HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the serum cytokine and chemokine levels in asymptomatic, mild, moderate, severe, and convalescent SARS-CoV-2-infected cases. Proinflammatory cytokine and chemokine production induced by SARS-CoV-2 were observed not only in symptomatic patients but also in asymptomatic cases, and returned to normal after recovery. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Moreover, a set of cytokine and chemokine profiles were significantly higher in SARS-CoV-2-infected male than female patients. The serum levels of MCP-1, G-CSF, and VEGF were weakly and positively correlated with viral titers. We suggest that combinatorial analysis of serum cytokines and chemokines with clinical classification may contribute to evaluation of the severity of COVID-19 and optimize the therapeutic strategies.

Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay.

BACKGROUND: As an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device). RESULTS: This assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples. CONCLUSIONS: Thus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Identification and genetic characterization of a novel circular single-stranded DNA virus in a human upper respiratory tract sample.

Metagenomic analysis through high-throughput sequencing is a tool for detecting both known and novel viruses. Using this technique, a novel circular single-stranded DNA (ssDNA) virus genome was discovered in respiratory secretions from a febrile traveler. The virus, named human respiratory-associated PSCV-5-like virus (HRAPLV), has a genome comprising 3,018 bases, with two major putative ORFs inversely encoding capsid (Cap) and replicase (Rep) protein and separated by two intergenic regions. One stem-loop structure was predicted in the larger intergenic region (LIR). The predicted amino acid sequences of the Cap and Rep proteins of HRAPLV showed highest identity to those of porcine stool-associated circular virus 5 isolate CP3 (PoSCV 5) (53.0% and 48.9%, respectively). The host tropism of the virus is unknown, and further study is warranted to determine whether this novel virus is associated with human disease.

Cytokine and chemokine levels in patients infected with the novel avian influenza A (H7N9) virus in China.

H7N9 avian influenza is an emerging viral disease in China caused by avian influenza A (H7N9) virus. We investigated host cytokine and chemokine profiles in serum samples of H7N9 patients by multiplex-microbead immunoassays. Statistical analysis showed that IP-10, IL-6, IL-17, and IL-2 were increased in H7N9 infected patients. Furthermore, IL-6 and the chemokine IP-10 were significantly higher in severe H7N9 patients compared to nonsevere H7N9 cases. We suggest that proinflammatory cytokine responses, characterized by a combined Th1/Th17 cytokine induction, are partially responsible for the disease progression of patients with H7N9 infection.

A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors.

Portable all-in-one microfluidic system for CRISPR-Cas13a-based fully integrated multiplexed nucleic acid detection.

Point-of-care testing of "sample in, answer out" is urgently needed for communicable diseases. Recently, rapid nucleic acid tests for infectious diseases have been developed for use in resource-limited areas, but they require types of equipment in central laboratories and are poorly integrated. In this work, a portable centrifugal microfluidic testing system is developed, integrated with magnetic bead-based nucleic acid extraction, recombinase-assisted amplification and CRISPR-Cas13a detection. The system, with the advantage of its power-supplied active rotating chip and highly programable flow control through integrated addressable active thermally-triggered wax valves, has a rapid turnaround time within 45 min, requiring only one user step. All reagents are preloaded into the chip and can be automatically released. By exploiting a multichannel chip, it is capable of simultaneously detecting 10 infectious viruses with limits of detection of 1 copy per reaction and 5 copies per reaction in plasmid samples and mock plasma samples, respectively. The system was used to analyse clinical plasma samples with good consistency compared to laboratory-based molecular testing. Moreover, the generalizability of our device is reported by successfully testing nasopharyngeal swabs and whole blood samples. The portable device does not require the operation of professional technicians, making it an excellent assay for on-site testing.

Serum microRNA expression profile as a biomarker for the diagnosis of pertussis.

Pertussis is a highly contagious, respiratory disease associated with substantial morbidity and mortality. A rapid and reliable diagnostic method is essential for appropriate treatment and prevention. Expression profiles of circulating microRNAs (miRNAs) have been proven as new non-invasive biomarkers for infectious diseases. We aimed to investigated the serum miRNA profile in pertussis patients and explored its potential as a novel diagnostic biomarker for pertussis. Among 664 different miRNAs analyzed using a miRNA array, 50 were overexpressed and 81 were underexpressed in the serum of pertussis patients. Expression levels of seven candidate miRNAs were further evaluated by real-time qRT-PCR. A panel of five miRNAs (miR-202, miR-342-5p, miR-206, miR-487b, miR-576-5p) was confirmed overexpressed in pertussis patients (p < 0.05). Risk score and receiver-operating characteristic (ROC) analysis showed that the area under the curve of the five-member miRNA profile was 0.980. At an optimal cutoff value (0.707), this panel of miRNAs yielded a sensitivity of 97.4 % and a specificity of 94.3 %. These data suggest that the five-member serum miRNA profile may serve as a new biomarker for pertussis diagnosis with high specificity and sensitivity.

Detection of severe fever with thrombocytopenia syndrome virus by reverse transcription-cross-priming amplification coupled with vertical flow visualization.

A virus known as severe fever with thrombocytopenia syndrome virus (SFTSV) was recently identified as the etiological agent of severe fever with thrombocytopenia syndrome (SFTS) in China. Reliable laboratory detection and identification of this virus are likely to become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method which incorporates reverse transcription-cross-priming amplification (RT-CPA) coupled with a vertical flow (VF) visualization strip for rapid detection of SFTSV. The RT-CPA-VF assay targets a conserved region of the M segment of the SFTSV genome and has a limit of detection of 100 copies per reaction, with no cross-reaction with other vector-borne bunyaviruses and bacterial pathogens. The performance of the RT-CPA-VF assay was determined with 175 human plasma specimens collected from 89 clinically suspected SFTS patients and 86 healthy donors. The sensitivity and specificity of the assay were 94.1% and 100.0%, respectively, compared with a combination of virus culture and real-time RT-PCR. The entire procedure, from specimen processing to result reporting, can be completed within 2 h. The simplicity and nearly instrument-free platform of the RT-CPA-VF assay make it practical for point-of-care testing.

Altered serum microRNAs as biomarkers for the early diagnosis of pulmonary tuberculosis infection.

BACKGROUND: Pulmonary tuberculosis (TB) is a highly lethal infectious disease and early diagnosis of TB is critical for the control of disease progression. The objective of this study was to profile a panel of serum microRNAs (miRNAs) as potential biomarkers for the early diagnosis of pulmonary TB infection. METHODS: Using TaqMan Low-Density Array (TLDA) analysis followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation, expression levels of miRNAs in serum samples from 30 patients with active tuberculosis and 60 patients with Bordetella pertussis (BP), varicella-zoster virus (VZV) and enterovirus (EV) were analyzed. RESULTS: The Low-Density Array data showed that 97 miRNAs were differentially expressed in pulmonary TB patient sera compared with healthy controls (90 up-regulated and 7 down-regulated). Following qRT-PCR confirmation and receiver operational curve (ROC) analysis, three miRNAs (miR-361-5p, miR-889 and miR-576-3p) were shown to distinguish TB infected patients from healthy controls and other microbial infections with moderate sensitivity and specificity (area under curve (AUC) value range, 0.711-0.848). Multiple logistic regression analysis of a combination of these three miRNAs showed an enhanced ability to discriminate between these two groups with an AUC value of 0.863. CONCLUSIONS: Our study suggests that altered levels of serum miRNAs have great potential to serve as non-invasive biomarkers for early detection of pulmonary TB infection.

Exosomal microRNA expression profiles derived from A549 human lung cells in response to influenza A/H1N1pdm09 infection.

Exosomes participate in intercellular communication by shuttling various small molecules from donor to recipient cells. We aimed to examine the role of exosomes and exosomal miRNAs in influenza virus infection. The results showed that influenza A/H1N1pdm09 infection could promote A549 cells to secrete exosomes, while blocking the generation of exosomes reduced viral RNA production. A total of 97 exosomal miRNAs with significantly altered expression were identified during influenza infection. Of 12 candidate miRNAs chosen for further validation, ten were confirmed by qRT-PCR. Among 5978 predicted target genes，we found 37 interferon pathway-related genes to be the potential targets of 29 differentially expressed miRNAs. Many target genes were annotated to various KEGG signaling pathways, some of which played important roles in influenza infection. These data will help to further understand the mechanism of influenza virus-host interactions, which is important for the development of preventative and therapeutic strategies against influenza virus.

Corrigendum: Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

[This corrects the article DOI: 10.3389/fmicb.2022.973367.].

Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

Whole genome sequencing provides rapid insight into key information about the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), such as virus typing and key mutation site, and this information is important for precise prevention, control and tracing of coronavirus disease 2019 (COVID-19) outbreak in conjunction with the epidemiological information of the case. Nanopore sequencing is widely used around the world for its short sample-to-result time, simple experimental operation and long sequencing reads. However, because nanopore sequencing is a relatively new sequencing technology, many researchers still have doubts about its accuracy. The combination of the newly launched nanopore sequencing Q20+ kit (LSK112) and flow cell R10.4 is a qualitative improvement over the accuracy of the previous kits. In this study, we firstly used LSK112 kit with flow cell R10.4 to sequence the SARS-CoV-2 whole genome, and summarized the sequencing results of the combination of LSK112 kit and flow cell R10.4 for the 1200bp amplicons of SARS-CoV-2. We found that the proportion of sequences with an accuracy of more than 99% reached 30.1%, and the average sequence accuracy reached 98.34%, while the results of the original combination of LSK109 kit and flow cell R9.4.1 were 0.61% and 96.52%, respectively. The mutation site analysis showed that it was completely consistent with the final consensus sequence of next generation sequencing (NGS). The results showed that the combination of LSK112 kit and flow cell R10.4 allowed rapid whole-genome sequencing of SARS-CoV-2 without the need for verification of NGS.

MicroRNA-195 suppresses enterovirus A71-induced pyroptosis in human neuroblastoma cells through targeting NLRX1.

Enterovirus A71 (EV-A71) emerged as a leading cause of virus derived infant encephalitis in most Asian countries. Some recent studies point out the critical role of microRNA (miRNA) in the regulation of pyroptosis. However, the role of miRNAs in the regulation of EV-A71 infection-induced pyroptosis was not previously explored. In this study, we utilized microRNA array and real-time PCR to verify that miR-195 significantly down-regulate in EV-A71-infected SH-SY5Y human neuroblastoma cells. An inverse correlation of NLRX1 with miR-195 expression in EV-A71-infected SH-SY5Y cells was found. Target prediction of miR-195 showed that NLRX1 could directly interact with miR-195. Results from luciferase reporter assays, qRT-PCR and western blotting demonstrated the negative regulation between miR-195 and NLRX1. Silencing NLRX1 expression with small interfering RNAs (siRNAs-NLRX1) and over-expression of miR-195 also attenuate the EV-A71 associated pyroptosis. Our findings provided evidence showed that miR-195 can regulate EV-A71 infection-induced pyroptosis, by directly targeting NLRX1.

Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification.

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

SARS-CoV-2 encoded microRNAs are involved in the process of virus infection and host immune response.

The outbreak of COVID-19 caused by SARS-CoV-2 is spreading worldwide, with the pathogenesis mostly unclear. Both virus and host-derived microRNA (miRNA) play essential roles in the pathology of virus infection. This study aims to uncover the mechanism for SARS-CoV-2 pathogenicity from the perspective of miRNA. We scanned the SARS-CoV-2 genome for putative miRNA genes and miRNA targets and conducted in vivo experiments to validate the virus-encoded miRNAs and their regulatory role on the putative targets. One of such virus-encoded miRNAs, MR147-3p, was overexpressed that resulted in significantly decreased transcript levels of all of the predicted targets in human, i.e., EXOC7, RAD9A, and TFE3 in the virus-infected cells. The analysis showed that the immune response and cytoskeleton organization are two of the most notable biological processes regulated by the infection-modulated miRNAs. Additionally, the genomic mutation of SARS-CoV-2 contributed to the changed miRNA repository and targets, suggesting a possible role of miRNAs in the attenuated phenotype of SARS-CoV-2 during its evolution. This study provided a comprehensive view of the miRNA-involved regulatory system during SARS-CoV-2 infection, indicating possible antiviral therapeutics against SARS-CoV-2 through intervening miRNA regulation.

An integrative drug repositioning framework discovered a potential therapeutic agent targeting COVID-19.

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires an urgent need to find effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). In this study, we developed an integrative drug repositioning framework, which fully takes advantage of machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 can interact with the nucleocapsid (N) protein of SARS-CoV-2 and is able to suppress the LPS-induced production of several inflammatory cytokines that are highly relevant to the prevention of immunopathology induced by SARS-CoV-2 infection.

Identification of microRNAs involved in the host response to enterovirus 71 infection by a deep sequencing approach.

Role of microRNA (miRNA) has been highlighted in pathogen-host interactions recently. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we performed a comprehensive miRNA profiling in EV71-infected Hep2 cells through deep sequencing. 64 miRNAs were found whose expression levels changed for more than 2-fold in response to EV71 infection. Gene ontology analysis revealed that many of these mRNAs play roles in neurological process, immune response, and cell death pathways, which are known to be associated with the extreme virulence of EV71. To our knowledge, this is the first study on host miRNAs expression alteration response to EV71 infection. Our findings supported the hypothesis that certain miRNAs might be essential in the host-pathogen interactions.

High-throughput sequencing of microRNAs in adenovirus type 3 infected human laryngeal epithelial cells.

Adenovirus infection can cause various illnesses depending on the infecting serotype, such as gastroenteritis, conjunctivitis, cystitis, and rash illness, but the infection mechanism is still unknown. MicroRNAs (miRNA) have been reported to play essential roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. We analyzed the miRNA expression profiles from adenovirus type 3 (AD3) infected Human laryngeal epithelial (Hep2) cells using a SOLiD deep sequencing. 492 precursor miRNAs were identified in the AD3 infected Hep2 cells, and 540 precursor miRNAs were identified in the control. A total of 44 miRNAs demonstrated high expression and 36 miRNAs showed lower expression in the AD3 infected cells than control. The biogenesis of miRNAs has been analyzed, and some of the SOLiD results were confirmed by Quantitative PCR analysis. The present studies may provide a useful clue for the biological function research into AD3 infection.

Identification of GRP75 as a novel PreS1 binding protein using a proteomics strategy.

The PreS1 region of the L protein is important in cell attachment and consequently in hepatitis B virus (HBV) infectivity. To identify novel PreS1 interacting protein, we performed Glutathione-S-transferase (GST) pull-down, two-dimensional gel electrophoresis (2-DE) and mass spectrometry assays. Glucose-regulated proteins (GRP) 78 and 75 were found to bind PreS1. The interactions between PreS1 and GRP75 were confirmed by a co-immunoprecipitation experiment. GRP78 and GRP75 may play important roles in mediating HBV virion entering into hepatocyte and regulating proper folding of the L protein due to their critical functions in protein folding and trafficking. The finding of novel PreS1 binding protein enriches our knowledge about molecular mechanism of HBV infection.