DETERMINATION OF THE 1L-5-6L MGF110 GENES INFLUENCE ON THE BIOLOGICAL PROPERTIES OF THE AFRICAN SWINE FEVER VIRUS (ASFARVIRIDAE; ASFIVIRUS) “VOLGOGRAD/14C” IN VIVO

African swine fever continues to pose a global agricultural problem due to the absence of vaccine prevention and the high cost of anti-epizootic measures. This study examines the functional role of the African swine fever virus (ASFV) 1L-5-6L multigene family 110 genes in vivo . Four clinically healthy Large White pigs were used in this study. Two groups of animals were inoculated with either the parental strain or the deletion variant, respectively. For subsequent challenge infection, the homologous virulent strain “Stavropol 01/08” was used. Blood samples were collected at specific time intervals. The ASFV infectious activity was determined by titration in porcine blood-derived macrophages. Virus-specific antibodies to the ASFV p30 protein were detected using an enzyme-linked immunosorbent assay (ELISA). The results of real-time polymerase chain reaction (PCR) showed a significant difference in Ct values between samples from the two groups of animals. The determination of ASFV infectious activity in blood samples demonstrated the presence of the virus in animals immunized with the parental strain. The virus was not detected in samples from animals immunized with the deletion strain. The ELISA method demonstrated the presence of p30 protein antibodies in serum samples from 10 to 14 days after immunization with the parental strain, while no antibodies were detected in serum samples from animals immunized with the deletion strain. The properties of the ASFV recombinant strain “Volgograd/D(1L-5-6L) MGF110” were studied in an in vivo experiment. It was found that the deletion strain does not reproduce in animals, unlike the parental strain.


INTRODUCTION
African swine fever (ASF) is a contagious viral disease of domestic pigs and wild boars characterized by hemorrhagic fever and mortality rates up to 100% [1].Currently, there are no specific methods for the prevention of this disease.The most promising approach is considered to be the creation of artificially attenuated ASF virus strains through the deletion of one or more genes [2,3].Despite many years of research by scientists worldwide, a safe and effective vaccine that fully protects the population from infection has not yet been developed.Therefore, further studies of the functions of individual ASF virus genes is necessary.The terminal regions of the virus genome, which mainly consist of multigene families containing several genes, are the most variable genome regions [4,5].However, the functions of individual genes within these multigene families have not been fully elucidated.
The aim of this study was to investigate the functional role of the MGF110 1L-5-6L genes in viral replication and their influence on the host immune response during African swine fever in an experiment on susceptible animals.
Pigs were inoculated intramuscularly with a dose of 10 3,0 HAU 50 см 3 on day 0. Control infection on day 28 after inoculation was carried out with the virulent strain of ASF virus "Stavropol 01/08" (genotype II).

Animals
The study used 4 clinically healthy Large White pigs aged 5 months and weighing 40-50 kg.The animals were randomly divided into 2 groups with 2 animals in each group and housed in separate rooms.The experimental plan with animals was approved by the Ethics Committee of the Federal Research Center for Virology and Microbiology, and permission was obtained to conduct the study (protocol 1 dated 10.02.2023).Clinical signs were assessed according to the method developed by King et al., 2011 [6].

Detection of the ASF virus genome by real-time PCR
Total DNA extraction from the tested samples was performed using the "DNA-sorb B" kit («Amplisense», Russia) according to the manufacturer's instructions.The presence of ASF virus genome fragments in the isolated total DNA samples was determined by real-time polymerase chain reaction (PCR) using the CFX 96 touch amplifier («BioRad», USA) according to the modified protocol recommended by OIE [7].

ELISA
Virus-specific antibodies to the ASF virus p30 protein were detected in blood serum samples using the "ASF-SEROTEST plus" enzyme immunoassay («Vetbiochem LLC», Moscow, Russia).

Determination of virus infectious activity
The ASF virus infectious activity was determined by titration in a 3-day culture of porcine blood derived macrophages.The results were evaluated based on the presence of specific hemadsorption within 7-10 days.Virus titers were calculated using the method described by B.A. Kerber modified by I.P. Ashmarin and expressed in 50% hemadsorbing units (HAU 50 см 3 ) [8].

RESULTS
After immunization on day 0, pig No. 2 from the group inoculated with the "Volgograd/14c" strain ("V") exhibited a short-term 2-day hyperthermia of 40.1 -40.3°C on days 6-7.No other clinical signs were observed in this animal later.The other animals remained clinically healthy throughout the 28-day period until the control infection.The clinical score for all pigs after inoculation and before the control infection did not exceed 2-3 points.The temperature data of the animals during the experiment are presented in Figure 1.
After the control infection with the "Stavropol 01/08" strain, animals in group "M" developed clinical signs characteristic of acute ASF on days 3-4.The disease was accompanied by hyperthermia of 40.6 -41.8°C, refusal of feed, apathy, cyanosis of the ear tips and underbelly, difficulty breathing and hind limb paralysis for 1-2 days before death.The disease duration was 3-5 days with the animals dying on days 7 and 8, respectively.The survival data of the animals during the experiment are presented in Figure 2. The maximum clinical scores after the control infection reached 17-19 points.Post-mortem examination of the deceased animals revealed characteristic pathological changes of acute ASF, including serosanguinous and hemorrhagic lymphadenitis of the submandibular, mediastinal, portal, gastric, mesenteric, and inguinal lymph nodes.Hemorrhages on the heart, congestive hyperemia and edema of the lungs, hemorrhages in the cortical and medullary layers of the kidneys, and splenic hemorrhage were also observed.
Pig No. 1 from group "V" exhibited a rise in body temperature of 40.3 -41.2°C on days 10-12 with subsequent minor increases on days 22, 23, and 27 after the control infection.Pig No. 2 from group "V" remained clinically healthy throughout the entire experiment (32 days).The clinical score for pigs immunized with the "Volgograd/14c" strain after the control infection did not exceed 2 and 5 points for pig No. 2 and pig No. 1, respectively (Figure 1).On day 32 after the control infection, the animals were euthanized.No visible pathological changes in the internal organs were observed during the necropsy.
The results of the real-time PCR study of DNA isolated from blood samples taken during the experiment are presented in Figure 3.   From the graph, it can be seen that the Ct values in samples from group "V" were initially higher than those in group "M" and did not change significantly throughout the experiment.In samples collected from animals immunized with the recombinant strain "Volgograd/D(1L-5-6L) MGF110", the Ct values were significantly lower compared to group "B", but after the control infection, a significant increase was observed until the animals' death.
Viremia in the blood of pigs inoculated with the ASFV "Volgograd/14c" strain was observed from 5-7 days in titers of 2.5 -3.0 lg HAU 50 см 3 .The maximum virus accumulation was observed on days 10-14 which reached 3.5 -4.75 lg HAU 50 см 3 .By days 21-28 viremia in animal No. 2 decreased, the titers were 2.5 -3.25 lg HAU 50 см 3 , while in pig No. 1 the virus was not detected on days 21 and 28 after infection.In animals of group No. 2, after immunization by the ASFV "Volgograd/D(1L-5-6L) MGF110" the infectious virus was not detected in the blood for 28 days (observation period) (Table 1).

DISCUSSION
The main method for elucidating the specific functions of ASFV genes is the development of deletion viruses.The scientific literature contains a wealth of data indicating the role of certain genes in the ASFV biology and describing their impact.[9;10].However, there is relatively limited information available on the functions of genes belonging to multigene families in the ASFV genome.According to the available data, the deletion of MGF360-9L and MGF505-7R genes contributes to virus attenuation, and immunization with the deletion virus protecting against infection with the parental strain [11].The deletion of the MGF100-1R gene does not lead to changes in the properties of the ASFV strain isolated in China [12].As for MGF 110, studies have shown that the 9L gene deletion also contributes to the virus attenuation [13].
In the work of Netherton et al., 2004 it was noted that the products of the MGF 110-4L and -6L genes are localized in pre-Golgi compartments and are involved in endoplasmic reticulum remodeling, disrupting its ability to synthesize proteins that stimulate cytokine production or participate in antigen presentation.Therefore, they do not affect the virus's ability to replicate in pig organisms but rather disrupt the immune response [14].This is further supported by the study by Ren et al., 2023 which investigated the impact of the 9L gene deletion which is part of MGF 110, by comparing the properties of the parental and deletion strains.The study revealed the 9L gene's influence on the degradation of TANK Binding Kinase 1 (TBK1), which affects the type I interferon production.The parental strain inhibits type I interferon production by degrading TBK1, while the deletion strain enhances type I interferon production by weakening TBK1 recovery, clarifying the mechanism by which the 9L gene deletion of MGF 110 ensures attenuated virulence in vitro [15,16].The deletion of the 11L gene of MGF110 has a weak effect on virus virulence, increases the induction of an immune response, and causes mild clinical signs [17].The 7L gene influence was studied in the work of Zhong et al., 2022.It was shown that MGF110-7L suppresses translation processes in infected cells and contributes to the stress granules formation.This occurs due to the phosphorylation of the eukaryotic initiation factor 2α (eIF2α) signaling pathway by Protein Kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK) [18].
In a previous study, the influence of the deletion of the 1L-5-6L MGF110 gene fragment on virus replication in COS-1 cells and its growth characteristics were investigated [19].It was found that these properties did not change during the cultivation of the recombinant strain "Volgograd/D(1L-5-6L) MGF110" in COS-1 cells.The data obtained in in vitro models do not always correspond to how the virus will manifest itself in the susceptible animal body.Based on the data obtained in this study, it can be concluded that there is a change in the accumulation level of the recombinant strain compared to the parental strain in vivo.This is indicated by significantly lower Ct values obtained when investigating DNA samples isolated from blood collected from animals during immunization before the control infection with the virulent ASFV strain "Stavropol 01/08".However, after the control infection, a significant increase is observed in the Ct values of samples from animals inoculated with the "Volgograd/ D(1L-5-6L) MGF110" strain (group "M") leading to death while Ct values in samples from animals inoculated with the parental strain "Volgograd/14c" (group "V") did not change significantly until the experiment end.This may indicate stable persistence of the parental strain "Volgograd/14c" in the animals' bodies.
These results are confirmed by the investigation of serum samples using the ELISA method, as well as the determination of the virus infectious activity in blood samples from immunized animals.The presence of antibodies to the p30 protein in serum samples and the active virus presence in blood samples from animals in group "V" confirm the reproduction of the virus strain "Volgograd/14c" in the animals' bodies.However, no antibodies or active virus of the deletion strain "Volgograd/D(1L-5-6L) MGF110" were detected in serum and blood samples from animals in group "M" indicating the absence of virus replication.
The results obtained in this experiment differ from those published by Ramirez-Medina et al., 2022 [20].In their study, the authors investigated the properties of the deletion strain "ASFV-G-ΔI177L/ΔMGF110-5L-6L" in which the I177L and MGF110-5L-6L genes are deleted.The authors claim that the MGF110-5L-6L deletion has no effect on the virus properties and growth characteristics both in vitro and in vivo and can serve as a DIVA marker for a candidate vaccine strain.Such discrepancies in the results can be explained by the fact that Ramirez-Medina et al., 2022 used the ASF virus virulent strain "Georgia" (ASFV-G), which is closest to the strains currently circulating in Europe and the Russian Federation.Furthermore, the strain they used was not adapted to immortalized cell lines and only replicated in primary pig leukocyte cell culture.In our study, the "Volgograd/14c" strain was used, which had undergone several passages on cell cultures, including immortalized cell lines, resulting in its partial attenuation.It is likely that the observed changes in properties exhibited by the virus in in vitro or in vivo conditions may be due not only to the specific gene deletion but also to a combination of changes that occurred in the virus genome as a result of adaptation to immortalized cell lines.Additionally, the "Volgograd/D(1L-5-6L) MGF110" strain had a larger deletion encompassing genes from 1L to 5-6L.

CONCLUSIONS
The experiment on susceptible animals revealed that the recombinant strain with a deletion of genes part from the multigene family 110 does not replicate in pig organisms.However, as shown in a previous study, there are no significant differences in growth characteristics and accumulation between the parental and deletion strains.
Based on this, it can be concluded that the 1L-5-6L genes of the multigene family 110 play a crucial role in the virus's adaptation to replicate in susceptible animal organisms.

Figure 2 .
Figure 2. Survival graph of the animals during the experiment.The red dashed line indicates the timing of the control infection of immunized animals.