1. Introduction
African swine fever (ASF) is a devastating infectious disease in pigs and wild boars characterized by viral hemorrhagic fever [
1]. The fatality rate of ASF has been reported to be as high as 100%, and it causes a great deal of economic damage globally [
2], being listed as one of the notifiable diseases by the World Organization for Animal Health (WOAH). Furthermore, since there is no vaccine for ASF, animal slaughter remains almost the only method to control the disease [
3].
ASFV first enters the pig’s body through the tonsils and/or dorsal pharyngeal mucosa. After passing through the mandibular or retropharyngeal lymph nodes, it spreads systemically through viremia. It can be detected in almost all pig tissues as it spreads [
4]. The virus has a restricted cellular tropism and replicates primarily in macrophages and monocytes, which are important for viral persistence and dissemination [
5,
6]. Host cytokines produced by these infected cells play a crucial role in ASFV pathogenesis [
7].
The clinical course of ASF can be influenced by various factors, such as the virus, the host, and the immunological status of the farm [
4]. The course of the disease in domestic pigs can be categorized as peracute, acute, subacute, or chronic [
4]. In Europe and Asia, the highly virulent strains responsible for ASF belong to genotype II, and they can cause acute to peracute disease with up to 100% lethality within 7–10 days. Clinical signs are typically non-specific and may include high fever, loss of appetite, gastrointestinal and respiratory symptoms, cyanosis, ataxia, and sudden death [
8]. However, the underlying factors that influence the ASFV-related disease outcome and course are still not well understood. Several studies have pointed out that further research should focus on host responses against ASFV [
8].
The gut microbiota is a complex and diverse microbial ecosystem that resides in the gastrointestinal (GI) tract of animals [
9]. It contains numerous microorganisms such as bacteria, viruses, fungi, protozoa, and archaea, and plays a significant role in the development and maintenance of the immune system [
10,
11,
12]. The absence of microbiota results in the incomplete development of the immune system, as seen in germ-free mice [
13,
14]. The gut microbiota can also regulate T-cell differentiation, mitigate excessive immune responses, and alleviate inflammation by producing potent metabolites, including short-chain fatty acids (SCFA) [
15]. On the other hand, when gut epithelial integrity is lost, gut microbiota and their toxins, such as lipopolysaccharide (LPS) and incompletely digested fats and proteins, enter the bloodstream, leading to systemic inflammatory responses and tissue damage [
16]. Thus, maintaining intestinal homeostasis and the microbial ecosystem is critical for the host’s overall health.
Increasingly, studies suggest that the disruption in the homeostasis of the GI microbiome and the host’s immune system can have negative effects on viral immunity [
17]. Viruses can change the host’s gut microbiome without directly infecting the GI tract, which has been shown to control the severity of the disease [
18,
19]. Thus, microbiome change after viral infection is an important characteristic of the pathogen in the context of viral–host interactions. It has not been determined how ASFV changes the gut microbiome in a pig’s intestines. The objective of this study is to analyze the gut microbiome of pigs following infection with highly virulent ASFV. The study involved administering a genotype II strain of ASFV to pigs that were free of specific pathogens and observing changes in the gut microbiome. The goal was to gain insight into how ASFV affects the pig’s gut microbiota and identify the features of the modified microbiome. This will help determine if the altered microbiome contributes to the development and outcome of ASF.
4. Discussion
ASF is a representative viral hemorrhagic fever (VHF) in animals. In humans, all causative agents of VHF are classified as RNA viruses and are categorized into four families: Arenaviridae, Bunyaviridae, Filoviridae, and Flaviviridae [
27]. However, in animals, VHFs are caused by a much wider variety of viruses, some of which have double-stranded RNA genomes and even DNA genomes [
28]. A common feature of VHFs is that the viruses infect and replicate in monocytes–macrophages, producing pro-inflammatory cytokines [
1,
27]. In addition, many of them present with GI signs rather than respiratory signs, along with high fever. Particularly, some VHFs, such as Ebola and Marburg fever, exhibit the bystander apoptosis of uninfected lymphocytes, which is an important feature of the pathology of ASF [
1,
29]. Since lymphocytes are the main inducers and effectors of GI immunity [
30,
31], the depletion of lymphoids is likely to alter the gut microbiome negatively. This may lead to a vicious cycle via the depression of gut microbiome function and increased intestinal permeability, strengthening the pathogenesis of the disease. Microbiome changes during VHF infection have scarcely been studied, and a fundamental discussion remains as to whether the host immune mechanism associated with the microbiome can affect the pathogenicity and severity of VHFs. This study investigated how ASFV changes a normal pig’s gut microbiome and whether the altered microbiome due to ASFV infection could function in a beneficial or harmful way in terms of the pathology of the disease.
ASFV usually infects mononuclear macrophages, and the development of the disease is induced by the cytokines they release. The key features of the pathogenesis of ASF in domestic swine are as follows: (a) severe lymphoid depletion, including lymphopenia and a state of immunodeficiency, and (b) vast hemorrhages [
32]. The GI tract contains a significant amount of lymphoid tissue, which is required to maintain gut immunity and homeostasis. This tissue is the most affected body site during ASFV infection. Accordingly, the intestinal environment is likely modified, resulting in alterations to the microbial ecosystem. The gut microbiome can play a positive role in developing and maintaining host immunity, or their opportunistic pathogenic properties can cause systemic inflammation as a double-edged sword [
33].
In this study, a remarkable change was observed in the normal pig’s gut microbiome during ASFV infection, wherein the host became potentially susceptible to inflammation and immunodeficiency. The clear separation between the NC and ASFV groups in terms of the PCoA adequately reflects the Anna Karenina principle, implying that dysbiotic individuals vary more in microbial community composition than healthy individuals—paralleling Leo Tolstoy’s dictum that “all happy families look alike; each unhappy family is unhappy in its own way” [
34]. This indicates that ASFV can induce certain perturbations within a healthy gut microbiome that generally require a lot of maintenance and result in time-course-varied patterns in individuals.
Wang et al. [
22] suggested that the course of acute ASFV infection could be divided into three phases: the primary phase (0–2 dpi) without changes in serum cytokine levels or clinical symptoms; the clinical phase (3–7 dpi) characterized by progressive clinical features, the upregulation of various pro-inflammatory cytokines (e.g., TNF-α, IFN-α, IL-1β, IL-6), and sustained fever; and the terminal phase, marked by an additional sharp increase in multiple cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-10) and the partial recovery of IFN-α. Our study was conducted according to these phases; the sequence samples were grouped into each phase according to the pig’s clinical characteristics and a few outliers were removed. The four phases were clearly distinguished on the PCoA, indicating that the gut microbiome may be associated with disease development. The detailed mechanisms remain to be further revealed. In addition, specific bacterial groups moved significantly during each phase: various SCFA-producing bacteria changed significantly. SCFA is mainly produced by some members of Firmicutes and Bacteroidetes, which metabolize indigestible polysaccharides. Acetate, propionate, and butyrate are the major SCFAs produced in the gut [
35]. SCFAs directly affect T-cell differentiation into effector T cells, such as Th1 and Th17 cells, as well as IL-10+ regulatory T cells (Treg), and have anti-inflammatory properties mediated through the G-protein-coupled receptor (GPCR) [
36,
37]. Butyrate, the main source of energy for colonic epithelial cells, inhibits the mRNA expression of pro-inflammatory cytokines in the mucosa by inhibiting NF-κB activation [
35,
38]. Butyrate, as a histone deacetylase inhibitor, can also alter gene expression, inhibit cell proliferation, and induce cell differentiation or apoptosis, leading to butyrate’s anti-inflammatory and anti-tumor properties [
39]. Therefore, a decrease in the microbiota that produces butyrate and other SCFAs is likely to be associated with host immune system abnormalities.
Overall, the major SCFA-producing bacteria
Firmicutes decreased during ASFV infection.
Ruminococcaceae, including a number of SCFA-producing bacteria, was the predominant family in all phases of ASFV infection. The relative abundance of
Ruminococcaceae progressively decreased, along with nine genera significantly reduced (FDR < 0.05).
Eubacterium_g23 was most involved in this change. The genus
Eubacterium is composed of phylogenetically and phenotypically diverse species, and many of them produce butyrate [
40].
Subdoligranulum, other butyrate producers within the same family, significantly decreased during ASFV infection as well (FDR < 0.05) [
41]. In the family
Lachnospiraceae, which is the second-largest portion in Firmicutes,
Blautia has been shown to significantly decrease during ASFV infection.
Blautia plays an important role in maintaining balance in the intestinal environment, upregulating intestinal Treg cells and preventing inflammation, and its reduced abundance has been associated with inflammatory bowel disease (IBD) patients [
15,
42]. In addition, the
Collinsella aerofaciens group, a unique butyrate-producing bacterium in the phylum
Actinobacteria, was also observed to decrease significantly during ASF infection [
43]. Overall, this decrease in butyrate-producing bacteria may be associated with the exacerbation of ASF.
Meanwhile, in the phylum
Bacteroidetes, some SCFA-producing bacteria such as
Alloprevotella, Bacteroides, and
Parabateroides were observed to increase significantly during ASFV infection.
Prevotella increased after ASFV infection and was the most abundant genus of
Bacteroidetes from the primary to terminal phases.
Alloprevotella is recognized as a beneficial bacteria and can produce SCFA-containing acetate and butyrate and promote an anti-inflammatory environment [
44,
45,
46].
Bacteroides and
Parabacteroides have similar physiological characteristics regarding carbohydrate metabolism and secreting SCFAs. They are considered to play a key role in regulating host immunity [
47]. For example,
B. fragilis expresses the capsular polysaccharide A (PSA) to induce CD4+ T-cell-dependent immune response and activates immunomodulatory IL-10, exhibiting anti-inflammatory effects during herpes simplex encephalitis [
48,
49].
P. distanosis can regulate innate inflammatory responses by locking the release of TNF-α, IL-6, IL-17, IL-12, or IFN-γ and protect intestinal permeability by promoting intestinal succinate and secondary bile acid production [
49]. These increases in beneficial bacteria suggest that they may be major symbiotic bacteria regulating immunity in the clinical and terminal phases of ASF. However, several microbes in
Bateroides and
Parabacteroides and their toxins have been pointed out as opportunistic pathogenic characteristics [
49,
50], and there is also a possibility that they will further worsen the disease progression of ASF. For instance,
Bacteroides spp. normally enters aseptic tissue through the intestinal mucosa, eventually causing other disease conditions and even forming abscesses in the central nervous system [
51,
52]. In addition,
Alloprevotella, Bacteroides, and
Parabacteroides are the main succinate producers in the host intestine [
45]. Succinate is recognized as a microorganism-derived metabolite associated with dysbiosis-related diseases such as obesity and IBD [
53]. As shown by the progressive increase in the
Phascalctobacterium succinatutens group after ASFV infection in DESeq2 analysis (FDR < 0.05), which only uses succinate as an energy source phase, the aforementioned bacteria can modify the intestinal environment to a succinate-rich environment during ASF.
One of the important results of this study is the microbiome change in the primary phase. The richness of the bacterial community significantly decreased in the primary phase of ASFV infection. For the cause of this observation, though environmental effects cannot be totally excluded, it is necessary to examine the possibility of the virus’s effects on the richness of the microbiome. It took only about 2 to 3 days for ASFV to be detected in the bloodstream and a few days more to observe the expression of host clinical signs, including fever [
54]. ASFV itself and/or immune cells affected by ASFV that reach the intestine via blood circulation may cause significant changes in the intestinal microbial ecosystem before host clinical symptoms appear. To the authors’ knowledge, this is the first evidence that a virus can change the gut microbiome during the incubation period of the disease.
The altered microbiome resulting from ASFV infection is similar to that observed with PRRSV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections reported elsewhere. The microbiome affected by the viruses shared several features regarding the increased abundance of
Proteobacteria and
Spirochaetes but also decreased SCFA-producing families of
Ruminococcaceae and
Lachnospiraceae [
18,
19]. These may be the major changes in which a pig’s gut microbiome is affected by viruses that infect immune cells. On the other hand, pig intestines affected by enteric viruses, such as porcine epidemic diarrhea virus (PEDV), were observed to have an increased abundance of
Escherichia-Shigella,
Enterococcus,
Fusobacterium, and
Veillonella and decreased
Bacteroidetes such as
Bacteroides and
Prevotella [
55,
56]. Therefore, the microbiome can be controlled according to the mechanism that the virus uses for its infection and proliferation. Furthermore, the PRRSV-infected pigs in previous studies have shown a different microbiome profile in a strain-virulence-dependent fashion [
19]. Future studies need to investigate the effect of the virulence of ASFV on the gut microbiome or vice versa.
In the predictive functional analysis performed using PICRUSt, the immune-related pathways of the gut microbiome in the ASFV group were significantly compromised, indicating that ASFV modified the gut microbiome, and it may be associated with the status of host immune suppression. ASFV has developed a variety of mechanisms to evade host immune responses, including immunodeficiency via weakening innate immunity, blocking molecular signaling, disturbing cytokine systems and lymphoid depletion, and so on [
22,
32]. Although detailing these mechanisms is needed in the future, the results of the current study provide evidence for understanding the ASFV–pig immune system interaction.
Additionally, the results of this study can provide evidence for host–viral interactions and immunopathology in human VHF. Human VHF usually requires BL-3 and BL-4 facilities, and most experimental studies use rodent models [
57]. On the other hand, pigs are very similar to humans in terms of anatomy and the functions of the immune system, e.g., the presence of tonsils, which are absent in rodents. The porcine immune system resembles humans for more than 80% of the analyzed parameters in contrast to mice with only about 10% [
56]. For this reason, this study provides useful information to help answer questions regarding immunity in human VHF. The new evidence from this study that a gut microbiome affected by VHF infection can degrade the host’s immune function during the early stage of infection may inspire research on VHF etiology.