LJUNGAN VİRUSUN (PARECHOVİRUS B) KEMİRİCİLERDE MOLEKÜLER YÖNTEMLE ARAŞTIRILMASI

Öz

Ljungan virus (LV), Picornaviridae ailesinde bulunan Parechovirus genusunda yer alan kemirici kökenli bir virustur. LV'nin, Kuzey Avrupa, Kuzey Amerika ve İtalya'da kemiricilerde saptanmış olması dünyada bu virusun geniş bir dağılımı olduğunu düşündürmektedir. Miyokarditli ve tip 1 diyabetli insanlarda LV antikorları gösterilmiş ve insanlarda intrauterin ölüm, ani bebek ölümü ve fetal santral sinir sistemi malformasyonlarında LV ilişkisi saptanmıştır. Bu çalışma ile Türkiye'de henüz araştırılmamış olan LV'nin Zonguldak ilindeki yabani kemiricilerdeki varlığı hakkında bilgi sahibi olunması ve varsa bölgeye özgü yeni LV suşunun/suşlarının belirlenmesi amaçlanmıştır. Araştırmada 85 adet Apodemus ve 35 adet Myodes örneklerinden oluşan toplam 120 adet kemiriciye ait beyin dokusu kullanılmıştır. Bu örneklerde, LV genomunda genetik çeşitliliğin yüksek olduğu VP1 bölgesini hedefleyen ters transkriptaz polimeraz zincir tepkimesi (PZT) yöntemi ile LV nükleik asit varlığı araştırılmıştır. Çalışma sonucunda örneklerde LV nükleik asit varlığı saptanmamıştır. Tek bir bölge ve iki kemirici cinsine ait örneklerinin analizleri sonucuna göre, Türkiye çapında LV varlığının bulunmadığını söylemek yeterli bir açıklama olmayacaktır. Virüs için uygun olmayan çevresel koşullar da virusun tespit edilememesinde rol oynamış olabilir. Türkiye’de LV durumunun ortaya koyulabilmesi için farklı bölgelerindeki kemiricilerde, daha büyük bir örneklem ile ve daha fazla sayıda kemirici türü ile daha fazla sayıda araştırma yapılması gerektiği sonucuna varılmıştır. Bu araştırma, Türkiye de Ljungan virüs varlığı ile ilgili yapılmış ilk çalışmadır.

Anahtar Kelimeler

• Apodemus
• Ljungan virus
• Myodes
• Türkiye

Destekleyen Kurum

Dokuz Eylül Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

2018.KB.SAG.067

Etik Beyan

Çalışma planı, Dokuz Eylül Üniversitesi, Hayvan Deneyleri Yerel Etik Kurulu (2011/07 numaralı karar) tarafından onaylanmıştır.

INVESTIGATION OF LJUNGAN VIRUS (PARECHOVIRUS B) IN RODENTS BY MOLECULAR METHODS

Öz

Ljungan virus (LV) is a rodent-borne virus in the genus Parechovirus of the Picornaviridae family. LV has been detected in rodents in Northern Europe, North America and Italy, suggesting a wide distribution of this virus in the world. LV antibodies have been demonstrated in humans with myocarditis and type 1 diabetes, and LV has been correlated with intrauterine death, sudden infant death and fetal central nervous system malformations in humans. This study aimed to obtain information about the presence of LV, which has not been investigated in Turkey, in wild rodents in Zonguldak province and to determine the new LV strain(s) specific to the region, if any. A total of 120 brain tissues from 85 Apodemus and 35 Myodes were used in the study. In these samples, the presence of LV nucleic acid was investigated by reverse transcriptase polymerase chain reaction (PCR) method targeting the VP1 region in the LV genome where genetic diversity is high. As a result of the study, the presence of LV nucleic acid was not detected in the samples. It will not be a sufficient explanation for the absence of LV presence in Turkey according to the analysis of samples from a single region and two rodent species. Unsuitable environmental conditions for the virus may also have played a role in the lack of virus detection. We concluded that further research on rodents in different regions, with a larger sample size and on more rodent species, is needed to reveal the occurrence of LV in Turkey. This is the first study on Ljungan virus presence in Turkey.

Anahtar Kelimeler

• Apodemus
• Ljungan virus
• Myodes
• Turkey

Destekleyen Kurum

Dokuz Eylül University Department of Scientific Research Projects

Proje Numarası

2018.KB.SAG.067

Etik Beyan

The study plan was approved by the Local Ethics Committee for Animal Experiments (decision number 2011/07) of Dokuz Eylül University.

Kaynakça

1. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraaction: twenty- something years on. Nat Protoc. 2006;1(2):581-5. doi:10.1038/nprot.2006.83
2. Çoğal M, Sözen M. Distribution of rodent species (Mammalia: Rodentia) in Zonguldak Province, Turkey. International Journal of Environment and Geoinformatics (IJEGEO). 2022;9(4):185-93. doi. 10.30897/ijegeo.1075643
3. Donoso Mantke O, Kallies R, Niklasson B, Nitsche A, Niedrig M. A new quantitative real-time reverse transcriptase PCR assay and melting curve analysis for detection and genotyping of Ljungan virus strains. J Virol Methods. 2007;141(1):71-7. doi:10.1016/j.jviromet.2006.11.029
4. Fevola C, Rossi C, Rosà R, et al. Distribution and seasonal variation of Ljungan virus in bank voles (Myodes glareolus) in Fennoscandia. J Wildl Dis. 2017;53(3):552-60. doi:10.7589/2016-06-145
5. Fevola, C, Rossi C, Rosso F, et al. Geographical Distribution of Ljungan Virus in Small Mammals in Europe. Vector Borne Zoonotic Dis. 2020;20(9):692-702. doi:10.1089/vbz.2019.2542
6. Forbes KM, Voutilainen L, Jääskeläinen A, et al. Serological survey of rodent-borne viruses in Finnish field voles. Vector Borne Zoonotic Dis. 2014;14(4):278-83.
7. Hauffe HC, Niklasson B, Olsson T, Bianchi A, Rizzoli A, Klitz W. Ljungan virus detected in bank voles (Myodes glareolus) and yellow-necked mice (Apodemus flavicollis) from Northern Italy. J Wildl Dis. 2010;46(1):262-6. doi:10.7589/0090-3558-46.1.262.
8. Jääskeläinen AJ, Kolehmainen P, Voutilainen L, et al. Evidence of Ljungan virus specific antibodies in humans and rodents, Finland. J Med Virol. 2013;85(11):2001-8. doi:10.1002/jmv.23681

9. Jääskeläinen AJ, Nurminen N, Kolehmainen P, et al. No Association between Ljungan Virus Seropositivity and the Beta-cell Damaging Process in the Finnish Type 1 Di-abetes Prediction and Prevention Study Cohort. Pediatr Infect Dis J. 2019;38(3):314-6.
10. Johansson S, Niklasson B, Maizel J, Gorbalenya AE, Lindberg AM. Molecular analysis of three Ljungan virus isolates reveals a new, close-to-root lineage of the Picornaviridae with a cluster of two unrelated 2A proteins. J Virol. 2002;76(17):8920-30. doi:10.1128/jvi.76.17.8920-8930.2002
11. Johansson ES, Niklasson B, Tesh RB, et al. Molecular characterization of M1146, an American isolate of Ljungan virus (LV) reveals the presence of a new LV genotype. J Gen Virol. 2003;84(Pt 4):837-44.
12. Kallies R. Ljungan virus: prevalence in rodents and virus pathogenesis. Dissertation. Robert Koch-Institute Berlin (2010).
13. Krous HF, Langlois NE. Ljungan virus: a commentary on its association with fetal and infant morbidity and mortality in animals and humans. Birth Defects Res A Clin Mol Teratol. 2010;88(11):947-52. doi: 10.1002/bdra.20728
14. Lundstig A, McDonald SL, Maziarz M, et al. Neutralizing Ljungan virus antibodies in children with newly diagnosed type 1 diabetes. J Gen Virol. 2021;102(5):001602.
15. Niklasson B, Hörnfeldt B, Lundman B. Could myocarditis, insulin-dependent diabetes mellitus, and Guillain-Barré syndrome be caused by one or more infectious agents carried by rodents? Emerg Infect Dis. 1998;4(2):187-93. doi:10.3201/eid0402.980206
16. Niklasson B, Kinnunen L, Hörnfeldt B, et al. A new picornavirus isolated from bank voles (Clethrionomys glareolus). Virology. 1999;255(1):86-93. doi:10.1006/viro.1998.9557
17. Niklasson B, Heller KE, Schønecker B, et al. Development of type 1 diabetes in wild bank voles associated with islet autoantibodies and the novel ljungan virus. Int J Exp Diabesity Res 2003;4(1):35-44. doi:10.1080/15438600303733
18. Niklasson B, Nyholm E, Feinstein RE, et al. Diabetes and myocarditis in voles and lemmings at cyclic peak densities-induced by Ljungan virus? Oecologia. 2006;150(1):1-7.
19. Niklasson B, Samsioe A, Papadogiannakis N, et al. Association of zoonotic Ljungan virus with intrauterine fetal deaths. Birth Defects Res A Clin Mol Teratol. 2007;79(6):488-93. doi:10.1002/bdra.20359
20. Niklasson B, Almqvist PR, Hörnfeldt B, Klitz W. Sudden infant death syndrome and Ljungan virus. Forensic Sci Med Pathol. 2009;5(4):274-90.
21. Niklasson B, Samsioe A, Papadogiannakis N, et al. Zoonotic Ljungan virus associated with central nervous system malformations in terminated pregnancy. Birth Defects Res Part A Clin Mol Teratol. 2009;85(6):542-5.
22. Romeo C, Ferrari N, Rossi C, et al. Ljungan virus and an adenovirus in Italian squirrel populations. J Wildl Dis. 2014;50(2):409-11.
23. Salisbury AM, Begon M, Dove W, Niklasson B, Stewart JP. Ljungan virus is endemic in rodents in the UK. Arch Virol. 2014;159(3):547-51. doi: 10.1007/s00705-013-1731-6
24. Samsioe A, Sjöholm A, Niklasson B, Klitz W. Fetal death persists through recurrent pregnancies in mice following Ljungan virus infection. Birth Defects Res B Dev Reprod Toxicol. 2008;83(5):507-10. doi:10.1002/bdrb.20169
25. Samsioe A, Papadogiannakis N, Hultman T, Sjöholm A, Klitz W, Niklasson B. Ljungan virus present in intrauterine fetal death diagnosed by both immunohistochemistry and PCR. Birth Defects Res A Clin Mol Teratol. 2009;85(3):2279. doi:10.1002/bdra.20554
26. Tapia G, Cinek O, Rasmussen T, Grinde B, Rønningen KS. No Ljungan Virus RNA in Stool Samples from the Norwegian Environmental Triggers of Type 1 Diabetes (MIDIA) Cohort Study. Diabetes Care. 2010;33(5):1069-71.
27. Tolf C, Gullberg M, Johansson ES, Tesh RB, Andersson B, Lindberg AM. Molecular characterization of a novel Ljungan virus (Parechovirus; Picornaviridae) reveals a fourth genotype and indicates ancestral recombination. J Gen Virol. 2009;90(Pt 4):843-53. doi:10.1099/vir.0.007948-0
28. Warvsten A, Bjornfors M, Arvidsson M, et al. Islet autoantibodies present in association with Ljungan virus infection in bank voles (Myodes glareolus) in northern Sweden. J Med Virol. 2017;89(1):24–31.
29. Zheng L, Wang F, Huang J, Xin H. Evaluation of the association of zoonotic Ljungan virus with perinatal deaths and fetal malformation. Birth Defects Res C Embryo Today. 2015;105(1):81-5.