However, study by EM and 3D reconstruction revealed differences between the persistently infected and the acutely infected ISE6 cells. The persistently infected tick cells had greater changes in ER structure and ER abundance (Fig. 7B-D) than the acutely infected cells, although viral titers (Fig. 1B & C) were similar in both settings. However, the most remarkable difference between acute and persistent infection was the number of tubular structures. These structures have been noted infrequently in infected cells [26, 30, 34] , and their relevance in flavivirus replication is uncertain. In our acute mammalian and tick cell experiments, we confirmed these observations and saw tubules only occasionally (Fig. 7D, 8 , and Movies S1, S2, and S3). However, in the persistently infected tick cells, the number of tubules increased dramatically (Fig. 7D, 8C , & Movie S3). While the diameter was similar to that seen in round vesicles (60-100 nm), the length was up to eight times as long. The tubules were often arranged in membrane-bound, fascicle-like bundles ( Fig. 8C and Movie S3). The 3D reconstructions demonstrated that the tubules were closed on each end and, although closely juxtaposed, were not connected by pores to other tubules or to the cytoplasm, unlike the round vesicles. The function of the tubules is obscure at this time, and it is unclear if they represent bona fide features of replication, aberrant structures, or the result of a cellular process to restrict infection. It is possible the tubules may play a role in initiation or maintenance of the persistent infections or if the apparent lack of pores in the tubules is consequential. It Figure 6 . Localization of LGTV TP21 replication complex to endoplasmic reticulum. Cells were infected at a MOI of 10, fixed, and costained for double-stranded RNA (dsRNA, red) and endoplasmic reticulum (protein disulfide isomerase (PDI), green). Nuclei counterstaining (DAPI, blue) is only shown in the merge panel. Scale bars, 10 um. doi:10.1371/journal.pone.0047912.g006 3D LGTV Infection in Mammalian and Tick Cells PLOS ONE | www.plosone.org may be that the increase in the number of tubules is the result of the higher number of defective virus particles, which are known to exist in persistently infected cells [60] [61] [62] . Tubules may be formed as a result of a failure to correctly gather the membrane to form the round vesicles and pores that are associated with the flavivirus replication complex. Perhaps the lack of pores prevents proper replication or packaging of the viral genome. Additional cell biology or biochemistry studies may shed light on the role of the tubules.