Molecular characterization of bovine leukemia virus reveals existence of genotype 4 in Chinese dairy cattle

Bovine leukemia virus (BLV) causes enzootic bovine leucosis and is widely spread worldwide, except several European countries, Australia and New Zealand. Although BLV is highly prevalent in China, information about the genetic diversity and evolutionary dynamics of BLV among Chinese dairy herds is still lacking. To determine the genetic variability of BLV, 219 cows from four cities of Ningxia province of China were screened for BLV infection by fluorescence resonance energy transfer (FRET)-PCR and sequencing, 16 selected positive samples were subjected to molecular characterization. Phylogenetic analysis using the neighbor-joining (NJ) method on complete sequences of envelope (env) gene of BLV obtained from China and those available in GenBank (representing BLV genotypes 1–10) revealed that those Chinese strains belonged to genotypes 4 and 6. Totally, 23 mutations were identified and 16 of them were determined to be unique mutations among Chinese strains. Alignment of the deduced amino acid sequences demonstrated six mutations in glycoprotein 51 (gp51) and three mutations in glycoprotein 30 (gp30) located in the identified neutralizing domain (ND), CD8+ T cell epitope, E-epitope, B-epitope, gp51N12 and cytoplasmic domain of transmembrane protein. This study reported for the first time the BLV genotype 4 in China, and further studies are warranted to compare its immunogenicity and pathogenicity with other BLV genotypes. Electronic supplementary material The online version of this article (10.1186/s12985-019-1207-8) contains supplementary material, which is available to authorized users.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), and approximately 30% of infected cattle develop persistent lymphocytosis (PL) while a small percentage of infected cattle die from malignant lymphoma. In recent years, a variety of methods have been applied for BLV genotyping [1][2][3]. Due to the biological functions, most of studies have primarily focused on the env gene. To date, at least 11 genotypes of BLV have been described based on the genetic polymorphism of the env gene [4,5]. Previous studies demonstrated that BLV was widely spread among dairy herds in China, and genotypes 6, 10 and 11 existed in Chinese dairy or yak herds [6,7].
From November 2018 to January 2019, bovine whole blood samples (n = 219) from four cities (Shizuishan, Yinchuan, Wuzhong and Zhongwei) of Ningxia province were submitted to Yangzhou University College of Veterinary Medicine for BLV identification. All samples were freshly collected in ethylenediaminetetraacetic acid (EDTA) blood collection tubes by Center for Animal Disease Control and Prevention of Ningxia province, and delivered on ice with next-day delivery. DNA was extracted from whole blood samples using commercial kit as previously described [8]. The FRET-qPCR targeting BLV pol gene (forward primer = 5′-CCTCAATTCCCTTT AAACTAGAACG-3′; reverse primer = 5′-ATGGGC TTTGTAAGAGCATTTGTA-3′; anchor probe = 5′-GACGGGCCAGGCAATAATCCAGT-(6-FAM)-3′; reporter probe = 5′-(LCRed640)-TTCCCGGTACGG AAACCAAATGG-phosphate-3′) was performed following the protocol previous described [9].In total, forty samples were identified to be positive from 219 whole blood samples. Copy numbers of BLV in positive cows ranged from 20 copies/ ml of whole blood to 362,936 copies/ ml of whole blood (mean 19,134 copies/ ml of whole blood and median 35 copies/ ml of whole blood) (Table 1), and those above 130 copies/ ml (n = 16) were further identified for genotyping based on the diversity of env gene.
Partial sequence of BLV env gene were amplified with an in-house regular PCR [6]. Amplicons were gel purified with the QIAquick Gel Extraction Kit and sequenced with both forward and reverse primers at the GenScript Biotech Corp. (Nanjing, China). Sequence data (PCR products based on the forward and reverse primers) obtained in this study were assembled with DNASTAR Lasergen 15.2 (DNASTAR Inc., Madison, WI) and aligned using CLUSTAL W in MEGA 7.0 (MEGA, Pennsylvania State University, University Park) along with those of BLV strains found on GenBank from around the world. A neighbor-joining (NJ) phylogenetic tree was constructed using the Tamura-Nei model [3,10] and the robustness of clusters was assessed by bootstrapping 1,000 replicates. Maximum-likelihood (ML) phylogenetic analysis was performed to confirm the results (Additional file 1 Figure S1).
Those sequences obtained in this study that were not identical to each other were submitted to GenBank with the GenBank accession numbers: MK820044 and MK840875-MK840880. A neighborjoining phylogenetic tree based on the env gene complete sequences (1,548 bp) of the Chinese strains and 37 reference strains representing BLV genotypes 1 to 10 from 14 countries demonstrated that three Chinese strains (MK820044, MK840877 and MK840879) belonged to genotype 4 and the remaining four (MK840875, MK840876, MK840878 and MK840880) belonged to genotype 6 ( Fig. 1).
For those three Chinese strains clustered into BLV genotype 4, the mean distance of the env nucleotides and the deduced amino acid (AA) were 0.003 ± 0.001 and 0.008 ± 0.004 between these strains, respectively ( Table 2). Compared with the BLV strains obtained from GenBank representing BLV genotypes 1 to 10, the Chinese strains had between 0.014 ± 0.002 and 0.038 ± 0.005 nucleotide distance. Similarly, the Chinese strains had between 0.012 ± 0.003 and 0.037 ± 0.008 deduced AA distance compared with these reference strains ( Table 2). The distance of nucleotide and deduced AA indicated that these three Chinese BLV strains were mostly similar to genotype 4 but distinct from genotype 5.
For the remaining four strains clustered into BLV genotype 6, the mean distance of the env nucleotides and the deduced AA were 0.004 ± 0.001 and 0.002 ± 0.002, respectively ( Table 2). Compared with the BLV strains obtained from GenBank, the Chinese strains had nucleotide distance between 0.020 ± 0.003 and 0.046 ± 0.005. Similarly, the Chinese strains had between 0.008 ± 0.003 and 0.037 ± 0.008 deduced AA distance compared with those reference strains ( Table 2). The distance of nucleotide and deduced AA indicated that the three Chinese BLV strains were mostly similar to genotype 6 but distinct from genotype 5. Although 10 genotypes of BLV have been discovered around the world, there is little information on Genotypes shown on the right are according to Yang et al. [6]. The numbers at the branches show bootstrap support (1000 replicates). The bar at the bottom of the figure denotes distance Table 2 Nucleotide and amino acid distances (distances ± SE) of BLV env gene between Chinese strains and the reference strains The values in bold along the diagonal are the distance (intra-genotype) of the nucleotides (above) and amino acids (below) between the Chinese strains and those in GenBank genetic diversity of BLV among Chinese dairy herds [11], until BLV genotype 6 was firstly identified in Yancheng, Shanghai, Yangzhou, Bengbu and Tianjin in 2019 [6]. The present study revealed the existence of BLV genotype 4 in China for the first time.
This study investigated the prevalence and genetic variability of BLV and identified the BLV genotype 4 in China for the first time. Together with our previous study [6] and studies conducted by Wang and Yu in 2018 and 2019 [5,7], BLV genotypes 1, 4, 6 and 10 were present in dairy cattle or yaks in China. BLV genotype 4 is the second most common genotype prevalent worldwide and was identified in Mongolia in 2016 [2]. The cow trade between China and Mongolia might contributed to the spread of BLV between the two countries. This study will help us to better understand the genetic diversity of BLV in China. However, further studies are needed to define the immunogenicity and pathogenicity between different genotypes of BLV.