Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.

Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.

Clinical presentation of patients with seasonal influenza and pandemic influenza A (H1N1-2009) requiring hospitalisation.

BACKGROUND: A sudden increase in the number of novel influenza A virus (pH1N1-2009) infection prompted us to compare the clinical presentation and outcomes of patients infected with pH1N1-2009 and seasonal influenza A virus during the post-pandemic phase. METHODS: During the period August 13 to September 27, 2010, case records of 106 patients with severe influenza like illness (ILI) and respiratory complications who underwent diagnostic testing by real-time polymerase chain reaction (RT-PCR) for confirmation of pH1N1-2009 were retrospectively studied. RESULTS: Nineteen (17.9%) patients were tested positive for pH1N1-2009 and 78 (73.6%) were tested positive for seasonal influenza A virus. The mean age of patients infected with pH1N1-2009 was 45.2 +/- 15.3 years (range of 22 to 80 years). Common presenting symptoms included fever in 17 (89.4%), cough in 16 (84.2%), myalgia in 15 (78.9%) and breathlessness in 10 (52.6%) patients. The most common comorbidities included bronchial asthma/bronchitis/chronic obstructive pulmonary disease (COPD) in 4 (21%); followed by hypertension in 3 (15.8%) and diabetes in 3 (15.8%) patients. Overall, of the 97 influenza infected patients, 9 (9.3%) needed hospitalisation to the intensive care unit (ICU); one patient with COPD died due to multi-organ failure. CONCLUSIONS: Both the pandemic and seasonal strains were found to be co-circulating in the community. Patients with severe hypoxia, hypertension, acute respiratory distress syndrome and shock required ICU care.