Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 27/10/2024 Aceptado: 25/12/2024 Publicado: 11/03/2025 hps://doi.org/10.52973/rcfcv-e35547 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 8 Isolaon and Molecular Characterizaon of Bovine Viral Diarrhea Virus in Calves with Congenital Malformaon in Türkiye Aislamiento y caracterización molecular del virus de la diarrea viral bovina en terneros con malformaciones congénitas en Turquía Sebahattin Akça 1,2 , Mehmet Özkan Timurkan 3 * , Ömer Faruk Küçükkalem 1 , Meryem Güloğlu 1,4 , Hakan Aydın 3 , İbrahim Sözdutmaz 5 1 Veterinary Control Instute, Republic of Türkiye Ministry of Agriculture and Forestry. Erzurum, Türkiye. 2 Atatürk University, Health Science Instute, Department of Virology, Erzurum, Türkiye 3Atatürk University, Faculty of Veterinary Medicine, Department of Virology. Erzurum, Türkiye. 4Atatürk University, Health Science Instute, Department of Pathology, Erzurum, Türkiye 5Erciyes University, Faculty of Veterinary Medicine, Department of Virology. Kayseri, Türkiye. * Corresponding author: momurkan@atauni.edu.tr ABSTRACT This study aims to idenfy the causave agent in cases of aboron on a cale farm in Erzurum province, Türkiye. Samples from the farm were sent to the Erzurum Veterinary Control Instute, Ministry of Agriculture and Forestry. The analysis revealed the presence of Bovine Viral Diarrhea Virus (BVDV) in a newborn calf exhibing congenital malformaons. Subsequently, blood and serum samples were collected for four weeks post-aboron to assess the acute/persistent infecon status on the farm. Using the ELISA method, angen, and anbody posivity were detected in both the cale and the aborted calf. Six blind passages were conducted in the MDBK cell line to isolate the virus from cerebrospinal fluid. Confirmaon of isolaon was carried out through regular CT increase in Real-Time RT-PCR due to the non-cytopathogenic nature of the detected virus. The isolate (EVE-BVDV-2023) was idenfied as belonging to the BVDV-1d genotype through paral genome analysis of the 5’UTR gene. This study conclusively confirms the presence of BVDV infecon in cale herds in the eastern region of Türkiye, parcularly in Erzurum province. Future studies should connue efforts to control and eradicate infecous agents in cale herds, with a parcular emphasis on addressing BVDV infecon. Key words: Abort; BVDV; isolaon; molecular characterizaon; Türkiye RESUMEN Este estudio tuvo como objevo idenficar el agente causal en casos de aborto en una granja de ganado en la provincia de Erzurum, Turquía. Las muestras de la granja se enviaron al Instuto de Control Veterinario de Erzurum, Ministerio de Agricultura y Silvicultura. El análisis reveló la presencia del virus de la diarrea viral bovina (BVDV) en un ternero recién nacido que presentaba malformaciones congénitas. Posteriormente, se recogieron muestras de sangre y suero cuatro semanas después del aborto para evaluar el estado de infección aguda/ persistente en la granja. Ulizando el método ELISA, se detectó posividad de angeno y ancuerpo tanto en el ganado como en el ternero abortado. Se realizaron seis pases ciegos en la línea celular MDBK para aislar el virus del líquido cefalorraquídeo. La confirmación del aislamiento se llevó a cabo mediante el aumento regular de CT en RT-PCR en empo real debido a la naturaleza no citopatógena del virus detectado. El aislado (EVE-BVDV-2023) se idenficó como perteneciente al genopo BVDV-1d a través del análisis parcial del genoma del gen 5’UTR. Este estudio confirma de manera concluyente la presencia de infección por BVDV en rebaños de ganado vacuno de la región oriental de Turquía, en parcular en la provincia de Erzurum. Los estudios futuros deben connuar los esfuerzos para controlar y erradicar los agentes infecciosos en los rebaños de ganado vacuno, con especial énfasis en abordar la infección por BVDV. Palabras clave: Abortar; BVDV; aislamiento; caracterización molecular; Turquía

Bovine Viral Diarrhea Virus in Calves in Turkey / Akça et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Bovine Viral Diarrhea Virus (BVDV) infecon is typically asymptomac in immunocompetent cale (Bos taurus). However, infecons caused by the cytopathogenic type of the virus, leading to mucosal disease (MD), can escalate to fatality, causing respiratory, digesve, and genital issues. This causave agent predominantly affects cale, sheep, and pigs, significantly disrupng animal producon and resulng in substanal economic losses [1 , 2]. BVDV, classical swine fever, and border disease belong to the Pesvirus genus within the Flaviviridae family [2 , 3 , 4 ,5]. The virus possesses a posive-strand RNA, approximately 12.3 kb in length [5]. A conserved non-coding 5’ untranslated region (UTR) region at the genome’s beginning and end serves as a highly conserved region used in DNA typing. The genome comprises a single open reading frame (ORF) encoding structural and non-structural proteins [6 ,7]. Transmission of BVDV infecon occurs both directly and indirectly. Indirect transmission involves secreons (nasal discharge, tears, saliva, genital discharge, etc.), semen, milk, and feces. The virus can localize in the genital tract, causing reproducve issues like aboron and inferlity [6]. Placental transmission to the fetus can result in aboron, malformaons, or persistent infecon in immune-tolerant young animals [6]. Infected animals may develop fatal mucosal disease (MD), either due to mutaons of the non-cytopathogenic biotype (NCP) or ingeson of cytopathogenic BVDV from external sources. In addion, the virus’s suppression of the immune system renders infected animals suscepble to secondary infecons. According to the Internaonal Commiee on Taxonomy of Viruses, pesviruses are categorized as follows: Bovine Viral Diarrhea Virus (BVDV), BVDV-1 (Pesvirus A), BVDV-2 (Pesvirus B), Classical Swine Fever (Pesvirus C), Border Disease (Pesvirus D) [8 , 9 , 10 , 11], Pronghorn Antelope Pesvirus (Pesvirus E), Porcine Pesvirus (Pesvirus F), Giraffe Pesvirus (Pesvirus G), HoBi-like Pesvirus (Pesvirus H), Aydin-like Pesvirus (Pesvirus I), Rat Pesvirus (Pesvirus J), and Atypical Porcine Pesvirus (Pesvirus K) [8 , 9 , 10]. BVDV is also divided into subtypes. The subtypes within BVDV-1 are classified into 21 (1a- 1u), BVDV-2 into 4 (2a-2d), and BVDV-3 into 4 (3a-3d) subtypes [5 , 6 ,7]. BVDV-1 and BVDV-2 genotypes exhibit two biotypes, non-cytopathogenic (NCP) and cytopathogenic (CP), based on their effects in cell culture [3 , 12 , 13 , 14 , 15]. The idenficaon of these subtypes relies on analyzing paral sequences of the 5’UTR, Npro, and E2 regions [3 , 6]. Three types of BVD have been documented in Türkiye (BVDV-1, BVDV-2, BVDV-3). However, the presence of BVDV-1 has been extensively reported [16]. In addion to acute and chronic clinical condions, diverse clinical manifestaons of BVDV have been observed, including connuous virus shedding by persistently infected (PI) calves, congenital anomalies, and aborons [17 , 18]. The severity of clinical signs and the localizaon of infecon vary. In pregnant cale, due to the insufficient connecon between the maternal epithelium and fetal trophoblasts during the first 30-35 days (d) of the BVD virus, vercal transmission occurs, and intrauterine infecon ensues with the development of cotyledons, resulng in early embryonic deaths [13 , 19]. Fetal deaths occur because the virus damages the maternal placenta directly, leading to a failure to transport substances vital to the fetus. Furthermore, due to the insufficient development of the fetus’s immune system during this period, it displays immune intolerance to the virus [13]. Consequently, beyond fetal death, mummificaon, premature birth, and aboron resulng from transplacental infecon in the first trimester of pregnancy, persistent infecon emerges in the fetus due to non-cytopathogenic (NCP) biotype infecon aſter 30 days of gestaon [3 , 13]. Persistently infected calves connuously shed the virus throughout their lives and remain constantly viremic [13]. Various diagnosc methods, including Peroxidase- Linked Anbody (PLA), Direct and Indirect Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF), Virus Neutralizaon Test (VNT), Neutralizing Peroxidase-Linked Anbody (NPLA), Reverse Transcriptase-Polymerase Chain Reacon (RT-PCR) [20], and Real-Time RT-PCR, are commonly employed to diagnose BVDV in clinical specimens [21]. This study aimed to idenfy the eology of the causave agent on a farm with a history of aboron cases with congenital anomalies. It also sought to isolate the idenfied pathogen and confirm the isolaon through real-me RT-PCR. The isolated virus was sequenced at the 5′UTR gene region to determine its relaonship with strains detected in Türkiye and globally for establishing control and management strategies. MATERIALS AND METHODS Ethical statement This research was conducted in accordance with ethical principles with due respect for animal rights. Approval was obtained from an ethics commission (Animal Experimental Local Ethics Commiee on 21 September 2023 (Number 13067196/124). Sample collecon Upon reviewing the anamnesis, it was determined that there were no previous instances of aboron. The herd’s feeding strategy involved grass, herbage, and culvated feed in pastures during summer and in barns during winter. According to reports, the herd received vaccinaon exclusively against foot-and- mouth disease. Addionally, it was noted that there are 66 farms with a total of 617 cale in the area, all of which are in good overall condion. This study included cale that had experienced aboron, along with calves displaying anomalies. The main subject of the invesgaon was a calf sample (protocol number 33, animal idenficaon number: TR254623145) sent from Aşkale district, Erzurum province, in January 2023; the sample was received by the Erzurum Veterinary Control Instute Directorate under protocol number EVE/2023/36 (FIG. 1). Whole blood and serum were also collected from the cale. 2 of 8

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Aboron bovine with congenital malformaons caused by BVDV-1d The virology laboratory of the instute conducted tests on the aborted material for various viral agents, including Akabane virus, Schmallenberg virus, Bovine Ephemeral Fever virus, Bovine Herpes virus-1 (BoHV-1), and BVDV. The eologic agent responsible for the aboron was idenfied as BVDV using Real- Time RT-PCR. To determine whether the aboron case caused by BVDV was an isolated incident or a herd-wide problem, a visit to the cale farm in queson was conducted; and, an invesgaon was performed, based on anamnesis informaon, for checking for other animals experiencing aboron, verifying vaccinaon pracces beyond the foot-and-mouth disease vaccine, confirming the supplementaon of vitamins and minerals, and assessing the nutrional status of the animals. Farms in the surrounding region were also evaluated. Macroscopic analysis Necropsy and macroscopic examinaon were performed on the abnormally aborted calf. Following the necropsy of the aborted sample, cerebrospinal fluid was collected for further analysis. A necropsy was conducted on the calf with anomalies that arrived at our instute for a thorough postmortem assessment. Macroscopically, an external hydrocephalus, resembling a sac filled with cerebrospinal fluid, was observed in the cranial region. The calvarium was cut easily, and the brain was rudimentary. Examinaon of the facial region revealed a short lower jaw (micrognathia inferior). In addion, incomplete development of teeth in the oral cavity was noted. While both anterior limbs were present, both posterior limbs were absent (amelia posterior). Furthermore, the anterior right leg was observed to be shorter than the leſt one. No internal organ formaon was detected in the peritoneal cavity of the calf (FIG. 1). The cale tested posive in both indirect and direct ELISA. Virological detecon (Angen ELISA and Real-Time RT-PCR) Angen ELISA The blood sample underwent centrifugaon at 1500 G for 10 minutes (min) using a Thermo centrifuge (SL16R, Thermo Scienfic, Germany) [22]. Subsequently, the buffy coat was subjected to direct angen (Ag) ELISA using the BVDV Angen Test Kit/Serum Plus (IDEEX, Germany). In addion, blood collected in an Ethylenediaminetetraacec acid (EDTA)-free tube was centrifuged at 1500 G for 10 min, and the serum sample was then analyzed through indirect anbody (Ab) ELISA using the BVDV/MD/BDV p80 Protein Anbody Test Kit (IDEXX, Germany). RNA Extracon Cerebrospinal fluid samples, stored in anbioc PBS (PBS, pH 7.4, Gibco, USA) (at +4°C, were centrifuged at 2000 G for 15 min. For nucleic acid isolaon, 200 µl of the supernatant prepared from cerebrospinal fluid and blood samples were collected, and RNA extracon was performed using a roboc isolaon device (Indimag 48S, Indical, Germany) with the Indical nucleic acid isolaon kit (Cat No: F202300039), following the kit procedure. The obtained RNAs were stored at -80°C (Ultra freezer, Lexicon, Esco, Singapore). The quality and quanty of the isolated RNAs were determined using a spectrophotometer (Nanodrop, Thermo Scienfic, Germany). Real-me RT-PCR and RT-PCR For real-me RT-PCR analysis (Rotor-Gene Q 5 plex, Qiagen, USA), we employed primers and probes reported by Baxi et al. [21]. For sequencing, BVDV-posive samples were analyzed by convenonal RT-PCR with the primers and methods reported by Vilcek et al. [23] in (TABLE I). TABLE I. Nucleode Informaon of Primers and Probes Used for Real-me and Convenonal RT-PCR Target PCR Primer-Prob Sequence (5’–3’) Referens No Real Time RT-PCR Forward CTA GCC ATG CCC TTA GTA G [21] Rewerse CGT CGA ACC AGT GAC GAC Probe (FAM/BHQ3) TAG CAA CAG TGG TGA GTT CGT TGG Probe (TXR/BHQ3) TAG CGG TAG CAG TGA GTT CGT TGG ATG GCC RT-PCR 324 ATG CCC W (AT) TA GTA GGA CTA GCA [23] 326 TCA ACT CCA TGT GCC ATG TAC 3 of 8

Bovine Viral Diarrhea Virus in Calves in Turkey / Akça et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Phylogenec analyses Samples from calves and cale that tested posive for BVDV via real-me RT-PCR underwent reanalysis through convenonal RT-PCR using 5’UTR gene-specific primers. Sequencing was performed on posive amplicons (TABLE I) [23]. Based on the obtained sequence data, we conducted phylogenec analysis, incorporang strains from GenBank and those documented from Türkiye in GenBank by Tamura et al. [24]. Virus isolaon Virus isolaon was carried out using the Madin-Darby Bovine Kidney (MDBK) cell line obtained from Erciyes University, Faculty of Veterinary Medicine, Department of Virology, Kayseri Türkiye. In brief, supernatants of ssue homogenates (Tissue Lyser LT, Qiagen, USA) were inoculated into a 25 cm 2 flask using the adsorpon method (1 h). Control wells were inoculated with PBS as a negave control. Following the adsorpon period, the flask was replenished with sufficient medium and incubated (Celculture CO 2 Incubatore, Esco, Singapore) at 37°C with 5% CO 2 for 6 days. Virus ters for each blind passage’s supernatant were tested by real-me RT-PCR (FIG. 2). The samples inoculated into the cell culture from the brain suspension were monitored for 6 d. This process was repeated through 6 serial blind passages. No cytopathic effect (CPE) formaon was observed at the end of the passages, indicang a non-cytopathogenic biotype. However, due to the non-cytopathogenic nature of the virus, Real-Time RT-PCR was performed to assess the reproducve status and isolaon of the virus. The Cycle Threshold (CT) values for each passage of the virus in cell culture were determined as follows: 25.94 (1st passage), 26.54 (2nd passage), 27.58 (3rd passage), 25.40 (4th passage), 26.34 (5th passage), and 26.76 (6th passage) (FIG. 2). Therefore, while the virus amount showed a relave decrease in the first 3 passages (indicang a new adaptaon of the virus to the cell culture), the increase in the CT value from the 4th passage suggested that the virus tended to mulply. FIGURE 2. Real-Time RT-PCR result (A1: 1 st passage, A2: 2 nd passage, A3: 3 th passage, A4: 4 th passage, A5: 5 th passage, A6: 6 th passage, A7: Maternal blood sample, A8: BVDV-1 PC, B1: BVDV-2 PC, B2: NC) RESULTS AND DISCUSSIONS Aſter PCR and electrophoresis, the obtained amplicons were sequenced. The raw data from the sequence analysis were compared with the nucleic acid sequence of reference strains obtained from the gene bank using the bioinformacs programs BioEdit version 7.0.5 and Clustral W soſtware. The isolate in the study was idenfied as BVDV-1d (FIG. 3). FIGURE 3. The MEGA v.11 was used to analyze BVDV 5’UTR gene sequences via the neigh- bor-joining method with 1,000 bootstrap datasets based on the ClustalW algorithm. A circular shape (●) indicates the phylogenec locaon of our study strain. BVD infecon is a global concern in cale, causing various reproducve and health issues, including aboron, sllbirth, fetal resorpon, congenital malformaons, fetal infecons, growth retardaon, decreased pregnancy rates, and the birth of underdeveloped calves. Moreover, it leads to respiratory and digesve tract infecons, making cale more suscepble to secondary agents due to immunosuppression worldwide [25 , 26]. A high incidence of the disease in cale populaons has been observed in various countries around the globe through serologic and virologic studies [27]. In a study by Deng et al. [28] in China, the surge in demand for beef and dairy products triggered significant investments in the cale industry, fostering rapid sector growth, resulng in increased cale transport and the swiſt spread of BVDV across the country due to a lack of effecve protecon (mandatory vaccinaon) and eradicaon efforts. Meanwhile, various European countries have iniated voluntary or mandatory BVD control programs, with some achieving disease-free status [29]. Hence, it is crucial for Türkiye to establish BVD infecon-free herds and implement prevenve control measures. Understanding Bovine Viral Diarrhea (BVD) is greatly aided by molecular typing studies. BVDV is classified into three genotypes 4 of 8

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico (BVDV-1, BVDV-2, BVDV-3), and extensive global research has focused on these genotypes. BVDV-1 is the most frequently studied genotype, with prevalent subgenotypes 1b, 1a, and 1c. European countries exhibit remarkable diversity in BVDV sub-genotypes. This suggests that there exists genec diversity throughout the connent [30], emphasizing the importance of molecular studies for effecve prevenon and control. In Europe, the BVDV-1d sub-genotype was idenfied in 281 out of 3,101 samples [30]. A country-specific study [31] revealed the dominance of subtypes 1b and 1d near the Danish border in Germany, 1d and 1f in Slovenia, 1b in Spain, 1a in Northern Ireland, and 1f, 1h, 1b, and 1d in Austria. The 1d genotype idenfied in this study aligns with strains recognized as subtype 1d in Europe. This underscores the need to consider imports of animals, animal products, and biological products from these countries. In a study by Kuta et al. [32] in Poland to determine BVDV types, all 65 BVDV samples were confirmed posive by RT-PCR. Of these samples, 31 were subtype 1b, 24 were 1d, 8 were 1f, and 2 were 1g. The herds with 1b and 1f types were vaccinated with BVDV-1a for 2 years, but by the end of vaccinaon, 1f type was found in calves. In their study in Poland, Mirosław and Polak [33] idenfied subtypes 1b, 1g, 1f, 1d, 1r, and 1e. Another study by Kucer et al. [34] in Croaa marked the first detecon of BVDV 1d in that country. Our idenficaon of type 1d in this study suggests that vaccines containing type 1a in Türkiye may be insufficient for protecon and control, and vaccines used in Türkiye should also include type 1d. Scharnböck et al. [35] analyzed 325 studies in 73 countries from 1961 to 2016 using the meta-analysis method and found that some animals infected with BVDV were Persistently Infected (PI), some were viremic, and some were anbody- posive. The prevalence of PI animals was higher in countries with fewer control/eradicaon studies. This study underscores the significance of control and eradicaon pracces in Türkiye, emphasizing the importance of symptomac and prophylacc treatment for animals at risk, since minimizing the prevalence of BVDV is crucial for herd health and welfare. In a review study of BVDV prevalence by Yeşilbağ et al. [30], 1d subtype was detected in 20 out of 91 samples on the African connent, with 1d posivity ranking second only to 1a and tying with 1c subtype. Fulton et al. [36] reported that the 1d subtype is predominant in southern Africa and causes respiratory tract infecons. Vuuren [37] suggested that BVDV-1 subtypes in Africa are similar to the classical BVDV-1 of American origin. This implies that BVDV-1 may have spread through animals and animal products (semen, biological products, etc.) transported from the USA to the African connent. In the Americas, subtype 1d was detected in 24 out of 1055 samples, ranking as the third most common subtype aſter 1b and 1a [30]. A study on a Angus herd in Brazil by Bianchi et al. [25] revealed that the herd grazed on natural pastures, established breeding condions through natural selecon and that the animals were housed in barns during winter; although the animals were vaccinated against foot-and-mouth disease (FMDV), rabies, and clostridia, they were not vaccinated against BVDV. The study reported an outbreak of mucosal disease caused by BVDV-1d in the herd, resulng in high morbidity and lesions limited to the upper digesve tract and skin (there were no lesions in the intesnes); the study also reported that the outbreak led to early embryonic loss, aborons, and sllbirths. In the current study, although no clinical findings were observed in the cale, in the aborted fetus experienced an anomaly. Nogueira et al. [38] conducted a study to uncover new aspects of pesvirus epidemiology in human in Brazil, and performed protein analysis on samples from the brains of infants who died of Zika virus disease and detected pepdes from the polyprotein of BVDV. The same study found BVDV RNA nucleic acid in amnioc fluid collected from mothers of infants with Zika and microcephaly during the Paraiba outbreak. This suggests that the Zika virus may not be the alone eologic agent, emphasizing the importance of considering the risks of BVDV, especially for veterinarians, veterinary technicians, and individuals involved in animal husbandry. Therefore, it should be kept in mind, as with any disease, that mixed infecons can always be detected when one agent is found in eologic invesgaons. In Australia, subtype 1d was not idenfied in any of the 439 samples [30]. Nevertheless, the potenal for interconnental transmission should not be ruled out. In Asia, subtype 1d was detected in 13 out of 1411 samples [30]. Numerous studies in Türkiye have explored the presence of BVDV, revealing a widespread distribuon of anbodies against pesviruses throughout the country. Alpay et al. [39] asserted that BVDV entered Türkiye through the trade of animals and animal products from other countries, this claim was also supported by another study [16] with the idenficaon of BVDV- 1c (Australia-specific strain) in a herd imported from Australia. Genec and molecular characterizaon studies in Türkiye have idenfied various BVDV subtypes, including BVDV-1a, 1b, 1d, 1f, 1h, 1i, 1l, as well as BVDV-2a, 2b, and BVDV-3. A study conducted between 2005 and 2007 by Oğuzoğlu et al . [40] found that the prevalent BVDV types were BVDV-1l (18/40), 1f (10/40), 1b (7/40), 1d (3/40), and 1a (2/40). Subtype 1l predominated in Ankara, BVDV-1f (8/10) in cale samples from the Marmara region, BVDV-1b (6/7) in the Central Anatolia region, BVDV-1d (3/3) in Şanlıurfa, and BVDV-1a (2/2) in the Marmara region. Timurkan and Aydın [10] idenfied 7 different subtypes (BVDV- 1l, -1a, -1d, -1b, -1c, -2a, -3) in aboron cases from Erzurum and Elazığ provinces. The commonality of the 1d subgenotype in our province, as observed by Timurkan and Aydın [10], suggests that this occurrence may be aributed to animal movements. Toker et al. [41] idenfied the presence of respiratory pathogens, including BPIV-3, BRSV, BVDV (1l and 1r), and BoHV- 1, in northwestern Turkish provinces (Bursa, Balıkesir, Kütahya, Bilecik, and Kocaeli) through phylogenec analyses conducted between 2012 and 2017. Their findings indicated similaries to other subtypes in Türkiye, excluding the detecon of the BVDV- 1d subtype. In a separate study, Cağırgan et al. [16] idenfied 9 BVDV-posive cases from 117 aborted fetal materials in western Türkiye between 2017 and 2020. Among them, 3 different BVDV- 1 types were detected, with 4 classified as the dominant type 1d, 3 as 1l, and 2 as 1f. Considering the prevalent circulaon of BVDV-1d and 1f types in European countries (Germany, Italy, Austria, and Poland), the study suggested allowing the import of animals and animal products from these countries aſter necessary analyses. Yeşilbağ et al. [2] conducted a study across 15 provinces of Türkiye between 1997 and 2005, focusing on cale and sheep, and found that out of the 30 virus isolates, 29 were from cale 5 of 8

Bovine Viral Diarrhea Virus in Calves in Turkey / Akça et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico [BVDV-1l (n=15), BVDV-1a (n=4), BVDV-1b (n=4), BVDV-1d (n=3), BVDV-1f (n=2), and BVDV-1h (n=1)], and 1 was BVDV-2 in sheep. While acknowledging that this study may not fully represent Türkiye, it is noteworthy that BVDV-1d was found in samples from other provinces. Sarıkaya et al. [7] idenfied 53 BVDV-posive cases (BVDV- 1a, BVDV-1b, and BVDV-2) out of 267 samples in their study conducted in the Central Anatolia region between 2009 and 2010. Although the BVDV-1d type was not detected, its absence in these regions doesn’t conclusively rule out its presence, more intensive surveillance studies could reveal the potenal existence of type 1d. Although type 1d has been sporadically idenfied in various surveillance studies in Türkiye, it has not been linked to reports of abnormalies; only aboron events have been menoned. This study sheds light on an important aspect of BVDV pathogenesis. Previous research has emphasized the significance of imported animal movements, emphasizing the need for more comprehensive invesgaons in this region. In this study, the non-cytopathogenic nature of BVDV prompted the use of the Real-Time RT-PCR method to assess virus reproducon accuracy through six passages in cell culture. While immunoperoxidase or IF methods generally yield more precise results, it was suggested that Real-Time RT-PCR could serve as an alternave method for reproducve control, a possibility that future studies may further support. Using the ELISA method, it was determined that the sample taken from the cale, who entered the facility aſter 4 weeks, tested posive for both angen and anbodies. Pesvirus biotypes, known to be both cytopathogenic (CP) and non- cytopathogenic (NCP), undergo biotype determinaon in cell culture. CP and NCP biotypes of pesviruses cause high morbidity, a short clinical course, lesions limited to the digesve system and skin, and aborons in both cale and sheep. The Flaviviridae family’s structural and non-structural proteins are crucial for viral replicaon [42]. Notably, the formaon of the non-structural protein NS3, angenically linked to NS2-3, is a significant genec difference observed only in cells infected with CP viruses [16 , 42]. In this study, samples from the cale and aborted calf were idenfied as BVDV-1d subtype and NCP biotype. Our results are compable with previous studies indicang that most aborons are caused by the NCP pesvirus biotype. CONCLUSION Consequently, it is crucial to conduct essenal analyses on imported animals, animal and biological products such as FBS, semen, vaccines etc. Connuous monitoring of BVDV outbreaks, systemac research, and classificaon of newly idenfied isolates can significantly improve discussions within the pesvirus family. These measures are necessary for future epidemiologic studies to be effecve. In addion, incorporang subtype 1d into vaccines used in Türkiye (BVDV-1a, BVDV-1b subtypes, and BVDV-2 subtypes) could contribute to defining new strategies for control and eradicaon programs. Author contribuons SA: Draſting manuscript, conceptualizaon, formal analysis, invesgaon, MÖT: Draſting manuscript, crical revisions, Conceptualizaon, Formal analysis, ÖFK: Conceptualizaon, Formal analysis, data collecon, MG: Formal analysis, data curaon HA: Crical revisions, IS: Crical revisions. Conflict of interest The authors declared no conflict of interest. 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