Confirmation PCRs, sequencing and phylogenetic studies PCR primers were designed based on the reads from the 454-sequencing run ( Table 2 ) and PCR assays were set up with the aim to look for each individual virus in each bushpig sera. For the RNA viruses cDNA synthesis was performed prior to PCR using Superscript III (Invitrogen, Carlsbad, CA, USA) and random primers according to the manufacturer's instructions. The PERV RNA was treated with DNase prior to the cDNA synthesis and both a "+" and a "-" RT cDNA synthesis reaction was performed. The PCR using each specific primer pair (Table 1) was performed according to the following procedure: 1x PCR buffer, 2.5 mM MgCl 2 , 1.0 mM dNTP, 0.4 μM forward primer and reverse primer each, and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). For each reaction, two μl DNA or cDNA from each respective bushpig was used. The amplification was performed with the following reaction conditions: a 12 minute enzyme activation step at 95°C followed by 39 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 90 seconds, finishing with one cycle for 10 minutes at 72°C. The PCR products were visualized on a 1.5% agarose gel. The PCR-positive products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and eluted in 25 μl EB. The purified products were sequenced with standard Sanger sequencing using Big Dye Termination kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The obtained chromatograms were edited using SeqMan (Lasergene 9, DNASTAR Inc., Madison, USA). Sequence identity plots were performed using the Bioedit software [28] . ClustalW as well as the phylogenetic analyses were carried out using Mega 5 [29] . The phylogenetic trees were constructed using the Neighbour-joining algorithm with p-distances and with a bootstrap value of 1000.  A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection. Vector-borne flaviviruses, such as Dengue (DENV), Yellow Fever, Japanese Encephalitis virus (JEV), and tick-borne encephalitis (TBEV) viruses are recognized as significant human pathogens and cause considerable mortality and morbidity worldwide. TBEV, a tick-borne flavivirus (TBFV), is responsible for 14,000 infections per year [1] and has a fatality rate of up to 40% [2] . Symptoms of TBEV infection can include fever, malaise, meningitis, and encephalitis. TBEV and other TBFV, such as Omsk Hemorrhagic Fever virus, are classified as NIAID Category C pathogens and are treated as biosafety level 4 agents in the United States. One TBFV, Langat virus (LGTV), is naturally attenuated [3, 4] , making it suitable for biosafety level 2 work and ideal for use in laboratory studies as a model for higher pathogenicity viruses.