Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs.

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.

Novel antibiotic-resistance markers in pGK12-derived vectors for Borrelia burgdorferi.

Extension of molecular genetics studies in Borrelia burgdorferi has been hampered by a lack of a variety of antibiotic resistance selective markers. Such markers are critical for isolation of B. burgdorferi strains with multiple mutants, for complementation with different cloning vectors, and for methods such as negative selection and reporter genes. To remedy this lack, resistance to various antibiotics of non-infectious (B31, 297) and infectious (N40) B. burgdorferi strains was examined and vectors incorporating appropriate antibiotic resistance genes as selective markers were developed. Minimal inhibitory concentrations for growth of B. burgdorferi on plates and in liquid media for aminoglycosides (kanamycin, gentamycin, sisomycin, amikacin, spectinomycin, neomycin), macrolides-lincosamids (erythromycin, lincomycin), coumarin derivatives (coumermycin A(1), novobiocin), glycopeptides (vancomycin, ristocetin), peptides (bacitracin, cycloserine), and chloramphenicol were found to differ significantly. There were also striking differences in resistance to these antibiotics between non-infectious and infectious B. burgdorferi strains. Antibiotic-resistance genes aph(3')-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA' from Staphylococcus aureus, and aac(3)-VIa from Enterobacter cloacae (conferring resistance to kanamycin, spectinomycin, lincomycin, and gentamycin/sisomycin, respectively) were subcloned either with their own promoters or under the control of the B. burgdorferi flaB promoter into pGK12 or its derivative pED1 to develop new cloning vectors for B. burgdorferi with the rationale that the absence of homologous regions between derived recombinant plasmids lacking the flaB promoter and the B. burgdorferi genome would permit avoidance of possible recombination with target DNA. Resistance to the corresponding antibiotic was conferred by vectors containing aph(3')-IIIa, aad9, linA' or aac(3)-VIa whether under the control of their own promoters or under the control of the flaB promoter. We conclude that these markers can be used for genetic study of B. burgdorferi and suggest they will be an important addition to the previously used coumermycin A(1), erythromycin and kanamycin in these studies.

Molecular signature of high yield (growth) influenza a virus reassortants prepared as candidate vaccine seeds.

BACKGROUND: Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral 'seeds' for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six 'internal' genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth in ovo. METHODOLOGY: The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry. RESULTS: Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA. SIGNIFICANCE: In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine 'seed' viruses.

Gene constellation of influenza A virus reassortants with high growth phenotype prepared as seed candidates for vaccine production.

BACKGROUND: Influenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo. METHODOLOGY: The genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers. SIGNIFICANCE: In this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6:2 and 5:3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.