关键词: 猪; 腹泻; 混合感染; 宏基因组学

Abstract: 【Objective】 This study was aimed to understand the prevalence of viral co-infections in diarrheic pig populations and the alterations in the gut microbiota community structure under viral infection. 【Method】 Seventy-three clinical porcine diarrhea samples were collected and subjected to metagenomic sequencing using single-end 100 bp read length (SE100).The raw data were quality-controlled using FastQC to obtain clean data,and host sequences were removed using BWA to generate unmapped data.Kraken 2 was used to align the unmapped data to an animal pathogen database for species classification annotation and to generate a species classification table.Principal component analysis (PCA) was performed for dimensionality reduction,and the data were normalized using reads per million (RPM).Z-score statistical methods were used to generate heatmaps for differential analysis visualization.MEGAHIT was employed for de novo assembly of viral sequences,and BWA was used to align unmapped data to contigs to assess assembly quality.High-quality contigs were selected and aligned to the NCBI database to find the most similar sequences.The unmapped data were aligned to these sequences to extract consensus sequences,which were used as the viral sequences.Phylogenetic analysis of Porcine epidemic diarrhea virus (PEDV) S gene and Porcine astrovirus (PAstV) ORF1a gene was performed using IQ-TREE. 【Result】 Various viruses were detected in 73 samples of pig diarrhea with the following positivity rates:PAstV 26.03%,Porcine kobuvirus (PKV) 20.55%,Porcine rotavirus (PoRV) 19.18%,PEDV 19.18%,Porcine sapporo virus (PSaV) 6.85% and Porcine deltacoronavirus (PDCoV) 4.11%.The mixed infection situation was complex,mainly consisting of a combination of PEDV and PAstV,accounting for 17.81% (13/73) of all samples and 30.95% (13/42) of mixed infection samples.The gut microbiota structure showed significant changes under different viral infections.The relative abundance of Lactobacillus was higher in PEDV-positive group than that in PEDV-negative group,and the relative abundances of Clostridium and Treponema were higher in PAstV-positive group than that in PAstV-negative group.Two complete PEDV genomes SE18 and SE22 were assembled,belonging to the GⅡa lineage,along with four PAstV ORF1a gene sequences SE18,PE31,PE36 and PE70,of which SE18 belonged to PAstV2 and the other three were closely related to HAstV-HMO-B strain. 【Conclusion】 This study revealed high positive rates and co-infection patterns of PAstV and PEDV in clinically diarrheic pig populations using metagenomic techniques,and also identified significant changes in gut microbiota composition due to PEDV and PAstV infections,providing critical data support for the prevention and control of mixed viral infections in pig diarrhea.

Key words: pig; diarrhea; mixed infection; metagenomics