Development and verification of plaque method for detection of tick-borne encephalitis virus titer / 中国生物制品学杂志

@#Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r = 0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.

Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology / Avaliação de três vacinas de Aeromonas hydrophila precipitadas com alúmen administradas a Labeo rohita, Cirrhinus mrigala e Ctenopharyngodon idella: imunocinética, desafio de imersão e histopatologia

Abstract Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.

Resumo Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes órgãos de peixes vacinados, enquanto pequenas alterações deteriorantes foram encontradas no grupo de controle e nos peixes vacinados com concentrações mais altas da vacina.

Analysis of the influential factors for influenza vaccination and their effects among elderly people of Shenzhen Pingshan district / 中国药房

OBJECTIVE To investigate the status and effect of influenza vaccination among some elderly population and their cognition of influenza vaccination in Shenzhen Pingshan district， and to provide reference for improving the vaccination willingness of the elderly population. METHODS Descriptive epidemiological method was used to analyze the status quo of influenza vaccination among some elderly populations in Pingshan district； the Logistic regression model was used to analyze the influential factors of influenza vaccination； the serum antibody levels of the elderly population were analyzed before and after influenza vaccination. RESULTS Totally 140 effective survey question naires were collected from elderly. The elderly who participated in the survey had a better cognition of the main transmission mode of the influenza virus， 122 people answered correctly， and the accuracy was 87.1%. Age， knowledge that influenza vaccine should be given once a year and knowledge of Shenzhen’s free vaccination policy were the promoting factors for influenza vaccination behavior among the elderly. The positive rates of the four influenza antibodies （type A H1， type A H3， type B Victoria， type B Yamagata） after inoculation were higher than before inoculation， with statistical significance. The antibody titer levels after vaccination were generally higher than before. CONCLUSIONS The positive rate of antibody testing and antibody titer in the elderly population after influenza vaccination are higher than before， which effectively protects the health of the elderly population. It is necessary to continuously improve the awareness of the elderly population about influenza vaccination and free vaccination policies.

Effect of ion-exchange chromatography on removal of fibronectin in von Willebrand factor / 中国输血杂志

【Objective】 To study the removal effect of fibronectin(Fn) from von willebrand factor(vWF) by ion-exchange chromatography through processing human coagulation factor Ⅷ chromatographic washing products, in order to select a method that can effectively reduce Fn without compromising the activity yield. 【Methods】 In a multi-batch process development experiment, Fractogel® EMD TMAE(M) strong anion filler produced by Merck(Germany) was used to conduct chromatography to investigate vWF ristomycins titer (vWF: RCof), vWF recovery, protein content and Fn content. 【Results】 During the development of vWF pilot purification process, the content of Fn in the samples can be effectively reduced by ion-exchange chromatography, with removal rate more than 87%, titer recovery of vWF more than 80%, and no significant change in other quality indexes. 【Conclusion】 The use of ion-exchange chromatography to purify vWF can effectively reduce the content of Fn, which has positive significance for developing new product process and improving the product quality of blood products manufacturers.

Study on the change of neutralizing antibody level in plasma donors after a third shot of inactivated novel coronavirus vaccine / 中国输血杂志

【Objective】 To investigate the trend of neutralizing antibody level in plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine. 【Methods】 Three commercial ELISA kits for novel coronavirus neutralization antibody detection, manufactured by Company A, B and C, were chosen and screened by Pseudotype Neutralization Test from December 2021 to June 2022. A total of 410 plasma samples from 64 plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine and there after donated plasma within six months were detected by the selected ELISA kit from July to October, 2022. The data were analyzed by Excel 2013 and SPSS 26 software. 【Results】 The high-throughput ELISA kit for SARS-CoV-2 neutralizing antibody detection, manufactured by Company A, was selected for further antibody titer detection. The mixed plasma titers were 1 337.34, 1 148.89, 852.19, 681.38, 556.44 and 457.19 U/mL from 1 to 6 months, respectively, after the 3rd shot of vaccine. The neutralizing antibody titer level began to increase around 7 days after the 3rd shot of vaccine injection and peaked (peak range: 264.07-2 208.39 U/mL, median: 569.34 U/mL) at 1 month (range: 9-43 days, median: 22 days), and then gradually decreased (P<0.05). 【Conclusion】 The neutralizing antibody titer of plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine began to rise around 7 days after vaccination, which reached the peak value at around 1 month and then gradually decreased.

Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology / Avaliação de três vacinas de Aeromonas hydrophila precipitadas com alúmen administradas a Labeo rohita, Cirrhinus mrigala e Ctenopharyngodon idella: imunocinética, desafio de imersão e histopatologia

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was [...].

Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes [...].

Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology

Abstract Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.

Resumo Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes órgãos de peixes vacinados, enquanto pequenas alterações deteriorantes foram encontradas no grupo de controle e nos peixes vacinados com concentrações mais altas da vacina.

Development and application of a detection method for varicella-zoster virus neutralizing antibody titer based on complement dependence / 中国生物制品学杂志

@#ObjectiveTo develop and apply a method for detecting the titer of varicella-zoster virus(VZV)neutralizing antibodies based on complement dependence,so as to improve the sensitivity of traditional plaque reduction neutralization assay for detection of the titer of VZV antibody.MethodsThe antigen(live attenuated varicella vaccine)and antibody(human VZV immunoglobulin)were mixed in different proportions and different incubation times. After neutralization,the antigen-antibody mixture was inoculated into human diploid cell 2BS strain cultured in a six-well plate. After 7 ～ 10 d of culture,the number of plaques was counted by Coomassie brilliant blue staining,and the 50% neutralizing antibody titer was calculated by Karber′s formula. Under the optimal neutralization conditions obtained,the effect of complement on the sensitivity of neutralization experiment was explored by changing the addition amount of complement(lyophilized guinea pig serum)to evaluate the optimal addition amount of complement. According to the determined neutralization test parameters,the neutralizing antibody titers of 12 anti-VZV mouse sera and 14 anti-VZV human sera were detected by using traditional plaque method and complement-dependent plaque method respectively.ResultsThe key parameters of the detection method were determined:the titer of VZV standard antigen was 500 ～ 1 000 PFU/mL;the proportion of complement added to the antigen-antibody neutralization system was 1∶10(v/v),and the neutralization condition was 37 ℃ for 1 h. Both the complement-dependent plaque method and the traditional plaque method were positive for anti-VZV mouse serum antibody,while the antibody titer detected by the traditional plaque method was generally lower,and the antibody level of mice inoculated with 2 doses of live attenuated varicella vaccine was significantly higher than that of mice inoculated with 1 dose(t = 0. 45,P < 0. 05);Both of the two methods were positive for anti-VZV human serum antibody.ConclusionA complement-dependent detection method for neutralizing antibody titer of VZV was established. The addition of complement significantly improved the sensitivity of neutralization detection. The evaluation of the titers of neutralizing antibodies in mouse serum with different immunization strategies by the method suggested that the immune effect of two doses of vaccine was better than that of one dose.

Development of national standards for genomic titer determination of recombinant type 5 adeno-associated virus / 中国生物制品学杂志

@#ObjectiveTo develop a national standard for genomic titer determination of recombinant type 5 adeno-associated virus(rAAV5).MethodsThe rAAV5-GFP stock solution prepared by the three-plasmid system was identified and verified for the appearance,pH,sterility,genomic titer,purity and infection titer according to the relevant requirements of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition),which was diluted and subpackaged to prepare candidate standards according to the results;The stability of candidate standards was investigated by thermal acceleration test;Three laboratories were organized to collaboratively calibrate the candidate standards using droplet digital PCR(ddPCR).ResultsAll the detection indexes of the candidate standard and the stock solution met the relevant requirements;The genomic titer showed no significant decrease at 25,4,-20,-40,-80 ℃ for 1,3,4,6 months;Through collaborative calibration by three laboratories,the candidate standard was assigned a value of 2. 56 × 10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL ～ 2. 64 × 10(12)copies/mL ～ 2. 64 × 10(12)copies/mL.ConclusionThe developed national standard for the determination of rAAV5 genomic titer had good stability and might be used for the quality evaluation of rAAV5 related products.

Correlation Analysis of Hemolytic Transfusion Reaction Induced by Low Titer Antibody / 中国实验血液学杂志

OBJECTIVE@#To establish the diagnostic process of low titer blood group antibody in the occurrence of adverse reactions of hemolytic transfusion.@*METHODS@#Acid elusion test, enzyme method and PEG method were used for antibody identification. Combined with the patient's clinical symptoms and relevant inspection indexes, the irregular antibodies leading to hemolysis were detected.@*RESULTS@#The patient's irregular antibody screening was positive, and it was determined that there was anti-Lea antibody in the serum. After the transfusion reaction, the low titer anti-E antibody was detected by enhanced test. The patient's Rh typing was Ccee, while the transfused red blood cells were ccEE. The new and old samples of the patient were matched with the transfused red blood cells by PEG method, and the major were incompatible. The evidence of hemolytic transfusion reaction was found.@*CONCLUSION@#Antibodies with low titer in serum are not easy to be detected, which often lead to severe hemolytic transfusion reaction.

Seroconversion and Side Effects After COVID Vaccination Among Persons with Type 2 Diabetes in Urban, Rural, and Tribal Areas in Kerala, India

Background: Persons with type 2 diabetes mellitus (T2DM) are at high?risk for COVID?19 infection and are a priority group for vaccination. Objectives: The objective of this study is to estimate the seroconversion and determine the side effects after COVID?19 vaccination among persons with T2DM in urban, rural, and tribal areas in Kerala. Methods: A cross-sectional study was conducted in urban, rural, and tribal field practice areas of a medical college in Central Kerala, among 396 persons with T2DM. The participants were selected by simple random sampling from the 200–250 diabetic patients visiting each health center. Qualitative and quantitative estimation of antibodies were done by WANTAI Ab enzyme?linked immunosorbent assay kit and Abbott SARS COV?2 IgG Quantitative assay, respectively. Results: The mean age of the respondents was 59.40 ± 12.25 years. A majority (65.5%) had received both doses of vaccine. About half (51.5%) experienced side effects after vaccination. Antibodies (IgG or IgM) were detected in 93.2% (95% confidence interval [CI] 90.2, 95.5) of participants. Those with a duration of diabetes ?5 years, with a single dose of vaccine, were five times (adjusted odds ratio [aOR] – 5.23,95% CI 1.86, 14.66) and four times (aOR – 4.11, 95% CI 1.66, 10.13) more likely, respectively, to be seronegative. Those who took medication for diabetes were protected against a no antibody (aOR – 0.05, 95% CI 0.02, 0.148) response. The median antibody titer in a subset (150) of participants was 365.2 (90–1587) AU/ml. Past COVID infection was an independent determinant of high IgG titers (aOR – 4.95, 95% CI 1.50, 16.36). Conclusion: Reinforcing the importance of vaccination particularly among those with longer duration of diabetes is imperative.

The Correlation Analysis between the Titer of IgG Anti-A/B Erythrocyte Antibody In Vivo of the Neonate and the Severity of Hemolytic Disease of Newborn / 中国实验血液学杂志

OBJECTIVE@#To investigate the titer of IgG anti-A/B erythrocyte antibody in vivo of the neonate with hemolytic disease of newborn(HDN), and explore its clinical valua in evaluating the severity of HDN.@*METHODS@#300 neonates with HDN, 50 neonates with neonatal hyperbilirubinemiain and 50 healthy neonates were selected as research object and Microtubes Gel Test was used to detect the titer of IgG anti-A/B erythrocyte antibody in vivo. Their clinical data and their mothers' prenatal examination data were retrospectively analyzed. Three hemolysis tests (direct antiglobulin test, free antibody test and release test), irregular antibody screening, and the titer of IgG anti-A/B blood group antibody was determined by serological method. Red blood cells(RBC), hemoglobin(Hb), reticulocytes(Ret) and nucleated red cells were detected by hematology analyzer. Indirect bilirubin and albumin(Alb) were detected by biochemical analyzer. The relationship between the titer of IgG anti-A/B erythrocyte antibody in vivo and the severity of HDN was analyzed.@*RESULTS@#There were six serological diagnosis modes in the HDN group,the difference between modes was statistically significant (P<0.05). The antibody titer relationship between HDN neonates and pregnant women was positive correlation(r=0.8302). The highest antibody titer of release test and free antibody test were 1∶32 and 1∶2, and the difference was statistically significant（P<0.05）. RBC, Hb and Alb in HDN patients were lower than those in neonatal hyperbilirubinemia patients and healthy neonates (P<0.05), and were negatively relevant with antibody titer in vivo (r=-0.8016). Bilirubin content in HDN patients were higher than those in neonatal hyperbiliru binemia patients and healthy neonates group(P<0.05), and was positively relevant with antibody titer in vivo (r=0.8731). The hospital day in HDN patients was significantly relevant with the antibody titer in vivo (r=0.8547), but not with the age, sex, weight and ABO blood types (P>0.05).@*CONCLUSION@#The detection of antibody titer in HDN patients can be used to evaluate the antibody concentration in vivo, predict the ability of antibody to induce erythrocyte hemolysis, and help to judge the serenrity and prognosis of HDN.

Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan

Abstract The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

Resumo O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Preparation of specific yolk immunoglobulin against Fusarium and evaluation of its anti- Fusarium effect / 中华实验眼科杂志

Objective:To prepare specific anti- Fusarium yolk immunoglobulin (IgY) and investigate its tolerance to temperature and pH and verify its antifungal effect against Fusarium. Methods:Eighteen 22-week-old Leghorn laying hens were selected and randomized into negative control group and experimental group according to the random number table method, with 9 hens in each group.The 2×10 7 colony forming units (CFU)/ml suspension of inactivated hyphae of Fusarium and Freund complete adjuvant was mixed in a 1∶1 ratio and emulsified.The hens in the experimental group were injected with 1 ml of the mixture to immunize and received 1 ml of Freund incomplete adjuvant as booster injection at two weeks after the initial injection.The egg yolk was collected from the 5th to 16th week after immunization.Specific anti- Fusarium IgY protein was prepared by salting out method using ammonium sulfate.The obtained protein solution was put into a freeze dryer and made into freeze-dried powder stored at 4 ℃.The hens in the negative control group were injected with 0.9% sodium chloride to prepare the non-specific antibody as the negative control.Bradford method was used to determine the concentration of specific egg IgY protein and the indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure its titer.The 1×10 5 CFU/ml and 1×10 3 CFU/ml Fusarium suspension were cultured with different concentrations of IgY and phosphate buffered saline (PBS) for 4 days, and the absorbance value at 600 nm was measured.The co-incubated PBS/negative IgY with Fusarium solution was set as blank control/negative control accordingly.The concentration-killing curve of anti- Fusarium IgY against Fusarium was obtained.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 7.4, and the diluted specific IgY solution was placed into the water bath for 30 minutes at 30, 40, 50, 60, 70, 80, 90 ℃, respectively, and was cooled down to room temperature.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, respectively, and the diluted specific IgY solution was placed at 4 ℃ for one hour.The activity of diluted specific IgY solution by different methods was measured by indirect ELISA, and the tolerance of IgY to various temperatures and pH was evaluated.Twelve 8-week-old SPF female C57BL/6 mice were selected and randomized into the PBS control group and specific IgY treatment group according to the random table method, with 6 mice in each group.The right eyes of the 12 mice were infected with Fusarium to establish mice model of fungal keratitis.One day after modeling, 200 mg/ml of anti- Fusarium IgY was dropped to the right eyes of mice in the specific IgY treatment group, and PBS was dropped to the right eyes of mice in the PBS control group.The corneas of mice in the two groups were observed under the slit lamp microscope at 1, 3 and 5 days following modeling, and the corneal ulcer was scored according to the grading scale for inflammation score.The use and care of experimental animals followed the Association for Research in Vision and Ophthalmology statement.This study protocol was approved by an Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL26168). Results:The IgY protein concentration from the 5th to 16th week after immunization was 1.57, 2.89, 24.98, 25.09, 23.89, 25.78, 21.57, 21.37, 18.98, 15.78, 14.67, 12.67 mg/ml, respectively.The titer of IgY was increased from the 5th week, and it reached the highest titer 1∶10 000 at the 7th week, which could be maintained until the 12th week after immunization before it dropped gradually.The concentration-killing curve showed that compared with the blank control group and negative control group, Fusarium grew slowly in the specific IgY treatment group.The specific IgY with a titer greater than 1∶10 000 had thermal stability below 60 ℃.The activity of specific IgY was highest at pH 4 to 6, which could be maintained above 70% at pH 3 to 9 and was further reduced with the decrease or increase of pH.At 1, 3 and 5 days after Fusarium infection, the inflammation scores were 3.50±0.55, 7.33±0.82, 4.00±0.63 in the PBS control group, and 3.33±0.82, 4.17±0.75, 2.50±0.55 in the specific IgY treatment group.There was a statistically significant overall difference in inflammation scores at various time points between the two groups ( Fgroup=247.35, P<0.05; Ftime=23.19, P<0.05). At 3 and 5 days after Fusarium infection, there was a smaller ulcer area and decreased inflammation scores in the specific IgY treatment group compared with the PBS control group, and the differences were statistically significant (all at P<0.05). Conclusions:The high titer specific IgY can be successfully prepared by salting out method using ammonium sulfate, which is with high stability, tolerance to temperature and pH.Moreover, it can alleviate the severity of corneal ulcers and reduce inflammation scores in the mouse model of fungal keratitis.

Advances in quantification of lentiviral vectors / 生物工程学报

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.

Improvement of natural product production in Streptomyces by manipulating pathway-specific regulators / 生物工程学报

Streptomyces are major sources of bioactive natural products. Genome sequencing reveals that Streptomyces have great biosynthetic potential, with an average of 20-40 biosynthetic gene clusters each strain. However, most natural products from Streptomyces are produced in low yields under regular laboratory cultivation conditions, which hamper their further study and drug development. The production of natural products in Streptomyces is controlled by the intricate regulation mechanisms. Manipulation of the regulatory systems that govern secondary metabolite production will strongly facilitate the discovery and development of natural products of Streptomyces origin. In this review, we summarize progresses in pathway-specific regulators from Streptomyces in the last five years and highlight their role in improving the yields of corresponding natural products.

Study on the stability of SARS-CoV-2 at different temperatures / 上海预防医学

Objective:To observe the stability of severe acute respiratory syrdrome coronavirus （SARS-CoV-2） in cell cultures at different temperatures so as to provide basic data and scientific basis for the research and control of COVID-19 epidemic. Methods:The Vero E6 cells inoculated with SARS-CoV-2. According to TCID50， SARS-CoV-2 with different dilution （10-1， 10-3， 10-5， 10-6）were stored at 37 °C， 22.5 °C， and 4 °C for one to seven days， and then infectious titer was determined by micro cytopathogenic effect assay， observing cytopathic effect （CPE）， and real-time fluorescence quantitative testing. Results:SARS-CoV-2 was stable under 4 °C. The infectivity of high concentration （10-1 dilution） under 22.5 °C for seven days gradually decreased， while lower concentration completely lost infectivity after one day. The virus lost infectivity when stored at 37 °C for more than one day. Conclusion:SARS-CoV-2 is highly stable at 4 °C， sensitive to heat， and related to virus concentration.

Clinical Analysis of Eight Cases of Pemphigus with High Titers of Anti-desmoglein Antibodies in Remission / 中国医学科学院学报

Objective To investigate the clinical manifestations,diagnosis,treatment,and laboratory examination characteristics of 8 pemphigus patients with high titers of anti-desmoglein antibodies in remission. Methods A retrospective study was conducted for the pemphigus patients diagnosed and treated in the department of dermatology from January 2013 to September 2020.The patients should have the serum anti-desmoglein antibodies ≥150 U/ml in remission or the antibody levels dropped less than 20%(calculated based on the maximum detection limit of 150 U/ml)of their initial ones detected before treatment,and the clinical and laboratory data of patients eligible for the inclusion criteria were collected. Results Among the 134 pemphigus patients with available follow-up data during this period,a total of 8 patients met the criteria,with the follow-up period of 21-85 months and the remission duration of 18-70 months.They all received less than or equal to 10 mg/d prednisone and had high titers of anti-desmoglein antibodies.At their first visit,the number of patients with positive anti-desmoglein 1/desmoglein 3 antibodies was 7.Two patients still had high titers of anti-desmoglein 1 antibodies 19 months and 21 months after they achieved remission,and 5 patients had high titers of anti-desmoglein 3 antibodies in 18-70 months.There was one patient showing high titers of both antibodies,especially for anti-desmoglein 1 antibodies.This patient relapsed after 19 months' remission while other patients were still in clinical remission. Conclusions Some pemphigus patients showed persistent high titers of anti-desmoglein antibodies in remission.Anti-desmoglein 3 antibodies were more common to keep positive,while high titer of anti-desmoglein 1 antibodies was less observed.The high titer of anti-desmoglein 1 antibodies had a correlation with recurrence.For the pemphigus patients with long-term clinical remission but high antibody titer,the dosages of corticosteroids should be adjusted carefully according to their actual clinical manifestations and the positive antibody type.For the patients with high titer of anti-desmoglein 1 antibodies,the dosage reduction of corticosteroids should be appropriately slower.