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Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology. ispartof: Science vol:344 issue:6182 pages:380-386 ispartof: location:United States status: published

The mortality rate of AHS is more than 90 % in fully susceptible horses. Outbreaks of AHS can spread rapidly and have severe economic consequences. Therefore, the disease is classified in "list A" by the OIE and the international movement of horses is subject to stringent zoosanitary regulations. Apart from prevention by large scale vaccinations, a good knowledge of the epidemiology of the disease together with accurate and rapid diagnostic procedures are the most important means of disease control. The use of genome segment 2 of the virus, in both diagnostic and epidemiological applications, was investigated. During this study it was shown that cDNA copies of genome segment 2 of the nine reference strain viruses of AHSV hybridize exclusively to viral RNA from their corresponding serotypes. This result provided proof of concept for the feasibility of developing nucleic acid based serotyping procedures. It was followed up by the development of the first two genome segment 2 based serotyping methods. The first is a method that uses a set of nine full-length AHSV genome segment 2 clones as probes to hybridize serotype-specifically to denatured dsRNA. The second method makes use of a universal RT-PCR primer set that was developed for the partial amplification of genome segment 2 of any AHSV serotype. The RT-PCR amplification product is labelled and used as a probe to hybridize with immobilized segment 2 cDNA of the nine serotypes of AHSV in a reverse line blot format. The reverse line blot hybridization method proved to be highly sensitive and accurate and can be used to serotype any AHSV with a single RTPCR and hybridization within one day. It was further shown that the method is sufficiently sensitive to be performed directly on clinical organ samples. A set of genome segment 2 clones was also used for the first ever segment 2-based AHSV phylogenetic analysis and molecular epidemiological investigation. Genome segment 2 sequence data was generated from AHSV 7 field cases that occurred in South Africa over a two year period which included an important outbreak in the African horsesickness free area in the Western Cape. The phylogenetic, classification was compared with historical and geographical data of the different disease cases. Results indicated that segment 2 nucleotide sequence data can be used to distinguish between different isolates/strains of one serotype and that the phylogenetic grouping of the isolates reflected their historical and geographical groupings. From this it can be concluded that genome segment 2 sequence data can be used in molecular epidemiological investigations to classify AHSVs according to geographical origin, or topotype. This information can be used to track the movement of a particular virus strain to its origin and implicate possible ways by which the disease is spread from endemic to non-endemic areas. With the new experimental diagnostic methods that have been developed, the virus can be serotyped directly from clinical organ samples within one day, compared to serological methods which take 2-3 weeks. The phylogenetic analysis demonstrated the advantage of a molecular epidemiological approach to provide definitive data for determining the origin of an AHS outbreak. In future, the use of these new serotyping procedures in conjunction with comprehensive molecular epidemiological data should significantly speed up AHSV diagnostics and contribute to more effective prevention, control and surveillance systems for AHS. Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.

MSc (Biochemie), North-West University, Potchefstroom Campus The expression of the proteins constituting the envelope of the hepatitis B virus was investigated. A novel human ovarian cancer cell line, UWOV2 (Sf), was tested for its ability to be transfected with and express the hepatits B virus surface antigen genes contained in a mammalian expression vector, using the standard protocols of calcium phosphate/DNA co-precipitation and lipofection. Confluent cell cultures were transfected with the vector which also carried the bacterial neomycin resistance gene and selection was applied with the antibiotic gentamycin. Both transfection procedures resulted in isolated foci of cells that survived the selection after all the cells in control cultures died. None of the cells in these foci survived long enough to be multiplied however, and no further analyses could be carried out it. Positive results were obtained in this laboratory expressing the envelope proteins of the hepatitis B virus in a baculovirus Sf 21-expression system (Strydom 1993 ). A series of fusion protein expression vectors were constructed for the expression of the three hepatitis B virus envelope proteins as His6-fusion proteins that could be isolated using affinity chromatography. The three open reading frames coding for the hepatitis B virus envelope proteins had already been cloned into pSP-vectors. These vectors were used to construct three pBlueBac His baculovirus transfer vectors further used in cotransfections. Recombinant baculovirus clones were prepared by cotransfecting linearized wild type Autographa californica virus genomic DNA with the three different transfer vectors respectively in Sf 21 cell cultures. Recombinant baculovirus clones were prepared and enriched for recombinants using the plaque-assay technique. Sf 21 cell cultures were infected with selected clones and used to quantify the production of HBsAg carrying proteins. An ELISA test kit was used to quantify both the HBsAg in the cell culture supematants as well as in cell lysates and in this way clones that showed the highest HBsAg expression were selected. In all cases the amounts of HBsAg were higher in the cell lysates than in the cell culture supernatants. Cell culture lysates were subjected to SDS-polyacrylamide gel electrophoresis and affinity chromatography on a Ni-NTA column. No fusion proteins could be isolated using this column but proteins with sizes corresponding to that of the envelope proteins of the hepatitis B virus were detected in the total cell culture lysates. In this study it was shown that all three the hepatitis B virus envelope proteins namely the large, medium and major polypeptides could be produced using insect cells infected with recombinant AcNPV clones. It was further shown that these proteins were produced in both the glycosilated and unglycosilated forms. Because the three open reading frames of the envelope protein genes were used seperately, the transcription of these could be compared. The results obtained showed that transcription did not exclusively start at the first initiation codon but in the case of the preS2-S and preS 1- preS2-S open reading frames, also at the second and third in fase initiation codons. Masters