Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk.

BACKGROUND: The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus. OBJECTIVE: Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased. METHODS: We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks. RESULTS: In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA. CONCLUSION: These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.

Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses.

BACKGROUND: Black elderberries (Sambucus nigra L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus. METHODS: The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays. RESULTS: For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses. CONCLUSION: Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product.