Genetic diversity and recombination of bovine enterovirus strains in China

Abstract

Bovine enterovirus (BEV) consisting of enterovirus species E (EV-E) and F (EV-F) is the causative agent associated with respiratory and gastrointestinal diseases in cattle. Here, we reported the characterization, genetic diversity, and recombination of novel BEV strains isolated from the major cattle-raising regions in China during 2012-2018. Twenty-seven BEV strains were successfully isolated and characterized. Molecular characterization demonstrated that the majority of these novel BEV strains (24/27) were EV-E, while only few strains (3/27) were EV-F. Sequence analysis revealed the diversity of the circulating BEV strains such as species and subtypes where different species or subtype coinfections were detected in the same regions and even in the same cattle herds. For the EV-E, two novel subtypes, designated as EV-E6 and EV-E7, were revealed in addition to the currently reported EV-E1-EV-E5. Comparative genomic analysis revealed the intraspecies and interspecies genetic exchanges among BEV isolates. The representative strain HeN-B62 was probably from AN12 (EV-F7) and PS-87-Belfast (EV-F3) strains. The interspecies recombination between EV-E and EV-F was also discovered, where the EV-F7-AN12 might be from EV-E5 and EV-F1, and EV-E5-MexKSU/5 may be recombined from EV-F7 and EV-E1. The aforementioned results revealed the genetic diversity and recombination of novel BEV strains and unveiled the different BEV species or subtype infections in the same cattle herd, which will broaden the understanding of enterovirus genetic diversity, recombination, pathogenesis, and prevention of disease outbreaks.

Importance: Bovine enterovirus (BEV) infection is an emerging disease in China that is characterized by digestive, respiratory, and reproductive disorders. In this study, we first reported two novel EV-E subtypes detected in cattle herds in China, unveiled the coinfection of two enterovirus species (EV-E/EV-F) and different subtypes (EV-E2/EV-E7, EV-E1/EV-E7, and EV-E3/EV-E6) in the same cattle herds, and revealed the enterovirus genetic exchange in intraspecies and interspecies recombination. These results provide an important update of enterovirus prevalence and epidemiological aspects and contribute to a better understanding of enterovirus genetic diversity, evolution, and pathogenesis.

Keywords: bovine enterovirus; cattle; isolation; molecular analysis; recombination.

Fig 1

Phylogenetic analyses of P1 on the novel bovine enterovirus strains. The reference sequences include the representative sequences of all known EV-E and EV-F types and the representative sequences of all 12 enterovirus species and 3 rhinovirus species in the Enterovirus genus were used as outgroup sequences. The amino acid sequences of P1 were used to construct the phylogenetic tree using the maximum likelihood method with 1,000 bootstrap replications. Bootstrap values of >50 are shown at the nodes. The scale bar represents 10% nucleotide sequence divergence for maximum likelihood methods. The analysis models for ML methods for the P1 amino acid are LG + G + I. Viruses are marked with symbols as follows: ● refers to the EV-E strains obtained in this study; ▲ refers to the EV-F strains obtained in this study; ◇ stands for the CEV-JL14 strain; ○ refers to the HY12 EV-E strain isolated from cattle; △ stands for the SD-S67 strain (an EV-F) isolated from goats.

Fig 2

Phylogenetic analyses of 2C + 3 CD on novel bovine enterovirus strains. The reference sequences include the representative sequences of all known EV-E and EV-F types and the representative sequences of all 12 enterovirus species and 3 rhinovirus species in the Enterovirus genus were used as outgroup sequences. The amino acid sequences of 2C + 3 CD were used to construct the phylogenetic tree using the maximum likelihood method with 1000 bootstrap replications. Bootstrap values of >50 are shown at the nodes. The scale bar represents 5% nucleotide sequence divergence for maximum likelihood methods. The analysis models for ML methods for the 2C + 3 CD amino acid is LG + G + I. Viruses are marked with symbols as follows: ● refers to the EV-E strains obtained in this study; ▲ refers to the EV-F strains obtained in this study; ◇ stands for the CEV-JL14 strain; ○ refers to the HY12 EV-E strain isolated from cattle; △ stands for the SD-S67 strain (an EV-F) isolated from goats.

Fig 3

Phylogenetic analyses for BEV subtyping. Phylogenetic analysis was performed to subtype the BEV strain based on the VP1 amino acid sequences. The reference sequences including the representative sequences of all known EV-E and EV-F types and the representative sequences of all 12 enterovirus species and 3 rhinovirus species within the Enterovirus genus were used as outgroup sequences. A phylogenetic tree was generated using the maximum likelihood method with 1000 bootstrap replications. The scale bar represents 10% nucleotide sequence divergence for maximum likelihood methods. The analysis model for ML methods for the VP1 amino acid is LG + G. Viruses are marked with symbols as follows: red characters refer to the EV-E and EV-F strains obtained in this study, and white characters in the EV-E1 subtype refer to the EV-E strains obtained in this study; the green character stands for the strains isolated in the laboratory. The subtypes of the strains are shown in the figure. R-A stands for rhinovirus A, R-B stands for rhinovirus B, R-C stands for rhinovirus C.

Fig 4

Recombination analyses of the HeN-B62 strain with EV-F types. The genome sequence of HeN-B62 (A) was used as query sequences in the bootscan analysis. The structural map of the bovine enterovirus genome is displayed below each panel. The possible crossover breakpoints were marked with a rectangular box with the enterovirus subtypes and represented by corresponding colors (B). Each point plotted is the percentage identity within a sliding window 200 nt wide centered on the position plotted, with a step size of 20 nt between points.

Fig 5

Recombination analyses between EV-E and EV-F types. The genome sequences of EV-F7-AN12 (A) and EV-E5-MexKSU/5 (B) were used as query sequences in the bootscan analysis. The structural map of the bovine enterovirus genome was displayed below each panel. The possible crossover breakpoints were marked with a rectangular box with the enterovirus subtypes and represented by corresponding colors. Each point plotted is the percentage identity within a sliding window 200 nt wide centered on the position plotted, with a step size of 20 nt between points.