Nitwara Wikan

Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes

Infections with dengue virus (DENV) are a significant public health concern in tropical and subtropical regions. However, little detail is known about how DENV interacts with the host-cell machinery to facilitate its translation and replication. In DENV-infected HepG2 cells, an increase in the level of LC3-II (microtubule-associated protein 1 light chain 3 form II), the autophagosomal membrane-bound form of LC3, was observed, and LC3 was found to co-localize with dsRNA and DENV NS1 protein, as well as ribosomal protein L28, indicating the presence of at least some of the DENV translation/replication machinery on autophagic vacuoles. Inhibition of fusion of autophagic vacuoles with lysosomes resulted in an increase in both intracellular and extracellular virus, and co-localization observed between mannose-6-phosphate receptor (MPR) and dsRNA and between MPR and LC3 identified the autophagic vacuoles as amphisomes. Amphisomes are formed as a result of fusion between endosomal and autophagic vacuoles, and as such provide a direct link between virus entry and subsequent replication and translation.

Zika virus: history of a newly emerging arbovirus

Zika virus was originally identified in a sentinel rhesus monkey in the Zika Forest of Uganda in 1947. The virus is a member of the family Flaviviridae, genus Flavivirus, and is transmitted to humans by Aedes species mosquitoes. The first report of Zika virus outside Africa and Asia was in 2007 when the virus was associated with a small outbreak in Yap State, part of the Federated States of Micronesia. Since then, Zika virus infections have been reported around the world, including in southeast Asia; French Polynesia and other islands in the Pacific Ocean; and parts of South, Central, and North America. Symptomatic infection in human beings normally results in a mild and self-limiting febrile disease, although recent reports have suggested a possible association with more serious sequelae such as Guillain-Barré syndrome, and microcephaly in newborn infants of mothers infected with Zika virus during pregnancy. In this Review, we summarise the history of Zika virus from its first detection to its current worldwide distribution.

A role for autophagolysosomes in dengue virus 3 production in HepG2 cells.

We have recently proposed that amphisomes act as a site for translation and replication of dengue virus (DENV)-2 and that DENV-2 entry and replication are linked through an ongoing association with membranes of an endosomal-autophagosomal lineage. In this report, we present the results of an investigation into the interaction between DENV-3 and the autophagy machinery. Critically, treatment with the lysosomal fusion inhibitor l-asparagine differentiated the interaction of DENV-3 from that of DENV-2. Inhibition of fusion of autophagosomes and amphisomes with lysosomes resulted in decreased DENV-3 production, implying a role for the autophagolysosome in the DENV-3 life cycle. Evidence based upon the co-localization of LC3 and cathepsin D with double stranded RNA and NS1 protein, as assessed by confocal microscopy, support a model in which DENV-3 interacts with both amphisomes and autophagolysosomes. These results demonstrate that the interactions between DENV and the host cell autophagy machinery are complex and may be determined in part by virus-encoded factors.

Identification and characterization of prohibitin as a receptor protein mediating DENV-2 entry into insect cells

Dengue is transmitted primarily by mosquitoes of the Aedes genus. Despite a number of studies, no insect dengue virus receptor protein has been clearly identified and characterized. Using a number of separation methodologies and virus overlay protein binding assays we identified a 35kDa protein that segregated with susceptibility to dengue serotype 2 (DENV-2) infection in two mosquito species and two mosquito cell lines. Mass spectroscopy identified the protein to be prohibitin, a strongly conserved and ubiquitously expressed protein in eukaryotic cells. Antibody mediated inhibition of infection and siRNA mediated knockdown of prohibitin expression significantly reduced infection levels and subsequent virus production in both Aedes aegypti and Aedes albopictus cell lines. Confocal microscopy showed a significant degree of intracellular colocalization between prohibitin and DENV-2 E protein, and coimmunoprecipitation confirmed that prohibitin interacts with dengue E. Prohibitin is the first characterized insect cell expressed dengue virus receptor protein.

Identification of prohibitin as a Chikungunya virus receptor protein

Chikungunya virus (CHIKV) has recently re‐emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two‐dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co‐localization, co‐immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses. J. Med. Virol. 84:1757–1770, 2012.

Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells.

Japanese encephalitis virus (JEV) a mosquito‐borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein‐binding assay (VOPBA) and liquid chromatography–mass spectrometry (LC/MS/MS) identified the 37/67 kDa high‐affinity laminin receptor protein and nucleolin as a potential JEV‐binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV‐2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti‐laminin receptor and anti‐CD4 antibodies significantly reduced virus entry while anti‐Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down‐regulation of CD4 and moderate down‐regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role. J. Med. Virol. 84:615–623, 2012.

Dengue infection of monocytic cells activates ER stress pathways, but apoptosis is induced through both extrinsic and intrinsic pathways

Monocytic cells are believed to be an important mediator of the pathology of dengue disease in cases of secondary infection where pre-existing antibodies from a first infection facilitate virus entry to Fc receptor bearing cells. In this study we assessed the induction of the ER stress in response to infection of monocytic U937 cells with all four DENV serotypes as well as the induction of apoptosis. Clear evidence of ER stress and the production of pro-apoptotic signals were observed, together with activation of caspase 9. Surprisingly, caspase 8 was also activated, independently of caspase 9, and this was accompanied by an increase in the expression of TNF-α, suggesting the simultaneous but independent activation of both extrinsic and intrinsic apoptosis pathways. Both the induction of ER stress and apoptosis were shown to be serotype independent.

Chikungunya virus infection of cell lines: analysis of the East, Central and South African lineage.

Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in Aedes mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution.

Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir.

Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, is a major cause of acute encephalitis, and neurons have been proposed to be the principle JEV target cells in the central nervous system. However, clinically, infection with JEV leads to increased levels of cytokines and chemokines in the serum and cerebrospinal fluid (CSF) the levels of which correlate with the mortality rate of patients. This research aimed to study the role of microglial cells in JEV infection. Mouse microglial cells (BV-2) supported the replication of JEV with extracellular production of virus by 10h post-infection, and virus titer reached a maximum (2.55x10(10)pfu/ml) by day 3 post-infection. While apoptosis was induced in response to virus infection, no alteration in nitric oxide production was observed. Microglial cells remained productively infected with JEV for up to 16 weeks without significant morphological alterations, and the released virions were infectious to mouse neuroblastoma (NA) cells. The high virus production and long persistence of JEV in microglial cells suggests that these cells may serve as viral reservoirs for the infection of neurons in the CNS.

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms.