49 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 Accepted: 31.01.2020 | Available on line: 27.07.2021 1Virology Department, Ankara University, Veterinary Medicine, Ankara, Turkey 2Virology Departmen, Hatay Mustafa Kemal University, Veterinary Medicine, Hatay, Turkey. 3Virology Department, Van Yuzuncuyıl University, Veterinary Medicine, Van Turkey. *Corresponding author at: Ankara University, Veterinary Medicine, Ankara, Turkey. E-mail: sevalbilge@hotmail.com. Seval Bilge Dağalp1*, Firat Dogan2, Ali Riza Babaoglu3, Touraj Aligholipour Farzani1 and Feray Alkan1 Keywords Bovine herpesvirus type 4, Glycoprotein B (gB), PCR, Sequencing, Thymidine kinase (TK), Virus isolation. Summary Bovine herpesvirus type 4 (BoHV-4) is a common virus in the world that is detected in clinically ill or in apparently healthy cattle. This study provides a molecular characterization of BoHV-4 strains from 24 cattle some showing respiratory and/or reproductive problems and some without any apparent clinical sign. This study also reported the growth properties of five BoHV-4 field isolates. The 24 sampled cattle came from 13 different herds in 10  provinces collected between 2007 and 2018. Phylogenetic analysis using partially amplified nucleotide sequences of ORF8 genes coding glycoprotein B (n = 24) and ORF3 genes coding thymidine kinase (n =  9), demonstrated genetic variability among the BoHV-4 strains analysed. The partial gB gene sequences clustered in three different genotypes (genotype I, II and III) were located within the genotype I cluster, such as Movar strain. The analysis of the five BoHV-4 strains isolated from vaginal swabs (n = 2), nasal swab (n = 1), and brain samples (n = 2) revealed no significant differences in their growth properties in MDBK cell culture. Genetic variability of bovine herpesvirus type 4 (BoHV-4) field strains from Turkish cattle herds of the virion, essential for viral infectivity (Little et  al. 1981, Lomonte et  al. 1997). It is one of the most studied genes because its region is one of the most highly conserved among Herpesviridae family members (Goltz et  al. 1994, Wellenberg et  al. 2001). Tissue tropism instead is most likely associated with variations in virion surface glycoproteins (Frazier et al. 2002, Wellenberg et al. 2001). Another frequently studied gene is ORF3, which codes for thymidine kinase (TK). It helps in understanding the precise regulation of specific genes, their promoter regions, and transactivation mechanisms (Kit et  al. 1986, Lomonte et al. 1992). In Turkey, previous studies have shown that BoHV-4 infection is prevalent in dairy cattle herds (Bilge Dağalp et  al. 2007, Bilge Dağalp et  al. 2010, Bilge Dağalp et  al. 2012, Gur and Dogan 2010, Kale et  al. 2011), with seropositivity levels reaching 56.8% and 44.9% in sampled herds with reproductive disorders and healthy appearance, respectively (Bilge Dağalp et  al. 2007). While several reports have detected BoHV-4 from cattle with or without clinical symptoms, the genetic characterization of local viruses is not yet well documented. The two Introduction Bovine herpesvirus type 4 (BoHV-4), a member of the Herpesviridae family, subfamily Gammaherpesvirinae in the Rhadinovirus genus, is found worldwide among cattle populations (Roizman et  al. 1992, Zimmerman et  al. 2001). The BoHV-4 genome is a double-stranded DNA of 144 ± 6kb and a long unique coding region (LUR) or light DNA (L-DNA) with a size of approximately 108 kb. Based on restriction endonuclease patterns, almost all American strains belong to the DN-599 group whereas European strains belong to the Movar 33/63 group (Bublot et  al. 1990). However, the restriction patterns of some strains do not completely match with those belonging to the DN-599 or Movar 33/63 groups. These are the LVR 140 strain, known as the Belgian reference strain (Thiry et  al. 1992, Wellemans et  al. 1986), and the Taiwan strain isolated from bovine vascular endothelial cells (Lin et al. 1997). Several BoHV-4 genes have been examined in detail by various researchers (Egyed et al. 1996, Goltz et al. 1994). BoHV-4 open reading frame 8 (ORF8) codes for glycoprotein B (gB), which is a major component 50 Genetic variability of BoHV-4 Bilge Dağalp et al. Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 Bilge Dağalp et al. 2012). Considering that the arrival dates of these samples are old, the specimens did not propagate in cell cultures and were discarded. As a result of these elimination only isolates and BoHV-4 suspected samples that were delivered to our laboratory in recent years were amplified and sequenced. They were selected as representative of different herds, clinical disorders, and sampling years. They included diagnostic samples originated from nasal and vaginal swabs, milk, and necropsy samples collected between 2007 and 2018 from cattle with severe reproductive disorders, abortions, and/or respiratory system disorders from 13 different dairy cattle herds of 10 provinces (Table I). PCR DNA extraction was carried out according to Sambrook and colleagues (Sambrook et  al. 1989). The detection of BoHV-4 DNA was performed using oligonucleotide primers specific for BoHV-4 ORF8 gene purposes of this study are therefore i) analysis of the gene regions coding gB and TK in local BoHV-4 strains from cattle suffering reproductive and respiratory disorders; ii) investigation of their growth characteristics in cell cultures. Materials and methods Samples For this study, 24 cattle samples in which BoHV-4 was identified were selected previously (Bilge Dağalp et al. 2010, Bilge Dağalp et  al. 2012). Those samples that were still positive when re-tested in this study were used for analysis of the genes coding gB. The field isolates (n = 5) and the samples that were delivered to our laboratory in the last years were also used. The sequences used in this study were obtained from the samples that were targeted for the amplification of the gB gene region or were found positive against gB in previous two projects (Bilge Dagalp et al. 2010, Table I. Details of the Bovine herpesvirus type 4 positive samples . Herd No/ Location Virus code Clinical signs Year Samples Sequences of gB genes Sequences of TK genes2 GenBank Acc.No Genotype GenBank Acc.No. I - Bursa K339 Metritis 2007 Vaginal swab1 EU055543 II JX644990 II - Aksaray KCVS12 Metritis, Upper respiratory disease 2007 Vaginal swab1 GQ246864 JX644991 KCORG Abortus 2011 Organ MK543547 ND KCB12 Neonatal death 2009 Brain1 JX644988 JX644992 KCVS2 Repeat breeder, upper respiratory disease 2009 Vaginal swab MK543548 ND III - Kars KBSB9 Abortus 2007 Brain GQ246865 ND IV - Kirklareli TG1 Metritis 2007 Leukocyte GQ246866 ND TGVS550 Repeat breeder 2011 Vaginal swab MK543551 ND TGVS549 Repeat breeder 2011 Vaginal swab MK543550 ND TGVS29 Abortus 2011 Vaginal swab MK543549 ND TG4 Metritis 2007 Vaginal swab GQ246863 ND V - Balikesir T6 Repeat breeder 2007 Leukocyte GQ246867 ND VI - Yozgat YL Abortus 2007 Leukocyte GQ375280 ND VII - Sivas U-NS66 Upper respiratory disease 2009 Nasal swab1 JX644989 JX644993 VIII - İzmir IZ1LK136 Abortus 2010 Leukocyte MK543546 ND IX - Ankara ANK.NS Repeat breeder, upper respiratory disease 2014 Nasal swab KX192364 ND ANK.Br. Neonatal death 2015 Brain1 KX192363 KX352279 X - Ankara ANK.Teat. Mammary lesions, mastitis 2014 Teat lesion KX192365 KX352280 XI - Kirklareli B1AC4 Upper respiratory disease, pneumonia 2017 Lung MH318579 MH614620 B2AC4 Upper respiratory disease, pneumonia 2017 Lung MH318580 MH614621 XII - Kastamonu KAS21 Repeat breeder 2017 Leucocyte MG181944 I Negative KAS24 Repeat breeder 2017 Leucocyte MG181945 Negative KAS25 Repeat breeder 2017 Leucocyte MG181946 MG242137 XIII - Ankara 9870MILK Mastitis 2018 Milk MH605274 III Negative 1 BoHV-4 field strains isolated from these samples; 2 All clustered in genotype I; ND = Not done 51 Bilge Dağalp et al. Genetic variability of BoHV-4 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 tested for BoHV-4 by direct immunofluorescence (IFA) technique, using a commercial conjugate (Bio028, BioX, Belqium) as described by Nettleton and colleagues (Nettleton et al. 1982) and/or PCR as described in the material and method section in this study. Samples showed cytopathic effects in MDBK cell cultures, 5-7 days after 3rd passage. After isolation and identification procedure, the supernatants from third passages of isolates (n = 5) were used for titration. The virus titer was calculated using the method of Reed and Muench (Reed and Muench 1938). To investigate the replication kinetics, five BoHV-4 strains isolated in this study were used. These BoHV-4 isolates (n = 5) were inoculated into MDBK cells grown in 24-well plates (Greiner Bio-one, Frickenhausen, Germany) at a multiplicity of infection (MOI) of 0.1. Cell cultures were incubated at 37 °C with 5% CO 2 atmosphere and observed daily for the presence of CPE. The supernatants were harvested at 24, 48, 72, 96, and 120 h post-infection (hpi) and frozen at -  80  °C for further viral quantitation, as described by Verna and colleagues (Verna et al. 2016). The percentage of CPE was recorded at the time of supernatant collection. Five replicates were performed to establish the kinetics of viral replication for each experiment (virus strain and harvest time points). Negative controls were included in each experiment. To determine the replication kinetics of these local isolates (n = 5), the infectivity titers were estimated using viral suspensions collected at 24, 48, 72, 96, and 120 hpi from every MDBK cell culture (Reed and Muench 1938). Results PCR, sequencing, and phylogenetic analysis After PCR, gB gene region of 24 samples and TK region of 9 of 24 samples were also amplified. In PCR, the local viruses produced the expected size of products (615 bp) for the gene coding gB and produced the expected size of products (567 bp) for the gene coding TK (Table I). All amplicons from the local strains along with the reference strain (DN-599) were commercially sequenced and their genome sequences were deposited in GenBank under the accession numbers in Table I. Phylogenetic analysis, performed using the gB coding gene sequences of the local viruses (n = 24) and BoHV-4 strains with different genotypes in GenBank, revealed that genotype II was prevalent in the local isolates (20/24, 83.3%) and sampled herds (11/13, 84.6%). The other two genotypes (genotype  I and III ) were identified in other two codifying gB (5’-CCCTTCTTTACCACCACCTACA-3’ and 5’-TGCCATAGCAGAGAAACAATGA-3’) (Goltz et  al. 1994), and for BoHV-4 ORF3 gene codifying TK (5’-GTTGGGCGTCCTGTATGGTAGC-3’ and 5 ’ - ATG TATG CCC A A A AC T TATA ATATG ACC AG - 3 ’ ) (Egyed et al. 1996) PCR was conducted as described by Wellenberg and colleagues (Wellenberg et  al. 2001) with some minor modifications. Briefly, 3  μl DNA was subjected to thermocycling in a 30  μl reaction mixture containing 2.5  U Taq DNA Polymerase (Fermentas, Lithuania), 3.5 mM dNTP mix (Fermentas, Lithuania), 10 pmol of each primer, 1.5 mM MgCl 2 , 1  X  PCR buffer, and 6% DMSO. Thermal cycling conditions were 6 min at 96 °C, followed by 40 cycles of 56 °C for 45 sec, 72 °C for 2 min, and 95 °C for 1 min. The reaction tubes were kept for a further 10 min at 72 °C for final extensions. PCR products were visualized in a transilluminator after electrophoresis in 1% agarose gel containing ethidium bromide. The North American (DN-599) reference strain of BoHV-4 was used as positive control. The reference virus strain was kindly provided by Dr. G.J. Wellenberg (Lelystad, Netherlands). Sequencing and phylogenetic analysis PCR amplicons with the expected sizes (615 bp and 567 bp for genes coding gB and TK, respectively) were subjected to sequence analysis. The amplicons were firstly gel purified using a commercial kit (GeneMark, Taiwan) and sequenced by a commercial enterprise. The gB and TK gene sequence assembly and editing were performed using Bioedit (Version 7.0.5.3) and Clustral W (Hall 1999). The sequences were then compared with those of the GenBank and sequenced database for similarities using the Basic Length Alignment Search Tool (BLAST) software of the National Center for Biotechnology Information (NCBI) (Althsul et al. 1997). The obtained analysis data were compared with different BoHV-4 strains by using neighbor joining method (1,000 replication) and they were phylogenetically analyzed in MEGA 6.0 software program under Kimura 2  parameter (Tamura et al. 2011). Virus isolation and determination of the replication kinetics of isolates in cell culture All samples (n = 24) were inoculated (100 µl) onto MDBK cell cultures for virus isolation. The cells were washed 3 times with PBS-M before adding medium. The monolayers were incubated at 37 °C in a 5% CO 2 atmosphere and checked daily for the appearance of cytopathic effects (CPE). The monolayers were passaged at weekly intervals for a total of three passages. The supernatants of all cell cultures were 52 Genetic variability of BoHV-4 Bilge Dağalp et al. Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 98% and 96.5%, respectively (Figure 5 and Table III). Considering the sequences for the two genes at aa and na level, the differences in ORF8 sequences were greater than the differences for the ORF3 gene sequences between our viruses and BoHV-4 from other countries, and also between our local viruses. Figure 6 shows the amino acid sequence alignments of ORF8 genes coding gB of our BoHV-4 samples and other strains, including the reference viruses (Movar and DN -599). Virus isolation and replication kinetics Out of 24 samples previously confirmed as positive for BoHV-4 by PCR on ORF8 gene amplification, five showed cytopathic effects in MDBK cell cultures, 5-7 days after the 3rd passages. These BoHV-4 strains were then successfully isolated and identified by immunofluorescence and/or PCR. The origin of these samples was as follows: two vaginal swabs (K339 and KC-VS12), one nasal swab (U-NS66-TR2009), and two brain samples (KC-B12 and ANK.Br.TR2015) from animals in herds  I, II, VII, and IX (Table I). Figure  7 shows the infectivity titers of 5 isolates which grew in cell culture (n = 5). Discussion BoHV-4 appears capable of significant genome drift, as demonstrated by the variability in the different herds (Figure 1 and Table I). The grouping in the phylogenetic tree is described by Verna and colleagues (Verna et al. 2012) and Areda and colleagues (Areda et al. 2018). The phylogenetic analysis of BoHV-4 based on sequences of the ORF3 gene coding TK revealed three distinct genotypes: genotype I, II, and III (Figure 2). As shown in Figure  2, phylogenetic comparisons with the previously described BoHV-4 TK gene sequences indicated that all of our BoHV-4 field viruses belonged to genotype  1, which includes the European (Movar-like) reference strains, along with the Italian, Japanese, and Argentinian BoHV-4 strains. We also compared the nucleic acid (na) sequences for the genes coding gB and TK of the local BoHV-4 samples both among themselves (Figures 3 and 4) and with the sequences deposited in GenBank from other countries (Tables II and III). Figure 5 shows the amino acid (aa) similarity rates for genes coding TK. For genotype I, the similarity rates for genes coding TK were 100% among the BoHV-4 strains detected in this study and 98.5-100% with the other viruses. The similarities with local BoHV-4 identified as genotype II and genotype III were MG181944KAS21-LOK-TR2017 MG181945KAS24-LOK-TR2017 MG181946KAS25-LOK-TR2017 MG264405 isolate D1611430-2.5 gB MG264400 D1611430-2.8 gB MG264401 D1611430-2.15 gB MG264402 D1611430-2.27 gB KP209018 isolate 08 209 gB MG264396 isolate D1300426-1.1 gB AF318573 gB MH318580 (B2 AC4-TR2017) MH318579 (B1 AC4-TR2017) GQ246867 T-6 KP209029 isolate DN 599 GQ246865 SB-9 AY847334 Movar33/63 Z15044 BoHV-4 gB KX192364 ANK-NS-TR2014 EU055543 K339 KX192365. ANK-Teat-TR2014 GQ246864 strain Kc-12 MK543547 TR2011-KCORG MK543546 TR2010-IZ1LK136 JX644989 U-NS66-TR2009 KX192363 ANK-Br-TR2015 JX644988 KC-B12-TR2009 MK543548 TR2009-KC-VS2 MK543549 TR2011-TGVS29 MK543550 TR2011-TGVS549 MK543551 TR2011-TGVS550 GQ375280 YL GQ246863 TG-4 GQ246866 strain TG-1 MG264399 isolate D1611430-1.88 gB MH605274 (9870MILK-TR2018) KP209016 isolate 07 435 gB KP209024 isolate 09 227 gB MG264397 isolate D1609492-1.6 gB MG264404 isolate D1611430-2.48 gB 46 54 99 63 60 83 58 64 82 65 Ge no ty pe I Ge no ty pe II Ge no ty pe II I 2 Figure 1. Phylogenetic tree based on nucleotide sequences of the gB gene of Turkish BoHV‑4 strains shown in round () shape and BoHV‑4 strains from various countries. AB035516 Japon 1999 Movar 33/63 AB035517 Japon 1999 AB035515 Japon 1999 MH614621 (B2AC4TK-TR2017) MH614620 (B1AC4TK-TR2017) KX352280 ANK-Teat-TR2014 TK KX352279 ANK-Br-TR2015 TK MG242137 KAS25TK-LOK-TR2017 JQ838059 Argentina 2012 JX644990 K339-TR2009 JX644992 KC-B12-TR2009 TK JX644991 KC-12-TR2007 TK JX644993.1 U-NS65-TR2009 TK KT166423 Italy 2015 TO-1 TK EU244699 Brazil 2007 EU244698 Brazil 2007 EU244697 Brazil 2007 EU244700 Brazil 2007 KP209015 Argentina 2014 KU180422 Argentina 2015 KU180419 Argentina 2015 KP209014 Argentina 2014 JQ838047 Argentina 2012 JQ838057 Argentina 2012 JQ838062 Argentina 2012 DN599 JQ838052 Argentina 2012 JQ838053 Argentina 2012 JQ838046 Argentina 2012 JQ838056 Argentina 20120.005 Ge no ty pe I Ge no ty pe II Ge no ty pe II I Figure 2. Phylogenetic tree based on the nucleotide sequences of the TK gene of Turkish BoHV‑4 strains showed in squared () shape and BoHV‑4 strains from various countries. 53 Bilge Dağalp et al. Genetic variability of BoHV-4 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 aborted foetus tissue samples, and dead calves (MK543547, JX644988, GQ246865, KX192363), and tissue and leucocyte samples from cattle with respiratory and/or fertility system disorders (MH31879-MH31880, KX192365, MK543546, GQ375280, GQ246866-GQ246867) (Table I). Our field viruses that clustered in genotype I (MG181944, MG181945, MG181946) came from the leucocyte samples of repeat breeder cows in one specific herd (herd no:  XI), which aligned with Argentinian and American BoHV-4 strains. For genotype III, only one field virus (MH605274) was detected in the milk sample from a cow with mastitis, which aligned with several North American BoHV-4 strains in the phylogenetic tree (Figure 1). Thus, the phylogenetic analysis shows that our local viruses sequences of several gene regions coding gB and TK in the BoHV-4 field isolates from Turkish cattle with different clinical signs (Frazier et al. 2002, Areda et al. 2018, Verna et al. 2016, Gagnon et al. 2017). Our molecular analysis of the partial gB gene sequences obtained from 24 local BoHV-4 strains revealed that Turkish BoHV-4 isolates clustered into three genotypes, with the separation of several viruses in different branches in genotype II (Figure 1). The majority of the local viruses were classified in genotype II, together with several European and North American BoHV-4 strains. Turkish BoHV-4 strains in genotype II (n = 20) were from various clinical samples, specifically nasal swabs (KX192364, JX644989), vaginal swabs (GQ246863, GQ246864, MK543548- MK543551, EU055543), Figure 3. Nucleotid identity (%) of gB gene sequences of some Turkish BoHV‑4. Figure 4. Nucleotide identity (%) of partial TK gene sequences of some Turkish BoHV‑4 strains. 54 Genetic variability of BoHV-4 Bilge Dağalp et al. Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 revealed three main clusters, which included European (Movar-like, genotype I) and American strains isolated from cattle, the African strain isolated from buffalo (DN599-like, genotype II), and Argentinean and Brazilian strains (genotype III) from cattle. The majority of samples (20/24) and herds (11/13) in this study were identified as genotype II. These herds were either small family herds of 1-10 cattle (III, VI, VIII, XII, XIII) or state farms (I, II, IV, V, VII, IX-XI) of about 1,000 cattle, which had been restocking from internal animal sources, transfering animals between herds, or occasionally introducing imported have high genetic similarities with BoHV-4 reported from different geographical regions worldwide, specifically Europe, and South and North America. Most of the local BoHV-4 strains with different clinical disorders were located in genotype II while BoHV-4 identified as genotype I and III were only detected in cows with repeat breeder and a cow with mastitis, respectively (Table I). Despite these findings, we cannot yet claim a definite association between the genotypes and clinical cases due to the differences in the numbers of BoHV-4 detected with different genotypes and in different clinical findings. The phylogenetic tree for the gene coding gB Table II. Nucleic acid identity (%) of gB gene sequences of Turkish BoHV‑4 strains and BoHV‑4 strains selected as representative for different genotypes from GenBank database. Genotype Virus Code Acc number Genotypes I II III MG264402 MG264401 KP209029 DN_599 AY847334 Movar33/63 MG264396 MG264404 MG264399 I KAS25 MG181946 100 100 99.7 99.3 99.7 92.7 92.3 KAS21 MG181944 100 100 99.7 99.3 99.7 92.7 92.3 KAS24 MG181945 100 100 99.7 99.3 99.7 92.7 92.3 MG264402 100 99.7 99.3 99.7 92.7 92.3 MG264401 100 99.7 99.3 99.7 92.7 92.3 II DN_599 KP209029 99.7 99.1 99.7 100 93.0 92.7 Movar33/63 AY847334 99.3 99.3 99.7 99.7 92.7 92.3 MG264396 99.7 99.7 100 99.7 93.0 92.7 U-NS66 JX644989 97.0 97.0 97.3 97.0 97.3 90.3 90.0 YL GQ375280 97.7 97.7 98.0 97.7 98.0 91.0 91.3 ANK-Br KX192363 99.0 99.0 99.3 99.0 99.3 92.3 92.7 KC-B12 JX644988 99.0 99.0 99.3 99.0 99.3 92.3 92.7 TG-4 GQ246863 98.7 98.7 99.0 98.7 99.0 92.0 92.3 TG-1 GQ246866 98.7 98.7 99.0 98.7 99.0 92.0 92.3 KC-12 GQ246864 99.3 99.3 99.7 99.3 99.7 92.7 92.3 ANK-Teat KX192365 99.3 99.3 99.7 99.3 99.7 92.7 92.3 K339 EU055543 99.3 99.3 99.7 99.3 99.7 92.7 92.3 ANK-NS KX192364 99.3 99.3 99.7 99.3 99.7 92.7 92.3 SB-9 GQ246865 99.3 99.3 99.7 99.3 99.7 92.7 92.3 T-6 GQ246867 99.7 99.7 100 99.7 100 93.0 92.7 B1 AC4 MH318579 99.7 99.7 100 99.7 100 93.0 92.7 B2 AC4 MH318580 99.7 99.7 100 99.7 100 93.0 92.7 IZ1LK136 MK543546 99.3 99.3 99.7 99.3 99.7 92.7 92.3 KCORG MK543547 99.3 99.3 99.7 99.3 99.7 92.7 92.3 KCVS2 MK543548 99.0 99.0 99.3 99.0 99.3 92.3 92.7 TGVS29 MK543549 98.7 98.7 99.0 98.7 99.0 92.0 92.3 TGVS549 MK543550 98.7 98.7 99.0 98.7 99.0 92.0 92.3 TGVS550 (MK543551) 98.7 98.7 99.0 98.7 99.0 92.0 92.3 III 9870MILK MH605274 91.7 91.7 92.0 91.7 92.0 99.0 98.7 MG264404 92.7 92.7 93.0 92.7 93.0 99.7 MG264399 92.3 92.3 92.7 92.3 99.7 55 Bilge Dağalp et al. Genetic variability of BoHV-4 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 Table III. Nucleic acid identity (%) of TK gene sequences of Turkish BoHV‑4 strains and BoHV‑4 strains selected as representative for different genotypes from GenBank database. Virus Code Acc number Genotypes I II III AB035516 Movar33/66 AB035517 JQ838059 JQ838062 DN-599 JQ838047 JQ838057 JQ838046 JQ838056 B1AC4 MH614621 99.5 100 100 98.0 98.0 98.0 96.5 96.5 B2AC4 MH614620 99.5 100 100 98.0 98.0 98.0 96.5 96.5 ANK-Teat KX352280 99.5 100 100 98.0 98.0 98.0 96.5 96.5 ANK-Br KX352279 99.5 100 100 98.0 98.0 98.0 96.5 96.5 U-NS66 JX644993 99.5 100 100 98.0 98.0 98.0 96.5 96.5 KC-12 JX644991 99.5 100 100 98.0 98.0 98.0 96.5 96.5 KC-B12 JX644992 99.5 100 100 98.0 98.0 98.0 96.5 96.5 K339 JX644990 99.5 100 100 98.0 98.0 98.0 96.5 96.5 KAS25 MG242137 99.5 100 100 98.0 98.0 98.0 96.5 96.5 Figure 5. Amino acid identity of TK gene sequences of Turkish BoHV‑4 and BoHV‑4 strains selected as representative for different genotypes from GenBank database. Figure 6. Amino acid differences of gB gene sequences of Turkish BoHV‑4 and BoHV‑4 strains selected as representative for different genotypes from GenBank database. 56 Genetic variability of BoHV-4 Bilge Dağalp et al. Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 then clinical cases (Figure 1 and Table I). We believe that further epidemiological studies of BoHV-4 will reveal the relationship between the genotypes and geographical regions, and the association between molecular characterization and pathogenesis of infection. This study was able to characterize nine BoHV-4 field strains for the sequences coding TK. Based on phylogenetic analysis of the TK gene sequences, these isolates (n = 9) were identified as genotype I, which includes the European (Movar-like) reference strains, together with the Italian, Japanese and Argentinian BoHV-4 strains (Figure 2). These field strains were detected in vaginal swabs (JX644990, JX644990), nasal swab (JX644993), teat lesion samples (KX352280), lungs (MH614620, MH614621), leucocytes from cows with respiratory and/or reproductive system disorders (MG242137), and brain tissue (KX352279 and JX644992) from calves that died after birth (Table I). Regarding the classification of the TK gene, the local strains were identical to each other (100%) and highly similar to other virus strains in genotype I (99.5-100%), genotype II (98%), and genotype III (96.5%) (Tables I and III, Figure  4). Consequently, these genotypes cannot imply a possible association with clinical signs. However, some researchers (Verna et al. 2016, Gagnon et al. 2017) have argued that the existence of different genotypes suggests some association between genetic variations and specific pathogenic potential. In our study, all BoHV-4 strains in genotype II and III, based on sequences for the ORF3 gene, came from Argentina or Brazil (Figure 2). This origin suggests an association between genotypes and geographic regions similar to that suggested for the gB gene sequences. This may be important for pursuing the traces of domestic or international trade. We found nine BoHV-4 strains with both the animals. There is clear animal trade between either small family herds and/or industrial herds. We can speculate that animal movements between different geographical regions in Turkey and also from other countries, such as Argentina or Uruguay, especially since 2010, may have influenced the distribution of the different genotype BoHV-4 strains and their genetic resemblances. It is noteworthy that all Turkish BoHV-4 samples classified in genotype  I and III, which were detected in diagnostic materials sampled in 2017 and 2018, showed close similarities with Argentinian and American BoHV-4 strains. In contrast, genotype II has been detected in cattle since at least 2007 (Table I). Given these results, the introduction into Turkey of live animals from other countries may be an important risk factor, as previously reported for BoHV-4 and several other infections (Fichtelova and Kovarcik 2010, Houe 1995). It should also be remembered that the gene region coding for gB is quite highly conserved among BoHV-4 strains (Campos et  al. 2014, Bellino et  al. 2015). It is therefore not useful for providing clues about the strains’ geographical origins. Thus, the gB gene sequences (GQ246866, MK543551, MK543550, MK543549, GQ246863) of BoHV-4 strains from cattle with reproductive disorders, sampled at two different time points (2007 and 2011) in herd IV were placed in the same branch in the genotype  II cluster (Figure 1), with the nucleotide similarities of  100%. This also supports claims that the gene coding gB is quite highly conserved, with similarities rates of (91.7-100%) among all the BoHV-4 strains analyzed in this study, despite being classified in three different genotypes (Figure 3). Additionally, the location on the different branches and/or clusters of the sequences of the samples in the phylogenetic tree from cattle with respiratory system and reproductive system disorders from different herds and years also supports claims that these genetic differences are more related to the herds sampled 24h ANK-Br-TR2015 6.0 5.5 5.25 5.25 4.75 5.75 4.25 3.75 4.75 4.25 3.75 4.75 4.5 4.0 3.25 4.75 3.5 4.0 3.0 KC-B12-TR2009 KC-VS12 K339/Bursa-TR2009 U-NS66-TR2009 7 6 5 4 3 2 1 0 48h 72h 96h 120h 5.5 4.0 Figure 7. Replication kinetics of cythopathic Bovine herpesvirus type 4 isolates in MDBK cell culture. 57 Bilge Dağalp et al. Genetic variability of BoHV-4 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 to respiratory and reproductive system disorders, and two brain tissue samples from calves (KC-B12 and ANK.Br.TR2015) were representative of the 24 different herds (herd no: I, II, VII, IX) and different sampling years (Table I). The replication kinetics of the BoHV-4 isolates showed some differences across the five sampling points. Of BoHV-4 isolates, four (KC-VS12, ANK-Br-TR2015, KC-B12 and U-NS66-TR2009) had the highest titers at 96 hpi whereas isolate K339’s titer increased constantly till 120 hpi. The highest virus titers were obtained with K339. Verna and colleagues (Verna et al. 2016) reported clear evidence that in  vitro characterization (biological and molecular) of viral replication in cell culture correlates with in  vivo virulence. Perez and colleagues (Perez et al. 2011) concluded that the viral strain is only weakly correlated with clinical manifestations. We therefore believe that the significant genetic variability in gB gene region sequences and the results on the in vitro biological behavior of our BoHV-4 field isolates provides an important resource for further studies into the relationship between pathogenesis and the molecular characterization of BoHV-4 from animals with different clinical signs or from apparently healthy cattle. Unfortunately, all the isolates that we used to investigate biological behavior in MDBK cells were genotype I for gB gene sequences, although we also identified BoHV-4 in genotypes II and III. Conclusions In conclusion, we provided a precise biological and molecular characterization of the ORF8 and ORF3 gene regions coding gB and TK of BoHV-4 field strains from Turkish cattle with different health status. We hope that these findings may help future researchers to understand more clearly the molecular epidemiology of BoHV-4 infection in cattle and the possible association between the virus and pathogenesis of infection. Acknowledgements The authors would like to thank G. Wellenberg for kindly providing the North American reference strain. Funding This study was supported by a grant from the Scientific Research Projects (Project No: 2004 0810 068 and 10B3338005), Ankara University. sequences for genes coding gB and TK. Of these, eight were identified as genotype II and one as genotype I for gB sequences while all were genotype I for TK genes (Table I). We hope that further molecular characterization studies using larger number of samples and targetting some other genes along with TK and gB genes will be able to evaluate the role of these gene(s) in the pathogenesis of BoHV-4 infection. In our study, the nucleotide and amino acid differences were more pronounced across the gB gene than the TK gene (Figures 3-6). All the local BoHV-4 samples located in genotype I had a change in the amino acid (aa) sequences of the gB gene at positions 44 (KQ), similar to other genotype  I BoHV-4 strains. The sequences (MH605274) of one field BoHV-4 sample from milk, which was the only strain clustered in genotype III, had changes at position 53.aa. (SD). Turkish BoHV-4 in genotype II had one aa change at 57.aa (AT). It was detected in seven strains (JX644989, MK543546, MK543547, GQ246864, KX192365, EU055543, KX192364). These formed a branch within genotype II in the phylogenetic tree. This is important given the variety of specimen types and their origin from seven different herds (Figure  6, Table I). The greatest aa change was detected in the gB sequences of BoHV-4 strains from a nasal swab (JX644989) and a leucocyte sample (GQ375280) from cattle in two different herds (Figure 6). These results regarding aa change, especially in the seven genotype II BoHV-4 samples, reveal traces of domestic and/or international trade, although the latter change (A57T) did not match the sequences of any BoHV-4 deposited in Genbank. The main conclusion from our results is that there are significant differences between the BoHV-4 strains circulating in Turkey, although it is not yet clear how clinical cases are related to their molecular genetic characteristics. Verna and colleagues (Verna et al. 2012) and Altamiranda and colleagues (Altamiranda et al. 2015) reported similar high variability among BoHV-4 strains in Argentina. Of the 24 samples used in this study, BoHV-4 was isolated in five of them. These were then included in a virus isolation study to investigate their biological behavior using MDBK cell cultures. Several factors may explain our failure to isolate the virus from 19 PCR-positive samples, including the poor quality of the samples or the inherent difficulty of isolating BoHV-4 from samples (Lin et  al. 1997, Wellenberg et  al. 2002). We were lucky that our five positive samples, including one nasal swab (U-NS66-TR2009) and two vaginal swab samples (K339 and KC-VS12) from cattle with different clinical disorders attributed 58 Genetic variability of BoHV-4 Bilge Dağalp et al. Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 Altamiranda E.G., Manrique J.M., Pérez S.E., Ríos G.L., Odeón A.C., Leunda M.R. & Verna A. 2015. Molecular characterization of the first bovine herpesvirus 4 (BoHV-4) strains isolated from in vitro bovine embryos production in Argentina. PloS One, 10 (7), e0132212. Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W. & Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25 (17), 3389-3402. Areda D., Chigerwe M. & Crossley B. 2018. Bovine herpes virus type-4 infection among postpartum dairy cows in California: risk factors and phylogenetic analysis. Epidemiol Infect, 146 (7), 904-912. Bellino C., Iussich S., Biasato I., Peletto S., Caruso C., Gianella P., Cagnasso A. & D’Angelo A. 2015. Potential pathogenetic role of bovine herpesvirus 4 in two dairy cows with dermatitis-pyrexia-hemorrhagic syndrome. J Clin Microbiol, 53 (8), 2763-2767. Bilge Dağalp S., Alkan F., Çalışkan E., Yıldırım Y., Oğuzoğlu Ç. & Burgu İ. 2012. The investigation of the herpesviruses (BoHV-1 and BoHV-4) on the occurrence of the reproductive disorders in dairy cattle herds, Turkey. Rev Med Vet, 163 (4), 206-211. Bilge Dağalp S., Güngör E., Demir A.B. & Alkan F. 2007. The seroprevalence of bovine herpes virus type 4 (BHV4) infection in dairy herds in Turkey and possible interaction with reproductive disorders. Rev Med Vet, 158 (4), 201-205. Bilge Dağalp S., Güngör E., Demir A.B., Pınar-Muz D., Yılmaz V., Oğuzoğlu T.Ç., Ataseven V.S. & Alkan F. 2011. The investigation of the presence of bovine herpesvirus type 4 (BoHV-4) in cows with metritis in a dairy herd. Vet J Ankara Univ, 57 (2), 87-91. Bublot M., Van Bressem M.F., Thiry E., Dubuisson J. & Pastoret P.P. 1990. Bovine herpesvirus 4 genome: cloning, mapping and strain variation analysis. J Gen Virol, 71, 133-142. Campos F.S., Franco A.C., Oliveira M.T., Firpo R., Strelczuk G., Fontoura F.E., Kulmann M.I., Maidana S., Romera S.A., Spilki F.R., Silva A.D., Hübner S.O. & Roehe P.M. 2014. Detection of bovine herpesvirus 2 and bovine herpesvirus 4 DNA in trigeminal ganglia of naturally infected cattle by polymerase chain reaction. Vet Microbiol, 171, 182-188. . Egyed J., Ballagi-Pordany A., Bartha A. & Belak S. 1996. Studies of in vivo distribution of bovine herpesvirus type 4 in the natural host. J Clin Microbiol, 34, 1091-1095. Fichtelova V. & Kovarcik K. 2010. Characterization of two BHV-4 strains isolated in the Czech Republic. Vet Med (Praha), 55 (3), 106-112. Frazier K., Baldwin C.A., Pence M., West J., Bernard J., Liggett A., Miller D. & Hines M. 2002. Seroprevalence and comparison of isolates of endometriotropic bovine herpesvirus-4. J Vet Diagn Invest, 14, 457-462. Gagnon C.A., Traesel C.K., Music N., Laroche J., Tison N., Auger J.P., Music S., Provost C., Bellehumeur C., Abrahamyan L., Carman S., Côteaux L. & Charette References S.J. 2017. Whole genome sequencing of a Canadian bovine gammaherpesvirus 4 strain and the possible link between the viral infection and respiratory and reproductive clinical manifestations in dairy cattle. Front Vet Sci, 4, 1-12. Goltz M., Broll H., Mankertz A., Weigelt W., Ludwig H., Buhk H.J. & Borchers K. 1994. Glycoprotein B of bovine herpesvirus type 4: its phylogenetic relationship to gB equivalents of the herpesviruses. Virus Genes, 9, 53-59. Gur S. & Dogan N. 2010. The possible role of bovine herpesvirus type-4 infection in cow infertility. Anim Sci J, 81, 304-308. Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98. Houe H. 1995. Epidemiology of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract, 11 (3), 521-547. Kale M., Ata A., Kocamüftüoglu M. & Hasırcıoglu S. 2011. Bovine herpesvirus type 4 (BHV-4) infection in relation to fertility in repeat breeder dairy cows. Acta Vet (Beograd), 61, 13-19. Kit S., Kit M., Ichimura H., Crandell R. & McConnel S. 1986. Induction of thymidine kinase activity by viruses with group B DNA genomes; bovine cytomegalovirus (bovine herpesvirus 4). Virus Res, 4, 197-212. Lin T.M., Shi G.Y., Tsai C.F., Su H.I., Guo Y.I. & Wu H.I. 1997. Susceptibility of endotelial cells to bovine herpesvirus type 4 (BHV4). J Virol Methods, 63, 219-225. Lomonte P., Bublot M., Pastoret P.P. & Thiry E. 1992. Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase genes of other herpesviruses. Arch Virol, 127 (1-4), 327-337. Nettleton P.F., Herring J.A. & Herring A.J. 1983. Evaluation of an immunofluorescent test for the rapid diagnosis of field infections bovine rhinotracheitis. Vet Rec, 112, 298-300. Pérez S.E., Verna A.E., Leunda M.R., Favier P.A., Ceriani M.C., Morán P.E. & Esteban E.N. 2011. Occurrence of bovine herpesvirus type 4 DNA in Argentinean Holstein cattle from Santiago del Estero, Argentina. Braz J Vet Res Anim Sci, 48 (6), 454-463. Reed L.J. & Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Epidemiol, 27, 493-497. Roizman B., Desroisers R.C., Fleckenstein B., Lopez C., Minson A.C. & Studdert M.J. 1992. The family herpesviridae: an update. Arch Virol, 123, 425-449. Sambrook J., Fritsch E.F. & Maniatis T. 1989. Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Tamura K., Dudley J., Nei M. & Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731-2739. Thiry E., Bublot M., Dubuisson J., Van Biessem M.F., Lequarre 59 Bilge Dağalp et al. Genetic variability of BoHV-4 Veterinaria Italiana 2021, 57 (1), 49-59. doi: 10.12834/VetIt.2095.11150.1 Wellemans G., Van Opdenbosch E. & Mammericks M. 1986. Experimental inoculation of bovine herpes virus  4 (Strain LVR 140) in pregnant and nonpregnant cows. Ann Rech Vet, 17 (1), 89-94. Wellenberg G.J., Verstraten E.R.A.M., Belak S., Verschuren F.A.M., Rijsewijk R., Peshev R. & Van Oirschot J.T. 2001. Detection of bovine herpes virus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk. J Virol Methods, 97, 101-112. Zimmerman W., Broll H., Ehlers B., Buhk H.J., Rosenthal A. & Goltz M. 2001. Genome sequence of bovine herpesvirus 4, a bovine Rhadinovirus, and identification of an origin of DNA replication. J Virol, 75, 1186-1194. A.S., Lomante P., Vanderplasschen A. & Pastoret P.P. Molecular biology of BHV4. Vet Microbiol, 33, 79-92. Verna A.E., Manrique J.M., Pérez S.E., Leunda M.R., Pereyra S.B., Jones L.R. & Odeón A.C. 2012. Genomic analysis of bovine herpesvirus type 4 (BoHV-4) from Argentina: high genetic variability and novel phylogenetic groups. Vet Microbiol, 160, 1-8. Verna A.E., Pérez S.E., Manrique J.M., Leunda M.R., Odeón A.C. & Jones L.R. 2016. Comparative study on the in vitro replication and genomic variability of Argentinean field isolates of bovine herpesvirus type 4 (BoHV-4). Virus Genes, 52, 372- 378.