Study of Dengue Virus Infection in SCID Mice Engrafted with Human K562 Cells

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.

Expression and membrane integration of SARS-CoV M protein

“…In our previous study [16], SARS-CoV M protein could not be detected in SDS-PAGE in regular boiling treatment. Therefore, non-heated treatments [18] were used in antigen preparations (sample buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1 % bromophenol blue, 10% glycerol; no boiling treatment) to detect the expression of SARS-CoV M protein. 4.5% (acrylamide percentage) gel was used as the stacking gel and 12% gel as the separating gel in this study.…”

Thermal aggregation of SARS-CoV membrane protein

“…For Western blotting analysis, cells were dissolved in sample preparation buffers after washing with PBS twice. Two sample preparation buffers were used: denaturing buffer containing ␤-mercaptoethanol (Ma et al, 2002;Makowski and Ramsby, 1997) and non-denaturing buffer without ␤mercaptoethanol (Lin et al, 1998). The samples were treated at room temperature or 100 • C in the sample buffer for 10 min before electrophoresis.…”

“…heating, 2-ME) would promote the unfolding of structural proteins in other coronaviruses (Callebaut and Pensaert, 1980;Deregt et al, 1987;Hogue and Brian, 1986;Sturman et al, 1980;Wege et al, 1979). Therefore, both denaturing (Ma et al, 2002;Makowski and Ramsby, 1997) and non-denaturing treatments (Lin et al, 1998) should be used for antigen preparations. Nucleocapsid and spike proteins can be detected using Western blotting analysis by either denaturing condition (sample buffer containing 67.5 mM Tris-HCl (pH 6.8), 5% 2-mercaptoethanol, 3% SDS, 0.1% bromophenol blue, 10% glycerol, treatment at 100 • C for 10 min) or non-denaturing conditions (sample buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol; no boiling treatment) while membrane protein could only be detected in non-denaturing but not denaturing condition .…”

Animal Models of Human Viral Diseases

“…The most well-defined model is AG129 which is deficient in IFNα/β and γ receptors and can recapitulate DHF/Dss if a mouse-adapted strain is utilized Yauch et al, 2009). sCID mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (Blaney et al, 2002;lin et al, 1998). DF symptoms developed after infection in NOD/sCID/Il2rγKO mice engrafted with CD34 + human progenitor cells (Mota and rico-Hesse, 2011).…”