Identification of bacteria associated with canine otitis externa based on 16S rDNA high-throughput sequencing.

Bacteria are regarded as predisposing and perpetuating factors causing otitis externa (OE), whereas auricular anatomy is a predisposing factor. This study aims to investigate bacterial populations in the external auditory canals of healthy dogs and dogs with OE. Four categories of ear swabs included healthy erect-ear dogs, erect-ear dogs with OE, healthy pendulous-ear dogs and pendulous-ear dogs with OE. After bacterial DNA extraction, 16S rDNAs were amplified using specific primers within a V3/V4 region. Following DNA library construction, high-throughput sequencing was performed on MiSeq (Illumina). CLC Microbial Genomics Module was used to determine the rarefaction curve, bacterial classification, relative abundance, richness and diversity index. The results demonstrated that healthy dogs had higher bacterial richness and diversity than the dogs with OE. Comparable with culture-dependent methods described previously, this study revealed predominant Corynebacterium spp., Pseudomonas spp., Staphylococcus spp., and Proteus spp. in OE cases. Furthermore, high-throughput sequencing might disclose some potential emerging pathogens including Tissierella spp., Acinetobacter spp., and Achromobacter spp., which have not been reported in previous canine OE cases. Nevertheless, larger sample sizes are further required for an extensive evidence-based investigation.

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.

Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens.

COVID-19 pandemic caused by SARS-CoV-2 infection continue to cause the morbidity and mortality in many countries. Limitations of the gold standard qRT-PCR for diagnosis of this infection includes need for expensive equipment, specialized molecular laboratory, and experienced staff. Currently, CRISPR-based diagnostic method was approved by the U.S. FDA for rapid detection. Several studies developed SARS-CoV-2 detection based on CRISPR-Cas12a platform; however, the validations with RNA extracted from clinical specimens were limited. Therefore, this study evaluated the clinical performance of previously described CRISPR-Cas12a based diagnostic assays for SARS-CoV-2. According to the results, the CRISPR-Cas12a assays on N1 and S genes provided diagnostic accuracy (≥ 95 %) comparable to the qRT-PCR results. The assays with E, N2 and S genes yielded acceptable sensitivity of detection (≥ 95 %) whereas N1 and S genes provided outstanding specificity of detection (100 %). Preferably, multiple target genes should be detected by using CRISPR-Cas12a to ensure the most effective SARS-CoV-2 detection. Therefore, the N1 and S genes would be attractive target genes for SARS-CoV-2 detection based on CRISPR-Cas12a.

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.

Hepatitis B Virus-Encoded MicroRNA (HBV-miR-3) Regulates Host Gene PPM1A Related to Hepatocellular Carcinoma.

BACKGROUND: Hepatitis B is a liver infection disease caused by the Hepatitis B Virus (HBV) that can become chronic and develop into hepatocellular carcinoma. HBV was classified as a double-stranded DNA virus. Currently, there is a report showing that HBV virus-encoded miRNA called HBV-miR-3 controls the replication of HBV. However, the regulation of HBV-miR-3 in host cells remains unclear. OBJECTIVE: This study aimed to investigate the regulation of HBV-miR-3 in host gene target which is related to chronic HBV infection and HCC process. METHODS: In this study, we analyzed the read count of HBV-miR-3 from next-generation sequencing of chronic hepatitis patients in Pegylated interferon alpha-2a (PEG-IFN-α-2a) treatment. To understand the regulation of HBV-miR-3 in host cells, the HBV-miR-3 recognition sites were predicted in host target genes using miRDB. The effect of HBV-miR-3 in host cells was examined using qPCR and 3' UTR dual luciferase assay. RESULTS: The read count of HBV-miR-3 was found in chronic hepatitis patients before treatment. Moreover, the decrease of HBV-miR-3 was correlated with response group of chronic hepatitis patients after treatment. On the other hand, the abundance of HBV-miR-3 showed no difference in nonresponse group of chronic patients after PEG-IFN-α-2a treatment. To study the role of HBV-miR-3 in patients, four HBV-miR-3 target regions from Protein phosphatase 1A (PPM1A) and DIX domain containing 1 (DIXDC1) were identified in the human genome using miRDB. Interestingly, we found that HBV-miR-3 hybridized with PPM1A mRNA. The mRNA expression from RT-qPCR showed no difference between HepG2 transfected with pSilencer_scramble or pSilencer_HBV-miR-3. However, the reporter assay showed that PPM1A mRNA was suppressed by HBV-miR-3. The protein expression of PPM1A showed a decrease in cells overexpressing HBV-miR-3. Finally, the HBV-miR-3 can promote cell proliferation in cells overexpressing HBV-miR-3. CONCLUSION: This study is the first report showed the HBV encoded miRNA can regulate host gene expression. HBV-miR-3 silenced PPM1A by inhibiting the translation process of PPM1A. The downregulation of PPM1A promotes cell proliferation related to HCC development.

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.

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.