Negative Regulation of Srebp-1 Fas

1 Negative regulation of SREBP-1/FAS signalling molecules activates the RIG-1/TBK1-mediated
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2 IFN-I pathway to inhibit BVDV replication
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3 Shanshan Liu1, An Luo1, Taolin Que1, Yuxin Liang1, Yuxin Song1, Tianyi Liu1, Jing Li1, Nan Li1,
4 Zechen Zhang1, Yu Liu1, 2, 3, Zecai Zhang1, 2, 3, Yulong Zhou1, 2, 3, Xue Wang1, 2, 3 and Zhanbo Zhu1, 2,
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5 3*
6 1College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University,
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7 Daqing, 163319, China
8 2Key Laboratory of Bovine Disease Control in Northeast China, Ministry of Agriculture and Rural
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Affairs, Daqing 163319, China
3Engineering
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Research Center for Prevention and Control of Cattle Diseases, Heilongjiang Province,
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11 Daqing 163319, China
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13 *Corresponding author: Zhanbo Zhu

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14 E-mail address: zhanbozhu@byau.edu.cn
15 Present address: College of Animal Science and Veterinary Medicine, HeiLongJiang BaYi
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16 Agricultural University, Daqing 163319, China. Tel.: +86-459-681-9969.
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18
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4823019
20 Abstract
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21 For many viruses, controlling the process of infection is largely dependent on the enzymes of the
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22 fatty acid synthesis (FAS) pathway. An appealing therapeutic target in antiviral research is fatty acid
23 synthetase (FASN), a crucial enzyme in the FAS pathway. Bovine viral diarrhea, caused by the
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24 Bovine viral diarrhea virus (BVDV), is a significant viral infectious disease posing a substantial
25 threat to global animal husbandry. Our study revealed that BVDV infection not only upregulates the
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26 expression of FAS-related enzymes in BT cells and the blood, liver, and spleen of mice but also
27 markedly enhances the accumulation of lipid droplets, free fatty acids, and triglycerides. The FAS
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pathway plays a pivotal role throughout the entire BVDV replication cycle. Additionally,
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29 administration of the FASN inhibitor C75 and Acetyl CoA carboxylase-1 (ACC-1) inhibitor TOFA
30 significantly reduced the BVDV content in both serum and organs, exhibiting inhibitory effects
31 across diverse viral strains. Intriguingly, We found that RIG-1/TBK1-mediated IFN-I signaling
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32 inhibits SREBP-1/FAS and reduces BVDV replication. Conversely, targeting a few essential
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33 enzymes of SREBP-1/FAS also activates IFN-I signaling. More importantly, FASN inhibitor led to
34 heightened expression of ISGs in mouse spleens by activating the RIG-1/TBK-1 pathway. These
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35 findings underscore the potential of FASN inhibitor in mitigating BVDV proliferation through the
36 activation of the RIG-1/TBK-1 pathway to induce ISGs, and offering a novel therapeutic approach
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37 for combating BVDV. Thus, it is crucial to negatively regulate SREBP-1/FAS signaling molecules in
38 order to create novel antiviral drugs that are safe, effective, and broad-spectrum.
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39 Keywords: Bovine viral diarrhea virus;fatty acid synthetase; IFN-I; ISGs; Antiviral drugs
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40 1. Introduction
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41 Bovine Viral diarrhea virus (BVDV) is classified as a distemper genus of Flaviviridae and causes
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42 bovine viral diarrhea/mucosal disease(Ridpath, 2008). In general, BVDV isolates are classified into
43 non-cytopathic and cytopathic biotypes based on their ability to cause significant cytopathic changes
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44 and cell death in cell cultures(Ammari et al., 2010; Ammari, 2012). Nucleotide sequence
45 comparisons of viral RNA reveal three BVDV genotypes: BVDV-1, BVDV-2, and BVDV-3, with
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47
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both cytopathic and non-cytopathic variants represented within each genotype(Mirosaw et al., 2017;
Rivas et al., 2022). The two main consequences of BVDV infection are persistent infection and
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48 immunosuppression(Walz et al., 2020). Mucosal disease is a rare form of infection in persistently
49 infected cattle and is usually fatal. Clinical symptoms vary from mild to severe, encompassing fever,
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50 depression, diminished milk production, abortion, diarrhea, and, ultimately, death(Lanyon et al.,
51 2014). BVDV infection is associated with elevated morbidity and mortality rates, alongside
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52 significant declines in cattle reproductive efficiency, resulting in substantial economic

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53 losses(Scharnbck et al., 2018). Hence, prioritizing biosecurity measures and implementing
54 vaccination strategies for infection prevention and control are imperative for safeguarding the
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55 economic sustainability of the global cattle industry.
56 Targeted inhibition of fatty acid synthetase (FASN) plays a crucial role in curbing viral replication.
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57 Lipids can act as direct receptors for all types of viruses on the cell surface or endosomes or as
58 cofactors within cells(Bagam et al., 2017; Taube et al., 2010). They also play an important role in the
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59 formation and function of viral replication complexes and the production of energy required for
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60 efficient viral replication(Nagy et al., 2016). In addition, lipids regulate not only the assembly,
61 transport and release of viral particles but also the distribution of viral proteins(Meyers et al., 2016;
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62 Ono et al., 2004; Zhang et al., 2000). Fatty acid (FA) or cholesterol synthesis uses citrate as the main
63 carbon source, which can usually cross the mitochondrial membrane and undergo cleavage into
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64 acetyl-CoA, with acetyl-CoA carboxylase (ACC) subsequently producing malonyl-CoA(Girdhar et
65 al., 2021a; Hodson and Gunn, 2020). Conversely, FASN catalyzes the production of palmitic acid
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(PA) (C16:0) from acetyl-CoA and malonyl-CoA(Sanchez and Lagunoff, 2015). Palmitic acid
undergoes further processing for utilization in cell membrane synthesis, lipid droplet storage, or
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68 palmitoylation of host and viral proteins. Additionally, FAS produces large amounts of ATP through
69 β -oxidative metabolism(Hardie and Pan, 2002). Cholesterol and fatty acids are essential for viral
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70 replication because they make up the main components of the viral envelope (Desselberger et al.,
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71 2022; Theken et al., 2021). Therefore, the metabolism of these lipids has been shown to be necessary
72 for the replication of various viruses. The FASN enzyme assumes a central role in this process,
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73 governing fatty acid synthesis and exerting regulatory control over viral replication. Certain viruses
74 exploit this mechanism by upregulating FASN expression and augmenting its activity(Heaton and
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75 Randall, 2011). Aerobic glycolysis, fatty acid synthesis, and glutamate breakdown are induced by
76 most flaviviruses, including hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), West
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77 Nile virus (WNV), Japanese encephalitis virus (JEV), and classical swine fever virus (CSFV), within
78 host cells. Inhibition of the fatty acid pathway has been demonstrated to reduce viral
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79 replication(Sumbria et al., 2021).
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80 Studies have shown that many viruses stimulate the synthesis of cholesterol and fatty acids, a process
81 counteracted by type I interferon signaling (IFN-I), which limits the synthesis of fatty acids and
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82 cholesterol(Fritsch and Weichhart, 2016). Moreover, studies highlight the role of type I IFN in
83 modulating cellular lipid metabolism by inhibiting de novo fatty acid synthesis and cholesterol
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84 synthesis while enhancing the uptake of exogenous lipids, thereby bolstering antiviral
85 resistance(Aliyari et al., 2022a; Palmer, 2022). Evidence suggests that intracellular lipid
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accumulation down-regulates the IFNAR1 chain of type I interferon receptors, resulting in impaired
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87 JAK-STAT signaling and diminished IFN- α antiviral response to HCV(Gunduz et al., 2012).
88 Investigations have demonstrated that CSFV-induced FFAs accumulation promotes viral infection,
89 and by inhibiting RLR signaling, CSFV can provide necessary ATP for viral replication while
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90 inhibiting type I IFN production, thus leading to sustained survival within host cells (Shengming et
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91 al., 2019). The induction of Fatty Acid Oxidation (FAO) to divert fatty acids from biosynthesis to
92 catabolism holds promise as a potential novel antiviral mechanism (Wu et al., 2016). However,
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93 additional investigations are imperative to elucidate the novelty and efficacy of this antiviral
94 approach. Notably, there are few studies on fatty acid synthesis and related molecular mechanisms of
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95 BVDV. Therefore, this study focuses on a mouse model exposed to BVDV to explore the molecular
96 intricacies of BVDV replication through the regulation of fatty acid synthesis. The objective is to
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97 pave the way for antiviral therapies targeting fatty acid metabolism as a novel strategy for treating
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98 viral infections.
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99 2. Materials and methods
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100 2.1 Virus
101 Bovine viral diarrhea virus BVDV-1a (strain NADL) and BVDV-1b (strain NY-1) were obtained
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102 from ATCC, and bovine viral diarrhea virus BVDV2 (strain YNJG) was sourced from Yunnan,
103 China.
104 2.2 BT Cells culture er

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105 BT cells were inoculated in a 12-well cell culture plate using DMEM (Gibco, Carlsbad, CA, USA)
106 supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum
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107 (FBS) (Gibco, Carlsbad, CA, USA) free of BVDV and antibodies at 37°C and 5% CO2. The cells
108 were infected with NADL, NY-1 and YNGJ at a multiplicity of infection (MOI) of 0.1.
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109 2.3 Animal care

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110 The experimental subjects comprised BALB/c mice, aged 6-8 weeks and weighing 18-20g, obtained
111 from Harbin Medical University. The mice were bred and raised in an individually ventilated cage
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112 (IVC) system with controlled environmental conditions: a 12-h light/dark cycle, ambient temperature
113 maintained at 20-23°C, relative humidity ranging from 40-60%, and ad libitum access to standard
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114 rodent food and water.

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115 2.4 Moral statement
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116 This study adhered to the principles outlined in the Basel Declaration and followed the guidelines
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117 established by the College of Animal Science and Veterinary Medicine of Heilongjiang Bayi
118 Agricultural University. The experimental protocol was approved by the Management Committee of
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119 the Experimental Animal Center of Heilongjiang Bayi Agricultural University (DWKJXY2022029).
120 Throughout the study, all standard biosafety and institutional safety procedures prescribed by the
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121 Experimental Animal Center were strictly observed.
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2.5 Animal experiment
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The BVDV mouse infection model was based on previous studies in our laboratory(Liu et al., 2021a).
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124 Each experimental group consisted of 6 mice, intraperitoneally infected with 105 copies of the NADL
125 strain (BVDV-1), NY-1 strain, and YNGJ strain (BVDV-2), while the control group received an
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126 equivalent dose of DMEM. C75 ( (HY-101068, MCE, USA) ) and TOFA (HY-12364A, MCE, USA)
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127 were initially dissolved in DMSO and subsequently diluted with DMEM. The DMSO control group
128 received treatment with an equal concentration of DMSO solution. Blood samples were collected
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129 from the tail vein on days 0, 1, 2, 3, 4, 5, 6, and 7 post-infection. Following the last drug treatment,
130 24 h later, blood was obtained by removing the eyeball. The resulting supernatant was collected and
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131 stored at -80 ℃.
132 2.6 Fluorescent quantitative PCR

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133 Mouse liver and spleen tissue (20 mg) were harvested and tissue homogenization was achieved by
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134 grinding, followed by the addition of 1 mL TRIzol reagent (15596026, Invitrogen, USA) for tissue
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135 homogenate splitting. Subsequently, reverse transcription was performed using the HiScript II Q RT
136 SuperMix for qPCR kit (R223-01, Vazyme, China) to synthesize cDNA. Real-time fluorescence
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137 quantitative PCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Q711-02,
138 Vazyme, China) on the CFX96TM Optics Module Real-Time PCR system (Bio-Rad, USA).
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139 Shanghai Sangong Biotechnology Company synthesized primers. Relative mRNA expression levels
140 were quantified using the 2-ΔΔCt method and normalized to the housekeeping gene beta-actin.The
141 gene primer sequences were listed in Table 1.

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142 2.7 Western blot
143 Mouse liver and spleen tissues (20 mg) were excised using ophthalmic scissors and placed in RIPA
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144 lysis buffer (P0013B, Beyotime, China) supplemented with 1 mM PMSF (ST506, Beyotime, China).
145 The extracted proteins were quantified using a BCA protein detection kit (P0012, Beyotime, China).
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146 Equal amounts of protein samples were separated on a 10% SDS-PAGE gel and transferred to a
147 polyvinylidene fluoride (PVDF) membrane for WB analysis. The main antibodies used include are
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148 Fatty Acid Synthase (#3108, 1:1000, Cell Signaling, USA), SREBF1 (14088-1-AP, 1:3000,
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149 Proteintech, China), PI3K (A11526, 1:1000, ABclonal, China), p-PI3K (A22996, 1:1000, ABclonal,
150 China), AKT (10176-2-AP, 1:1000, Proteintech, China), p-AKT (Ser473) (80455-1-RR, 1:1000,
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151 Proteintech, China), RIG-1 (20566-1-AP, 1:4000, Proteintech, China), MDA5 (66770-1-Ig, 1:4000,
152 Proteintech, China), TBK1 (A3458, 1:1000, ABclonal, China), p-TBK1 (AP1026, 1:1000, ABclonal,
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153 China), IRF3 (A19717, 1:1000, ABclonal, China), p-IRF3 (AP0623, 1:1000, ABclonal, China) and
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154 GAPDH (#M20006, 1:8000, Abmart, China). The secondary antibodies utilized are
155 peroxidase-coupled Goat anti-mouse IgG (H+L) (ZB-2305, 1:800, ZSBG-Bio, China) and
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156 peroxidase-coupled Goat anti-rabbit IgG (H+L) (ZB-2301, 1:800, ZSBG-Bio, China).
157 2.8 Free fatty acids (FFAs) and triglycerides (TG)
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158 Mouse serum was separated and stored in equal parts at -20°C or -80°C for later use. The contents of
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160
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FFAs and TG in serum of mice were detected according to the instructions provided by FFAs and TG
detection kit (YX-060601Y, China). Optical density (OD) at 450 nm was measured using a
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161 microplate reader.
162 2.9 Oil red O

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163 Following blood extraction, the chest cavity was opened, and the liver and spleen were fixed in 4%
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164 paraformaldehyde. Subsequently, the samples were immersed in oil red working solution for 8-10
165 min (away from light), followed by washing. Differentiation was achieved with 75% alcohol,
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166 followed by additional washing. Nuclei were restained with hematoxylin for approximately 3-5 min,
167 washed with tap water, and rapidly differentiated with hydrochloric acid alcohol. After further
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168 washing with tap water, the sections were restored to a blue with ammonia aqueous solution and
169 washed again with tap water. Surrounding water was absorbed using filter paper, and the sections
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170 were sealed with glycerin gelatin. Observation of the sections was conducted under a white light
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171 microscope (ECLIPSE CI-L, Nikon, Japan).
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172 2.10 H&E
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173 The liver and spleen were fixed in 4% paraformaldehyde. Tissue samples were then excised with a
174 scalpel as per experimental requirements, dehydrated using an automated dehydration machine, and
175 embedded in paraffin using a paraffin embedding machine. Sections with a thickness of 3-5µm were
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176 dewaxed, stained with hematoxylin and eosin dye, dehydrated, and sealed. The sections were
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178
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observed under a white light microscope (ECLIPSE CI-L, Nikon, Japan).
2.11 Statistical analysis

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179 Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA,
180 USA). The unpaired two-tailed student's t-test was used for comparison between the two groups.
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181 One-way analysis of variance (one-way ANOVA) was used. Data are represented using standard
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182 deviation (±SD) and mean value. Statistical significance was defined as p<0.05.
3. Result
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183
184 3.1 BVDV infection alters the metabolic pathway of BT cells

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185 Studies have shown that viruses can manipulate host metabolism to promote viral replication by
186 disrupting key metabolic pathways and targeting major regulatory proteins of metabolism(Girdhar et
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187 al., 2021b). To investigate the regulation of BT cell metabolism during BVDV infection, we infected
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188 BT cells with different strains of BVDV (NADL, NY-1 and YNGJ, MOI=0.1) for 24h, and collected
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189 cell samples for non-target metabolism detection. KEGG pathway analysis showed that BVDV
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190 infection caused changes in A variety of metabolic pathways, including fatty acid synthesis, amino
191 acid synthesis, pyruvate metabolism and arachidonic acid metabolism (FIG.1 A-C). Studies have
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192 shown that many viruses use fatty acid synthesis to promote their own replication. Subsequently, we
193 analyzed the differential metabolites and found that after BVDV infected BT cells, the differential
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194 metabolites included many fatty acids (FIG.1 D-F). Among them, 18 fatty acids were up-regulated
195 and 2 fatty acids down-regulated in NADL-infected BT cells (FIG.1 G). In NY-1-infected BT cells, 6
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fatty acids were up-regulated and 1 fatty acid was down-regulated (FIG.1 H). In YNGJ-infected BT
cells, 17 fatty acids were up-regulated and 3 fatty acids down-regulated (FIG.1 I).
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198 3.2 BVDV infection upregulates the expression of genes associated with the fatty acid synthesis
199 pathway in vivo/in vitro

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200 To investigate the relationship between BVDV replication and fatty acid synthesis, we initially
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201 assessed the expression of SREBP1 (an important transcriptional regulator that regulates fatty acid
202 synthesis) and enzymes (FASN, ACC-1, and SCD-1) associated with the fatty acid synthesis pathway
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203 in BT cells and mice infected with three BVDV strains (NADL, NY-1, YNGJ). Our results revealed a
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204 significant increase in both mRNA of SREBP-1, FASN, ACC-1, and SCD-1 in BT cells (FIG.2 A-D)
205 and in the blood, liver, and spleen of mice (FIG.2 G-I) during BVDV infection. Subsequently, the
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206 protein levels of fatty acid synthase (FASN), revealing that BVDV infection significantly enhanced
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207 FASN protein expression in BT cells (FIG.2 E) and in the spleen of mice (FIG.2 J). Concurrently, the
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208 transcription factor SREBP, positioned upstream of the cholesterol and fatty acid biosynthesis
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209 pathway, exhibited a substantial increase in BVDV-infected BT cells and in the spleen of mice
210 compared to the control group (FIG.2 F, K). In summary, our findings indicate that BVDV infection
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211 promotes the expression of genes related to the fatty acid synthesis pathway and activates this
212 pathway in mice.
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213 3.3 BVDV infection promotes the accumulation of FFAs, TG and LDs
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Next, we further investigated the impact of BVDV infection on the accumulation of fatty acid
synthesis products (fatty acids and triglycerides). Our investigation revealed that BVDV infection led
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216 to a heightened accumulation of lipid droplets (LDs) in BT cells within 24 h (FIG.3 A). Additionally,
217 oil red O staining of liver and spleen tissues from BVDV-infected mice demonstrated a notable
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218 increase in LDs content in the liver compared to the control group (FIG.3 B, C). However, due to the
219 abundance and density of lymphocytes in the spleen, the effect of oil red staining was less obvious,
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220 with no evident staining observed (FIG.3 I). Nevertheless, we observed a significant elevation in
221 FFAs and TG levels in the serum, liver, and spleen on day 7 post-BVDV infection compared to the
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222 control group (FIG.3 D-I).

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223 3.4 FASN and ACC-1 participate in the BVDV replication cycle
224 To elucidate the specific stage at which FAS contributes to viral replication, we initially examined
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225 whether FAS affects BVDV binding and entry into cells. BT cells were pretreated with different
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226 concentrations of C75 (FASN inhibitor) and TOFA (ACC-1 inhibitor) for 4 h, followed by
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227 inoculation with BVDV (MOI=10) at 4°C for 1 h, and subsequently incubated at 37°C for 0 or 1 h to
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228 assess adhesion and invasion, respectively. Our results revealed that both C75 and TOFA inhibited
229 BVDV adhesion and invasion (FIG.4 A, B). Furthermore, Our findings demonstrated that C75 and
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230 TOFA exerted inhibitory effects throughout the entire cycle of BVDV replication (FIG.4 C, D). To
231 verify the direct inhibition of C75 and TOFA on BVDV viral particles, BVDV particles were
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232 exposed to different concentrations of C75 and TOFA at 37°C for 8 h and then inoculated into cells at
233 37°C for 1 h. After 24 h, cells were collected, and RT-qPCR was performed. Our results indicated
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that C75 and TOFA affected the invasion of BVDV particles into cells (FIG.4 E, F). In summary, our
results suggest that FASN plays a crucial role in the adhesion, invasion, and replication of
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236 BVDV-infected cells.
237 3.5 Pharmacological inhibitors of FASN and ACC reduce BVDV load in vitro/in vivo
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238 To investigate whether inhibiting fatty acid synthesis could inhibit BVDV proliferation, we used the
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239 C75 and TOFA to target FAS pathway. Our results indicated that both C75 and TOFA effectively
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240 reduced BVDV mRNA and E2 protein levels at both 24 and 48 h post-treatment (FIG.5 A, B).
241 Indirect immunofluorescence results further demonstrated significant inhibition of BVDV infection
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242 by C75 and TOFA (FIG.5 C). Subsequently, we assessed the BVDV load in the serum and organs of
243 mice. Notably, we observed a significant decrease in BVDV content in the serum at days 3, 5, and 7
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244 post-treatment (FIG.5 D). Furthermore, examination of BVDV mRNA levels in different BVDV
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245 strains and various organs revealed a substantial reduction in BVDV content in mice following
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246 targeted inhibition of FASN and ACC1 (FIG.5 E-G).
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247 3.6 Over-expression of RIG-1, STING and TBK1 inhibits FAS and reduces BVDV replication
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248 Earlier studies have indicated that the antiviral activity of type I IFN (IFN-I) is linked to the
249 inhibition of cholesterol and fatty acid synthesis(Aliyari et al., 2022a). RNA viruses induce IFN-I by
250 mediating downstream signal transduction of RIG-1 and MDA-5, while cGAS-STING mediated
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251 sensing of cytosolic DNA(Zevini et al., 2017). Therefore, in order to further study the signaling
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pathway of IFN-I induced by BVDV, we transfected over-expression plasmid of RIG-1, STING and
downstream TBK1 into BT cells, and found that the over-expression of RIG-1, STING and TBK1 in
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254 BT cells significantly inhibited the expression of FAS key enzymes (FIG.6 A). However, only
255 over-expression of RIG-1 and TBK1 significantly reduced the mRNA level of BVDV (FIG.6 B). In
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256 addition, over-expression of RIG-1, STING and TBK1 significantly inhibited the protein expression
257 of FASN and SREBP-1 (FIG.6 C-E), while only over-expression of RIG-1 and TBK1 significantly
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258 inhibited the expression of BVDV E2 (FIG.6 F). These data suggest that RIG-1/TBK1-mediated
259 IFN-I signaling can inhibit FAS and reduce BVDV replication.
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260 3.7 Targeting SREBP-1, FASN, and ACC-1 activates IFN-I signaling and inhibits BVDV
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261 replication
262 Studies have shown that FASN is an IFN-I suppressed gene(Aliyari et al., 2022b). To further
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263 investigate whether targeting SREBP-1, FASN and ACC-1 can activate IFN-1 signaling to inhibit
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264 BVDV replication. sgSREBP-1, sgFASN and sgACC-1 plasmids constructed by CRISPR/Cas9
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265 technology were transfected into BT cells for 24h and infected with BVDV NADL strain (MOI=0.1).
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266 The results showed that the mRNA expression of SREBP-1, FASN, ACC-1 and BVDV was
267 significantly decreased in BT cells transfected with sgSREBP-1, sgFASN and sgACC-1 plasmids
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268 (FIG.7 A-D). Subsequently, in the condition of BVDV infection, we found that transfection of
269 sgSREBP-1, sgFASN and sgACC-1 plasmid significantly promoted the expression of RIG-1 and
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270 MDA5 (FIG.7 E-G), resulting in significantly higher phosphorylation levels of TBK1 and IRF3 than
271 vector control group (FIG.7 E, H, I), and inhibited the proteins expression of FASN and BVDV E2
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(FIG.7 E, J, K). This evidence suggests that targeting SREBP-1-regulated fatty acid synthesis can
inhibit BVDV replication by activating RIG-1/MDA5/ TBK1-mediated IFN-Ⅰ signaling.

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274 3.8 Fatty acid synthesis inhibits BVDV replication by regulating RIG-1/TBK-1-mediated IFN-I
275 in mice
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276 To assess whether FASN and ACC-1 inhibitors enhance the body's anti-BVDV abilities by
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277 promoting the IFN-I response, we processed spleen samples from BVDV-infected mice into protein
278 lysates for analysis. Our results showed that the expression of RIG-1 and MDA-5 in the groups
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279 treated with the C75 and TOFA inhibitors was significantly higher than in the untreated group (FIG.8
280 A-C). The activation of RIG-1 and MDA-5 subsequently induced the phosphorylation of TBK1
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281 (FIG.8 A, E) and prompted the expression of IRF3 (FIG.8 A, D). This cascade of events, in turn,
282 stimulated the production of antiviral factors, including ISGs, IFN-α, and IFN-β (FIG.8 G, H),
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283 ultimately inhibiting the expression of the BVDV E2 protein (FIG.8 A, F). These data demonstrate
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284 that the targeted inhibition of FASN impedes BVDV replication by activating the RIG-1/TBK-1
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285 pathway-mediated IFN-I response.
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286 4. Discussion
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287 Studies have shown that viral infections promote fatty acid synthesis. For instance, HCMV induces a
288 shift in glucose metabolism to fatty acid synthesis in HFF cells(Vastag et al., 2011). In the case of
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289 HCV, the increased abundance and activity of FASN contribute to the production of fatty acids and
290 neutral lipids induced by the virus(Nasheri et al., 2013). Long-chain fatty acids are involved in the
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synthesis of triglycerides for energy storage and transportation(Wilfling et al., 2014). Free fatty acids
play a variety of roles in cellular energy and signaling and are key molecules in a variety of
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293 biological states (Mok et al., 2016). Our findings reveal that both the mRNA and protein expressions
294 of genes related to fatty acid synthesis in BVDV-infected BT cells and mouse models were
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295 significantly higher compared to uninfected groups. Simultaneously, we observed a significant
296 increase in the levels of triglycerides (TG) and free fatty acids (FFAs) following BVDV infection.
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297 Lipid droplets serve as storage organelles for lipids, triglycerides, and sterols, contributing to cellular
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298 energy storage. The formation of lipid droplets signifies heightened fatty acid synthesis, preparing the
299 cell for rapid membrane formation and sustaining energy reserves(Sanchez and Lagunoff, 2015).
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300 Dias et al. reported a substantial accumulation of lipid droplets in human neuroblastoma SH-SY5Y
301 cells and neural stem cells (NSC) upon Zika virus (ZIKV) infection(Qin et al., 2022). Our findings
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302 further corroborate this trend, demonstrating increased lipid droplet accumulation in BVDV-infected
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303 BT cells, as well as in the liver of mice. Collectively, this evidence strongly suggests that BVDV
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304 infection induces elevated fatty acid synthesis in mice.
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305 RNA viruses manipulate cytoplasmic lipids during replication to establish a favorable replication
306 milieu(Yang et al., 2008). Fatty acid synthase plays a key role in regulating the entry and production
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307 of HCV(Tongluan et al., 2017a). Similarly, FASN is implicated in the replication of CSFV, where it
308 localizes to the CSFV replication site within the endoplasmic reticulum (ER) and interacts with
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309 NS4B to modulate CSFV replication(Liu et al., 2021b). Additionally, human cytomegalovirus
310 (HCMV) and varicella-zoster virus (VZV) induce FAS activity to generate components found in their
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virion envelopes or glycoproteins(Koyuncu et al., 2013). This reveal that FASN participates
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312 throughout the entire replication cycle of BVDV, including adhesion, entry, and replication.
313 Moreover, when FASN and ACC-1 inhibitors are co-incubated with BVDV, they may disrupt the
314 lipid composition of virions, impairing BVDV replication in BT cells. In summary, FASN is
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315 involved in the entire life cycle of BVDV replication and may be related to the lipid structure on the
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316 surface of BVDV particles.

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317 Numerous studies have demonstrated the reliance of viruses on host cellular metabolic pathways,
318 including nucleotide biosynthesis, glucose metabolism, and lipid synthesis, to facilitate their
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319 replication(Girdhar et al., 2021a). Some viruses even manipulate the metabolic machinery of their
320 host cells to support their own life cycle. For instance, TVB-3166, an inhibitor of FASN and
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321 palmitate synthesis, has been demonstrated to curtail the progeny production of respiratory syncytial
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322 virus (RSV B) and exhibit broad activity against other respiratory viruses, such as human
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323 parainfluenza 3 (PIV3) and human rhinovirus 16 (HRV16)(Ohol et al., 2015). In particular,
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324 pharmacological inhibitors of fatty acid synthesis (TVB-3664, TVB-2640 and orlistat) can block
325 emerging infectious viruses such as SARS-CoV-2(Chu et al., 2021). Studies have shown that C75
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326 can reduce the replication of HCV(Nasheri et al., 2013), dengue virus (DENV)(Tongluan et al.,
327 2017b), CSFV(Liu et al., 2021b) and other flaviviruses by inhibiting FASN. In this study, we found
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328 that the FASN inhibitor C75, along with the ACC-1 inhibitor TOFA, not only attenuated BVDV
329 replication in murine blood but also substantially reduced BVDV levels in various organs compared
330
331
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to the untreated group. More importantly, C75 and TOFA showed inhibitory effects on different
strains of BVDV, including NADL (BVDV-1), NY-1 (BVDV-1), and YNGJ (BVDV-2). Our study
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332 shows that the FASN inhibitor C75 can inhibit BVDV replication in mice.
333 Research indicates that FASN can damage innate antiviral immunity, particularly IFN-I
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334 responses(Martino et al., 2021). Innate antiviral immunity is mainly mediated by IFN-I dependent
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335 induction of ISG, and ISGs co-promote antiviral status. Studies on the novel coronavirus have shown
336 that FASN is inhibited not only in an IFNAR-dependent manner but also in a STAT1-dependent
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337 manner(Aliyari et al., 2022a). Moreover, the expressions of IFN signaling molecules and
338 pro-inflammatory cytokines in SGIV-infected cells were also significantly increased after FAS
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339 inhibitor treatment (Zheng et al., 2022). Transcriptomic analyses have further suggested that FASN
340 serves as a negative regulator of IFN-I responses(Aliyari et al., 2022a). In our study, We found that
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341 when RIG-1/TBK1-mediated IFN-I signaling is activated, it inhibits the expression of FAS related
18
342 genes and the replication of BVDV. Conversely, targeting FAS also activates RIG-1/TBK1-mediated
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343 IFN-I signaling to inhibit BVDV replication. It was demonstrated that RIG-1/TBK1-mediated IFN-I
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344 signaling interacts with FAS to regulate BVDV replication. we also observed activation of the
345 RIG-1/TBK-1 pathway within the IFN-I cascade following intraperitoneal injection of C75 and
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346 TOFA in a BVDV-infected mouse model. This activation led to increased production of IFN-α,
347 IFN-β, and ISGs, thus augmenting the antiviral response. Therefore, we demonstrate that the FASN
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348 inhibitor C75 can inhibit the proliferation of BVDV in mice by activating the RIG-1/TBK1 pathway.
349 5. Conclusion
350
er
Our investigation reveals the fatty acid synthesis is involved in BVDV binding, invasion and RNA
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351 replication. In addition, inhibiting key enzymes FASN and ACC-1 of the fatty acid synthesis pathway
352 can block BVDV replication by inducing RIG-1/MDA-5 dependent IFN-I responses. These findings
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353 offer insights for the development of antiviral drugs and establish a foundation for the prevention and
354 treatment of bovine viral diarrhea.

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355 Funding Statement

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356 This study was supported by the Heilongjiang Province's "Jie Bang Gua Shuai" Science and
357 Technology Research Project (2023ZXJ02B03), National Natural Science Foundation of China
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358 (32072896), Heilongjiang Postdoctoral Found (LBH-Q20054, LBH-Z22233), Natural Science

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359 Foundation of Heilongjiang Province (LH2021C072), Heilongjiang Bayi Agricultural University
360 Talent Research Start-up Fund (XYB202107, XYB202014).

19
361 Disclosure statement
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362 No potential conflict of interest was reported by the author(s).
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363 Author contributions
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364 Z.Z.(Zhanbo Zhu), Y.L. Z.Z. (Zecai Zhang) and Z.Z. (Zhanbo Zhu) contributed to the conception,
365 design and guidance of the study. S.L., T.Q., A.L., Y.L., Y.S., participated in animal experiments and
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366 sample collection. S.L., A.L., Y.L., Y.S., and N.L. conducted the experiments. Acquisition, analysis,
367 or interpretation of data: S.L. Y.L., and Z.Z. (Zhanbo Zhu) drafted the manuscript. All authors have
368 read and agreed to the published version of the manuscript. er

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369 Data availability statement
370 The authors confirm that the data supporting the findings of this study are available within the article
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371 [and/or] its supplementary materials.

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372 References
373 Aliyari, S.R., Ghaffari, A.A., Pernet, O., Parvatiyar, K., Wang, Y., Gerami, H., Tong, A.J., Vergnes, L., Takallou, A., Zhang, A.,
rin
374 Wei, X., Chilin, L.D., Wu, Y., Semenkovich, C.F., Reue, K., Smale, S.T., Lee, B. & Cheng, G., 2022b. Suppressing fatty acid
375 synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2. Acta
376 Pharm Sin B 12, 1624-1635. https://doi.org/10.1016/j.apsb.2022.02.019.
377 Ammari, Mais, McCarthy, Fiona, M., Nanduri, Bindu, Pinchuk & Lesya, M., 2010. Analysis of Bovine Viral Diarrhea
ep
378 Viruses-infected monocytes: identification of cytopathic and noncytopathic biotype differences. BMC Bioinformatics.
379 Ammari, M.G., 2012. https://doi.org/10.1186/1471-2105-11-S6-S9
380 Bagam, P., Singh, D.P., Inda, M.E. & Batra, S., 2017. Unraveling the role of membrane microdomains during microbial
381 infections. Cell Biology and Toxicology 33, 429-455. https://doi.org/10.1007/s10565-017-9386-9.
Pr
20
382 Chu, J., Xing, C., Du, Y., Duan, T., Liu, S., Zhang, P., Cheng, C., Henley, J., Liu, X. & Qian, C., 2021. Pharmacological
383 inhibition of fatty acid synthesis blocks SARS-CoV-2 replication. Nature Metabolism, 3.
d
384 https://doi.org/10.1038/s42255-021-00479-4
385 Desselberger, U., Pohl, C.H. & O'Neill, H.G., 2022. Editorial: Significance of Cellular Lipids for Viral Replication and
we
386 Pathogenesis. Front Physiol 13, 906205. https://doi.org/10.3389/fphys.2022.906205.
387 Fritsch, S.D. & Weichhart, T., 2016. Effects of Interferons and Viruses on Metabolism. Front Immunol 7, 630.
388 https://doi.org/10.3389/fimmu.2016.00630.
389 Girdhar, K., Powis, A., Raisingani, A., Chrudinová, M., Huang, R., Tran, T., Sevgi, K., Dogru, Y.D. & Altindis, E, 2021.
vie
390 Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism. Annu Rev Virol. Sep
391 29;8(1):373-391. https://doi.org/10.1146/annurev-virology-091919-102416
392 Girdhar, K., Powis, A., Raisingani, A., Chrudinová, M., Huang, R., Tran, T., Sevgi, K., Dogus Dogru, Y. & Altindis, E., 2021b.
393 Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism. Annu Rev Virol 8, 373-391.
re
394 https://doi.org/10.1146/annurev-virology-091919-102416.
395 Gunduz, F., Aboulnasr, F.M., Chandra, P.K., Hazari, S. & Dash, S., 2012. Free fatty acids induce ER stress and block
396 antiviral activity of interferon alpha against hepatitis C virus in cell culture. Virology Journal 9, 1-12.
397 https://doi.org/10.1186/1743-422X-9-143
398
399
400
er
Hardie, D.G. & Pan, D.A., 2002. Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase.
Biochemical Society Transactions 30, 1064-1070. https://doi.org/10.1042/bst0301064
Heaton, N.S. & Randall, G., 2011. Multifaceted roles for lipids in viral infection. Trends in Microbiology 19, 368-375.
pe
401 Hodson, L. & Gunn, P.J., 2020. Publisher Correction: The regulation of hepatic fatty acid synthesis and partitioning: the
402 effect of nutritional state. Nature Reviews Endocrinology 16.https://doi.org/10.1016/j.tim.2011.03.007
403 Kanno, T., Nakajima, T., Yokoyama, S., Asou, H.K., Sasamoto, S., Kamii, Y., Hayashizaki, K., Ouchi, Y., Onodera, T.,
404 Takahashi, Y., Ikeda, K., Hasegawa, Y., Kinjo, Y., Ohara, O., Nakayama, T. & Endo, Y., 2021. SCD2-mediated
405 monounsaturated fatty acid metabolism regulates cGAS-STING-dependent type I IFN responses in CD4(+) T cells.
ot
406 Commun Biol 4, 820. https://doi.org/10.1038/s42003-021-02310-y.

407 Koyuncu, E., Purdy, J.G., Rabinowitz, J.D., Shenk, T. & Damania, B., 2013. Saturated Very Long Chain Fatty Acids Are
408 Required for the Production of Infectious Human Cytomegalovirus Progeny. PLOS Pathogens,9,5(2013-5-16) 9,
tn
409 e1003333. https://doi.org/10.1371/journal.ppat.1003333

410 Lanyon SR, Hill FI, Reichel MP, Brownlie J, 2014. Bovine viral diarrhoea: pathogenesis and diagnosis. Vet J.
411 Feb;199(2):201-9. https://doi.org/10.1016/j.tvjl.2013.07.024
rin
412 Liu, C., Liu, Y., Liang, L., Cui, S. & Zhang, Y., 2019. RNA-Seq based transcriptome analysis during bovine viral diarrhoea
413 virus (BVDV) infection. BMC Genomics 20, 774. https://doi.org/10.1186/s12864-019-6120-4.
414 Liu, Y., Wu, C., Chen, N., Li, Y., Fan, C., Zhao, S., Bai, T., Zhao, Z., Chen, J., Su, S., Zhang, Z., Zhou, Y. & Zhu, Z., 2021a. PD-1
415 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c
ep
416 Mouse Model of CP BVDV Acute Infection. Front Immunol 12, 727254. https://doi.org/10.3389/fimmu.2021.727254.
417 Liu YY, Liang XD, Liu CC, Cheng Y, Chen H, Baloch AS, Zhang J, Go YY, Zhou B, 2021. Fatty Acid Synthase Is Involved in
418 Classical Swine Fever Virus Replication by Interaction with NS4B. J Virol. 10;95(17):e0078121.
419 https://doi.org/10.1128/jvi.00781-21
Pr
21
420 Martino, M.D., Daviaud, C. & Vanpouille-Box, C., 2021. Abstract PR-007: Targeting FASN improves type I interferon
421 responses in irradiated glioblastoma, Abstracts: AACR Virtual Special Conference on Radiation Science and Medicine;
d
422 March 2-3.https://doi.org/10.1158/1557-3265.RADSCI21-PR-007
423 Matsuhashi, T., Maruyama, S., Uemoto, Y., Kobayashi, N., Mannen, H., Abe, T., Sakaguchi, S. & Kobayashi, E., 2011.
we
424 Effects of bovine fatty acid synthase, stearoyl-coenzyme A desaturase, sterol regulatory element-binding protein 1, and
425 growth hormone gene polymorphisms on fatty acid composition and carcass traits in Japanese Black cattle. J Anim Sci
426 89, 12-22. https://doi.org/10.2527/jas.2010-3121.
427 Meyers, N.L., Fontaine, K.A., Kumar, G.R. & Ott, M., 2016. Entangled in a membranous web: ER and lipid droplet
vie
428 reorganization during hepatitis C virus infection. Current Opinion in Cell Biology 41,
429 117-124.https://doi.org/10.1016/j.ceb.2016.05.003
430 Mirosaw, P., Antos, A. & Polak, M., 2017. Genetic diversity of bovine diarrhea and mucosal disease virus. Postepy
431 Mikrobiologii 56, 389-394. https://doi.org/10.3389/fvets.2022.1028866
re
432 Mok, H.J., Lee, J.W., Bandu, R., Kang, H.S., Kim, K.H. & Kim, K.P., 2016. A rapid and sensitive profiling of free fatty acids
433 using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) after chemical
434 derivatization. RSC Advances, 6. https://doi.org/10.1039/C6RA01344A
435 Nagy, P.D., Strating, J.R.P.M., Van, K.F.J.M. & Condit, R.C., 2016. Building Viral Replication Organelles: Close Encounters
436
437
438
er
of the Membrane Types. Plos Pathogens 12, e1005912. https://doi.org/10.1371/journal.ppat.1005912
Nasheri, N., Joyce, M., Rouleau, Y., Yang, P., Yao, S., Tyrrell, D.L. & Pezacki, J.P., 2013. Modulation of Fatty Acid Synthase
Enzyme Activity and Expression during Hepatitis C Virus Replication. Chemistry & Biology 20, 570-582.
pe
439 https://doi.org/10.1016/j.chembiol.2013.03.014
440 Ohol, Y.M., Zhaoti, W., George, K., Gregory, D. & Santanu, B., 2015. Direct Inhibition of Cellular Fatty Acid Synthase
441 Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses. Plos One 10, e0144648.
442 https://doi.org/10.1371/journal.pone.0144648
443 Ono, A., Ablan, S.D., Lockett, S.J., Nagashima, K. & Freed, E.O., 2004. Phosphatidylinositol (4,5) bisphosphate regulates
ot
444 HIV-1 Gag targeting to the plasma membrane. Proceedings of the National Academy of Sciences of the United States of
445 America 101, 14889-14894. https://doi.org/10.1073/pnas.0405596101
446 Palmer, C.S., 2022. Innate metabolic responses against viral infections. Nat Metab 4, 1245-1259.
tn
447 https://doi.org/10.1038/s42255-022-00652-3.
448 Qin, Z.L., Yao, Q.F., Ren, H., Zhao, P. & Qi, Z.T., 2022. Lipid Droplets and Their Participation in Zika Virus Infection. Int J
449 Mol Sci 23. https://doi.org/10.3390/ijms232012584.
rin
450 Ridpath, J.F., 2008. Bovine Viral Diarrhea Virus. Encyclopedia of Virology 24, 374-380.
451 https://doi.org/10.1016/j.cvfa.2009.10.007
452 Rivas, J., Hasanaj, A., Deblon, C., Gisbert, P. & Garigliany, M.-M., 2022. Genetic diversity of Bovine Viral Diarrhea Virus in
453 cattle in France between 2018 and 2020. Frontiers in Veterinary Science 9. https://doi.org/10.3389/fvets.2022.1028866.
ep
454 Sanchez, E.L. & Lagunoff, M., 2015. Viral activation of cellular metabolism. Virology s 479–480, 609-618.
455 https://doi.org/10.1016/j.virol.2015.02.038
456 Scharnbck, B., Roch, F.F., Richter, V., Funke, C., Firth, C.L., Obritzhauser, W., Baumgartner, W., Ksbohrer, A. & Pinior, B.,
457 2018. A meta-analysis of bovine viral diarrhoea virus (BVDV) prevalences in the global cattle population. Nature
Pr
458 Publishing Group. https://doi.org/10.1038/s41598-018-32831-2
22
459 Shengming, Qian, Mao, Wenxian, Chen, Mengpo, Zhao, Keke, Dan & Song, 2019. Serum Lipidomics Analysis of Classical
460 Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro. Frontiers in
d
461 cellular and infection microbiology 9, 410-410. https://doi.org/10.3389/fcimb.2019.00410
462 Sumbria, D., Berber, E., Mathayan, M. & Rouse, B.T., 2021. Virus Infections and Host Metabolism—Can We Manage the
we
463 Interactions? Frontiers in Immunology 11, 594963. https://doi.org/10.3389/fimmu.2020.594963
464 Taube, S., Jiang, M. & Wobus, C.E., 2010. Glycosphingolipids as Receptors for Non-Enveloped Viruses. Viruses 2,
465 1011-1049. https://doi.org/10.3390/v2041011
466 Theken, K.N., Tang, S.Y., Sengupta, S. & FitzGerald, G.A., 2021. The roles of lipids in SARS-CoV-2 viral replication and the
vie
467 host immune response. J Lipid Res 62, 100129. https://doi.org/10.1016/j.jlr.2021.100129.
468 Tongluan, N., Ramphan, S., Wintachai, P., Jaresitthikunchai, J., Khongwichit, S., Wikan, N., Rajakam, S., Yoksan, S.,
469 Wongsiriroj, N. & Roytrakul, S., 2017a. Involvement of fatty acid synthase in dengue virus infection. Virology Journal 14.
470 https://doi.org/10.1186/s12985-017-0685-9.
re
471 Tongluan, N., Ramphan, S., Wintachai, P., Jaresitthikunchai, J., Khongwichit, S., Wikan, N., Rajakam, S., Yoksan, S.,
472 Wongsiriroj, N., Roytrakul, S. & Smith, D.R., 2017b. Involvement of fatty acid synthase in dengue virus infection. Virology
473 Journal 14, 28. https://doi.org/10.1186/s12985-017-0685-9.
474 Vastag, L., Koyuncu, E., Grady, S.L., Shenk, T.E., Rabinowitz, J.D. & Lagunoff, M., 2011. Divergent Effects of Human
475
476
477
https://doi.org/10.1371/journal.ppat.1002124 er
Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism. PLoS Pathogens 7, e1002124.
Walz, P.H., Chamorro, M.F., Falkenberg, S.M., Passler, T. & Woolums, A.R., 2020. Bovine viral diarrhea virus: An updated
pe
478 American College of Veterinary Internal Medicine consensus statement with focus on virus biology, hosts,
479 immunosuppression, and vaccination. Journal of Veterinary Internal Medicine. https://doi.org/10.1111/jvim.15816
480 Wilfling, F., Haas, J.T., Walther, T.C. & Jr, R.V.F., 2014. Lipid droplet biogenesis. Current Opinion in Cell Biology 29, 39-45.
481 Wu, D., Sanin, D.E., Everts, B., Chen, Q., Qiu, J., Buck, M.D., Patterson, A., Smith, A.M., Chang, C.H., Liu, Z., Artyomov,
482 M.N., Pearce, E.L., Cella, M. & Pearce, E.J., 2016. Type 1 Interferons Induce Changes in Core Metabolism that Are Critical
ot
483 for Immune Function. Immunity 44, 1325-1336. https://doi.org/10.1016/j.immuni.2016.06.006.

484 Yang, W., Hood, B.L., Chadwick, S.L., Liu, S., Watkins, S.C., Luo, G., Conrads, T.P. & Wang, T., 2008. Fatty acid synthase is
485 up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production. Hepatology.
tn
486 Zevini, A., Olagnier, D. & Hiscott, J., 2017. Crosstalk between Cytoplasmic RIG-I and STING Sensing Pathways. Trends
487 Immunol 38, 194-205. https://doi.org/10.1016/j.it.2016.12.004.
488 Zhang, J., Pekosz, A. & Lamb, R.A., 2000. Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the
rin
489 Cytoplasmic Tails of the Spike Glycoproteins. Journal of Virology 74. https://doi.org/10.1128/jvi.74.10.4634-4644.2000
490 Zheng, Q., Huang, Y., Wang, L., Zhang, Y., Guo, X., Huang, X. & Qin, Q., 2022. SGIV Induced and Exploited Cellular De
491 Novo Fatty Acid Synthesis for Virus Entry and Replication. Viruses 14. https://doi.org/10.3390/v14020180.
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492 FIG.1 BVDV infection alters the metabolic pathway of BT cells
493 After infecting BT cells with BVDV-1 (NADL (CP type), NY-1 (NCP type)) and BVDV-2 (YNGJ
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494 (NCP type)) (MOI=0.1) for 24h, BT cells were collected for non-target metabolism detection. (A-C)
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495 KEGG pathway analysis of BVDV (A: NADL, B: NY-1, C: YNGJ) infected-BT cells. (D-F) Cluster
d
496 analysis of differential metabolites in BVDV (D: NADL, E: NY-1, F: YNGJ) infected-BT cells. (G-I)
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497 Foldchange of fatty acids in BVDV infected-BT cells (G: NADL, H: NY-1, I: YNGJ).
498 FIG.2 BVDV infection up-regulates the expression of genes related to fatty acid synthesis
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499 pathway in vivo/in vitro
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500 After infecting BT cells with BVDV-1 (NADL, NY-1 and YNGJ) (MOI=0.1) for 12h, 24h and 36h,
501 BT cells were lysed with TRZOL to extract RNA. (A-D)The mRNA expression of SREBP-1, FASN,
502
503
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ACC-1 and SCD-1 was detected by RT-qPCR. (E, F) After BT cells were infected with different
BVDV strains (MOI=0.1) for 24h, the cells were lysed with protein lysate, and the protein levels of
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504 FASN and SREBP-1 were detected by Western-blot. (G-I) Mice infected with different BVDV
505 strains (1×105 copies/mL) were infected on the 7 d. Blood, spleen and liver were collected and then
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506 ground with a grinding rod. TRZOL was used to split blood and tissue to extract RNA. The mRNA
507 expression of SREBP-1, FASN, ACC-1 and SCD-1 was detected by RT-qPCR. (J,K) Tissue was
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508 cleaved by protein lysate, and protein levels of FASN and SREBP-1 were detected by protein
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509 imprinting. All data were analyzed by GraphPad Prism, mean±SD. *, p<0.05, **, p<0.01, ***,
510 p<0.001.
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511 FIG.3 BVDV infection promoted the accumulation of FFAs, TG and LDs
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512 (A) After BT cells were infected with different BVDV strains for 12h, oil red dye was added
513 according to the instructions of the oil red staining kit to detect the accumulation of lipid droplets in
24
514 the cells. (B, C) After 7 days of infection with different BVDV strains (1×104 copies/mL), spleen and
d
515 liver were collected and fixed with 4% paraformaldehyde, and then oil red working fluid was added
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516 for tissue oil red O staining. (D, E) Serum was collected and TG and FFAs contents were detected
517 according to TG and FFAs ELISA kit instructions. (F-I) Spleen and liver were collected, and PBS
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518 was added for grinding. Homogenate concentration was detected using BCA kit, TG and FFAs
519 ELISA kit were used to detect TG and FFAs contents. All data were analyzed by GraphPad Prism,
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520 mean±SD. *, p<0.05, **, p<0.01, ***, p<0.001.
521 FIG.4 FASN and ACC-1 participate in the BVDV replication cycle
522
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(A,B) FASN and ACC-1 inhibitors C75 and TOFA were added to pre-treated cells 6h before BVDV
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523 (NADL) (MOI=0.1) infection of BT cells, within 1h incubation of the virus, 1h after incubation of
524 the virus and the whole process of virus infection, and were collected 24h after infection. The
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525 expression of BVDV mRNA was detected by RT-qPCR. (C,D) After pretreating BT cells with
526 different concentrations of C75 and TOFA for 6 h, the cells were inoculated with BVDV (NADL)
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527 (MOI=10) at 4◦C for 1h, and then transferred to 37◦C for incubation for 0h (binding) and 1h (entry).
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528 The expression of BVDV mRNA was detected by RT-qPCR. (E, F) BVDV (NADL) was incubated
529 with different doses of C75 and TOFA at 37◦C for 8h and inoculated into cells at 37◦C for 1h to
ep
530 collect cells. The expression of BVDV mRNA was detected by RT-qPCR. All data were analyzed by
531 GraphPad Prism, mean±SD. *, p<0.05, **, p<0.01, ***, p<0.001.

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532 FIG.5 FASN and ACC pharmacological inhibitors reduce BVDV load in vitro/in vivo
25
533 (A) BT cells infected with BVDV (NADL) (MOI=0.1) were treated with C75 and TOFA. The cells
d
534 were collected at 24h and 48h, and the mRNA expression of BVDV was detected by RT-qPCR. (B)
we
535 The expression of BVDV E2 protein was detected by Western-blot. (C) BVDV (NADL) (MOI=0.1)
536 infected BT cells were treated with C75 and TOFA for 24h, and the number of BVDV infected cells
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537 was detected by IFA (200×). (D) C75 and TOFA were injected intraperitoneally into mice infected
538 with BVDV, and serum was collected every 3d to detect TCID50. (E-G) Mouse organ tissues were
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539 collected, ground into homogenate, RNA was extracted, and the expression of BVDV mRNA was
540 detected by RT-qPCR. All data were analyzed by GraphPad Prism, mean±SD. *, p<0.05, **, p<0.01,
541 ***, p<0.001.

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542 FIG.6 Over-expression of MDA5, STING and TBK1 inhibits FAS and reduces BVDV
543 replication
ot
544 The over-expressed plasmids of RIG-1, STING and TBK1 were transfected for 24 h, and infected
545 with BVDV NADL strains for 24 h. (A) Cell samples were collected to extract RNA, and mRNA
tn
546 expressions of FAS related genes SREBP-1, ACC-1, FASN and SCD-1 were detected by RT-qPCR.
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547 (B) The mRNA expression of BVDV was detected by RT-qPCR. (C) The cells were lysed with
548 protein lysate, and the protein levels of FASN and SREBP-1 were detected by Western-blot.(D)
ep
549 Analyzing the protein level of FASN. (E) Analyzing the protein level of SREBP-1. (F) Analyzing the
550 protein level of BVDV E2. All data were analyzed by GraphPad Prism, mean±SD. *, p<0.05, **,
Pr
551 p<0.01, ***, p<0.001.
26
552 FIG.7 Targeting SREBP-1, FASN, and ACC-1 activates IFN-1 signaling and inhibits BVDV
d
553 replication
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554 The plasmids of sgSREBP-1, sgFASN and sgACC-1 were transfected for 24 h, and infected with
555 BVDV NADL strains for 24 h. (A) Cell samples were collected to extract RNA, and mRNA
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556 expressions of SREBP-1 was detected by RT-qPCR. (B) mRNA expressions of ACC-1 was detected
557 by RT-qPCR. (C) mRNA expressions of FASN was detected by RT-qPCR. (D) The mRNA
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558 expression of BVDV was detected by RT-qPCR. (E) The cells were lysed with protein lysate, and the
559 protein levels of genes associated with IFN-1 signaling pathway were detected by Western-blot.
560 er
(F-K) Analyzing the protein level of RIG-1, MDA5, p-TBK-1/TBK-1, p-IRF-3/IRF-3, BVDV E2,
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561 FASN. All data were analyzed by GraphPad Prism, mean±SD. *, p<0.05, **, p<0.01, ***, p<0.001.
562
ot
563 FIG.8 Fatty acid synthesis inhibits BVDV replication by regulating RIG-1/ TBK1-mediated
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564 IFN-1
565 (A) C75 and TOFA were injected intraperitoneally into mice infected with BVDV, spleen was
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566 collected on day 7 and ground into homogenate. (B) Adding lysate to extract protein and conducting
567 protein imprinting to analyze the protein level of RIG-1 (C) MDA5. (D) Analyzing the protein level
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568 of p-TBK-1/TBK-1. (E) Analyzing the protein level of p-IRF-3/IRF-3. (F) Adding TRIZOL to
Pr
569 extract RNA. The mRNA expression of IFN-α and IFN-β was detected by RT-qPCR. (G) mRNA
570 level of antiviral factor ISGs. All data were analyzed by GraphPad Prism, mean±SD. *, p<0.05, **,
27
571 p<0.01, ***, p<0.001.
d
572
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573
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574
575
re
576
577 er
pe
578
579
ot
580
tn
581
rin
582
583
ep
584
Pr
585
28
586
d
587 FIG.1
we
vie
re
er
pe
ot
tn
588
589
rin
590
ep
591
Pr
592
29
593
d
594 FIG.2
we
vie
re
er
pe
ot
tn
595
596
rin
597
ep
598
Pr
599
30
600
d
601 FIG.3
we
vie
re
er
pe
ot
tn
602
603
rin
604
ep
605
Pr
606
31
607
d
608 FIG.4
we
vie
re
er
pe
ot
tn
609
610
rin
611
ep
612
Pr
613
32
614
d
615 FIG.5
we
vie
re
er
pe
ot
tn
616
617
rin
618
ep
619
Pr
620
33
621
d
622 FIG.6
we
vie
re
er
pe
ot
tn
623
624
rin
625
ep
626
Pr
627
34
628
d
629 FIG.7
we
vie
re
er
pe
ot
tn
630
631
rin
632
ep
633
Pr
634
35
635
d
636 FIG.8
we
vie
re
er
pe
ot
tn
637
638
rin
639
ep
640
Pr
641
36
642
d
643 Table 1. Primer sequence
we
Gene Primer sequence (5′-3′)
Bovine-SREB
vie
Forward ATGCCATCGAAACGCTAC
P-1
Bovine-SREB
Reverse GTCCGCAGACTCAGGTTCTC
P-1
re
Bovine-FASN Forward GGTGCGTCCTGGTGTCTAA
Bovine-FASN Reverse CCTCGGGTGAGGACATTTAT
Bovine-ACC-
1
Forward GCTATGGAAGTCGGCTGTGGAAG er (Matsuhashi et al.,
2011)
pe
Bovine-ACC-
Reverse GGAAGAGGCGGATGGGAATTGC
1
Bovine-SCD-
Forward CTACACAACCACCACCACCATCAC
ot
Bovine-SCD-
Reverse CTCTCATTTCAGGGCGGATGTCTTC
tn
Bovine-ISG20 Forward GGCCCGAGGGTGACATCAC
Bovine-ISG20 Reverse CAGCGCCTTGAAGTCGTGCT

rin
Bovine-ISG15 Forward TCGCCCAGAAGATCAATGTG
Bovine-ISG15 Reverse AGCACCTCCCTGCTGTCAAG

(Liu et al., 2019)
ep
Bovine-IFITM
Forward CTACCGCCAAGTGCCTGAA
1
Bovine-IFITM
Pr
Reverse AATGAGAAGAACGATCGATCCAA
1
37
Bovine-IFITM
Forward GCTTAAGGAGGAGCACGAGG
d
3
we
Bovine-IFITM
Reverse TGAACAGGGACCACACGATG
3
Bovine-MX1 Forward ACATGATCGTCAAGTGCCGT
vie
Bovine-MX1 Reverse AAGGTCCCTGAAATGTGCGT
Bovine-OAS1 Forward CTCACAGAGTTCGGGTGTCC
Bovine-OAS1 Reverse TGCCGTTTCTGGACCTCAAA
re
Bovine-IFNA Forward GTGAGGAAATACTTCCACAGACTCACT
Bovine-IFNA Reverse TGAGGAAGAGAAGGCTCTCATGA
Bovine-IFNB Forward CCTGTGCCTGATTTCATCATGA
Bovine-IFNB
Mouse-SREB
Reverse GCAAGCTGTAGCTCCTGGAAAG er
pe
Forward ATCGGCGCGGAAGCTGTCGGGGTAGCGTC
P-1
Mouse-SREB
Reverse ACTGTCTTGGTTGTTGATGAGCTGGAGCAT
P-1
ot
Mouse-FASN Forward GCTGCTGTTGGAAGTCAGC
Mouse-FASN Reverse AGTGTTCGTTCCTCGGAGTG

tn
Mouse-ACC-1 Forward CCAATGGTTGAGTGGGTTTT

(Kanno et al., 2021)
Mouse-ACC-1 Reverse CGGGTATTCCCCCTAACCT
rin
Mouse-SCD-1 Forward TGGTTTCCATGGGAGCTG
Mouse-SCD-1 Reverse TTGATGTGCCAGCGGTACT
Mouse-IFIT3 Forward TGAACTGCTCAGCCCACA

ep
Mouse-IFIT3 Reverse AGAGATTCCCGGTTGACCTC
Mouse-IFIT1 Forward AGCAGAGAGTCAAGGCAGGTTTC

Pr
Mouse-IFIT1 Reverse TGGTCACCATCAGCATTCTCTCC
38
Mouse-Mx1 Forward TTCAAGGATCACTCATACTTCAGC
d
Mouse-Mx1 Reverse GGGAGGTGAGCTCCTCAGT
we
Mouse-Oasl1 Forward GGCCAACCAGTGTCTGAAA
Mouse-Oasl1 Reverse TGGATATCGGGTGCTCTCTT
Mouse-IFNB Forward CTTCTCCGTCATCTCCATAGGG
vie
Mouse-IFNB Reverse CACAGCCCTCTCCATCAACT
Mouse-ISG15 Forward TCCTGGTGAGGAACAAAAGG
Mouse-ISG15 Reverse CGCAAATGCTTGATCACTGT
re
Mouse-ISG20 Forward CAATGCCCTGAAGGAGGATA
Mouse-ISG20 Reverse TGTAGCAGGCGCTTACACAG
Mouse-IFNA Forward TACTCAGCAGACCTTGAACC
Mouse-IFNA
Q-BVDV
Reverse GGTACACAGTGATCCTGTGG
Forward ATGCCCATAGTAGGACTAGCA
er
pe
Q- BVDV Reverse TCAACTCCATGTGCCATGTAC
644
ot
tn
rin
ep
Pr
39

Negative Regulation of Srebp-1 Fas

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Negative Regulation of Srebp-1 Fas

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1 Negative regulation of SREBP-1/FAS signalling molecules activates the RIG-1/TBK1-mediated

13 *Corresponding author: Zhanbo Zhu

14 E-mail address: zhanbozhu@byau.edu.cn

16 Agricultural University, Daqing 163319, China. Tel.: +86-459-681-9969.

52 significant declines in cattle reproductive efficiency, resulting in substantial economic

53 losses(Scharnbck et al., 2018). Hence, prioritizing biosecurity measures and implementing

55 economic sustainability of the global cattle industry.

104 2.2 BT Cells culture er

109 2.3 Animal care

114 rodent food and water.

131 stored at -80 ℃.

132 2.6 Fluorescent quantitative PCR

141 gene primer sequences were listed in Table 1.

157 2.8 Free fatty acids (FFAs) and triglycerides (TG)

162 2.9 Oil red O

2.11 Statistical analysis

184 3.1 BVDV infection alters the metabolic pathway of BT cells

199 pathway in vivo/in vitro

212 pathway in mice.

222 control group (FIG.3 D-I).

inhibit BVDV replication by activating RIG-1/MDA5/ TBK1-mediated IFN-Ⅰ signaling.

295 significantly higher compared to uninfected groups. Simultaneously, we observed a significant

316 surface of BVDV particles.

354 treatment of bovine viral diarrhea.

355 Funding Statement

358 (32072896), Heilongjiang Postdoctoral Found (LBH-Q20054, LBH-Z22233), Natural Science

359 Foundation of Heilongjiang Province (LH2021C072), Heilongjiang Bayi Agricultural University

360 Talent Research Start-up Fund (XYB202107, XYB202014).

368 read and agreed to the published version of the manuscript. er

371 [and/or] its supplementary materials.

406 Commun Biol 4, 820. https://doi.org/10.1038/s42003-021-02310-y.

409 e1003333. https://doi.org/10.1371/journal.ppat.1003333

458 Publishing Group. https://doi.org/10.1038/s41598-018-32831-2

483 for Immune Function. Immunity 44, 1325-1336. https://doi.org/10.1016/j.immuni.2016.06.006.

492 FIG.1 BVDV infection alters the metabolic pathway of BT cells

531 GraphPad Prism, mean±SD. *, p<0.05, **, p<0.01, ***, p<0.001.

541 ***, p<0.001.

551 p<0.01, ***, p<0.001.

Bovine-FASN Reverse CCTCGGGTGAGGACATTTAT

Bovine-ISG20 Forward GGCCCGAGGGTGACATCAC

Bovine-ISG20 Reverse CAGCGCCTTGAAGTCGTGCT

Bovine-ISG15 Forward TCGCCCAGAAGATCAATGTG

Bovine-ISG15 Reverse AGCACCTCCCTGCTGTCAAG

Bovine-MX1 Forward ACATGATCGTCAAGTGCCGT

Bovine-OAS1 Forward CTCACAGAGTTCGGGTGTCC

Bovine-OAS1 Reverse TGCCGTTTCTGGACCTCAAA

Bovine-IFNA Reverse TGAGGAAGAGAAGGCTCTCATGA

Bovine-IFNB Forward CCTGTGCCTGATTTCATCATGA

Mouse-FASN Forward GCTGCTGTTGGAAGTCAGC

Mouse-FASN Reverse AGTGTTCGTTCCTCGGAGTG

Mouse-ACC-1 Forward CCAATGGTTGAGTGGGTTTT

Mouse-SCD-1 Forward TGGTTTCCATGGGAGCTG

Mouse-SCD-1 Reverse TTGATGTGCCAGCGGTACT

Mouse-IFIT3 Forward TGAACTGCTCAGCCCACA

Mouse-IFIT3 Reverse AGAGATTCCCGGTTGACCTC

Mouse-IFIT1 Forward AGCAGAGAGTCAAGGCAGGTTTC

Mouse-IFIT1 Reverse TGGTCACCATCAGCATTCTCTCC

Mouse-Oasl1 Reverse TGGATATCGGGTGCTCTCTT

Mouse-IFNB Forward CTTCTCCGTCATCTCCATAGGG

Mouse-ISG15 Forward TCCTGGTGAGGAACAAAAGG

Mouse-ISG15 Reverse CGCAAATGCTTGATCACTGT

Mouse-ISG20 Reverse TGTAGCAGGCGCTTACACAG

Mouse-IFNA Forward TACTCAGCAGACCTTGAACC

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531 GraphPad Prism, mean±SD. , p<0.05, , p<0.01, , p<0.001.