a); Biii-ANAND/IN/2016 (Supplementary Figure S3b); Bi-GDYHTJ718-6/CN/2018 (Supplementary Figure S3c); 18-0942/CA-SK/18 (Supplementary Figure S3d); 19-0935/CA-SK/19 (Supplementary Figure S3e); CC_CkAstV/US/2014 (Supplementary Figure S3f); and Bii-GA2011/US/2011 (Supplementary Figure S3g).