Sulfated Polysaccharide, Curdlan Sulfate, Efficiently Prevents Entry/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection In Vitro: A Possible Candidate for Clinical Application

10.1371/journal.pntd.0002188PLoS Neglected Tropical Diseases74

Immature Dengue Virus: A Veiled Pathogen?

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level

A new insect-specific flavivirus from Northern Australia suppresses replication of West Nile Virus and Murray Valley Encephalitis Virus in co-infected mosquito cells

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10-43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date