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

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

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\u27)-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA\u27 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\u27)-IIIa, aad9, linA\u27 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

Restriction Fragment Length Polymorphism Analysis of NYMC X-197 Glycoproteins, Matrix and Nonstructural Gene Segments.

<p>NYMC X-197 derived the HA and NA gene segments from the wt virus and the M and NS gene segments from A/PR/8/1934. <b>A.</b> HA: lane 1: A/PR/8/1934 undigested; 2 and 3: A/PR/8/1934 digested with <i>Pvu</i>II and <i>Hind</i>III, respectively; 4: A/Brisbane/11/2010 undigested; 5 and 6: A/Brisbane/11/2010 digested with <i>Pvu</i>II and <i>Hind</i>III, respectively; 7: NYMC X-197 undigested; 8 and 9: NYMC X-197 digested with <i>Pvu</i>II and <i>Hind</i>III, respectively. <b>B.</b> NA: lane 1: A/PR/8/1934 undigested; 2 and 3: A/PR/8/1934, digested with <i>Bsg</i>I and <i>Eco</i>57I, respectively; 4: A/Brisbane/11/2010 undigested; 5 and 6: A/Brisbane/11/2010 digested with <i>Bsg</i>I and <i>Eco</i>57I, respectively; 7: NYMC X-197 undigested; 8 and 9: NYMC X-197 digested with <i>Bsg</i>I and <i>Eco</i>57I, respectively. <b>C.</b> M: lane 1: A/PR/8/1934 undigested; 2: A/PR/8/1934 digested with <i>Bsg</i>I; 3: A/Brisbane/11/2010 undigested; 4: A/Brisbane/11/2010 digested with <i>Bsg</i>I; 5: NYMC X-197 undigested; 6: NYMC X-197 digested with <i>Bsg</i>I. <b>D.</b> NS: lane 1: A/PR/8/1934 undigested; 2 and 3: A/PR/8/1934 digested with <i>Sml</i>I and <i>Xmn</i>I, respectively; 4: A/Brisbane/11/2010 undigested; 5 and 6: A/Brisbane/11/2010 digested with <i>Sml</i>I and <i>Xmn</i>I, respectively; 7: NYMC X-197 undigested; 8 and 9: NYMC X-197 digested with <i>Sml</i>I and <i>Xmn</i>I, respectively.</p

Oligonucleotide Primers used in RT-PCR.

<p>F: forward primers; R: reverse primers used for PCR amplification.</p

Restriction Fragment Length Polymorphism Analysis of NYMC X-197 Polymerase and Nucleoprotein Gene Segments.

<p>NYMC X-197 generated from A/Brisbane/11/2010 (H3N2)×A/PR/8/1934 (H1N1) has a 5∶3 gene ratio, the PB1 gene segment was derived from wt virus; PB2, PA and NP gene segments were derived from A/PR/8/1934. <b>A.</b> PB2: lane 1: A/PR/8/1934 undigested; 2: A/PR/8/1934 digested with <i>Pvu</i>II; 3: A/Brisbane/11/2010 undigested; 4: A/Brisbane/11/2010 digested with <i>Pvu</i>II; 5: NYMC X-197 undigested; 6: NYMC X-197 digested with <i>Pvu</i>II. <b>B.</b> PB1: lane 1: A/PR/8/1934 undigested; 2: A/PR/8/1934 digested with <i>Pvu</i>II; 3: A/Brisbane/11/2010 undigested; 4: A/Brisbane/11/2010 digested with <i>Pvu</i>II; 5: NYMC X-197 undigested; 6: NYMC X-197 digested with <i>Pvu</i>II. <b>C.</b> PA: lane 1: A/PR/8/1934 undigested; 2 and 3: A/PR/8/1934 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively; 4: A/Brisbane/11/2010 undigested; 5 and 6: A/Brisbane/11/2010 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively; 7: NYMC X-197 undigested; 8 and 9: NYMC X-197 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively. <b>D.</b> NP: lane 1: A/PR/8/1934 undigested; 2 and 3: A/PR/8/1934 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively; 4: A/Brisbane/11/2010 undigested; 5 and 6: A/Brisbane/11/2010 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively; 7: NYMC X-197 undigested; 8 and 9: NYMC X-197 digested with <i>Hind</i>III and <i>Xmn</i>I, respectively.</p

Restriction Enzymes used for RFLP.

<p>Gene segments PB2, PB1 and M were digested by a single enzyme.</p><p>*<i>Sml</i>I was used to digest 2009 H1N1pdm M gene segment.</p

Frequency of Origin of Internal Genes in High Yield Reassortants.

<p>Frequency of Origin of Internal Genes in High Yield Reassortants.</p

HY Reassortants: Gene Constellations and Fold Increase in HA Titer.

<p>*HA Titer is given as reciprocal of viral dilution at titration end point.</p><p>**[FI]: fold increase in HA titer over wt parent virus.</p>a<p>Used in seasonal influenza vaccine production.</p>b<p>Used in 2009 H1N1pdm vaccine production.</p><p>P: hy donor virus A/PR/8/1934 gene. <i>WT</i>: wild type virus gene.</p

Gene Constellations Present in Hy Reassortants.

<p>P: hy donor virus A/PR/8/1934 gene. <i>WT</i>: wild type virus gene.</p