Analysis of Changes in African Swine Fever Virus Genetic Structure and Biological Properties During Adaptation to Continuous Cell Culture

Abstract

Functions of many African swine fever virus genes and multigene family members have not been yet understood. In particular, no virus genes directly associated with pig virulence have been identifed. Identifcation of such genes will enable preparation of deletion mutant ASF virus strains as well as development and testing of pilot safe vaccines based on the said virus strains. Comparative analysis of the virus biological characteristics and detection of differences in its genome structure affecting certain phenotypic features is a main method used for the virus basic pathogenicity and immunogenicity examination. The most interesting and effective approach to addressing this problem is an analysis of changes in the gene structure during ASF virus adaptation to replication in continuous cell culture. The said factors have made continuous cell culture-adapted variant ASF virus preparation necessary. Variant viruses with modifed biological features were prepared during adaptation of ASFV Odintsovo 02/14 isolate to replication in CV-1 cell culture. Lethality level was 16.7% when pigs were infected with adapted variant virus at 30th passage and survived animals became resistant to reinfection with homologous virulent ASFV Arm07 isolate. It should be noted that the virus passage in non-permissive cell culture up to 30 serial passages did not result in changes in its genotype; however, a large 3,000 bp deletion similar to that one in continuous Vero-cell culture-adapted BA71V strain genome appeared in right terminal variable region of the genome.

Identification and Isolation of Two Different Subpopulations Within African Swine Fever Virus Arm/07 Stock

“…For instance, sequencing of the whole genome of Ba71-adapted in Vero cells revealed a multitude of changes compared to its parental strain [ 6 , 57 ]. Other ASFV strains have also undergone genetic mutation when grown in certain cell lines, such as Vero or CV1 [ 58 , 59 ]. Importantly, we have verified that isolation of Arm/07/CBM/c2 by three passages in COS-1 did not generate variations in its sequence.…”

Rapid CRISPR/Cas9 Editing of Genotype IX African Swine Fever Virus Circulating in Eastern and Central Africa

“…We chose the WSL cell because it is suitable for productive replication of ASFV (Portugal et al, 2012;Keil et al, 2014;Hübner et al, 2018), and it does not interfere with genome integrity of the investigated ASFV strain (unpublished result), whereas after adaptation of ASFV to Vero cells, large deletions resulting in loss of virulence and protective capabilities were frequently observed (Tabarés et al, 1987;Krug et al, 2015;Rodríguez et al, 2015). Similar deletions and re-arrangements in ASFV genome have also been observed for viruses passaged in PIPEC (Borca et al, 2021) and CV-1 (Mazloum et al, 2019) cells. Prior to transfection, we determined that 1 µg/ml of puromycin dihydrochloride was the suitable concentration for selecting resistant clones using dose-response evaluation.…”

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

“…Поскольку ранее установлено, что адаптация возбудителя АЧС к росту в перевиваемой культуре клеток приводит к значительным изменениям в генетическом материале и снижению вирулентности [3,7], наиболее информативным подходом в изучении механизмов патогенности вируса АЧС следует признать анализ происходящих при его адаптации изменений в геноме. Указанные предпосылки обусловливают необходимость получения варианта вируса, адаптированного к росту в перевиваемой КК.…”

“…Определены биологические свойства и полноразмерная последовательность геномов этих штаммов, полученных на уровне 30 и 50 пассажей и адаптированных к клеточной культуре (ASFV/ARRIAH/CV-1/30 и ASFV/ARRIAH/CV-1/50 соответственно). Иссле-дования выявили существенное снижение вирулентности изолята ASFV/ARRIAH/CV-1/30 (~ <20 %), однако его геном в постадаптационный период не был удовлетворительно охарактеризован [3].…”

“…Поскольку в ходе адаптации к росту в перевиваемых КК вирулентность возбудителя снизилась [3], целью данной работы являлся сравнительный анализ полногеномного сиквенса 2 штаммов вируса АЧС, полученных на уровне 30 и 50 пассажей в культуре клеток CV-1, с генетическим материалом исходного изолята.…”