The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 μm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 μm/s at 16 hpi compared to 0.22 μm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence. IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae. This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders’ coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. Importance Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin D, confirming they were the site of de novo RNA synthesis, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders’ coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs are sites of de novo RNA synthesis, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM.ImportanceMembers of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

word count: 250 12 Text word count: 4,977 13 14 Abstract 15The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, are 16 non-enveloped viruses with a segmented double-stranded (ds)RNA genome that replicate in 17 discrete cytoplasmic virus factories (VFs). Reassortment is common, however, the underlying 18 mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to 19 provide new information on VF trafficking during dsRNA virus co-infection, we rescued two 20 recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split 21 present address: Mars Inc. 42dsRNA virus during a co-infection. Discrete VFs initially formed from each virus that 43 3 subsequently coalesced from 10 hours pi. We hypothesise that VF coalescence is required for 44 the reassortment of dsRNA viruses and the potential for reassortment increases later in the 45 replication cycle. This study provides new information that adds to our understanding of dsRNA 46 virus VF trafficking. 47 48