Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging

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Abstract

Introduction. African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4–6 hours and the late 8–20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results.

Objective. Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain.

Materials and methods. For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum.

Results. The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented.

Conclusion. A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

About the authors

Natalia N. Vlasova

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: vlanany@yandex.ru
ORCID iD: 0000-0001-8707-7710

Dr of Biol. Sci., Principal Researcher of the Laboratory of Biochemistry and Molecular Biology

Russian Federation, 109428, Moscow

Oleg Yu. Chernykh

Department of Veterinary Medicine of the Krasnodar Territory

Email: gukkvl50@kubanvet.ru
ORCID iD: 0000-0001-8584-8251

Dr of Vet. Sci., Professor, Director

Russian Federation, 350051, Krasnodar Territory, Krasnodar

Roman A. Krivonos

Department of Veterinary Medicine of the Krasnodar Territory

Email: uv@krasnodar.ru
ORCID iD: 0009-0008-4580-6776

Ph.D. of Vet. Sci., Head

Russian Federation, 350051, Krasnodar Territory, Krasnodar

Oleg A. Verkhovsky

Research Institute for Diagnosis and Prevention of Human and Animal Diseases

Email: info@dpri.ru
ORCID iD: 0000-0003-0784-9341

Dr of Biol. Sci., Professor, President

Russian Federation, 123098, Moscow

Taras I. Aliper

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: aliper@vetbio.ru
ORCID iD: 0000-0003-2696-1363

Dr. of Biol. Sci., Professor, Head of the Laboratory of Epizootology, diagnostics and prevention of viral diseases of pigs

Russian Federation, 109428, Moscow

Afshona M. Anoyatbekova

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: aafshona@mail.ru
ORCID iD: 0000-0002-6579-779X

Ph.D.of Vet. Sci., Senior Researcher, Laboratory of Biochemistry and Molecular Biology

Russian Federation, 109428, Moscow

Elena V. Zhukova

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: evz-sk@mail.ru
ORCID iD: 0000-0001-7423-6102

Ph.D. Sc., Leading Researcher, Laboratory of Biochemistry and of Molecular Biology

Russian Federation, 109428, Moscow

Oksana D. Kucheruk

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: kussike17@yandex.ru
ORCID iD: 0000-0002-2161-9406

Ph.D. of Vet. Sci., Leading Researcher, Laboratory of Biochemistry and Molecular Biology

Russian Federation, 109428, Moscow

Anton G. Yuzhakov

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Author for correspondence.
Email: anton_oskol@mail.ru
ORCID iD: 0000-0002-9354-6824

Ph.D. of Biol. Sci., Head of the Laboratory of Biochemistry and Molecular Biology

Russian Federation, 109428, Moscow

Mikhail I. Gulyukin

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: admin@viev.ru
ORCID iD: 0000-0002-7489-6175

Dr. of Vet.Sci., Academician of RAS, Head of Scientific Areas Center in the administration

Russian Federation, 109428, Moscow

Aleksey M. Gulyukin

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences

Email: admin@viev.ru
ORCID iD: 0000-0003-2160-4770

Dr. of Vet. Sci., Corresponding Member RAS, Director

Russian Federation, 109428, Moscow

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2. Fig. 1. Haemadsorption upon the continuous cell lines of porcine origin infected by ASF virus isolate ASFV/Znaury/dp-22. a – PPK66b – day 2; b – ST – day 4; c – PK – day 6; magnification ×100.

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3. Fig. 2. Phylogenetic tree based on the analysis of the nucleotide sequences of the B646L (p72) gene of various strains of genotype II and a new isolate from the v. Nog of the Znaury district of the Republic of South Ossetia.

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4. Fig. 3. Nucleotide sequence alignment of ASFV genotype II isolates with and without a 10-nucleotide insert in the I73R/I329L intergenic region.

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Copyright (c) 2023 Vlasova N.N., Chernykh O.Y., Krivonos R.A., Verkhovsky O.A., Aliper T.I., Anoyatbekova A.M., Zhukova E.V., Kucheruk O.D., Yuzhakov A.G., Gulyukin M.I., Gulyukin A.M.

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