A study of Identification and verification of the role of key metabolites and metabolic pathways on ASFV replication, Zunji shi et.al

Description

African swine fever (ASF), one of the most harmful diseases prevalent in pig farms nowadays, caused by the African swine fever virus (ASFV) that cause enormous economic losses. Viremia usually develops within a few days after ASFV infection. However, the metabolic changes in pig serum after ASFV infection remain unclear. In this study, serum samples collected from ASFV-infected pig at different times were analyzed using pseudotargeted metabolomics method. Metabolomic analysis revealed 225 metabolites decreased and 65 metabolites increased in serum collected in ASFV infected 5 days post-infection (dpi), 254 metabolites decreased and 58 metabolites increased in serum collected ASFV infected 10 dpi. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the significant changed metabolites indicated that dopaminergic synapse has the highest rich factor in both ASFV5 (ASFV infected 5 dpi) VS ASFV0 (ASFV infected 0 dpi) and ASFV10 (ASFV infected 10 dpi) VS ASFV0.

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Metabolite Extraction Take out the sample from the -80°C refrigerator and thaw it on ice, 20 mg of sample was mix with 400 μL 70% methanol water internal standard extractant homogenize with a steel ball using a ball mill at 30 HZ for 20 s, then centrifuged at 3000 rpm for 30 s at 4°C. Subsequently, add 400 μL of 70% methanol water internal standard extractant, shook 5 min with 1500 rpm and placed on ice for 15 min. The samples were centrifuged at 12000 rpm for 10 min at 4°C, the supernatant (300 μL) was transferred to a new Eppendorf tube and incubated at -20°C for 30 min. Finally, the samples were centrifuged at 12000 rpm for 3min at 4°C, and take the supernatant for further analysis. UHPLC-TQMS Analysis The collected supernatant, 2ul sample volume, was injected into a 100 × 2.1 mm2, 1.8 µm HSS T3 column (Waters, Milford, MA) held at 40°C using an Agilent 1290 Infinity UPLC system (ExionLC AD). The mobile phase consisted of A (water, containing 0.1% formic acid) and B (acetonitrile, containing 0.1% formic acid). The gradient elution procedure was as followes: 95:5 A/B at 0 min, 10:90 A/B at 10.0 min, 10:90 A/B at 11.0 min, 95:5 A/B at 11.1 min, 95:5 A/B at 14.0 min. The flow rate was 0.4 ml/min. The AB Triple TOF 6600 mass spectrometer (AB SCIEX, USA) was used for its ability to acquire MS/MS spectra on an information dependent basis (IDA). In this mode, the acquisition software (TripleTOF 6600, AB SCIEX) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, 12 precursor ions whose intensity greater than 100 were chosen for fragmentation at collision energy (CE) of 30 V (12 MS/MS with product ion accumulation time of 50 msec each). Electrospray ionization (ESI) worked in positive and negative ion modes, The parameters were set as follows: ion source gas 1 as 50 Psi, ion source gas 2 as 50 Psi, curtain gas as 25 Psi, source temperature 500 °C, ion Spray voltage floating (ISVF) 5500 V or -4500 V in positive or negative modes, respectively.

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