Osteoclastogenesis induced by CHIKV-infected fibroblast-like synoviocytes: a possible interplay between synoviocytes and monocytes/macrophages in CHIKV-induced arthralgia/arthritis.

Fibroblast-like synoviocytes are known to migrate from joint to joint and are proposed to be one of the key players in the inflammatory cascade amplification in rheumatoid arthritis patients. In the recent CHIKV epidemic, patients developed arthritis-like syndrome and the synoviocyte is one of the suspected players in CHIKV-induced polyarthritis. Thus, to learn more on this syndrome, the responses of fibroblast-like synoviocytes to chikungunya virus (CHIKV) infection, and the interaction between CHIKV-infected synoviocytes and phagocytes, were investigated. Primary human fibroblast-like synoviocyte (HFLS) cultures were infected with clinical isolates of CHIKV at an MOI of 0.001pfu/cell. Data indicated that HFLS are permissive to CHIKV replication, generating peak titers of 10(5)-10(6)pfu/ml. Interestingly, CHIKV-infected HFLS cultures secreted mainly the mediators that are responsible for phagocytes recruitment and differentiation (RANKL, IL-6, IL-8 and MCP-1) but not arthritogenic mediators (TNF-α, IL-1ß, MMP-1, MMP-2 or MMP-13). The interaction between CHIKV-infected synoviocytes and phagocytes was studied using UV-irradiated, CHIKV-infected HFLS supernatant. Data revealed that supernatants from CHIKV-infected HFLS cultures not only induced migration of primary human monocytes, but also drove monocytes/macrophages into osteoclast-like cells. These differentiated osteoclast-like cells produced high levels of TNF-α and IL-6, principal mediators of arthritis. This data suggests a potential interplay between infected HFLS and recruiting phagocytes which may responsible for the arthralgia/arthritis in CHIKV-infected patients.

The difference in IL-1beta , MIP-1alpha, IL-8 and IL-18 production between the infection of PMA activated U937 cells with recombinant vaccinia viruses inserted 2004 H5N1 influenza HA genes and NS genes.

BACKGROUND: The severity of avian influenza H5N1 disease is correlated with the ability of the virus to induce an over production of proinflammatory cytokines from innate immune cells. However, the role of each virus gene is unknown. To elaborate the function of each virus gene, the recombinant vaccinia virus inserted HA and NS gene from the 2004 H5N1 virus were used in the study. METHODS: U937 cells and PMA activated U937 cells were infected with recombinant vaccinia virus inserted with HA or NS gene. The expressions of HA and NS proteins in cells were detected on immunofluorescence stained slides using a confocal microscope. The cytokine productions in the cell supernatant were quantitated by ELISA. RESULTS: The recombinant vaccinia virus inserted with HA genes induces the production of IL-1beta, MIP-1alpha, IL-8 and IL-18 cytokines from PMA activated U937 cells significantly more than cells infected with wild type vaccinia, whereas the recombinant vaccinia virus inserted with NS genes it was similar to that with the wild type vaccinia virus. However, there was no synergistic nor antagonistic effect of HA genes and NS genes in relation to cytokines production. CONCLUSION: Only the HA gene from the 2004 H5N1 virus induces IL-1beta, MIP-lalpha, IL-8 and IL-18 cytokine productions from activated U937 cells. The same HA gene effect may or may not be the same in respiratory epithelial cells and this needs to be explored.

Clinical isolates of dengue virus with distinctive susceptibility to nitric oxide radical induce differential gene responses in THP-1 cells.

In the present study, 10 clinical isolates of dengue virus were selected according to their susceptibility to the inhibitory effect of nitric oxide radical, NO. Five of them are nitric oxide-susceptible viruses while the other five are nitric oxide-resistant viruses. These isolates were investigated to identify genetic factors that are responsible for the different phenotypes. Due to the evidence showing that NO suppresses DENV RNA polymerase activity, we, therefore, hypothesized that the RdRp domain of NS5 may responsible for NO inhibition. To answer this question, sequences of NS5 gene of NO-susceptible viruses and NO-resistant viruses were compared. We found that these two groups of viruses contain different amino acid sequence at position 621 to 646 in the active site of NS5. These data suggested that response to the inhibitory effect of nitric oxide radical may, at least in part, be regulated by NS5. The effect of these two different phenotypes of viruses on host cells was studied using cDNA array screening. The cDNA array analysis demonstrated that the nitric oxide-resistant group had a stronger influence on host cells since it induced changes in the expression of a greater number of genes than did the nitric oxide-susceptible group, 97 genes versus 71 genes, respectively. The NO-resistant virus also stimulated cytokines known to be virulent factors, such as IL 6, IL 8, RANTES, and the inflammatory factors. In conclusion, our data demonstrated that dengue viruses isolated from patients show genotypic and phenotypic differences which may correlate with virulence.

Differences in global gene expression in peripheral blood mononuclear cells indicate a significant role of the innate responses in progression of dengue fever but not dengue hemorrhagic fever.

BACKGROUND: Dengue virus infection causes an array of symptoms ranging from dengue fever (DF) to dengue hemorrhagic fever (DHF). The pathophysiological processes behind these 2 clinical manifestations are unclear. METHOD: In the present study, genomewide transcriptomes of peripheral blood mononuclear cells (PBMCs) collected from children with acute-phase DF (i.e., DF PBMCs) or acute-phase DHF (i.e., DHF PBMCs) were compared using microarray analysis. Results of genome screening were validated at the genomic and proteomics levels. RESULTS: DHF had stronger influences on the gene expression profile than did DF. Of the affected genes, metabolic gene expression was influenced the most. For the immune response category, 17 genes were more strongly up-regulated in DF PBMCs than in DHF PBMCs. Eight of the these 17 genes were categorized as belonging to the interferon (IFN) system. The up-regulation of IFN-related genes was accompanied by strong expression of CD59, a complement inhibitor. DHF PBMCs expressed genes involved in T and B cell activation, cytokine production, complement activation, and T cell apoptosis more strongly than did DF PBMCs. CONCLUSION: We hypothesize that, during DF, genes in the IFN system and complement inhibitor play a role in lowering virus production and reducing tissue damage. In patients with DHF, the dysfunction of immune cells, complement, and cytokines increases viral load and tissue damage.

Attenuating characteristics of DEN-2 PDK53 in flavivirus-naïve peripheral blood mononuclear cells.

A live-attenuated DEN-2 virus, DEN-2 strain 16681-PDK53, has been found to be attenuated for both humans and mice with an unknown mechanism. To partially answer this question, responses of flavivirus-naïve primary human PBMC to infection with attenuated DEN-2 PDK53 (D2/IC-VV45R) virus and its parental, virulent DEN-2 16681 virus (D2/IC-30P-A) were investigated at the cellular and genetic levels using cDNA array analysis. Both DEN-2 viruses produced similar replication kinetics in flavivirus-naïve PBMC. In contrast, virulent DEN-2 virus caused a higher percentage of apoptotic death. A macro-array analysis showed that the virulent D2/IC-30P-A virus induced changes in the expression of a greater number of genes than did the attenuated D2/IC-VV45R virus, 31 genes versus 19 genes, respectively, by 24 h post-infection. Interestingly, both viruses stimulated cytokines known to be virulence factors for DEN virus infection, such as IL-1beta, IL-6, IL-8, IL-10, MIP-1beta, and MIP-1alpha. The virulent virus additionally up-regulates immune suppression factors and down-regulates immune activator and growth factors. In conclusion, our data demonstrated that D2-PDK53 effected less change in PBMC than D2-16681 in terms of observable cellular effect and expression of cytokine and chemokine related genes.

Screening of upregulated genes in suckling mouse central nervous system during the disease stage of rabies virus infection.

The pathogenesis of hydrophobia remains unclear. The aim of this study was to identify the differentially upregulated genes that correlated with disease development in an experimental mouse model to provide better understanding of pathological mechanisms in rabies. The present work employed Clontech mouse array 1.2 II containing 1,176 gene transcripts. Suckling mice were intracerebrally infected with canine rabies virus. The gene expression profiles on day 2, 4 and 6 post inoculation were followed. The results show genes whose expression increased at least twofold above the control, mock-infected brain. The numbers of genes showing altered expression level were 29, 109 and 98 genes on day 2, 4 and 6, respectively. The genes with altered expression were classified into eight major groups, namely immune response, metabolism, receptor and transporter, growth factors, death mediated factors, transcription and translation factors, proteases, and kinases. The numbers of upregulated genes during the disease stage was much higher than during the asymptomatic stage. This suggested that direct interaction between RABV and target cells induced massive destruction of a cellular homeostasis which may lead to functional termination of the CNS.

Screening of pro-apoptotic genes upregulated in an experimental street rabies virus-infected neonatal mouse brain.

Rabies virus (RABV) is able to induce apoptotic death of target cells. The molecular pathway of RABV-induced cell death is partially known. In the present study, cDNA array analysis was used as a tool to screen for pro-apoptotic genes that may be involved in RABV induction. RNA was extracted from the infected CNS and from mock-infected controls. When the mean gene expression was compared between the infected group and controls, 21 potential apoptotic genes were identified that exhibited more than 2.5-fold difference in their expression levels. These 21 genes can be grouped into two groups, those genes that participate in the commitment phase and those that play a role as executioners. Examples of genes in commitment phase were death receptors (Fas-L receptor, TNF-receptor), lysosomal proteases, calpain, caspase-1, signaling molecules (ERK, p38MAPK) and bcl-2 family members. Cytochrome c and caspase-3 were representatives of executioners. Based on types of genes activated during the commitment phase, two independent apoptotic mechanisms may be activated in response to the RV infection. The first is immune-mediated death which may operate through the receptor-ligand pathway activated by caspase-1 and the pro-inflammatory cytokine, IL-1beta. The other mechanism is a protease-mediated process which involves lysosomal proteases and calcium-dependent neutral proteases. These two stimulating pathways were followed by Bad, Bak, Bid activation and subsequently the upregulation of cytochrome c and caspase-3. In addition, mobilization of K+ ion and other accessory apoptotic genes such as annexins and clusterin were also upregulated.

Reactivation of Nedd-2, a developmentally down-regulated apoptotic gene, in apoptosis induced by a street strain of rabies virus.

A laboratory strain of rabies virus has been reported to induce apoptosis in experimental animals. The present study demonstrated that a bat strain and a primary canine rabies virus isolate also induced apoptosis in vivo. This death process involved reactivation of the caspase gene, Nedd-2, a developmentally down-regulated apoptotic gene. Expression of Nedd-2 was significantly up-regulated in infected adult and suckling mice. Reactivation of Nedd-2 in infected adult mice started at around day 3 and was prominent on day 5. The level of expression was constantly high up to the time that mice showed signs of illness. Expression of Nedd-2 correlated with the appearance of apoptotic nuclei within the infected brain, suggesting that reactivation of a developmentally down-regulated gene, Nedd-2, may be required for apoptotic elimination of cells damaged by infection.