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Delayed antibody dependent enhancement of low passage dengue virus 4 isolates.

Wikan N, Libsittikul S, Yoksan S, Auewarakul P, Smith DR - BMC Res Notes (2015)

Bottom Line: The concept of antibody dependent enhancement (ADE) of dengue virus (DENV) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates.This study utilized two low passage DENV 4 isolates and a laboratory adapted DENV 4 isolate to investigate the potential of low passage isolates to undergo ADE.These results show that low passage DENV 4 viruses undergo ADE mediated infection, but that the process is significantly temporally delayed as compared to laboratory adapted DENV 4.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand. nitwara@hotmail.com.

ABSTRACT

Background: The concept of antibody dependent enhancement (ADE) of dengue virus (DENV) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates. This study utilized two low passage DENV 4 isolates and a laboratory adapted DENV 4 isolate to investigate the potential of low passage isolates to undergo ADE.

Results: Little or no ADE of infection was observed on day 2 post infection with low passage isolates, while high enhancement of infection was seen with the laboratory adapted virus. However, both of the low passage isolates showed high levels of infection (60-100%) by day 5 post infection.

Conclusions: These results show that low passage DENV 4 viruses undergo ADE mediated infection, but that the process is significantly temporally delayed as compared to laboratory adapted DENV 4.

No MeSH data available.


Related in: MedlinePlus

Analysis of ADE infection of U937 cells with high and low passage DENVs. DENV 4 LAB, DENV 4 DF or DENV 4 DHF were incubated with a 1:200 dilution of monoclonal antibody HB114 (+HB 114) or with an isotype matched anti-alphavirus antibody (+alpha) control as appropriate, or with an equivalent volume of medium before incubation with U937 cells for 2 h after which cells were a washed with PBS and incubated with normal growth media (RPMI supplemented with 10 % FBS) with daily addition of normal medium to the cells, or b incubated with normal growth media with daily addition of normal medium to the cells or d incubated with normal growth media with daily addition of normal growth medium containing a 1:1000 dilution of HB 114 antibodies. Percentage infection was determined by flow cytometry and all experiments were undertaken independently in triplicate. Error bars show SEM M mock infection, DH (DENV 4 DHF) DF (DENV 4 DF), DL (DENV 4 LAB). c Plaque assay of supernatants from (b) assayed on days “0” (immediately after infection) 1, 2, 3 and 5 post infection. Input virus titer (−2 h) is also displayed. Plaque assays were undertaken independently in triplicate with duplicate assay of titer
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Fig2: Analysis of ADE infection of U937 cells with high and low passage DENVs. DENV 4 LAB, DENV 4 DF or DENV 4 DHF were incubated with a 1:200 dilution of monoclonal antibody HB114 (+HB 114) or with an isotype matched anti-alphavirus antibody (+alpha) control as appropriate, or with an equivalent volume of medium before incubation with U937 cells for 2 h after which cells were a washed with PBS and incubated with normal growth media (RPMI supplemented with 10 % FBS) with daily addition of normal medium to the cells, or b incubated with normal growth media with daily addition of normal medium to the cells or d incubated with normal growth media with daily addition of normal growth medium containing a 1:1000 dilution of HB 114 antibodies. Percentage infection was determined by flow cytometry and all experiments were undertaken independently in triplicate. Error bars show SEM M mock infection, DH (DENV 4 DHF) DF (DENV 4 DF), DL (DENV 4 LAB). c Plaque assay of supernatants from (b) assayed on days “0” (immediately after infection) 1, 2, 3 and 5 post infection. Input virus titer (−2 h) is also displayed. Plaque assays were undertaken independently in triplicate with duplicate assay of titer

Mentions: To address this issue, an optimized condition of ADE of multiplicity of infection (m.o.i.) 1 and a 1:200 dilution of antibody was selected and after the 2 h incubation of cells with the virus–antibody complex, cells were washed to remove un-internalized virus complexes and subsequently incubated in standard medium. A marked reduction in the level of DENV 4 LAB infection was seen, but only minimal levels (<20 %) of infection were seen with the two low passage isolates (Fig. 2a), even by day 5 post infection. When the washing step was omitted, high levels of infection were again seen with DENV 4 DF by day 3 and DENV 4 DHF by day 5 (Fig. 2b). Control experiments using an isotyped matched anti-alphavirus antibody showed no evidence of infection above levels seen with no antibody (Fig. 2b). These experiments show that it is not virus replication that is delayed in the low passage isolates, but the infection process itself. In parallel with these experiments, cells were incubated with virus but with no antibody. These are effectively an assessment of the ability of the viruses to directly enter into U937 cells in the absence of antibody–virus complex formation. Some (<20 %) direct infection was observed with DENV 4 LAB was observed, but no direct entry was seen with the low passage isolates.Fig. 2


Delayed antibody dependent enhancement of low passage dengue virus 4 isolates.

Wikan N, Libsittikul S, Yoksan S, Auewarakul P, Smith DR - BMC Res Notes (2015)

Analysis of ADE infection of U937 cells with high and low passage DENVs. DENV 4 LAB, DENV 4 DF or DENV 4 DHF were incubated with a 1:200 dilution of monoclonal antibody HB114 (+HB 114) or with an isotype matched anti-alphavirus antibody (+alpha) control as appropriate, or with an equivalent volume of medium before incubation with U937 cells for 2 h after which cells were a washed with PBS and incubated with normal growth media (RPMI supplemented with 10 % FBS) with daily addition of normal medium to the cells, or b incubated with normal growth media with daily addition of normal medium to the cells or d incubated with normal growth media with daily addition of normal growth medium containing a 1:1000 dilution of HB 114 antibodies. Percentage infection was determined by flow cytometry and all experiments were undertaken independently in triplicate. Error bars show SEM M mock infection, DH (DENV 4 DHF) DF (DENV 4 DF), DL (DENV 4 LAB). c Plaque assay of supernatants from (b) assayed on days “0” (immediately after infection) 1, 2, 3 and 5 post infection. Input virus titer (−2 h) is also displayed. Plaque assays were undertaken independently in triplicate with duplicate assay of titer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556410&req=5

Fig2: Analysis of ADE infection of U937 cells with high and low passage DENVs. DENV 4 LAB, DENV 4 DF or DENV 4 DHF were incubated with a 1:200 dilution of monoclonal antibody HB114 (+HB 114) or with an isotype matched anti-alphavirus antibody (+alpha) control as appropriate, or with an equivalent volume of medium before incubation with U937 cells for 2 h after which cells were a washed with PBS and incubated with normal growth media (RPMI supplemented with 10 % FBS) with daily addition of normal medium to the cells, or b incubated with normal growth media with daily addition of normal medium to the cells or d incubated with normal growth media with daily addition of normal growth medium containing a 1:1000 dilution of HB 114 antibodies. Percentage infection was determined by flow cytometry and all experiments were undertaken independently in triplicate. Error bars show SEM M mock infection, DH (DENV 4 DHF) DF (DENV 4 DF), DL (DENV 4 LAB). c Plaque assay of supernatants from (b) assayed on days “0” (immediately after infection) 1, 2, 3 and 5 post infection. Input virus titer (−2 h) is also displayed. Plaque assays were undertaken independently in triplicate with duplicate assay of titer
Mentions: To address this issue, an optimized condition of ADE of multiplicity of infection (m.o.i.) 1 and a 1:200 dilution of antibody was selected and after the 2 h incubation of cells with the virus–antibody complex, cells were washed to remove un-internalized virus complexes and subsequently incubated in standard medium. A marked reduction in the level of DENV 4 LAB infection was seen, but only minimal levels (<20 %) of infection were seen with the two low passage isolates (Fig. 2a), even by day 5 post infection. When the washing step was omitted, high levels of infection were again seen with DENV 4 DF by day 3 and DENV 4 DHF by day 5 (Fig. 2b). Control experiments using an isotyped matched anti-alphavirus antibody showed no evidence of infection above levels seen with no antibody (Fig. 2b). These experiments show that it is not virus replication that is delayed in the low passage isolates, but the infection process itself. In parallel with these experiments, cells were incubated with virus but with no antibody. These are effectively an assessment of the ability of the viruses to directly enter into U937 cells in the absence of antibody–virus complex formation. Some (<20 %) direct infection was observed with DENV 4 LAB was observed, but no direct entry was seen with the low passage isolates.Fig. 2

Bottom Line: The concept of antibody dependent enhancement (ADE) of dengue virus (DENV) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates.This study utilized two low passage DENV 4 isolates and a laboratory adapted DENV 4 isolate to investigate the potential of low passage isolates to undergo ADE.These results show that low passage DENV 4 viruses undergo ADE mediated infection, but that the process is significantly temporally delayed as compared to laboratory adapted DENV 4.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand. nitwara@hotmail.com.

ABSTRACT

Background: The concept of antibody dependent enhancement (ADE) of dengue virus (DENV) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates. This study utilized two low passage DENV 4 isolates and a laboratory adapted DENV 4 isolate to investigate the potential of low passage isolates to undergo ADE.

Results: Little or no ADE of infection was observed on day 2 post infection with low passage isolates, while high enhancement of infection was seen with the laboratory adapted virus. However, both of the low passage isolates showed high levels of infection (60-100%) by day 5 post infection.

Conclusions: These results show that low passage DENV 4 viruses undergo ADE mediated infection, but that the process is significantly temporally delayed as compared to laboratory adapted DENV 4.

No MeSH data available.


Related in: MedlinePlus