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The role of gastropods in African swine fever virus ecology

Virology Journal volume 21, Article number: 180 (2024) Cite this article

Abstract

The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.

Introduction

African swine fever (ASF) virus is a DNA virus of the Asfarviridae family that causes a lethal disease in both domestic and wild Eurasian pigs. Transmission of the ASF virus in domestic pigs and wild boars occurs mainly by contact, inhalation and ingestion. In a large region of East, Southern and the southern part of Central Africa, the circulation of the virus is between the Ornithodoros moubata ticks and the virus-resistant common warthog Phacochoerus africanus. This forms a stable sylvatic cycle that maintains the circulation of the virus in nature, with occasional spill-over to domestic pigs via the bite of an infected tick [1,2,3]. Even though no similar reservoirs have yet been found in Eurasia, the virus has shown the ability to survive for a long time outside it’s natural reservoirs [4]. In Eurasian conditions, wild boars can transmit the virus to domestic pigs via contact. Another known mechanism for transmission of the virus is anthropogenic, associated with the transportation of pig products and hunting [5, 6]. So, the search for possible reservoirs of the ASF virus in Eurasia still remains important.

African swine fever (ASF) virus is the only DNA-containing arbovirus with a complex epidemiology. The natural reservoir hosts of African swine fever virus are warthogs (Phacochoerus africanus) and bushpigs (Potamchoerus porcus). Soft ticks (often referred to as O. moubata or O. moubata porcinus) of the genus Ornithodoros belonging to the Argasidae family act as reservoir hosts and biological vectors for ASF virus. This may indicate the plasticity of the viral genome, allowing it to survive in various environmental niches. The ASF virus can propagate both directly and indirectly. Direct transmission occurs via contact between infected and healthy pigs. Indirect transmission can occur by biological vectors such as soft ticks belonging to the Ornithodoros genus, or through a variety of mechanical vectors. In addition, ASF virus can persist in uncooked pork products for extended periods (for 3–6 months), contributing to its spread through fomites such as premises, vehicles, implements, and clothing. Within the tick vector, ASF virus can be transmitted transstadially (between life stages), transovarially (from female ticks to offspring), and sexually during mating, highlighting the diverse pathways through which this highly contagious virus can infect swine populations.

The ASF virus is well known for its environmental stability, which aids in its persistence and dissemination. The ASF virus is very resistant to changes in pH and temperature because of its intricate multilayered structure. ASF virus can survive and spread in different seasons/climates and pH conditions. In addition, the presence of organic matter, such as blood, serum, and uncooked meat or meat products, increases the stability and survival time of the virus.

Overall, the ability of ASF virus to persist in both biological and environmental reservoirs plays a crucial role in maintaining its presence in affected regions and facilitating its spread among swine populations. Understanding and managing these persistence mechanisms are essential in efforts to control and prevent outbreaks of African Swine Fever.

The virus is classified in the family Asfarviridae and is the only virus in this family. In 2019 it was reported about abalone Asfarvirus-like virus (AbALV), which infects marine gastropods (Haliotis diversicolor) and causes amyotrophia. Then, in 2020 complete genome of this virus was published. Comparison of genomes of ASF virus and AbALV (not yet classified) revealed that this virus is closely related to ASF virus [7,8,9]. Thus, relying on that study, we decided to delve deeper into it by further studying the possibility that the ASF virus might also be able to infect gastropods as AbALV.

Previously, we reported the possibility of long-term survival of the ASF virus in air-breathing land snails (Xeropicta derbentina). Moreover, the transcription of at least some viral genes in the body of snails was described [10]. In this study we aimed to get more insights into the potential relevance of freshwater snails.

Materials and methods

Animal husbandry

Snail stocks were obtained from a local aquarium shop. Snails used in the experiment were never used for breeding afterward. A list of freshwater gastropods is presented in Fig. 1 and Table 1. The sizes and exteriors of the freshwater gastropods used in the project corresponded to the world Mollusca database (https://www.academia.edu/32919684/WMSDB_Worldwide_Mollusc_Species_Data_Base_Checklist_of_Phylum_MOLLUSCA) [11].

Table 1 Freshwater snails used for experiments
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Fig. 1
figure 1

Freshwater gastropods cocultivated with African swine fever virus

1.Faunus ater (Family: Pachychilidae) Genus: Faunus. 2.Brotia herculea white (Family: Pachychilidae) Genus: Brotia. 3.Pomacea bridgesii (Family: Ampullariidae) Genus: Pomacea. 4.Physa fontinalis (Family: Physidae Genus: Physa). 5.Tarebia granifera (Family: Thiaridae) Genus: Tarebia. 6.Melanoides tuberculata (Family: Thiaridae Genus: Melanoides). 7.Anentome helena (Family: Nassariidae) Genus: Anentome. 8.Planorbarius corneus (Family: Planorbidae) Genus: Planorbarius. 9.Asolene spixii (Family: Ampullariidae) Genus: Asolene

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Snails were kept under a 12 h/12 h light/dark regime. The stock and experimental tanks were kept in a climate room at 25 °C (± 2 °C). Water tanks were covered to prevent evaporation. All water tanks were equipped with filters, and the power of the filters was selected according to the size of the tank. Water was changed 10% every four weeks to ensure good water quality. In the pre-experimental period (1 month), snails were kept under the same conditions to adapt and exclude bacterial diseases.

Virus

The African swine fever virus Armenia08 strain was used in all experiments [12]. The virus (Armenia08) was first isolated in 2007 from the spleen of an ASFV-infected swine.

To demonstrate infection with noncytopathogenic as well as cytocidal viruses, hemadsorption assay is used. The ASF virus titration was performed as described previously and expressed by hemadsorption units (HADU) - as log10 HADU50/ml for non-adapted cells [13,14,15]. Also, virus genome copies were measured by qRT-PCR.

The infection dose of the virus was determined based on two parameters: concentration of the virus in water 103 HADU/ml and body weight of snails 103 HADU/gram (the latter parameter may have fluctuated slightly due to difficulties in determining the ratio of mineral shell mass to body mass). The virus was then added in all nine aquariums, corresponding with the above-mentioned conditions [14, 15]․

Sampling

All the nine species of chosen gastropods were kept in nine separate aquariums (conditions table). Water was collected from all experimental containers with gastropods. The water was mixed before sampling. After sampling, the water was frozen at -30 °C. Gastropod faeces were collected every week after the start of the experiment using a sterile pipette. After collection, the samples were centrifuged at 2,000 rpm, the water was removed and the sediment was frozen at -30 °C.

The gastropods were frozen in equal quantities at 4, 12, and 20 weeks after the start of the experiment. Measurements were performed 4 weeks after the start of the experiment.

For the virus hemadsorption assay, inactivated ASF virus was used as a negative control. The virus was inactivated in a water bath at 65 °C for 10 min. After heat inactivation, the virus was tested for infectivity on porcine alveolar macrophages (PAM) with hemadsorption assay.

ASF virus transcriptional activity evaluation

To assess the level of transcriptional activity of ASF virus genes in snails, we compared the level of cDNA obtained from all experimental groups with dilution standards of cDNA obtained from ASF virus-infected PAMs [10]. cDNA was obtained through reverse transcription of isolated viral RNA from snail bodies, faeces and water. Undoubtedly, such a study gives only a very rough estimate, but it makes it possible to determine and evaluate the phenomenon as a whole.

Gene expression analysis by quantitative real-time PCR

To determine ASF virus expression in PAMs (from both: to investigate possibility of infection of the snail derived ASF virus and for comparison of gene expression in PAMs and snails) total viral RNA/DNA was isolated using the HiGene™ Viral RNA/DNA Prep kit (BIOFACT, Yuseong-gu, Darjeon, Republic of Korea). RNA/DNA samples were then reverse transcribed using the FIREScript® RT cDNA synthesis kit (Solis Biodyne, Tartu, Estonia). Both methods were conducted following the manufacturer’s instructions. DNA/RNA concentrations were measured using a NanoDrop® ND-1000, UV-Vis Spectrophotometer (Waltham, Massachusetts, USA) including the measurement of A260/280 values (~ 1.8 for DNA; a ratio of ~ 2.0 for RNA). Quantitative real-time PCR was performed using the SYBR green method previously described [16, 17] on Bio-Rad CFX 96 Real-Time PCR system (Bio-Rad Laboratories, Inc. Hercules, California, USA). Each reaction mixture (20 µL) was composed of 4 µL of 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis BioDyne, Tartu, Estonia), 0.2 µL of each specific primer (initial concentration − 100 pmoL/uL), 4 µL of template DNA /cDNA, and 11.6 µL of ddH2O. DNA isolated from an ASFV-infected pig’s spleen was used as a positive control. ddH2O was used as a negative control. Reactions were carried out in the following conditions: polymerase activation: 95 °C for 12 min, 40 cycles: 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s. Standard curves were created using serial 10-fold dilutions of viral DNA. The fluorescence threshold value (Ct) was calculated using the CFX Maestro Software (Bio-Rad Laboratories, Inc. Hercules, California, USA). For ASF virus genes transcription evaluation the viral genes normalization method has been used (for standard dilutions the viral essential genes were used for example: K196R, R298L). Primers used for amplification were designed based on ASFV Georgia 2007/1 sequence (Gene bank: FR682468.2) genes in FASTA format and ordered from Integrated DNA Technology-IDT (https://eu.idtdna.com/pagesas , accessed on 11 May 2019) [18].

All primers used in the experiments are presented in Table 2, and are divided into 3 groups according to gene expression timing – early, late, and ambivalent [19]. Ambivalent genes are not specifically related to replication time.

Table 2 Oligonucleotide primers employed in qRT-PCR assay
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For alignment of the cDNA plots, Cq-values were rescaled after comparing with the amount of viral genome copies and modified in absolute amounts along the y-axis for better visualization.

ASF virus infection of the porcine alveolar macrophage (PAM) culture

Primary alveolar macrophage cells (PAM) were obtained during bronchoalveolar lavage of 3-month-old porcine lungs, and were resuspended in sterile Hank’s balanced salt solution. They were centrifuged at 600 g for 10 min and resuspended in RPMI 1640 medium with 5% fetal bovine serum at a cell concentration of 0.5-1 × 106 ml. After keeping the adhered cells for 3 h at 37 °C in a humidified incubator containing CO2, they were washed three times with RPMI medium to remove contaminating non-adherent cells and then incubated in RPMI 1640 medium with 10% FBS [20].

The infection of PAM cells was performed by 1 × 10− 1 HADU50/ml logarithmic dilutions of all gastropod faeces. After infection virus doses were measured by HADU and qRT-PCR.

Transmission of African swine fever virus in the process of natural feeding

In order to assess the infectivity of ASF virus from ASFV-infected snails to pigs three pigs were fed a mixed diet containing two bodies of frozen Melanoides tuberculate and Melanoides tuberculate cultivation water samples (samples were used from the 10th week of experiment). The choice of Melanoides tuberculate is due to the high titers of the virus both in the bodies and in the water where the snails were cultured. Each pig received at least 30 ml of water “cultivated with” Melanoides tuberculate.

Immunofluorescence

Deparaffinized slides were incubated in citrate buffer overnight within a water bath at 60 °C, followed by cooling to room temperature (RT). After cooling, the slides were washed with dH2O and carefully dried. A hydrophobic barrier (wax) was drawn around the region of tissue sections, and blocking buffer (PBS + 5% BSA + 0.3% Triton X-100) was applied to the tissue sections. The slides were incubated in a humidified chamber for 1 h at RT. Slides were then washed using dH2O. Primary mouse anti-ASFV P30 protein (MyBioSource) was added to the tissues at 1:500 in staining buffer (PBS + 1% BSA + 0.3% Triton X-100) and incubated in a humidified chamber for 1 h at RT. Slides were washed 3x with washing buffer (PBS + 0.05% Tween-20) using a shaker and donkey anti-mouse IgG secondary antibodies (Abcam) conjugated with AlexaFluor® 488 (1:400 in staining buffer) were then added and incubated for 1 h at RT in a dark humidified chamber. After the staining procedure, the slides were washed 3x with washing buffer on a shaker, and counterstained with Hoechst 33342 (Thermo Scientific). Finally, the slides were covered with coverslips and interpreted using the Cytation C10 confocal imaging reader (Agilent) equipped with Gen5 v3.14 software.

Statistical analysis

All experiments were repeated three times and statistical analysis was performed using the Student’s t-test. An error bar was used to demonstrate standard deviations. SPSS version 17.0 software (SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis.

Results

Detection of ASFV in gastropod excretions in water, bodies of the gastropods, and feaces

The first step of our study was to determine the possibility of infection of freshwater gastropods with the ASF virus as well as the survival of the virus in the presence of freshwater snails.

In water at a temperature of 22–24 °C, the virus can maintain a detectable level expressed in hemadsorption units, for 3 weeks after the start of the experiment (Fig. 2A). Copies of the genomic DNA of the virus persist almost twice as long (Fig. 2A). The duration of the virus persistence in the water with the presence of the snails Pomacea bridgesii, Tarebia granifera, and Asolene spixii is significantly longer. This effect is manifested both in the measurements of qRT-PCR (Fig. 2B) and in the analysis of HADU (Fig. 2C). The duration of the persistence of the virus in the presence of Physa fontinalis snails revealed only a trend towards an increase in the survival time of the ASF virus (p < 0.1).

Fig. 2
figure 2

Detection of African swine fever virus levels in water. A African swine fever virus levels in water without freshwater gastropods. B African swine fever virus levels in water with different gastropods measured by HADU. C African swine fever virus DNA genome copies in water with different gastropods measured by qRT-PCR; gene: K196R (* significant, p < 0.05; ** tendency, p < 0.1)

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Measurements of virus levels in the bodies of various gastropods are presented in Fig. 3. These measurements were taken at the end of the 4th week after the start of the experiment. This period was chosen because by this time there were no detectable particles and copies of the virus genomes left in the water (at the same temperature). As can be seen from Fig. 4, the data on the number of copies of the viral genome (Fig. 3A) measured by qRT-PCR were very similar to the data levels of infectious particles (Fig. 3B) measured by HADU. It should be noted that the virus was preserved in all gastropod bodies except Brotia herculea white and Anentome helena.

Fig. 3
figure 3

Detection of African swine fever virus levels in bodies of freshwater gastropods on 28th day of incubation. A African swine fever virus levels in bodies of different gastropods measured by HADU. B African Swine Fever Virus DNA genome copies in bodies of different gastropods measured by qRT-PCR; gene: K196R (* some of the probes were negative)

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Fig. 4
figure 4

Detection of African swine fever virus levels in faeces of freshwater gastropods. A African swine fever virus levels in faeces of different gastropods measured by HADU. B African swine fever virus DNA genome copies in faeces of different gastropods measured by qRT-PCR; gene: K196R (* significant, p < 0.05; ** tendency, p < 0.1)

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In the faeces of freshwater snails, the ASF virus persists much longer (Fig. 4A, B). The duration of the virus persistence (expressed in hemadsorption unit (HADU)) in the faeces of the Pomacea bridgesii and Asolene spixii is up to 10 weeks after the start of cocultivation. In faeces of Tarebia granifera, Melanoides tuberculate, and Physa fontinalis it survived for up to 8 weeks after the start of cocultivation. The duration of the virus persistence (expressed by the qRT-PCR) in the faeces of the snails is longer up to 18 weeks.

In water, where freshwater gastropods were cultivated, copies of the ASF virus genomes persisted much longer than in water (at the same temperature) without snails. A similar result was obtained in the study of viral levels by the HADU method.

Following four weeks of cocultivation, the transcriptional activity of the ASF virus genes in freshwater snails is illustrated in Figs. 5, 6, and 7.

Fig. 5
figure 5

Expression of early genes of African swine fever virus in bodies of freshwater gastropods. A Anentome Helena; B Melanoides tuberculate; C Tarebia granifera; D Pomacea bridgesii; E Physa fontinalis; F Planorbarius corneus; G Asolene spixii; H Faunus ater; I Brotia herculean * significant, p < 0.05; ** tendency, p < 0.1

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Fig. 6
figure 6

Expression of ambivalent genes of African swine fever virus in bodies of freshwater gastropods. A Anentome Helena; B Melanoides tuberculate; C Tarebia granifera; D Pomacea bridgesii; E Physa fontinalis; F Planorbarius corneus; G Asolene spixii; H Faunus ater; I Brotia herculean * significant, p < 0.05; ** tendency, p < 0.1

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Fig. 7
figure 7

Expression of late genes of African swine fever virus in bodies of freshwater gastropods. A Anentome Helena; B Melanoides tuberculate; C Tarebia granifera; D Pomacea bridgesii; E Physa fontinalis; F Planorbarius corneus; G Asolene spixii; H Faunus ater; I Brotia herculean * significant, p < 0.05; ** tendency, p < 0.1

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Fig. 8
figure 8

Infection levels of African swine fever virus in faecal samples of gastropods titrated on PAMs after 5 weeks of cultivation. All groups with gastropods are significantly higher than control indicators (p < 0.01)

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Figure 5 shows data on the transcriptional activity of early genes of the ASF virus in various species of freshwater snails. As follows from the data in the figure, in the snails Anentome helena (Fig. 5A), Faunus ater (Fig. 5H), Brotia herculea (Fig. 5I), the transcriptional activity of viral genes is either absent or at a very low level. In the snails Melanoides tuberculata (Fig. 5B), Tarebia granifera (Fig. 5C), Pomacea bridgesii (Fig. 5D), Physa fontinalis (Fig. 5E), the transcriptional activities of all or almost all studied genes were determined, and the transcriptional activity of ASF virus genes in snails Planorbarius corneus (Fig. 5F) and Asolene spixii (Fig. 5G) have intermediate values.

Figure 6 presents data on the transcriptional activity of ambivalent ASF virus genes in various species of freshwater snails. As follows from the data in the Fig. 6, in the snails Anentome helena (Fig. 6A), Asolene spixii (Fig. 6G), Faunus ater (Fig. 6H), Brotia herculea (Fig. 6I), the transcriptional activity of viral genes is either absent or at a very low level. In the snails Melanoides tuberculata (Fig. 6B), Pomacea bridgesii (Fig. 6D), Physa fontinalis (Fig. 6E), the transcriptional activities of all or almost all studied genes were determined, and the transcriptional activity of the ASF virus in the snails Tarebia granifera (Fig. 6C), Planorbarius corneus (Fig. 6F) was noticed only in certain genes.

Figure 7 shows data on the transcriptional activity of late genes of the ASF virus in various species of freshwater snails. As follows from the data in the Fig. 7, in the snails Anentome helena (Fig. 7A), Faunus ater (Fig. 7H), Brotia herculea (Fig. 7I), the transcriptional activity of viral genes is either absent or at a very low level. In the snails Melanoides tuberculata (Fig. 7B), Tarebia granifera (Fig. 7C), Pomacea bridgesii (Fig. 7E), Physa fontinalis (Fig. 7E) and Planorbarius corneus (Fig. 7F), the transcriptional activities of all or almost all studied genes were determined, and the transcriptional activity of the ASF virus in the Asolene spixii snails (Fig. 7G) was noted only in certain genes.

The ability of ASF virus isolated from gastropods to infect PAMs

These experiments were designed to determine whether ASF virus (virus obtained from faeces of gastropods) is capable of subsequent productive infection to normal cells. Primary cultures of PAMs were infected with the highest dilutions (1 × 10− 1) of the gastropod’s faeces. These experiments were performed after the 5th week of incubation of the ASF virus with gastropods., all faecal samples were able to cause productive infection of the ASF virus in the PAM. Control samples of ASF virus in the water were free of infectious ASF virus (Fig. 8).

The ability of virus transmission from gastropods to pigs after natural feeding

Experimental studies of whether freshwater gastropods could subsequently transmit the virus through natural feeding of pigs have been ineffective; all three experimental pigs were free of ASF virus after 3 weeks of feeding.

IFA results for anti-p30 monoclonal antibodies in gastropods infected with ASF virus

The localization of the ASF virus in the tissues of freshwater gastropods was determined using IFA. The p30 protein in the tissues of Melanoides tuberculata could be detected on day 36 after infection.

The predominant localization of the ASF virus in the digestive system of gastropods is shown in Fig. 9. The location of p30 in Melanoides tuberculate’s stomach, as well as in the odontophore (Fig. 9A) and surrounding muscle tissue, is depicted in Fig. 9A, B, C, and D. Presence of p30 described in tissue compared with control (Fig. 9B), which was free from specific fluorescence (Fig. 9C, D).

Fig. 9
figure 9

IFA results for anti-p30 monoclonal antibodies in control and ASFV-infected gastropods. A Intestine of Melanoides tuberculate. Staining H.E. Scale bar 20 μm. B Intestine of control Melanoides tuberculate with IFA for anti-p30 monoclonal antibodies. Scale bar 20 μm. C Intestine of Melanoides tuberculate with IFA for anti-p30 monoclonal antibodies 20 μm. D Odontophore and muscles of the odontophore of Melanoides tuberculate with IFA for anti-p30 monoclonal antibodies. Scale bar 40 μm

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Discussion

The ASF virus is the sole member (ASFV-like Abalone virus still needs to be classified as shown by Matsuyama et al., 2020 [7]) of the genus Asfivirus within the family Asfarviridae. Unlike most arboviruses, it can be easily transmitted from one host to another without a biological vector. It is the only DNA virus that is transmitted by a vector. It has several unique genes that code for proteins involved in metabolism [21].

Several metagenomic studies show a presence of ASF-like sequences with aquatic ecosystems, for example, an association with freshwater reservoirs has been known for a long time [22], and their presence in marine ecosystems has also recently been identified [23]. Also, some authors [24] identified ASF virus in freshwater samples before the ASF epidemic in China in 2018. Data from previous publications showed that not only aquatic but also terrestrial gastropods can theoretically be hosts of the ASF virus [10]. The ASF-like virus has recently been described as infecting the marine gastropod abalone [7] and it’s structure and genome is closely related to ASF virus [8, 9].

This study investigated the survival of the ASF virus in the presence of freshwater gastropods. Data from the co-cultivation of 9 species of freshwater gastropods with ASF virus show a longer survival in the environment in the presence of most gastropods studied. Except for the species Brotia herculea and Anentome helena, all gastropods extended the circulation of the virus (both copies of the genome and haemadsorbtion units) by approximately 2–3 times compared to the freshwater environment without gastropods. Moreover, the virus was also isolated from gastropod bodies and faeces in large quantities and for a longer time than in culture water.

The possibility of long-term survival of the ASF virus in the bodies of snails can have a few possible reasons. This may be the result of some snails that allow the virus to be successfully preserved for a relatively long time because of some substances produced in the water with snails. Alternatively, some freshwater snails may support ASF virus replication.

Analysis of the transcriptional activity of ASF genes in the bodies of freshwater snails revealed similar data on the activation of early, ambivalent, and late genes of the virus. Most of the genes studied demonstrate significant levels of expression when co-located in the bodies of the snails Melanoides tuberculata, Tarebia granifera, Pomacea bridgesii, and Physa fontinalis. The virus isolated from all gastropod faeces was able to cause a productive infection in the PAM at a time when the virus was absent in the control population (water without gastropods) for at least 2 weeks. However, attempts to infect pigs were unsuccessful. This was probably due to the infectious dose used [25].

Gastropods occupy a fairly important place in the diet of pigs, especially among wild boars, and probably with free grazing of domestic pigs [26]. Therefore, it is impossible to exclude the possibility of transmission of the virus with a larger number of gastropods in the diet of pigs.

Conclusion

The data of our analysis suggest that gastropods theoretically can be the amplifying or maintenance hosts of ASF virus and represent an ecological niche for the long-term survival of the ASF virus.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We are thankful to the Directorate of the Institute of Molecular Biology of NAS for all the technical support and the Science Committee of RA.

Funding

The work was supported partly by the Science Committee of RA in the frames of the research project NO 21APP-1F007.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology and Virology, Institute of Molecular Biology of NAS RA, Yerevan, Armenia

    Arpine Poghosyan, Sona Hakobyan, Hranush Avagyan, Aida Avetisyan, Nane Bayramyan, Lina Hakobyan, Liana Abroyan, Bagrat Baghdasaryan, Elina Arakelova, Elena Karalova & Zaven Karalyan

  2. Experimental Laboratory of Yerevan State Medical University after M. Heratsi, Yerevan, Armenia

    Hranush Avagyan, Aida Avetisyan & Elena Karalova

  3. A.B. Nalbandyan Institute of Chemical Physics, NAS RA, Yerevan, Armenia

    Aram Davtyan

  4. Yerevan State Medical University after M. Heratsi, Yerevan, Armenia

    Zaven Karalyan

  5. Laboratory of Molecular and Cellular Immunology, Institute of Molecular Biology NAS RA, Yerevan, Armenia

    Davit Poghosyan

Authors
  1. Arpine Poghosyan
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  2. Sona Hakobyan
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  9. Davit Poghosyan
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  12. Elena Karalova
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  13. Zaven Karalyan
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Contributions

Conceptualization, Z.K. and H.A.; methodology A.A., L.H., L.A., A.D., E.A., D.P. and E.K.;software Z.K., H.A. and A.D.; validation, H.A., A.P. and Z.K.  formal analysis, Z.K.;investigation, S.H., N.B., A.P., B.B. and H.A.; resources, H.A. and Z.K. data curation, Z.K.; writing—original draft preparation, Z.K.; writing—review and editing, S.H.,A.P.; visualization, S.H., A.A., A.P. and H.A.; supervision, Z.K.; projectadministration Z.K., A.A. and H.A.; funding acquisition, H.A., Z.K.

Corresponding author

Correspondence to Zaven Karalyan.

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Institutional review board statement

The studies were reviewed and approved by the Ethics Committee of the Institute of Molecular Biology NAS RA (IRB 00004079, 2013; Protocol N5 from 25 May 2018). The animal study protocol was approved by the Ethics Committee of the Institute of Molecular Biology NAS RA (IRB 06042021/1, 2021).

Competing interests

The authors declare no competing interests.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Poghosyan, A., Hakobyan, S., Avagyan, H. et al. The role of gastropods in African swine fever virus ecology. Virol J 21, 180 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-024-02444-5

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Virology Journal

ISSN: 1743-422X