Discovery and development of novel 10,12-disubstituted aloperine derivatives against HCoV-OC43 by targeting allosteric site of host TMPRSS2.

By inducing steric activation of the 10CH bond with a 12-acyl group to form a key imine oxime intermediate, 20 novel (10S)-10,12-disubstituted aloperine derivatives were successfully synthesized and assessed for their antiviral efficacy against HCoV-OC43. Of them, compound 3i exhibited the moderate activities against HCoV-OC43, as well as against the SARS-CoV-2 variant EG.5.1 with the comparable EC50 values of 4.7 and 4.1 µM. A mechanism study revealed that it inhibited the protease activity of host TMPRSS2 by binding to an allosteric site, rather than the known catalytic center, different from that of camostat. Also, the combination of compound 3i and molnupiravir, as an RdRp inhibitor, showed an additive antiviral effect against HCoV-OC43. The results provide a new binding mode and lead compound for targeting TMPRSS2, with an advantage in combating broad-spectrum coronavirus.

Evolution and Discovery of Matrine Derivatives as a New Class of Anti-Hepatic Fibrosis Agents Targeting Ewing Sarcoma Breakpoint Region 1 (EWSR1).

A series of new tricyclic matrinane derivatives were continuously synthesized and evaluated for their inhibitory effects on genes and proteins related to hepatic fibrosis at the cellular level, including collagen type I α1 chain (COL1A1), α smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and matrix metalloprotein 2 (MMP-2). Among them, compound 6k exerted an appealing potency and significantly reduced liver injury and fibrosis in both bile duct ligation (BDL) rats and Mdr2 knockout mice. An activity-based protein profiling (ABPP) assay indicated that 6k might directly bind to Ewing sarcoma breakpoint region 1 (EWSR1) to inhibit its function and affect the expression of downstream liver fibrosis-related genes and thus regulate liver fibrosis. These results provided a potential novel target for the treatment of liver fibrosis and powerful information for the development of tricyclic matrinanes into promising anti-hepatic fibrosis agents.

Stimulus-Responsive Metal-Organic Framework Signal-Reporting System for Photoelectrochemical and Fluorescent Dual-Mode Detection of ATP.

Dual-mode bioanalysis integrating photoelectrochemical (PEC) and other modes is emerging and allows signal cross-checking for more reliable results. Metal-organic frameworks (MOFs) have been shown to be attractive materials in various biological applications. This work presents the utilization of MOF encapsulation and stimuli-responsive decapsulation for dual-mode PEC and fluorescence (FL) bioanalysis. Photoactive dye methylene violet (MV) was encapsulated in zeolitic imidazolate framework-90 (ZIF-90) to form an MV@ZIF-90 hybrid material, and MV could be released by adenosine triphosphate (ATP)-induced ZIF-90 disintegration. The released MV not only had FL emission but also had a sensitization effect on the ZnIn2S4 (ZnInS) photoanode. Based on the MV-dependent sensitization effect and FL emission characteristic, a dual-mode PEC-FL strategy was established for ATP detection with low detection limits, that is, 3.2 and 4.1 pM for PEC and FL detection, respectively. This study features and will inspire the construction and implementation of smart MOF materials for dual-mode bioanalysis.

The difference in CD4+ T cell immunity between high- and low-virulence Tembusu viruses is mainly related to residues 151 and 304 in the envelope protein.

Tembusu virus (TMUV) can result in a severe disease affecting domestic ducks. The role of T cells in protection from TMUV infection and the molecular basis of T cell-mediated protection against TMUV remain largely uncharacterized. Here, we used the high-virulence TMUV strain Y and the low-virulence TMUV strain PS to investigate the protective role for TMUV-specific CD4+ and CD8+ T cells. When tested in a 5-day-old Pekin duck model, Y and PS induced comparable levels of neutralizing antibody, whereas Y elicited significantly stronger cellular immune response relative to PS. Using a duck adoptive transfer model, we showed that both CD4+ and CD8+ T cells provided significant protection from TMUV-related disease, with CD8+ T cell conferring more robust protection to recipient ducklings. For TMUV, CD4+ T cells mainly provided help for neutralizing antibody response, whereas CD8+ T cells mainly mediated viral clearance from infected tissues. The difference in T cell immunity between Y and PS was primarily attributed to CD4+ T cells; adoptive transfer of Y-specific CD4+ T cells resulted in significantly enhanced protective ability, neutralizing antibody response, and viral clearance from the brain relative to PS-specific CD4+ T cells. Further investigations with chimeric viruses, mutant viruses, and their parental viruses identified two mutations (T151A and R304M) in the envelope (E) protein that contributed significantly to TMUV-specific CD4+ T cell-mediated protective ability and neutralizing antibody response, with more beneficial effects being conferred by R304M. These data indicate T cell-mediated immunity is important for protection from disease, for viral clearance from tissues, and for the production of neutralizing antibodies, and that the difference in CD4+T cell immunity between high- and low-virulence TMUV strains is primarily related to residues 151 and 304 in the E protein.

An Antibody Neutralization Determinant on Domain III and the First α-Helical Domain in the Stem-Anchor Region of Tembusu Virus Envelope Protein.

Previous studies identified three neutralizing epitopes on domains I, II, and III of the Tembusu virus (TMUV) envelope (E). More evidence is needed to understand the molecular basis of Ab-mediated neutralization and protection against TMUV. In this study, we observed a neutralizing mAb, 6C8, that neutralized TMUV infection primarily by inhibiting cell attachment. In immunofluorescence assays, 6C8 recognized the premembrane and E proteins coexpressed in HEK-293T cells, but failed to react with premembrane or E expressed individually. Epitope mapping identified nine E protein residues positioned on BC/EF loops and F/G strands in domain III and the first α-helical domain in the stem region. Further investigation with mutant viruses showed that 6C8 pressure resulted in mutations at residues 330 of BC loop and 409 of the first α-helical domain, although 6C8 only exhibited a moderate neutralizing activity in BHK-21 cells and a weak protective activity in BALB/c mice and Shaoxing duck models. Mutations A330S and T409M conferred high- and low-level 6C8 resistance, respectively, whereas the combination of A330S and T409M mutations conferred moderate-level 6C8 resistance. As a result, a quasispecies comprising three groups of antigenic variants appeared in BHK-21 cell-derived viral stocks after repeated passages of TMUV strain Y in the presence of 6C8 treatment. Taken together, these findings have raised a concern about Ab-induced antigenic variations in vivo, and they have revealed information concerning the conformational structure of the 6C8 epitope and its role in constraint on antigenic variations. The present work contributes to a better understanding of the complexity of the TMUV immunogen.

Antibody prophylaxis against Tembusu virus-associated disease.

Earlier studies have shown that Tembusu virus (TMUV) can elicit high levels of neutralizing antibodies, but the ability of antibodies to protect against TMUV-associated disease and to inhibit replication of TMUV in vivo remains to be investigated. Here, we tested the prophylactic efficacy of TMUV immune serum directly using a 2-day-old Pekin duck model. Passive administration of the immune serum prior to challenge protected ducklings against morbidity and mortality, substantially reduced TMUV-caused tissue injury, and significantly decreased TMUV levels in the periphery and central nervous system. These findings demonstrate that antibodies play a dominant protective role in controlling TMUV-associated disease.

A quasispecies in a BHK-21 cell-derived virulent Tembusu virus strain contains three groups of variants with distinct virulence phenotypes.

Previous studies resulted in the isolation of a low-virulence plaque-purified variant from the third passage (P3) in BHK-21 cells of a Tembusu virus (TMUV) isolate, suggesting the presence of viral quasispecies in the P3 culture. To confirm this notion, the fourth passage virus (P4) was prepared by infecting BHK-21 cells with P3 for isolation of more variants. We isolated 10 plaque-purified viruses. Comparative genome sequence analysis identified six of the 10 viruses as genetically different variants, which harbored a total of eight amino acid differences in the envelope, NS1, NS3, and NS5 proteins. When tested in a 2-day-old Pekin duck model, P4 caused 80 % mortality, belonging to a high-virulence TMUV strain. Out of the six genetically different variants, two presented high-virulence, one exhibited moderate-virulence, and three displayed low-virulence, causing 60 %-70 %, 40 %, and 10 % mortalities, respectively. These results demonstrate that P4 contains at least three groups of variants with distinct virulence phenotypes. Analysis of links between the eight residues and virulence of the six variants identified NS1 protein residue 183 and NS5 protein residues 275 and/or 287 as novel determinants of TMUV virulence. The analysis also provided a new clue for future studies on the molecular basis of TMUV virulence in terms of genetic interaction of different proteins. Overall, our study provides direct evidence to suggest that TMUV exists in in vitro culture of a virulent isolate as a quasispecies, which may enhance our understanding of molecular mechanism of TMUV virulence.

Mapping of a unique epitope on domain III of the envelope protein of Tembusu virus.

We recently developed a Tembusu virus (TMUV)-specific monoclonal antibody (MAb) 12F11, which was found to recognize a long amino acid sequence between residues 8 and 77 of domain III of the envelope protein (EDIII). Here, the epitope recognized by MAb 12F11 was mapped using alanine substitutions combined with dissociation constant analysis. The findings, and prediction of tertiary structure of TMUV EDIII, showed that the MAb 12F11 epitope contained one critical residue and 13 peripheral residues. Moreover, the antigenic site was shown to span four loops (N-terminal region, AB, BC, and CD) and three ß-strands (A, B, and D). The present work contributes to the understanding of antigenic structure of TMUV envelope protein.

Substantial Attenuation of Virulence of Tembusu Virus Strain PS Is Determined by an Arginine at Residue 304 of the Envelope Protein.

The Tembusu virus (TMUV) PS strain, derived by several passages and plaque purifications in BHK-21 cells, displays markedly lower virulence in Pekin ducklings relative to a natural isolate of TMUV, but the potential virulence determinants and the in vivo mechanisms for substantial virulence attenuation of the passage variant remain unknown. Here, we constructed a series of chimeric and mutant viruses and assessed their virulence using a 2-day-old Pekin duckling model. We showed that residue 304 in the envelope (E) protein is the molecular determinant of TMUV virulence. Further investigations with mutant and parental viruses demonstrated that acquisition of positive charges at E protein residue 304 plays a critical role in substantial attenuation of neurovirulence and neuroinvasiveness, which is linked to enhanced binding affinity for glycosaminoglycans (GAGs). In Pekin ducklings infected by subcutaneous inoculation, an Arg at residue 304 in the E protein was shown to contribute to more rapid virus clearance from the circulation, markedly reduced viremia, and significantly decreased viral growth in the extraneural tissues and the central nervous system, relative to a Met at the corresponding residue. These findings suggest that the in vivo mechanism of virulence attenuation of the TMUV passage variant closely resembles that proposed previously for GAG-binding variants of other flaviviruses. Overall, our study provides insight into the molecular basis of TMUV virulence and the in vivo consequences of acquisition of a GAG-binding determinant at residue 304 in the E protein of TMUV.IMPORTANCE TMUV-related disease emerged in 2010 and has a significant economic impact on the duck industry. Although the disease was originally recognized to affect adult ducks, increasing evidence has shown that TMUV also causes severe disease of young ducklings. It is, therefore, essential to investigate the pathogenesis of TMUV infection in a young duckling model. The significance of our studies is in identifying E protein residue Arg304 as the molecular determinant for TMUV virulence and in clarifying the crucial role of positive charges at E protein residue 304 in virulence attenuation of a TMUV passage variant. These data will greatly enhance our understanding of the pathogenesis of TMUV infection in ducklings and have implications for development of a safe and efficient vaccine.

The Neutralizing Antibody Response Elicited by Tembusu Virus Is Affected Dramatically by a Single Mutation in the Stem Region of the Envelope Protein.

Tembusu virus (TMUV) is a mosquito-borne flavivirus that most commonly affects adult breeder and layer ducks. However, a TMUV-caused neurological disease has also been found in ducklings below 7 weeks of age, highlighting the need to develop a safe vaccine for young ducklings. In this study, a plaque-purified PS TMUV strain was attenuated by serial passage in BHK-21 cells. Using 1-day-old Pekin ducklings as a model, the virus was confirmed to be attenuated sufficiently after 180 passages, whereas the neutralizing antibody response elicited by the 180th passage virus (PS180) was substantially impaired compared with PS. The findings suggest that sufficient attenuation results in loss of immunogenicity in the development of the live-attenuated TMUV vaccine. Comparative sequence analysis revealed that PS180 acquired one mutation (V41M) in prM and four mutations (T70A, Y176H, K313R, and F408L) in the envelope (E) protein. To identify the amino acid substitution(s) associated with loss of immunogenicity of PS180, we rescued parental viruses, rPS and rPS180, and produced mutant viruses, rPS180-M41V, rPS180-A70T, rPS180-H176Y, rPS180-R313K, rPS180-L408F, and rPS180-M5, which contained residue 41V in prM, residues 70T, 176Y, 313K, and 408F in E, and combination of the five residues, respectively, of PS in the backbone of the rPS180 genome. The neutralizing antibody response elicited by rPS180-L408F and rPS180-M5 was significantly higher than those by other mutant viruses and comparable to that by rPS. Furthermore, we produced mutant virus rPS-F408L, which contained residue 408L of PS180 in the backbone of the rPS genome. The F408L mutation conferred significantly decreased neutralizing antibody response to rPS-F408L, which was comparable to that elicited by rPS180. Based on homologous modeling, residue 408 was predicted to be located within the first helical domain of the stem region of the E protein (EH1). Together, these data demonstrate that a single mutation within the EH1 domain exerts a dramatical impact on the TMUV neutralizing antibody response. The present work may enhance our understanding of molecular basis of the TMUV neutralizing antibody response, and provides an important step for the development of a safe and efficient live-attenuated TMUV vaccine.

Pathogenicity of a Jinding duck-origin cluster 2.1 isolate of Tembusu virus in 3-week-old Pekin ducklings.

Tembusu virus (TMUV) infection most commonly affects breeder and layer ducks during laying period, and can also affect young ducks below 7 weeks of age. Here, we report our investigation of a TMUV-caused fatal disease of Jingding ducklings (Anas platyrhynchos domesticus) in Northeast China. The disease resulted in mortalities of up to 40 % in 2 to 4-week-old ducks, up to 25 % in 5 to 6-week-old ducks, and less than 10 % in 7 to 8-week-old ducks. Using a TMUV-specific reverse transcription-PCR assay, all 44 ducks collected from 10 different farms were found positive for TMUV. Phylogenetic analysis of the E nucleotide sequence revealed that five of the six TMUV strains detected from three young ducks and three laying ducks were grouped within cluster 2.1. Inoculation of the liver sample of a 40-day-old sick duck in BHK-21 cells resulted in isolation of cluster 2.1 TMUV strain H. In experimental infections performed using 3-week-old Pekin ducklings (Anas platyrhynchos domesticus) (n = 30; 10 birds/group), high mortality (60 %) was caused by strain H, in sharp contrast with a very low mortality (10 %) caused by strain Y which was isolated during outbreaks of the TMUV-related disease of young Jinding ducks in 2014 in the same region. These findings clearly demonstrated that the cluster 2.1 TMUV strain H is more pathogenic for 3-week-old ducklings as compared to the cluster 2.2 TMUV strain Y. The present study may enhance our understanding of pathogenicity of TMUV in young ducks, and will stimulate further studies on the pathogenesis of TMUV infection.

Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

Detection of Neutralizing Antibodies to Tembusu Virus: Implications for Infection and Immunity.

Neutralizing antibodies are the key mediators of protective immune response to flaviviruses after both infection and vaccination. Plaque reduction neutralization test (PRNT) is considered the "gold standard" for measurement of the immunity. To date, little is known regarding neutralizing antibody response to Tembusu virus (TMUV), a novel flavivirus emerging in ducks in 2010. Here, we developed a PRNT for detection of TMUV neutralizing antibodies. Following optimization and validation, the PRNT was applied to test serum samples from different flocks of ducks. Using sera prepared in experimental conditions, the levels of 50% end point titer (neutralizing dose, ND50) generated from positive sera (5,012-79,433) were significantly higher than those from mock-infected sera (10 to 126), indicating that the test can be used in the detection of TMUV-specific neutralizing antibodies. Dose-dependent efficacy test of a cell-derived 180th passage of a plaque-purified virus of the PS TMUV isolate (PS180) in combined with immunization-challenge experiments revealed that ND50 titer of ~1,258 is the minimum capable of providing adequate protection against challenge with virulent TMUV. In the investigation of serum samples collected from three flocks infected by TMUV and four flocks vaccinated with a licensed attenuated vaccine (the 120th passage virus), ND50 titers peaked at 1 week after both disease onset (7,943-125,893) and vaccination (3,612-79,432), and high levels of ND50 titer were detected in sera collected at 15 weeks after disease onset (5,012-63,095) and 17 weeks after vaccination (3,981-25,119). Together these findings demonstrated that spontaneous and experimental infections by TMUV and vaccination with the licensed TMUV attenuated vaccine elicit high, long-lasting neutralizing antibodies. The highest ND50 titer of neutralizing antibodies elicited by PS180 was determined to be 3,162, suggesting that attenuation of TMUV by more passages has a dramatic impact on the neutralizing antibody response of the virus.

Host Differences Affecting Resistance and Susceptibility of the Second Generation of a Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Earlier work suggested the possibility to anti duck hepatitis A virus genotype 3 (DHAV-3) using the resistance breeding strategy. Here, we report the creation of the second generations of a resistant Pekin duck flock (designated Z8R2) and a highly susceptible Pekin duck flock (designated Z8S2) and the investigation of their responses to DHAV-3. Experimental infection with DHAV-3 at 7 days of age resulted in a high mortality (66.3%) in 11 susceptible Z8S2 families and an extremely low mortality rate (2.67%) in 32 Z8R2 families, indicating that Z8R2 exhibits strong resistance to DHAV-3, while Z8S2 is highly susceptible to the virus. Detection of DHAV-3 in the liver between 1 and 60 hours post inoculation (hpi) suggests that DHAV-3 can be replicated rapidly and efficiently in the liver of Z8S2, whereas the replication of the virus in the liver of Z8R2 is suppressed greatly. High levels of serum biochemical markers (e.g., ALT, AST, ALP and GGT) were detected in Z8S2 at 24 hpi, which were significantly higher than those in Z8R2. Analysis of transcripts in the liver revealed that the expression levels of several pattern recognition receptors (PRRs) (e.g., TLR4/7, RIG-1 and MDA5) and cytokines (e.g., IL-2, IL-6, IL-8, IFN-α, and IFN-γ) in Z8S2 were significantly higher than those in Z8R2 at 12 and 24 hpi. Together these findings suggest that Z8R2 and Z8S2 Pekin ducks, which were derived from the same Z8 line, exhibit disparate pathogenic outcomes following DHAV-3 infection. Therefore, it is possible to select a Pekin duck flock resistant to DHAV-3 employing the strategy described here. It is likely that the high viral load and the strong inflammatory response correlate with the high susceptibility of Z8S2 Pekin ducks to DHAV-3.

Identification of a New Broadly Cross-reactive Epitope within Domain III of the Duck Tembusu Virus E Protein.

In 2010, a pathogenic flavivirus termed duck Tembusu virus (DTMUV) caused widespread outbreak of egg-drop syndrome in domesticated ducks in China. Although the glycoprotein E of DTMUV is an important structural component of the virus, the B-cell epitopes of this protein remains uncharacterized. Using phage display and mutagenesis, we identified a minimal B-cell epitope, 374EXE/DPPFG380, that mediates binding to a nonneutralizing monoclonal antibody. DTMUV-positive duck serum reacted with the epitope, and amino acid substitutions revealed the specific amino acids that are essential for antibody binding. Dot-blot assays of various flavivirus-positive sera indicated that EXE/DPPFG is a cross-reactive epitope in most flaviviruses, including Zika, West Nile, Yellow fever, dengue, and Japanese encephalitis viruses. These findings indicate that the epitope sequence is conserved among many strains of mosquito-borne flavivirus. Protein structure modeling revealed that the epitope is located in domain III of the DTMUV E protein. Together, these results provide new insights on the broad cross-reactivity of a B-cell binding site of the E protein of flaviviruses, which can be exploited as a diagnostic or therapeutic target for identifying, studying, or treating DTMUV and other flavivirus infections.

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

BACKGROUND: Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. METHODS AND RESULTS: To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. CONCLUSIONS AND SIGNIFICANCE: We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.