@phdthesis{Adenugba2021,
  author    = {Adenugba, Akinbami Raphael},
  title     = {Functional analysis of the gene organization of the pneumoviral attachment protein G},
  doi       = {10.25972/OPUS-12814},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128146},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The putative attachment protein G of pneumonia virus of mice (PVM), a member of the Pneumoviruses, is an important virulence factor with so far ambiguous function in a virus-cell as well as in virus-host context. The sequence of the corresponding G gene is characterized by significant heterogeneity between and even within strains, affecting the gene and possibly the protein structure. This accounts in particular for the PVM strain J3666 for which two differing G gene organizations have been described: a polymorphism in nucleotide 65 of the G gene results in the presence of an upstream open reading frame (uORF) that precedes the main ORF in frame (GJ366665A) or extension of the major G ORF for 18 codons (GJ366665U). Therefore, this study was designed to analyse the impact of the sequence variations in the respective G genes of PVM strains J3666 and the reference strain 15 on protein expression, replication and virulence. First, the controversy regarding the consensus sequence of PVM J3666 was resolved. The analysis of 45 distinct cloned fragments showed that the strain separated into two distinct virus populations defined by the sequence and structure of the G gene. This division was further supported by nucleotide polymorphisms in the neighbouring M and SH genes. Sequential passage of this mixed strain in the cell line standardly used for propagation of virus stocks resulted in selection for the GJ366665A-containing population in one of two experiments pointing towards a moderate replicative advantage. The replacement of the G gene of the recombinant PVM 15 with GJ366665A or GJ366665U, respectively, using a reverse genetic approach indicated that the presence of uORF within the GJ366665A significantly reduced the expression of the main G ORF on translational level while the potential extension of the ORF in GJ366665U increased G protein expression. In comparison, the effect of the G gene-structure on virus replication was inconsistent and dependent on cell line and type. While the presence of uORF correlated with a replication advantage in the standardly used BHK-21 cells and primary murine embryonic fibroblasts, replication in the murine macrophage cell line RAW 264.7 did not. In comparison, the GJ366665U variant was not associated with any effect on replication in cultured cells at all. Nonetheless, in-vivo analysis of the recombinant viruses associated the GJ366665U gene variant, and hence an increased G expression, with higher virulence whereas the GJ366665A gene, and therefore an impaired G expression, conferred an attenuated phenotype to the virus. To extend the study to other G gene organizations, a recombinant PVM expressing a G protein without the cytoplasmic domain and for comparison a G-deletion mutant, both known to be attenuated in vivo, were studied. Not noticed before, this structure of the G gene was associated with a 75\% reduction in G protein expression and a significant attenuation of replication in macrophage-like cells. This attenuation was even more prominent for the virus lacking G. Taking into consideration the higher reduction in G protein levels compared to the GJ366665A variant indicates that a threshold amount of G is required for efficient replication in these cells. In conclusion, the results gathered indicated that the expression levels of the G protein were modulated by the sequence of the 5' untranslated region of the gene. At the same time the G protein levels modulated the virulence of PVM.},
  subject      = {G glycoprotein},
  language  = {en}
}
@phdthesis{Schneiderbanger2010,
  author    = {Schneiderbanger, Daniel},
  title     = {Molekulare Epidemiologie des Respiratory Syncytial Virus bei Kindern mit Atemwegsinfektionen im Zeitraum von 2002 bis 2006},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51984},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Hintergrund: Infektionen mit dem Respiratory Syncytial Virus (RSV) sind die h{\"a}ufigste virale Ursache f{\"u}r respiratorische Erkrankungen bei S{\"a}uglingen und Kleinkindern. Reinfektionen treten lebenslang auf. Es wurden zwei Typen (A und B) und mehrere Genotypen beschrieben. Die vorliegenden Daten {\"u}ber die molekulare Epidemiologie von RSV in Deutschland sind nur begrenzt. Material und Methoden: Zwischen Januar 2002 und Juli 2006 wurden 221 Nasenrachensekrete (NRS) von Kindern, welche in der Universit{\"a}tskinderklinik W{\"u}rzburg behandelt wurden, durch Routine-Untersuchung mit einem Immunfluoreszenztest auf RSV-Antigen positiv befunden. Die phylogenetische Analyse wurde aus Restmaterial von 211 NRS durchgef{\"u}hrt, indem die zweite variable Region des G-Gens amplifiziert und sequenziert wurde. Ergebnisse: Insgesamt war die Pr{\"a}valenz von Typ A-Viren mit 69,5 \% gr{\"o}ßer als die der Typ B-Viren mit 30,5 \%. RSV Typ A war das dominierende Virus in allen Saisons außer in der Saison 2002-2003. {\"U}ber den gesamten Beobachtungszeitraum traten drei A-Genotypen (GA2, GA5 und GA7) und vier B-Genotypen (GB3, SAB3, BA und ein neuer Genotyp) auf. Die Genotypen GA2, GA5, SAB3 und BA waren am h{\"a}ufigsten im Umlauf und in beinahe allen Saisons pr{\"a}valent. Unter den B-Genotypen nahm der Anteil des Genotyps BA von 25 \% (2002) auf 92 \% (2005-2006) zu. Drei Typ B-Sequenzen wurden einem neuen Genotyp zugeordnet, welcher BWUE benannt wurde. Es wurde eine Reinfektion mit demselben Genotyp (GA5) bei einem Kind beobachtet, welches im Alter von 12 und 28 Monaten mit einer RSV-Infektion hospitalisiert war. Schlußfolgerung: Die Ergebnisse unserer Studie stehen in Einklang mit der molekularen Epidemiologie von RSV in anderen geographischen Regionen. Wir beobachteten sowohl Genotypen, welche {\"u}ber mehrere Saisons pr{\"a}valent waren, als auch Genotypen, welche {\"u}ber den beobachteten Zeitraum zunehmend dominanter werdend andere Genotypen verdr{\"a}ngten. Zudem wurde ein neuer B-Genotyp entdeckt.},
  subject      = {RSV},
  language  = {de}
}