Blood Metagenomic Analysis And Emerging Pathogens Of Healthy Blood Donors In Some Areas Of China

Objective:The objective of this study was to map a comprehensive description of microbiome present in blood of healthy blood donors in five regions in China based on high-throughput sequencing and metagenomics analysis.Quantitative Real-time PCR(qPCR)and enzyme-linked immunosorbent assays(ELISA)were performed to assess the prevalence of nucleic acids and antibodies of potential pathogens.The aim of this study was to assess the risk of potential pathogen infection in healthy individuals of five regions in China.Methods:(1)Samples processing for high-throughput sequencing A total of 9 113 healthy blood samples were collected from Liangshan Yi Autonomous Prefecture,Wenshan Zhuang and Miao Autonomous Prefecture,Wuhan city,Kunming city and Nanjing city.Individual serum from each region was pooled into a master pool separately.Total viral nucleic acids were extracted from each master pool.10?L of each nucleic acid samples were transcribed into cDNA separately.High-throughput sequencing of ten nucleic acid samples was performed on the Illumina NovaSeq 6000 platform.The sequenced data were mapping with the sequences of microbial genome to annotate the classification of microbe.(2)HSV detection based on SYBR Green qPCR system The whole genome sequences of HSV-1 and HSV-2 were searched in NCBI and the consensus sequence was compared by BLAST.Then universal primers were designed according to the consensus sequences.Primers and plasmid standards were synthesized.Primers,plasmid,and optimized reaction and cycling conditions used for qPCR assays.(3)Molecular epidemiological investigation of HSV The serum of 2 779 healthy blood donors were randomly selected from 5 regions to screen for specific antibodies against herpes simplex virus type 1 and 2 by enzyme-linked immunosorbent assay.Samples from Wuhan and Kunming were pooled using three-stage hierarchical configuration,and samples from Liangshan and Nanjing followed a rectangular array-based pooling strategies.All mini pools and suspicious individual samples were detected by qPCR respectively.The prevalence of HSV in healthy blood donation population was compared between/among different genders,ages,occupations,education levels,and frequency of donation.Results:(1)Based on metagenomic analysis of the blood microbiome of Liangshan,Wenshan,Wuhan,Kunming and Nanjing.The reads(percentage)of viruses in DNA pools were 15(0.01%),0,164(0.02%),12 702(0.35%)and 0 respectively,and those of bacteria were 213 057(94.24%),653 972(98.25%),1 011 139(98.87%),3 648 382(99.43%)and 96 327(92.00%),and those of eukaryotes were 10 623(4.70%),8 657(1.30%),8274(0.81%),3 750(0.10%)and 3 511(3.37%);The reads(percentage)of viruses in cDNA pools of Liangshan,Wenshan,Wuhan,Kunming and Nanjing were 0,0,0,6 966(0.13%)and 9 558(1.51%),and those of bacteria were 4 105 600(98.15%),4 417 409(98.17%),4 718 190(98.68%),5 117 838(98.84%)and 527 378(83.27%)respectively,and those of eukaryotes were 18 446(0.44%),16 487(0.37%),8 669(0.18%),10 395(0.20%)and 37920(5.22%).(2)The detection method of HSV based on SYBR Green qPCR was successfully established.The detection limit of qPCR for HSV DNA was 101 copies/mL.(3)The prevalence of total antibodies against HSV-1 was 8.70%(113/1 299),39.13%(72/184),29.35%(27/92),38.36%(89/232)and 32.76%(76/232)in Liangshan,Wuhan,Kunming,Nanjing and Dehong by ELISA;and that of total antibodies against HSV-2 was 18.32%(238/1 299),38.04%(70/184),36.96%(34/92),40.09%(93/232)and 16.8%(40/232)respectively.The positive rates of HSV DNA in healthy blood donors in Liangshan,Wuhan,Kunming,Nanjing and Dehong were 0.77‰(1/1 299),0,0.2%(2/1000),0.25‰(1/4 039)and 0 respectively.The positive rates of HSV-1 and Hsv-2 were significantly different in Liangshan.Among healthy blood donors,the highest positive rates were found in unmarried Yi men aged 18-24.Conclusion:High-throughput sequencing is an effective and well-established method for screening for unknown pathogens.A large number of microorganisms are present in the blood samples of healthy blood donors.HSV has a certain prevalence of antibodies in Liangshan,Wuhan,Kunming,Nanjing and Dehong.The prevalence of HSV DNA is low(less than 0.2%).Our results provide a reference for the study of microbiome in healthy individuals and the improvement of blood screening strategies in blood transfusion services.