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. 2016 Sep 14:16:487.
doi: 10.1186/s12879-016-1794-6.

Monocytes and B cells support active replication of Chandipura virus

Soumen Roy  1 , Daya Pavitrakar  1 , Rashmi Gunjikar  1 , Vijay M Ayachit  1 , Vijay P Bondre  1 , Gajanan N Sapkal  2
Affiliations

Monocytes and B cells support active replication of Chandipura virus

Soumen Roy et al. BMC Infect Dis. .

Abstract

Background: Interaction between immune system and Chandipura virus (CHPV) during different stages of its life cycle remain poorly understood. The exact route of virus entry into the blood and CNS invasion has not been clearly defined. The present study was undertaken to assess the population in PBMC that supports the growth of virus and to detect active virus replication in PBMC as well as its subsets.

Methods: PBMC subsets viz.: CD3(+), CD14(+), CD19(+), CD56(+)cells were separated and infected with CHPV. The infected cells were then assessed for transcription (N gene primer) and replication (NP gene primer) of CHPV by PCR. The supernatant collected from infected cells were titrated in Baby Hamster Kidney (BHK) cells to assess virus release. The cytokine and chemokine expression was quantified by flow cytometry.

Results: Amplification of N and NP gene was detected in CD14(+) (monocyte) and CD19(+) (B cell), significant increase in virus titre was also observed in these subsets. It was observed that, although the levels of IL-6 and IL-10 were elevated in CD14(+) cells as compared to CD19(+)cells, the differences were not significant. However the levels of TNFα and IL-8 were significantly elevated in CD14(+) cells than in CD19(+)cells. The levels of chemokine (CXCL9, CCL5, CCL2, CXCL10) were significantly elevated in CHPV infected PBMC as compared to uninfected cells. CCL2 and CXCL9 were significantly increased in CHPV infected CD14(+)cells as compared to CD19(+) cells.

Conclusion: CD14(+)and CD19(+)cells support active replication of CHPV. High viral load was detected in CD14(+) cells infected with CHPV hence it might be the primary target cells for active replication of CHPV. An elevated levels of cytokines and chemokines observed in CD14(+) cells may help in predicting the pathogenecity of CHPV and possible entry into the central nervous system.

Keywords: B cells; Chandipura virus; Chemokine; Cytokine; Monocytes; PBMC.

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Figures

Fig. 1
Fig. 1
Detection of transcriptive and replicative form of CHPV in human PBMC. The PBMC’s were isolated from the whole blood cells of healthy donors and infected with MOI = 5 CHPV in complete medium (RPMI + 10 % FCS). Cells were harvested at different hours time interval (1, 2, 3 , 8, 12 and 24 h) and total RNA was extracted by Trizol method. The transcriptive form (N gene) and replicative form (NP gene) was detected by RT-PCR. The assay was performed in triplicates
Fig. 2
Fig. 2
Replication kinetics of CHPV in human PBMC. The PBMC’s from healthy donors were infected with CHPV at MOI = 1, MOI = 2 and MOI = 5 along with the controls. The culture supernatant was collected at different time interval (0, 12, 24 and 48 h) post infection. The release of progeny virus was quantitated by calculating TCID50 titre in BHK cells
Fig. 3
Fig. 3
Detection of transcriptive and replicative form of CHPV in human PBMC subsets. The PBMC was isolated from the whole blood cells healthy donors. CD3+,CD14+,CD19+ cells were separated from PBMCs by MACS separation method and were infected with CHPV at MOI = 5 in complete medium (RPMI + 10 % FCS). Cells were harvested at 24 h pi and total RNA was separated by Trizol method. The transcriptive form (N gene) and replicative form (NP gene) was detected by RT-PCR. The assay was performed in triplicates. Cell control is represented as C in the figure
Fig. 4
Fig. 4
Replication kinetics of CHPV in human PBMC subsets (formula image) PBMC, (formula image) CD14+cells, (formula image) CD19+cells and (formula image) CD3+cells infected with MOI = 5 of CHPV. The culture supernatant was collected at different time interval. The release of progeny virus was quantitated by calculating TCID50 titre in BHK cells. The assay was performed in triplicates
Fig. 5
Fig. 5
Cytokine and Chemokine levels in CHPV infected human PBMC subsets. a Pro-inflammatory cytokine levels (IL-10, IL-8) in CHPV infected PBMC, CD14+cell, CD19+cell supernatant. b Pro-inflammatory cytokine levels (TNFα, IL-6, IL-1β) in CHPV infected PBMC, CD14+cell, CD19+cell supernatant and respective un infected cell control (CC) at 24 h post infection (pi). c Chemokine levels (CCL2, CCL5, CXCL9, CXCL10) in CHPV infected PBMC, CD14+cell, CD19+cell supernatant and respective un infected cell control (CC) at 24 h post infection. The box plot represents minimum, first quartile, median, third quartile and maximum. Total of 9 donors were used in the experiment. Statistical analysis was performed by Wilcoxon signed rank test. Paired test was applied to compare cytokine and chemokine levels among CHPV infected PBMC, CD14+cell, CD19+cell supernatant and respective uninfected cell control (CC). p value < 0.05 (*) significant, (**) 0.005 moderately significant and (***) 0.0005 highly significant among groups
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