Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause acute respiratory disease and multiorgan failure. Finding human host factors that are essential for SARS-CoV-2 infection could facilitate the formulation of treatment strategies. Using a human kidney cell line-HK-2-that is highly susceptible to SARS-CoV-2, we performed a genome-wide RNAi screen and identified virus dependency factors (VDFs), which play regulatory roles in biological pathways linked to clinical manifestations of SARS-CoV-2 infection. We found a role for a secretory form of SARS-CoV-2 receptor, soluble angiotensin converting enzyme 2 (sACE2), in SARS-CoV-2 infection. Further investigation revealed that SARS-CoV-2 exploits receptor-mediated endocytosis through interaction between its spike with sACE2 or sACE2-vasopressin via AT1 or AVPR1B, respectively. Our identification of VDFs and the regulatory effect of sACE2 on SARS-CoV-2 infection shed insight into pathogenesis and cell entry mechanisms of SARS-CoV-2 as well as potential treatment strategies for COVID-19.

High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis.

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 "ESBL-positive" isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

Human tryptophanyl-tRNA synthetase is an IFN-γ-inducible entry factor for Enterovirus.

Enterovirus A71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand, foot, and mouth disease and life-threatening encephalitis. We identified an IFN-γ-inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pulldown, antibody/antigen blocking, and CRISPR/Cas9-mediated deletion. Hyperexpression and plasma membrane translocation of hWARS were observed in IFN-γ-treated semipermissive (human neuronal NT2) and cDNA-transfected nonpermissive (mouse fibroblast L929) cells, resulting in their sensitization to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to those seen in patients with severe EV-A71 infection. Expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important coxsackievirus A16 (CV-A16) and EV-D68. This is the first report to our knowledge on the discovery of an entry factor, hWARS, that can be induced by IFN-γ for EV-A71 infection. Given that we detected high levels of IFN-γ in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to entry factor expression upon IFN-γ stimulation and the therapeutic options for treating severe EV-A71-associated complications.

Integrated analysis of mRNA-seq and miRNA-seq for host susceptibilities to influenza A (H7N9) infection in inbred mouse lines.

Host genetic factors play an important role in diverse host outcomes after influenza A (H7N9) infection. Studying differential responses of inbred mouse lines with distinct genetic backgrounds to influenza virus infection could substantially increase our understanding of the contributory roles of host genetic factors to disease severity. Here, we utilized an integrated approach of mRNA-seq and miRNA-seq to investigate the transcriptome expression and regulation of host genes in C57BL/6J and DBA/2J mouse strains during influenza virus infection. The differential pathogenicity of influenza virus in C57BL/6J and DBA/2J has been fully demonstrated through immunohistochemical staining, histopathological analyses, and viral replication assessment. A transcriptional molecular signature correlates to differential host response to infection has been uncovered. With the introduction of temporal expression pattern analysis, we demonstrated that host factors responsible for influenza virus replication and host-virus interaction were significantly enriched in genes exhibiting distinct temporal dynamics between different inbred mouse lines. A combination of time-series expression analysis and temporal expression pattern analysis has provided a list of promising candidate genes for future studies. An integrated miRNA regulatory network from both mRNA-seq and miRNA-seq revealed several regulatory modules responsible for regulating host susceptibilities and disease severity. Overall, a comprehensive framework for analyzing host susceptibilities to influenza infection was established by integrating mRNA-seq and miRNA-seq data of inbred mouse lines. This work suggests novel putative molecular targets for therapeutic interventions in seasonal and pandemic influenza.

PacBio But Not Illumina Technology Can Achieve Fast, Accurate and Complete Closure of the High GC, Complex Burkholderia pseudomallei Two-Chromosome Genome.

Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68-69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201-366 contigs), sharing only 92.2-100% and 92.0-100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.

MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes sporadic zoonotic disease and healthcare-associated outbreaks in human. MERS is often complicated by acute respiratory distress syndrome (ARDS) and multi-organ failure(1,2). The high incidence of renal failure in MERS is a unique clinical feature not often found in other human coronavirus infections(3,4). Whether MERS-CoV infects the kidney and how it triggers renal failure are not understood(5,6). Here, we demonstrated renal infection and apoptotic induction by MERS-CoV in human ex vivo organ culture and a nonhuman primate model. High-throughput analysis revealed that the cellular genes most significantly perturbed by MERS-CoV have previously been implicated in renal diseases. Furthermore, MERS-CoV induced apoptosis through upregulation of Smad7 and fibroblast growth factor 2 (FGF2) expression in both kidney and lung cells. Conversely, knockdown of Smad7 effectively inhibited MERS-CoV replication and protected cells from virus-induced cytopathic effects. We further demonstrated that hyperexpression of Smad7 or FGF2 induced a strong apoptotic response in kidney cells. Common marmosets infected by MERS-CoV developed ARDS and disseminated infection in kidneys and other organs. Smad7 and FGF2 expression were elevated in the lungs and kidneys of the infected animals. Our results provide insights into the pathogenesis of MERS-CoV and host targets for treatment.

Treatment With Lopinavir/Ritonavir or Interferon-ß1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe disease in human with an overall case-fatality rate of >35%. Effective antivirals are crucial for improving the clinical outcome of MERS. Although a number of repurposed drugs, convalescent-phase plasma, antiviral peptides, and neutralizing antibodies exhibit anti-MERS-CoV activity in vitro, most are not readily available or have not been evaluated in nonhuman primates. We assessed 3 repurposed drugs with potent in vitro anti-MERS-CoV activity (mycophenolate mofetil [MMF], lopinavir/ritonavir, and interferon-ß1b) in common marmosets with severe disease resembling MERS in humans. The lopinavir/ritonavir-treated and interferon-ß1b-treated animals had better outcome than the untreated animals, with improved clinical (mean clinical scores ↓50.9%-95.0% and ↓weight loss than the untreated animals), radiological (minimal pulmonary infiltrates), and pathological (mild bronchointerstitial pneumonia) findings, and lower mean viral loads in necropsied lung (↓0.59-1.06 log10 copies/glyceraldehyde 3-phosphate dehydrogenase [GAPDH]; P < .050) and extrapulmonary (↓0.11-1.29 log10 copies/GAPDH; P < .050 in kidney) tissues. In contrast, all MMF-treated animals developed severe and/or fatal disease with higher mean viral loads (↑0.15-0.54 log10 copies/GAPDH) than the untreated animals. The mortality rate at 36 hours postinoculation was 67% (untreated and MMF-treated) versus 0-33% (lopinavir/ritonavir-treated and interferon-ß1b-treated). Lopinavir/ritonavir and interferon-ß1b alone or in combination should be evaluated in clinical trials. MMF alone may worsen MERS and should not be used.

Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging. MATERIALS AND METHODS: Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions. RESULTS: The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3. CONCLUSIONS: C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways.

Effects of Bai-Hu decoction on fever induced by lipopolysaccharide.

This study was designed to investigate the antifebrile effect and mechanisms of Bai-Hu decoction (BHD), a traditional Chinese medical (TCM) prescription. The rabbits used in this study received an intravenous injection of lipopolysaccharide (LPS) after being orally administered with BHD, ibuprofen, or saline, and their rectal temperatures were monitored by a copper-constantan thermocouple. Concentrations of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in serum and hypothalamus were assayed using the commercially available rabbit IL-1ß and TNF-α enzyme-linked immunosorbent assay kits following the manufacturer's instructions. The BHD treatment group exhibited a significant fall in body temperature in both peaks compared with the LPS group (p<0.05). BHD reduced the concentrations of IL-1ß and TNF-α in serum, and of TNF-α in hypothalamus to control the febrile responses at 1 hour. Besides the levels of IL-1ß in hypothalamus and serum, the concentration of TNF-α in hypothalamus was decreased remarkably in the BHD group than in the LPS group at 3 hours. The main findings, the partial mechanisms of BHD in reducing biphasic fever elicited by LPS, were that treatments with the crude extract of BHD could remarkably reduce the increased concentrations of IL-1ß and TNF-α, not only in serum but also in hypothalamus. The results indicated that BHD would be a valuable candidate for further investigation as a traditional antifebrile and anti-inflammatory natural drug.