OBJECTIVETo produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities.

METHODSFive BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody.

RESULTSFour strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay.

CONCLUSIONNS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Dengue Virus ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; Rabbits ; Viral Envelope Proteins ; immunology ; Viral Nonstructural Proteins ; immunology

OBJECTIVETo achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris.

METHODSEDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification.

RESULTSThe recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera.

CONCLUSIONDENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

Dengue Virus ; genetics ; Genetic Vectors ; Pichia ; metabolism ; Recombinant Proteins ; genetics ; Viral Envelope Proteins ; secretion

OBJECTIVETo establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue.

METHODSThe DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA.

RESULTSThe sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000).

CONCLUSIONDENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.

Antibodies, Viral ; blood ; Dengue ; diagnosis ; immunology ; virology ; Dengue Virus ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin G ; blood ; Protein Structure, Tertiary ; Sensitivity and Specificity ; Seroepidemiologic Studies ; Viral Envelope Proteins ; immunology

OBJECTIVE: This study is to obtain precise data on iron physiological requirements in Chinese children using single stable isotope tracer technique. METHODS: Thirty boys (10.6 ± 0.2 years) and 27 girls (10.4 ± 0.2 years) were received oral 6 mg ⁵⁷Fe each day for 5 consecutive days. Venous blood samples were subsequently drawn to examine the change of total iron concentration and ⁵⁷Fe abundance at day 0, 14, 28, 60, 90, 180, 360, 450, 540, 630, 720. The iron physiological requirement was calculated by iron loss combined with iron circulation rate once ⁵⁷Fe abundance stabilized in human body. RESULTS: The iron physiological requirement was significantly lower in boys than those values in girls (16.88 ± 7.12 vs. 18.40 ± 8.81 μg/kg per day, P < 0.05). Correspondingly, the values were calculated as 722.46 ± 8.43 μg/day for boys and 708.40 ± 7.55 μg/day for girls, respectively. Considering nearly 10% iron absorption rate, the estimated average iron physiological requirement was 6.0 mg/day in boys and 6.2 mg/day in girls. CONCLUSION This study indicate that iron physiological requirement could require more daily iron intake in girls as compare with the values in boys having the same body weight. These findings would be facilitate to the new revised dietary reference intakes.
Body Weight ; Child ; China ; Female ; Humans ; Iron ; Isotopes ; Male ; Nutritional Status

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.
Animals ; Mice ; Antiviral Agents/pharmacology* ; COVID-19 ; Hepatitis B virus ; Interferon Type I/metabolism* ; SARS-CoV-2/drug effects* ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/antagonists & inhibitors*

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.
Animals ; Coronavirus Papain-Like Proteases/antagonists & inhibitors* ; Cricetinae ; Humans ; Mice ; Pandemics ; SARS-CoV-2/enzymology* ; COVID-19 Drug Treatment