Comparison of methods for detection of antibody level in serum immunized with SARS-CoV-2 mRNA vaccine / 中国生物制品学杂志

@#Objective To compare three methods for detection of antibody level in serum immunized with SARS-CoV-2mRNA vaccine. Methods Enzyme-linked immunosorbent assay(ELISA),pseudo virus-based neutralization assay(PBNA)and micro-cytopathic effect neutralization test(MCPENT)were used to detect the antibody levels of a total of 120 serum samples(40 before immunization and 80 after immunization)before and after 2 doses of mRNA vaccine immunization,and the consistency and correlation of the three methods were analyzed. Results The consistency rates of the three methods detecting 120 serum samples were all over 90%,the Kappa coefficients were all more than 0. 7,and each P was less than0. 01. The correlation coefficient(r)between the antibody potency results of positive serum samples detected by the three methods was 0. 825～0. 902,and each P was less than 0. 01. Conclusion The three methods have good consistency and correlation in detecting antibody level of serum immunized with SARS-CoV-2 mRNA vaccine.

Cytopathic effect of rotavirus NSP4 on rat neurons and its influence on intracellular calcium homeostasis / 中华微生物学和免疫学杂志

Objective To investigate the cytopathic effect of amino acid residues 86 to 175 of rotavirus nonstructural protein 4 (NSP486-175) on rat neurons and to analyze the underlying mechanism.MethodsPrimary cultured rat neurons were treated with NSP486-175 and the morphological changes induced in rat neurons were observed.Lactate dehydrogenase (LDH) activity in the culture supernatant of NSP486-175 treated-neurons was measured.Laser scanning confocal microscope was used to detect the concentration of intracellular Ca2+ labeled with Fluo-3 AM.Results Exogenous addition of NSP486-175 induced obvious cytopathic effect on rat neurons.The LDH activity in the culture supernatant of treated-neurons was stronger than that of the control group.The intensity and the distribution of fluorescence in neurons were altered after stimulation with NSP486-175.Conclusion NSP486-175 can induce the damage of rat neurons, which may be related to its role in increasing the concentration of intracellular Ca2+.This study may provide certain theoretical basis for understanding extra-intestinal spread and pathogenesis of rotavirus infection.

Construction of RIP140 recombinant adenovirus and its expression in neonatal rat cardiomyocytes / 中国药理学通报

Aim The limited transfection efficiency for plasmid in primary neonatal rat cardiomyocytes,which are terminal differentiated cells,and long foreign DNA (the RIP140 gene sequence are as long as 3.5 kb) cause us to choose a better system to study RIP140 gene expression in primary non-replicative cells. Methods Full-length of RIP140 was cloned into pAdTracker-CMV shuttle vector,and then recombined with virus backbone pAdEasy-1 vector in BJ5183 bac-teria.Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determina-tion,and then transfected into AD293 cells for amplifi-cation.Titers of virus particles were determined by Tis-sue Culture Infectious Dose 50 (TCID50 )method and cell vitality was analyzed by CCK-8 kit in cardiomyo-cytes.RIP140 gene was identified by Western blot. Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into AdEasyTM sys-tem.Virus titers of Ad-RIP140 and Ad-GFP were 1011.3 and 1011.7 PFU·mL-1 ,respectively.Cell vitali-ty was not affected when the Multiplicity of Infection (MOI)was lower than 200.Green fluorescent protein (GFP)and Western blot analysis showed RIP140 gene was remarkably increased in cardiomyocytes for 12h in-fection by Ad-RIP140 (P<0.05 ).Conclusion Re-combinant adenovirus containing RIP140 gene was suc-cessfully constructed and effectively expressed in car-diomyocytes.These will be helpful for further research on the function of RIP140 in cardiomyocytes.

Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines.

Purpose: Many virulence factors are involved in the pathomechanism of infection caused by Helicobacter pylori. Toxins such as vacuolating cytotoxin, encoded by the vacA gene and the immunogenic protein cagA, encoded by the cagA gene (cytotoxin-associated gene) are major factors conferring the property of virulence. The current study is aimed at isolation of H. pylori and separation of its toxin from antral biopsies of patients. Materials and Methods: The following cell lines were used to demonstrate the cytopathic effect (CPE) of the separated toxin: African green monkey kidney (Vero), baby hamster kidney, human lung carcinoma (LLC-MK2), and human epithelial. Results: H. pylori was isolated from 27 out of 45 patients (60%) selected for the study. CPE of H. pylori toxin was highly signifi cant on Vero cells than other cell lines used as it reached a high dilution titer of toxin (1/16) in 13 isolated strains (48.15%). No signifi cant difference in CPE of toxin in different dilutions was detected among other cell lines used in different groups. H. pylori toxin could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis as a distinct band with a molecular weight ranging between 66 and 97 kDa and closely related to 87 kDa. Conclusion: H. pylori vacuolating cytotoxin plays a vital role in the pathogenesis of gastroduodenal diseases (gastritis, gastric ulcer, duodenal ulcer, and gastric cancer). The Vero cell lines were found to be the most suitable form of tissue culture when compared with other cell lines used in our study for demonstrating the activity of H. pylori toxin.

The antiviral effect of jiadifenoic acids C against coxsackievirus B3

Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0-6 h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.

Inhibition of EV71 replication in vitro by polysaccharides from Selaginella tamaritcina / 国际药学研究杂志

Objective To evaluate the inhibitory effect of polysaccharides from Selaginella tamariscina on Enterovirus 71 (EV71) replication in vitro. Methods For detecting the cytotoxicity, series of doses of polysaccharides from Selaginella tamariscina were added in RD cells, cell survival rates were evaluated by observing the cytopathic effect (CPE) and using cell counting Kit-8 (CCK8) assay, then the half toxic concentration(CC50) of the drugs were calculated. For studying the antiviral activity, the cellular model was established by infecting RD cells with EV71. Several groups were set in the experiments: normal control group, virus-infected group, positive drug treated EV71 -infected group(ribavirin 3. 2 mg/L) and series of doses of polysaccharides treated EV71 -infected groups. The CPE inhibitions were determined by using CCK8 assay and the half inhibitory concentrations of drugs(IC50) were calculated. The inhibitiory effect of polysaccharides on EV71 RNA were determined by using real-time RT-PCR methods for detecting the virus RNA levels in cell cultures. Results The CC50 of 30% and 50% alcohol precipitated polysaccharides from Selaginella tamariscina on RD cells were 396 and 142 mg/L, respectively; the IC50 calculated according to the CPE inhibitory rates were respectively 40. 8 and 26. 2 mg/L. Additionally, these two polysaccharides significantly reduced viral RNA copies in EV71-infected RD cells. Conclusion Selaginella tamariscina together with its 30% and 50% alcohol precipitated polysaccharides can be developed as potential new anti - E V 71 drugs.

Broad reactive monoclonal antibodies for rapid identification of enteroviruses show cross-reactivity with chikungunya virus infected cells

In the past decade, enterovirus 71 (EV71) and chikungunya (CHIK) virus have re-emerged periodically causing serious public health problems in Malaysia, since their first emergence in 1997 and 1998 respectively. This study demonstrates that CHIK virus causes similar patterns of cytopathic effect in cultured Vero cells as some enteroviruses. They also show positive cross-reaction on direct immunofl uorescence staining using monoclonal antibodies meant for typing enteroviruses. Without adequate clinical and epidemiological information for correlation, CHIK virus isolated from patients with acute febrile rash can be wrongly reported as untypeable enterovirus due to its cross-reactivity with commercial pan-enterovirus monoclonal antibodies. This is due to the diagnostic laboratory being unaware of such cross-reactions as it has not been reported previously. Final identifi cation of the virus could be determined with specific antibodies or molecular typing using specifi c oligonucleotide primers for the CHIK virus.

Propiedades biológicas de cepas venezolanas de DENV-2 aisladas de pacientes con fiebre de dengue y fiebre hemorrágica del dengue / Biological properties of Venezuelan DENV-2 isolated from patients with Dengue Fever and Dengue Hemorraghic Fever

INTRODUCCIÓN: el virus del dengue es responsable de un creciente problema de salud pública, sin que se haya dilucidado aún el papel de algunos atributos biológicos en la virulencia y(o) patogenicidad de los serotipos virales. OBJETIVO: evaluar propiedades biológicas in vitro e in vivo de 4 cepas venezolanas de DENV2 (LAR1693, LAR19094, LAR2303, INH35262) causantes de fiebre de dengue y fiebre hemorrágica del dengue y la cepa (16681) aislada de un paciente con fiebre hemorrágica del dengue /síndrome de choque del dengue. MÉTODOS: las cepas evaluadas fueron aisladas de sueros de pacientes virémicos con fiebre de dengue y fiebre hemorrágica del dengue. La titulación se realizó mediante el ensayo de titulación en placas en la línea celular BHK-21, esta metodología permitió determinar el tamaño de placa y la cinética de multiplicación viral. Se determinó el tiempo y la intensidad del efecto citopático e inmunofluorescencia específica en la línea celular C6/36-HT mediante las técnicas de microscopia óptica y de fluorescencia, respectivamente. De manera adicional, se emplearon las metodologías de citometría de flujo para cuantificar la replicación viral en las células C6/36-HT y la inoculación en ratones lactantes para la determinación de neurovirulencia. RESULTADOS: las cepas, excepto 16681, produjeron placas pequeñas (m.o.i.: 0,01). La replicación viral en C6/36-HT fue mayor para 16681. El efecto citopático permitió clasificar las cepas en baja (LAR19094 y LAR2303), moderada (INH35262 y 16681) y alta citopatogenicidad (LAR1693). El primer día posinoculación se detectó inmunofluorescencia entre 5 y 50 %, excepto para 16681. La neurovirulencia en ratones lactantes inoculados con 10(4) ufp/mL causó 77,5 % (INH35262) a 100 % (LAR1693 y 16681) de mortalidad, con variabilidad en los días de supervivencia. La replicación viral (24, 36 y 48 h posinoculación) mediante citometría de flujo fue desde 3,33 % hasta 90,3 %. CONCLUSIONES: los resultados permitieron concluir que el comportamiento de las cepas virales no fue evidencia suficiente para correlacionar estos atributos biológicos in vitro e in vivo con la severidad de los cuadros clínicos.

INTRODUCTION: the dengue virus causes a growing public health problem, but it has not been possible yet to determine the role of some biological attributes in the virulence and /or pathogeneity of viral serotypes. OBJECTIVE: to evaluate the in vitro and in vivo biological properties of 4 Venezuelan DENV-2 strains (LAR1693, LAR19094, LAR2303, IHN35262) responsible for dengue fever and dengue hemorrhagic fever and the reference strain (16681) isolated from a patient suffering dengue hemorrhagic fever and dengue shock syndrome. METHODS: the evaluated strains were isolated from sera of viremic patients with dengue fever and dengue hemorrhagic fever. They were then subjected to the plaque titration assay in BHK-21 cell line in order to determine the plaque size and kinetics of viral replication. The length of time and intensity of cytopathic effect and specific immunofluorescence on the C6/36-HT cell line was evaluated by optical microscopy and fluorescence respectively. Additionally, the flow cytometry to quantify viral replication in C6/36-HT cells and the intracerebral inoculation in newborn mice to find out neurovirulence were both used. RESULTS: all strains, except for 16681, showed small plaques at 0.01 m.o.i. The viral replication in C6/36-HT was higher for 16681 strain. Through cytophatic effect observations the strains were classified with low (LAR19094 and LAR2303), mild (INH35262 y 16681) and high (LAR1693) cytophatogeneity. The specific immunofluoresce ranged 5 to 50 % in the first day post inoculation, except for 16681 strain. The neurovirulence in newborn mices inoculated with 10(4) pfu/mL yielded 77.5 (INH35262) to 100% (LAR1693 y 16681) mortality rate and variability during survival days. The viral replication at 24.36 and 48 hours after inoculation was assessed by flow cytometry, ranging from 3.33 to 90.3% CONCLUSIONS: the results led to the conclusion that viral strain behaviors were not sound evidence to correlate these in vitro e in vivo biological attributes with the severity of the clinical pictures.

Preparation of recombinant human metapneumovirus expressing green fluorescent protein / 中华微生物学和免疫学杂志

Objective To construct the recombinant human metapneumovirus(hMPV) (defined as rhMPV NL/1/00 GFP) in vitro by reverse genetics technique. Methods BSR-T7 cells were transfected using LipofectAMINE 2000 with the full-length cDNA plasmid, and four major protein expressing plasmids, pCITE-N, pCITE-P, pCITE-M2.1 and pCITE-L. After 3 d, cells were subjected to one -80℃ freeze-thaw cycle to prepare lysates. The supernatant of lysate was used to inoculate Vero-E6 cells. After 1-4 d, cells were found for the obvious development of cytopathic effects under light microscope and green fluoroscopic signals under fluorescence microscope, and were observed up to 10 d. The supernatant were collected to de-tect virus titer. Viral RNA was extracted from the supernatant and reverse transcriptase polymerase chain re-action (RT-PCR) was used to amplify N, F and G genes of rescued virus. Results Cytopathic effects and green fluoroscopic signals was readily and obviously observed after 1-4 d post-inoculation in Vero-E6 cells, then cytopathic effects got worse and green fluoroscopic signals became stronger gradually up to 10 d. The ti-ters of the 1st, 5th, 10th,15th and 20th generation virus ranged from 105.0 to 106.5 TCID50/ml. Amplicons with size of 910 bp (N), 450 bp (F) and 980 bp (G) by RT-PCR were accordant with expectant. Nucleotide sequence analysis of above cDNA fragments showed 100% similarity with reported sequence of hMPV NI/1/00 strain. The recombinant virus was genetically constant and GFP-labeled after 20 passages in Vero-E6 cells. Conclusion Recombined hMPV was successfully rescued by reverse genetics technique. This study lays ground for exploring pathogenesis of hMPV infection and development of hMPV attenuated vac-cines.

Inhibitory effects of novel heteropoly compounds on influnenza virus (H1N1) in vitro / 吉林大学学报(医学版)

Objective To investigate the toxicities and inhibitory effects of three kinds of kegin-type novel heteropoly compounds which were synthesised by molecular assembly interactions on influnenza A virus(H1N1) in vitro.Methods The toxicities and anti-influnenza virus activities were tested by methods of CPE-MTT and chick embryos culture.Results TC_(50) in MDCK cells and LD_(50) in chick embryos of three kinds of kegin-type novel heteropoly compounds were higher than those of amantadine.TI in MDCK cells and chick embryos were both higher than that of amantadine. Conclusion The three kinds of kegin-type novel heteropoly compounds have lower toxicities and higher anti-virus effects on influnenza A virus(H1N1) than amantadine.Through chemic modifications,the toxicity of amantadine can be decreased and the activity of amantadine can be increased.

Production and Activity of Serum Neutralizing Antibody in Different Animals Undergoing Long-term Toxicity Study with Recombinant Consensus Interferon ? / 中国药房

0.05).In1?g/kg dosage group,no neutralizing activity was detected at the end of convalescent period.CONCLUSION:The neutralizing antibodies can be detected in the serum of both hamsters and rhesus monkeys after repeating injection of recombinant consensus interferon?.The titers of antibody are in direct ratio with the duration and the injected dose.

Study on the antiviral effects of Stichopus japonicus Selenka glycosaminoglycans (SJ-GAG) on HSV-Ⅰ / 中国海洋药物

Objective To study the antiviral effects of SJ-GAG on herpes simplex virus Ⅰ(HSV-Ⅰ) in vitro as well as its protective effect on infected mice.Methods The Vero cells were exposed to HSV-Ⅰ SM44 and different concentrations of SJ-GAG,respectively.Cytopathic effect(CPE) was observed and qunatitative PCR was used to evaluate the antiviral effect of SJ-GAG.In vivo experiment,the mice were infected with HSV1 intracranial vaccination and followed SJ-GAG intragastric administration 4h later to test the protection of SJ-GAG on HSV1 infected mice.Results When the concentration of SJ-GAG was above 1.6mg?mL-1,it showed cytotoxicity.When the concentration was among 0.25～0.2mg?mL-1,it expressed marked antiviral effect without cytotoxicity.SJ-GAG could prolong the survival duration of infected mice and decrease the mortality rate significantly.The protection of SJ-GAG showed a dose-effect relationship.Conclusion SJ-GAG has antiviral effect and shows some protective effect on HSV-1 infected mice.

ANTIVIRAL TESTS OF METHYLENE BLUE INJECTION ON VZV IN VITRO / 药物分析学报

Methylene blue injection has a stronger direct inactivation on VZV in vitro. When the injection was diluted from 1:16 to 1:128, which was obvious (P＜0.01 and P＜0.05). The MIC was 1:222, the IC50 was 1:135 and IC90 was 1:77. The results of microcellculture method showed when the injection was diluted from 1:16 to 1:64, it also effectively inhibited the proliferation of VZV in WISH continuous cell-lines (P＜0.01 and P＜0.05). The MIC was 1:95, the IC50 was 1:45 and the IC90 was 1:21.