RESEARCH ARTICLE J Res Vet Med. 2022: 41(2) 80-87 DOI:10.30782/jrvm.11082913 First Isolation And Characterization Of Bovine Herpesvirus 1.2b (BoHV-1.2b ) Strain From Upper Respiratory Tract Of Cattle In Türkiye Gizem Aytogu1, Eda Baldan Toker1, Berfin Kadiroglu1, Ozer Ates1, Pelin Tuncer Goktuna2, Kadir, Yesilbag*1 1 Department of Virology, Bursa Uludag University, Faculty of Veterinary Medicine, 16059 Bursa, Türkiye 2 Pendik Veterinary Control Institute İstanbul, Türkiye Received 04-03-2022 Accepted 25-07-2022 Abstract Analyzing fingerprints by Restriction Length Polymorphism (RFLP) analysis of complete virus genome which is laborious and time-consum- ing classifies Bovine Herpervirus-1 (BoHV-1) strains into three subtypes, BoHV-1.1, 1.2a, 1.2b. These subtypes can also being referred accord- ing to clinical futures, however, no clear relation was shown. Mostly BoHV-1.2b is associated with genital disease conditions. In this study, BoHV-1 isolate was obtained in a nasal swab sample taken from respiratory tract disease. In this study also, phylogenetic analysis which was targeting UL44 (Glycoprotein C) region of the genome, subtyping was carried out by a recently developed multiplex PCR targeting UL39 and US3 region followed by a RFLP analysis using Hind III enzyme. Also, success of isolation was compared in two continuous cell lines. SFT-R cell line found more susceptible for BoHV-1 field sample isolation than MDBK. The obtained isolate (ID:8640) was serologically undistinguishable from Cooper strain while molecular analysis classified as BoHV-1.2b. Current study reports the first isolation of BoHV-1.2b in Turkey as well as infrequent BoHV-1.2b isolation from clinical case of respiratory illness. Results also highlights the efficiency of PCR based RFLP analysis for easy and quick subtyping but demonstrates the requirement of more investigation to reveal differences based on genetic diversity of BoHV-1 field isolates. Keywords: BoHV-1 subtyping, molecular characterization, restriction endonuclease analysis, virus isolation. Introduction are associated with the viral envelope 4. According to com- position and restriction site analysis of the genome, BoHV- Bovine herpesvirus type 1 (BoHV-1) is associated with 1 strains are classified into three subtypes. BoHV-1.1 is various clinical disorders in cattle comprising respiratory generally responsible for infections in respiratory tissues and reproductive systems. Clinical signs of the infection as well as genital tract. Thus, the respiratory disease called involves nasal discharge, conjunctivitis, depression, fever, infectious bovine rhinotracteitis (IBR) is generally caused milk yield reduction, genital disorders like abortion and by this subtype 5. Subtype BoHV-1.2, generally accepted to vulvovaginitis in female cattle and balanoposthitis in bulls. be responsible for genital infections known as “infectious Although several European countries have successfully pustular vulvovaginitis (IPV) and infectious pustular bala- eradicated BoHV-11, introduction of the virus to all the noposthitis (IPB)”. BoHV-1.2 subtype also further divided continents had been reported2 and has been found to be into two groups of variants (genotype) namely BoHV-1.2a the cause of mass economical losses in cattle industry 3. and BoHV-1.2b, based on genetic and antigenic differenc- BoHV-1, classifies in the Varicellovirus genus of the sub- es. BoHV-1.2a strains can cause abortion in cattle while family Alphaherpesvirinae of Herpesviridae. Virus has BoHV-1.2b is not assumed to be abortifacient 6 despite an enveloped icosahedral symmetry containing double causing common genital infections. Possibility of respira- stranded viral DNA which is about 135 kb in length and tory infections caused by BoHV-1.2b strains that is less vir- codes for 33 structural proteins. At least 13 of mentioned ulent compared to BoHV-1.1, is also suggested 5,7. A former * Corresponding author: Kadir Yeşilbağ, kyesilbag@uludag.edu.tr, (+90 224) 2941295 80 Aytogu et al 2022 subtype (BoHV-1.3) causing encephalitis in cattle is cur- fuged at +4 ºC, 3000 rpm for 10 minutes and the superna- rently classified as a distinct virus species of BoHV-5 .8 Despite tants obtained were filtered via 0,2 µm and treated with an- genetic variations, BoHV-1 subtypes are closely related to tibiotics (penicillin 100 IU /ml and streptomycin 1 µg /ml) each other in serological testing. Since BoHV-5, BoHV-1 and antifungals (amphotericine B 2,5 µg /ml) solutions. All and its subtypes share antigenically very similar epitopes, the samples and inoculum were either directly included in determination of virus species and subtypes by serologi- the test protocol or stored at -80 ºC up to testing. cal differention is laborious. Differentiation of BoHV-1 subtypes was entirely performed by restriction site anal- Cell line and the test virus: Madin Darby bovine kidney ysis using different DNA digestion enzymes9. Though this (MDBK) and Sheep fetal thymus (SFT-R) cell lines were analysis are still employed for the same purposes, so far, used for virus inoculation and serum neutralization stud- there are many sequences from different parts of BoHV-1 ies. Both of the cell line were grown in Dulbecco’s modified genome deposited in the GenBank for various strains of minimal essential medium (DMEM) supplemented with the virus 10. Hence, phylogenetic analysis on the basis of 10% fetal calf sera. Cell lines and calf sera were tested by selected genes is a reliable method for subtyping BoHV-1 PCR and virus isolation protocols to be free of Pestivirus isolates. One of the most common part of viral DNA used contamination before use. BoHV-1 strain Cooper propa- for characterization and differentiation of BoHV-1 isolates gated in MDBK cell line was used as positive control virus is the gene coding for glycoprotein C (gC, UL44) encode in PCR and neutralization tests. for envelope glycoprotein 11 which is responsible for attach- ment of the virions onto cells. Virus isolation: All the samples taken from total of 36 an- imals were inoculated onto MDBK and SFT-R cells pre- The agent, BoHV-1, leads to a latent infection localized pared in 24 well plates. Cells cultivated in 75 cm2 flasks in sensory ganglia after primary infection that persists were suspended as containing 1x105 cells /mL and 1 mL for lifelong. Clinical symptoms and virus shedding are of this suspension was distributed to each well of 24 well induced in latently infected animals affected by various plates for using in virus isolation procedure. Culture media stressors or subjected to corticosteroid application 2. Labo- were changed after 24 h and samples were inoculated. All ratory diagnosis for virus detection can be achieved using the wells were daily screened under inverted light micro- different methods including PCR, antigen ELISA and vi- scope during 3 blind passages each for 7 days. The culture rus isolation in cell culture. Cell lines from different tissue media from the wells in which cytopathogenic effect (CPE) origin are susceptible for BoHV-1 propagation with visual was observed was individually collected and stored at -80 ºC. cytopathogenic effect (CPE) 2. At the end of each blind passage (day 7 pi), inoculated cell cultures in 24 well plates were freeze-thawed for 3 times The aim of this study was to present the first isolation of and the media from each well was subjected to following BoHV-1.2b from natural case of bovine respiratory infec- test step. tion, and its molecular characterization consisting of both phylogenetic analysis and PCR-RFLP method which is less ELISA: The sample (8640) producing CPE in cell culture preferred in BoHV isolates. At the same time, the success was subjected to commercial antigen ELISA protocols de- of the BoHV-1 isolation from field samples in two continu- tecting BoHV-1 (Pulmotest, Bio-X diagnostics, Belgium) ous cell lines, MDBK and SFT-R was compared. and bovine viral diarrhea virus (Herdcheck, IDEXX, Swit- zerland). While applying the commercial BVDV ELISA Material and Methods method originally validated for detection of persistently Samples: In September 2014, non-clothed blood samples infected carriers, for confirmation of the validity of applied from 37 beef cattle with lumpy skin disease (LSD)-like method, BVDV NADL strain produced in cell culture in findings were submitted for laboratory diagnosis. There the laboratory was also used as an external control, apart was no history of BoHV-1 vaccination in the herd. Skin from the positive control included in the kit and it was samples from 2 of these animals and additional nasal swab found to comply with the validation criteria. sample (sample ID: 8640) from one animal exhibiting na- sal discharge was also submitted. Thus, the total number of Both of ELISA protocols were applied as described by the samples handled in this study was 39. All the samples were manufacturers. The test plates were read on the ELISA subjected to nucleic acid isolation for testing LSD virus by reader (Thermo-Multiskan EX, Finland) at a wavelength PCR. In the meantime, inocula prepared from samples for of 450 nm. the purpose of virus isolation in cell culture were centri- 81 Aytogu et al 2022 Polymerase chain reaction: Because the sample 8640 sis was applied on the sequences truncated as to be coding produced positive result in BoHV-1 antigen ELISA it was region. Neighbor-Joining method and bootstrap analysis further confirmed in PCR. For that purpose both the orig- with 1000 replicates were chosen in Mega 7 software. For inal swab sample directly taken from the animal and cell phylogeny tree, reference BoHV-1 and BoHV-5 sequences culture supernatant from 4th passage level were subjected were obtained from GenBank. Accession numbers for the to testing. Viral nucleic acid was isolated using a commer- reference strains are shown on Fig.1. cial kit (NucleoSpin Virus, Macharey-Nagel, Germany). The primer pair P1-forward (5’-CACGGACCTGGTG- GACAAGAAG-3’) and P2-revers (5’-CTACCGTCAC- GTGCTGTGTACG-3’) targeting the glycoprotein B (gB, UL26) gene of BoHV-1 12 was employed. The 50 µL of reac- tion mixture was consisted of nuclease free water (39,8 µL), MgCl2 (25mM, 2 µL), 10 x PCR buffer (5 µL), forward and revers primers (50pmol, 0,5 µL each), dNTPs (10mM each, 1 µL), Taq DNA polymerase (0,2 U) and sample DNA (1 µL). Applied thermal profile was as follows: 94ºC for 5 min; 35 cycles of 94ºC for 1 min, 61ºC for 1 min, 72 ºC for 1 min and final extension at 72 ºC for 10 min. The 468 bp PCR products were visualized in 2% agarose gel electrophoresis by Red-SafeTM (ABC Scientific, CA, USA) staining. Serum neutralization assay: For serological identifica- tion, viral isolate from sample 8640 was tested in serum neutralization assay using polyclonal serum yielded against BoHV-1 Cooper strain (BoHV-1.1). Animal experiments to obtain hyperimmune sera were conducted according to Fig. 1 Phylogenetic tree based on maximum likelihood method rules by national and local ethical commitee (HADYEK). for glycoprotein C gene of BHV-1 field strain of 8640 Briefly, 2 series of 2 fold diluted hiperimmunised sera were The tree was displayed using Mega10 software. The numbers at prepared in DMEM as duplicates of 96 well plates in a vol- branches indicates the frequency after 1000 bootstrap evalua- ume of 50 µL. BoHV-1 Cooper strain and viral isolate from tion. Other sequences compared with 8640 isolate were obtained sample 8640 were prepared in the titer of 100TCID50 and from the NCBI Pubmed website. 50 µl volume of virus suspension was added onto each step of the serum dilutions in different series. After incuba- tion at 37ºC in a 5% CO2 atmosphere for 2 hours, 50 µl of Restriction Fragment Length Polymorphism (RFLP): MDBK cell suspension including 3x105 cells /ml was added Restriction site analysis were performed as described by to each well and incubated in the same conditions for 5 Maidana et al. 14. Multiplex PCR was carried out by using days by daily evaluation. Inhibition of the cytopathogenic two different primer sets targeting UL39 open reading virus growth was recorded as positive reaction for neutral- frame and the US3 upstream intergenic regions. Primers ization and the antibody titer was evaluated as the highest set RS1 (US3; F: 5'-TCGTCGAAGAGCGTCCACACA-3', dilution where the last positive result was observed. R: 5'-ACCGCGCTGTACCGGCAGCT-3') and RS2, (US3, Sequencing and phylogenetic analysis: For genetic char- F: 5'-TACAAATCGGCGGCGCCAAA-3', R: 5'-TTGTT- acterization of the isolate, a different PCR protocol tar- GACGGCCAAGTATAA-3') amplified the fragments 493 geting gC region of the genome was used. Test protocol bp and 700 bp, respectively. The multiplex PCR amplifi- including the primer pairs PF (5’-CGGCCACGACGCT- cation was performed in a final volume of 50 μL, contain- GACGA-3’) and PR (5’-CGCCGCCGAGTACTACCC-3’) ing; 25 μL Maxima Hot Start 2x Green PCR Master Mix were applied as described elsewhere 11. Reaction conditions (Thermo Scientific™, K1061), 2,5 μL RNAse/DNAse free were applied as given above. The 572-575 bp PCR products water, 5 μL of 10 pmol each sense and antisense primers, 1 were visualized in 1% agarose gel electrophoresis by Red- μL extracted DNA and 1,5 μL DMSO. PCR products were SafeTM (ABC Scientific, CA, USA) staining. PCR products directly cleaved with Hind III enzyme according to recom- were submitted for sequencing (Genmar, Turkey) and ob- mended conditions (FastDigest HindIII, Thermo Scientif- tained sequence data were aligned by ClustalW Multiple ic™) without a purification step. The digested products were alignment tool using BioEdit software 13. Phylogeny analy- separated in 1,5 % agarose gel using 1xTAE buffer. BoHV- 82 Aytogu et al 2022 1.1 reference strain Cooper was used as control virus both ing gB region of BoHV-1 genome. Nucleotide sequence for multiplex PCR and digestion. analyses of gC region conducted to identify the genetic similarities between 8640 isolate (Access no. MW207646) Results and with subtype classification realized BoHV-1 virus- Virus isolation and ELISA: Although samples refer to the es. BoHV-5 and Bubaline alphaherpesvirus-1 (BuHV-1) laboratory with LSD-like clinical findings, characteristic sequences were also included in phylogenetic analyses. CPE form encountered in cell cultures and number of days Aligned nucleotide sequences revealed high degrees of for CPE detection suggested bovine herpesvirus infection. identity in herpesviruses included in the assessment, but By virus isolation studies conducted on 39 samples, the not for the BoHV-1 subtype characterization. The phyloge- nasal swab sample (ID: 8640) and one blood sample (data netic analysis showed the BoHV-1, BoHV-5 and BuHV-1 not shown) produced positive result as indicated by visual isolates grouped in distinct branches (Fig.1). The strains of CPE characterized by rounding, aggregation and lysis of BoHV-1.1 and BoHV-1.2 grouped together in two different the infected cells in the culture. Those of effects were ob- branches. The sequences analyzed for the BoHV-1.2a and served both in MDBK and SFT-R cell cultures at 5th day BoHV-1.2b strains are also intertwined in two different pi in the first round of blind passages, while an obvious branches. Isolate 8640 revealed between BoHV-1.2 isolates virus growth was recorded starting from 24th hours post but mostly similar with K22 strain (BoHV-1.2b) subtype infection by the second passage (Fig.2). Further 5 passag- and SM023 (BoHV-1.2b) subtype. Genomes alignment was es were achieved to confirm cytopathogenic growth of the also verified using BLAST database (NCBI, nucleotide se- isolates. The isolate produced a higher viral titer (DKID50 quence blast). BLAST analysis revealed that this sequence 105.75) in the culture of SFT-R cells comparing to MDBK homologies in the context of query cover was mostly seen between strain 8640, strain B589 (BHV-1.2b), and strain K22 (BHV-1.2b), respectively. While nucleotide sequence of strain 8640 was compared, glycoprotein C region was mostly conserved according to sequence of BoHV-1.2b K22 reference strain (Fig.3). Multiple sequence alignments showed single substitutions at nucleotide position 17589 which differs from other aligned sequences of BoHV-1.2. Also another substitution revealed at 17360 position iden- tified only in K22 strain sequence. Fig. 2 Virus propogation in SFT cells Arrows: Foci from virus growth areas (cytopathogenic effect, isolate 8640, 2nd passage, d.1 pi) Fig. 3 Multiple nucleotide sequence alignment of the isolate 8640 to strains from BoHV-1 subtypes cells (DKID 105.2550 ). All the final confirmatory tests were Multiple alignment was conducted between isolates 8640 detect- performed on cell culture supernatant from 4th passage of ed in this study and 6 other strains retrieved from GeneBank the original sample and positive results obtained in BoHV- [ BoHV-1.2b K22 reference strain (KM258880.1), BHV-1.2b 1 Ag ELISA (OD:0.248). Beside sample was found negative B589 strain (KM258881.1), BHV-1.2b SM023 (KM258882.1), by BVDV ELISA in which BVDV NADL strain produced BHV-1.2b SP1777 (KM258883.1), BHV-1.2a Halk-THYM-Thm- in cell culture in the laboratory was used as an external TR1993 (KY748021.1), BoHV-1.1 Cooper (DQ173733) ]. Num- control. bers on the sequence indicate nucleotide positions in the glyco- PCR and Phylogenetic analysis: BVDV negatif status of protein C gene for each virus. Dots indicate that nucleotides in the sample was further confirmed by RT-PCR. LSD PCR that line are identical in all sequences in the alignment. test performed on the samples was determined negative. However, 486 bp amplicons were yielded by PCR target- 83 Aytogu et al 2022 Restriction Fragment Length Polimorphism (RFLP): century, respiratory form has recognized in North Amer- For RFLP; multiplex PCR products were identical for ica. Acute respiratory disease form, known as Infectious BoHV-1.1 cooper strain. Both the UL39 and the US3 frag- bovine rhinotracheitis (IBR) quickly spread to Europe 15. ments were amplified as it was expected, and the isolate Causative agents of those two different clinical conditions 8640 was detected positive for UL39 region. However, were not antigenically separated from each other by cross several repeats were conducted in different reaction con- neutralization assays 17. Analyzing fingerprints by restric- ditions, no amplification was observed for 8640 isolate by tion endonuclease of viral DNA allowed subtype classifi- using RS2 primer set. The restriction patterns obtained by cation. Due to the mostly isolation of BoHV-1.1 from re- Hind III digestions are shown in Fig.4. The fingerprints spiratory tract disease or abortion and BoHV-1.2 in genital identical to BoHV-1.1 Cooper strain showed the RS1 and lesions, these subtypes can also being referred according RS2 fragments without cleavage. For the isolate 8640, RS1 to clinical futures 8. However, no clear relation was shown amplicon was cleaved into 2 sub-fragments. According to between tropism of respiratory or genital tract infections recommended RS1 sub-fragment patterns the field isolate and subtypes 18,19. Although BoHV-1.2b mostly associated 8640 was determined as BoHV-1.2b. with genital diseases in accordance with some of previous reports 5,10,20, the present study also reveals the molecular characterization of BoHV-1.2b isolate obtained from nasal swab sample at respiratory disease. In this study, the phylogenetic analysis based on gC gene region clearly grouped the BoHV-1, BoHV-5 and BuHV-1 isolates into different clusters. Unlike the results of some researches which also targets gC region 11,21,22 the phyloge- ny tree constructed based on nucleotide sequences in this study did not show consistent branches between BoHV-1 subtypes. The isolate 8640 sequence identity was found mostly close to BoHV-1.2b reference strain K22 and strain SM023 (BoHV-1.2b). In a study 23 some of the Chinese BoHV-1.2b isolates have been shown to be grouped sep- Fig. 4 Genomic fingerprints of the strain 8640 obtained after arately in phylogenetic investigations and suspected as RFLP analysis of PCR products from UL39 “atypical” BoHV-1.2 strains with UY2002 strain 23. The Line 1: ladder; Line 2: BHV-1.1 strain Cooper undigested PCR closest sequence of those three samples (MT179808, amplicons of both primer sets (RS1 = amplicon of 493 bp; RS2 MT179819, MT179820) was also identified as UY2002. But = amplicon of 700 bp) Line 3: strain 8640, digested amplicons of mentioned Chinese sequences which was referred as BHV- RS1; Line 4: negative control (DNase, RNase free water). 1.2b, has fallen into a different branch separated from 8640. In subtyping BoHV, it is accepted as the gold standard to Virus neutralization assay: The viral isolate taken from examine the complete genome of the virus using the Hind nasal swab sample (BoHV-1 8640/Tur/2014) was also III enzyme by RFLP method. BoHV-1, BoHV-5 differenti- identified as to be BoHV-1 using serum-virus neutraliza- ation and even subtype restriction endonuclease analysis tion assay beside BoHV-1 antigen ELISA and PCR. Serum profiles has distinct fingerprints 24–26. But this technique neutralization assay resulted with a neutralization reaction needs large quantity of purified viral DNA to display pat- up to 1:32 serum dilution similar to BoHV-1 strain Cooper terns after virus isolation which is laborious and time con- used as control virus. suming. Besides this classic application, alternative meth- ods which enables restriction of BoHV PCR products have Discussion and Conclusion been developed for alphaherpesviruses 14,27–29. PCR-RFLP BoHV-1 was first recognized in Germany in early 19th method is promising for rapid and easy implementation of century, characterized by clinical signs associated with detection and differentiation of BoHV-1 subtypes. In this genital tissues, known as “infectious pustular vulvovagi- study, in addition to PCR sequencing assay, one of those nitis” (IPV) in cows and “infectious pustular balanopos- proposed techniques was used for subtyping. PCR-RFLP thitis” (IPB) in bulls 15. Strain K22 was the first isolate 16 performed successfully for BoHV-1.1 strain Cooper. But and today subtyped as BoHV-1.2. After half of the 19th isolate 8640 showed amplification only for the RS1 primer 84 Aytogu et al 2022 pair. Failure of amplification using RS2 primers was pro- which reports genomic characterization of 13 field strains posed for BoHV-5 and BuHV-1 strains by the authors 14. only 4 could be adapted to the MDBK cell line as intend- However RS1 cleavage was only proposed for BoHV-1.2b ed quantity (3). Regarding the data from the current study subtypes, not for the BoHV-1.1, BoHV-1.2a, BoHV-5 and SFT-R cell lines can be realized as satisfied alternative to BuHV. Comparing with the phylogenetic analysis, results MDBK cell line for BoHV-1 isolation. was not surprising. Although it is not considered a valid method for subtype characterization, the results of phylo- In the present study, the isolation and molecular character- genetic analyses show that the isolate 8640 is not BoHV- ization of BoHV-1 strain 8640 were performed from nasal 5 or BuHV-1. And partial RFLP results obtained in this swab sample of a cattle. These findings are valuable for en- study supports digestion of RS1 amplicons, which classifies suring interior or cross-country epidemiological tracking. isolate 8640 as subtype BoHV-1.2b. It could be assumed But further research are needed to determine the preva- that possible mutations can be responsible for the failure of lence of BoHV-1.2b subtype in the field. Though standard- RS2 amplification. Though restriction patterns for BoHV- ization of laborless molecular techniques are also needed 1 by PCR-RFLP or selected amplification regions further to provide opportunity for extensive research in this di- needs to be investigated in order to enable the evaluation rection. Increased complete genome sequence investments of possible mutations between field isolates. may reflect the genetic diversity and contribute diagnostic improvements. BoHV infections are common in Turkey where the stud- ies mainly involve serological investigations and limited Financial Support number of molecular detections 30,31, and only one covers This study was financially supported by Bursa Uludağ detailed characterization 21. Beside the predominant preva- University Research Fund (BUU-BAP), Project No: OUA- lence of BoHV-1.1 or few BoHV-1.2a detection, according P(V)-2020/7. Dr. E.B. Toker is also granted for postdoc to our knowledge BoHV-1.2b strain has not been reported position by Turkish Scientific and Technological Research in Turkey so far. BoHV-1.2b has been isolated in Europe, Council (TUBİTAK) Project No: 119 O 571. America, Australia and lately in China 10,23. Hence the iso- late 8640 is the first BoHV-1.2b isolate reported from Tur- Ethical Statement key. Our results do not provide to determine the origin of This study was approved by the Bursa University Animal the current isolate, however, it should be noted that live Experiments Local Ethics Committee (2020 – 09 /10). animal importation can be an effective route to introduce such new viral subtypes in to country. Conflict of Interest The authors declare that they have no conflicts of interest. We also compared the cross neutralization antibody reac- tions between hyperimmune sera for Cooper strain and References isolate 8640 (BoHV-1.2b). There was no difference be- 1. Raaperi K, Orro T, Viltrop A. Epidemiology and con- tween the neutralization capacities of hyperimmune se- trol of bovine herpesvirus 1 infection in Europe. Vet J. rum against both the viruses tested. It was also previously 2014;201(3):249-256. doi:10.1016/j.tvjl.2014.05.040 reported to recognize BoHV1.2b isolate by BoHV-1.1 hy- 2. Nandi S, Kumar M, Manohar M, Chauhan RS. 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