Cloning and sequencing gB, gD, and gM genes to perform the genetic variability of bovine herpesvirus-1 from Indonesia

Aim: Previous research has shown that bovine herpesvirus-1 (BHV-1) in Indonesia was closely related to subtype-1 based on glycoprotein D genes. This study aimed to analyze the genetic variability of the BHV-1 isolated from the recent case in Indonesia not only based on gD but also other genes such as gB and gM and to study the homology and similarity of the sample to other BHV-1 isolated in other countries or regions. Materials and Methods: Samples were drawn from the tracheal organ in recent field case and prepared for DNA extraction. The gB, gD, and gM were amplified using nested polymerase chain reaction (nPCR) with our specifically designed primer pair and based on the specified bands of 350 bp gB, 325 bp gD, and 734 bp gM confirmed as BHV-1. The PCR product was ligated into pGEM-T and transformed into competent Escherichia coli. The purified plasmid was subsequently sequenced. Results: The virus sample isolated from the recent field case of infectious bovine rhinotracheitis (IBR) from Indonesia showed variability based on the gB, gD, and gM sequences. However, all of the genes had high similarity (98-100%) to BHV-1.2. Conclusion: The recent field case of IBR in Indonesia was similar to BHV-1.2.


Introduction
The genome Bovine herpesvirus-1 (BHV-1) consisted of two segments, which were unique long (UL) and unique short (US) [1]. The genome herpesvirus is large, double-stranded, and linear [2]. The BHV-1 included in class D Herpesvirus [3]. The US located between internal repeat and terminal repeat and had two alternative directions toward the UL. Inversely, the UL was fixed orientation [4,5]. Each segment encoded some glycoproteins, such as gB, gD, and gM. The glycoproteins played an important role in virus-cell interaction [6]. Glycoproteins B, D, and M were identified in the virion [7]. The glycoproteins B and D have been extensively studied [8]. The glycoprotein D played an important role in virus cell as binding protein receptors [9,10]. The glycoprotein B had significant variability used in describing molecular evolutionary [11,12]. Glycoprotein M was the only conserved glycoprotein among Herpesviridae [13][14][15]. The glycoprotein M was inessential for lytic replication [16] and increased by dexamethasone treatment [17].
The BHV-1 has been divided into three subtypes, which were BHV-1.1, BHV-1.2a, and BHV-1.2b [18]. BHV-5 used to be classified as BHV-1.3. There were several methods that have been used to differentiate the subtypes, such as a monoclonal antibody of specific antigen and DNA fingerprinting by visualizing the presence of the restriction site [19][20][21]. Recently, sequencing of the whole genome was necessary to differentiate the BHV-1 field strain from vaccine strain based on SNPs [22,23]. Some BHV-1 strains such as Cooper strain had the pattern of genome mapping that used restriction enzyme [24]. The gC mapping could differentiate the BHV-1 from BHV-5, which had different patterns in the site of restriction enzyme [25]. There was a restriction fragment length polymorphism that could differentiate BHV-1.1, BHV-1.2, and BHV-5 [26]. The homology of the BHV-1.1 and BHV-1.2 was about 95% [20], and that of the gD of the BHV-1.1 and the BHV-1.2 was 98.1% [27] Meanwhile, the similarity of the BHV-1 and BHV-5 was 82% [9,10,28] Available at www.veterinaryworld.org/Vol.11/September-2018/9.pdf evolution mechanism [21,29]. Molecular recombination frequently occurred during the replication cycle of the BHV-1 [30]. Recombination might modify the virulence of alpha herpesvirus [29] and gave different isomeric forms of the virus [31]. The recombination could occur both in vivo and in vitro [32]. Some studies have examined the function of recombinant viruses with a mutation such as deletions or replacements of individual genes in the developing vaccines [33][34][35].
This study aimed to perform to test of the characteristic of BHV-1 found in the field case of infectious bovine rhinotracheitis (IBR) disease and to study the homology and similarity of the samples to other BHV-1 isolated in other countries of the region using some genes both from UL and US segments.

Materials
The samples of the study were drawn from the tracheal section in the field cases. They were obtained from the Animal Disease Investigation Center, Lampung, of the Ministry of Agriculture of the Republic of Indonesia.

The preparation of samples
The tracheal samples were washed using phosphate-buffered saline (PBS) 3 times. The mixture of 1000 IU/ml Penicillin (Meiji Pharmaceutical Industries, Indonesia), 1000 µg/ml Streptomycin-sulfate Penicillin (Meiji Pharmaceutical Industries, Indonesia), 250 µg/ml Kanamycin Penicillin (Meiji Pharmaceutical Industries, Indonesia), and the PBS was used in the first washing. And then, they were washed 3 times using pure PBS. Subsequently, they were crashed out using DMEM and quartz sand and centrifuged at 1000 rpm for 5 min. The suspension was filtered using Sartorius Filter 0.2 mµ. The filtered fluid was prepared for DNA extraction. Viral DNA was extracted using QIAamp DSP DNA Mini Kit (Qiagen). The DNA eluted with 200 µl AE Buffer that contained 10 mM Tris-Cl and 0.5 mM EDTA pH 9.0. The quality of DNA extraction was measured using BioSpec-nano spectrophotometer (Shimadzu Biotech, Japan).

Mapping and primer designing for gB, gM, and gD gene amplification
We designed two pairs of primer to amplify gB, gD, and gM in examining the variability of Indonesian BHV-1 sample. The sequence of these primers is shown in Table-1 [36]. Mapping of the amplified fragment within the whole genome of BHV-1 is shown in Figure-1.

Alignment, amplification, and cloning the targeted gene
The study intended to identify the characteristics of Indonesian BHV-1 samples based on gB, gM, and gD . The alignment was determined using DNAStar program with eight (n=8) reference genes obtained from GenBank with accession numbers (AJ004801. The polymerase chain reaction (PCR) assay proceeded in two rounds. Each round consisted of 2.5 mM MgCl 2 , 0.3 mM for each of dNTPs, 1 U of DNA Polymerase, 10 pmol forward and reverse primer, and template DNA of <100 ng per reaction until total volume was up to 50 µl for PCR grade water (KAPA HiFi HotStart ReadyMix, KAPABiosystem).
The optimization of the PCR of gB was performed as follows: Initial denaturation for 3 min at 95°C, denaturation for 20 s at 98°C, annealing for 15 s at 53°C which were run in 35 cycles, while the extension lasted for 15 s at 72°C and the final extension The PCR product of the second round was ligated into plasmid pGEM ® -T (Promega) using T4 ligation enzyme (Promega) once it has been purified in FastGene purification (NIPPON Genetics) and attached using poly A at the 3'-end with 10x A-attachment (TOYOBO). The ligated plasmid was transformed into competent Escherichia coli C3040 (NEB ® stable, High efficiency) and kept in LB agar overnight at 37°C. The single white colony was picked up and transferred into LB broth media containing 100 µl ampicillin per mL and incubated at 37°C with constant shaking overnight. The recombinant cloned plasmid was prepared to release the DNA fragment by chemistry lysis [37]. Moreover, it was confirmed by gel agarose, and any appropriate cloned plasmid was subsequently subjected to PCR amplification using the designated primers. Only the positive inserted cloned plasmid was subsequently subjected to PCR sequencing.

The establishment of cloned plasmid and nt sequencing
The cloned plasmid was purified using FastGene (NIPPON Genetics) and subjected to sequencing with Sanger methods. The cycling of the forward and reverse PCR sequencing was based on the protocol of BigDye Terminator v3.1 Cycle Sequencing Kit at the annealing temperature of 50°C using M13. The Sanger sequencing was run using 3130 Genetic Analyzer (Applied Biosystems). The data were analyzed using DNAStar Lasergene software. The resulting consensual sequence of the forward and reverse sequence was submitted and compared to the reference genes retrieved from GenBank with accession numbers (AJ004801.1, KU198480.1,

Sequence alignment
Sequence results were analyzed using SeqMan (DNAStar Lasergene, version 7). All of the assembled sequences were aligned using Clustal W method packaged in MegAlign software (DNAStar Lasergene, version 7) based on multiple sequence alignments [38,39].

The sequence of gD between BHV-1 and BHV-5
The glycoprotein D gene of Indonesian sample was sequenced, which covered 323 bp. This segment has been used for the detection and monitoring of IBR diseases in Indonesia. We analyzed the results and aligned them with the reference genes of BHV-1.  (Table-2). SNPs occurred in 29 points between BHV-1 and BHV-5. Transversion occurred in 6 points. The transition occurred in 23 points. The base mutation between adenine and guanine occurred in almost the same number of points as the base mutation between cytosine and thymine. The previous study used gD to examine the difference between BHV-1 and BHV-5. The similarity between the samples and reference genes was identical with BHV-1.2 strain B589 (99.7%). The samples had about 92.3% similarity to BHV-5 and 98. .1% similarity to other BHV-1.1. The similarity between BHV-1 and BHV-5 was about 92.0-92.9%. gB between BHV-1.1 and BHV-1.2 The sequences of glycoprotein B gene of the sample (L/9) covered 335 bp nt. The alignment was made between the sample L/9 and the reference genes of BHV-1.1 (n=3) and BHV-1.2 (n=4) using Clustal W (MegAlign software, DNAStar Lasergene). The results showed that the BHV-1.1 gB reference position was 55924-56227, while the BHV-1.2 reference position was 55652-55955. These sequences resulted in the deletion and substitution as summarized in Table-3. There were 18 nt deletions occurring in the sequences between BHV-1.1 and BHV-1.2 strain B589 at nt 259-276. The substitution (the transitions of adenine to guanine and thymine to cytosine) was observed at the sequence in the positions 304, 321, and 438. The samples L/9 did not have any deletion. The sample L/9 showed about 99.1% similarity with BHV-1.1 and 99.7-100% similarity with BHV-1.2. It showed that the sample L/9 was closely related to BHV-1.2.

The sequence of gM between BHV-1.1 and BHV-1.2
The sample (L/9) covered 689 bp of gM segment. The alignment was made between the sample L/9 and the reference genes of BHV-1.1 (n=3) and BHV-1.2 (n=4) using Clustal W (MegAlign software, DNAStar Lasergene, USA). The results showed that the BHV-1.1 gM reference position was 85089-85735, while the BHV-1.2 reference position was 84874-85520. The sequences resulted in the substitution mutation as summarized in Table-4. There were three mutation sites found in the sequences between BHV-1.1

Discussion
The first case of IBR in Indonesia was in 1981. The predicted causal factor was due to live cattle importation [40,41]. Previous research reported that only BHV-1.1 was found in the field case [42]. The case of IBR in Indonesia escalated. This study was a molecular study of the recent field case of IBR. The molecular analysis was based on sequencing the analysis of Table-3: The alignment results of gB between BHV-1.1 and BHV-1.2 There were 18 nt deletions except the sequence of BHV-1.2 strain B589. The sample L/9 did not have any deletion site. There were four single nt polymorphisms occurring at 251, 304, 321, and 438. The point of mutation was substitution transition (A to G and T to C) covered with blue box that differentiated BHV-1.1 and BHV-1.2.  6 7 7 7 7 7 7 7 2 3 5  1 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 2 9 6 BHV=Bovine herpesvirus, A to G=Adenine to guanine, T to C=Thymine to cytosine, nt=Nucleotides
Based on the alignment of gM, the sequence contained substitution (only transition). The gM served the function as secondary envelopment and formed with gN that inhibited transporter-associated processing [43]. The gM fragment was more conserved than gB and gD to measure the evolutionary molecular genetics. The sample L/9 had close similarity to BHV-1.2 strain SP 1777 (KM 258883.1) and BHV-1.2 strain SM 023 (KM 258882.1).
This molecular study showed that there was variability of the samples drawn from field case among the reference genes (BHV-1.1 and BHV-1.2 BHV-5). The use of gB, gD, and gM led to the differentiation of BHV-1.1 and BHV-1.2 and particularly BHV-1.2 strain B589.

Conclusion
The sequence variability within gD, gB, and gM genes of BHV-1 isolated from Indonesian samples are mostly due to transition and transversion mutation. The result also indicated that the causative agent for recent IBR cases in Indonesia is related to BHV-1.2.