Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Precision tailoring of laser beam propagation in a single crystalline diamond tool for in situ laser-assisted diamond turning.

The characteristics of laser beam propagation within a diamond tool critically influence the applied thermal softening capability of in situ laser-assisted diamond turning (In-LAT). In the present work, we perform optical geometric analysis, optical simulation and experimental validation to propose a novel diamond tool configuration for precisely tailoring laser beam propagation in In-LAT. First, the characteristics of laser beam propagation in the current In-LAT diamond tool are theoretically and experimentally explored. Second, according to the issues discovered in the current In-LAT diamond tool, an improved tool configuration based on the total internal reflection of a laser beam within the diamond tool is proposed, aiming for promoting refraction of the laser beam from the rake face of the diamond tool as well as eliminating the reflection of laser beam to tool holder. Finally, the optimization of laser beam incident position is carried out for achieving the superior profile and intensity of the emitted laser spot. Current work provides rational laser beam propagation for improving the thermal-softening capability of an In-LAT diamond tool.

Reduced neutralization and Fc effector function to Omicron subvariants in sera from SARS-CoV-1 survivors after two doses of CoronaVac plus one dose subunit vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron harbors more than 30 mutations of the spike protein and exhibits substantial immune evasion. Although previous study indicated that BNT162b2 messenger RNA vaccine induces potent cross-clade pan-sarbecovirus neutralizing antibodies in survivors of the infection by SARS-CoV-1, the neutralization activity and Fc-mediated effector functions of these cross-reactive antibodies elicited in SARS-CoV-1 survivors to Omicron subvariants still remain largely unknown. In this study, the neutralization activity and Fc-mediated effector functions of antibodies boosted by a third dose vaccination were characterized in SARS-CoV-1 convalescents and healthy individuals. Potent cross-clade broadly neutralizing antibodies were observed in SARS-CoV-1 survivors who received a three-dose vaccination regimen consisting of two priming doses of CoronaVac followed by one booster dose of the protein subunit vaccine ZF2001. However, the induced antibodies exhibited both reduced neutralization and impaired Fc effector functions targeting multiple Omicron subvariants. Importantly, the data also support the notion that immune imprints resulted from SARS-CoV-1 infection may exacerbate the impairment of neutralization activity and Fc-mediated effector functions to Omicron subvariants and provided invaluable information to vaccination strategy in future.

Evaluation of a high-sensitivity SARS-CoV-2 antigen test on the fully automated light-initiated chemiluminescent immunoassay platform.

OBJECTIVES: To describe a high-sensitivity SARS-CoV-2 antigen test that is based on the fully automated light-initiated chemiluminescent immunoassay (LiCA®), and to validate its analytical characteristics and clinical agreement on detecting SARS-CoV-2 infection against the reference molecular test. METHODS: Analytical performance was validated and detection limits were determined using different types of nucleocapsid protein samples. 798-pair anterior nasal swab specimens were collected from hospitalized patients and asymptomatic screening individuals. Agreement between LiCA® antigen and real-time reverse transcription polymerase chain reaction (rRT-PCR) was evaluated. RESULTS: Repeatability and within-lab precision were 1.6-2.3%. The C5∼C95 interval was -5.1-4.6% away from C50. Detection limits in average (SD) were 325 (±141) U/mL on the national reference panel, 0.07 (±0.04) TCID50/mL on active viral cultures, 0.27 (±0.09) pg/mL on recombinant nucleocapsid proteins and 1.07 (±1.01) TCID50/mL on inactivated viral suspensions, respectively. LiCA detected a median of 374-fold (IQR 137-643) lower levels of the viral antigen than comparative rapid tests. As reference to the rRT-PCR method, overall sensitivity and specificity were determined to be 97.5% (91.4-99.7%) and 99.9% (99.2-100%), respectively. Total agreement between both methods was 99.6% (98.7-99.9%) with Cohen's kappa 0.98 (0.96-1). A positive detection rate of 100% (95.4-100%) was obtained as Ct≤37.8. CONCLUSIONS: The LiCA® system provides an exceptionally high-sensitivity and fully automated platform for the detection of the SARS-CoV-2 antigen in nasal swabs. The assay may have high potential use for large-scale population screening and surveillance of COVID-19 as an alternative to the rRT-PCR test.

LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2.

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2.

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

Entecavir resistance mutations rtL180M/T184L/M204V combined with rtA200V lead to tenofovir resistance.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) imposes a high genetic barrier to drug resistance and potently inhibits replication of multidrug-resistant hepatitis B virus. Few clinical cases with confirmed TDF-resistance have been reported to date. METHODS AND RESULTS: Here, we report viral rebound in a patient with chronic hepatitis B who underwent TDF monotherapy and harboured a quadruple mutant consisting of classic entecavir (ETV)-resistance mutations (rtL180M/T184L/M204V) together with an rtA200V mutation in the reverse transcriptase gene. Sequencing analysis revealed that this quadruple mutant emerged as a major viral population. In vitro phenotyping demonstrated that the rtL180M/T184L/A200V/M204V mutant had moderate resistance to TDF treatment, with a 4.52-fold higher half maximal effective concentration than that of wild-type virus. Importantly, this patient with TDF resistance achieved virological suppression after TDF/ETV combination rescue therapy. CONCLUSION: An rtL180M/T184L/A200V/M204V mutant with moderate resistance to TDF monotherapy was selected during sequential nucleoside analogue (NA) treatment in a stepwise manner. ETV/TDF combination therapy effectively suppressed replication of this TDF-resistant mutant. Our studies provide novel insights into the treatment of NA-naïve patients as well as patients with TDF resistance.

Direct Nanomachining on Semiconductor Wafer By Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) is one of the most important instrumental methods of modern electrochemistry due to its high spatial and temporal resolution. We introduced SECM into nanomachining by feeding the electrochemical modulations of the tip electrode back to the positioning system, and we demonstrated that SECM is a versatile nanomachining technique on semiconductor wafers using electrochemically induced chemical etching. The removal profile was correlated to the applied tip current when the tip was held stationary and when it was moving slowly (<20 µm s-1 ), and it followed Faraday's law. Both regular and irregular nanopatterns were translated into a spatially distributed current by the homemade digitally controlled SECM instrument. The desired nanopatterns were "sculpted" directly on a semiconductor wafer by SECM direct-writing mode. The machining accuracy was controlled to the sub-micrometer and even nanometer scales. This advance is expected to play an important role in electrochemical nanomachining for 3D micro/nanostructures in the semiconductor industry.

Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses.

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

Activation of Stimulator of Interferon Genes in Hepatocytes Suppresses the Replication of Hepatitis B Virus.

Induction of interferon and proinflammatory cytokines is a hallmark of the infection of many different viruses. However, hepatitis B virus (HBV) does not elicit a detectable cytokine response in infected hepatocytes. In order to investigate the molecular mechanism underlying the innate immune evasion, a functional cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway was reconstituted in a human hepatoma cell line supporting tetracycline-inducible HBV replication. It was demonstrated that induction of HBV replication neither activated nor inhibited this cytosolic DNA sensing pathway. However, human hepatoma cells, as well as immortalized mouse hepatocytes, express low levels of STING, which upon activation by cGAMP, the natural ligand of STING, led to induction of a proinflammatory cytokine response. Treatment of immortalized mouse hepatocytes supporting HBV replication with either cGAMP or a small molecule pharmacologic STING agonist significantly reduced viral DNA in a STING- and Janus kinase 1-dependent manner. Moreover, cGAMP treatment was able to induce inflammatory cytokine gene expression and inhibit the transcription of covalently closed circular DNA in HBV-infected human hepatoma cells expressing sodium taurocholate cotransporting polypeptide, an essential receptor for HBV infection of hepatocytes. The studies reported here and previously (F. Guo et al., Antimicrob Agents Chemother 59:1273-1281, 2015, https://doi.org/10.1128/AAC.04321-14) thus support the notion that pharmacological activation of STING in macrophages and hepatocytes induces host innate responses that can efficiently control HBV replication. Hence, despite not playing a significant role in host innate immune response to HBV infection of hepatocytes, STING is potentially a valuable target for immunotherapy of chronic hepatitis B.

Interferon induction of IFITM proteins promotes infection by human coronavirus OC43.

IFNs are a family of cytokines that are essential for the antiviral response in vertebrates. Not surprisingly, viruses have adapted to encode virulence factors to cope with the IFN response. Intriguingly, we show here that all three types of interferons, IFN-α, IFN-γ, and IFN-λ, efficiently promote infection by a human coronavirus, HCoV-OC43, one of the major etiological agents of common cold, through the induction of IFN-inducible transmembrane (IFITM) proteins. IFITMs typically exert their antiviral function by inhibiting the entry of a broad spectrum of viruses into their host cells, presumably by trapping and degrading invading virions within the endocytic compartments. In contrast, HCoV-OC43 uses IFN-induced human IFITM2 or IFITM3 as an entry factor to facilitate its infection of host cells. Reverse genetics analyses suggest that the structural motifs critical for the IFITM proteins' enhancement of HCoV-OC43 infection are distinct from those required for inhibiting infection by other viruses. We also present evidence showing that IFITM family members work as homo- and hetero-oligomers to modulate virus entry. The observed enhancement of HCoV-OC43 infection by IFNs may underlie the propensity of the virus to invade the lower respiratory tract under inflammatory conditions.

The Interferon-Inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion.

UNLABELLED: Interferon alpha (IFN-α) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-α elicits a pleiotropic antiviral state in hepatitis B virus (HBV)-infected hepatocytes, but whether or not IFN-α impedes the late steps of the HBV life cycle, such as HBV secretion, remains elusive. Here we report that IFN-α treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon-stimulated gene(s) that is responsible for the reduction of HBV virion release, we found that tetherin, a broad-spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-α in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, GPI anchor-truncated tetherin exhibited a dominant-negative effect and was incorporated into the liberated virions. We also found colocalization of tetherin and HBV L protein at the intracellular multivesicular body, where the budding of HBV virions takes place. In line with this, electron microscopy demonstrated that HBV virions were tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knockdown of tetherin or overexpression of dominant negative tetherin attenuated the IFN-α-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-α inhibits HBV virion egress from hepatocytes through the induction of tetherin. IMPORTANCE: Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface with its membrane anchor domains. Here we report that interferon directly and selectively inhibits the secretion of HBV virions, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to liberated HBV virions. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virions occurs in the intracellular cisterna and that tetherin colocalizes with HBV virions on the multivesicular body, which is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.

Inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus NL63 spike protein-mediated entry by altering the glycan processing of angiotensin I-converting enzyme 2.

Endoplasmic reticulum (ER)-resident glucosidases I and II sequentially trim the three terminal glucose moieties on the N-linked glycans attached to nascent glycoproteins. These reactions are the first steps of N-linked glycan processing and are essential for proper folding and function of many glycoproteins. Because most of the viral envelope glycoproteins contain N-linked glycans, inhibition of ER glucosidases with derivatives of 1-deoxynojirimycin, i.e., iminosugars, efficiently disrupts the morphogenesis of a broad spectrum of enveloped viruses. However, like viral envelope proteins, the cellular receptors of many viruses are also glycoproteins. It is therefore possible that inhibition of ER glucosidases not only compromises virion production but also disrupts expression and function of viral receptors and thus inhibits virus entry into host cells. Indeed, we demonstrate here that iminosugar treatment altered the N-linked glycan structure of angiotensin I-converting enzyme 2 (ACE2), which did not affect its expression on the cell surface or its binding of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein. However, alteration of N-linked glycans of ACE2 impaired its ability to support the transduction of SARS-CoV and human coronavirus NL63 (HCoV-NL63) spike glycoprotein-pseudotyped lentiviral particles by disruption of the viral envelope protein-triggered membrane fusion. Hence, in addition to reducing the production of infectious virions, inhibition of ER glucosidases also impairs the entry of selected viruses via a post-receptor-binding mechanism.

STING agonists induce an innate antiviral immune response against hepatitis B virus.

Chronicity of hepatitis B virus (HBV) infection is due to the failure of a host to mount a sufficient immune response to clear the virus. The aim of this study was to identify small-molecular agonists of the pattern recognition receptor (PRR)-mediated innate immune response to control HBV infection. To achieve this goal, a coupled mouse macrophage and hepatocyte culture system mimicking the intrahepatic environment was established and used to screen small-molecular compounds that activate macrophages to produce cytokines, which in turn suppress HBV replication in a hepatocyte-derived stable cell line supporting HBV replication in a tetracycline-inducible manner. An agonist of the mouse stimulator of interferon (IFN) genes (STING), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), was found to induce a robust cytokine response in macrophages that efficiently suppressed HBV replication in mouse hepatocytes by reducing the amount of cytoplasmic viral nucleocapsids. Profiling of cytokines induced by DMXAA and agonists of representative Toll-like receptors (TLRs) in mouse macrophages revealed that, unlike TLR agonists that induced a predominant inflammatory cytokine/chemokine response, the STING agonist induced a cytokine response dominated by type I IFNs. Moreover, as demonstrated in an HBV hydrodynamic mouse model, intraperitoneal administration of DMXAA significantly induced the expression of IFN-stimulated genes and reduced HBV DNA replication intermediates in the livers of mice. This study thus proves the concept that activation of the STING pathway induces an antiviral cytokine response against HBV and that the development of small-molecular human STING agonists as immunotherapeutic agents for treatment of chronic hepatitis B is warranted.

Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

Sulfamoylbenzamide derivatives inhibit the assembly of hepatitis B virus nucleocapsids.

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.

Validation of 12 Rapid Antigen Tests for the Detection of SARS-CoV-2.

The rapid identification SARS-CoV-2 virus has become the basis for the control of the COVID-19 outbreak. The rapid antigen tests for SARS-CoV-2 are quick, widely available, and inexpensive. Rapid antigen tests have gradually replaced the time-consuming and costly RT-PCR. Currently, although several RAT kits have been extensively used for the diagnosis of COVID-19, validity data are limited due to the inconsistent sensitivity and poor reproducibility. Meanwhile, WHO does not recommend specific commercial RAT kits. Therefore, it is crucial to establish a method to evaluate the effectiveness of different rapid antigen tests kits. This study aimed to develop an evaluation system for rapid antigen tests to provide an efficient and accurate technique for screening SARS-CoV-2 antigen detection kits. Given large number of rapid antigen tests kits available, this study only focused on those that are representative and commonely used in China. By minimzing biases through randomization, concealment, and blinding, we eventually found that the Test 1 had the lowest sensitivity and the Test VI had the highest sensitivity. This study provided an evaluation platform that can potentially serve as a reference for COVID-19 diagnostic strategies.

A leveling method based on current feedback mode of scanning electrochemical microscopy.

Substrate leveling is an essential but neglected instrumental technique of scanning electrochemical microscopy (SECM). In this technical note, we provide an effective substrate leveling method based on the current feedback mode of SECM. By using an air-bearing rotary stage as the supporter of an electrolytic cell, the current feedback presents a periodic waveform signal, which can be used to characterize the levelness of the substrate. Tuning the adjusting screws of the tilt stage, substrate leveling can be completed in minutes by observing the decreased current amplitude. The obtained high-quality SECM feedback curves and images prove that this leveling technique is valuable in not only SECM studies but also electrochemical machining.

Investigation of Surface Integrity of Selective Laser Melting Additively Manufactured AlSi10Mg Alloy under Ultrasonic Elliptical Vibration-Assisted Ultra-Precision Cutting.

Additive manufacturing technology has been widely used in aviation, aerospace, automobiles and other fields due to the fact that near-net-shaped components with unprecedented geometric freedom can be fabricated. Additively manufactured aluminum alloy has received a lot of attention, due to its excellent material properties. However, the finished surface of additively manufactured aluminum alloy with nanoscale surface roughness is quite challenging and rarely addressed. In this paper, a novel machining technology known as ultrasonic elliptical vibration-assisted cutting (UEVC) was adopted to suppress the generation of cracks, improve the surface integrity and reduce tool wear during the ultra-precision machining of selective laser melting (SLM) additively manufactured AlSi10Mg alloy. The experimental results revealed that, in the conventional cutting (CC) process, surface defects, such as particles, pores and grooves, appeared on the machined surface, and the machined surface rapidly deteriorated with the increase in cumulative cutting area. In contrast, an almost flawless machined surface was obtained in the UEVC process, and its roughness value was less than 10 nm. Moreover, the tool wear of the CC tool was remarkably greater than that of the UEVC tool, and the standard flank wear width of the CC tool was more than twice that of the UEVC tool. Therefore, the UEVC technology is considered to be a feasible method for the ultra-precision machining of SLM additively manufactured AlSi10Mg alloy.

Interferon Control of Human Coronavirus Infection and Viral Evasion: Mechanistic Insights and Implications for Antiviral Drug and Vaccine Development.

Recognition of viral infections by various pattern recognition receptors (PRRs) activates an inflammatory cytokine response that inhibits viral replication and orchestrates the activation of adaptive immune responses to control the viral infection. The broadly active innate immune response puts a strong selective pressure on viruses and drives the selection of variants with increased capabilities to subvert the induction and function of antiviral cytokines. This revolutionary process dynamically shapes the host ranges, cell tropism and pathogenesis of viruses. Recent studies on the innate immune responses to the infection of human coronaviruses (HCoV), particularly SARS-CoV-2, revealed that HCoV infections can be sensed by endosomal toll-like receptors and/or cytoplasmic RIG-I-like receptors in various cell types. However, the profiles of inflammatory cytokines and transcriptome response induced by a specific HCoV are usually cell type specific and determined by the virus-specific mechanisms of subverting the induction and function of interferons and inflammatory cytokines as well as the genetic trait of the host genes of innate immune pathways. We review herein the recent literatures on the innate immune responses and their roles in the pathogenesis of HCoV infections with emphasis on the pathobiological roles and therapeutic effects of type I interferons in HCoV infections and their antiviral mechanisms. The knowledge on the mechanism of innate immune control of HCoV infections and viral evasions should facilitate the development of therapeutics for induction of immune resolution of HCoV infections and vaccines for efficient control of COVID-19 pandemics and other HCoV infections.