Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag

No / Yellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae. A fragment of the coat protein and readthrough domain of SCYLV was expressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.