Epidemiology and evolution of novel deltacoronaviruses in birds in central China

Abstract

The variety and widespread of coronavirus in natural reservoir animals is likely to cause epidemics via interspecific transmission, which has attracted much attention due to frequent coronavirus epidemics in recent decades. Birds are natural reservoir of various viruses, but the existence of coronaviruses in wild birds in central China has been barely studied. Some bird coronaviruses belong to the genus of Deltacoronavirus. To explore the diversity of bird deltacoronaviruses in central China, we tested faecal samples from 415 wild birds in Hunan Province, China. By RT-PCR detection, we identified eight samples positive for deltacoronaviruses which were all from common magpies, and in four of them, we successfully amplified complete deltacoronavirus genomes distinct from currently known deltacoronavirus, indicating four novel deltacoronavirus stains (HNU1-1, HNU1-2, HNU2 and HNU3). Comparative analysis on the four genomic sequences showed that these novel magpie deltacoronaviruses shared three different S genes among which the S genes of HNU1-1 and HNU1-2 showed 93.8% amino acid (aa) identity to that of thrush coronavirus HKU12, HNU2 S showed 71.9% aa identity to that of White-eye coronavirus HKU16, and HNU3 S showed 72.4% aa identity to that of sparrow coronavirus HKU17. Recombination analysis showed that frequent recombination events of the S genes occurred among these deltacoronavirus strains. Two novel putative cleavage sites separating the non-structural proteins in the HNU coronaviruses were found. Bayesian phylogeographic analysis showed that the south coast of China might be a potential origin of bird deltacoronaviruses existing in inland China. In summary, these results suggest that common magpie in China carries diverse deltacoronaviruses with novel genomic features, indicating an important source of environmental coronaviruses closed to human communities, which may provide key information for prevention and control of future coronavirus epidemics.

Keywords: bird deltacoronavirus; common magpie; genome recombination; spike; viral genome.

FIGURE 1

The maximum likelihood tree of C‐terminal domain fragment (261 amino acids) in RNA‐dependent RNA polymerase (RdRp). The tree was constructed using IQ‐treewith LG + I + G4 substitution model and 10,000 ultrafast bootstraps. The scale bar indicates amino acid substitutions per site. The four newly identified HNU CoVs are shown in triangle and bold

FIGURE 2

Genome organization of members of Deltacoronavirus. Open reading frames downstream of spike (S) gene are magnified to show the differences among the genomes of the 15 CoVs. S, envelope (E), membrane (M) and nucleocapsid (N) genes are represented by green boxes. Putative accessory proteins are represented by blue boxes. The novel HNU CoVs discovered in this study are shown in bold.

FIGURE 3

The maximum likelihood tree of amino acid sequences of chymotrypsin‐like protease (3CL^pro), helicase (Hel), polyprotein 1ab (ORF1ab), spike (S) protein and nucleocapsid (N) protein. These trees were constructed using IQ‐tree with 10,000 ultrafast bootstraps, and their substitution models were as follows: LG + G4 (3CL^pro), LG + F + R2 (Hel), LG + F + R5 (ORF1ab), WAG + I + G4 + F (S), LG + F + G4 (N). The scale bar indicates amino acid substitutions per site. The four newly identified HNU CoVs are shown in triangle and bold.

FIGURE 4

Detection of potential recombination events of novel HNU CoVs. (a) The possible recombination events in HNU1‐1 or HNU1‐2 strain. (b) The possible recombination events in HNU2. Both of Similarity Plot and Bootscan method in Simplot v3.5.1 were performed with an F84 distance model, a window size of 800 base pairs and a step size of 100 base pairs. The bootstrap replicates of Bootscan were set to 1,000.

FIGURE 5

Bayesian phylogeographic analysis of 24 bird deltacoronaviruses strain sequences based on RdRp gene. Maximum clade credibility (MCC) tree with ancestral state reconstruction according to a discrete trait model, the substitution model was GTR + F + R3 which obtained by Model Finder under the BIC standard, the colours of branch and internal nodes indicated the most probable state reconstruction. The numbers on branch indicated posterior probability, and the time of divergence estimated was marked near the internal node. The root state posterior probabilities estimated for each regions of Bayesian phylogeographic MCC tree, which were inferred using an asymmetric substitution model.