Mosquitoes of five genera were assessed including (six species), (seven species), (two species), (1 species) and (one species) (data not shown). 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 your 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 much like 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 mosquitoes. The new computer virus is tentatively named Palm Creek computer virus (PCV) after its place of isolation. We also exhibited that prior contamination of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10C43 fold (1 to 1 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. Introduction Flaviviruses are responsible for a number of important mosquito-borne diseases of humans and animals in Australia, including dengue, Murray Valley encephalitis and Japanese encephalitis (JE) [1]. Dengue, JE, yellow fever and West Nile fever are also major medical problems around the world [2]. Flaviviruses are a group of small, enveloped viruses that contain a positive-sense RNA genome with a single open reading frame (ORF) which is usually flanked by 5 and 3 untranslated regions (UTRs). The ORF is usually translated as a single polyprotein, which is usually cleaved by viral and cellular proteases into Bergaptol three structural (C, prM and E) and seven non-structural proteins (NS1-NS5). Flaviviruses are usually transmitted between arthropods and vertebrates and rely on replication in both of these hosts for their natural transmission cycle. In 1975, Stollar and Thomas reported the isolation of an unusual computer virus (cell fusing agent computer virus; CFAV) from mosquito cell cultures [3]. Further analysis revealed that CFAV is usually a distant relative of members of the flavivirus genus, but did not replicate in vertebrate cells. CFAV and comparable viruses – Kamiti River computer virus (KRV) and Culex flavivirus (CxFV) – were subsequently isolated from mosquitoes in the wild and shown to belong to a distinct insect-specific flavivirus (ISF) lineage [4]C[6]. With the introduction of improved molecular Bergaptol tools for viral detection, several new Bergaptol species of ISF including Aedes flavivirus (AeFV [7], [8]), Quang Binh computer virus (QBV [9]), Nakiwogo computer virus (NAKV [10]), Chaoyang computer virus (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ883471″,”term_id”:”227937394″,”term_text”:”FJ883471″FJ883471 C Wang et al., 2009), Lammi computer virus [11], Nounan computer virus [12], Calbertado computer virus [13] and Culex theileri flavivirus (CTFV [14]), have since been isolated from numerous regions of the world. Data from several studies indicates that at least some ISFs are managed in nature in the absence of a vertebrate host by vertical transmission from female mosquitoes to their progeny [15]C[17]. A lack of a direct association of these viruses with disease has largely seen ISFs ignored to date, however, recent reports by Kent et al. (2010) [18] and Bolling et al. (2012) [17] suggesting that co-infection with CxFV may enhance or suppress transmission of Bergaptol West Nile computer virus (WNV) in some vectors has created intense desire for the conversation of ISFs with other flaviviruses in mosquito cells. In this paper, we statement the isolation and phylogenetic analysis of a new ISF detected in mosquitoes from northern Australia and the generation of ISF-specific recombinant proteins and monoclonal antibodies. We also provide evidence of super-infection exclusion of heterologous flaviviruses in cell cultures previously infected with this new computer virus. Materials and Methods Ethics Statement The mouse work in this study was carried out under conditions approved by The University or college of Queensland Animal Ethics Committee (Animal Ethics Number 299/10). Surgery was Rabbit Polyclonal to PTPN22 performed under ketamine/Xylazine and all efforts were made to minimize suffering. No specific permits were required for the explained field studies and no specific permissions were required for the locations/activities for mosquito trapping because they are public lands and are not privately owned or protected in any.