IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BOVINE HERPERVIRUSES ( BoHV ) DNA TERMINASE PARTIAL GENE IN ACEH CATTLE

Bovine Herpesvirus (BoHV) is a member of Herpesviridae family caused infectious bovine rhinotracheitis (IBR) among cattles, resulting in economic loss in cattle industry. BoHV-1 infection in cows is closely related to abortion, respiratory infection, reduction of milk production, infertility, and low birth weight. The aims of this study were to identify and characterize the molecular of BoHV-1 and other types of BoHV and other Herpesviridae family using PCR to amplify DNA terminase gene. Four out of 210 nasal swab samples were positive for herpes virus based on DNA terminase gene detection. Further characterization of these positive samples showed 99-100% identities to BoHV-1 and BoHV-6 sequences. Genetic distance between genera of BoHV-1 and BoHV-6 was 0.518 and within genera was 0.001 and 0.044. According to phylogenetic tree analysis of DNA terminase gene, 3 samples were clustered with BoHV-1 in the genus of Varicellovirus; meanwhile one sample was clustered with BoHV-6 in Macavirus genus. This study provides scientific information on molecular characteristics of Herpesviridae family, especially BoHV-1 in Aceh province, Indonesia. ____________________________________________________________________________________________________________________

Several diseases caused by Bovine Herpesvirus-1 (BoHV-1) are infectious bovine rhinotrachaetis (IBR), infectious pustular vulvovaginalis (IPV), and infectious pustular balanoposthitis (IPB) (Ackermann dan Engels, 2006;Muylkens et al., 2007).An investigation in the first and second trimester of abortion among cattle revealed that BoHV-1 was the caused of abortion (Yildirim et al., 2011).An analysis model was used in dairy farms to see the impact of BoHV-1 infection on milk production.The result showed a reduction in milk production on average 0.92 kg/day/head (van Schaik et al., 1999).Due to the challenges and losses caused by BoHV, it is important to control diseases caused by BoHV, including surveillance and serological and/or molecular identification of the virus.This is in accordance to the Minister of Agriculture Decree No.4026/kpts/OT.140/4/2013 (2013), on diagnosis of strategical infectious animal diseases and exotic diseases stated that IBR is one of the 22 diseases that must be controlled and mitigated by the central government, provincial government, and local district/municipality government in accordance to their authority.Moreover, IBR is one of disease that must be eradicated from breeding and artificial insemination centers in Indonesia.
The BoHV tipe which is found in Indonesia based on molecular characteristic of glycoprotein D (gD) gene is Bovine Herpesvirus-1.1 (BoHV-1.1)(Saepulloh et al., 2009).Currently, there is no official report of the existence of other BoHV types (BoHV-2, BoHV-3, BoHV-4, BoHV-5, dan BoHV-6) in Indonesia, neither from serological test nor agent identification.This study aimed to detect the presence of BoHV-1 and other herpes virus family among local Aceh cattles, to determine molecular characterization of DNA terminase gene partially on detected herpes virus family, and also to analyze and to identify genetic relationship between the detected viruses.

MATERIALS AND METHODS
Nasal swab samples of this study were archive samples collected from Aceh cattle in Indrapuri district, Aceh Besar Regency.The samples were kept in a 4 C and transported to laboratory for storage at -40 C until further analysis.Total samples in this study were 210 samples.
This study was conducted in the Virology Laboratory of Medan Diseases Investigation Center and Biotechnology Laboratory of The Primate Research Center, Institute for Research and Community Services, Bogor Agricultural University (PSSP LPPM-IPB).

Molecular Detection Using Degenerate Primer
DNA was extracted from nasal swab samples using QIAamp DNA Blood mini Kit (Qiagen, Hilden, Germany) according to the guideline from manufacturer.The concentration and purity of DNA were measured and then the sample was stored at -40 C or used directly for PCR.
PCR amplification was carried out using nested PCR method and degenerate primer according to PREDICT protocol (Anthony et al., 2013).In this study two sets of primers were used (Table 1) to amplify the DNA terminase target gene.Amplification process was carried out using PCR machine (VeritiThermal Cycler, Applied Biosystem, USA) referring to van Devanter et al. (1996) and Chmielewicz et al. (2001).PCR product was analyzed on 1.8 % agarose gel electrophoresis containing 1 µg/ml ethidium bromide (EtBr).Electrophoresis result was further visualized in GelDoc using Quantity One program (BioRad).

Sequencing and Molecular Analysis
Positive samples based on PCR result were sequenced.Sequencing result was analyzed using MEGA 6.0 (Molecular Evolutionary Genetics Analysis 6) (Tamura et al., 2013) and aligned using ClustalW to determine the variance in nucleotide sequence.The alignment result was further analyzed for identity tracing (% identity) using Basic Local Alignment Search Tool (BLAST) (McGinnis and Madden, 2004) and BioEdit program (Hall, 2011).Geneious program was used to translate the nucleotide sequences into amino acid.MEGA 6.0 software was used to determine genetic distance and to construct the phylogenetic tree (Tamura et al., 2013).

DNATerminase Gene Amplification
DNA terminase gene amplification resulted positive band around 416 bp using TS-TERM-708s and TS-TERM-708as primers (Figure 1).The result was similar to Chmielewicz et al. (2001), whereby herpes virus detection using degenerate primer for DNA terminase gene as the target would yield an amplicon 419 bp.The different amplicon length between our result and the reference was probably caused by species difference.The analyzed sample sequence was identical to BoHV-1 whereas the reference sequence was Alcelaphine herpesvirus 1 (AlHV-1).Chmielewicz et al. (2001) also detected possibly novel gamma herpes virus from goats which yielded an amplicon with several base pairs different.Similar detection was carried out by Kleiboeker et al. (2002), in deers suspected of Malignant Catarrhal Fever (MCF) due to Deer herpesvirus.Detection using degenerate primer also produced an amplicon with several base pairs different.

Herpes Virus Identification based on DNA Terminase Sequence
The detection and identification of DNA terminase revealed a similarity between subfamilies of Alpha herpesvirinae and Gamma herpesvirinae.After further analysis on genus level, Gamma herpesvirinae subfamily was found to be similar in 1 sample (DIC1.6)only to 1 genus, namely Macavirus which includes BoHV-6 species.This is only detected in one sequence sample specifically DIC1.6.Alpha herpesvirinae subfamily was similar to only 1 genus, namely Varicellovirus, which includes BoHV-1 species, detected in 3 sample sequences, namely DIC 1.1, DIC 1.2 and DIC 1.7 (Tabel 2).The similarity between species was analysed using BLAST with the highest similarity value compared to other species.Two species of herpes virus, BoHV-1 and BoHV-6, was detected from DNA terminase gene.This indicates that DNA terminase is a conserved area and could be used to detect herpes virus at family level.This was in line with Przech et al. (2003), who found that DNA terminase gene is a conserved area which is used by the virus during viral genomic cleavage dan packaging.Several researches have used DNA terminase gene to detect herpes virus existence in animals (Kurobe et al., 2008;Kleiboeker et al., 2002;Chmielewicz et al., 2001).

DNATerminase Matrix Identity
Results of matrix identity analysis of DNA terminase gene sequence showed similarity in identity value using Bioedit and BLAST software.The identity value of the samples was 99-100% towards the reference viral sequence.The obtained value was the highest identity percentage compared to the reference viral sequence in GenBank database with query cover value 100% (Table 3).This result is consistent with Saepulloh et al. (2009), who stated that the matrix identity of BoHV-1 isolates in Indonesia was 98.8-100 % compared to Cooper strain Bovine herpesvirus 1.The result indicated that the sample sequence analyzed was identical to the reference.

Genetic Distance and Phylogenetic Construction of DNA Terminase Gene
The genetic distance between genera Varicellovirus and Macavirus on DNA terminase target gene was 0.518.Large difference between genera indicates different genetic characteristic, which could be seen on phylogenetic tree.The phylogenetic tree of the samples formed two different genera cluster.The first cluster was members of Alpha herpesvirinae subfamily which consisted of DIC1.1, DIC1.2, and DIC1.7.The second cluster was Gamma herpesvirinae subfamily which consisted of DIC 1.6 sequence sample.
Within genera distance of Macavirus and Varicellovirus were 0.044 and 0.001, respectively (Table 4).The small genetic distance of Varicellovirus indicated that its members is genetically more stable compared to members of Macavirus.Previous study on diversity of virus Suid herpesvirus-1 (SuHV-1) evolution stated that smaller within genera genetic distance is more stable compared to other groups with larger value (Fonseca et al., 2016).
DNA terminase phylogenetic tree was constructed by comparing 5 other genus as the outgroup, namely genus Radhinovirus and Lymphocryptovirus of Gamma herpesvirinae subfamily, genus Roseolovirus and Cytomegalovirus of Beta herpesvirinae subfamily, and genus Simplexvirus of Alpha herpesvirinae subfamily.The outgroups were clustered based on each genus characteristic and described the relationship between our samples and some reference of herpes virus (Figure 2).

Table 4 .
Genetic distance within genera and between genera on DNA terminase target gene