PiRNA Omics Study Of HCV-infected Human Hepatoma Cells Huh7.5.1 And Preliminary Evaluation Of Recombinant Zika Vaccine Immunology

Objective:Hepatitis C virus(HCV),an enveloped positive-strand RNA virus,is the only member of the hepatitis virus genus of the Flaviridae family.HCV infection has become a global health problem due to the large number of infected people and its serious consequences.Piwi-interactiing RNA(piRNA)is a class of small non-coding RNAs with a length of about 24nt to 32nt,which regulates the expression of target genes by interacting with PIWI proteins.However,the characterization of the piRNA expression profile during HCV infection remains unknown.This study aims to reveal the characterization of the piRNA expression profile of HCV-infected cells,screen the differentially expressed piRNA and analyze their potential biological functions through the small RNA omics analysis of HCV-infected human liver cancer cell Huh7.5.1,and provide new clues for understanding the molecular mechanism of HCV-infected liver disease.Methods:The small RNA sequencing dataset GSE74014 of HCV-infected human hepatoma cell Huh7.5.1 was downloaded from the GEO database.Firstly,a variety of software is used to quality control the original data to ensure the reliability of the data.Then,a series of bioinformatics analysis was conducted on the effective data after quality control filtering,including comparing and annotation with reference genome,miRBase and piRBase database,to obtain known piRNA and conduct length distribution and base bias statistics.Then,TPM(transcript per million)was used to calculate the known piRNA expression level,and the differences between HCV infected samples and control samples were compared.The criteria of the piRNA screening is that Fold Change value is greater than or equal to 2(or(|log2FC| greater than or equal to 1)and p value is less than 0.05.The Miranda software was used to predict the target genes of differential piRNA,and GO and KEGG enrichment analysis were conducted to analyze the gene ontology and pathway of the predicted target genes,so as to further search for differential piRNA with potential research value.Results:The small RNA sequencing data set GSE74014 was filtered by quality control.The clean reads of each sample in the control group and the experimental group were distributed in the range of 9.0M to 16.02M,and the comparison rate with the reference genome was distributed in the range of 87.39%to 91.95%,and the length distribution of small RNA was unimpeak at 22nt.It suggests the data is reliable.Through comparing and annotation of piRNA database,the known piRNA length distributed between 15nt to 32nt and presented a double peak,with the peak value at 18nt and 25nt respectively.The first base of piRNA is known to have a significant uracil bias.Visualization of known piRNA expression level indicated that the expression patterns of samples within the group were highly similar,while the expression patterns of samples between the groups were low,which proved that the piRNA expression levels of samples in the control group and the experimental group were comparable.Compared with the control group,the total number of differentially expressed piRNA was 87 in HCV-infected cells,among which 63 piRNA were significantly upregulated(log2FC>1,P<0.05).Twenty-four piRNA were significantly down-regulated(log2FC<-1,P<0.05).In addition,HCV infection induced the expression of piR-hsa-23821 and piR-hsa-11817 in Huh7.5.1,while inhibited the expression of piR-hsa-28522 and piR-hsa-32260.Functional enrichment analysis of GO and KEGG indicated that differential target genes of piRNA were mainly enriched in the biological processes of transcription,transcriptional regulation and signal transduction,and were involved in a number of tumor-related pathways,lipid metabolism pathways and immune pathways.Conclusion:Compared with uninfected control cells,HCV-infected Huh7.5.1 cells resulted in differential expression of 87 piRNAs,including 63 piRNAs significantly upregulated and 24 piRNAs significantly down-regulated.We identified two HCV-induced piRNAs(piR-hsa-23821,piR-hsa-11817)and two HCV-completely inhibited piRNAs(piR-hsa-28522 and piR-hsa-32260).Functional enrichment analysis of these target genes differentially expressing piRNA suggested that they might play an important role in HCV infection and metabolic disorders and liver diseases related to HCC cell proliferation,which need to be further studied.Objective: Zika virus(ZIKV)is one of the arbo-bome flaviviruses.ZIKV infection can cause not only self-limiting symptoms,but also neurological disorders and autoimmune complications,including microcephaly and Guillain-Barre syndrome.There have been several outbreaks of ZIKV in history,and the virus continues to threaten human life and health.However,for a variety of reasons,there is currently no approved vaccine available to prevent ZIKV infection.We need to develop a safe and effective vaccine to deal with the ZIKV epidemic that China may face in the future.The purpose of this study was to conduct a preliminary immunological evaluation of a new recombinant Zika vaccine constructed by our research group,and this study includes immunogenicity evaluation and the preparation for efficacy evaluation.The recombinant Zika vaccine uses T7 phage as the vaccine carrier,and the third domain of ZIKV envelope protein as the design target of the vaccine.The recombinant phage can display the ZIKV ED ? protein on the surface to induce the immune response.The preliminary immunological evaluation of the recombinant Zika vaccine in this study will be conducive to the development of this new and cost-effective Zika vaccine.Methods:In this study,recombinant phages preserved by our group were used as vaccine seeds.After successful resuscitation and PCR identification,further culture was expanded.The successful recombinant phages were rapidly proliferated in coli.BLT5403 in logarithmic growth phase,and then purified by PEG8000 precipitation and high speed centrifugation.Bacteriophages with titers of 5x10⁹pfu/mL measured by double agar sandwich method were inactivated by formaldehyde and prepared into vaccine for use.BALB/c female mice aged 6-8 weeks were immunized intraperitoneally with the immunization dose of 10⁹ pfu,and the immunization was strengthened in the second week after the first immunization.The no-load phage group and the ED? protein group were set as controls and the same immunization procedure was performed.At 1th,4th,8th,12 th,16th,20 th and 24 th weeks after the first immunization,the serum of the tail vein of mice was collected and isolated,and the level of specific anti-ED? IgG in the immune serum was detected by indirect ELISA method.At the 9th week after the first immunization,eyeball blood of mice was collected and sacrificed.Spleens of mice were taken for ELISPOT test to detect IL-4 and IFN-y secreted by spleen cells of mice after immunization,so as to evaluate the level of cellular immunity induced by Zika vaccine.The titer and viral load of the concentrated Zika virus were determined by CCID50 and qPCR.Results: The recombinant phages stored in our group were successfully revived and incubated at 37°C for 4-6h to form phage plaques.The correct recombinant phage was identified by PCR and expanded in host bacteria to a titer of 5×10⁹pfa/ml.After formaldehyde inactivation,it became the vaccine to be used.Immunization of BALB/c mice with a low dose(10⁹pfu)of recombinant Zika vaccine without adjuvant induced the production of specific anti-ED? IgG antibodies.The antibody with geometric mean titer of 1:400 was detected from the 4th week after the first immunization,and then the antibody level increased and reached the peak of 1:975,21 at the 8th week.After that,the antibody level showed a slight fluctuation until the 24 th week after the first immunization,and the antibody titer induced by the recombinant vaccine remained at 1:883.27.The ELISPOT test was conducted in the 9th week after the first immunization of mice.The results showed that the secretion of cytokines IL-4 and IFN-y was detected in spleen cells of mice immunized with recombinant Zika vaccine after stimulated by ED? protein,and the number of spots in unstimulated pores was smaller than that in stimulated pores stimulated by ED? protein.Two-way ANOVA analysis showed that the influence of stimulated and unstimulated factors on the number of spots was statistically significant(IL-4,P =0.0180;IFN-y,P = 0.0123),but Multiple comparisons showed P values greater than 0.05 for each of the two groups,indicating that there was no significant difference between the recombinant vaccine group,phage load group and ED? protein group,suggesting that the recombinant Zika vaccine could not induce a cellular immune response.In preparation for the efficacy and protection evaluation of the recombinant Zika vaccine,ZIKV was successfully amplified and concentrated in C6/36 cells.The resulting venom was identified by PCR and sequencing,and its titer was determined to be 10⁶³¹³ CCID50/ml and viral load was 4.211x10¹¹ncopies/ml.The standard curve was successfully constructed for the determination of ZIKV viral load by the absolute quantitative qPCR method,and the standard equation was y=-4.1898x+59.528.Conclusion: After immunizing BALB/c mice with a low dose(10⁹pfu)of the recombinant Zika vaccine without adjuvant,the humoral immune response was mainly induced,and specific anti-EDHI IgG antibody was induced,but the cellular immune response was not significantly induced.The concentration of Zika virus with measured titer and load can be used for subsequent effectiveness evaluation.