Natural Oil Blend Formulation (NOBF) as an anti- African Swine Fever Virus (ASFV) agent in Primary Porcine Alveolar Macrophases (PAM) cells of Swine

African swine fever is one of the severe pathogens of swine. It has a significant impact on production and on economics. So far, there are no known remedies, such as vaccines or drugs, reported. The natural oil blend formulation (NOBF) successfully tested against the African swine fever virus (ASFV) in in vitro conditions. The natural oil blend formulation (NOBF) combines Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia. We used a two-fold serial dilution to test the NOBF formulation dose. The in vitro trial results demonstrated that NOBF up to dilution 13 or 0.000625 ml deactivates the lethal dose 105HAD50 of ASFV. There was no hemadsorption (Rosetta formation) up to dilution 12 or 0.00125 ml of NOBF. The Ct value of the NOBF group at 96 hours post-infection was the same as the initial value or lower (25), whereas the Ct value of positive controls increased several folds (17.84). The in vitro trial demonstrated that NOBF can deactivate the African swine fever virus. HIGHLIGHTS The natural oil blend formulation (NOBF) was formulated using three natural oils, i.e., Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia. The in vitro trial was conducted using porcine alveolar macrophages (PAMs) and further passaged in the PAMs; the stock used in the present study was that obtained after the 15th passage. The natural oil blend formulation (NOBF) showed protection against ASF virus up to a dilution of 13 or 0.000625 of a dilution of 16 or 0.000078 ml that was tried. The real-time PCR analysis showed that the virus did not replicate in the NOBF group, which implies that either ASFV growth was inhibited in the presence of NOBF or that it was inactivated. The in vitro trial outcome showed that NOBF has anti-ASFV properties.


INTRODUCTION
African Swine Fever Virus (ASFV), reported deadly for pigs. It is listed as a "notifiable disease" by the OIE due to high illness rates and a high mortality rate, up to 100%, and substantial financial losses (Penrith et al., 2013and Rowlands et al. 2008, Halasa et al. 2016).
Further spread of ASF to China has had disastrous consequences, especially in lieu of the fact that China contains more than half of the world's pig population (Sánchez-Cordón et al., 2018).
To date, as far as Vietnam is concerned, ASF has appeared in all 63 provinces of Vietnam, has destroyed more than 5.6 million pigs (more than 20% of total pigs), has decreased pork production by 8.3%, and has affected mainly small-scale farms (WOAH 2019).
The typical signs and symptoms of ASF are high fever, decreased appetite and weakness, difficulty in standing, red or blue blotches on the skin (particularly around ears and snout), and especially in sows, the symptoms of miscarriage, stillbirths, and weak litters can occur (WOAH the Asfarviridae family's icosahedral morphology.
The virus can persist for a long time in the environment, carcasses, and various swine products.
The role of natural oils as antiviral components is well known. In both crude and pure forms, as a standardized compound, natural products are significant components with antiviral properties (Jassim and Naji, 2003). A formulation was developed by blending three natural oils, Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia, with antiviral properties.
Cineole, the significant component of eucalyptus oil, has potent anti-inflammatory and antimicrobial properties (Jun et al. 2013). Cineole is also known to treat the respiratory tract's primary viral infections (Muller et al. 2016 andLi et al. 2016). Linalool, a major component of lavender oil, has shown antiviral activities (Choi 2018 andTakizawa et al. 2001). Isobornyl acetate extracted from pine oil has anti-microbial properties (Li et al. 2016).
In the present study, the efficacy of the natural oil bled formulation was evaluated against African swine fever using porcine alveolar macrophage (PAM) cells of swine in an in vitro medium.

Trial Station
In this trial, we selected 7-10 weeks-old healthy pigs negative for ASFV, PCV2, CSFV, PRRSV as well as negative for ASFV Ab. The animals were housed and used in isolated area in the Biosecurity Animal Facility center of the Vietnam National University of Agriculture (VNUA), Hanoi, Vietnam. The Ministry of Agriculture and Rural Development approved by the VNUA Animal Care and Use Committee granted permission for this trial. The study period was between October 2019 and January 2020.

Natural oil blend Formulation (NOBF)
A formulation was developed by mixing an essential oil blend using three oils, Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia, in a determined concentration. The formulation was pre-tested on animals for toxicity and tolerance level. We followed a twofold dilution procedure to obtain the optimum dose of the application. The natural oil blend formulation (NOBF) was serially diluted from 2.5 ml to 0.000078 ml (dilution 1 to dilution 16) to perform the in vitro trials.

Toxicity Test of Natural oil blend Formulation (NOBF)
The toxicity level of NOBF was tested at two levels, first level test was performed in the PAM cells whereas the tolerance level of NOBF was tested in live pigs. The NOBF in 16 double-fold dilutions of 2.5 ml to 0.000078 ml were mixed with the PAM cells and the ASFV. The cell viability was tested under a microscope at every 24-hour interval.

Active ingredient identification in NOBF
The gas chromatography technique was used to extract and identify the active ingredients

Primary porcine alveolar macrophages (PAM) cells
Our team collected the primary porcine alveolar macrophages (PAMs) from 7 weeks-old healthy pigs (negative for ASFV, PCV2, CSFV, PRRSV). We maintained the cells in the growth medium, including an RPMI 1640 medium (Gibco, USA) supplemented with 10% fatal calf serum (FCS; Gibco, USA), 1% penicillin-streptomycin solution (Gibco, USA) at 37°C with 5% CO 2 . We also prepared red blood cells from EDTA-treated swine blood by using Percoll (GE Healthcare, USA) and kept it in an RPMI 1640 medium (Gibco, USA), 1% penicillin-streptomycin solution, and maintained it at 4 o C until use.

African swine fever virus preparation
The VNUA-ASFV-L01/HN/04/19 virus strain was isolated from a pig with apparent symptoms in Thai Binh province, Vietnam. The virus strain was purified and quantified in the molecular biology laboratory of VNUA, Vietnam. The lethal and sublethal doses were optimized in the controlled conditions. The ASFV strain VNUA01/04.2019 was adapted to grow in porcine alveolar macrophages (PAMs), and further passaged in PAMs. The stock used in the present study was that obtained after the 15 th passage. Briefly, the PAMs were infected at a multiplicity of infection (MOI) of 0.1 with VNUA-ASFV-L01/HN/04/19 in the growth medium, including an RPMI 1640 medium supplemented with 10% fatal calf serum (FCS) and 1% penicillin-streptomycin solution. We added to each well the maintenance medium containing an RPMI 1640 medium supplemented with 5% fatal calf serum (FCS), 1% penicillin-streptomycin solution, and 2% suspension of red blood cells.
The team performed the ASFV titration on the PAMs cultures in 96-well plates. The presence of ASFV was assessed by hemadsorption (HAD). The observation of HAD was for 5 days, and the calculation of 50% HAD observation (HAD50) was performed by using the method described by Reed and Muench (1938).

Anti-viral activity of natural oil blend formulation (NOBF) using an in vitro medium
The PAMs were grown on 48-well tissue culture plates using a growth medium, including an RPMI 1640 medium supplemented with 10% fatal calf serum (FCS) and 1% penicillinstreptomycin solution. The natural oil blend formulation (NOBF) was serially two-fold diluted (  At the 10 5 HAD50 at the ratio of 1:1, duplicate cultures were infected with the corresponding virus in a diluted volume of a medium containing the NOBF at 37 o C in an atmosphere of 5% CO 2 for 1 hour (Table 1 and Figure 1). Cultures were added to the maintenance medium and incubated until a massive cytopathic effect, such as hemadsorption (HAD) or rosette formation.
Rosette formation was observed daily by an inverted microscope for 4-5 days. After four freeze-thaw cycles, the supernatants were assessed for the ASFV virus by real-time PCR using the recommendations in the OIE manual described by King et al. 2003.

RESULTS AND DISCUSSION
African swine fever is a highly contagious fatal acute hemorrhagic viral disease of pigs that currently has no treatment or vaccination protocol. It threatens the pig industry worldwide.

The active ingredient natural oil blend formulation (NOBF)
The gas chromatography technique measures the active ingredients present in the blended oil. By comparing with chromatographs of pure standards, GC analysis identified the presence of anti-viral compounds, namely, A) Cineole B) Linalool and C) Isobornyl acetate in the NOBF (Figure 2). The result shows that Cineole was present at a retention time of 3.048 minutes with a relative percentage area of peak around 99.4%, which makes it the primary compound in the NOBF. The second compound identified is Linalool at a retention time of 5.966 minutes and a relative percentage area of peak around 0.31%. The minor compound identified is Isobornyl acetate at a retention time of 3.767 and a relative percentage area of peak around 0.28% (Table 2).

Toxicity test and efficacy of natural oil blend formulation (NOBF) in PAMs culture against African swine fever virus
The in vitro level trials using the natural oil blend formulation were conducted to evaluate camphor are also present in low to moderate qualities. Linalol has antimicrobial, anti-inflammatory, and mood alleviating effects (Shellie et al. 2002, Johanna et al. 2014. Pine oil consists mainly of alpha-terpineol or cyclic terpene alcohols and isobornyl acetate. Pine oil is a phenolic disinfectant that is mildly antiseptic and has anti-fungal, anti-bacterial, and anti-viral properties due to isobornyl acetate (Shellie et al. 2002, Johanna et al. 2014.
Eucalyptus oil has a history of full application as a pharmaceutical, antiseptic, repellent, flavoring, fragrance, and for industrial use. Eucalyptus oil has anti-bacterial, anti-viral, and anti-inflammatory effects; pre-clinical results also show that eucalyptus oil stimulates the innate cell-mediated immune response by its effects on the phagocytic ability of human monocyte-derived macrophages ( Shellie et al. 2002, Johanna et al. 2014, Juergens et al. 2003and 2004, Serafino et al. 2008and Gobel et al. 1994.  The PAM cells were isolated from the pathogen-free clean piglets. The characteristic feature of the ASF virus cells is to adsorb swine erythrocytes (hemadsorption) on its surface. This property has been successfully exploited to differentiate the ASF virus from other agents that produce diseases with symptoms likely to be confused with those observed in ASF and to develop specific techniques for ASF virus titration (Galindo et al. 2000). Erythrocyte rosettes formation around infected blood swine monocytes is a characteristic feature of ASFV-infected cells (Hess andDe Tray 1960, Malmquist andHay 1960). It is considered the standard hemadsorption test for many ASFV isolates (Enjuanes et al. 1976, Galindo et al. 2000. The detailed stepwise observation at 96 hours post-infection occurred as follows: In vitro observation showed that the natural oil blend formulation (NOBF) up to dilution 13 or 0.000625 ml could inhibit or degenerate ASFV at a titer of 105HAD50 in the PAMs culture (Table 1)

Real-time PCR analysis of in vitro trial
After four freeze-thaw cycles of PAMs, the supernatants were collected and applied for total DNA extraction, then used for real-time PCR. The real-time PCR results (Ct value) indicated that the virulent ASFV strain was unable to replicate or was denatured in the PAMs cultures (Table 3). No difference in Ct value was observed in the initial ASFV input control as compared to the natural oil blend group at dilutions 10, 11, and 12 after 130 hours post-infection in PAMs (Table 3). Remarkably, the difference in the Ct value of the natural oil blend group was statistically significant compared with the ASFV positive control group (Table 3).

HAD β /real-time PCR No Groups Dilution Rosetta formation
Real-time PCR Ct value (mean) 7 Input control of ASFV α 25.74 α Initial ASFV dose that is used to apply all groups except negative control group.

CONCLUSION
Although ASF was first described almost a century ago, controlling the disease has proven to be a challenge, mainly because no effective treatment or vaccine is available. Live-attenuated vaccines that are widely used experimentally protect some animals against infection challenges with a homologous virus, but not with a heterologous one, and most of them become carriers of the ASF virus. Inactivated vaccine or viral protein vaccines do not appear to induce any significant protection. ASF virus genes may play a role in the modulation of protection that has been researched. However, until now, the only treatment for ASF is eradication, based on the control of animal transport and vectors and the early detection and slaughter of infected and carrier animals. The role of natural oil extracts in virus eradication has been recognized and considered. In this study, we try to demonstrate that the natural oil blend formulation can degenerate the African Swine Fever Virus (ASFV).
The trial outcome can be summarized in the following way: the natural oil blend formulation (NOBF) application deactivated the ASF virus at the lowest concentration of 0.000635 ml.
As a continuation of the study, the next step is to conduct in vivo trials to optimize the dose and delivery route of the natural oil blend formulation (NOBF) and to establish it as an anti-ASFV candidate.