Determining influenza virus shedding at different time points in madin-darby canine kidney cell line

Monitoring of influenza virus shedding and optimization of multiplicities of infection [MOI] is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney [MDCK] cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI [0.01] could result in accurate, precise quantitative assays in virus diagnosis and titration methods

[Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus]

In this study, two conserved genes [M1 and NP] of influenza virus were expressed in a bicistronic vector in order to develop a universal gene based vaccine. Plasmids M1-pIRES2-EGFP, pIRES2-NP were constructed by cloning the PCR products of M1 and NP genes which were amplified from the A/Peurto Rico/8/34 [H1N1] influenza virus strain into the plasmid expression vector pIRES2-EGFP, respectively. For construction of M1-pIRES2-NP bicistronic plasmid, M1 gene was extracted from M1-pIRES-EGFP plasmid and subcloned into pIRES2-NP construct. Finally, simultaneous expression of both genes was assessed by transient transfection of bicistronic plasmid into BHK-21 cell lines and subsequent immunofluorescence staining. The results of enzymatic double digestions on the constructed plasmids and sequencing demonstrated the success of cloning processes of above mentioned genes. Correct expression of these genes was confirmed by M1-pIRES2-NP plasmid expression in BHK-21 cell lines confirmed by immunofluoresence microscopy. Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing "IRES" sequence is achievable

[Evaluation of intramuscular versus intra-dermal inoculation of bacterial plasmid encoding nucleoprotein of influenza virus]

The use of bacterial plasmids carrying specific genes of pathogens as genetic vaccines is a relatively new technique for induction of cellular immune responses against microbial pathogens. Mechanisms of production of specific immune responses against these vaccines are not still completely understood. Therefore, it is necessary to examine various routes of inoculation to find the best way of immunization for specific antigens. In this research, intramuscular method of inoculation of influenza vaccine nucleoprotein [NP] encoding vector was compared with that of intra-dermal method. In this study, the ability of two different methods of immunization [intramuscular and intra-dermal] in induction of CTL responses as well as their efficiency in clearance of influenza virus from the lung of BALB/c mice was compared. Female BALB/c mice were immunized with influenza virus NP expressing plasmids on days 0, 14 and 28. CTL activity of mice was evaluated by lactate dehydrogenase technique two weeks after the last inoculation. In addition, the mice were challenged by live influenza virus and the viral titer was measured 4 days postchallenge in the lungs of animals. The results of experiments demonstrated that intramuscular immunization of mice induces a stronger CTL response. Mice immunized by intramuscular route also showed a higher ability in virus clearance from the lungs. Results of this study showed that different routes of immunization of influenza NP genetic vaccine induce different levels of cell-mediated immune responses and protection from the live virus