Objective To develop a capture enzyme-linked immunosorbent assay (ELISA) for influenza A virus nonstructural protein NS1 using two anti-NS1 monoclonal antibodies(McAb) that map to distinct sites on the protein and to detect the NS1 in the culture supernatant and cell lysis supernatant of influenza virus. Methods Among the McAb against influenza A virus NS1, antibody pairs were analyzed to choose the optimal coating McAb and detecting McAb by determinating the detection sensitivity and specificity. Results The capture ELISA efficiently detected as little as 240 pg/ml of recombinant NS1 protein and exhibited no cross-reactivity for influenza B virus, parainfluenza virus NS1. Conclusion A sensitive and specific NS1-based capture ELISA for influenza A virus NS1 was successfully established, which could be an important tool for diagnosis of influenza A virus infection.

Objective:To ascertain whether the immune complexes (ICs) formed by Dengue virus 1 non-structure protein 1 (DENV1 NS1)and its IgG antibodies could mediate passive systemic anaphylaxis (PSA) and to explain the pathogenesis of Dengue hemorrhagic fever or Dengue shock syndrome (DHF/DSS).Methods:The monoclonal antibodies (mAbs) or mAb cocktails from 20 IgG mAbs of DENV1 NS1 prepared in this lab were screened to initiate PSA and passive cutaneous anaphylaxis (PCA) in mice.Meanwhile, the effects of GdCl3 and platelet activating factor ( PAF) antagonist CV-3988 on PSA induced by the NS1-IgG ICs were observed.Results:Two groups of monoclonal antibody cocktails with purified NS 1 were proved to be capable of provoking PCA and PSA in mice,whereas the other mAbs or mAb cocktails could be not .The murine PSA initiated by NS1-IgG(5D25+3B1) ICs could be sig-nificantly inhibited by in vivo treatment with GdCl3 or PAF antagonist CV-3988.Conclusion: The NS1-IgG ICs formed with DENV1 NS1 and IgG mAb cocktails can mediate PSA and PCA ,but not all of ICs formed by DENV 1 NS1 mAbs or mAb cocktails with DENV 1 NS1 can induce PSA ,indicating that it may be related to the special epitopes of DENV 1 NS1.The monocyte/macrophages and PAF may be as major effector cells and the major mediator for PSA induced by NS 1-IgG ICs,respectively.

Objective:To express SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus Expression System and analyze the antigenicity of the recombinant protein.Methods: The SARS-CoV nucleocapsid gene was amplified by PCR.The PCR product digested with BamHⅠand SalⅠrestriction endonucleases was cloned into vector pFastBac HTC of Bac-to-Bac Baculovirus expression system.Recombinant plasmid was transformed DH 10Bac cells to obtain the recombinant Bacmid DNA.Recombinant Bacmid DNA was transferred into Sf9 cells which were inducted to express the recombinant protein in High Five cells.After purified by Ni affinity chroma-tography ,the antigenicity of the recombinant protein was analyzed by Western blot and ELISA.Results:Recombinant plasmid was con-structed successfully.The recombinant protein with the relative molecular mass of 48 kD was efficiently expressed in High Five cells and purified successfully by Ni affinity chromatography.Western blot and ELISA analysis showed that the recombinant protein could be spe -cifically recognized by the monoclonal antibody to SARS-CoV N protein and immune serum from rabbits ,respectively.The recombinant protein can specifically reacted with serum from SARS patients ,not with serum from healthy persons and patients infected with hCoV-229 E and hCoV-OC43.Conclusion: SARS-CoV nucleocapsid protein has been expressed successfully in the Bac-to-Bac Baculovirus Expression System ,and obtained good antigenicity.It is preliminary deemed that it can't reacted with serum from patients infected with hCoV-229E and hCoV-OC43.

Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.

Objective: To evaluate an assay permitting amplification of target 5′-untranslated region (5′-UTR) sequences directly from clinical specimens and distinction among serotypes of enterovirus (EV). Methods: A total of 518 rectal swabs and 148 nasal swabs tested positive by pan-enterovirus real-time PCR were collected. 5′-UTR and the viral protein 1 (VP1) gene fragments were amplified and sequenced separately for serotyping. The inconsistent samples by 5′-UTR and VP1 serotyping were further determined by using the serotype-specific RT-PCR. Results: A total of 553 (83.0%) samples were detected by 5′-UTR serotyping and 318 (47.7%) were detected by VP1 serotyping in all 666 positive specimens, and there was significant difference in the detection rates between two methods in rectal and nasal swabs (P<0.001). For the rectal swabs, the mainly detected serotypes were CoxA6 (217), CoxA16 (88), EVA71 (40), CoxA10 (28) and CoxA4 (27) by 5′-UTR serotyping. Compared with the VP1 serotyping, the sensitivity and specificity of 5′-UTR serotyping were 57.1%-100% and 67.4%-98.1% respectively, with varied consistence with serotypes (kappa value 0.214-0.283). For the nasal swabs, the most frequently detected serotype was EVD68, with the sensitivity of 100%, the specificity of 91.1%, and the poor consistence (kappa value 0.217). CoxA6, CoxA16, EVA71, CoxA10 and EVD68 were further confirmed by serotype-specific RT-PCR. Using VP1 serotyping combined with serotype-specific RT-PCR as a reference method , the effect of performance of 5′-UTR serotyping on diagnosis was increased. Conclusions The performances of 5′-UTR serotyping in enterovirus vary with serotypes. The application of 5′-UTR serotyping should be considered comprehensively according to the purpose of the study.

Objective:To compare the accuracy of IOLMaster 700 and IOLMaster 500 in intraocular lens (IOL) power calculation.Methods:A cross-sectional study was conducted.Two hundred and sixty-two eyes of 262 patients who underwent phacoemulsification combined with IOL implantation at the Eye Hospital of Wenzhou Medical University from November 2018 to November 2019 were enrolled.Preoperative biometry for cataract surgery was performed using IOLMaster 700 and IOLMaster 500.IOL power was calculated through the built-in formulas, Haigis, Holladay Ⅰ, Hoffer Q and SRK/T of the two devices.The difference in IOL power calculation between the two devices was analyzed through the prediction error of IOL power calculation using different formulas across different axial length (AL) ranges.This study complied with the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of the Eye Hospital of Wenzhou Medical University (No.2020-038-K-33). Written informed consent was obtained from each patient before the surgery.Results:There was no significant difference in mean absolute error (MAE) between IOLMaster 700 and IOLMaster 500 using Haigis, Hoffer Q and SRK/T over the entire AL range (all at P >0.05). The MAE of IOLMaster 500 was 0.47 (0.24, 0.90) D, which was significantly lower than 0.50 (0.28, 0.99) D of IOLMaster 700 using Holladay Ⅰ formula ( Z=-3.120, P=0.002). When AL was <22.0 mm and ≥24.5 mm-<26.0 mm, there was no significant difference in MAE between the two devices using the four formulas (all at P >0.05). When AL was ≥22.0 mm-24.5 mm, there was no significant difference in the MAE between the two devices using Haigis, Hoffer Q and SRK/T (all at P >0.05), but 0.42 (0.18, 0.75) D from IOLMaster 500 was smaller than 0.45 (0.25, 0.79) D from IOLMaster 700 using Holladay Ⅰ, showing a statistically significant difference ( Z=-3.487, P <0.001). But the difference was negligible and therefore was of no clinical significance.When AL was ≥26.0 mm, there was no statistically significant difference in the MAE between the two devices using Haigis, Holladay Ⅰ and SRK/T, but 0.66 (0.38, 1.00) D from IOLMaster 500 was significantly smaller than 0.98 (0.62, 1.32) D from IOLMaster 700 using Hoffer Q ( Z=-3.046, P=0.002). Conclusions:The refractive prediction accuracy of IOLMaster 700 and IOLMaster 500 using Haigis, Hoffer Q and SRK/T is similar over the entire AL range.For patient with long AL, the IOL calculation from IOLMaster 700 using Hoffer Q is significantly larger than that from IOLMaster 500, which requires extra caution in clinical practice.The accuracy of IOLMaster 700 and IOLMaster 500 for IOL prediction is very similar.

OBJECTIVETo investigate the characteristics and dynamic changes of serum neutralizing antibody response in patients with primary infection of dengue virus type 1 (DENV-1).

METHODSSerum samples were obtained from the same patients with primary infection of DENV-1 within 2 weeks after symptom onset in 2006 and in 2010. A group-specific DENV NS1 capture ELISA-based micro-neutralizing test (ELISA-MNT) capable of detecting neutralizing antibodies against all the 4 serotypes of DENV was used to test the neutralizing antibody titers against DENV in the serum samples. The neutralizing antibody titers against a standard strain and 2 clinically isolated strains of DENV-1 were detected in serum samples collected in 2010.

RESULTSCross-reactive neutralizing antibody response against all the 4 serotypes of DENV was found in both of the serum samples collected in 2006 and 2010, but the samples collected in 2006 showed stronger cross-reactive neutralizing antibody responses. The neutralizing antibody against DENV-2, rather than the anticipated DENV-1 antibody, had the highest titer in the samples collected in 2006, whereas the antibody against homologous DENV-1 had the highest titer in the samples obtained in 2010. The neutralizing antibody titers against the homologous DENV-1 was significantly higher in samples collected in 2010 (U=86.500, P=0.000), which also demonstrated significantly different neutralizing antibody titers against the 3 different strains of DENV-1 (Χ(2)=12.123, P=0.002).

CONCLUSIONThe production of cross-reactive neutralizing antibodies between the 4 serotypes of DENV is a characteristic of DENV infection, particularly during early infection, but only the homologous neutralizing antibody increases obviously over time. The titers of the neutralizing antibodies against different strains, even of the same serotype, may differ distinctly.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Cross Reactions ; Dengue ; blood ; immunology ; Dengue Virus ; classification ; immunology ; Humans ; Neutralization Tests

Objective: By analyzing the clinical features of patients with dengue fever in Guangzhou from 2015 to 2018 to furnish the reference evidences for the diagnoses and treatment of dengue fever. Methods: A total of 406 dengue fever patients admitted to Zhujiang hospital during 2015 to 2018 were analyzed for the clinical manifestations and laboratory examination results, retrospectively. ZIKV, CHIKV and the serotypes of DENV were detected in some samples. Results: DENV serotypes were tested in 96 dengue fever patients and 69 cases were positive. Among them, 58 cases (84.1%) were DENV-1, 10 cases (14.5%) were DENV-2, 1 case (1.5%) was DENV-3, DENV-4 was negative and no co-infection with different serotypes of dengue virus was found. Of all the 406 patients, 371 (91.4%) were diagnosed as dengue fever and 35 (8.6%) were severe cases. The most common manifestations included fever, weakness and rash. Significantly higher incidence (P<0.05) of bleeding, coughing, expectoration, vomiting and abdominal pain were found in severe dengue fever. The most common laboratory findings were leucopenia and thrombocytopenia. Significant increase(P<0.05) of total bilirubin, aspartate aminotransferase, serum creatinine, high-sensitivity C-reactive protein and procalcitonin were observed. ZIKV and CHIKV nucleic acids were tested in 380 dengue fever patients and the result were negative for both. Conclusions The incidence of dengue fever in Guangzhou increased in 2017 and 2018.Most cases of dengue fever were mild and DENV-1 was the dominant serotype.