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Changes in the genome of African swine fever virus (Asfarviridae: Asfivirus: African swine fever virus) associated with adaptation to reproduction in continuous cell culture

https://doi.org/10.36233/0507-4088-50

Abstract

Introduction. African swine fever virus (ASFV) is a large, double-stranded DNA virus in the Asfarviridae family. It is the causative agent of African swine fever (ASF). Only the genome of BA71V strain, adapted to Vero cell culture, was fully analyzed.

The aim of this study was analyzing the complete genome sequence of two strains of adapted to the growth in CV-1 cell culture (CC) ASFV obtained after 30 and 50 passages, in comparison to the parental virus.

Material and methods. ASFV isolate Odintsovo 02/14 (parental), ASFV adapted variants ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 were all used to extract genomic DNA (gDNA). Sequencing library was constructed using the «Nextera XT DNA library preparation kit» («Illumina», USA).

Results. Genomes of ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 consisted of 186 529 bp and 186 525 bp, respectively. Total 78 single nucleotide polymorphisms (SNPs) were identified between the parental Odintsovo 02/14 and the two high passaged strains, as well as a 2947 bp large-size deletion in the 3’ variable region of adapted viruses was detected.

Discussion. ASFV as a DNA-containing virus may not have a very high level of mutation, but this is the second study showing that adaptation to growth in continuous CC leads to large deletions in the genome of the virus.

Conclusion. Mutations in the protein-coding regions of the genome can be synonymous and non-synonymous, i.e. leading to amino acid substitution. Additional research is needed to understand the influence of the mutations described in the adaptation process on the reproduction of the virus and its virulence.

For citation:


Mazloum A., Igolkin A.S., Zinyakov N.G., van Schalkwyk A., Vlasova N.N. Changes in the genome of African swine fever virus (Asfarviridae: Asfivirus: African swine fever virus) associated with adaptation to reproduction in continuous cell culture. Problems of Virology. 2021;66(3):211-216. https://doi.org/10.36233/0507-4088-50

Introduction

African swine fever virus (ASFV) is a large (175–215 nm), double-stranded DNA virus in the Asfarviridae family. It is the causative agent of African swine fever (ASF), a haemorrhagic disease with high mortality rates in domestic pigs.

To study the foundations of the pathogenicity and immunogenicity of the ASFV, it is necessary to carry out a comparative analysis of the biological properties of the virus and the structure of its genes. ASFV is able to replicate in porcine primary cell cultures (CC), such as porcine leukocytes CC, alveolar macrophages, etc. However, these cultures are difficult to standardize because their properties differ depending on the characteristics of the animal used, which makes it much more difficult to obtain a genetically uniform sample of virus suitable for analysis. To address this problem, various ASFV isolates have been adapted to grow in continuous cell culture, ex. Georgia 2007/1 and BA71 in Vero cell culture. As a result of this adaptation, ASFV-G/V strain and the BA71V strain were obtained [1][2]. Although, both these strains were reported to be avirulent following adaptation, only the genome of BA71V was sequenced and analyzed [2].

In the process of adaptation to growth in continuous cell cultures, the virulence of the ASFV decreased [3]. The purpose of this work was a comparative analysis of the nucleotide sequence obtained as a result of the whole genome sequencing of two strains of the ASFV obtained after 30 and 50 passages in the CV-1 cell culture, with the genome of the original isolate.

Previous studies indicated a decrease in virus virulence of isolate ASFV/ARRIAH/CV-1/30, yet the genome of this strain following adaptation was not well characterized [3]. In this study, we investigate how ASFV adaptation to growth in continuous cell culture might influence the virus’s genome, and identify the resulting mutations.

Material and methods

Strains ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 were propagated in CV-1 cell culture, as described previously [4]. Genomic DNA (gDNA) of the ASFVs were extracted using the previously described phenol-chloroform method and the resulting gDNA pellets diluted in nuclease-free water [5]. A sequencing library was constructed using the «Nextera XT DNA library preparation kit» (Illumina, USA) and Next Generation Sequencing (NGS) was performed using a «MiSeq reagent kit version 2» with 2 × 250-bp paired-end sequencing on a MiSeq benchtop sequencer (Illumina, USA). In order to assemble each of the genomes, reads were mapped to the reference genome (FR682468.1_ASFV/Georgia 2007/1) and a consensus sequence was generated for each of the isolates (CLC Genomics Workbench v.9.5.2 (QIAGEN, Aarhus; www.clcbio.com). The original set of reads were subsequently mapped to the newly assembled virus genome, with an average coverage depth of 45× and an average read length of 250 nucleotides (nt). Open reading frames (ORFs) were predicted using GATU software. The complete genome sequences of both high passage strains were deposited in GenBank with accession numbers MW528217 and MW528218.

The complete genome sequences of previously characterized ASFV isolate Odintosovo 02/14 (KP843857.1) and the genotype II reference, ASFV/Georgia 2007/1 (FR682468.1), were included in subsequent genetic analysis. The sequences of two reference and the two cell culture adapted strains were used to generate an alignment, which in turn was used to detect single nucleotide polymorphisms (SNPs). Both the construction of the alignment and SNPs detections were performed using CLC Genomics Workbench v.9.

Results

Since the biological and genetic characteristics of the ASFV isolate Odintsovo 02/14 has been studied, the isolate was used during progressive adaption of the virus to continuous cell culture using fibroblast-like pseudodiploid cells from the African green monkey (Cercopithecus aethiops) (CV-1) [6]. The complete genomes of cell culture adapted variants from passage 30 and 50 (referred to as: ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV1/50 respectively) were determined.

The complete genomes of the analyzed viruses differ in length with ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 consisting of 186,529 bp and 186,525 bp respectively, while in comparison the reference strain Georgia 2007/1 and parental isolate Odintsovo 02/14 was 189,344 bp and 189,122 bp respectively. This indicates a large deletion within the genomes of the highly passaged strains. Total 78 SNPs were identified between the parental Odintsovo 02/14 and the two high passaged strains. These could be further classified as 13 SNPs within the intergenic regions and 5 associated with homopolymer repeat sequences. The SNPs within open reading frames were characterized as 8 synonymous SNPs and 52 non-synonymous SNPs. All 60 synonymous and non-synonymous SNPs are listed in Table 1.

Table 1. Genes of different strains of the African swine fever virus encoding proteins with synonymous and non-synonymous single nucleotide polymorphisms


Note
. The amino acid predicted for each strain and its position are listed in the column «Predicted amino acid substitutions in the protein»; the positions affected in ASFV/ARRIAH/CV-1/30 or Odintsovo 02/14 strains are also listed there. Synonymous polymorphisms are highlighted in light gray; nucleotide changes that led to amino acid substitutions are marked in dark gray;
* – the first letter represents the amino acid of the wild type gene (the numbers indicate the position in the amino acid sequence of the protein), and the second letter represents the amino acid after the mutation.

Predicted proteins with conservative non-synonymous single nucleotide polymorphisms and synonymous in light grey. The amino acid predicted for each isolate and strain is listed as well as its position. Amino acid exchanges marked with dark grey indicate positions where either ASFV/ARRIAH/CV-1/30 or Odintsovo 02/14 were affected.

The majority (n = 36) of the non-synonymous SNPs are conservative amino acid exchanges, while 14 resulted in an exchange of a charged amino acid with either an amino acid with a different or no charge. Two (n = 2) of the non-synonymous SNPs resulted in early termination of the predicted protein (Table 1 and Table 2). The predicted proteins affected by the early termination as well as the non-conservative amino acid exchanges are listed in Table 2.

Table 2. List of predicted amino acid substitutions with either charge changes or generation of stop-codon


Note
. Mutations unique to the genome of the Odintsovo 02/14 strain are marked in gray;
* – the first letter represents the amino acid of the wild type gene (the numbers indicate the position in the amino acid sequence of the protein), and the second letter represents the amino acid after the mutation.

The presented results clearly demonstrate that the majority of amino acid exchanges occurred in the genome of the adapted ASFV/ARRIAH/CV-1/50 and/or ASFV/ARRIAH/CV-1/30 variants, with the exception of the mutations highlghted in gray, which were unique to the genome of isolate Odintsovo 02/14.

Discussion

Under certain conditions, ASFV, a double-stranded DNA virus, may not have a high level of genome variability, but since no effective vaccine have been developed till the moment against African swine fever, it is of high importance to understand and study the relation between certain biological properties and changes in the virus genome.

Since it was previously described how adaptation of the ASFV to growth in a continuous cell culture leads to significant changes in its genome and a decrease in virulence [3][7], complete genome sequencing and analysis is the most informative approach to understand the effect of these mutations on the pathogenesis of the virus.

In the presented study, it is clearly demonstrated that adaptation of the ASFV to growth in CV-1 continuous cell culture leads to the appearance of a large deletion in the 3’ variable region of the genome. Comparative analysis of the genome of the original isolate and the adapted variants also revealed 78 oligonucleotide polymorphisms that influenced or did not affect the predicted amino acid sequence of the encoded protein.

Only 2 oligonucleotide substitutions in the MGF_110_1L and B354L genes led to the formation of stop codons, which makes it possible to exclude the possibility of a complete synthesis of proteins encoded by these genes when pigs are infected with ASFV/ARRIAH/CV1/50 strain.

Conclusion

The most significant modification in the genomes of ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 was a large-size (2947 bp) deletion in the right variable region of adapted viruses, between 181,980–184,929 bp. A similar deletion was described in the genome of another cell culture adapted strain, BA71V, where the same seven predicted proteins were affected (Table 2) [2]. The importance of each of the described mutations on cell adaptation, virus growth and virulence requires additional investigations. 

References

1. Krug P.W., Holinka L.G., O’Donnell V., Reese B., Sanford B., Fernandez-Sainz I., et al. The progressive adaptation of a Georgian isolate of African swine fever virus to Vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome. J. Virol. 2015; 89(4): 2324–32. https:// doi.org/10.1128/jvi.03250-14

2. Rodríguez J.M., Moreno L.T., Alejo A., Lacasta A., Rodríguez F., Salas M.L. Genome sequence of african swine fever virus BA71, the virulent parental strain of the nonpathogenic and tissue-culture adapted BA71V. PLoS One. 2015; 10(11): e0142889. https://doi. org/10.1371/journal.pone.0142889

3. Mazloum A., Zinyakov N.G., Pershin A.S., Shevchenko I.V., Zhukov I.Yu., Fedoseeva D.N., et al. Analysis of changes in African swine fever virus genetic structure and biological properties during adaptation to continuous cell culture [Analiz izmeneniy geneticheskoy struktury i biologicheskikh svoystv virusa afrikanskoy chumy sviney pri adaptatsii k perevivaemoy kul’ture kletok]. Veterinariya segodnya. 2018; (4): 21–5. https://doi.org/10.29326/2304- 196X-2018-4-27-21-25 (in Russian)

4. Mazloum A., Sharipova D.V., Gavrilova V.L. Guidelines for the isolation and titration of African swine fever virus in porcine spleen cell culture [Metodicheskiye rekomendatsii po vydeleniyu i titrovaniyu virusa afrikanskoy chumy sviney v kul’ture kletok selezenki sviney]. Vladimir; 2019. (in Russian)

5. Szpara M.L., Tafuri Y.R., Enquist L.W. Preparation of viral DNA from nucleocapsids. J. Vis. Exp. 2011; (54): 3151. https://doi. org/10.3791/3151

6. Elsukova A.A., Shevchenko I.V., Varentsova A.A., Puzankova O.S., Zhukov I.Y., Pershina A.S., et al. Biological properties of African swine fever virus Odintsovo 02/14 isolate and its genome analysis. Int. J. Env. Agricult. Res. 2017; 3(10): 26–37. https://doi. org/10.25125/agriculture-journal-IJOEAR-OCT-2017-15

7. Tabares E., Olivares I., Santurde G., Garcia M.J., Martin E., Carnero M.E. African swine fever virus DNA: deletions and additions during adaptation to growth in monkey kidney cells. Arch. Virol. 1987; 97(3): 333–46. https://doi.org/10.1007/bf01314431


About the Authors

A. Mazloum
FSBI «Federal Centre for Animal Health» («ARRIAH»)
Russian Federation

Mazloum Ali, Ph.D. (Biol.), Leading Veterinarian.

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia



A. S. Igolkin
FSBI «Federal Centre for Animal Health» («ARRIAH»)
Russian Federation

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia



N. G. Zinyakov
FSBI «Federal Centre for Animal Health» («ARRIAH»)
Russian Federation

600901, Vladimir region, Vladimir, Yuryevets microdistrict, Russia



A. van Schalkwyk
Onderstepoort Veterinary Institute
South Africa

100 Old Soutpan road, Onderstepoort 0110



N. N. Vlasova
Onderstepoort Veterinary Institute
Russian Federation

100 Old Soutpan road, Onderstepoort 0110



For citation:


Mazloum A., Igolkin A.S., Zinyakov N.G., van Schalkwyk A., Vlasova N.N. Changes in the genome of African swine fever virus (Asfarviridae: Asfivirus: African swine fever virus) associated with adaptation to reproduction in continuous cell culture. Problems of Virology. 2021;66(3):211-216. https://doi.org/10.36233/0507-4088-50

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ISSN 0507-4088 (Print)
ISSN 2411-2097 (Online)