Effect of temperature on virus eradication and growth of infected tissue cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses were eradicated or their concentrations greatly diminished when infected meristem tips were grown in static or shake culture at 32 or 34 ^oC. Similar cultures grown at 22 ^oC remained infected. Tobacco mosaic virus (TMV) was not eradicated from shake cultures at 32 ^oC and the concentration of TMV in such cultures at 32 ^oC was sometimes higher than at 22 ^oC. The concentration of CMV and AlfMV was considerably less in cultured tissues than in infected leaf material, but that of TMV was similar. Cultures infected with CMV or TMV grew more slowly than healthy cultures. Growth of AlfMV infected and healthy cultures was similar.     

Reversible stages of the low-pH-triggered conformational change in influenza virus hemagglutinin

The refolding of the prototypic fusogenic protein hemagglutinin (HA) at the pH of fusion is considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. Here, we show that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH-activated HA returns from the conformations wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on the pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. The existence of the identified reversible stage of refolding can be crucial for allowing multiple copies of HA to synchronize their release of conformational energy, as required for fusion.     

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.