ABSTRACT
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
Subject(s)
Animals , Virus Shedding , Madin Darby Canine Kidney Cells , Cell LineABSTRACT
There are many effective chemotherapeutic agents used in influenza disease which some of them inhibit virus replication by interfering with FluV [influenza virus] viral binding or its penetration into cell membrane. A series of polyoxometalates compounds such as POM-523 and PM-504 have been synthesized and have showed inhibitory effects on viruses. In this study we examined anti influenza activity of a novel polyoxometalate derivative [POM-4960] synthesized in the Faculty of Chemistry of Damghan University of Basic Sciences. To evaluate the anti-influenza activity of POM, following the treatment of FluV with POM at different temperatures and incubation periods, viral titer reduction was assessed by haemaglutination assay [HA]. The 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay was used to determine TCID50 [tissue culture infective dose] of virus, CC50 [median cytotoxic concentration] of POM, protection percentage and antiviral activity of POM in cell culture. RT-PCR and direct Immunofluorescent assays were performed to evaluate the effect of POM on viral infection and viral RNA load, respectively. POM reduced HA titer near to zero in all cell culture specimens and showed high protection against viral infection of the cells. Reduction in viral infection was confirmed by RT-PCR and Immunofluorescent staining methods. Moreover, this POM derivative has a dual [cumulative] effect on attachment and penetration inhibition compared to other POM's with just one inhibitory effect. POM-4960 could be considered as a powerful antiinfluenza agent with low toxicity and high antiviral potency.
ABSTRACT
Influenza virus is a major infectious pathogen of the respiratory system causing a high degree of morbidity and mortality annually. The worldwide vaccines are decided and produced annually by The Worldwide vaccines are decided and produced annually by World Health Organization and licensed companies based on the samples collected from all over the world. The aim of this study was to determine phylogenecity and heterogenecity of the circulating influenza isolates during 2008-2009 outbreaks in Tehran, compare them with the vaccine strains that were recommended by WHO for the same period. Nasopharyngeal swabs [n=142] were collected from patients with influenza and influenza-like illness. Typing and subtyping of the isolates were performed using multiplex RT-PCR and phylogenetic analysis was carried out for hemagglutinin genes of the isolates. Fifty out of 142 samples were positive for influenza A virus, and no influenza B virus was detected. Phylogenetic analyses revealed that the A/H1N1 isolates were related closely to A/Brisbane/59/2007, and the A/H3N2 isolates were close to A/Brisbane/10/2007 vaccine strains. The findings of the present study demonstrate that the A/H1N1 was the predominant subtype of human influenza virus among the patients studied in Tehran during 2008-2009 winter seasons. In addition, some amino acid variation was found in Tehran/2008/H1N1 isolates from the 2008-2009 vaccine strain, but the H3N2 isolates showed higher genetic resemblance to the vaccine strain