Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus.

In mammals, lipid droplets (LDs) are ubiquitous organelles that modulate immune and inflammatory responses through the production of lipid mediators. In insects, it is unknown whether LDs play any role during the development of immune responses. We show that Aedes aegypti Aag2 cells - an immune responsive cell lineage - accumulates LDs when challenged with Enterobacter cloacae, Sindbis, and Dengue viruses. Microarray analysis of Aag2 challenged with E.cloacae or infected with Dengue virus revealed high transcripts levels of genes associated with lipid storage and LDs biogenesis, correlating with the increased LDs numbers in those conditions. Similarly, in mosquitoes, LDs accumulate in midgut cells in response to Serratia marcescens and Sindbis virus or when the native microbiota proliferates, following a blood meal. Also, constitutive activation of Toll and IMD pathways by knocking-down their respective negative modulators (Cactus and Caspar) increases LDs numbers in the midgut. Our results show for the first time an infection-induced LDs accumulation in response to both bacterial and viral infections in Ae. Aegypti, and we propose a role for LDs in mosquito immunity. These findings open new venues for further studies in insect immune responses associated with lipid metabolism.

Pro-inflammatory response resulting from sindbis virus infection of human macrophages: implications for the pathogenesis of viral arthritis.

Several viruses cause acute and chronic joint inflammation in humans, and among them, the alphaviruses are of special interest due to the increasing number of outbreaks in which they are the etiological factor. Sindbis virus (SinV), a member of the Alphavirus genus, is the most widely distributed of all known arboviruses. Although SinV causes arthritis in humans, the molecular and cellular factors that contribute to the pathogenesis of this disease are almost completely unknown. Despite the crucial role of macrophages in the development of arthritis, these cells have not been recognized as potential targets for viruses causing arthritis. In this study, replication of SinV in human macrophages was demonstrated. The infection promoted macrophage activation, leading to the release of macrophage migration inhibitor factor (MIF) from intracellular stores and inducing the expression and secretion of TNF-alpha, IL-1beta, and IL-6. Production of these cytokines was followed by the expression of matrix metalloproteinases (MMPs) 1 and 3, which could be involved in the articular damage that has been observed in disease induced by SinV. The use of different strategies to block MIF action, including an anti-MIF antibody, the MIF inhibitor ISO-1 and knockout mice for the MIF gene, showed that cytokine secretion and MMP expression during infection were regulated by MIF, suggesting that this cytokine acts in autocrine and paracrine manner upstream in the macrophage activation cascade. Thus, these are remarkable similarities between macrophage responses induced by SinV infection and those observed in rheumatoid arthritis, despite the different etiologies of infectious and autoimmune arthritides.

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.

Murine T-helper cell immune response to recombinant vaccinia-Venezuelan equine encephalitis virus.

The T-helper (Th) cell immune response following immunization of C3H (H-2k) mice with a recombinant vaccinia (VAC) virus (TC-5A) expressing the structural proteins (capsid, E1 and E2) of the attenuated vaccine strain (TC-83) of Venezuelan equine encephalitis (VEE) virus was compared with the immune response induced in mice after immunization with TC-83 virus. TC-5A virus elicited Th cells that strongly recognized both VAC and TC-83 viruses in in vitro lymphoblastogenesis tests. Th-cell activation was associated with elevated levels of interleukin-2. TC-5A virus induced long-term humoral immunity; VEE virus-binding and neutralizing antibodies were detected in mouse sera collected from mice 16 months after a single immunization.

Radial haemolysis in gel used in serological studies on Venezuelan equine encephalomyelitis virus.

Radial haemolysis in gel was successfully used for the detection of antibody in convalescents after laboratory infection with Venezuelan equine encephalomyelitis (VEE) virus at remote intervals after infection (up to 23 years). The antibodies reacted only with VEE antigen but not with Sindbis and Chikungunya alphavirus antigens. Radial haemolysis in gel was sensitive as the haemagglutination inhibition test but the antibody could be titrated by the diameter of the haemolysis zone omitting the serum dilutions. Slight deviations in the virus dose used for sensitization of erythrocytes or in pH values were not critical. For the test, the sera were heated at 56 degrees C for 30 min. Both sera stored for long periods and those collected on paper disks could be used.