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Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains
Virus Research 253 (2018) 103–111
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Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains
T
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Hironobu Murakamia, , Jumpei Uchiyamab, Chihiro Suzukia, Sae Nikaidoa, Kaho Shibuyaa, Reiichiro Satoc, Yosuke Maedad, Michiko Tomiokad, Shin-nosuke Takeshimae,f, Hajime Katog, Masahiro Sakaguchib, Hiroshi Sentsuih, Yoko Aidae, Kenji Tsukamotoa a
Laboratory of Animal Health II, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Veterinary Microbiology I, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan c Laboratory of Farm Animal Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan d Laboratory of Clinical Veterinary Medicine for Large Animal, School of Veterinary Medicine, Kitasato University, Higashi 23bancho 35-1, Towada, Aomori, 034-8628, Japan e Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan f Department of Food and Nutrition Faculty of Human Life, Jumonji University, 2-1-28, Sugasawa, Niiza, Saitama, 352-8510, Japan g Southern Nemuro Operation Center, Hokkaido Higashi Agricultural Mutual Aid Association, 119, Betsukai-Midorimachi, Betsukai, Notsuke-gun, Hokkaido 086-0292, Japan h Laboratory of Veterinary Epizootiology, School of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bovine leukemia virus Phylogenetic analysis Genetic variation Molecular clone Proviral load Viral property
Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.
1. Introduction Bovine leukemia virus (BLV), which belongs to the family Retroviridae genus Deltaretrovirus, is the etiologic agent of enzootic bovine leukosis (EBL), which is a lethal infectious disease of cattle. The BLV infection results not only in EBL development but also in reductions in lifetime milk production, reproductive efficiency, and lifespan (Brenner et al., 1989; Nekouei et al., 2016; Polat et al., 2017b; Schwartz and Levy, 1994). In addition, the prevalence of BLV infection is high in several regions worldwide (Ott et al., 2003) and is thus responsible for economic losses throughout the livestock industry. However, BLV in infected cows cannot be technically eliminated because BLV integrates
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Corresponding author. E-mail address: [email protected] (H. Murakami).
https://doi.org/10.1016/j.virusres.2018.06.005 Received 6 May 2018; Received in revised form 14 June 2018; Accepted 14 June 2018 Available online 18 June 2018 0168-1702/ © 2018 Elsevier B.V. All rights reserved.
into the host genomic DNA of peripheral blood cells as a provirus (Murakami et al., 2011). Therefore, the prevention of BLV infection spread and EBL development would help in reducing the economic losses due to BLV infection in the livestock industry worldwide. Useful information for controlling of infectious diseases can be obtained by analyzing factors involved in transmissibility and pathogenesis. Previous studies have demonstrated that the host factors, such as bovine leukocyte antigen (BoLA) and immunoreaction (Aida et al., 2013; Kabeya et al., 2001; Lewin and Bernoco, 1986; Lewin et al., 1988; Miyasaka et al., 2013; Murakami et al., 2004; Nishimori et al., 2017), affect BLV transmissibility and pathogenesis of BLV, whereas induced mutations in BLV genome regions can also have an effect (Florins et al.,
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presence of precipitation lines that indicate the presence of BLV-specific antibodies was determined by eye. For PCR, a partial pol gene from the BLV genome, which is highly conserved, was amplified using Go-Taq Green Master Mix (Promega Corporation), as described previously (Murakami et al., 2016).
2006, 2007; Gillet et al., 2016; Inoue et al., 2011, 2013; Watanabe et al., 2015). The BLV genome is relatively well conserved and the genetic variations are limited (Mansky and Temin, 1994), while the analyses of host factors are more advanced than those of viral factors. The changes in viral properties resulting from mutations that affect transmissibility and pathogenesis have mainly been analyzed by inducing mutations. Thus, it remains ambiguous whether spontaneous variations in the BLV genome affect viral properties that are related to viral transmissibility and pathogenesis. The BLV genome contains a variety of functional genes and regions. The BLV proviral genome encodes a long terminal repeat (LTR) at both the 5′ and 3′ termini, and the gag-pro-pol, env, and nonstructural genes are encoded between the two LTRs. The nonstructural genes AS1, R3, G4, tax, and rex are reportedly encoded within the pX region, which is located between the env and 3′ LTR (Aida et al., 2013; Durkin et al., 2016). In addition, five micro RNAs (miRNAs) are encoded between the env and R3 genes (Kincaid et al., 2012; Rosewick et al., 2013), and the noncoding RNA, AS2, is encoded on the minus strand of the gag-pro-pol gene (Durkin et al., 2016). In our previous study, a small spontaneous deletion mutation in the G4 gene resulted in low virus production and static proviral load (PVL), which is closely related to transmissibility and pathogenesis (Jimba et al., 2010; Juliarena et al., 2016; Ohno et al., 2015; Somura et al., 2014), in a BLV-infected cow (Murakami et al., 2016). In addition, BLV strains harboring spontaneous nonsense mutation were identified in BLV-infected cows, and spontaneous mutations in limited regions may be related to BLV pathogenesis (de Brogniez et al., 2015; Inoue et al., 2011, 2013; Matsumura et al., 2011; Moratorio et al., 2013; Watanabe et al., 2015; Willems et al., 2000). These previous reports suggest that these spontaneous limited variations in the BLV genome may have the potential to change viral properties relating to transmissibility and pathogenesis. Thus, we hypothesized that each wild-type strain harboring spontaneous nucleotide substitutions in BLV genome has different viral properties. In this study, we examined the association between genetic variations over the whole BLV genome and viral properties based on virus activity in vitro and the PVL in BLVinfected cows.
2.4. Sequencing of the BLV proviral genome BLV genomes were sequenced, as described previously (Murakami et al., 2016). The whole BLV genome was amplified using PrimeSTAR GXL polymerase (Takara Bio, Inc., Shiga, Japan) and primers (Supplementary Table S1). The PCR products from each sample were electrophoresed in 1% agarose gels and purified from the gels using the Wizard SV Gel and PCR Clean-Up System (Promega Corporation). The purified PCR products were used as templates for sequencing. The sequences of the products were determined using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems), according to the manufacturers’ instructions. The whole BLV genome sequences of the 27 strains identified in this study were deposited in the GenBank database under the accession numbers AP018006 to AP018032. 2.5. Phylogenetic analysis The genome sequences were aligned by ClustalW, and maximumlikelihood trees were constructed, using MEGA 7.0 software (Kumar et al., 2016), as described previously (Polat et al., 2016). The reliability of the phylogenetic relationships was evaluated using nonparametric bootstrap analysis with 1000 replicates. Partial sequences (400 bp) of the env gene and whole BLV genome were used because the method described by Rola-Luszczak et al. (2013) can correctly identify each BLV genotype using a short sequence of the env gene sequence. 2.6. Alignment of amino acid and nucleotide sequences
2. Materials and methods
Editing and alignment of nucleotide and amino acid sequences were performed using UGENE (Okonechnikov et al., 2012) and MEGA 7.0 software (Kumar et al., 2016).
2.1. Collection of samples
2.7. Typing of the BoLA-DRB3 gene
Blood samples were collected from Holstein–Friesian cows from dairy farms in Japan. Thirteen tumor samples were provided by a meat inspection center and other farms in Japan. Detailed information for each sample is provided in Table 1.
Polymorphisms of the BoLA-DRB3 gene in BLV-infected cows were identified using a PCR sequence-based typing method as described previously (Takeshima et al., 2011). Briefly, the partial BoLA-DRB3 gene fragments of the cows listed in Table 1 were amplified by PCR. The sequences of the amplified PCR products were determined by direct sequencing. Based on the sequence data, BoLA-DRB3 alleles were identified using ASSIGN 500ATF software (Conexio Genomics PTY, Fremantle, Australia).
2.2. Extraction of DNA from blood and tumor samples Genomic DNA was extracted from whole blood and tumor samples using the Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) and the Get pureDNA Kit-Cell, Tissue (Dojindo Molecular Technologies, Rockville, MD, USA), respectively, according to the manufacturers’ instructions.
2.8. Construction of BLV molecular clones and plasmids Cloning of the whole BLV genome was performed as described previously (Murakami et al., 2016). Briefly, the BLV proviral genome was amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara Bio, Inc.) and cloned into the pSMART LC Amp vector (Lucigen Corporation, Milwaukie, WI, USA). The BLV molecular clones were transfected into Escherichia coli (E. coli) strain Stbl3 cells. The constructed molecular clones were confirmed by sequencing using primers described in Supplementary Table S2. The BLV U3 promoter sequence and firefly luciferase sequences were amplified by PCR using the genomic DNA of FLK-BLV and the pCMV-Luc plasmid vector (Promega), respectively, as a template DNA. The amplified U3 promoter sequence was replaced with the EF1α promoter region of pBApo-EF1α-Pur (Takara Bio, Shiga, Japan), and the amplified firefly luciferase sequence was cloned downstream of the
2.3. Detection of BLV infection in cows These sera isolated from blood and genomic DNA extracted from tumors were subjected to the agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR) analysis, respectively. The AGID test was performed as described previously (Murakami et al., 2016). Briefly, a gel with one central well and six surrounding wells was prepared. The BLV antigen and the positive reference serum were placed into the central well and into two symmetrical outer wells, respectively, whereas the serum samples, which were prepared by centrifugation of whole blood, were placed into the remaining four wells. After incubation in a humidified chamber at room temperature for 48 h, the 104
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Table 1 Cows and BLV genotype 1 strains used in this study. Cow AF060 AF076 AF193 AF245 AF266 AF293 AF438 AF481 AF513 AF746 AF784 AF805 AF902 AF982 AK001 AK006 AK007 AK011 AN003 AN004 AN006 AN008 AN009 AN011 AN013 AN014 AN015 AN903 a b c
Breed Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Japanese Black Japanese Black Japanese Black Japanese Black Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Collection Date December 2013 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 2013 and 2015 December2014 June 2015 December 2016 July 2016 July 2009 October 2009 2009 Unknown Unknown May 2007 May 2007 June 2007 June 2007 September 2009
Region Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Aomori, Japan Aomori, Japan Aomori, Japan Aomori, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan
Agea 85 48 59 38 76 2 111 38 9 64 45 29 24 41 117 99 68 49 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Symptom Non-EBL→EBLb Non-EBL Non-EBL Non-EBL Non-EBL→EBLb Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL
Groupc A C B A A B C C A B C A C A B B B B A A A B A B B B B A
Age in months at first sampling. Cows AF060 and AF266 developed EBL in September 2014 and November 2014, respectively. The group of strain isolated from the infected cow.
cells were measured using the quantitative PCR (qPCR) and syncytium assay, as described previously (Murakami et al., 2017, 2016). Briefly, 293 T cells were transfected with the molecular clones together with the pTK-Luc vector using the FuGENE HD Transfection Reagent (Promega Corporation). At 24 h post-transfection, the cells were washed twice with DMEM and the growth medium was replaced. At 48 h posttransfection, measurements of luciferase activity and viral titer were performed using the cells and cell supernatants after removal of cell debris by centrifugation, respectively. To determine transfection efficiency, the luciferase activity of the cells was measured using the Pikka Gene Kit (Toyo Ink America LLC), according to the instruction manual. For qPCR, first, vRNA was extracted from the supernatant using the High Pure Viral Nucleic Acid Kit (Roche, Penzberg, Germany), according to the manufacturer's instructions. cDNA was then synthesized from the vRNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan), according to the manufacturer's instructions. The qPCR was performed using GoTaq Probe qPCR Master Mix (Promega Corporation) and a 7500 Real-Time PCR system (Applied Biosystems), as previously reported (Murakami et al., 2016). For the syncytium assay, CC81 cells were cultured for 24 h in 96-well plates (5 × 103 cells/well). The supernatants of the 293 T cells transfected with each clone were serially diluted. The CC81 cells were cultured in the serially diluted supernatants supplemented with polybrene at a final concentration of 4 μg/ml. The CC81 cells were cultured until confluent and syncytia were visualized by Giemsa stain and light microscopy. Syncytia were defined as cells containing more than five nuclei. The syncytium-forming units were normalized to the transfection efficiency as described above.
U3 promoter sequence. The plasmid vector harboring the U3 promoter and firefly luciferase gene, which was named pU3-Luc, was transfected into E. coli strain DH5α. 2.9. Cell lines and culture conditions Human kidney 293 T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; DS Pharma Biomedical Co. Ltd., Osaka, Japan). FBS and 2.95% tryptose phosphate broth solution were added to Eagle’s minimum essential medium (Nissui Pharmaceutical) at concentrations of 5% and 10%, respectively, for maintaining of feline kidney CC81 cells. 2.10. Measurement of viral RNA (vRNA) in the supernatant of molecular clone-transfected 293 T cells The amount of virus obtained from 293 T cells transfected with each molecular clone and the previously constructed pTK-Luc vector was measured using primers and probe sets described elsewhere (Murakami et al., 2017, 2016). The pTK-Luc vector, which contains the firefly luciferase gene under the control of the herpes simplex virus thymidine kinase promoter, was used to estimate transfection efficiency. Firefly luciferase activity in transfected cells was measured using the Pikka Gene Kit (Toyo Ink America LLC, Wood Dale, IL, USA), according to the manufacturer's instructions. This activity was used as a marker for transfection efficiencies in the molecular clone-transfected cells. Transfection efficiency was calculated by setting the firefly luciferase activity in pBLV-AN060 to 1, and viral copy numbers were normalized using the relative firefly luciferase activity to eliminate bias from transfection efficiency.
2.12. Measurement of viral transactivation Each BLV molecular clone together with pU3-Luc and pRL-TK (Toyo Ink, Tokyo, Japan) were transfected into the 293 T cells using the FuGENE HD Transfection Reagent (Promega). The pU3-Luc and pRL-TK were used to measure viral transactivation by Tax and transfection
2.11. Measurement of viral titers Titers of virus produced from molecular clone-transfected 293 T 105
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substitutions using MEGA 7.0 software. Consequently, the nucleotide substitution rate between each strain was 0.92%–0.01%. As expected, this rate was low. We next analyzed the location of each nucleotide substitution to identify specific sites accumulated these substitutions. To search for unique substitutions among the 28 strains, the consensus genome sequence of the 28 BLV genomes was generated, and nucleotide substitutions were extracted by UGENE software. The result showed that no specific sites were present in the BLV genome (Fig. 2A). Because the nucleotide substitutions were sporadically located in whole BLV genome, we performed BLV strain categorization using each whole BLV genome. The 28 whole BLV genomes were phylogenetically analyzed using MEGA 7.0 software and, except for strains isolated from cow AN008 (pvAN008) strain, these genomes could be divided into three groups. These were named groups A, B, and C (Fig. 2B) and contained 11 (pvAF060, pvAF266, pvAF805, pvAF513, pvAN004, pvAN903, pvAN009, pvAN003, pvAN006, pvAF245, and pvAF982), 11 (pvAK007, pvAK011, pvAK001, pvAN014, pvAK006, pvAN011, pvAN013, pvAN015, pvAF293, pvAF193, and pvAF746) and 5 BLV strains (pvAF076, pvAF481, pvAF902, pvAF784, and pvAF438), respectively. The pvAN008 strain was classified separately because it harbored unique nucleotide substitutions (Supplementary Fig. S1) and could not be classified into these groups. The groups categorized were not biased toward any regions nor to any periods of sample collection (Table 1). On the other hand, BLV strains derived from EBL cows were categorized into group A or B, and all strains belonging to group C were derived from non-EBL cows (Fig. 2B). This suggested that the EBL-derived strains might be genetically similar strains, regardless of region. Next, to explore which substitutions determined the three groups, we searched for group-specific substitutions using multiple alignment of whole genome generated by MEGA 7.0 software. The specific nucleotide substitutions of each group are showed in Supplementary Fig. S1. Consequently, several group specific substitutions were found. The nucleotide position at 5528 in the env gene and at 6485 on upstream of miRNA were found to be common mutation of groups C and A, respectively. The nucleotide position at 7153 on the genes for G4 and AS1 genes was found in group B and two strains of group C (Supplementary Fig. S1). In the non-coding region, the group-specific substitutions were located in the LTR responsible for viral transcriptional activity, AS2, which is a putative noncoding RNA region, and upstream/downstream of the miRNA-coding region (Supplementary Fig. S2). Although the miRNA coding regions BLV-miR-B1, 2, 3, 4, and 5-3/5p, which are associated with tumorigenesis, were relatively conserved, as reported previously (Gillet et al., 2016; Rosewick et al., 2013), we identified the insertion of a thymidine residue into the B2-3p region in addition to substitutions in regions B3-3p and B5-3p. Furthermore, the AS2 region harbored the largest number of substitutions among the noncoding regions. These substitutions at nucleotides 2031, 2313, and 2594 of the AS2 regions were observed in most of the group A strains, whereas substitutions at nucleotides 2082 and 2301of the AS2 region were observed in most of the group C strains. In addition, several group-specific nucleotide substitutions located at genes resulted in amino acid mutation in the gag-pro-pol gene (Supplementary Fig. S3), env gene (Supplementary Fig. S4), and the pX region, which encodes nonstructural proteins (Supplementary Fig. S5). The substitution at amino acid 52 of the G4 protein located in the pX region was adjacent to an important sequence (amino acids 48–51; RLPL) related to virus productivity (Murakami et al., 2017) and was specific to group B and C strains. Several nucleotide and amino acid substitutions were located at or near functional domains and might be associated with viral properties, although most of the spontaneous substitutions were not located within the functional sites or domains of the BLV genome. This suggests that above specific nucleotide substitutions were accumulated in genome and generated each group classified in this study.
efficiency, respectively. After 48 h post-transfection, the cells were analyzed for firefly and Renilla luciferase activities using the Pikka Gene Dual Assay Kit (Toyo Ink), as previously report (Murakami et al., 2016). 2.13. Measurement of PVL in BLV-infected cows The BLV provirus in blood was measured with the BLV Coordination of Common Motifs qPCR-2 method (BLV-Co-Co-Mo-qPCR-2; Riken Genesis, Yokohama, Japan), as described previously (Jimba et al., 2012). The analysis was performed to quantify viral and BLV proviral genomes in all samples using GoTaq Probe qPCR Master Mix (Promega Corporation) and the 7500 Real-Time PCR system (Applied Biosystems). 2.14. Statistical analysis Comparison of two and three groups were performed using Student’s t test and one-way analysis of variance with Tukey’s post hoc test. All the statistical analyses were performed using R version 3.3.3 statistical software (Team, 2017). 3. Results and discussion 3.1. Identification of whole BLV genome sequences and genotypes Thirteen tumor samples and 14 blood samples were collected from cows with and without EBL (EBL and non-EBL cows, respectively) between 2007 and 2015, and BLV infection was confirmed as infection with BLV by PCR analysis and the AGID test, respectively. A tumor sample from an EBL cow (cow AN903), which has been used in our previous study (Murakami et al., 2016), was also included in this study. The 14 non-EBL dairy cows were kept on the same farm in Japan, whereas each of the EBL cows were each from a different Japanese farm (Table 1). Two dairy cows (pre-EBL cows; cows AF060 and AF266) had not developed EBL at the time of blood sampling time, but both developed the disease in 2014. Among the EBL cows, four cows (cows AK001, AK006, AK007, and AK011) were beef cattle, but we could not obtain information as to whether the other 10 cows (cows AN003, AN004, AN006, AN008, AN009, AN011, AN013, AN014, AN015, and AN903) were used for dairy or beef production (Table 1). All the 27 BLV strains were identified using whole genome sequences from direct sequencing. It is thought that the dominant strain in the BLV-infected cows was identified in this study because it was not possible to detect viral heterogeneity. To confirm the genotypes of the 27 BLV strains and a strain harboring cow AN903 identified in the present and previous studies, respectively, we phylogenetically analyzed them together with 90 registered BLV strains and identified their genotypes using the 400 bp BLV env gene sequence of total 118 BLV strains containing 28 strains used in this study, as described previously (Rola-Luszczak et al., 2013). Consequently, all the 28 BLV strains (Supplementary Table S3) clustered only with genotype 1 strains (Fig. 1); because genotype 1 is a common genotype in Japan and worldwide (Inoue et al., 2011; Matsumura et al., 2011; Rodriguez et al., 2009), this result was as expected. On the other hand, the 28 strains were generated subclusters with GenBank deposited strains, such as strains isolated in Argentina, the U.S.A., or Iran. Therefore, all 28 BLV strains used in this study were of genotype 1 but had limited genetic variations. 3.2. The categorization of the 28 BLV strains based on limited genetic variations The 28 strains were named as pvX, where X is the identification number of the cow. To examine a genetic variation among the 28 strains, the 28 BLV genome sequences were analyzed for nucleotide 106
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Fig. 1. Phylogenetic analysis by maximum-likelihood of 118 partial BLV env gene sequences from different geographical locations worldwide. The phylogenetic tree was constructed from 118 distinct 400 bp BLV env gene sequences, including the 28 BLV strains derived from BLV-infected cattle listed in Table 1 and the 90 BLV strains registered in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). The accession numbers and country of origin of these 90 strains are shown. Ten genotypes (G1–10) are indicated by numbers around the circumference of the figure.
specific group was isolated from cows carrying a specific BoLA-DRB3 allele. Thus, the BoLA-DRB3 allele of infected cows did not appear to select for any of the BLV genetic variants identified. These results showed that BLV genetic variations in BLV were independent of BoLADRB3, and that group A and B strain-infected EBL cows were not affected by host genetics. Therefore, these limited genetic variations could be associated with viral properties independent of BoLA-DRB3.
3.3. Association between the selection of BLV strains and the BoLA-DRB3 allele of host cows Because the BoLA-DRB3 allele is a determinant of pathogenesis in BLV-infected cows (Miyasaka et al., 2013; Takeshima and Aida, 2006), we thought this allele could influence the selection of BLV genetic variants of BLV. To evaluate whether strains from specific groups were selected by the BoLA-DRB3 type of an individual cow, we determined the BoLA-DRB3 allele of each infected cow used in this study using a PCR sequence-based typing method. The results showed that the BoLADRB3 alleles identified in this study were not biased toward a specific allele (Supplementary Table S4). Examination of the relationship between genetic variants of the BLV genome and the BoLA-DRB3 allele indicated that infection with a specific group of viruses categorized in this study was not related to the BoLA-DRB3 allele. In addition, no
3.4. Dependence of virus activity in vitro on the groups categorized in this study To evaluate the dependence of a viral property on a genetic variant, virus production was measured in vitro using BLV molecular clones from each strain analyzed in this study. First, 26 molecular clones were constructed from the 26 BLV-infected cows infected with group A, B, 107
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Fig. 2. Analyses of whole BLV genomes using 28 strains. (A) Summary of the substitution ratio throughout the BLV genome. The substitution ratio of nucleotides is shown with the BLV genome map. The consensus reference genome sequence generated from the 28 strains used in this study was compared with the individual sequences of the 28 strains. The top graph shows the ratio of mutations in each nucleotide over the whole genome. On the lower genome map, the right- and leftfacing arrows indicate the genes encoded on the sense and antisense strands, respectively. The square on the genome map shows the promoter region, LTR, or noncoding RNA. (B) Phylogenetic analysis of 28 whole BLV genome sequences. A maximum-likelihood phylogenetic tree was constructed from 28 distinct whole BLV genome sequences (the assigned accession numbers are shown in Supplementary Table S3). The dots show strains derived from pre-EBL (cows AF060 and AF266) or EBL cows. The bar on the figure denotes distance. 108
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Fig. 3. Virus replication activity in molecular clone-transfected 293 T cells. The viral in the supernatant of 293 T cells transfected with each molecular clone and pTKLuc were measured by (A) qPCR and (B) syncytium assay. Viral RNAs and infectious viruses were measured by qPCR and the syncytium assay, respectively, and normalized to the transfection efficiency measured by luciferase activity. (C) The transcriptional activity of 293 T cells transfected with each molecular clone, pU3Luc, and pRL-TK. Transcriptional activity was measured using firefly luciferase activity and normalized to the transfection efficiency determined by Renilla luciferase activity. The experiments were conducted in triplicate. Significant differences are indicated by single and double asterisks (*P < 0.05 and **P < 0.01, respectively, one-way analysis of variance).
transmissibility but also the risk of the EBL development. Thus, the measurement of PVL could be an indication of potential for transmissibility and pathogenesis in BLV-infected cows. First, we measured the PVL in blood samples collected from 18 cows (cows AF060, AF076, AF193, AF245, AF266, AF293, AF438, AF481, AF513, AF746, AF784, AF805, AF902, AF982, AK001, AK006, AK007, and AK011), which we could obtain blood sample at first sampling time. Six (cows AF060, AF245, AF266, AF513, AF805, and AF982), seven (cows AF193, AF293, AF746, AK001, AK006, AK0007, and AK011), and five cows (cows AF076, AF438, AF481, AF784, and AF902) were infected with group A, B, and C strains, respectively. The mean of PVL in group A, B, and C strain-infected cows were 23016.6, 18435.9, and 10038 copies/105 cells, respectively (Supplementary Fig. S6), and PVL was high in the order of group A, B, and C strain-infected cows, but these differences were not statistically significant. We next evaluated PVL transition, because a previous study reported that the PVL fluctuates in BLV-infected cows (Murakami et al., 2016). The PVLs of BLV-infected cows were additionally measured by qPCR in November 2015. Among the 18 cows with available blood samples, six were collected from the EBL cows, which included cow AF060 and AF266, and four from the nonEBL cows were slaughtered before analysis. Therefore, the PVLs of these cows could not be measured. However, the PVLs of eight non-EBL cows (i.e., cows AF076, AF245, AF293, AF481, AF513, AF784, AF902, and AF982) were measured in 2013 and 2015. The BLV-infected cows showed different patterns of change in PVLs between 2013 and 2015. Cow AF245 exhibited a significant increase in the PVL, whereas cows AF513 and AF982 had consistently high PVLs. Cows AF293 and AF784 exhibited significant decreases in the PVL, and cows AF076, AF481, and AF902 had consistently low PVLs (Fig. 4). Overall, these results, with regard to the categorizations described above, indicated that the PVLs in cows infected with group A strains increased significantly or remained high, whereas those in cows infected with group B and C strains decreased significantly or remained low, suggesting that virus productivity dependent on the limited genetic variations associated with PVL. In support of this finding, our previous study also showed that low virus productivity caused by a spontaneous deletion mutation occurred in a cow with a static and low PVL (Murakami et al., 2016), although BLV mainly propagates in vivo via mitosis (Rodriguez et al., 2011). Thus, the group A strains may have the potential to maintain a high PVL via high productivity. Although it is unknown whether the high virus production was directly related to a high PVL, high virus productivity is likely a determinant of a high PVL.
and C strains. Sequencing of these clones confirmed that the whole genome sequences were identical to each strain sequence identified in this study. Thus, the dominant stain in each infected with each cow could be isolated as a BLV molecular clone. In addition, clone pBLVAN903, which was cloned whole genome of pvAN903 strain, was previously constructed from the BLV-infected cow AN903 (Murakami et al., 2016) (Supplementary Table S3). These 27 molecular clones were transfected into cell line, and viral replication activity was measured using qPCR, syncytium assay, and luciferase assay. Next, the level of viral RNA in the supernatants of cell lines transfected with each clone was evaluated. Because 293 T cells are suitable for estimating virus production (Murakami et al., 2017), this cell line was used in this study. The results showed that the copy numbers of viral RNA were significantly higher in group A than in the groups B or C. Group B strains exhibited higher copy numbers than group C strains, but the difference was not statistically significant (Fig. 3A). Next, to validate these data, the production of infectious virus was measured using the syncytium assay. The results were very similar to those for the viral RNA copy number: the strains belonging to group A produced significantly higher titers of infectious virus than those belonging to groups B and C (Fig. 3B). Thus, the groups identified by the maximum-likelihood phylogenetic tree were reflected at the level of virus production in vitro. Next, to examine whether the transcriptional activity was related to virus production, we measured virus transcriptional activity using a luciferase assay. Each molecular clone together with pU3-Luc and pRLTK, which were used for measuring LTR transcriptional activity of BLV and transfection efficiency, respectively, were transfected into 293 T cells. After 48 h post-transfection, the transcriptional activities of each molecular clone were measured. Consequently, group A strains showed significantly higher transcriptional activity than group B and C strains, reflecting the results of virus production (Fig. 3A and B). These results showed that the limited genetic variation affected virus productivity in vitro, as assessed based on transcriptional activity, and suggested that the limited substitutions identified in this study would be functional site or essential sites for virus activity.
3.5. Relationship between PVL transition and the groups categorized in this study PVL is defined as the proviral copy number in blood and is dependent of viral propagation in BLV-infected cows (Aida et al., 2013). In addition, EBL cows tend to have a higher PVL than non-EBL cows. Thus, PVL measurement can be a useful tool to assess not only the 109
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Fig. 4. PVL of BLV in eight cows infected with group A, B or C strains. The solid and open bars show the PVL of BLV-infected cows in 2013 and 2015, respectively. All experiments were performed in triplicate. Results are shown as means with standard error. Significant differences are indicated by single and triple asterisks (*P < 0.05 and ***P < 0.001, respectively, Student’s t-test).
3.6. Viral properties affected by the spontaneous genetic variations
Acknowledgments
Previous studies have shown that the genetic distances between BLV genomes revealed by phylogenetic analysis are related to their geographical locations (Lee et al., 2016; Pandey et al., 2017; Polat et al., 2017a, 2016; Rodriguez et al., 2009; Yang et al., 2016). In this study, the phylogenetic tree based on the BLV genomes showed that virus production in vitro and the PVL were greater in cows infected with group A strains than in those infected with group C strains (Figs. 2B, 3A, B, and 4). These results indicate that similarities of genetic variations in BLV may be associated with similar virus production in vitro and virus propagation in vivo and that limited genetic substitutions could be related to these viral properties. Thus, the limited substitutions seem to be important for viral transmissibility and pathogenesis. Our study focused on genetic variations in BLV that may be related to viral activity and PVL transition. The results showed that a general concept for RNA virology, that is, the effect of genetic variations on viral properties (Domingo et al., 2012), could be adapted to BLV studies. In addition, BLV transmissibility and pathogenesis are affected by various factors, including the integration site of the provirus, immunoreaction, and the genetic backgrounds of the host and virus (Kabeya et al., 2001; Murakami et al., 2016; Rosewick et al., 2017; Takeshima and Aida, 2006; Takeshima et al., 2017). Based on the findings of the present and previous studies, it is conceivable that genetic variations in the virus are also associated with BLV transmissibility and pathogenesis via changes in viral properties. However, because only genotype 1 strains and small sample sizes were used in this study, this study is merely the first step in understanding BLV transmissibility and pathogenesis in terms of limited genetic variations throughout the whole BLV genome. Thus, additional analyses are needed to reveal differences in virulence based on genetic variations. We believe that the generation of further information about the relationship between genetic variations and the biological properties of BLV could lead to new approaches to control BLV infection.
This work was partially supported by a research project grant awarded by the Azabu University Research Services Division and JSPS KAKENHI Grant Number 17K15385. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2018.06.005. References Aida, Y., Murakami, H., Takahashi, M., Takeshima, S.N., 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front. Microbiol. 4, 328. Brenner, J., Van-Haam, M., Savir, D., Trainin, Z., 1989. The implication of BLV infection in the productivity, reproductive capacity and survival rate of a dairy cow. Vet. Immunol. Immunopathol. 22 (3), 299–305. de Brogniez, A., Bouzar, A.B., Jacques, J.R., Cosse, J.P., Gillet, N., Callebaut, I., Reichert, M., Willems, L., 2015. Mutation of a single envelope n-linked glycosylation site enhances the pathogenicity of bovine leukemia virus. J. Virol. 89 (17), 8945–8956. Domingo, E., Sheldon, J., Perales, C., 2012. Viral quasispecies evolution. Microbiol. Mol. Biol. Rev. 76 (2), 159–216. Durkin, K., Rosewick, N., Artesi, M., Hahaut, V., Griebel, P., Arsic, N., Burny, A., Georges, M., Van den Broeke, A., 2016. Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs. Retrovirology 13 (1), 33. Florins, A., Gillet, N., Asquith, B., Debacq, C., Jean, G., Schwartz-Cornil, I., Bonneau, M., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2006. Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep. J. Virol. 80 (24), 11998–12008. Florins, A., Gillet, N., Boxus, M., Kerkhofs, P., Kettmann, R., Willems, L., 2007. Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep. J. Virol. 81 (18), 10195–10200. Gillet, N.A., Hamaidia, M., de Brogniez, A., Gutierrez, G., Renotte, N., Reichert, M., Trono, K., Willems, L., 2016. Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis In Vivo. PLoS Pathog. 12 (4) e105588. Inoue, E., Matsumura, K., Maekawa, K., Nagatsuka, K., Nobuta, M., Hirata, M., Minagawa, A., Osawa, Y., Okazaki, K., 2011. Genetic heterogeneity among bovine leukemia viruses in Japan and their relationship to leukemogenicity. Arch. Virol. 156 (7), 1137–1141. Inoue, E., Matsumura, K., Soma, N., Hirasawa, S., Wakimoto, M., Arakaki, Y., Yoshida, T., Osawa, Y., Okazaki, K., 2013. L233P mutation of the tax protein strongly correlated with leukemogenicity of bovine leukemia virus. Vet. Microbiol. 167 (3-4), 364–371. Jimba, M., Takeshima, S.N., Matoba, K., Endoh, D., Aida, Y., 2010. BLV-CoCoMo-qPCR: quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm. Retrovirology 7, 91. Jimba, M., Takeshima, S.N., Murakami, H., Kohara, J., Kobayashi, N., Matsuhashi, T., Ohmori, T., Nunoya, T., Aida, Y., 2012. BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status. BMC Vet. Res. 8, 167. Juliarena, M.A., Barrios, C.N., Ceriani, M.C., Esteban, E.N., 2016. Hot topic: bovine leukemia virus (BLV)-infected cows with low proviral load are not a source of infection for BLV-free cattle. J. Dairy. Sci. 99 (6), 4586–4589. Kabeya, H., Ohashi, K., Onuma, M., 2001. Host immune responses in the course of bovine leukemia virus infection. J. Vet. Med. Sci. 63 (7), 703–708. Kincaid, R.P., Burke, J.M., Sullivan, C.S., 2012. RNA virus microRNA that mimics a b-cell
Conflict of interest The authors declare that there are no conflicts of interest.
Ethics statement This study was conducted in accordance with the Guidelines for Laboratory Animal Welfare and Animal Experiment Control set out by School of Veterinary Medicine, Azabu University [permit numbers: 161121-2].
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Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains
T
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Hironobu Murakamia, , Jumpei Uchiyamab, Chihiro Suzukia, Sae Nikaidoa, Kaho Shibuyaa, Reiichiro Satoc, Yosuke Maedad, Michiko Tomiokad, Shin-nosuke Takeshimae,f, Hajime Katog, Masahiro Sakaguchib, Hiroshi Sentsuih, Yoko Aidae, Kenji Tsukamotoa a
Laboratory of Animal Health II, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Veterinary Microbiology I, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan c Laboratory of Farm Animal Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan d Laboratory of Clinical Veterinary Medicine for Large Animal, School of Veterinary Medicine, Kitasato University, Higashi 23bancho 35-1, Towada, Aomori, 034-8628, Japan e Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan f Department of Food and Nutrition Faculty of Human Life, Jumonji University, 2-1-28, Sugasawa, Niiza, Saitama, 352-8510, Japan g Southern Nemuro Operation Center, Hokkaido Higashi Agricultural Mutual Aid Association, 119, Betsukai-Midorimachi, Betsukai, Notsuke-gun, Hokkaido 086-0292, Japan h Laboratory of Veterinary Epizootiology, School of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bovine leukemia virus Phylogenetic analysis Genetic variation Molecular clone Proviral load Viral property
Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.
1. Introduction Bovine leukemia virus (BLV), which belongs to the family Retroviridae genus Deltaretrovirus, is the etiologic agent of enzootic bovine leukosis (EBL), which is a lethal infectious disease of cattle. The BLV infection results not only in EBL development but also in reductions in lifetime milk production, reproductive efficiency, and lifespan (Brenner et al., 1989; Nekouei et al., 2016; Polat et al., 2017b; Schwartz and Levy, 1994). In addition, the prevalence of BLV infection is high in several regions worldwide (Ott et al., 2003) and is thus responsible for economic losses throughout the livestock industry. However, BLV in infected cows cannot be technically eliminated because BLV integrates
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Corresponding author. E-mail address: [email protected] (H. Murakami).
https://doi.org/10.1016/j.virusres.2018.06.005 Received 6 May 2018; Received in revised form 14 June 2018; Accepted 14 June 2018 Available online 18 June 2018 0168-1702/ © 2018 Elsevier B.V. All rights reserved.
into the host genomic DNA of peripheral blood cells as a provirus (Murakami et al., 2011). Therefore, the prevention of BLV infection spread and EBL development would help in reducing the economic losses due to BLV infection in the livestock industry worldwide. Useful information for controlling of infectious diseases can be obtained by analyzing factors involved in transmissibility and pathogenesis. Previous studies have demonstrated that the host factors, such as bovine leukocyte antigen (BoLA) and immunoreaction (Aida et al., 2013; Kabeya et al., 2001; Lewin and Bernoco, 1986; Lewin et al., 1988; Miyasaka et al., 2013; Murakami et al., 2004; Nishimori et al., 2017), affect BLV transmissibility and pathogenesis of BLV, whereas induced mutations in BLV genome regions can also have an effect (Florins et al.,
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presence of precipitation lines that indicate the presence of BLV-specific antibodies was determined by eye. For PCR, a partial pol gene from the BLV genome, which is highly conserved, was amplified using Go-Taq Green Master Mix (Promega Corporation), as described previously (Murakami et al., 2016).
2006, 2007; Gillet et al., 2016; Inoue et al., 2011, 2013; Watanabe et al., 2015). The BLV genome is relatively well conserved and the genetic variations are limited (Mansky and Temin, 1994), while the analyses of host factors are more advanced than those of viral factors. The changes in viral properties resulting from mutations that affect transmissibility and pathogenesis have mainly been analyzed by inducing mutations. Thus, it remains ambiguous whether spontaneous variations in the BLV genome affect viral properties that are related to viral transmissibility and pathogenesis. The BLV genome contains a variety of functional genes and regions. The BLV proviral genome encodes a long terminal repeat (LTR) at both the 5′ and 3′ termini, and the gag-pro-pol, env, and nonstructural genes are encoded between the two LTRs. The nonstructural genes AS1, R3, G4, tax, and rex are reportedly encoded within the pX region, which is located between the env and 3′ LTR (Aida et al., 2013; Durkin et al., 2016). In addition, five micro RNAs (miRNAs) are encoded between the env and R3 genes (Kincaid et al., 2012; Rosewick et al., 2013), and the noncoding RNA, AS2, is encoded on the minus strand of the gag-pro-pol gene (Durkin et al., 2016). In our previous study, a small spontaneous deletion mutation in the G4 gene resulted in low virus production and static proviral load (PVL), which is closely related to transmissibility and pathogenesis (Jimba et al., 2010; Juliarena et al., 2016; Ohno et al., 2015; Somura et al., 2014), in a BLV-infected cow (Murakami et al., 2016). In addition, BLV strains harboring spontaneous nonsense mutation were identified in BLV-infected cows, and spontaneous mutations in limited regions may be related to BLV pathogenesis (de Brogniez et al., 2015; Inoue et al., 2011, 2013; Matsumura et al., 2011; Moratorio et al., 2013; Watanabe et al., 2015; Willems et al., 2000). These previous reports suggest that these spontaneous limited variations in the BLV genome may have the potential to change viral properties relating to transmissibility and pathogenesis. Thus, we hypothesized that each wild-type strain harboring spontaneous nucleotide substitutions in BLV genome has different viral properties. In this study, we examined the association between genetic variations over the whole BLV genome and viral properties based on virus activity in vitro and the PVL in BLVinfected cows.
2.4. Sequencing of the BLV proviral genome BLV genomes were sequenced, as described previously (Murakami et al., 2016). The whole BLV genome was amplified using PrimeSTAR GXL polymerase (Takara Bio, Inc., Shiga, Japan) and primers (Supplementary Table S1). The PCR products from each sample were electrophoresed in 1% agarose gels and purified from the gels using the Wizard SV Gel and PCR Clean-Up System (Promega Corporation). The purified PCR products were used as templates for sequencing. The sequences of the products were determined using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems), according to the manufacturers’ instructions. The whole BLV genome sequences of the 27 strains identified in this study were deposited in the GenBank database under the accession numbers AP018006 to AP018032. 2.5. Phylogenetic analysis The genome sequences were aligned by ClustalW, and maximumlikelihood trees were constructed, using MEGA 7.0 software (Kumar et al., 2016), as described previously (Polat et al., 2016). The reliability of the phylogenetic relationships was evaluated using nonparametric bootstrap analysis with 1000 replicates. Partial sequences (400 bp) of the env gene and whole BLV genome were used because the method described by Rola-Luszczak et al. (2013) can correctly identify each BLV genotype using a short sequence of the env gene sequence. 2.6. Alignment of amino acid and nucleotide sequences
2. Materials and methods
Editing and alignment of nucleotide and amino acid sequences were performed using UGENE (Okonechnikov et al., 2012) and MEGA 7.0 software (Kumar et al., 2016).
2.1. Collection of samples
2.7. Typing of the BoLA-DRB3 gene
Blood samples were collected from Holstein–Friesian cows from dairy farms in Japan. Thirteen tumor samples were provided by a meat inspection center and other farms in Japan. Detailed information for each sample is provided in Table 1.
Polymorphisms of the BoLA-DRB3 gene in BLV-infected cows were identified using a PCR sequence-based typing method as described previously (Takeshima et al., 2011). Briefly, the partial BoLA-DRB3 gene fragments of the cows listed in Table 1 were amplified by PCR. The sequences of the amplified PCR products were determined by direct sequencing. Based on the sequence data, BoLA-DRB3 alleles were identified using ASSIGN 500ATF software (Conexio Genomics PTY, Fremantle, Australia).
2.2. Extraction of DNA from blood and tumor samples Genomic DNA was extracted from whole blood and tumor samples using the Wizard genomic DNA purification kit (Promega Corporation, Madison, WI, USA) and the Get pureDNA Kit-Cell, Tissue (Dojindo Molecular Technologies, Rockville, MD, USA), respectively, according to the manufacturers’ instructions.
2.8. Construction of BLV molecular clones and plasmids Cloning of the whole BLV genome was performed as described previously (Murakami et al., 2016). Briefly, the BLV proviral genome was amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara Bio, Inc.) and cloned into the pSMART LC Amp vector (Lucigen Corporation, Milwaukie, WI, USA). The BLV molecular clones were transfected into Escherichia coli (E. coli) strain Stbl3 cells. The constructed molecular clones were confirmed by sequencing using primers described in Supplementary Table S2. The BLV U3 promoter sequence and firefly luciferase sequences were amplified by PCR using the genomic DNA of FLK-BLV and the pCMV-Luc plasmid vector (Promega), respectively, as a template DNA. The amplified U3 promoter sequence was replaced with the EF1α promoter region of pBApo-EF1α-Pur (Takara Bio, Shiga, Japan), and the amplified firefly luciferase sequence was cloned downstream of the
2.3. Detection of BLV infection in cows These sera isolated from blood and genomic DNA extracted from tumors were subjected to the agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR) analysis, respectively. The AGID test was performed as described previously (Murakami et al., 2016). Briefly, a gel with one central well and six surrounding wells was prepared. The BLV antigen and the positive reference serum were placed into the central well and into two symmetrical outer wells, respectively, whereas the serum samples, which were prepared by centrifugation of whole blood, were placed into the remaining four wells. After incubation in a humidified chamber at room temperature for 48 h, the 104
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Table 1 Cows and BLV genotype 1 strains used in this study. Cow AF060 AF076 AF193 AF245 AF266 AF293 AF438 AF481 AF513 AF746 AF784 AF805 AF902 AF982 AK001 AK006 AK007 AK011 AN003 AN004 AN006 AN008 AN009 AN011 AN013 AN014 AN015 AN903 a b c
Breed Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Holstein–Friesian Japanese Black Japanese Black Japanese Black Japanese Black Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Collection Date December 2013 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 2013 and 2015 December 2013 2013 and 2015 December 2013 2013 and 2015 2013 and 2015 December2014 June 2015 December 2016 July 2016 July 2009 October 2009 2009 Unknown Unknown May 2007 May 2007 June 2007 June 2007 September 2009
Region Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Tokyo, Japan Aomori, Japan Aomori, Japan Aomori, Japan Aomori, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan Tochigi, Japan
Agea 85 48 59 38 76 2 111 38 9 64 45 29 24 41 117 99 68 49 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Symptom Non-EBL→EBLb Non-EBL Non-EBL Non-EBL Non-EBL→EBLb Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL Non-EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL EBL
Groupc A C B A A B C C A B C A C A B B B B A A A B A B B B B A
Age in months at first sampling. Cows AF060 and AF266 developed EBL in September 2014 and November 2014, respectively. The group of strain isolated from the infected cow.
cells were measured using the quantitative PCR (qPCR) and syncytium assay, as described previously (Murakami et al., 2017, 2016). Briefly, 293 T cells were transfected with the molecular clones together with the pTK-Luc vector using the FuGENE HD Transfection Reagent (Promega Corporation). At 24 h post-transfection, the cells were washed twice with DMEM and the growth medium was replaced. At 48 h posttransfection, measurements of luciferase activity and viral titer were performed using the cells and cell supernatants after removal of cell debris by centrifugation, respectively. To determine transfection efficiency, the luciferase activity of the cells was measured using the Pikka Gene Kit (Toyo Ink America LLC), according to the instruction manual. For qPCR, first, vRNA was extracted from the supernatant using the High Pure Viral Nucleic Acid Kit (Roche, Penzberg, Germany), according to the manufacturer's instructions. cDNA was then synthesized from the vRNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan), according to the manufacturer's instructions. The qPCR was performed using GoTaq Probe qPCR Master Mix (Promega Corporation) and a 7500 Real-Time PCR system (Applied Biosystems), as previously reported (Murakami et al., 2016). For the syncytium assay, CC81 cells were cultured for 24 h in 96-well plates (5 × 103 cells/well). The supernatants of the 293 T cells transfected with each clone were serially diluted. The CC81 cells were cultured in the serially diluted supernatants supplemented with polybrene at a final concentration of 4 μg/ml. The CC81 cells were cultured until confluent and syncytia were visualized by Giemsa stain and light microscopy. Syncytia were defined as cells containing more than five nuclei. The syncytium-forming units were normalized to the transfection efficiency as described above.
U3 promoter sequence. The plasmid vector harboring the U3 promoter and firefly luciferase gene, which was named pU3-Luc, was transfected into E. coli strain DH5α. 2.9. Cell lines and culture conditions Human kidney 293 T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; DS Pharma Biomedical Co. Ltd., Osaka, Japan). FBS and 2.95% tryptose phosphate broth solution were added to Eagle’s minimum essential medium (Nissui Pharmaceutical) at concentrations of 5% and 10%, respectively, for maintaining of feline kidney CC81 cells. 2.10. Measurement of viral RNA (vRNA) in the supernatant of molecular clone-transfected 293 T cells The amount of virus obtained from 293 T cells transfected with each molecular clone and the previously constructed pTK-Luc vector was measured using primers and probe sets described elsewhere (Murakami et al., 2017, 2016). The pTK-Luc vector, which contains the firefly luciferase gene under the control of the herpes simplex virus thymidine kinase promoter, was used to estimate transfection efficiency. Firefly luciferase activity in transfected cells was measured using the Pikka Gene Kit (Toyo Ink America LLC, Wood Dale, IL, USA), according to the manufacturer's instructions. This activity was used as a marker for transfection efficiencies in the molecular clone-transfected cells. Transfection efficiency was calculated by setting the firefly luciferase activity in pBLV-AN060 to 1, and viral copy numbers were normalized using the relative firefly luciferase activity to eliminate bias from transfection efficiency.
2.12. Measurement of viral transactivation Each BLV molecular clone together with pU3-Luc and pRL-TK (Toyo Ink, Tokyo, Japan) were transfected into the 293 T cells using the FuGENE HD Transfection Reagent (Promega). The pU3-Luc and pRL-TK were used to measure viral transactivation by Tax and transfection
2.11. Measurement of viral titers Titers of virus produced from molecular clone-transfected 293 T 105
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substitutions using MEGA 7.0 software. Consequently, the nucleotide substitution rate between each strain was 0.92%–0.01%. As expected, this rate was low. We next analyzed the location of each nucleotide substitution to identify specific sites accumulated these substitutions. To search for unique substitutions among the 28 strains, the consensus genome sequence of the 28 BLV genomes was generated, and nucleotide substitutions were extracted by UGENE software. The result showed that no specific sites were present in the BLV genome (Fig. 2A). Because the nucleotide substitutions were sporadically located in whole BLV genome, we performed BLV strain categorization using each whole BLV genome. The 28 whole BLV genomes were phylogenetically analyzed using MEGA 7.0 software and, except for strains isolated from cow AN008 (pvAN008) strain, these genomes could be divided into three groups. These were named groups A, B, and C (Fig. 2B) and contained 11 (pvAF060, pvAF266, pvAF805, pvAF513, pvAN004, pvAN903, pvAN009, pvAN003, pvAN006, pvAF245, and pvAF982), 11 (pvAK007, pvAK011, pvAK001, pvAN014, pvAK006, pvAN011, pvAN013, pvAN015, pvAF293, pvAF193, and pvAF746) and 5 BLV strains (pvAF076, pvAF481, pvAF902, pvAF784, and pvAF438), respectively. The pvAN008 strain was classified separately because it harbored unique nucleotide substitutions (Supplementary Fig. S1) and could not be classified into these groups. The groups categorized were not biased toward any regions nor to any periods of sample collection (Table 1). On the other hand, BLV strains derived from EBL cows were categorized into group A or B, and all strains belonging to group C were derived from non-EBL cows (Fig. 2B). This suggested that the EBL-derived strains might be genetically similar strains, regardless of region. Next, to explore which substitutions determined the three groups, we searched for group-specific substitutions using multiple alignment of whole genome generated by MEGA 7.0 software. The specific nucleotide substitutions of each group are showed in Supplementary Fig. S1. Consequently, several group specific substitutions were found. The nucleotide position at 5528 in the env gene and at 6485 on upstream of miRNA were found to be common mutation of groups C and A, respectively. The nucleotide position at 7153 on the genes for G4 and AS1 genes was found in group B and two strains of group C (Supplementary Fig. S1). In the non-coding region, the group-specific substitutions were located in the LTR responsible for viral transcriptional activity, AS2, which is a putative noncoding RNA region, and upstream/downstream of the miRNA-coding region (Supplementary Fig. S2). Although the miRNA coding regions BLV-miR-B1, 2, 3, 4, and 5-3/5p, which are associated with tumorigenesis, were relatively conserved, as reported previously (Gillet et al., 2016; Rosewick et al., 2013), we identified the insertion of a thymidine residue into the B2-3p region in addition to substitutions in regions B3-3p and B5-3p. Furthermore, the AS2 region harbored the largest number of substitutions among the noncoding regions. These substitutions at nucleotides 2031, 2313, and 2594 of the AS2 regions were observed in most of the group A strains, whereas substitutions at nucleotides 2082 and 2301of the AS2 region were observed in most of the group C strains. In addition, several group-specific nucleotide substitutions located at genes resulted in amino acid mutation in the gag-pro-pol gene (Supplementary Fig. S3), env gene (Supplementary Fig. S4), and the pX region, which encodes nonstructural proteins (Supplementary Fig. S5). The substitution at amino acid 52 of the G4 protein located in the pX region was adjacent to an important sequence (amino acids 48–51; RLPL) related to virus productivity (Murakami et al., 2017) and was specific to group B and C strains. Several nucleotide and amino acid substitutions were located at or near functional domains and might be associated with viral properties, although most of the spontaneous substitutions were not located within the functional sites or domains of the BLV genome. This suggests that above specific nucleotide substitutions were accumulated in genome and generated each group classified in this study.
efficiency, respectively. After 48 h post-transfection, the cells were analyzed for firefly and Renilla luciferase activities using the Pikka Gene Dual Assay Kit (Toyo Ink), as previously report (Murakami et al., 2016). 2.13. Measurement of PVL in BLV-infected cows The BLV provirus in blood was measured with the BLV Coordination of Common Motifs qPCR-2 method (BLV-Co-Co-Mo-qPCR-2; Riken Genesis, Yokohama, Japan), as described previously (Jimba et al., 2012). The analysis was performed to quantify viral and BLV proviral genomes in all samples using GoTaq Probe qPCR Master Mix (Promega Corporation) and the 7500 Real-Time PCR system (Applied Biosystems). 2.14. Statistical analysis Comparison of two and three groups were performed using Student’s t test and one-way analysis of variance with Tukey’s post hoc test. All the statistical analyses were performed using R version 3.3.3 statistical software (Team, 2017). 3. Results and discussion 3.1. Identification of whole BLV genome sequences and genotypes Thirteen tumor samples and 14 blood samples were collected from cows with and without EBL (EBL and non-EBL cows, respectively) between 2007 and 2015, and BLV infection was confirmed as infection with BLV by PCR analysis and the AGID test, respectively. A tumor sample from an EBL cow (cow AN903), which has been used in our previous study (Murakami et al., 2016), was also included in this study. The 14 non-EBL dairy cows were kept on the same farm in Japan, whereas each of the EBL cows were each from a different Japanese farm (Table 1). Two dairy cows (pre-EBL cows; cows AF060 and AF266) had not developed EBL at the time of blood sampling time, but both developed the disease in 2014. Among the EBL cows, four cows (cows AK001, AK006, AK007, and AK011) were beef cattle, but we could not obtain information as to whether the other 10 cows (cows AN003, AN004, AN006, AN008, AN009, AN011, AN013, AN014, AN015, and AN903) were used for dairy or beef production (Table 1). All the 27 BLV strains were identified using whole genome sequences from direct sequencing. It is thought that the dominant strain in the BLV-infected cows was identified in this study because it was not possible to detect viral heterogeneity. To confirm the genotypes of the 27 BLV strains and a strain harboring cow AN903 identified in the present and previous studies, respectively, we phylogenetically analyzed them together with 90 registered BLV strains and identified their genotypes using the 400 bp BLV env gene sequence of total 118 BLV strains containing 28 strains used in this study, as described previously (Rola-Luszczak et al., 2013). Consequently, all the 28 BLV strains (Supplementary Table S3) clustered only with genotype 1 strains (Fig. 1); because genotype 1 is a common genotype in Japan and worldwide (Inoue et al., 2011; Matsumura et al., 2011; Rodriguez et al., 2009), this result was as expected. On the other hand, the 28 strains were generated subclusters with GenBank deposited strains, such as strains isolated in Argentina, the U.S.A., or Iran. Therefore, all 28 BLV strains used in this study were of genotype 1 but had limited genetic variations. 3.2. The categorization of the 28 BLV strains based on limited genetic variations The 28 strains were named as pvX, where X is the identification number of the cow. To examine a genetic variation among the 28 strains, the 28 BLV genome sequences were analyzed for nucleotide 106
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Fig. 1. Phylogenetic analysis by maximum-likelihood of 118 partial BLV env gene sequences from different geographical locations worldwide. The phylogenetic tree was constructed from 118 distinct 400 bp BLV env gene sequences, including the 28 BLV strains derived from BLV-infected cattle listed in Table 1 and the 90 BLV strains registered in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). The accession numbers and country of origin of these 90 strains are shown. Ten genotypes (G1–10) are indicated by numbers around the circumference of the figure.
specific group was isolated from cows carrying a specific BoLA-DRB3 allele. Thus, the BoLA-DRB3 allele of infected cows did not appear to select for any of the BLV genetic variants identified. These results showed that BLV genetic variations in BLV were independent of BoLADRB3, and that group A and B strain-infected EBL cows were not affected by host genetics. Therefore, these limited genetic variations could be associated with viral properties independent of BoLA-DRB3.
3.3. Association between the selection of BLV strains and the BoLA-DRB3 allele of host cows Because the BoLA-DRB3 allele is a determinant of pathogenesis in BLV-infected cows (Miyasaka et al., 2013; Takeshima and Aida, 2006), we thought this allele could influence the selection of BLV genetic variants of BLV. To evaluate whether strains from specific groups were selected by the BoLA-DRB3 type of an individual cow, we determined the BoLA-DRB3 allele of each infected cow used in this study using a PCR sequence-based typing method. The results showed that the BoLADRB3 alleles identified in this study were not biased toward a specific allele (Supplementary Table S4). Examination of the relationship between genetic variants of the BLV genome and the BoLA-DRB3 allele indicated that infection with a specific group of viruses categorized in this study was not related to the BoLA-DRB3 allele. In addition, no
3.4. Dependence of virus activity in vitro on the groups categorized in this study To evaluate the dependence of a viral property on a genetic variant, virus production was measured in vitro using BLV molecular clones from each strain analyzed in this study. First, 26 molecular clones were constructed from the 26 BLV-infected cows infected with group A, B, 107
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Fig. 2. Analyses of whole BLV genomes using 28 strains. (A) Summary of the substitution ratio throughout the BLV genome. The substitution ratio of nucleotides is shown with the BLV genome map. The consensus reference genome sequence generated from the 28 strains used in this study was compared with the individual sequences of the 28 strains. The top graph shows the ratio of mutations in each nucleotide over the whole genome. On the lower genome map, the right- and leftfacing arrows indicate the genes encoded on the sense and antisense strands, respectively. The square on the genome map shows the promoter region, LTR, or noncoding RNA. (B) Phylogenetic analysis of 28 whole BLV genome sequences. A maximum-likelihood phylogenetic tree was constructed from 28 distinct whole BLV genome sequences (the assigned accession numbers are shown in Supplementary Table S3). The dots show strains derived from pre-EBL (cows AF060 and AF266) or EBL cows. The bar on the figure denotes distance. 108
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Fig. 3. Virus replication activity in molecular clone-transfected 293 T cells. The viral in the supernatant of 293 T cells transfected with each molecular clone and pTKLuc were measured by (A) qPCR and (B) syncytium assay. Viral RNAs and infectious viruses were measured by qPCR and the syncytium assay, respectively, and normalized to the transfection efficiency measured by luciferase activity. (C) The transcriptional activity of 293 T cells transfected with each molecular clone, pU3Luc, and pRL-TK. Transcriptional activity was measured using firefly luciferase activity and normalized to the transfection efficiency determined by Renilla luciferase activity. The experiments were conducted in triplicate. Significant differences are indicated by single and double asterisks (*P < 0.05 and **P < 0.01, respectively, one-way analysis of variance).
transmissibility but also the risk of the EBL development. Thus, the measurement of PVL could be an indication of potential for transmissibility and pathogenesis in BLV-infected cows. First, we measured the PVL in blood samples collected from 18 cows (cows AF060, AF076, AF193, AF245, AF266, AF293, AF438, AF481, AF513, AF746, AF784, AF805, AF902, AF982, AK001, AK006, AK007, and AK011), which we could obtain blood sample at first sampling time. Six (cows AF060, AF245, AF266, AF513, AF805, and AF982), seven (cows AF193, AF293, AF746, AK001, AK006, AK0007, and AK011), and five cows (cows AF076, AF438, AF481, AF784, and AF902) were infected with group A, B, and C strains, respectively. The mean of PVL in group A, B, and C strain-infected cows were 23016.6, 18435.9, and 10038 copies/105 cells, respectively (Supplementary Fig. S6), and PVL was high in the order of group A, B, and C strain-infected cows, but these differences were not statistically significant. We next evaluated PVL transition, because a previous study reported that the PVL fluctuates in BLV-infected cows (Murakami et al., 2016). The PVLs of BLV-infected cows were additionally measured by qPCR in November 2015. Among the 18 cows with available blood samples, six were collected from the EBL cows, which included cow AF060 and AF266, and four from the nonEBL cows were slaughtered before analysis. Therefore, the PVLs of these cows could not be measured. However, the PVLs of eight non-EBL cows (i.e., cows AF076, AF245, AF293, AF481, AF513, AF784, AF902, and AF982) were measured in 2013 and 2015. The BLV-infected cows showed different patterns of change in PVLs between 2013 and 2015. Cow AF245 exhibited a significant increase in the PVL, whereas cows AF513 and AF982 had consistently high PVLs. Cows AF293 and AF784 exhibited significant decreases in the PVL, and cows AF076, AF481, and AF902 had consistently low PVLs (Fig. 4). Overall, these results, with regard to the categorizations described above, indicated that the PVLs in cows infected with group A strains increased significantly or remained high, whereas those in cows infected with group B and C strains decreased significantly or remained low, suggesting that virus productivity dependent on the limited genetic variations associated with PVL. In support of this finding, our previous study also showed that low virus productivity caused by a spontaneous deletion mutation occurred in a cow with a static and low PVL (Murakami et al., 2016), although BLV mainly propagates in vivo via mitosis (Rodriguez et al., 2011). Thus, the group A strains may have the potential to maintain a high PVL via high productivity. Although it is unknown whether the high virus production was directly related to a high PVL, high virus productivity is likely a determinant of a high PVL.
and C strains. Sequencing of these clones confirmed that the whole genome sequences were identical to each strain sequence identified in this study. Thus, the dominant stain in each infected with each cow could be isolated as a BLV molecular clone. In addition, clone pBLVAN903, which was cloned whole genome of pvAN903 strain, was previously constructed from the BLV-infected cow AN903 (Murakami et al., 2016) (Supplementary Table S3). These 27 molecular clones were transfected into cell line, and viral replication activity was measured using qPCR, syncytium assay, and luciferase assay. Next, the level of viral RNA in the supernatants of cell lines transfected with each clone was evaluated. Because 293 T cells are suitable for estimating virus production (Murakami et al., 2017), this cell line was used in this study. The results showed that the copy numbers of viral RNA were significantly higher in group A than in the groups B or C. Group B strains exhibited higher copy numbers than group C strains, but the difference was not statistically significant (Fig. 3A). Next, to validate these data, the production of infectious virus was measured using the syncytium assay. The results were very similar to those for the viral RNA copy number: the strains belonging to group A produced significantly higher titers of infectious virus than those belonging to groups B and C (Fig. 3B). Thus, the groups identified by the maximum-likelihood phylogenetic tree were reflected at the level of virus production in vitro. Next, to examine whether the transcriptional activity was related to virus production, we measured virus transcriptional activity using a luciferase assay. Each molecular clone together with pU3-Luc and pRLTK, which were used for measuring LTR transcriptional activity of BLV and transfection efficiency, respectively, were transfected into 293 T cells. After 48 h post-transfection, the transcriptional activities of each molecular clone were measured. Consequently, group A strains showed significantly higher transcriptional activity than group B and C strains, reflecting the results of virus production (Fig. 3A and B). These results showed that the limited genetic variation affected virus productivity in vitro, as assessed based on transcriptional activity, and suggested that the limited substitutions identified in this study would be functional site or essential sites for virus activity.
3.5. Relationship between PVL transition and the groups categorized in this study PVL is defined as the proviral copy number in blood and is dependent of viral propagation in BLV-infected cows (Aida et al., 2013). In addition, EBL cows tend to have a higher PVL than non-EBL cows. Thus, PVL measurement can be a useful tool to assess not only the 109
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Fig. 4. PVL of BLV in eight cows infected with group A, B or C strains. The solid and open bars show the PVL of BLV-infected cows in 2013 and 2015, respectively. All experiments were performed in triplicate. Results are shown as means with standard error. Significant differences are indicated by single and triple asterisks (*P < 0.05 and ***P < 0.001, respectively, Student’s t-test).
3.6. Viral properties affected by the spontaneous genetic variations
Acknowledgments
Previous studies have shown that the genetic distances between BLV genomes revealed by phylogenetic analysis are related to their geographical locations (Lee et al., 2016; Pandey et al., 2017; Polat et al., 2017a, 2016; Rodriguez et al., 2009; Yang et al., 2016). In this study, the phylogenetic tree based on the BLV genomes showed that virus production in vitro and the PVL were greater in cows infected with group A strains than in those infected with group C strains (Figs. 2B, 3A, B, and 4). These results indicate that similarities of genetic variations in BLV may be associated with similar virus production in vitro and virus propagation in vivo and that limited genetic substitutions could be related to these viral properties. Thus, the limited substitutions seem to be important for viral transmissibility and pathogenesis. Our study focused on genetic variations in BLV that may be related to viral activity and PVL transition. The results showed that a general concept for RNA virology, that is, the effect of genetic variations on viral properties (Domingo et al., 2012), could be adapted to BLV studies. In addition, BLV transmissibility and pathogenesis are affected by various factors, including the integration site of the provirus, immunoreaction, and the genetic backgrounds of the host and virus (Kabeya et al., 2001; Murakami et al., 2016; Rosewick et al., 2017; Takeshima and Aida, 2006; Takeshima et al., 2017). Based on the findings of the present and previous studies, it is conceivable that genetic variations in the virus are also associated with BLV transmissibility and pathogenesis via changes in viral properties. However, because only genotype 1 strains and small sample sizes were used in this study, this study is merely the first step in understanding BLV transmissibility and pathogenesis in terms of limited genetic variations throughout the whole BLV genome. Thus, additional analyses are needed to reveal differences in virulence based on genetic variations. We believe that the generation of further information about the relationship between genetic variations and the biological properties of BLV could lead to new approaches to control BLV infection.
This work was partially supported by a research project grant awarded by the Azabu University Research Services Division and JSPS KAKENHI Grant Number 17K15385. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2018.06.005. References Aida, Y., Murakami, H., Takahashi, M., Takeshima, S.N., 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front. Microbiol. 4, 328. Brenner, J., Van-Haam, M., Savir, D., Trainin, Z., 1989. The implication of BLV infection in the productivity, reproductive capacity and survival rate of a dairy cow. Vet. Immunol. Immunopathol. 22 (3), 299–305. de Brogniez, A., Bouzar, A.B., Jacques, J.R., Cosse, J.P., Gillet, N., Callebaut, I., Reichert, M., Willems, L., 2015. Mutation of a single envelope n-linked glycosylation site enhances the pathogenicity of bovine leukemia virus. J. Virol. 89 (17), 8945–8956. Domingo, E., Sheldon, J., Perales, C., 2012. Viral quasispecies evolution. Microbiol. Mol. Biol. Rev. 76 (2), 159–216. Durkin, K., Rosewick, N., Artesi, M., Hahaut, V., Griebel, P., Arsic, N., Burny, A., Georges, M., Van den Broeke, A., 2016. Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs. Retrovirology 13 (1), 33. Florins, A., Gillet, N., Asquith, B., Debacq, C., Jean, G., Schwartz-Cornil, I., Bonneau, M., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2006. Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep. J. Virol. 80 (24), 11998–12008. Florins, A., Gillet, N., Boxus, M., Kerkhofs, P., Kettmann, R., Willems, L., 2007. Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep. J. Virol. 81 (18), 10195–10200. Gillet, N.A., Hamaidia, M., de Brogniez, A., Gutierrez, G., Renotte, N., Reichert, M., Trono, K., Willems, L., 2016. Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis In Vivo. PLoS Pathog. 12 (4) e105588. Inoue, E., Matsumura, K., Maekawa, K., Nagatsuka, K., Nobuta, M., Hirata, M., Minagawa, A., Osawa, Y., Okazaki, K., 2011. Genetic heterogeneity among bovine leukemia viruses in Japan and their relationship to leukemogenicity. Arch. Virol. 156 (7), 1137–1141. Inoue, E., Matsumura, K., Soma, N., Hirasawa, S., Wakimoto, M., Arakaki, Y., Yoshida, T., Osawa, Y., Okazaki, K., 2013. L233P mutation of the tax protein strongly correlated with leukemogenicity of bovine leukemia virus. Vet. Microbiol. 167 (3-4), 364–371. Jimba, M., Takeshima, S.N., Matoba, K., Endoh, D., Aida, Y., 2010. BLV-CoCoMo-qPCR: quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm. Retrovirology 7, 91. Jimba, M., Takeshima, S.N., Murakami, H., Kohara, J., Kobayashi, N., Matsuhashi, T., Ohmori, T., Nunoya, T., Aida, Y., 2012. BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status. BMC Vet. Res. 8, 167. Juliarena, M.A., Barrios, C.N., Ceriani, M.C., Esteban, E.N., 2016. Hot topic: bovine leukemia virus (BLV)-infected cows with low proviral load are not a source of infection for BLV-free cattle. J. Dairy. Sci. 99 (6), 4586–4589. Kabeya, H., Ohashi, K., Onuma, M., 2001. Host immune responses in the course of bovine leukemia virus infection. J. Vet. Med. Sci. 63 (7), 703–708. Kincaid, R.P., Burke, J.M., Sullivan, C.S., 2012. RNA virus microRNA that mimics a b-cell
Conflict of interest The authors declare that there are no conflicts of interest.
Ethics statement This study was conducted in accordance with the Guidelines for Laboratory Animal Welfare and Animal Experiment Control set out by School of Veterinary Medicine, Azabu University [permit numbers: 161121-2].
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