Multiplicity of infection

Add 100 pil of the recombinant virus stock, keeping the multiplicity of infection below one. 

Add high-titre virus stock to the plates so that multiplicity of infection is between 3 and 10. 

Calculate the volume of recombinant virus required to infect at a multiplicity of infection of 3-10 by the equation ... 

In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed. The issues addressed include particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process. Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes. In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters. Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression. 

MOI(t) multiplicity of infection at time t (pfu/cells) P(t) cumulative probability that cells are infected... 

Wong et al. [100] reported that very low multiplicities of infection could be used with the model (3-Galactosidase baculovirus/insect cell system. They proposed the Cell Yield Concept stating that choosing the appropriate TOI (time of infection) it is possible to achieve the optimal cell yield for whatever MOI is chosen. 

Where MOIq represents the multiplicity of infection at time 0. Comparing the Licari and Bailey parameter a with the Dee and Shuler expression means that ... 

DIP formation in virus stocks can be avoided by infecting insect cells at a low multiplicity of infection (MOI). According to Wickham et al. (1991), a low MOI is used to minimize the probability of a DIP entering in a cell along with an intact helper virion. As a result, some cells receive only intact virus, others only defective virus, or no virus at all. Non-infected cells or cells infected only with defective virus will not produce any more virus, while cells infected with intact virus will produce more intact and infectious viruses than the co-infected cells. Therefore, the DIP fraction will decrease compared with the original inoculum. This causes an increase of virus titer. 

Wickham TJ, Davis T, Granados RR, FFammer DA, Shuler ML, Wood FFA (1991), Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection, Biotechnol. Lett. 13 483-488. 

In order to study the virus growth curve a one-step growth cycle is performed. A high multiplicity of infection (m.o.i.) is used to ensure every cell is infected — usually 10 plaque forming units (p.f.u.) per cell is adequate. For virus production, however, the infection is prolonged under conditions where secondary infection can occur and a low m.o.i. is recommended especially where there is a tendency for defective virus particles to be produced. 

MNNG m.o.i. MOPC A-methyl-iV-nitro-jV-nitrosoguanidine multiplicity of infection mineral oil-induced plasmacytoma... 

Remove medium by vacuum aspiration, and add 0.4 mL of Ex-Cell 400 medium. Add 0.1 mL of PI virus stock (approx 2 1 x 107 pfu/mL) or appropriate control wild-type virus (approx multiplicity of infection [MOI] of 1 to 10). 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

Infect the cells with either cell-free virus at the desired multiplicity of infection, or with an infected cell culture at a ratio of 1 part infected cell culture to 10 parts uninfected cell culture. 

Remove the medium and add a virus inoculum at a multiplicity of infection (moi) of 0.002 plaque forming units (PFU)/cell. 

Significant internal p24 staining was not detectable until after 36 h of infection in the flow cytometric assay using C8166 cells infected with a HIV-1 clinical isolate at a multiplicity of infection of 1 500. Experimental conditions will require optimization according to the virus/cell model used. 

Cool dishes on ice, in a cold room. Inoculate with 50-100 pL of virus, at an multiplicity of infection of approx 5. 

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