Neutralización del virus SARS-CoV-2 (linaje B.1.1) por suero hiperinmune de llama (Lama glama) en cultivo de células Vero / Neutralization of SARS-CoV-2 (lineage B.1.1) by hyperimmune llama (Lama glama) serum in Vero cell culture

Objetivo. Evaluar la respuesta serológica de anticuerpos de una llama (Lama glama) a la inmunización del virus SARS-CoV-2 (linaje B.1.1) y la capacidad neutralizante del suero de llama hiperinmune frente al virus SARS-CoV-2 (linaje B.1.1) en células Vero. Materiales y métodos. Se inmunizó una llama con el virus SARS-CoV-2 inactivado (Linaje B.1.1) y se analizaron muestras de suero para evaluar el nivel de anticuerpos mediante ELISA, así como la reactividad a antígenos de SARS-CoV-2 mediante Western Blot. Además, se evaluó la neutralización viral en cultivos celulares por la Prueba de Neutralización por Reducción de Placas (PRNT, por sus siglas en inglés). Resultados. Se observó un aumento en la serorreactividad en la llama inmunizada desde la semana 4 en adelante. Los títulos de anticuerpos fueron más elevados en el séptimo refuerzo de inmunización. Los resultados de Western Blot confirmaron los hallazgos positivos del ELISA, y los anticuerpos del suero inmune reconocieron varias proteínas virales. El ensayo de neutralización (PRNT) mostró una neutralización viral visible, concordante con los resultados de ELISA y Western Blot. Conclusiones. Los hallazgos sugieren que el suero hiperinmune de llama podría constituir una fuente de anticuerpos terapéuticos contra las infecciones por el virus SARS-CoV-2 (linaje B.1.1) y que deberá ser evaluado en estudios posteriores.

Objective. To evaluate the serological antibody response of a llama (Lama glama) to SARS-CoV-2 (B.1.1 lineage) immunization and the neutralizing capacity of hyperimmune llama serum against SARS-CoV-2 virus (B.1.1 lineage) in Vero cells. Materials and methods. A llama was immunized with inactivated SARS-CoV-2 (B.1.1 lineage). Serum samples were analyzed to evaluate the level of antibodies by ELISA, as well as reactivity to SARS-CoV-2 antigens by Western Blot. In addition, viral neutralization in cell cultures was assessed by the Plate Reduction Neutralization Test (PRNT). Results . Seroreactivity increased in the immunized llama from week 4 onwards. Antibody titers were the highest after the seventh immunization booster. Western blot results confirmed the positive ELISA findings, and immune serum antibodies recognized several viral proteins. The neutralization assay (PRNT) showed visible viral neutralization, which was in accordance with the ELISA and Western Blot results. Conclusions. The findings suggest that hyperimmune llama serum could constitute a source of therapeutic antibodies against SARS-CoV-2 infections (lineage B.1.1), and should be studied in further research.

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay / 中华预防医学杂志

Objective@#To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.@*Methods@#HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.@*Results@#The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05).@*Conclusion@#We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Serum levels of antibody against enterovirus 71 in healthy children at Shanghai in 2011 / 中华传染病杂志

Objective To test the levels of enterovirus 71 (EV71) antibody among children of different ages in Shanghai in 2011,and to investigate the relationship between antibody levels and virus infection.Methods EV71 antibody was detected by microneutralization assay from the serum specimens of healthy children of different ages collected during July to August,2011.The results were analyzed by t test for quantitative data with normal distribution,and by x2 test for count data.Results The positive rate of EV71 antibody among the 93 serum specimens was 58.1％ (54/93).The geometric mean titer (GMT) of EV71-specific neutralizing antibody was 1 ∶ 14.48.The positive rate of EV71 antibody in infants less than 6 months old was 87.5％ (21/24),and the GMT was 1∶29.56.In children aged 2 to 3 years,the positive rate of EV71 antibody decreased to 3.7％ (1/27),and GMT decreased to 1∶4.21,which were both statistically significantly lower than those less than 6 months old (x2 =36.37,t=7.58; both P＜0.01).The positive rate of EV71 antibody increased to 83.3％ (20/24) in children aged 5 to 6 years,with GMT reaching 1∶21.74.Whereas in children aged 7 to 8 years,the positive rate was 66.7％ (12/18) and GMT was 1∶20.76,without statistically significant difference compared with those aged 5 to 6 years (x2 =1.58,t=0.597; both P＞0.05).No statistically significant difference was found between boys and girls in positive rate of EV71 antibody [62.7 ％ (32/51) vs 52.4 ％ (22/42),x2 =1.02,P＞0.05] or GMT (1 ∶ 16.23 vs 1 ∶ 12.61,t=0.881,P＞0.05).Conclusions Children aged 2 to 3 years were at higher risk for EV71 infection,with EV71 antibody level significantly lower than other age groups.

Evaluation of immunogenicity of the 2008-2009 seasonal influenza vaccines by microneutralization test / 소아과

PURPOSE: For evaluating the immunogenicity of an influenza vaccine, the microneutralization (MN) test has a higher sensitivity and specificity as compared to the hemagglutination inhibition (HI) test. However, the MN test is more time consuming and is difficult to standardize. We performed the MN test to determine its usefulness as an alternative or complementary test to the HI test for evaluating the immunogenicity of influenza vaccines. METHODS: We compared the MN test with the HI test using 50 paired samples taken from a previous clinical study (2008-2009) in Korean children under 18 years of age. RESULTS: The linear correlation coefficients of the 2 tests for H3N2, H1N1, and influenza B were 0.69, 0.70, and 0.66, respectively. We identified a high index of coincidence between the 2 tests. For an influenza vaccine, the postvaccination seroprotection rates and seroconversion rates determined by the MN test were 78.0% and 96.0%, 90% and 42.0%, and 42.0% and 48.0% for H3N2, H1N1, and influenza B, respectively. Geometric mean titer fold increases of H3N2, H1N1, and influenza B were 2.89, 5.04, and 4.29, respectively, and were 2.5-fold higher. We obtained good results in the evaluation of the immunogenicity of the 2008-2009 seasonal influenza vaccines. CONCLUSION: We found that the MN test was as effective as the HI test. Therefore, we suggest that the MN test can be used as an alternative or complementary test to the HI test for evaluating the immunogenicity of influenza vaccines.

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1 (DHAV-1) Isolates in Shandong Province of China / 中国病毒学·英文版

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8％-99.7％ similarity at the nucleotide level and 92.4％-99.6％ at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.

Neutralización por reducción en placas como método específico para el Diagnóstico Serológico de Fiebre Amarilla

Objetivos: Estudiar la capacidad neutralizante de muestras séricas humanas para ser empleada como prueba de confirmación frente a otras técnicas y evaluar la relación de neutralización en diferentes poblaciones para cepas de FA: 287-78 (nativa) y 17D (referencial). Materiales y métodos: Se empleó la cepa 17D para la estandarización de la prueba en células VERO utilizando dos métodos (sólido y semi-líquido); además, se seleccionaron muestras séricas humanas en base al antecedente vacunal (presente o ausente), a la procedencia (endémica y no endémica) y la positividad a virus del dengue, las cuáles fueron evaluadas por ELISA. Las poblaciones seleccionadas se enfrentaron por la prueba estandarizada (PRNT) frente a las cepas de FA: 17D (cepa vacunal derivada del prototipo Asibi) y 287-78 (primer aislamiento de FA del Instituto Nacional de Salud del Perú). Resultados: Se obtuvieron buenos resultados con el método semi-líquido, siendo el séptimo día el óptimo de coloración de las placas. De los cuatro grupos seleccionados, tres fueron positivos por ELISA y los resultados por PRNT fueron para la suspensión 17D, 29 negativos y 67 positivos con títulos entre 1:10 y 1:2560; y para la cepa 287-78, 17 negativos y 79 positivos con títulos entre 1:10 y 1:2560. Conclusiones: La técnica semi-líquida con tiempo de coloración al séptimo día ofrece óptimos resultados en plaqueo y neutralización. Además, el ELISA resultó ser menos específico frente al PRNT y se encontró variación en relación a títulos y capacidad neutralizante en algunos sueros para ambas cepas.

Objectives: To study the neutralizing capability of human serum samples in order to be used as a confirmation test compared to other techniques, and to assess the neutralization relationship in different populations for 287-78 (native) and 17D (referential) YF strains. Materials and methods: The 17D strain was used to standardize the test in VERO cells using two methods (solid and semi-liquid). Additionally, human serum samples selected on the basis of having received YF vaccine (yes/no), their origin, and being positive for Dengue virus, and these samples were assessed using an ELISA. The selected populations were confronted using the standardized test (PRNT) with YF 17D strains (vaccinal strain derived from the Asebi prototype) and with YF 287-78 (the first YF strain isolated by the Peruvian National Institute of Health). Results: Accurate results were obtained with the semi-liquid technique, the seventh day was the optimum time for plate staining. Three of the four selected groups were positive for ELISA. The findings for PRNT were: using YF 17D strain, 29 negatives and 67 positives, with titers between 1:10 and 1:2560; and for YF 287-78, 17 negatives and 79 positive, with titers between 1:10 and 1:2560. Conclusions: The semi-liquid technique with the staining time at the seventh day showed up optimal results for plating and neutralization. Additionally, the ELISA method turned out to be less specific compared to PRNT and variations related to titers and neutralizing capability in some sera for both strains were detected.

Comparison of gene expression and DNA immunization efficacy encoding prME and E proteins derived from Japanese encephalitis virus / 中华传染病杂志

3 ?g/well.All BALB/c mice immunized with pJME by im.and gene gun injection,and JE inactivated vaccines by ip.injection survived for 21 days.BALB/c mice immunized with pJE by im.injection and gene gun injection partially survived for 21 days.Titres of neutralization antibody produced with pJME were higher than pJE.Protective immunity and titre of neutralization antibody produced by im injection was the same as gene gun injection(im/gene gun injection: 1∶320/1∶320) at day 21.The antibody from BALB/c mice sera after twice pJME immunization only reacts with JEV-E protein. Conclusions Expression efficacy of proteins encoded by pJME and pJE in transfected cells is different.Expression level of related proteins was dependant on recombinant amount for transfection in a certain degree.Immunity effect induced with pJME was higher than pJE.The efficacy of DNA immunization produced by gene gun injection was higher than im.injection.Titres of neutralization antibody induced by DNA immunization were correlated to efficacy of protective immunity.Neutralization antibody from BALB/c mice sera produced by pJME immunization contained anti-E antibody against JEV-E protein.

Little role of anti-gB antibodies in neutralizing activity of patient's sera with human cytomegalovirus (HCMV) infection

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.

Serologic study on the outbreak of acute respiratory infection caused by Adenovirus 3 / 中华传染病杂志

Objective To finally diagnose the outbreak of acute respiratory infection caused by Adenovirus 3 in students of the north of Jiangsu province by serologic study. Methods An enzyme-linked immunosorbent assay (ELISA) has been used to detect the adenovirus IgM in the serum of acute phase and the adenovirus IgG in the serum of recovery phase. A neutralization test has been used to detect the neutralization antibody in the serum of acute and recovery phase from cases and in the serum from the control. The SPSS11.0 has been applied to the statistical analysis of the data. Results The positive rate of IgM, IgG and recovery phase neutralization antibody of cases are 3.7%, 44.4% and 59.5% respectively, while those of control are 0%, 8.3% and 33.3% respectively, and the P values of Chi-Square are 0.510, 0.018 and 0.226 respectively. The neutralization test of paired serum of 6 in 9 cases showed that the antibody titers was obviously increased. The concordance between IgG detected by ELISA and neutralization antibody detected by neutralization test is 61.4% and the P value of Kappa is 0.070. Conclusion By the serologic study, we can finally diagnose that the outbreak of acute respiratory infection was caused by Adenovirus 3.

Analysis of viral etiology of sporadic viral meningitis / 中华传染病杂志

0.05).) Conclusions Coxsackie virus is the most common pathogen in patients with viral meningitis in Chaoshan district, and the organization of disease prevention and scientific research and clinical medical should attach importance to it.