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    Akabane virus infection in Eastern Mediterranean Region in Turkey: Culicoides (Diptera: Ceratopogonidae) as a possible vector
    (Springer, 2021) Dagalp, Seval Bilge; Dik, Bilal; Dogan, Firat; Farzani, Touraj Aligholipour; Ataseven, Veysel Soydal; Acar, Gulizar; Sahinkesen, Ilker
    Akabane virus (AKAV), which causes Akabane disease, is an arthropod-borne virus (arbovirus) transmitted by Culicoides biting midges and mosquitoes. AKAV is an important pathogen that causes abortion and congenital anomalies in ruminants. In this study, we determined the prevalence of AKAV infection and identified possible viral vectors in Turkey's Eastern Mediterranean region. The presence and prevalence of AKAV infection were assessed using serological and virological methods. Serologically, the prevalence of AKAV antibodies in cattle, sheep and goats were 44.74% (400/894), 22.90% (60/262) and 14.52% (63/434), respectively, while the total prevalence was 32.89% (523/1590). AKAV-specific nucleic acid amplicons were obtained by real-time RT-PCR from 1.13% (9/799) and 1.74% (5/288) of the cattle and sheep tested, respectively. No goats were positive for AKAV RNA. Overall, AKAV-specific nucleic acid amplicons were detected in 0.87% (14/1604) of the sampled ruminants. In addition, specimens of the assumed vector, Culicoides, were caught using light traps and identified. Ten Culicoides species were detected in the area, of which Culicoides schultzei complex was the dominant species although 32 specimens could not be identified at the species level. These were defined as Culicoides spp. AKAV nucleic acid was detected in C. schultzei, Culicoides longipennis and Culicoides circumscriptus. Phylogenetic analysis indicated two different AKAV genogroups (genogroups Ib and genogroups II) while potential AKAV vectors in this region are C. schultzei complex, C. longipennis and C. circumscriptus.
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    Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?
    (Elsevier, 2020) Dogan, Firat; Dagalp, Seval Bilge; Dik, Bilal; Farzani, Touraj Aligholipour; Alkan, Feray
    Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.
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    Development of a BoHV-4 viral vector expressing tgD of BoHV-1 and evaluation of its immunogenicity in mouse model
    (Springer, 2021) Bilge-Dagalp, Seval; Farzani, Touraj Aligholipour; Dogan, Firat; Yoldar, Zeynep Akkutay; Ozkul, Aykut; Alkan, Feray; Donofrio, Gaetano
    In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD increment TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgD Delta TK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.
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    Genetic variability of bovine herpesvirus type 4 (BoHV-4) field strains from Turkish cattle herds
    (Ist Zooprofilattico Sperimentale Abruzzo & Molise G Caporale-Izs A&M, 2021) Dagalp, Seval Bilge; Dogan, Firat; Babaoglu, Ali Riza; Farzani, Touraj Aligholipour; Alkan, Feray
    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. Phylogeneticanalysis 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 1,11and111) 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.
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    Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey
    (Springer, 2020) Dagalp, Seval Bilge; Farzani, Touraj Aligholipour; Dogan, Firat; Alkan, Feray; Ozkul, Aykut
    The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.
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    Prevalance of Schmallenberg orthobunyavirus (SBV) infection in sampled ruminants in Turkey's Eastern Mediterranean region between 2015 and 2017
    (Elsevier Sci Ltd, 2022) Dogan, Firat; Dik, Bilal; Bilge-Dagalp, Seval; Farzani, Touraj Aligholipour; Ataseven, Veysel Soydal; Acar, Gulizar; Sahinkesen, Ilker
    Schmallenberg orthobunyavirus (SBV), first discovered in 2011, belongs to the Orthobunyavirus genus of the Peribunyaviridae family. SBV, which predominantly infects ruminants, can cause severe fetal malformations when pregnant animals are infected during a critical phase of gestation. In this study, 1590 blood serum samples from cattle, sheep, and goats were obtained for serological investigation and 1604 specimens for virological investigation (including 1414 whole blood with EDTA, 165 vaginal swab samples from aborting animals, and tissue samples from 25 dead and/or aborted fetuses) in private and family-type ruminant establishments in Turkey's Eastern Mediterranean region. All the blood serum samples were tested for the presence of antibodies using ELISA, which showed SBV antibodies in 29.11% (95% CI: 26.89%-31.35%). The virological samples were tested using real-time RT-PCR for SBV nucleic acid presence, which showed 3.17% (95% CI:2.32%-4.04%) were positive. Finally, 10 different Culicoides species (a total of 29,156 Culicoides, including 16,005 females and 13,151 males) were tested to identify the vectors thought to carry infections in the region. However, no SBV nucleic acid was detected in the Culicoides pools.
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    Solunum sistemi problemli kedilerde Feline Herpesvirus 1 (FHV-1) ve Feline Calicivirus varlığının moleküler olarak araştırılması
    (Selçuk Üniversitesi, 2019) Bilge Dağalp, Seval; Doğan, Fırat; Farzani, Touraj Aligholipour; Babaoğlu, Ali Rıza; Acar Kırmızı, Gülizar; Çabalar, Mehmet
    Amaç: Bu çalışmada FHV (Feline Herpesvirus) ve FCV (Feline Calicivirus) şüpheli örneklerde söz konusu enfeksiyonların varlığı/yaygınlığının araştırılması, bildirilen semptomlarla ilişkilendirilmesi ve bu virusların moleküler karakterizasyonu amaçlandı. Gereç ve Yöntem: Bu amaçla klinik olarak solunum sistemi problemi gösteren toplam 70 kediden 31 nasal, 30 konjunktival, 8 oral, 7 orafarengeal ve 11 rektal swap ile 32 EDTA’lı kan örneği olmak üzere toplam 119 örnek alınmıştır. Alınan örneklerden viral nükleik asit ekstraksiyonu yapıldı ve söz konusu enfeksiyonların varlığı PCR ile araştırıldı. Pozitif bulunan örnekler moleküler karakterizasyon için dizin analizi işlemine tabi tutuldu. Bulgular: Örneklenen kedilerin toplam %45,71’i (32/70) FHV-1 ve %10’u (7/70) FCV nükleik asit varlığı yönünden pozitif olarak tespit edildi. Toplam pozitifliği bildirilen bu kedilerin, %4,29’i (3/70) her iki etken için birlikte pozitif olarak değerlendirildi. Örneklenen solunum sistemi bulgulu kedilerde FHV-1 enfeksiyonun daha yaygın olduğu görüldü; yaş bilgisi olan kedilerde enfeksiyonların her yaş grubunda oluştuğu gözlenirken; özellikle oral ve/veya orafarengeal swap örneklerinin her iki enfeksiyonun teşhisinde de duyarlı örnekler olduğu tespit edildi. Öneri: Bu enfeksiyonların, enfekte kedilerde görülmesinin yanı sıra sağlıklı görünümlü hayvanlarda da tespit edilebildiği ve söz konusu enfeksiyonların bulaşında bu durumun göz ardı edilmemesi gerekliliği ortaya çıkmaktadır. Bu nedenle klinik belirtilerin şiddetinin azaltılması, saçılımın ve persistansın en aza indirgenmesi adına özellikle toplu yaşayan, dışarıda bir arada bulunan kedilerin düzenli aşılanması üzerinde durulmalıdır. Ayrıca FHV ve FCV dışındaki etkenlerin de (M.felis, C.felis, B.bronchoseptica gibi bakterilerIer) kedilerde solunum, göz ve oral lezyonlarda bulunabileceği unutulmamalı ve bu etkenler de enfekte kedilerde kontrol edilmelidir.