Pre incuba tion of cells with extract, followed by virus infection, or post exposure of the infected cells to EF, inhibited virus replication MG132 side effects to a lesser extent. To investi gate this in more detail, several experiments were per formed with KAN 1 to determine the effect of adding EF at different times relative to virus infection of the cells. Complete inhibition was achieved by incubating KAN 1 and EF together before adding to the cells. However other combinations of pre and post exposure to EF resulted in only partial Inhibitors,Modulators,Libraries reduction in virus production, compared to untreated. These results suggest that EF was acting either directly on the virus or at a very early stage in the replica tion cycle. It is noteworthy to mention, that removal of EF containing Inhibitors,Modulators,Libraries medium 6. 5 hours p. i.
and further incubation in normal medium for 1. 5 hours in order to prevent an exposure of newly formed virions to EF prior to titration, did not change this result. Intra cellular RNP localization Next, the production and intra cellular localization of viral RNP were determined by immunofluorescence, in MDCK cells infected with KAN 1, with and without EF treatment. Inhibitors,Modulators,Libraries In normally infected cells, the nucleocapsid Inhibitors,Modulators,Libraries protein, which is the main com ponent of the RNPs, appeared initially in the nucleus Inhibitors,Modulators,Libraries followed by migration to the cytoplasm. The same pattern was seen in EF treated cells infected with untreated virus, and in cells exposed to EF after infection. However, when cells were infected with EF treated virus, the overall number of posi tive cells was significantly reduced.
Nevertheless, the amount and the localization of RNPs detected in cells infected with pre treated IV was the same as for untreated cells infected with untreated virus. It should be noted that the treatment of infected cells at different time points p. i. did not affect the number of cells positive sellekchem for NP staining. These results suggest that EF affects a very early stage before replication, but once the virus has entered the cells its replication and spread are not affected. Interaction of Echinaforce with Viral HA The first step in entry of IV into cells depends on the inter action between the viral HA and a specific cellular sialic acid containing receptor. If EF could inhibit this interac tion by binding to the HA, then entry of virus might be prevented. Receptor binding of functional HA can be measured by its ability to agglutinate chicken erythro cytes, which can be easily enumerated visually. Direct interaction between virus and EF was therefore examined by inspecting viral hemagglutination activity in the presence and absence of EF. Results for the pandemic S OIV and two HPAIV are shown in Table 2. EF inhibited HA activity for all 3 virus strains, in a con centration and time dependent manner.