Tilapia Lake Virus Does Not Hemagglutinate Avian and Piscine Erythrocytes and NH4Cl Does Not Inhibit Viral Replication In Vitro

Abstract

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH 4 Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH 4 Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH 4 Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

Effects and mechanism of Aβ1−42 on EV-A71 replication

“…In the penetration-inhibition assay, we further studied the effect of Aβ 1–42 on the EV-A71 RNA level at 1 h post-virus attachment (MOI = 2.5). Figure 3 c shows that consistent with NH 4 Cl treatment [ 23 ], Aβ 1–42 efficiently downregulated the level of VP1 RNA after EV-A71 attachment in all three cell lines.…”

“…In the penetration-inhibition assay, we further studied the effect of Aβ 1-42 on the EV-A71 RNA level at 1 h post-virus attachment (MOI = 2.5). Figure 3c shows that consistent with NH 4 Cl treatment [23], Aβ 1-42 efficiently downregulated the level of VP1 RNA after EV-A71 attachment in all three cell lines. Taken together, these results demonstrated that Aβ 1-42 primarily targeted EV-A71 attachment, internalization, and uncoating stage to inhibit virus replication.…”

Tilapia lake virus: The story so far

“…In addition to the liver and brain, positive hybridization signals were also detected in the anterior kidneys, gills, gastrointestinal tract and spleen, as well as in the connective tissues of infected fish (Bacharach et al., 2016; Jaemwimol et al., 2018; Jansen et al., 2019). A recent study demonstrated that a lysosomotropic agent had no effect on TiLV replication, suggesting that TiLV can replicate without acidic pH in contrast to other orthomyxoviruses (Chengula, Mutoloki, Evensen, & Munang’andu, 2019).…”

“…Other serological applications, such as haemagglutinin (HA) test, were carried out for TiLV but the results demonstrated that TiLV did not agglutinate turkey ( Meleagris gallopavo ), Atlantic salmon ( Salmo salar L ) and Nile tilapia ( O. niloticus ) red blood cells. Interestingly, this suggests that TiLV likely uses other mechanisms to bind and enter into fish cells (Chengula et al., 2019), and such mechanisms need to be further investigated.…”

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification

“…Despite being an orthomyxo-like virus, the mode of TiLV entry into the cells is largely unknown. Differences from other orthomyxoviruses, including the prototypic influenza viruses and the infectious salmon anemia virus (ISAV), have been suggested, as TiLV does not hemagglutinate erythrocytes, and as ammonium chloride does not inhibit its replication ( Chengula et al, 2019 ). Here, we employed different inhibitory treatments to dissect the cellular requirements for TiLV entry and replication in tilapia cells.…”