Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing.

High-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer, but low-risk HPV strains can sometimes also be involved. Although HPV genotyping techniques used in clinical diagnosis cannot detect low-risk HPV, next-generation sequencing (NGS) can detect both types. However, DNA library preparation is complicated and expensive. The aim of this study was to develop a simplified, cost-effective sample preparation procedure for HPV genotyping based on next-generation sequencing (NGS). After DNA extraction, a first round of PCR was performed using modified MY09/11 primers specific for the L1 region of the HPV genome, followed by a second round of PCR to add the indexes and adaptors. Then, the DNA libraries were purified and quantified, and high-throughput sequencing was performed using an Illumina MiSeq platform. The sequencing reads were compared with reference sequences for HPV genotyping. The limit of detection for HPV amplification was 100 copies/µl. Analysis of the correlation of pathological cytology with the HPV genotype in individual clinical samples showed that HPV66 was the most common genotype found in the normal stage, whereas HPV16 was the main genotype found in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer. This NGS method can detect and identify several HPV genotypes with 92% accuracy and 100% reproducibility, and it shows potential as a simplified and cost-effective technique for large-scale HPV genotyping in clinical samples.

IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production.

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7-/ - MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7-/ - MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7-/ - MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7-/ - MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7-/ - MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7-/ - MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7-/ - MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.

Enhancing the yield of seasonal influenza viruses through manipulation of microRNAs in Madin-Darby canine kidney cells.

Annual influenza vaccine is recommended to reduce the occurrence of seasonal influenza and its complications. Thus far, Madin-Darby canine kidney (MDCK) cell line has been used to manufacture cell-based influenza vaccines. Even though host microRNAs may facilitate viral replication, the interaction between MDCK cells-derived microRNAs and seasonal influenza viruses has been less frequently investigated. Therefore, this study highlighted microRNA profiles of MDCK cells to increase the yield of seasonal influenza virus production by manipulating cellular microRNAs. MDCK cells were infected with influenza A or B virus at a multiplicity of infection (MOI) of 0.01, and microRNA collections were then subjected to MiSeq (Illumina) Sequencing. The validated profiles revealed that cfa-miR-340, cfa-miR-146b, cfa-miR-197, and cfa-miR-215 were the most frequently upregulated microRNAs. The effect of candidate microRNA inhibition and overexpression on viral replication was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The hybridization pattern between candidate miRNAs and viral genes was performed using miRBase and RNAhybrid web-based programs. Moreover, the predicted microRNA-binding sites were validated by a 3'-UTR reporter assay. The results indicated that cfa-miR-146b could directly target the PB1 gene of A/pH1N1 and the PA gene of B/Yamagata. Furthermore, cfa-miR-215 could silence the PB1 gene of A/pH1N1 and the PB1 gene of B/Victoria. However, the PB2 gene of the A/H3N2 virus was silenced by cfa-miR-197. In addition, the HA and NA sequences of influenza viruses harvested from the cell cultures treated with microRNA inhibitors were analyzed. The sequencing results revealed no difference in the antigenic HA and NA sequences between viruses isolated from the cells treated with microRNA inhibitors and the parental viruses. In conclusion, these findings suggested that MDCK cell-derived microRNAs target viral genes in a strain-specific manner for suppressing viral replication. Conversely, the use of such microRNA inhibitors may facilitate the production of influenza viruses.

Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a.

Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 103, and 103 copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.

Hsa-miR-30e-3p inhibits influenza B virus replication by targeting viral NA and NP genes.

Influenza B virus is a member of the Orthomyxoviridae family which can infect humans and causes influenza. Although it is not pandemic like influenza A virus, it nevertheless affects millions of people worldwide annually. MicroRNAs are small non-coding RNAs regulating gene expression at posttranscriptional level. They play various important roles in cellular processes including response to viral infection. MiRNA profiles from our previous study suggested that miR-30e-3p was one of the upregulated miRNAs that responded to influenza B virus infection. In this study, in silico prediction and in vitro investigation proved that this miRNA can directly target NA and NP genes of the influenza B virus and inhibit its replication. This finding might be useful for using miRNA as an alternative therapeutics for influenza virus infection.

High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses.

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin-Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3'-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

Serum miR-29a and miR-122 as Potential Biomarkers for Non-Alcoholic Fatty Liver Disease (NAFLD).

BACKGROUND: Non-Alcoholic Fatty Liver Disease (NAFLD) is an over accumulation of triglyceride in the liver without alcohol consumption. Its major cause is insulin resistance. Patients with NAFLD can develop liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are non-coding RNAs that regulate post-transcriptional gene silencing. Previous research reported that miR-29 family (a, b and c) and miR-122 have an important role in regulating insulin resistance related to NAFLD. OBJECTIVE: The purpose of this study was to investigate that miR-29 and miR-122 can be possible biomarkers for non-invasive diagnosis of NAFLD. METHOD: Serum samples were collected from 58 NAFLD patients and 34 healthy controls. MiRNAs were extracted from serum by using microRNA purification kit followed by polyuridylation, reverse transcription and quantitative real-time PCR. Also, we analyzed the correlation between miR-29 and miR-122 and level of liver inflammation in NAFLD patients. RESULTS: We found that the serum miR-29a levels in NAFLD patients were significantly lower (P = 0.006) than the control group, while miR-29c levels were unchanged, and miR-29b levels were undetectable. However, we found that serum miR-122 levels in NAFLD patients were significantly higher (P < 0.001) than those found in the control group. For miR-29a, the area under curve (AUC) was 0.679 (P = 0.0065) with 60.87% sensitivity and 82.35% specificity. For miR-122, the AUC was 0.831 (P < 0.0001) with 75.00% sensitivity and 82.35% specificity. Interestingly, the levels of serum miR- 122 were significantly different between patients without steatohepatitis (NAS < 4) and steatohepatitis (NAS ≥ 4), indicating that the levels of miR-122 were related to the severity of NAFLD. CONCLUSION: The levels of miR-29a and miR-122 might be beneficial and compelling as possible biomarkers for non-invasive diagnosis of NAFLD.

Human MicroRNAs Expression Profiles in Influenza B Virus-Infected Cells based on Illumina MiSeq Platform.

BACKGROUND: Influenza B virus causes influenza-like illness in humans. MicroRNAs (miRNAs) are small non-coding RNAs regulating gene expression through mRNA degradation or translational repression. MiRNAs have evolved to regulate many cellular processes including the viral infection response. OBJECTIVE: This study aims to investigate the miRNA profiles of human cells infected with influenza B virus. METHODS: A549 cells were infected with influenza B viruses (MOI = 0.5). MiRNAs were extracted at 24 and 48 hours post-infection. MiRNAs were used to construct four DNA libraries: influenza Binfected and an uninfected control for both time points. Then high-throughput sequencing was performed using the Miseq platform (Illumina). Sequencing data were analyzed by Miseq reporter software. The miRNAs were categorized and counted based on the frequency of reads. All filtered contigs were aligned with data from miRbase. The relative expression of each miRNA between uninfected and influenza B-infected cells was calculated. RESULTS: There were 13 down-regulated miRNAs and 21 up-regulated miRNAs observed in influenza B infected cells at 24 hours post infection. At 48 hours post infection, 14 miRNAs were downregulated, whereas 8 miRNAs were up-regulated. CONCLUSION: This study suggested that miRNAs may play important roles in host gene regulation in response to viral infection.

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus-host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3'-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3'-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.