Occurrence of astrovirus in young racing pigeons and genome characterization of 2 new astrovirus genomes representing 2 new species

Abstract

Enteropathies are a serious concern in racing pigeons as they significantly impair performance in races and their training, and viruses are suspected to be one of the main factors. Astroviruses are well-known to be responsible for causing enteric disease in humans and various other animals including birds, although their prevalence and pathogenicity in pigeons is poorly understood. In this study, we investigated 2 groups of young racing pigeons (sick-study group and healthy-control group) to assess the correlation between the number of astrovirus genome copies in cloacal swabs and the occurrence of enteropathy. To determine this, we developed a novel TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for astrovirus detection and absolute quantitative analysis. We also performed high-throughput sequencing to obtain the complete genome sequences and establish the genetic similarity of the obtained strains to known astroviruses of poultry and other avian species. Two new complete genome sequences of pigeon astroviruses in the Avastrovirus genus were identified, representing 2 new species. These were found most closely related to astroviruses identified in Columbidae species and chickens. They share an average of 75.8% genome-wide pairwise identity and 57.6% and 64.6% capsid protein sequence identity with other unclassified columbid avastrovirus sequences in GenBank. Although the difference in prevalence of astrovirus in the study and control group was found statistically insignificant, there was a significant difference between the number of genome copies in positive samples from both groups. These unambiguous results leave the role of astroviruses as enteropathogenic factors in pigeons still undetermined.

Keywords: astrovirus; complete genome; digital droplet PCR; enteropathy; pigeon.

Figure 1

The standard curve (A) and amplification curves (B) of the TaqMan quantitative PCR (qPCR) sensitivity test. The individual curves represent consecutive tenfold dilutions (2.358 × 10⁸-2.358 × 10²). The flat line in lilac represents the negative control.

Figure 2

(A) Results of digital droplet (ddPCR) quantification of astrovirus genetic material in both examined groups of pigeons expressed as mean genome copy number per 20 μL of the sample. Hollow squares represent mean values for each group. Symbols “#” and “*” indicate a statistically significant difference in astrovirus genome copy numbers between the groups (P < 0.05). (B) Visual presentation of example samples, as shown in QuantaSoft software (Bio-Rad, Hercules, California). From top to bottom—a sample with high, moderate and low number of genome copies.

Figure 3

Schematic illustration of genome organization of obtained pigeon astrovirus sequences.

Figure 4

(A) Neighbor-joining phylogenetic tree of twenty-eight astrovirus sequences retrieved from GenBank database and 2 astrovirus sequences obtained in this study. All sequences used are complete genome sequences, with the exception of 4 (accession numbers FR727146-FR727149) consisting of partial RNA-dependent RNA polymerase (RdRp) and complete capsid protein CDs. The tree was built with the use of MEGA 11 software using Tamura-Nei genetic distance model with distances calculated on the basis of the number of base substitutions per site, the bootstrap number of 1,000 repetitions, gamma distribution of 1 and pairwise deletion as a missing data treatment (Saitou and Nei, 1987; Tamura and Nei, 1993; Tamura et al., 2021). Individual sequences are labelled with an accession number and the host name. The sequences obtained in this study are additionally labelled with the strain name. (B) Pairwise identity matrix of the 2 astrovirus sequences obtained in this study with other avian astrovirus sequences retrieved from GenBank database and used for phylogenetic analysis. All sequences used are complete genome sequences, with the exception of 4 (accession numbers FR727146-FR727149) consisting of partial RNA-dependent RNA polymerase (RdRp) and complete capsid protein CDs.

Figure 5

(A) Neighbor-joining phylogenetic tree of twenty-eight ORF2 amino acid sequences of astroviruses retrieved from GenBank database and of 2 astroviruses obtained in this study. The tree was built with the use of MEGA 11 software using Tamura-Nei genetic distance model with distances calculated on the basis of the number of base substitutions per site, the bootstrap number of 1,000 repetitions, gamma distribution of 1 and pairwise deletion as a missing data treatment (Saitou and Nei, 1987; Tamura and Nei, 1993; Tamura et al., 2021). Individual sequences are labelled with an accession number and the host name. The sequences obtained in this study are additionally labelled with the strain name and underlined. (B) Pairwise identity matrix of the ORF2 amino acid sequences of the 2 astroviruses obtained in this study and of other avian astrovirues retrieved from GenBank database and used for phylogenetic analysis.