Evaluation of passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells of serum-free suspension culture / 中国生物制品学杂志

@#Objective To evaluate the passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells(sMDCK)of serum-free suspension culture.Methods H5N1(NIBRG-14) influenza virus working-bank vaccine strains were passed 15 consecutive times in sMDCK cells.The 8-segment nucleotide sequences(HA,NA,M,NP,NS,PA, PB1 and PB2 genes) of the main-bank,working-bank,virus of P1,P2,P3,P5,P10 and P15 generations were detected for genetic stability by second and first generation sequencing.The stability of amino acid sequences of hemagglutinin(HA)and neuraminidase(NA),the main antigens of the working-bank,P5 and P15 generation viruses,were evaluated by using peptide coverage as indicators;Influenza vaccine was prepared with working-bank,P5 and P15 generation viruses,with which the female BALB/c mice were immunized i.m.with 10 in each group,15 μg HA per mouse,and boosted 28 d later at the same dosage and route.At 28,42 and 56 d after the primary immunization,the mice were detected for the titer of neutralizing antibody in serum to evaluate the stability of immunogenicity.Results No segment insertion or deletion was detected in each generation of influenza virus,and the nucleotide sequence was completely consistent with the main-bank;Single nucleotide polymorphism(SNP) mutations did not occur in the main-bank,working-bank,P1,P2,P3,P5 and P10 generations of viruses,while the possibility of SNP mutation showed in many gene loci of P15 generation virus,with heterozygous SNP accounting for 91.62%.The coverage rate of HA and NA protein peptides of P5 and P15 generation viruses ranged from96.7% to 100%.There was no significant difference in serum neutralizing antibody titer of mice in the working-bank,P5 and P15 groups(H=2.253,2.029 and 1.408,P=0.324,0.363 and 0.495,respectively) at 28,42 and 56 d after the first immunization.Conclusion H5N1(NIBRG-14) influenza virus vaccine strain has good genetic stability in sMDCK cells,which is expected to be used in the production of sMDCK cell matrix pandemic influenza vaccine.

Preliminary analysis of isolation effect of influenza virus by recombinant MDCK cells stably expressing trypsinogen / 中国生物制品学杂志

@#ObjectiveTo investigate the isolation effect of influenza virus by recombinant MDCK cells(MTY6 cells)stably expressing trypsinogen.MethodsAccording to the virus isolation method recommended by the World Health Organization(WHO)Global Influenza Surveillance Network(GISN)and the National Influenza Centers(NICs),a total of 20 throat swab specimens containing positive nucleic acid for H1N1,H3N2 and B influenza virus were isolated simultaneously using MDCK and MTY6 cells. Guinea pig red blood cells and chicken red blood cells were used for agglutination test respectively and the agglutination effects of different types of red blood cells,the positive rate of virus and the titer of hemagglutinin isolated from different cells were statistically compared.ResultsThe agglutination effect of the same virus isolate on the two types of red blood cells was different. The complete agglutination time of guinea pig red blood cells was about 2 times that of chicken red blood cells,and the deposition shape showed a ring shape. The average hemag-glutinin titer was 23. 6 ± 1. 2times that of chicken red blood cells. Under the same conditions,3 samples were negative for both types of cells,11 samples were positive for both types of cells,and the other 6 samples were negative for MDCK cells while positive for MTY6 cells. The positive rate of MTY6 cells was 30% higher than that of MDCK cells. The isolated positive samples included 8 cases of H1N1 subtype and the hemagglutinin titer of virus isolated by MTY6 cells was significantly higher than that by MDCK cells[13. 0(1. 7,23. 0)times on average]. 2 cases of H3N2 and 2 cases of B were isolated,the hemagglutinin titer of each virus isolated by MTY6 cells was 11. 3 and 32. 0 times higher than that by MDCK cells on average respectively.ConclusionIn conclusion,guinea pig red blood cells were superior to chicken red blood cells for influenza virus detection by cell isolation. Under the same conditions,MTY6 cells were more sensitive than MDCK cells for influenza virus isolation,and had the potential to be used as a high-quality cell matrix for influenza virus isolation.

Anti-Acanthamoeba castellanii activity of alkaloid-enriched extracts and lycorine from the Amaryllidaceae species

Abstract Free-living amoebae of the genus Acanthamoeba are the causative agents of granulomatous encephalitis and keratitis, severe human infections. Bioactive compounds from plants are recognized as an alternative source for the development of new drugs. The Amaryllidaceae is a botanical family able to synthesize a very specific and consistent group of biologically active isoquinoline-like alkaloids. The alkaloidal fractions from the Brazilian species Hippeastrum canastrense, H. diniz-cruziae, H. puniceum, and Crinum x amabile, along with the alkaloid lycorine, were investigated against Acanthamoeba castellanii. The in vitro assays were performed with distinct concentrations of lycorine and alkaloidal fractions, while the cell viability was evaluated by the MTT method upon MDCK cells. Chlorhexidine 0.02% was used as the positive control. The effect of alkaloid fractions was concentration dependent, and 2000 µg mL-1 of H. canastrense and H. diniz-cruziae provided a 100% inhibition. At concentrations of 250, 500, and 1000 µg mL-1, the H. diniz-cruziae alkaloidal fraction showed the lowest cytotoxic effect (5%-7%) and remarkable anti-amoebic activity, demonstrating values of IC50 285.61 µg mL-1, low cytotoxicity (5%-7%), and selectivity index (7.0). Taken together, the results are indicative of the great potential that the alkaloids from H. diniz-cruziae have as new candidates for anti-amoebicidal compounds

Determination of the bioaccessibility of cadmium and arsenic in earthworms by PBET digestion in vitro / MDCK cell model with risk assessment / 药学学报

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the content of cadmium (Cd) and arsenic (As) in earthworms. A physiologically-based extraction test (PBET) digestion in vitro /MDCK cell model was established to investigate the bioaccessibility of Cd and As in earthworms. The hazard index (HI) method and the margin of exposure (MOE) method were used to assess the risks of the total content and the bioaccessible content of Cd and As. The results showed that the total content of Cd and As in six batches of earthworms ranged from 8.319 to 33.606 mg·kg-1 and from 0.532 to 16.412 mg·kg-1, respectively. After uptake by MDCK cells, the bioaccessibility of Cd in earthworms ranged from 10.13% to 64.16%, and the bioaccessibility of As was from 2.72% to 46.57%. The results of risk assessment showed that before uptake by MDCK cells, the MOE values of As and HI values of Cd for all batches of earthworms were greater than 1, which suggests that the risks of As are acceptable but the risks of Cd are unacceptable. After transportation by MDCK cells, except for one batch of earthworms, the HI values of Cd in the other five batches were less than 1, which suggests that the risks are at a safe level. This study provides important technical support for a more objective and scientific assessment of the health risks of heavy metals in traditional Chinese medicines, and for a more scientific and reasonable standard limit of heavy metals.

Cloning and analyzing of MDCK cells for H5N1 influenza vaccine production / 中华微生物学和免疫学杂志

Objective To screen a Madin-Darby canine kidney (MDCK) cell line for H5N1 influ-enza virus isolation and to evaluate its safety in vaccine production. Methods MDCK cells were cloned by the method of limiting dilution. Hemagglutination test was used to screen MDCK cells that were suitable for H5N1 influenza virus production. Tests for analyzing the characteristics, extraneous agents, endogenous agents and tumorigenicity of MDCK cells were performed according to Chinese Pharmacopeia Volume Ⅲ. Results A total of 108 MDCK cell lines were obtained and three of them were selected after hemagglutina-tion test. G1 cells were chosen following further screening with tumorigenicity test and receptor abundance analysis. The average number of chromosomes of the MDCK-G1 cells was 78±4. No bacteria, fungi or myco-plasma contamination was detected. In experimental group, each nude mouse was injected with 1×107/ml viable cells to observe their tumorigenicity. Twelve weeks after cell injection, no node was found at injection sites or in gross anatomy. There was no significant difference between the experimental and negative control groups. The result of the tumorigenicity test was negative. No node formation was found after injecting nude mice with cell lysate or cellular DNA collected from equivalent amount of cells. It was indicated that the MDCK-G1 cells were of low-oncogenic potential. Conclusions The MDCK-G1 cell line could be used as a substrate to produce H5N1 influenza virus vaccine.

Effect and mechanism of Mahuang Tang against influenza A/H1N1 virus / 中国中药杂志

To study the effect and underlying mechanism of Mahuang Tang against influenza A virus ， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹， ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.

Study on the Transport Mechanism of Punicalagin in MDCK Cells Monolayer Model / 中国药师

Objective:To investigate the transport mechanism of punicalagin in MDCK monolayer model .Methods:The safe con-centration of punicalagin in MDCK cells was determined by CCK8 assay.Millicell -ERS was used to measure cell monolayer TEER value to determine the integrity of the cell monolayer .The effects of direction , drug concentration , time, P-gp inhibitor and EDTA-Na2 on the absorption and transport of punicalagin were studied systematically .And then the drug concentration was analyzed by HPLC to calculate the apparent permeability coefficient (Papp) and efflux ratio(ER).Results: Punicalagin transport in MDCK cells was time and concentration dependent .Punicalagin showed poor absorption in MDCK cells .Papp from apical to basolateral side ( AP-BL) within the concentration range of 100-300μg· ml-1 was (6.13 ±0.12) ×10 -7 cm· s-1 , (6.96 ±0.26) ×10 -7 cm· s-1 and (5.94 ±0.10) ×10 -7 cm· s-1 , respectively .P-gp inhibitor and EDTA-Na2 could significantly increase the transport of punicalagin in AP-BL direc-tion, while the transport decreased at 4℃.Conclusion:The transport mechanism of punicalagin might be passive diffusion as the dom-inating process involving active transportation .Punicalagin is one of P-gp substrates with exocytosis and absorbed via the paracellular route.

Study on the Anti-influenza A Virus H1N1 Effect of Aqueous Extraction of Lonicerae Flos in vitro / 中国药房

OBJECTIVE:To investigate the effect of aqueous extraction of Lonicerae Flos (SYHW) on anti-influenza A virus H1N1 (H1N1 virus) in vitro. METHODS:Using Madin-Darby canine kid ney (MDCK) cells cultured in vitro by H1N1 virus, half of the tissue culture infection dose(TCID50)was calculated. Culturing MDCK cells for 24 h with different mass concentrations of SYHW,the maximum non-toxic concentration was investigated. And then test was divided into normal cell group,virus control group,SYHW preventive administration group,therapeutic administration group and direct killing group (given SYHW of maxi-mum non-toxic concentration,infecting cells by 100 TCID50 H1N1 virus),and antiviral effective rate (ER) of SYHW was deter-mined. Test was divided into normal cell group,virus control group,SYHW therapeutic group and direct killing group (the same administration and infection as above),changes of cell proliferation index (PI) and cell apoptosis rate were respectively deter-mined. RESULTS:100 TCID50 of H1N1 virus was 1.26×10-7,and the maximum non-toxic concentration of SYHW on MDCK cells was 50 μg/mL(cell survival rate was 91.3%). ERs of preventive administration group,therapeutic administration group and direct killing group were 0,80.3% and 52.7%,respectively. Compared with normal cell group,PI value in virus control group was sig-nificantly reduced (P<0.05),early and late apoptotic rates were significantly increased (P<0.05). Compared with virus control group,PI value in directly killing group was significantly increased(P<0.05),and early apoptotic rate was significantly reduced (P<0.05);early apoptotic rates in therapeutic administration group were significantly reduced (P<0.05). CONCLUSIONS:SY-HW shows anti-H1N1 virus effect in vitro,therapeutic administration and directly killing are preferred in antiviral effect.

In vitro validation of self designed “universal human Influenza A siRNA”.

The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.

Application of the 4℃refrigerator in the isolation of influenza B virus / 中国基层医药

Objective To explore the effect of cryopreserved canine kidney cells (MDCK)single -layer on the isolation and proliferation of influenza virus B.Methods Revived P17 MDCK cells were passage for 2 -4 gener-ations,and subsequently preserved in 4℃ refrigerator for 3,6 and 9 days,respectively.Under same experimental con-ditions,the 4℃ refigerater preserved cells were co -incubated with influenza -like illness(ILI)throat swab speci-mens.Cytopathic effect (CPE)was observed,and the proliferation of virus was determined using real -time PCR and the hemagglutinin titers were determined by serological test.Results (1)CPE:The CPE of the MDCK cells pre-served in 4℃ refrigerator for 3 or 6 days had no significant differences compared with that in the control group,while the cell preserved in 4℃ refrigerator for 9 days showed CPE fastly and maintained for a short time.(2)Real -time PCR:the proliferation of influenza virus B in the MDCK cells preserved with 4℃refrigerator for 3 or 6 days (25.86 × 105 -30.25 ×106 ,26.31 ×105 -30.54 ×106 )had on difference compared with that of the control group (24.82 × 105 -29.86 ×106 ),with the proliferation rate of 105 to 106 times,while the proliferation cell with 4℃ cryopreserved for 9 days(19.72 ×104 -28.34 ×105 )the proliferation in cells preserved for 9 days was sharply decreased,with pro-liferation rate of 104 to 105 times.(3)The HA titer:The virus strains with hemagglutination titer above or equal to 116 (P >0.05)isolated with MDCK cells preserved in 4℃ refrigerator 3 or 6 days were not significantly different from that of the control group (10.92 ±0.79).And the cells with 4℃ cryopreserved for 9 days were significantly dicreased (P <0.01).Conclusion No significant effects on the isolation and proliferation of influenza virus B using MDCK cell preserved in 4℃ frigerator for near one week were observed in the present study.

In silico designing of siRNA targeting PB 1 gene of Influenza A virus and in vitro validation.

Genomic variability makes Influenza A virus (IAV) ‘the least susceptible’ to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silents the gene by cleaving mRNA. Present study aimed to develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. Total 147 strains worldwide and 42 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). To choose the most efficient siRNA, three levels screening method was developed. Finally one pair of siRNA was chosen due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line propagating two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/ 2011(H3N2)] and Influenza A/pandemicH1N1 [A/India/LKO2151/2012(H1N1)]. The longest ORF was targeted by the selected siRNA, which showed 57 % inhibition in replication of Influenza A/pdmH1N1 and 60.6 % inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.

Study on isolating iufluenza viruses from MDCK cell / 实用医学杂志

Objective To summarize the experience of isolating influenza virus from MDCK cell , and to optimize the isolation method for influenza isolation and raise the ability of influenza surveillance. Methods Sixty-one cases of influenza viruses positive specimens identified with PCR method by the influenza network laboratory in Xianyang CDC were used to infect the MDCK cells. The influenza viruses-infected MDCK cells were collected and characterized by hemagglutination test and hemagglutination inhibition assay. Meanwhile , different concentrations of glutamine liquid were added to the medium to investigate the effect of glutamine on the growth of MDCK cells. Results Twenty five flu strains were isolated from 61 cases influenza viruses positive specimens , of which 18 were A (H3N2) subtype strains and 7 were A (H1N1) subtype strains. The titer of HA was higher than 1∶16, and the titer of HI was higher than 1 ∶ 640. The proliferation rate of MDCK cells was low, and the cell activity was also decreased at 48 h after incubation with glutamine concentration lower than 0.1%. Conclusions The influenza viruses were isolated from the MDCK cells. The glutamine concentration is strongly associated with the activity of MDCK cells and cell apoptosis.

Research on multiplication of the H9N2 subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system / 中国人兽共患病学报

To explore the regularity for the multiplication of avian influenza virus subtype H9N2 in large-scale microcarrier-based MDCK cell culture system, and to determine the optimal proliferation conditions. H9N2 subtype of avian influenza virus was inoculated into the MDCK cell growing on 24 well plate, and the HA titers of virus at different time were detected in the conditions of different infectious doses,different concentrations of TPCK- trypsin and different pH. The optimal conditions were determined. Then the H9N2 subtype avian influenza virus was grown in microcarrier-based MDCK cell in 250mL and 5L roller bottles. It was demonstrated that high viruse yield with a hemagglutination unit of 9 log2(1:512) could be obtained under the optimal conditions of multiplication . The result indicated the H9N2 subtype avian influenza virus could be produced in microcarrier-based MDCK cell in a large-scale culture system with a high virus yield and demonstrates the feasibility of the development of mammalian cell-based in influenza vaccine in microcarrier culture systems.

The C-terminal variable domain of LigB from Leptospira mediates binding to fibronectin

Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.

Changes of Tight Junction and Epithelial Cells after Treatment of Cadmium / 체질인류학회지

Cadmium (Cd) affects cell proliferation, differentiation, apoptosis and other cellular activities and can cause numerous molecular lesions that would be relevant to carcinogenesis. The mechanism of adverse effects of Cd has been poorly understood and, especially on the tight junction. Since there is rare information about the effect of Cd on tight junction protein, we here investigated whether Cd can alter the localization of the proteins. This study examined Cd effects on of tight junction (occludin, ZO-1, and ZO-2) using MDCK cell culture. The change of MDCK cell and tight junction was investigated after treatment of cadmium with phase contrast microscopy, TEER, cell viability, Transmission electron microscopy and confocal laser microscopy. After treatment of cadmium, transendothelial electrical resistance decreased with time and concentration dependent manner. AlamarBlue assay revealed that decreased cell viability also decreased with time and concentration dependent manner. The tight junction moved down between intercellular spaces with decreased density and the cellular thickness around cell junctions decreased with increasing concentration and exposure time of CdCl2. The MDCK cells eventually showed cell death with. Confocal laser microscopy revealed that immunofluorescent reaction of occludin, ZO-1 and ZO-2 decreased. Occludin, ZO-1 and ZO-2 were disrupted at tight junction. These data suggest that after treatment of Cd, increased permeability of MDCK cell monolayer increased. This might be accompanied with disruption of occludin, ZO-1 and ZO-2.

Transcytosis mechanism of chlorogenic acid across cell monolayer model / 中国药理学通报

Aim To investigate the transcytosis mechanism of chlorogenic acid(CGA)by using Caco-2 and MDCK(Madin Darby canine kidney) monolayers models.Method ① Caco-2 and MDCK cell models:Caco-2 cell(105 cells/cm2) and MDCK cell(5?104 cells/cm2) were inoculated in Millicell-CM culture plate inserts,and the TEER of cell monolayer were detected to make sure the models are available for experiments.② Permeating experiments: to measure the value of OD of CGA and calculate the cumulative amount.Result CGA could be Absorbed and secreted on two monolayer models.Verapamil could inhibit the secretion at lower concentration of CGA on MDCK monolayer model.P-pg could partly act on the secretion of CGA on Caco-2 and MDCK cell models.Conclusion CGA can secrete and Absorb at the same time across Caco-2 and MDCK cell monolayers,P-pg partly involving in the secretion of CGA.

In Vitro Cultivation Model of Cryptosporidium parvum in MDCK Cells and its Development / 中国寄生虫学与寄生虫病杂志

Objective To develop an in vitro culture system for Cryptosporidium parvum in Madin-Darby canine kidney(MDCK) cell and observe its life cycle(from desquamate to oocyst).Methods Oocysts of C.parvum were co-cultured with MDCK cells in vitro.Culture condition was optimized and the life cycle of C.parvum investigated.Results The optimal culture conditions for C.parvum in MDCK cells were 2.0?105 cells cultured for 12 h, and infected by 1.0?105 oocysts in the Dulbecco′s Modified Eagle Medium with 5% FBS.Following 72 h co-culture, desquamate, sporozoites, trophozoites, meronts, microgametocytes, macrogametocytes, zygote, thin-wall oocyst, and thick-wall oocyst appeared orderly.Between the 60th and 72th hour, many oocysts emerged.Inoculated by the C.parvum-infected cell culture supernatant at the 48th hour, the immunosuppressed mice became infected.Conclusion The culture system provides a model for propagation of the parasites and demonstrates a complete in vitro life cycle of C.parvum.