Human DC-SIGN mediates dengue virus infection of mouse B lymphocytes
Date Issued
2006
Date
2006
Author(s)
Lin, Ta-Chun
DOI
en-US
Abstract
Dengue virus is an arthropod-borne flavivirus that causes dengue fever and heamorrhagic fever in humans. It is an emerging and volatile public health concern, but the pathogenic mechanism has not been elucidated. An animal model is needed to study the pathogenesis of dengue virus infection.
It has been reported that human C-type lectin DC-SIGN that is expressed on human monocyte-derived dendritic cells binds to dengue virus. Thus, it is my speculation that animals expressing hDC-SIGN will facilitate dengue virus entering target cells and allow virus replication. The long term goal of our laboratory is to generate transgenic mouse lines to study the immunopathogenic mechanism of dengue. As a first step to reach that goal, the first aim of my study was to investigate whether mouse cells expressing hDC-SIGN became more susceptible to dengue virus infection. Next, I designed and constructed a plasmid to generate hDC-SIGN transgenic mice for the study of dengue immunopathogenesis.
First, I compared human B cell lines Raji and transfected Raji-DC-SIGN for their susceptibility to dengue virus infection and found that Raji-DC-SIGN cells were susceptible to dengue virus infection, verifying results reported in the literature. Later, I transfeced mouse B cell line M12 with hDC-SIGN and selected three stable clones, all with >90% of hDC-SIGN expression. Comparing hDC-SIGN expressing M12 with untrasnfected parental M12, I found that after infection dengue virus core gene mRNA and 13 kDa viral proteins were detected in hDC-SIGN-M12 but not in parental M12 at 12h. The virus copy numbers in the supernatants of hDC-SIGN-M12 culture also gradually increased at 12 h on after infection, indicating dengue virus replicates in the hDC-SIGN-transfected M12 cells. These results demonstrate that hDC-SIGN mediates dengue virus entry into mouse B cells and transfected mouse B cells support dengue virus replication. Furthermore, cellular response of hDC-SIGN-M12 to dengue virus was monitored. The results showed that infection by dengue virus upregulated the expressions of CD69 and CD86 and the production of TNF-α and IL-6 in hDC-SIGN-M12 cells. The results of these in vitro studies indicate that mouse cells can support the replication of and mount cellular responses to dengue virus, providing that hDC-SIGN is expressed.
I went on to construct a plasmid (pK14Tyr-PolIIhDC-SIGN-IN2B) that hDC-SIGN is under the control of polymerase II promoter, followed by a polyA tail and two copies of insulators. In vitro study showed that 293T cells transfected with pK14Tyr-PolIIhDC-SIGN-IN2B plasmid expressed hDC-SIGN on the surface. Offspring of the fertilized eggs whose pronuclei were injected of the plasmid were screened for transgene expression. Screening of the genomic DNA by PCR, I found two transgenic founders carried the hDC-SIGN transgene and the peripheral blood leukocytes of one of them expressed hDC-SIGN on the cell surface. These results showed that the strategy of plasmid construction works. The transgenic progeny will be ready for the study of the immunopathogenic mechanisms of dengue in the near future.
Subjects
登革熱病毒
dengue virus
DC-SIGN
SDGs
Type
other
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