Antibodies against thrombin in dengue patients contain both anti-thrombotic and pro-fibrinolytic activities.

Dengue virus (DENV) infection may result in severe life-threatening Dengue haemorrhagic fever (DHF). The mechanisms causing haemorrhage in those with DHF are unclear. In this study, we demonstrated that antibodies against human thrombin were increased in the sera of Dengue patients but not in that of patients infected with other viruses. To further characterise the properties of these antibodies, affinity-purified anti-thrombin antibodies (ATAs) were collected from Dengue patient sera by thrombin and protein A/L affinity columns. Most of the ATAs belonged to the IgG class and recognized DENV nonstructural protein 1 (NS1). In addition, we found that dengue patient ATAs also cross-reacted with human plasminogen (Plg). Functional studies in vitro indicated that Dengue patient ATAs could inhibit thrombin activity and enhance Plg activation. Taken together, these results suggest that DENV NS1-induced thrombin and Plg cross-reactive antibodies may contribute to the development of haemorrhage in patients with DHF by interfering with coagulation and fibrinolysis.

Type I interferons protect mice against enterovirus 71 infection.

In this study, the contribution of type I interferons (IFNs) to protection against infection with enterovirus 71 (EV71) was investigated using a murine model where the virus was administrated to neonatal Institute of Cancer Research (ICR) mice by either the intraperitoneal (i.p.) or the oral route. In i.p. inoculated mice, post-infection treatment of dexamethasone (5 mg kg(-1) at 2 or 3 days after infection) exacerbated clinical symptoms and increased the tissue viral titre. In contrast, polyriboinosinic : polyribocytidylic acid [poly(I : C); 10 or 100 microg per mouse at 12 h before infection], a potent IFN inducer, improved the survival rate and decreased the tissue viral titres after EV71 challenge, which correlated with an increase in serum IFN-alpha concentration, the percentage of dendritic cells, their expression of major histocompatibility complex class II molecule and IFN-alpha in spleen. Treatment with a neutralizing antibody for type I IFNs (10(4) neutralizing units per mouse, 6 h before and 12 h after infection) resulted in frequent deaths and higher tissue viral load in infected mice compared with control mice. In contrast, an early administration of recombinant mouse IFN-alphaA (10(4) U per mouse for 3 days starting at 0, 1 or 3 days after infection) protected the mice against EV71 infection. In vitro analysis of virus-induced death in three human cell lines showed that human type I IFNs exerted a direct protective effect on EV71. It was concluded that type I IFNs play an important role in controlling EV71 infection and replication.

Human endothelial cell activation and apoptosis induced by enterovirus 71 infection.

Enterovirus 71 (EV71), a neurotropic virus, its infection is transmitted mainly by the oral-fecal route. However, it is unclear how EV71 is disseminated/spread from initial replication sites to the central nervous system. Since endothelial cells form the interface between blood and tissues throughout the body, it is likely that EV71 can infect and then exit endothelial cells to establish infection. In this study, human endothelial cells were examined for susceptibility to EV71 infection using human microvascular endothelial cell line (HMEC-1 cell). Immunofluorescence assay confirmed EV71 infection of HMEC-1. Viable viruses were cultured from both the culture supernatant and the cell lysate. Live but not UV-inactivated EV71 induced HMEC-1 to secrete IL-6, macrophage migration inhibition factor, and macrophage chemo-attractant protein 1, and to express toll-like receptor 4. In addition, EV71 decreased the viability and increased the apoptosis of HMEC-1 cells after 36-48 hr of infection. These results demonstrate that EV71 is able to infect, activate, and induce apoptosis of endothelial cells, which may play a role in the pathogenesis of EV71 infection.

A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection.

A mouse-adapted enterovirus 71 (EV71) strain with increased virulence in mice, MP4, was generated after four serial passages of the parental EV71 strain 4643 in mice. Strain MP4 exhibited a larger plaque size, grew more rapidly, and was more cytotoxic in vitro than strain 4643. Although strains 4643 and MP4 both induced apoptosis of SK-N-SH human neuroblastoma cells, MP4 was more virulent than 4643 in 1-day-old mice (50% lethal doses, 10(2) and 10(4) PFU/mouse, respectively). Strain MP4 (5 x 10(6) PFU/mouse), but not 4643, could orally infect 7-day-old mice, resulting in rear-limb paralysis followed by death 5 to 9 days after inoculation with the virus. Histopathologically, neuronal loss and apoptosis were evident in the spinal cords as well as the brain stems of the infected mice. The limb muscles displayed massive necrosis. There was early and transient virus replication in the intestines, whereas the spinal cord, brain, and muscle became the sites of viral replication during the late phase of the infection. Virus transmission occurred among infected and noninfected cagemates, as demonstrated by the occurrence of seroconversion and the presence of viable viruses in the stool samples of the latter. Protection against EV71 challenge was demonstrated following administration of hyperimmune serum 1 day after inoculation with the virus. Nucleotide sequence analysis of the genome of EV71 strain MP4 revealed four nucleotide changes on the 5' untranslated region, three on the VP2 region, and eight on the 2C region, resulting in one and four amino acid substitutions in the VP2 and 2C proteins, respectively.