Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR)

Erkan Anıl Aydin; Cihan İnan

doi:10.35229/jaes.1559848

EN TR

Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR)

Abstract

Poxviruses are large double-stranded DNA (dsDNA) viruses that infects both human, animals and insects. Most known member of this viruses is Variola virus that caused pox disease and eradicated by vaccination. While other members still infects their hosts it is important to detect, isolate and fight with this viruses. For this purpose determining the titers of viruses is important. Virus titers are used for vaccine development, antiviral activity, genetic engineering, gene therapy and disease treatment studies. Despite all these uses, the methods used to determine virus titer are tedious and time-consuming. We developed a method using quantitative real-time PCR to improve the speed and efficiency of titer determination. In this method, we used a known single-copy gene from AMEV virus, cloned it into a plasmid and generated standard graphs. We measured the virus whose titer was unknown using the standard graph. As a result, a method was developed that can be used to determine the titer of Poxviruses in a simple, fast and effective way.

Keywords

qPCR , Poxvirus , AMEV , Virus Titer , DNA

Entomopoxvirüslerin Viral Titresinin Kantitatif Gerçek Zamanlı PCR (qPCR) ile Belirlenmesi

Öz

Poksvirüsler, hem insanları hem hayvanları hem de böcekleri enfekte eden büyük çift sarmallı DNA (dsDNA) virüsleridir. Bu virüslerin en bilinen üyesi, çiçek hastalığına neden olan ve aşılama ile ortadan kaldırılan Variola virüsüdür. Diğer üyeler hala konakçılarını enfekte ederken, bu virüsleri tespit etmek, izole etmek ve onlarla savaşmak önemlidir. Bu amaçla virüslerin titrelerini belirlemek değerlidir. Virüs titreleri aşı geliştirme, antiviral aktivite, genetik mühendisliği, gen terapisi ve hastalık tedavi çalışmaları için kullanılır. Tüm bu kullanımlara rağmen, virüs titresini belirlemek için kullanılan yöntemler sıkıcı ve zaman alıcıdır. Bu çalışmada titre belirlemenin hızını ve verimliliğini artırmak için kantitatif gerçek zamanlı PCR kullanan bir yöntem geliştirilmiştir. Bu yöntemde, AMEV virüsünden bilinen tek kopyalı bir gen kullanılarak, bir plazmite klonlandı ve standart grafikler oluşturuldu. Titresi bilinmeyen virüsü standart grafik kullanarak analiz edildi. Sonuç olarak Poxvirüslerin titresi basit, hızlı ve etkili bir şekilde belirlenebileceği bir metod geliştirildi.

Anahtar Kelimeler

qPCR , Poksvirus , AMEV , Virus Titresi , DNA

References

Baer, A. & Kehn-Hall, K. (2014). Viral concentration determination through plaque assays: Using traditional and novel overlay systems. Journal of Visualized Experiments, 93, e52065, 1-10. DOI: 10.3791/52065
Bawden, A.L., Glassberg, K.J., Diggans, J., Shaw, R., Farmerie, W. & Moyer, R.W. (2000). Complete genomic sequence of the Amsacta moorei entomopoxvirus: Analysis and comparison with other poxviruses. Virology, 274, 120-139. DOI: 10.1006/viro.2000.0449
Bhat, T., Cao, A. & Yin, J. (2022). Virus-like particles: measures and biological functions. Viruses, 14, 383. DOI: 10.3390/v14020383
Darling, A. J., Boose, J. A. & Spaltro, J. (1998). Virus assay methods: accuracy and validation. Biologicals : journal of the International Association of Biological Standardization, 26(2), 105-110. DOI: 10.1006/biol.1998.0134
Granados, R.R. & Naughton, M. (1975). Development of Amsacta moorei entomopoxvirus in ovarian and hemocyte cultures from estigmene acrea larvae. Intervirology 5, 62-68. DOI: 10.1159/000149881
Guo, F.B. & Yu, X.J. (2007). Re-prediction of protein-coding genes in the genome of Amsacta moorei entomopoxvirus. J Virol Methods, 146, 389-392. DOI: 10.1016/j.jviromet.2007.07.010
Hall, R. L. & Hink, W. F. (1990). Physical mapping and field inversion gel electrophoresis of Amsacta moorei entomopoxvirus DNA. Archives of virology, 110(1-2), 77-90. DOI: 10.1007/BF01310704
Haller, S.L., Peng, C., McFadden, G. & Rothenburg, S. (2014). Poxviruses and the evolution of host range and virulence. Infection, Genetics and Evolution, 21, 15-40. DOI: 10.1016/j.meegid.2013.10.014
Inan, C., Muratoglu, H., Arif, B.M. & Demirbag, Z. (2018). Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248). Virus Genes 54, 438-445. DOI: 10.1007/s11262-018-1561-4
Itakura, Y., Matsuno, K., Ito, A., Gerber, M., Liniger, M., Fujimoto, Y., Tamura, T., Kameyama, K. ichiro, Okamatsu, M., Ruggli, N., Kida, H. & Sakoda, Y. (2020). A cloned classical swine fever virus derived from the vaccine strain GPE− causes cytopathic effect in CPK-NS cells via type-I interferon-dependent necroptosis. Virus Res., 276. DOI: 10.1016/j.virusres.2019.197809

Marlow, S.A., Billam, L.J., Palmer, C.P. & King, L.A. (1993). Replication and morphogenesis of Amsacta moorei entomopoxvirus in cultured cells of Estigmene acrea (salt marsh caterpillar). The Journal of general virology, 74(Pt 7), 1457-1461. DOI: 10.1099/0022-1317-74-7-1457
Moss B. (2006). Poxvirus entry and membrane fusion. Virology, 344(1), 48-54. DOI: 10.1016/j.virol.2005.09.037 Muratoğlu, H., Nalçacıoğlu, R. & Demirbağ, Z. (2010). Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk). Ann. Microbiol., 60, 523-530. DOI: 10.1007/s13213-010-0082-8
Palacios, J. del C.A., Barreto, C.A.B., Lara, J.S.M. & Navas, Á.M.L. (2017). Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use. Open Med. Chem. J., 11, 66-80. DOI: 10.2174/1874104501711010066
Palmer, C.P., Miller, D.P., Marlow, S.A., Wilson, L.E., Lawrie, A.M. & King, L.A. (1995). Genetic modification of an entomopoxvirus: deletion of the spheroidin gene does not affect virus replication in vitro. The Journal of general virology, 76(Pt 1), 15-23. DOI: 10.1099/0022-1317-76-1-15
Perera, S., Krell, P., Demirbag, Z., Nalçacioǧlu, R. & Arif, B. (2013). Induction of apoptosis by the Amsacta moorei entomopoxvirus. Journal of General Virology 94, 1876-1887. DOI: 10.1099/vir.0.051888-0
Plesa, G., McKenna, P.M., Schnell, M.J. & Eisenlohr, L.C. (2006). Immunogenicity of cytopathic and noncytopathic viral vectors. Journal of virology, 80(13), 6259-6266. DOI: 10.1128/JVI.00084-06
Reed, L.J. & Muench, H. (1938). A Simple Method of Estimating Fifty Percent Endpoints. American Journal of Hygiene, 27, 493-497.
Sezen, K. & Demirbağ, Z. (2005). Entomopoksvirüsler ve Biyolojik Kontrol. Turkish Journal of Parasitology, 29(4), 280-286.
Sliva, K., Erlwein, O., Bittner, A. & Schnierle, B.S. (2004). Murine leukemia virus (MLV) replication monitored with fluorescent proteins. Virol. J., 1. DOI: 10.1186/1743-422X-1-14
Fajardo, T. V. M., Vanni, M. F. & Nickel, O. (2017). Absolute quantification of viruses by TaqMan real-time RT-PCR in grapevines. Ciência Rural, 47(6), 1–5. https://doi.org/10.1590/0103-8478cr20161063
Jiang, S. S., Chen, T. C., Yang, J. Y., Hsiung, C. A., Su, I. J., Liu, Y. L., Chen, P. C. & Juang, J. L. (2004). Sensitive and Quantitative Detection of Severe Acute Respiratory Syndrome Coronavirus Infection by Real-Time Nested Polymerase Chain Reaction. Clinical Infectious Diseases, 38(2), 293–296. https://doi.org/10.1086/380841
Jiang, Y., Zhang, S., Qin, H., Meng, S., Deng, X., Lin, H., Xin, X., Liang, Y., Chen, B., Cui, Y., Su, Y. H., Liang, P., Zhou, G. Z. & Hu, H. (2021). Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus. European Journal of Medical Research, 26(1), 1-7. DOI: 10.1186/s40001-021-00608-5
Keyaerts, E., Vijgen, L., Maes, P., Duson, G., Neyts, J. & Van Ranst, M. (2006). Viral load quantitation of SARS-coronavirus RNA using a one-step real-time RT-PCR. International Journal of Infectious Diseases, 10(1), 32-37. DOI: 10.1016/j.ijid.2005.02.003
Lo, H. R. & Chao, Y. C. (2004). Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction. Biotechnology progress, 20(1), 354-360. DOI: 10.1021/bp034132i
Thomas, M.A., Lichtenstein, D.L., Krajcsi, P. & Wold, W.S. (2007). A real-time PCR method to rapidly titer adenovirus stocks. Methods in molecular medicine, 130, 185-192. DOI: 10.1385/1-59745-166-5:185

Details

Primary Language

English

Subjects

Animal Cell and Molecular Biology

Journal Section

Research Article

Authors

Erkan Anıl Aydin
0009-0003-3117-3844
Türkiye

Cihan İnan ^*
0000-0003-0288-5917
Türkiye

Early Pub Date

December 17, 2024

Publication Date

December 31, 2024

Submission Date

October 2, 2024

Acceptance Date

November 18, 2024

Published in Issue

Year 2024 Volume: 9 Number: 4

DOI

https://doi.org/10.35229/jaes.1559848

IZ

https://izlik.org/JA96RU32MB

APA

Aydin, E. A., & İnan, C. (2024). Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR). Journal of Anatolian Environmental and Animal Sciences, 9(4), 570-576. https://doi.org/10.35229/jaes.1559848

EBSCOHost Scilit CABI

JAES/AAS-Journal of Anatolian Environmental and Animal Sciences/Anatolian Academic Sciences&Anadolu Çevre ve Hayvancılık Dergisi/Anadolu Akademik Bilimler-AÇEH/AAS

Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR)

Abstract

Keywords

Entomopoxvirüslerin Viral Titresinin Kantitatif Gerçek Zamanlı PCR (qPCR) ile Belirlenmesi

Öz

Anahtar Kelimeler

References

Details

Primary Language

Subjects

Journal Section

Authors

Early Pub Date

Publication Date

Submission Date

Acceptance Date

Published in Issue

DOI

IZ

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