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. 1998 Dec;72(12):9729-37.
doi: 10.1128/JVI.72.12.9729-9737.1998.

Study of Dengue virus infection in SCID mice engrafted with human K562 cells

Y L Lin  1 , C L Liao, L K Chen, C T Yeh, C I Liu, S H Ma, Y Y Huang, Y L Huang, C L Kao, C C King
Affiliations

Study of Dengue virus infection in SCID mice engrafted with human K562 cells

Y L Lin et al. J Virol. 1998 Dec.

Abstract

Here we report that severe combined immunodeficient (SCID) mice engrafted with human K562 cells (K562-SCID mice) can be used as an animal model to study dengue virus (DEN) infection. After intratumor injection into K562 cell masses of PL046, a Taiwanese DEN-2 human isolate, the K562-SCID mice showed neurological signs of paralysis and died at approximately 2 weeks postinfection. In addition to being detected in the tumor masses, high virus titers were detected in the peripheral blood and the brain tissues, indicating that DEN had replicated in the infected K562-SCID mice. In contrast, the SCID mice were resistant to DEN infection and the mock-infected K562-SCID mice survived for over 3 months. These data illustrate that DEN infection contributed directly to the deaths of the infected K562-SCID mice. Other serotypes of DEN were also used to infect the K562-SCID mice, and the mortality rates of the infected mice varied with different challenge strains, suggesting the existence of diverse degrees of virulence among DENs. To determine whether a neutralizing antibody against DEN in vitro was also protective in vivo, the K562-SCID mice were challenged with DEN-2 and received antibody administration at the same time or 1 day earlier. Our results revealed that the antibody-treated mice exhibited a reduction in mortality and a delay of paralysis onset after DEN infection. In contrast to K562-SCID, the persistently DEN-infected K562 cells generated in vitro invariably failed to be implanted in the mice. It seems that in the early stage of implantation, a gamma interferon activated, nitric oxide-mediated anti-DEN effect might play a role in the innate immunity against DEN-infected cells. The system described herein offers an opportunity to explore DEN replication in vivo and to test various antiviral protocols in infected hosts.

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Figures

FIG. 1
FIG. 1
(A) Demonstration of the specificity of anti-DEN MAbs used in this study. Lysates from 35S-labeled DEN-2 PL046-infected C6/36 cells were immunoprecipitated with MAbs as indicated on the top of the gel. Culture supernatant collected from NS-1 myeloma cells was used as the negative control (lane 1). The numbers on the left side denote the positions of molecular mass standards. Positions of E, NS1 (monomer), and NS3 proteins are indicated by arrows on the right side of the gel. (B) Detection of DEN antigens from different K562 cells by Western immunoblotting assay. The lysates of DEN-2 PL046-infected K562 cells (lane 1), DEN-2 PL046-inoculated K562 tumor mass of the SCID mouse (lane 2), and K562 cells alone (lane 3) were immunoblotted with MAbs specific for DEN E, NS1, or NS3 protein as described in the Materials and Methods. The numbers on the left side denote the positions of molecular mass standards. Positions of E, NS1 (dimer) (NS12), and NS3 proteins are indicated by arrows on the right side of the gel.
FIG. 2
FIG. 2
(A) Distribution of DEN-2 PL046 in blood and different organs of K562-SCID mice after infection. Various organs and blood from four DEN-2 PL046-inoculated K562-SCID mice were individually obtained for virus titration when the animals showed signs of disease at the times postinfection indicated in the figure. Virus titers were determined by a standard plaque assay on BHK-21 cells as described in Materials and Methods. (B) Kinetics of DEN-2 PL046 replication in different organs of the infected K562-SCID mice. Two mice were sacrificed at the specified days postinfection for virus titration.
FIG. 3
FIG. 3
(A) Survival patterns of K562-SCID mice following i.t. challenge with different DEN strains. (B) Neurovirulence of different DEN strains examined in SCID mice by i.c. inoculation (B).
FIG. 4
FIG. 4
In vitro effects of the addition of antibody specific for viral E protein on DEN infection. U937 cells (A) or K562 cells (B) were infected with DEN-2 PL046 which had been preincubated with serially diluted MAb-containing ascitic fluid at 37°C for 60 min. Virus titers in the culture supernatants were determined at day 4 (for U937 cells) or day 6 (for K562 cells) p.i. Ab, antibody.
FIG. 5
FIG. 5
In vivo effects of a neutralizing antibody on DEN infection in K562-SCID mice. (A) K562-SCID mice were infected by 107 PFU of DEN-2 PL046 that was pretreated with either PBS or 100-fold- or 2-fold-diluted ascitic fluid containing MAb 56-3.1 at 37°C for 60 min. (B) K562-SCID mice were passively transferred with either 0.5 ml of PBS or undiluted or 10-fold diluted MAb 56-3.1 1 day prior to the challenge of 107 PFU of DEN-2 PL046. The survival of mice was checked daily up to 30 days p.i.
FIG. 6
FIG. 6
Inhibition of DEN-2 PL046 replication in human K562 cells (A) or murine neuronal N18 cells (B) by coculture with IFN-γ-activated murine RAW 264.7 cells in the presence or absence of the NO synthase inhibitor l-NMA. The virus titers in the culture supernatants were measured by plaque assays. The amounts of NO2− production in culture media from the tested cells were measured by Griess assay as described in Materials and Methods.
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