The problem of the universality of biological models for in vitro replication of virulent and attenuated strains of African swine fever virus (Asfarviridae: Asfivirus), classical swine fever virus (Flaviviridae: Pestivirus), and porcine reproductive and respiratory syndrome virus (Arteriviridae: Betaarterivirus)

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Introduction

Russia is currently among the top five largest pork producers and among the top ten leaders in pork exports. According to Rosstat, in 2025, Russian farms of all ownership types held approximately 28.4 million pigs. At the same time, smallholder farms and private subsistence farms in the country have seen a noticeable decline in the number of domestic pigs in recent years. Thus, as of early 2025, pig farms in Compartments III–IV (farms with a high level of biosecurity) accounted for 94.3% of production, private subsidiary plots for 5.3%, and peasant farms for only 0.3% [1]. The trend toward declining pork production in small-scale private farms and collective farms is linked to direct losses of livestock and reduced animal productivity during outbreaks of infectious diseases, as well as to the high costs of specific prevention and eradication measures.

Among the many pathogens, the greatest damage to pig farming is caused by the African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) viruses. According to the recommendations of the World Organization for Animal Health (OIE), laboratory diagnosis for the above-mentioned diseases should be aimed at isolating the virus in susceptible cell cultures¹,²,³. Despite the labor-intensive nature of the method and the time required to carry it out, virus isolation in cell cultures remains a crucial step in obtaining relevant diagnostic, production, and control strains. Sequential passaging allows for the accumulation of the virus to assess its cultural properties (cytopathic activity, replication kinetics, cellular tropism, and infectivity in various biological systems), and also provides a high-quality substrate for the production of vaccine raw materials, subsequent viral attenuation, and high-throughput genome sequencing. However, there are currently a number of challenges associated with the cultivation of ASF, CSF, and PRRS viruses.

ASFV is caused by an enveloped virus with double-stranded DNA, a member of the genus Asfivirus in the family Asfarviridae [2]. Due to the strict specificity of the virus for replication in specialized cells of the family Suidae, primary cell cultures—in which mononuclear cells predominate—are most commonly used for virus isolation: porcine alveolar macrophages (PAM), porcine bone marrow cells (pBMC), porcine spleen (PS), and porcine leukocytes (PL) [3–6]. However, there have been numerous research efforts to use cell cultures for working with field isolates of the ASF virus [7]. Successful replication of the ASF virus has been observed in monkey cell lines (Vero, CV-1, COS-1, MA-104); however, no infectious activity was detected in hamster (VNK-21, CHO), mouse (A9, 3T6, NP3, L929), or dog (MDCK) cell cultures [8]. Adapting the ASF virus to passaged cell lines is a rather complex and time-consuming process. Nevertheless, identifying permissive cell lines for its replication will allow us to model (accelerate) the mutation process during virus passage in non-natural host cells and, as a result, obtain detailed information about the mechanism of its attenuation.

The etiological agent of CSFV is an enveloped, positive-sense RNA-containing Pestivirus suis of the genus Pestivirus in the family Flaviviridae [9]. Due to the absence of cytopathic effects (CPE) in cell culture, naturally susceptible animals were long used for the accumulation and isolation of the CSF virus [10, 11]. However, following the introduction of methods for identifying non-cytopathic viruses into laboratory practice (immunofluorescence assay (IFA) and reverse transcription polymerase chain reaction (RT-PCR)), in vitro isolation became possible. The CSF virus is capable of replicating both in primary cultures (swine testis (ST)) and in passaged cell cultures (piglet kidney (PK-15)) [12, 13]. In studies by O.L. Kolbasova, 12 sublines of RK-15 passaged cells were evaluated, and a high level of spontaneous karyotypic variability was established. Heterogeneity was also evident in terms of permissiveness to the CSF virus, as indicated by significant variability in accumulation titers—ranging from 2.5 to 7.5 log₁₀ TCID₅₀/cm³ [14]. This points to significant genetic heterogeneity in this cell culture.

In Russia, lapinized strains of subgenotypes 1.1 and 1.2 are traditionally used for the production of live vaccines against CSFV. The strains are adapted for replication in primary and subcultures of lamb testis cells (LT-1) [15]. The use of the LT-1 subculture entails certain challenges: The Hayflick limit, which requires constant and labor-intensive preparation of new primary cultures and limits production volume; and difficulties in standardizing cultivation processes.

Since the establishment of primary cell cultures is labor-intensive, and testing the sensitivity of PK-15 sublines requires ongoing virological studies, investigating the susceptibility of cell subcultures and other passaged lines to the CSF virus may contribute to the improvement of diagnostic procedures and methods for producing vaccine raw materials.

PRRS is caused by a single-stranded positive-sense RNA virus belonging to the family Arteriviridae, genus Betaarterivirus. The virus is divided into European (subgenus Eurpobartevirus, species Betaarterivirus suid 1 (Lelystad reference strain)) and American species (subgenus Ampobartevirus, species Betaarterivirus suid 2 (VR-2332reference strain)) [16, 17]. The two distinct species of the PRRS virus are most commonly referred to in the literature as types or genotypes [18]. Primary PAM cell culture is recognized as the standard method for isolating PRRS viruses, while the passaged green monkey cell line (Marc-145) is used for adaptation. This is due to the presence of the transmembrane receptor CD163, which is responsible for viral adsorption onto target cells [19]. Information on the permissiveness of certain primary cell cultures (ST, PS, PL) that are potentially capable of supporting PRRS replication has not been published in the open literature. In turn, heterogeneous cultures of ST, PL, and PS contain a large number of mononuclear cells in which viral replication is possible.

Thus, the aim of the study was to identify a universal biological model for the in vitro study of ASF, CSF, and PRRS viruses, based on the results of an analysis of the degree of permissiveness of various cell cultures (primary cultures, subcultures, and passaged cell lines) and the use of real-time PCR (qPCR) to detect viral replication.

Materials and methods

Viruses. The following strains were used in the study: the virulent reference strain of genotype II, Arm 07, and the ASF/CV60/2020 strain of the African swine fever virus, which was attenuated in green monkey cell culture (CV-1); a virulent reference strain of the Ximing subgenotype 1.1 and a vaccine strain SK of the subgenotype 1.2 of the CSF virus; the Lelystad strain of subgenotype 1.1, adapted to Marc-145 cell culture, and the virulent BR-01 strain of subgenotype 1.1 of the European-type PRRS virus; the virulent Irkutsk 2007/1 strain and the laboratory variant Irkutsk 2007/1/Marc-145/35 of the American-type PRRS virus, adapted to Marc-145 cell culture [20–23].

Working seed material for all strains was obtained from susceptible primary cell cultures (ASF virus–in PS cell culture; CSF virus–in ST cell culture; PRRS viruses–in PAM cell culture).

Cell cultures. Primary cell cultures were used: pBMC, PS, ST, PL, and porcine kidney (PK). The organs were obtained at the laboratory and animal facility complex of the Federal State Budgetary Institution ARRIAH from clinically healthy donor piglets (4 weeks old, weighing up to 8 kg) purchased from farms free of African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS). The pBMC culture was obtained by washing bone marrow cells from large tubular bones, while PS, ST, and PK were obtained by trypsinization; the PL cell culture was obtained by centrifuging blood with the addition of an anticoagulant (sodium citrate) in a ficoll density gradient [24, 25]. The cell lines used in this study were obtained from the cell culture collection of the Federal State Budgetary Institution ARRIAH: pig kidney (IB-RS-2), SPEV hybrid (pig embryonic kidney, line B) and pig splenocytes (A₄C₂), Siberian mountain goat kidney (PSGK-30), green monkey kidney (MA-104 and Marc-145), African green monkey kidney (BGM and COS-1), pig kidney (RK-15 (ATSS), RK-15 (Hungary), RK-15 (ARRIAH), and a re-inoculated renal culture (RRC). Subcultures of the synovial membrane from a kid at passages 5, 8, 10, 19, 21, and 23 (MSC/ARRIAH) and of the synovial membrane from a lamb at passages 3, 8, 10, 23, 25, and 27 (MSO/ARRIAH) were obtained by culturing tissue explants from a kid/lamb followed by in vitro passage.

Cell culture contamination. Experiments for each strain and cell line were conducted in three replicate tests (n = 3). Virus-containing material was applied to a confluent or subconfluent (in the case of pBMC, PS, ST, and PAM) monolayer of the test cultures–after the conditioned medium had been removed–at the following doses: for ASF virus–MOI 0.5 HAdU₅₀/cell (hemadsorbing units); for CSF virus–0.5 TCID₅₀/cell (cell culture infectious doses); for PRRS virus–0.01 TCID₅₀/cell (tissue cytopathic doses). The cells were incubated for 1 hour at an average temperature of 37.0 ± 0.5 °C, after which DMEM medium containing 10% bovine fetal serum and a 1X mixture of antibiotics and antifungals was added. The ASF virus was allowed to accumulate for 144 hours, while the CSF and PRRS viruses were allowed to accumulate for 96 hours at a temperature of 37.0 ± 0.5 °C. Upon completion of cultivation, the virus-containing material was frozen at −50 ± 5 °C, then thawed at room temperature and used for the next passage on test cultures.

qPCR. Viral genomes were extracted using the affinity sorption method in accordance with the instructions for the Ribo-Sorb kit (Central Research Institute of Epidemiology, Moscow, Russia). For qPCR, the ASF, CSF, and PRRS test systems manufactured by AmpliSens (Central Research Institute of Epidemiology, Moscow, Russia) were used.

Interpretation of results. The reaction was assessed visually using light microscopy. The first assessment was conducted 24 hours after inoculation, and subsequently on a daily basis throughout the entire cultivation period. For the ASF virus, the presence or absence of the hemadsorption phenomenon was assessed; for the PRRS viruses, the presence or absence of CPE was assessed. In the case of CSF virus cultivation, the possible effect of idiopathic cytotoxicity was taken into account. The virus-containing suspension was then tested by qPCR for the presence of the genomes of the corresponding viruses.

The dynamics of viral replication in the cell culture under study were assessed semi-quantitatively (based on changes in the cycle threshold (Ct) value) according to four possible scenarios:

– high permissiveness – a decrease in the Ct value is observed with each passage, or the value remains stable (plateau effect, statistically insignificant) throughout all 5 passages;

– satisfactory permissiveness – an increase in the Ct value is possible during the first 3 passages, but a decrease in the Ct value is observed starting from the 3rd–4th passage, followed by stability;

– low permissiveness – an increase in the Ct value is possible during the first 3 passages, but stability is observed starting at least from the 3rd passage;

– lack of permissiveness – an increase in Ct values is observed, up to the point where no Ct value is detected.

Statistical processing of experimental results. The nonparametric Mann–Whitney U test was used to compare two independent samples based on the level of a quantitative variable. This method was chosen due to the small sample size (n = 3) and the fact that the distribution of the analyzed variables in the study groups may deviate from a normal distribution. The statistical significance of the differences was assessed by comparing the calculated empirical value of the U-statistic with the critical value from the Mann–Whitney tables for the given sample sizes and a significance level of p = 0.05. Differences were considered statistically insignificant if p > 0.05.

Ethical statement. The authors confirm adherence to institutional and national standards regarding the use of laboratory animals, in accordance with the Consensus author guidelines for animal use (IAVES, July 23, 2010). The study protocol was approved by the Ethics Committee of the Federal State Budgetary Institution “ARRIAH” (Protocol No. n/a dated February 7, 2025).

Results

Assessment of Cell Culture Susceptibility to the ASF Virus

To study susceptibility to the virulent strain Arm 07 (Fig. 1) and the attenuated strain ASF/CV60/2020 (Fig. 2), we used primary porcine cell cultures: pBMC, PS, ST, PL, PAM, and PK; re-inoculated cell lines: IB-RS-2, PSGK-30, A₄C₂, BGM, Marc-145, PK-15 (ARRIAH subline), MA-104, COS-1; and subcultures: MSC/ARRIAH and MSO/ARRIAH.

 

Fig. 1. Permissiveness of various cell cultures to the virulent reference strain Arm 07 of the ASFV genotype II (n = 3). Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 1. Пермиссивность различных культур клеток к вирулентному референс-штамму Arm 07 вируса АЧС II генотипа (n = 3). Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

Fig. 2. Permissiveness of various cell cultures to the attenuated ASF/CV60/2020 strain of the ASFV II genotype (n = 3). Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 2. Пермиссивность различных культур клеток к аттенуированному штамму ASF/CV60/2020 вируса АЧС II генотипа (n = 3). Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

Primary cell cultures showed high susceptibility to strain Arm 07: pBMC – 6.6 ± 2.6 Ct, PAM – 7.3 ± 1.8 Ct, PS – 8.8 ± 2.4 Ct, PL – 9.5 ± 2.1 Ct, ST – 10.7 ± 3.5 Ct, with hemadsorption observed 24 hours after inoculation throughout all 5 passages. The primary PK cell culture showed satisfactory susceptibility: Ct was 15.28 ± 1.7, observed during passages 1–3. For most of the subcultured cell lines–Marc-145, MA-104, COS, BGM, PSGK-30, A₄C₂, IB-RS-2, and the MSC/ARRIAH and MSO/ARRIAH subcultures–a lack of permissiveness was demonstrated.

The following samples showed high susceptibility to the ASF/CV60/2020 strain, as assessed by qPCR: primary PS cell cultures–7.3 ± 1.25 Ct, pBMC – 7.5 ± 2.9 Ct, PAM – 7.8 ± 2.7 Ct, PL – 8.8 ± 2.8 Ct, PT – 11.7 ± 4.7 Ct, PK – 12.6 ± 1.2 Ct. Passaged cell lines from the African green monkey, to which the virus had previously been adapted, showed satisfactory permissiveness: Marc-145 – 16.06 ± 2.78 Ct, MA-104 – 15.5 ± 3.29 Ct. With high and satisfactory permissivity, the phenomenon of hemadsorption was observed throughout all passages. Other cell cultures used demonstrated a lack of permissiveness to the ASF/CV60/2020 strain; the hemadsorption effect was observed in MSC/ARRIAH and MSO/ARRIAH subcultures exclusively during the first two passages.

Assessment of Cell Culture Susceptibility t o the CSF Virus

The permissiveness of primary (ST, PS, PK) and passaged (RRC, A₄C₂, IB-RS-2, PSGK-30) cell cultures, as well as the PK-15 sublines (ATCC, Hungary, ARRIAH) and subcultures (MSC/ARRIAH and MSO/ARRIAH) to the virulent Shi-min strain (Fig. 3) and the PS vaccine strain (Fig. 4) over the course of 5 consecutive passages.

 

Fig. 3. Permissiveness to the virulent Shi-min strain of CSFV over five consecutive passages. Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 3. Пермиссивность к штамму Ши-мынь вируса КЧС на протяжении 5 последовательных пассажей. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

Fig. 4. Permissiveness to the CK strain of the CSFV over five consecutive passages. Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 4. Пермиссивность к штамму СК вируса КЧС на протяжении 5 последовательных пассажей. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

The cell lines most sensitive to the Shi-min strain were ST – 9.9 ± 1.1 Ct, PAM–10.47 ± 3.7 Ct, PS–11.7 ± 2.1 Ct, PL—12.31 ± 1.8 Ct, PK – 16.03 ± 3.04 Ct, RRC – 16.3 ± 1.79 Ct, and PK-15 – 17.8 ± 6.71 Ct (ATCC subline). At the same time, the IB-RS-2 cell culture (21.5 ± 4.13 Ct) exhibited low permissiveness, while the PK-15 cell cultures (Hungarian and ARRIAH sublines), PSGK-30, and A₄C₂ did not exhibit permissiveness to the CSF virus.

High permissiveness was observed in the MSO/ARRIAH subcultures (3, 8, 10p)–14.5 ± 2.1 Ct, MSO/ARRIAH (23, 25, 27p) – 14.9 ± 4.5 Ct, and MSC/ARRIAH (5, 9, 10p) – 12.5 ± 2.2 Ct, MSC/ARRIAH (19, 21, 23p)–13.4 ± 3.1 Ct–for the PK vaccine strain, which had been preliminarily adapted for replication in a lamb testis cell subculture (LT-1). Primary cell cultures of PAM – 11.85 ± 1.7 Ct, ST – 13.2 ± 3.1 Ct, PL – 13.75 ± 2.37 Ct, and PS – 15.63 ± 1.37 Ct showed high permissiveness. Among the re-cultured cell lines, the RRC cell culture was found to be sensitive—16.86 ± 1.87 Ct. Satisfactory permissiveness was demonstrated by the cell lines IB-RS-2–21.6 ± 2.8 Ct, PK-15 (ATCC) – 21.8 ± 2.2 Ct, PK – 22 ± 3.51 Ct. The cell lines PSGK-30, RK-15 (Hungary), PK-15 (ARRIAH), and A₄C₂ showed no permissiveness.

Assessment of Cell Culture Susceptibility to PRRS Viruses

To study susceptibility to the virulent strain Irkutsk 2007/1 of the American-type PRRS virus (Fig. 5), primary (PAM, PL, ST, PS), passaged (Marc-145, MA-104), as well as MSC/ARRIAH and MSO/ARRIAH subcultures, were tested to study permissivity toward the virulent Irkutsk 2007/1 strain of the American-type PRRS virus (Fig. 5). High permissiveness was observed in PAM (13.2 ± 1.97 Ct) and PL (14.25 ± 1.7 Ct). The other cell lines tested showed no susceptibility to this strain of the American-type PRRS virus.

 

Fig. 5. Susceptibility of cell cultures to the virulent strain Irkutsk 2007/1 of the American type PRRSV. Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 5. Результаты чувствительности культур клеток к вирулентному штамму Иркутский 2007/1 вируса РРСС американского типа. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

High permissiveness toward the Irkutsk 2007/1/Marc-145/35 variant of the American-type PRRS virus (Fig. 6) was observed in PAM–12.5 ± 2.94 Ct, PL – 13.11 ± 2.1 Ct, and MA-104 – 11.2 ± 2.5 Ct. The remaining cell lines, including Marc-145, showed no permissiveness, reducing viral replication over the course of 5 passages.

 

Fig. 6. Susceptibility of cell cultures to the laboratory variant Irkutsk 2007/1/Marc-145/35 of the American type PRRSV. Statistically insignificant results between the next and the previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive cultures).

Рис. 6. Результаты чувствительности к лабораторному варианту Иркутский 2007/1/Marc-145/35 вируса РРСС американского типа. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

To study susceptibility to the virulent strain BR-01 of the European-type PRRS virus (Fig. 7), primary cultures of PAM, PL, PS, and ST, as well as passaged cell lines (Marc-145, MA-104) and subcultures MSC/ARRIAH and MSO/ARRIAH, were tested. High permissiveness was observed in the PAM cell culture (11.2 ± 2.5 Ct) and the PL cell culture (12.81 ± 3.1 Ct). No permissiveness was observed in the primary ST and PS cell cultures or in the MSC/ARRIAH and MSO/ARRIAH subcultures.

 

Fig. 7. Susceptibility of cell cultures to the virulent strain BR–01 of the European type PRRSV. Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 7. Результаты чувствительности культур клеток к вирулентному штамму БР-01 вируса РРСС европейского типа. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

The Lelystad strain of the European-type PRRS virus, adapted to Marc-145 cell culture (Fig. 8), showed high permissivity in Marc-145 (10.4 ± 1.9 Ct), PAM–11.2 ± 2.4 Ct, and PL–11.64 ± 2.3 Ct; however, MSC/ARRIAH, MSO/ARRIAH, ST, and PS showed no permissiveness. When this virus was passaged in MA-104 cell culture, reproductive stability was observed during the first 3 passages, followed by a decrease in infectious activity during the 4th–5th passages.

 

Fig. 8. Permissiveness of the adapted Lelystad strain of the European type PRRSV to Marc-145 cell culture. Statistically insignificant results between the next and previous passages (p > 0.05) are marked on the graph with a star (in the case of permissive culture).

Рис. 8. Пермиссивность адаптированного штамма Lelystad вируса РРСС европейского типа к культуре клеток Marc-145. Статистически незначимые результаты между последующим и предыдущим пассажами (p > 0,05) отмечены на графике звездочкой (в случае пермиссивных культур).

 

Discussion

This study demonstrated that the selection of a cell culture is a critically important step for the isolation and adaptation of the ASF virus to that culture, given the virus’s evolutionarily established tropism for porcine monocyte cells. Primary cell cultures with a predominant population of macrophages (pBMC, PAM, and PS) are suitable for these purposes. The study results also confirmed high permissiveness of PL; however, it should be noted that after isolation from defibrinated blood, monocytes account for approximately 5–10% of the total cell population [26]. Numerous studies confirm that macrophages are the primary target cells for ASFV replication and exhibit high permissiveness in vitro (virus titers can reach 6–7 log₁₀ HAdU₅₀/cm³) [27, 28].

A clone of the А₄С₂/9к cell line infected with the ASF virus exhibited peak infectivity on days 6–7, with a viral titer of 6.5 log₁₀ HAdU₅₀/cm³ [29]. Other researchers have reported viral replication in COS-1 cell culture with a pronounced cytopathic effect [30]. The present study did not confirm the permissiveness of A₄C₂ and COS-1 to the ASF virus. This may be due to the heterogeneity of these cell lines, which necessitates the creation of clonal, highly permissive sublines [31].

In studies by A. Rai et al. (2022), the MA-104 cell line proved to be a promising substrate for isolating the ASF virus from clinical samples, demonstrating sensitivity comparable to that of porcine macrophages [32]. In contrast, the Marc-145 line exhibited low permissiveness–ASFV replication was possible only upon inoculation with high doses [33]. The present study showed that, through successive passages, the field ASF virus does not adapt to just any MA-104 line. This requires alternating passages between two cultures: a highly permissive one (PS, pBMC, or PAM) and a target one (MA-104 or any other monkey kidney line). With this approach, it is possible to obtain attenuated strains capable of replicating in MA-104 and Marc-145 (for example, strain ASF/CV60/2020, as shown in this study). However, for the isolation of ASF virus from field samples, monkey kidney cell cultures are not a universal model.

Working with primary macrophages presents various challenges, which has spurred the search for immortalized cell lines. The IPKM line (immortalized porcine kidney macrophages) showed high susceptibility to ASF virus (strain Arm 07 induced CPD 48 hours after infection and reached titers of up to 7.5 log₁₀ TCID₅₀/cm³) [34]. The immortalized line of porcine alveolar macrophages 3D4/21 supported ASFV replication; however, abortive infection was partially observed, characterized by a defect in late protein synthesis and the absence of infectious particle formation [33, 35]. In Russia, immortalized lines of porcine mononuclear cells are not used. However, for the purpose of efficiently isolating the ASFV pathogen, it is necessary to establish cell lines that, as a result of passage, do not alter the viral genome and, in this respect, do not differ from the primary cell culture.

Historically, most field isolates of the CSF virus have not been cytopathic. For a long time, this hindered the process of isolating the virus in cell cultures. Currently, with the availability of methods for identifying and replicating the virus (RPIF, PCR), the RK-15 cell line is recognized as the most studied and widely used cell culture for CSF diagnosis; however, according to the results of this study and those obtained by O.L. Kolbasova et al. (2000), the heterogeneity of this cell line with respect to permissiveness to the CSF virus has been confirmed. The optimal solution is to create clonal sublines of RK-15 that are highly permissive to the CSF virus.

In this study, the MSC/ARRIAH and MSO/ARRIAH subcultures–which had not previously been used for the propagation of swine viruses–demonstrated high permissiveness for the vaccine strain SK (subgenotype 1.2) of the CSF virus and may also be considered as alternative culture systems for the production of vaccine raw materials.

It has previously been established that PAM is a highly permissive cell culture for the propagation of the PRRS virus, but the process of obtaining it is labor-intensive. This study did not confirm the hypothesis that all primary cultures with a predominance of porcine mononuclear cells are susceptible to the PRRS virus. Thus, PS and ST did not demonstrate permissiveness to the PRRS virus, whereas PAM and PL showed a high degree of susceptibility to both the European and American strains of the virus.

The reference strain ATCC VR-2332 of the American-type PRRS virus was successfully isolated from diseased piglets in the CL2621 cell line (subclone MA-104) [36]. Subsequent studies confirmed that VR–2332 can be propagated in MA-104 cells to a titer of up to 7.0 log₁₀ TCID₅₀/cm³ [37]. Further cloning of the MA-104 line led to the creation of one of the world’s most widely used lines for PRRS cultivation–Marc-145–in which the virus titer reached 8.5 log₁₀ TCID₅₀/cm³, which is significantly higher than in the parent MA-104 line [38]. According to our study, the Marc-145 cell line showed no susceptibility to American-type PPV (the virulent strain Irkutsk 2007/1 also failed to replicate in the MA-104 cell line). The observed phenomenon of differential susceptibility of the MA-104 and Marc-145 cell lines to different variants of the PRRS is a direct consequence of their long-term divergent evolution in vitro and the directed adaptation of the viral strains. Thus, in this study, the adapted Lelystad strain demonstrated sensitivity only to the Marc-145 cell culture. The parental MA-104 line and its derivative clone Marc-145, which have been cultured independently for decades, have undergone significant genomic and epigenomic drift, leading to changes in the expression profiles of key cellular factors (receptors such as CD163 and components involved in the viral replication cycle) [39]. Thus, the difference in permissiveness between MA-104 and Marc-145 serves as an experimental marker of their phenotypic divergence, and the selectivity of laboratory-adapted strains confirms the role of directed viral evolution in narrowing cellular tropism.

Despite the success achieved with Marc-145, there remained a need for porcine cell lines that would better model natural infection with the PRRS virus. Early attempts to accumulate the PRRS virus in immortalized porcine mononuclear cells (AMC), such as 3D4/2, 3D4/21, and 3D4/31 were unsuccessful [35]. The problem of cell line resistance turned out to be the absence of the CD163 receptor [40]. Only the creation of transgenic lines expressing CD163 helped resolve the issue. Thus, an immortalized line of porcine alveolar macrophages (mPAM) that stably expresses CD163 demonstrated the ability to support replication of the PRRS virus, with a titer of 5.5 log₁₀ TCID₅₀/cm³ [40]. In numerous studies, the insertion of the CD163 gene into the genomes of cells that were initially non-susceptible to the PRRS virus (RK-15, NEC-293T, VNK-21) led to high levels of transgene expression and, as a result, the acquisition of permissiveness to this virus by the transformed cell lines [41].

Conclusion

This study provided a comparative assessment of the susceptibility of a wide range of cell cultures (primary cultures, subcultures, and passaged lines) to ASF, CSF, and PRRS viruses using virulent and attenuated strains.

According to the study results, primary cultures—in which porcine mononuclear cells (PMCs) predominate—remain promising for the isolation of field isolates of the ASF virus (6.6 ± 2.6 Ct), PAM (7.3 ± 1.8 Ct), SS (8.8 ± 2.4 Ct), LS (9.5 ± 2.1 Ct), TS (10.7 ± 3.5 Ct), which demonstrated high permissiveness and the phenomenon of hemadsorption both during the replication of the virulent strain Arm 07 and the attenuated ASFV/CV60/2020—SS (7.3 ± 1.25 Ct), pBMC (7.5 ± 2.9 Ct), PAM (7.8 ± 2.7 Ct), PL (8.8 ± 2.8 Ct), ST (11.7 ± 4.7 Ct), and PK (12.6 ± 1.2 Ct). Most of the tested standard re-inoculation lines (MA-104, Marc-145, COS-1) do not support the reproduction of strains that have not been previously adapted to them.

For the CSF virus, heterogeneity in the widely used PK-15 cell line has been confirmed based on permissivity, which requires the use of clonal sublines or alternative cultures. Primary cultures of PAM (7.8 ± 2.7 Ct), PL (8.8 ± 2.8 Ct), ST (9.9 ± 1.1 Ct), PS (11.7 ± 2.1 Ct), PK (16.03 ± 3.04 Ct), as well as the RRC line (16.3 ± 1.79 Ct) and PK-15 (17.8 ± 6.71 Ct) (ATCC subline) showed high sensitivity to the virulent Shi-min strain. Based on these findings, RRC can be recommended for the isolation of field isolates of the CSF virus, provided that the method is validated during its adaptation to laboratory conditions. For the SK vaccine strain of the CSF virus, primary PAM cell cultures (11.85 ± 1.7 Ct), ST (13.2 ± 3.1 Ct), PL (13.75 ± 2.37 Ct), and PS (15.63 ± 1.37 Ct), as well as MSO/ARRIAH subcultures (3, 8, 10p) – 14.5 ± 2.1 Ct, MSO/ARRIAH (23, 25, 27p) – 14.9 ± 4.5 Ct, and MSC/ARRIAH (5, 9, 10p) – 12.5 ± 2.2 Ct, MSC/ARRIAH (19, 21, 23p) – 13.4 ± 3.1 Ct, which, given their higher processability, opens up the possibility of using them as alternative biological systems for the production of vaccine raw materials.

The primary cultures of PAM and PL are highly permissive biological models for isolating the PRRS virus from field samples; for the American-type virulent strain, the mean Ct values were 13.2 ± 1.97 and 14.25 ± 1.7 Ct, respectively, while for the European-type virulent strain, they were 11.2 ± 2.5 Ct and 12.81 ± 3.1 Ct, respectively. The MA-104 and Marc-145 lines may be genetically heterogeneous across different cell culture collections, and as a result, may differ in their permissiveness to the PRRS virus. Using PRRS virus strains adapted to Marc-145 as an example, significant differences in the degree of permissiveness between the parental line MA-104 and its derivative (daughter form) Marc-145 have been demonstrated. For the adapted American-type PRRS virus strain—MA-104 (11.2 ± 2.5 Ct)–and for the European-type PRRS virus strain–Marc-145 (10.4 ± 1.9 Ct).

The results obtained in the course of these studies are of scientific and practical significance for laboratory work in selecting in vitro biological models to optimize the replication processes of the viruses under study. In turn, this selection should be determined by specific objectives: isolation of a field isolate, production of vaccine raw materials, attenuation to create vaccine strains, or obtaining adapted variants of the virus for basic research. According to the findings of this study, universal biological models for the in vitro replication of both virulent and attenuated/vaccine strains of ASF, CSF, and PRRS viruses are certain primary cultures dominated by mononuclear cells, such as PAM and PL.

Global experience shows that the limitations associated with the low susceptibility of passaged cell lines to ASF, CSF, and PRRS viruses can be overcome by using immortalized porcine mononuclear cell cultures. Thus, the development and implementation of such cell lines in laboratory practice are extremely important tasks for further research.

 

¹ WOAH – World Organization for Animal Health. Terrestrial Manual. Chapter 3.8.1. African swine fever (Infection with African Swine Fever Virus). Available at: https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.08.01_ASF.pdf (access date: October 25, 2025).

² WOAH – World Organization for Animal Health. Terrestrial Manual. Chapter 3.9.3. Classical Swine Fever (Infection with Classical Swine Fever Virus). Available at: https://www.woah.org/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_cards/CLASSICAL_SWINE_FEVER.pdf (access date: November 15, 2025).

³ WOAH – World Organization for Animal Health. Terrestrial Manual. Chapter 3.9.6. Porcine Reproductive and Respiratory Syndrome (Infection with Porcine). Available at: https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.09.06_PRRS.pdf (access date: December 1, 2025).

About the authors

Danila S. Moiseenko

Federal Center for Animal Health Protection

Author for correspondence.
Email: moiseenko_ds@arriah.ru
ORCID iD: 0009-0003-4465-0179

a postgraduate student, a veterinarian at the reference laboratory for African swine fever

Russian Federation, 600901, Vladimir

Anastasia S. Sadchikova

Federal Center for Animal Health Protection

Email: sadchikova@arriah.ru
ORCID iD: 0009-0001-0801-2394

veterinarian at the reference laboratory for African swine fever

Russian Federation, 600901, Vladimir

Elena V. Chernyshova

Federal Center for Animal Health Protection

Email: chernishova@arriah.ru
ORCID iD: 0000-0003-4054-8532

Candidate of Veterinary Sciences, Head of the Reference Laboratory for Rabies and BSE of the Laboratory Diagnostic Center

Russian Federation, 600901, Vladimir

Alexey S. Igolkin

Federal Center for Animal Health Protection

Email: igolkin_as@arriah.ru
ORCID iD: 0000-0002-5438-8026

Candidate of Veterinary Sciences, Deputy Head of the Center – Head of the reference laboratory for African swine fever at the Laboratory Diagnostic Center

Russian Federation, 600901, Vladimir

Natalia I. Bakhtieva

Federal Center for Animal Health Protection

Email: bahtieva@arriah.ru
ORCID iD: 0009-0006-4370-7366

leading biologist at the Rabies Reference Laboratory and the BSE Laboratory Diagnostic Center

Russian Federation, 600901, Vladimir

Elena G. Kuznetsova

Federal Center for Animal Health Protection

Email: kuznetsova_eg@arriah.ru
ORCID iD: 0009-0008-6851-9323

Candidate of Biological Sciences, Leading Researcher in the Cell Culture Sector of the Cell Culture Department of the Scientific and Production Service (CCC OCC NPS)

Russian Federation, 600901, Vladimir

Nikolay A. Kolchanov

Federal Center for Animal Health Protection

Email: koichanov@arriah.ru
ORCID iD: 0000-0003-3651-8517

employee of the Cell culture Sector of the Cell Culture Department of the Scientific and Production Service (CCC OCC NPS)

Russian Federation, 600901, Vladimir

Elena A. Trofimova

Federal Center for Animal Health Protection

Email: trofimova_ea@arriah.ru
ORCID iD: 0009-0009-9235-0959

Head of the Cell Culture Sector of the Cell Culture Department of the Scientific and Production Service

Russian Federation, 600901, Vladimir

Irina V. Vologina

Federal Center for Animal Health Protection

Email: vologina@arriah.ru
ORCID iD: 0000-0001-7536-6275

Candidate of Veterinary Sciences, Head of the Cell Culture Department

Russian Federation, 600901, Vladimir

Vera L. Gavrilova

Federal Center for Animal Health Protection

Email: gavrilova_vl@arriah.ru
ORCID iD: 0000-0001-5843-2779

Candidate of Biological Sciences, Researcher, Reference Laboratory for African swine fever

Russian Federation, 600901, Vladimir

Olga S. Puzankova

Federal Center for Animal Health Protection

Email: puzankova@arriah.ru
ORCID iD: 0000-0003-1584-3169

Candidate of Veterinary Sciences, Senior Researcher, Reference Laboratory for African Swine Fever

Russian Federation, 600901, Vladimir

Roman S. Chernyshev

Federal Center for Animal Health Protection

Email: chernishev_rs@arriah.ru
ORCID iD: 0000-0003-3604-7161

Candidate of Biological Sciences, Junior Researcher at the Reference Laboratory for African Swine Fever

Russian Federation, 600901, Vladimir

References

Supplementary files