Date Published: April 4, 2017
Publisher: Public Library of Science
Author(s): Mingyue Wang, Jia He, Haibing Lu, Yajing Liu, Yingrui Deng, Lisai Zhu, Changming Guo, Changchun Tu, Xinping Wang, Dong-Yan Jin.
The Enterovirus genus of the family of Picornaviridae consists of 9 species of Enteroviruses and 3 species of Rhinoviruses based on the latest virus taxonomy. Those viruses contribute significantly to respiratory and digestive disorders in human and animals. Out of 9 Enterovirus species, Enterovirus E-G are closely related to diseases affecting on livestock industry. While enterovirus infection has been increasingly reported in cattle and swine, the enterovirus infections in small ruminants remain largely unknown.
Virology, molecular and bioinformatics methods were employed to characterize a novel enterovirus CEV-JL14 from goats manifesting severe diarrhea with morbidity and mortality respectively up to 84% and 54% in China.
CEV-JL14 was defined and proposed as a new Enterovirus species L within the genus of Enterovirus of the family Picornaviridae. CEV-JL14 had a complete genome sequence of 7461 nucleotides with an ORF encoding 2172 amino acids, and shared 77.1% of genomic sequence identity with TB4-OEV, an ovine enterovirus. Comparison of 5’-UTR and structural genes of CEV-JL14 with known Enterovirus species revealed highly genetic variations among CEV-JL14 with known Enterovirus species. VP1 nucleotide sequence identities of CEV-14 were 51.8%-53.5% with those of Enterovirus E and F, 30.9%-65.3% with Enterovirus G, and 43.8–51. 5% with Enterovirus A-D, respectively. CEV-JL14 was proposed as a novel species within the genus of Enterovirus according to the current ICTV demarcation criteria of enteroviruses.
CEV-JL14 clustered phylogenetically to neither Enterovirus E and F, nor to Enterovirus G. It was defined and proposed as novel species L within the genus of Enterovirus. This is the first report of caprine enterovirus in China, the first complete genomic sequence of a caprine enterovirus revealed, and the unveiling of significant genetic variations between ovine enterovirus and caprine enterovirus, thus broadening the current understanding of enteroviruses.
The genus Enterovirus within the family of Picornaviridae consists of 9 species (A, B, C, D, E, F, G, H and J) of Enteroviruses (EV) and 3 species (A, B, C) of Rhinoviruses based on the latest virus taxonomy. Those viruses are the etiological agents contributing to neurological, respiratory and digestive disorders in human and animals[2–8]. Out of 9 Enterovirus species, Enterovirus E (EV-E), Enterovirus F (EV-F) and Enterovirus G (EV-G) are closely related to diseases affecting on the livestock industry, where EV-E and EV-F (formerly named bovine enterovirus A and B) are the causative agents of enterovirus infections in cattle manifesting clinical signs varying from respiratory diseases to enteritic, reproductive disease and infertility[4, 5, 7–11]. The EV-G (formerly named porcine enterovirus B) is the causative agents of swine enterovirus infections[6, 12, 13]. It has been demonstrated that EV-E and EV-F were usually isolated from the cattle manifesting symptoms of digestive and respiratory diseases, suggesting the potential pathogenicity of enterovirus related to those infections. EV-E and EV-F were also occasionally isolated or detected from cattle of subclinical infection or environment[14–17], which leads to the conclusion that EV-E and EV-F were not significant agents to the livestock industry. With increasing number of enteroviruses isolated from animals developing respiratory and digestive diseases, the pathogenicity of enteroviruses to animal has been drawn much attention[7, 8, 11, 13, 18]. While infection of enteroviruses and their sequences have been increasingly reported in cattle and swine recently, the enterovirus infections in small ruminants such as sheep or goat remain largely unknown. Recently, the first complete genome sequence of an ovine enterovirus (TB4-OEV) was reported in 2012, a partial 5’-UTR sequence of a caprine enterovirus in Japan and several BEV-like 5’-UTR sequences in Thailand were determined from diarrheal goats or unidentified goats[8, 19]. Although the phylogenetic analysis has clustered those sequences to clades of BEV-like based on the 5’-UTR sequences, such an analysis need to be reevaluated since the paucity of enough enterovirus sequences from petite ruminants including sheep and goats. It was reported that TB4-OEV strain is likely an interspecies recombinant between bovine enterovirus and porcine enterovirus based on the virus genome sequence analysis, where its 5’-UTR sequence has a high sequence identity with EV-E and EV-F, and the rest of its genome has a high sequence identity to EV-G. Phylogenetic analysis grouped the TB4-OEV as a member of the species EV-G currently.
In this study, we have isolated and characterized a novel enterovirus from goats characterized by severe diarrhea with a high morbidity and mortality. Sequence analysis clearly distinguishes the CEV-JL14 from the EV-E, EV-F, EV-G, and other known enterovirus species based on the current criteria for enterovirus classification. Our phylogenetic analysis showed that CEV-JL14 clustered to neither EV-E, EV-F, EV-G nor other enterovirus species, it is clustered to a novel distinct clade together with TB4-OEV and Caprine enterovirus G1 from Japan and recently reported enterovirus in goats from Thailand. Pursuant to current criteria for classification of enterovirus species, where a range from 50 to 55% for heterologous species, 70 to 85% for heterologous serotypes/homologous species and greater than 90% for homologous serotypes, we proposed that CEV-JL14, together with TB4-OEV and other ovine/caprine enterovirus strains, was classified to a novel enterovirus species L (EV-L). This proposal is based on our following findings: 1) the nucleotide sequence identity of VP1 for CEV-JL14 was only 51.8–53.5% to EV-E and EV-F, 30.9%-65.3% to EV-G, and 43.8–51.5% to EV-A-D, which is well-matched with the criteria that 50–55% of nucleotide sequence belongs to heterogeneous species; 2) the deduced VP1 amino acid sequence were only 48.5%-53.3% to EV-E and EV-F, 57.0%-68.0% to EV-G, and 37.0–45.2% to EV-A-D, which is also consistent with the enterovirus species criteria; 3) CEV-JL14, together with TB4-OEV, was clustered to a distinct clade from EV-E, EV-F, EV-G, and EV-A-D based on the 5’-UTR sequence analysis and their secondary structure prediction. 4) The P1 amino acid sequence identity of CEV-JL14 was 81.1% to TB4-OEV, 61.0–63.1% to EV-E and EV-F, 69.7–74.7% to EV-G, and 45.7–52.9% to EV-A-D. 5) the percentage difference of G+C content for CEV-JL14 and TB4-OEV to EV-G is more than 2.5%, suggesting that CEV-JL14 and TB4-OEV should not listed as member of EV-G. 6) The proteolytic cleavage sites between CEV-JL14 and TB4-OEV is highly conserved, while they are highly variable between CEV-JL14/TB4-OEV and EV-G. Those results support our claim that CEV-JL14, together with other ovine/caprine enterovirus strains, ought to be considered as new species in the genus of Enterovirus. Although cladograms variations were revealed employing different capsid genes for the analysis, similar cladogram patterns were observed even under circumstance of the lack of enough virus isolates. With more and more caprine or ovine enterovirus sequences revealed, the clade will become much clearer.