African swine fever (ASF) is one of the most important viral diseases of wild boar and domestic pigs, causing significant socio-economic losses in affected countries . Depending on host characteristics and the circulating viral isolate, clinical signs may vary from the highly lethal form with 100% mortality to sub-clinical and a clinical form [9, 19–21]. Since the developed vaccines against ASFV have been failing to induce effective protection, it is reasonable to evaluate antiviral agents against this virus [7, 8, 10]. The aim of this study focused on (i) characterization of ASFV strain isolated from the capital Hanoi outbreak in Red River Delta region of Vietnam and (ii) investigation of the antiviral activities of MCFAs at different composing materials, including C8 (caprylic acid), C8-C6-C10 (caprylic acid-caproic acid-capric acid) and C8-C10-C12 (caprylic acid-capric acid-lauric acid), against ASFV infection in vitro. We firstly screened the cytotoxicity of MCFAs on PAMs cells, the permissive cells for ASFV infection in vitro, by using the MTT assay according to the manufacturer's protocols. As shown in Fig. 1B, at the dose of 200 µg/ml, products 1 and 3 have shown a less cytotoxic effect when compared to product 2 on PAM cells, suggesting the anti-proliferative effects of C6 component in product 2. All products do not induce the cytotoxicity at the dose of 100 µg/ml (Fig. 1B), suggesting that all products are suitable for further in vitro examination. It has been reported previously that C6 and C10 compounds at low concentration may cause an alteration in cell membrane structures. However, the cytotoxic effects on mammalian cells may not reflect the adverse effects in humans and animals because of many physiological factors, including mucins and serum .
In order to examine antiviral effects of chemical/disinfectants, information of viral strain used in whole experiments is very important, including genetic characterization. In order to evaluate the antiviral effects of MCFAs, an ASFV isolated from an outbreak in the capital Hanoi of Vietnam was employed. Some virus assays, including virus isolation in combination with HAD assay and conventional PCR for genotyping and serotyping have been performed to confirm this strain. In Fig. 2A, a strong positive HAD was observed in culture, indicating the success of viral isolation. This isolate was named as Pig/Hanoi/2019/01. Further conventional PCR was conducted for verification of HAD positive data as recommended by OIE and genetic characterization of Pig/Hanoi/2019/01 based on the sequencing analysis of the p72, CD2v, and TRS region gene. As expected, positive 478-bp, 816-bp, and 356-bp bands of PCR products were appeared on gel, respectively (Fig. 2B).
Nucleotide sequence comparisons using the Basic Local Alignment Search Tool (http://blast.ncbi. nlm.nih.gov/Blast.cgi) revealed that the p72 and CD2v sequences of Pig/Hanoi/2019/01 were 100% identical to those of China isolates of ASFV SY18, Pig/HLJ/2018, AnhuiXCGQ, and CN201801 [3, 23, 24]. Phylogenetic analysis of p72 and CD2v of ASFV strain Pig/Hanoi/2019/01 demonstrated that the ASFV, Pig/Hanoi/2019/01, belongs to genotype II and serotype 8, and closely related with ASFVs isolated from China in 2018 , Russia in 2012 and 2017  and Estonia 2014  (Fig. 3A-B). On the other hand, the TRS regions in the IGR between I73R and I329L has been extremely useful for resolving epidemiologic complexities at the genotype, region, and country levels [27–29], such that additional genome markers are required to evaluate the origin and to map the outbreak of closely related ASF isolates circulating in Viet Nam. Our results showed that the Pig/Hanoi/2019/01 strain had an intergenic region II variant with an additional 10 nucleotide (5′-GGAATATATA-3′) into the TRS region between the I73R and I329L genes (Fig. 3C). The intergenic region II variant of the Pig/Hanoi/2019/01 strain was identified to those described in isolates Ukr12/Zapo, Belgium 2018/1 . The same TRS insertion was also found in China isolates of ASFV SY18, Pig/HLJ/2018, AnhuiXCGQ, and CN201801 [3, 23, 24] and differ from ASFV isolated in Georgia in 2007 . The sequences of p72, CD2v, and TRS genes of Pig/Hanoi/2019/01 strain have been deposited in GenBank as the accession numbers of MT332151-3, respectively.
Recent research suggested that the susceptibility to chemicals/disinfectants depends on viral characteristics in which non-enveloped viruses are more resistant than enveloped viruses . Moreover, some chemical synthesis or extract from a plant such as an acacetin, apigenin, genkwanin, rhoifolin, vitexin, and vitexin 2-O-rhamnoside are able to inhibit or reduced ASF virus-specific protein synthesis and viral factory formation in Vero cell line system, in which apigenin showed potent inhibition of ASF virus-infected Vero cells with not display a cytopathic effect [17, 18, 32]. However, there is no evidence of the survivability of ASFV in feed and water after pre-treatment with MCFAs. On the other hand, a recent report demonstrated that the activity of MCFAs could enhance the RNA degradation and mitigating of PEDV in swine feed and ingredients . To investigate the potent effects of MCFAs to reduce Pig/Hanoi/2019/01 infectivity, we used both in vitro feed and water models in which MCFA pre-treated feed and water were spiked to Pig/Hanoi/2019/01 isolate at 105 and 101HAD50, respectively and the samples were collected after 24 h post-inoculation. Real-time PCR amplification of the p72 gene was performed to detect the presence of viral DNA in feed and water based on OIE recommended protocol. A recent in vivo report has indicated that the minimum infectious dose of ASFV in feed is 104TCID50 in which 40% of pigs orally exposed to ASFV have shown s positive result, while other study suggested that minimum dose of ASFV oral exposure should be 105 (Niederwerder et al., 2019). The results of MCFA pre-treated feed exposed to ASFV at the dose of 105HAD50 are summarized in Fig. 4. No Cq value was obtained in the negative control group, while feed exposed to ASFV without MCFA treatment as a positive control group shown the Cq value ranged from 25.85 ± 0.67 to 26.52 ± 0.82 after 24 h. It is indicated that the ASFV could survive in the feed consumption and can be spread the ASFV in the new population of the pigs industry [11, 33, 34]. In feed experiment group, a strong anti-ASFV effect was observed in all product treatment after 24 h post-inoculation. Pre- treatment with product 1 significantly reduced ASFV replication at two highest doses of 0.375 and 0.5% (P < 0.01) (Fig. 4). Product 2 has shown the potential susceptibility to ASFV isolated in Vietnam in which all doses (0.125% and 0.25% with P < 0.05; 0.375%, and 0.5% with P < 0.01) induced a statistical increase in Cq value when compared to a positive control. On the other hand, at the dose of 0.25%, product 3 caused significantly enhancement at the levels of the Cq value when compared to a positive control (P < 0.05) and a significant increase in dose- dependence was recognized at doses 0.375 and 0.5% in product 3 group (P < 0.01) (Fig. 4). These results demonstrated that all products of MCFAs significantly increased in the Cq value when compared to the positive control (P < 0.01) at the highest doses of 0.375% and 0.5% at 24 h after virus incubation (Fig. 4). Additionally, our findings also indicated that MCFAs in synergistic forms have shown promising candidates to reduce ASFV infectivity in which product 2 reduced ASFV infectivity at the lowest dose of 0.125%, while a significant increase in Cq value was noted in product 3 group at a dose of 0.25% (P < 0.05), suggesting that synergistic MCFA C6-C8-C10 is the most potential MCFAs against ASFV in feed model. It is clear that the antiviral effects of MCFAs on the reduction of ASFV infectivity in feed depends on the composing materials in the mixture and the dose. Recent study in the effects of MCFAs on PEDV infectivity has indicated that C8 component has shown the strongest antiviral effect as an individual component and MCFA blend (C8-C6-C10) is promising product to reduce PEDV in feed. An agreement between our data on ASFV and previous study on PEDV in feed was recognized in this study.
One of the most important steps when performing “feed and water biosecurity” is disinfection, and it may reduce or inhibit the risk of the ASFV contamination in the environment or other pathogens . Recent report demonstrated that the ASFV could be directly transmitted to contract pigs by contaminated fomites, drinking water, facilitate pig industry , excretions , and carriers . The minimum infectious doses of ASFV for water via the oronasal route was estimated to be 100TCID50, respectively, suggesting that the small amount of infective material could lead to ASFV transmission to the healthy pig industry . To further evaluation of MCFA- activities to reduce ASFV infectivity in swine water consumption, the effect of MCFAs at dose- and component- dependence were also tested. These results shown in Fig. 5 indicated that all products have shown a strong antiviral effect against ASFV infectivity at doses of 0.375% and 0.5% (P < 0.05) in water assays. At the dose of 0.25%, pre-treatment with product 2 and 3 induced significant increases at the level of Cq value, while at lowest dose of product 3 (0.125%), a statistical enhancement was noted after 24 h post incubation, suggesting that product 3 (C8-C10-C12) is most potential candidate as MCFA additives to reduce ASFV contamination in water model at the time point of 24 h. Further studies in time dependence should be performed to obtain the best understanding of MCFA blend activities against ASFV in swine feed and water consumption. Interestingly, a good correlative result obtained in both in vitro feed and water experiments was observed in which MCFAs, including product 1, product 2, and product 3 at doses of 0.375% and 0.5% have reduced markedly the survival of ASFV after 24-hour post-inoculation at ASFV titration of 105HAD50 and 101HAD50, respectively and MCFAs in synergistic forms are most promising candidates to reduce ASFV infectivity in swine feed and water consumption at the dose of 0.25%. Additionally, the potential effects of MCFAs, individual and synergistic forms, on ASFV infectivity are dose- and component- dependence. It has been indicated that, MCFAs consists of four chemical structures, including caproic acid (C6), caprylic acid (C8), capric acid (C10) and lauric acid (C12). The antimicrobial activities of MCFA have been reported previously. Some studies have suggested the potential antiviral effects of caprylic acid (C8) and/or capric acid (C10) on vesicular stomatitis virus or visa virus . Additionally, the synergistic effects of MCFAs have shown more effective against pathogens when compared to their individual form in which combined effects of C8 and C10 caused a significant decline of bacterial flora, while no efficacy was found by individual C8 or C10 . As a shortest member of MCFA family, caproic acid C6 and its antimicrobial effects on bacterial growth have been reported in previous study in which at the dose of 25 µg/mL this acid has shown a completed inhibition on actinobacillus actinomycetemcomitans growth, a gram-negative bacterium . The significant activities of C6 in combination with C8 and C10 against PEDV in previous study and our current ASFV experiment indicated that, this compound possessed both anti-bacterial and viral properties. Also, other studies have demonstrated that among all MCFAs, C12 component is majority of antibacterial activities [37, 38]. The role of C12 component in synergistic MCFAs was markedly noted in our feed and water experiment, suggesting the susceptibility of lauric acid C12 to viral pathogens. Further studies are essential to elucidate the chemical properties and antiviral activities of this compound. The anti-bacterial effects on MCFAs have been reported in both in vitro and in vivo studies, however the study of their effects on viral pathogens are limited. Although, it is difficult to be definitive regarding to molecular mechanism underlying MCFA, both individual and synergistic forms, activities against ASFV infectivity, it could be investigated in further studies, particularly in vivo model.