Molecular Characterization of Bovine Foamy Virus and Its Association with Bovine Leukemia Virus Infection in Vietnamese Cattle
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NOTE Virology Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle Dung Thi LE1)#, Son Vu NGUYEN2,3)#, Mari OKAMOTO1), Nanako YAMASHITA-KAWANISHI1), Tung Duy DAO4,5), Vuong Nghia BUI4,5), Haruko OGAWA4), Kunitoshi IMAI4) and Takeshi HAGA1)* 1)Division of Infection Control and Disease Prevention, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2)Department of Veterinary Pathology, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi 100000, Vietnam 3)Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 4)Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan 5)National Institute of Veterinary Research, 86 Truong Chinh, Dong Da, Hanoi 100000, Vietnam ABSTRACT. The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using J. Vet. Med. Sci. conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine 83(8): 1273–1277, 2021 leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene doi: 10.1292/jvms.21-0190 was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This Received: 27 March 2021 study provides information regarding BFV infection and confirms the existence of two BFV clades Accepted: 10 June 2021 among Vietnamese cattle for the first time. Advanced Epub: KEY WORDS: bovine foamy virus (BFV), bovine leukemia virus (BLV), cattle, Vietnam 28 June 2021 Foamy viruses (FVs) belong to the subfamily Spumaretrovirinae within the family Retroviridae. FVs are ubiquitous in a wide range of mammals, including nonhuman primates (Simian FV, or SFV), chimpanzees (Prototype FV, or PFV), bats (Chiropteran FV, or CFV), cats (feline FV, or FFV), horses (equine FV, or EFV), and cattle (bovine FV, or BFV). In their natural hosts, they establish a lifelong, persistent infection [21, 28]. Although FVs have been known to cause a pronounced cytopathic effect in many tissue culture cells in vitro [21, 31], these viruses have not been shown to be associated to a defined disease with obvious clinical signs. However, the pathogenic potential of FVs was suggested. A previous study demonstrated the transient depression of the cell- mediated immune response occurred in rabbits infected by SFV [10]. Another study showed that chronic progressive polyarthritis of cats has been associated with FFV [24]. In humans, among patients with subacute granulomatous thyroiditis (de Quervain) and autoimmune hyperthyroidism (Grave’s disease), PFV was commonly found [17, 32]. Besides, mice transgenic expressing the partial or complete genome of PFV were shown to develop progressive encephalopathy and myopathy [1, 4]. BFV was first isolated from lymphosarcomatous and apparently healthy cattle in 1969 [19]. Subsequently, BFV infection was confirmed in livestock cattle in different parts of the world [2, 3, 5, 11, 12, 14, 20, 23, 27, 33]. Moreover, the previous studies showed the concomitant infection of BFV with either bovine leukemia virus (BLV) or bovine immunodeficiency virus, and suggested the presence of BFV in the host may contribute to diseases caused by other pathogens [2, 12, 13]. The BFV genome is composed of gag, pol, and env structural genes flanked by the long terminal repeats (LTRs) and additional accessory genes designated as tas and bet [26]. The previous results of phylogenetic analysis suggested that BFV could be classified into two distinct clades, the European clade and the non-European clade [7, 9, 23]. The European clade comprised the two European isolates, including BFV Reims and BFV 100, and the non-European clade comprising the BFV isolates from China, USA, and Japan [7, 9, 23]. *Correspondence to: Haga, T.: [email protected] #These authors contributed equally to this work. (Supplementary material: refer to PMC https://www.ncbi.nlm.nih.gov/pmc/journals/2350/) ©2021 The Japanese Society of Veterinary Science This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) 1273 D. T. LE ET AL. Although BFV has been detected worldwide, epidemiology and molecular characteristics of BFV remain to be the least analyzed among the retroviruses. This study aimed to determine for the first time, the presence of BFV and its molecular characteristic in Vietnamese cattle. Additionally, we investigated the association between BFV and BLV infection. The present study will update and contribute to enriching the knowledge in the prevalence and molecular characteristics of BFV. In this study, 243 blood samples were collected from 67 beef and dairy cattle farms located in the three Vietnamese provinces, namely, Hanoi, Vinhphuc, and Bacninh, from February 2017 to June 2018. All investigated cattle did not show any symptoms at the time of sampling. Peripheral blood was obtained from the jugular vein and used for DNA extraction. The blood samples were collected following the guidelines of National technical regulation on Animal diseases–General requirements for sample collection, storage, and shipment (QCVN01-83:2011/BNNPTNT). Permission was obtained from the farm owners before collecting the samples. Total DNA was extracted from each EDTA-treated whole blood sample using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) and High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s instructions. DNA samples were used to confirm the existence of BFV proviral DNA by PCR assays targeting gag and pol genes. The partial sequence of pol and gag genes of the BFV provirus were amplified by PCR using the primer sets (Table 1). Simultaneously, we used the extracted DNA samples to screen the prevalence of BLV infection and the results were published [6, 18]. In the present study, additional primer sets suggested by Dao et al. [6] targeting to env-gp51 gene were used to confirm the results obtained in the previous studies (Table 1). Each PCR reaction mixture contained 10 µl of Buffer KOD FX Neo 2×, 4 µl of distilled water, 4 µl of dNTPs 2 mM, 0.8 µl of primers mixture, 0.4 µl of KOD FX Neo Polymerase (Toyobo, Osaka, Japan) and 0.8 µl of extracted DNA/the first round PCR product. Distilled water was used as a negative control for PCR. The thermal cycling conditions for both PCR reactions targeting BFV pol and gag genes were as follows: an initial denaturation at 94°C for 2 min; 40 cycles of denaturing at 98°C for 10 sec, annealing at 64.5°C for 30 sec, and extension at 68°C for 15 sec. The thermal cycle reactions for the first round PCR to amplify the BLV env gene were carried out in the following conditions: an initial denaturation at 94°C for 2 min; followed by 40 cycles of denaturing at 98°C for 10 sec, annealing at 57°C for 30 sec, and extension at 72°C for 1 min; an additional cycle was run at 72°C for 10 min. The reaction parameters for the second round PCR were 2 min at 94°C for initial denaturation, followed by 35 cycles consisting of denaturation at 98°C for 10 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min; the last cycle was done at 72°C for 10 min. The amplification reactions were performed in a S1000TM Thermal Cycler (Bio Rad, Hercules, CA, USA). The PCR products were analyzed by electrophoresis on 2% agarose gel containing Gel Red (Biotium, Fremont, CA, USA). The BFV pol and gag positive PCR products were purified using NucleSpin Gel and PCR Clean up kit (Macherey-Nagel GmbH & Co., KG, Duren, Germany) following the manufacturer’s recommendations. The purified PCR products were subsequently sequenced using ABI 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Vietnamese BFV sequences were obtained by MEGA 7.0 software [16]. The nucleotide sequences of Vietnamese strains were aligned together with BFV pol and gag sequences obtained from the GenBank of National Center for Biotechnology Information by BioEdit version 7.2.5 [8]. Phylogenetic analysis of the partial sequences of the pol and gag genes were performed using MEGA 7.0 software [16]. Phylogenetic trees were constructed using the maximum likelihood (ML) algorithm with the Kimura-2 parameter model (K2+G) of nucleotide substitution. The reliability of phylogenetic relationships was evaluated using bootstrap analysis with 1,000 replicates. The statistically significant association between BLV and BFV infection was determined by Pearson’s 2χ test. PCR test targeting BFV gag gene identified 10 out of 243 samples (4.1%) as positive, whereas PCR assay targeting pol gene identified eight (3.3%) (Table 2). Of 67 investigated herds, the existence of BFV was confirmed in ten herds (14.9%). BFV was prevalent among the cattle in the three investigated provinces. BFV-positive cattle were confirmed in seven out of 45 farms in Hanoi, in two out of 14 farms in Vinhphuc and in one out of eight farms in Bacninh. Simultaneously, BLV was detected in 65 out of 243 cattle (26.7%) in the same group of animals.