The overall aim of this project was to develop a sensitive, specific and reliable molecular assay for the detection of human pathogenic viruses in shellfish. In initial studies, 'viral surrogates' were used to evaluate two different assay formats, the reverse transcriptase-polymerase chain reaction-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and the enzyme-labelled deoxynucleic acid-enzyme-linked coagulation assay (EDNA-ELCA). Each format was tested for ease of use, reliability and sensitivity, when compared with ethidium bromide gel detection. The RT-PCR-ELISA proved to be a successful alternative to ethidium bromide gel electrophoresis and studies involving virus detection in contaminated environmental samples were performed. The newly developed ELISA method was able to successfully detect enterovirus (EV) and Norwalk-like viruses (NLVs) in artificially, and naturally contaminated shellfish. In shellfish studies the ELISA had a detection sensitivity of 10-100, and 100-1000 TCID50 PV respectively, when a traditional elution/precipitation, and an immunocapture procedure were employed. The assay could also successfully detect virus in trout kidney samples, artificially contaminated with infectious pancreatic necrosis virus (IPNV), with detection sensitivities of 105, and 107 pfu reported when the elution/precipitation, and immunocapture procedures were used. In naturally contaminated shellfish samples, positive NLV and EV detection were achieved using the ELISA method. The ability of the ELISA to positively detect virus in environmental samples was compared to the TaqMan quantitative PCR system, an alternative detection method. Both methods were used to screen various contaminated environmental samples, including faeces and shellfish. The ELISA performed well, and unlike the TaqMan system no optimisation for each sample matrix tested was required. Overall the ELISA was shown to be a very robust and sensitive method. The technique was easily established in a new laboratory and no specialised equipment was required to perform the assay. The method has a high sample throughput, capable of screening 96 samples per run. Each sample takes only approximately 50 s to screen, making the technique extremely time efficient. The ELISA is a safe, quick, reliable technique, which has great potential for use as a standard virus detection method in a standard equipped laboratory.

No description available.

OBJETIVO: Determinar la prevalencia de neonatos perinatalmente expuestos al Virus de Inmunodeficiencia Humana en el Hospital Nacional Hipólito Unanue durante los periodos 2010 – 2014 MATERIALES Y MÉTODOS: Se realizó un estudio de tipo Observacional – Transversal – Retrospectivo - Descriptivo. Nuestra muestra, coincide con la población, 290 neonatos perinatalmente expuestos al VIH. El método que se realizó para la recolección de datos, fue el uso de un instrumento AD – HOC, el cual fue llenado con datos recogidos del Registro de PROCITTS (Programa de control de infecciones de transmisión sexual y SIDA) del HNHU. RESULTADOS: Se encontró un 16.2 % de prevalencia de neonatos perinatalmente expuestos al VIH durante el año 2010, 14.1 % durante el año 2011, 19.3 % durante el año 2012, 27.2 % durante el año 2013 y un 23.1 % durante el año 2014. CONCLUSIONES: La prevalencia de neonatos perinatalmente expuestos al VIH, ha tenido un leve incremento durante los años estudiados, con una cobertura profiláctica en más de la mitad de la población (94.8 %)

Two Tobravirus expression vectors were evaluated for the use as a rapid screening method for anti-nutritional proteins against plant pathogens. Accumulation of green fluorescent protein (GFP) and snowdrop lectin gene (Galanthus nivalis agglutinin, LECGNA2, M55556) in Nicotiana benthamiana by Tobacco rattle virus expression vectors was characterized. Virally expressed proteins were detected in leaves (3-14 days post-inoculatiion) and roots (6-24 dpi) by UV (GFP), western blotting and tissue printing. 25 -50 ng of GNA was detected in root extracts. Cross protection was induced by TRV-GFP. Foreign genes inserted in place of TRV RNA2 non-structural genes (2b and 2c) were stably maintained over serial passages. But recombination at remaining 'cross-over' sites may occur. 2D iso-electricfocusing detected a 50-kDa GNA molecule in root and leaf extract. GNA did not confer resistance to root-knot nematodes, although gall by root-knot nematodes (mixed Meloidogyne spp. and M.javanica Crete line 17) were significantly reduced by 22% in roots infected by TRV-GNA (3.83 sqrt galls and 4.5 sqrt galls respectively) compared to virus-free roots treatment (4.94 sqrt galls, sed 0.398; p<.025 and 5.273 sqrt galls, sed 0.2403; <.003 respectively). Effects of GNA on Aulacorthum solani was delayed to the second nymph generation (N2). Mean N2 weights feeding on TRV-GNA (0.246 mg ±0.0159; p <05) and TRV-fsGNA (0.212 mg ±0.018; p<.001) infected plants were significantly smaller by 15.2% and 26.6% respectively, compared to virus-free treatments (0.290 mg ±0.014). Similar trends were detected for total nymph weights. Low toxicity was related to high quality phloem and ingestion of smaller volumes for normal development (i.e. concentration effect). Decrease in gall by the mixed Meloidogyne population and an unexpected toxicity to A solani indicated that truncated GNA was a protein with merolectin properties. The viability of this system as a rapid 'm planta' expression system is discussed.

Describe el aislamiento y caracterización molecular de un nuevo flavivirus únicamente de mosquito, aislado de Culex (Melanoconion) occossa colectados en 2009 de un área urbana de Iquitos, Perú, ubicada en la cuenca del Amazonas, en la región noreste del Perú. La evidencia del Flavivirus es detectada mediante ensayo de inmunofluorescencia indirecta (IFI) en sobrenadante de cultivo celular de las células infectadas C6/36 usando anticuerpos policlonales grupo de Flavivirus y confirmada por RT-PCR. En comparación por pares de las secuencias de la región ENV, la más alta de nucleótidos (47,4%) y de aminoácidos (39,8%) la identidad se observa en el virus Nounané (NOUV). En comparación por pares de la región NS5, la identidad de nucleótidos más alta se observa en el virus Spondweni (65,9%), el virus de Iguape (IGUV; 65,7%) y el virus de Kedougou (65,6%); sin embargo, en el nivel de aminoácidos, la más alta identidad en pares de bases se observa en IGUV (69,8%), virus Naranjal (69,6%) y el virus Bussuquara (69,3%). El análisis filogenético utilizando ENV parcial y secuencias de aminoácidos NS5 revela que este Flavivirus forma un clado con NOUV. Para investigar la gama de huéspedes del nuevo Flavivirus, se inoculan una variedad de células de mamíferos (Vero 76, Vero E6, BHK, LLCMK y MDCK) con el tercer pasaje del aislamiento C6/36 y monitorea el efecto citopático (EC). No se detecta EC, y todas las líneas de células de mamífero son negativas para el antígeno Flavivirus por IFI y ARN Flavivirus por RT-PCR luego de catorce días de incubación. Propone que este Flavivirus genéticamente diferente sea llamado Nanay Virus (NANV), debido a la zona de Iquitos, Perú, donde fue detectado por primera vez.

No description available.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / N6-Metiladenosina (m6A) es la modificación química más abundante encontrada en los ARN mensajeros (ARNm) de células eucariontes. Esta modificación regula el metabolismo de los ARNm a diferentes niveles incluidos el corte y empalme de exones, traducción, estabilidad y degradación. Existen tres grupos de proteínas que regulan estos procesos: aquellas que catalizan la adición de la modificación, las proteínas metiltransferasas METTL3 y METTL14, las que remueven la metilación, las desmetilasas FTO y ALKBH5, y finalmente, las que reconocen m6A y actúan como efectoras de esta modificación, las proteínas lectoras YTHDF1,2 y 3. Recientemente se ha descrito la presencia de m6A en el ARN de diferentes virus que replican en el citoplasma, como los virus de la hepatitis C, virus Zika e influenza A, donde la modificación m6A regula la expresión de proteínas virales, estabilidad de los ARN virales (genómicos y mensajeros) y por ende la replicación viral. Sin embargo, se desconoce su rol en la replicación de otros virus de importancia clínica, como el virus respiratorio sincicial humano (VRSh). VRSh es uno de los principales causantes de enfermedades respiratorias en menores de 3 años, adultos mayores e inmunodeprimidos. Es un virus que infecta células del tracto respiratorio replicando en el citoplasma de estas. Posee un ARN genómico de simple hebra de polaridad negativa y actualmente no existen vacunas licenciadas ni tratamientos antivirales para combatir su infección. El objetivo de este trabajo fue determinar si las proteínas que participan en la adición, remoción y reconocimiento de m6A, tienen un impacto en la replicación del VRSh. Para esto, se sobreexpresaron las proteínas metiltransferasas, desmetilasas y lectoras en células HEK293T infectadas con VRSh y se estudió la expresión de la proteína viral de fusión (F) por Western Blot (WB). Además, se cuantificó los niveles del ARNm de F y el ARN genómico intra y extracelular mediante RT-qPCR bajo las mismas condiciones. Los resultados obtenidos muestran que la sobreexpresión de las metiltransferasas disminuyó la expresión de la proteína F y los niveles del ARNg viral, mientras que la sobreexpresión de las desmetilasas tuvo el efecto contrario, sugiriendo que m6A afecta de forma negativa la expresión de proteínas virales y la replicación del genoma de VRSh. Por otra parte, la sobrexpresión de las proteínas YTHDF1, 2 y 3 aumentó los niveles del ARNm de la proteína F, aunque disminuyó los niveles del ARNg del virus. Además, mediante ensayos de inmunoprecipitación de estas proteínas se observó que tanto YTHDF1, 2 y 3 se unen al ARNg de VRSh, sugiriendo que la modificación m6A se encuentra en el ARNg de VRSh. En conjunto, estos resultados sugieren la presencia de m6A en el ARN de VRSh, posiblemente regulando la síntesis de ARN mensajeros y ARN genómico, al igual que la expresión de proteínas virales, mediado por la actividad de las proteínas YTHDF1, 2 y 3. / Octubre 2019

A Dissertation submitted to the Faculty of Medicine, University of the witvmtersrand, Johannesburg, in fulfilment of the requirements for the Degree of Master of Science in Medicine. / The potential of Aedes aegypti to act as a vector of dengue (DEN) viruses was assessed in the tropical and subtropical regions of South Africa. The prevalence in bamboo pot ovitraps, utilization of artificial larval habitats and antihropophflism of Ae. aegypti was measured in fifteen populations from coastal Natal and five from the Transvaal. Pot indices generally ranged from 40-100 per cent (x±s;c= 60,3±9,8), with a mean abundance index of 0,43±0,15 (sx) mosquitoes/pot/day. Artificial container indices ranged from 11-83 per cent (X±si = 56,8±5,6). Biting rates ranged from 0,2-29,0 per manhour, in direct relationship to the level of urbanization. The feral Nduntupopulation was least anthropophilic, although pot and abundance indices were high. To investigate the presence of subspp. aegypti and formosus, the pale scales on tergite-1 were counted in ten or more siblings from each of 196 families, representing eighteen populations. At least 118 of these families were heterogeneous, each containing some siblings with no pale scales and others with pale scales on tergite-1, thus invalidating this character for distinguishing between the subspp. Isozyme electrophoresis 'did not provide diagnostic electromorphs for distingllishing between different populations. Allelomorph frequencies and the numberof pale scales on tergite-l and tergite-2 differed significantly in individual populations but not between anthropophilic and non-anthropophilic populations. As there was no correlation between tergal morphology, isozymes and anthropophilism, the populations could not be resolved into the two subspp. Based on the only morphological character that appears to be reliable, viz. the blackness of the background scales, all of the populations are probably Ae. aegypti formosus. The vector competence of five populations for DEN-1 and DEN-2 viruses was tested in the laboratory after allowing mosquitoes to feed on an infective blood-virus mixture. Viral antigen was detected by indirect fluorescent antibody test. Head-squash infection rates (HSIRs)ranged from 11-54 per cent for DEN-land from 19- 46 per cent for DEN...2. Transmission rates (TRs) were determined by in vitro capillary method and ranged from 67-100 per cent for DEN-1 and from 11-86 pier cent for DEN-2.Meanvector competence indices (calculated from HSIRsand TRS) ware 0,13-0,41 for DEN-l and 0,,18-0,34 for DEN-2.It is concluded that, should DEN be reintroduced to South Africa via the shipping or tourist industries, Ae. aegypti would be an efficient urban vector. The Durban population is of particular epidemiological importance because it was highly anthropophilic and was the most vectorially competent of the South African populations. / Andrew Chakane 2018

M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Hepatitis B virus (HBV) and human immunodeficiency virus (Thakur et al.) are endemic in South Africa. There no data on the HBV genotypes prevailing in HIV-infected South Africans. The aim this study was to determine the HBV genotypes in HIV-infected patients and to identify mutations occurring in the basic core promoter/preccore (BCP/PC) and complete S regions. Twenty six HBV isolates from HBV-HIV co-infected individuals from Helen Joseph urban hospital prior to and at six month after initiation of HAART were analyzed. Three hundred HBV isolates from the rural Shongwe Hospital were recruited prior to treatment. Restriction fragment length polymorphism (RFLP) together with sequencing of the BCP/PC and complete surface region amplicons were employed for genotyping and analysis. There was no significant difference in the HBV genotypes from both cohorts. Subgenotype A1 was the predominant genotype. HBV DNA was detected in 13/26 (50 %) Helen Joseph patients and 72/300 (24%) HBV DNA was detected in patients from Shongwe cohort: 28/300 (9.3%) HBsAg-positive and 44/300 (14.7%) HBsAg-negative. The BCP/PC region of HBV isolates from both cohorts showed mutations that could account for the HBeAg negativity, although in the case of the Helen Joseph cohort the HBeAg status was unknown because of depletion of serum. The HBeAg negativity in 44/49 Shongwe patients (89,7%) could be accounted for by the following mutations: 1) the basic core promoter mutations T1753C A1762T G1764A, which down regulate transcription of precore mRNA; 2) the Kozak sequence mutants that affect HBeAg translation, 3) the G1862T mutation, which interferes with post-translational modification of the HBeAg-precursor, and 4) the classical G1896A stop codon mutation with C1858T. The G1862T mutation occurred in HBV from 24.1% HBsAg-positive Shongwe patients but in none of the HBsAg-negative patients (p<0.05). PreS deletion mutants were found in isolates from both cohorts. These have previously been described in hepatocellular carcinoma patients. The Helen Joseph HBV isolates had the “a” determinant and immune/vaccine escape mutants. In addition to these, Shongwe isolates had HBV reactivation markers mutations V168A + S174N in 23.8% of the HBsAg-negative and in none of the HBsAg-positive infections. Drug resistant mutations were found in three Shongwe HBV isolates from ART naïve individuals and in none Helen Joseph HBV isolates. In conclusion, South African HIV-infected individuals were predominantly infected with subgenotype A1 of HBV. Mutations in the BCP and precore region of HBV isolates could account for the HBeAg negativity in the majority of patients. A number of HBV isolates displayed both immune escape and drug resistance mutations that were responsible of the HBsAg-negativity in patients from which they were isolated.

Members of the family Hepadnaviridae have been detected in both avian and mammalian species. They have a very limited host range, and among the nonhuman primates, have been found to occur naturally in chimpanzees, gorillas, gibbons, orang-utans and woolly monkeys. The human hepatitis B virus (HBV) has been shown to infect chimpanzees, Barbary macaques and tree shrews. During the course of a previous study, to determine the susceptibility of baboons (Papio ursinus orientalis) to HBV infection, HBV DNA was detected in the serum of 2 baboons prior to their inoculation with HBV-positive human serum, raising the possibility that baboons are naturally infected with a hepadnavirus. Therefore the aim of this study was to determine the prevalence of HBV in wildcaught baboons and to molecularly and functionally characterise the virus isolated from these baboons. DNA was extracted from the sera of wild-caught baboons and four separate regions of the HBV genome amplified by nested polymerase chain reaction (PCR). Samples were only considered to be positive for HBV if at least three of these regions amplified. DNA was extracted from the liver tissue of one of the HBV DNA-positive baboons using a proteinase K digestion followed by a phenolchloroform extraction and ethanol precipitation. From this extract, the complete HBV genome was amplified by nested PCR of eight overlapping subgenomic fragments, and sequenced. This sequence was analysed phylogenetically using both the PHYLIP and Simmonic software packages. A selective real time PCR using SYBR®-green detection was used to detect covalently closed circular (ccc) DNA. RNA was extracted from the baboon liver tissue using a guanidinium-acidphenol extraction method, reverse transcribed and portions of the HBV genome amplified by nested PCR. Transmissibility of the virus was tested by injecting four experimentally naïve baboons individually with serum from four HBV DNApositive baboons and followed for 26 weeks. HBV was detected in the serum of 5/69 (7,2%) wild-caught baboons by Southern hybridization and in 11/49 (22,4%) adult and 4/20 (20,0%) juvenile wild caught baboons. This gave an overall prevalence of 21,7% in the baboon population surveyed. Serologically, the baboon sera were negative for all markers of HBV infection and alanine aminotransferse (ALT) levels were normal. In the one baboon liver tissue available, HBcAg was detected by immunohistochemical staining in some of the hepatocyte nuclei, but HBsAg was not detected. Phylogenetic analysis of the complete genome of the HBV isolate found it to cluster with subgenotype A2, a surprising result considering that subgenotype A1 predominates in South Africa. However, unlike other subgenotype A2 isolates, the basic core promoter had the G1809T / C1812T double mutation characteristic of subgenotypes A1 and A3 and the precore region had the G1888A mutation unique to subgenotype A1. These mutations in the basic core promoter and precore regions have previously been shown to reduce the expression of the precore and core proteins. Four additional mutations in the polymerase, surface, X and core open reading frames (ORFs) further differentiated the baboon HBV strain form the majority of previously sequenced subgenotype A2 isolates. cccDNA was detected at low levels in the baboon liver tissue. Regions of the precore/core and surface ORFs were amplified off reverse transcribed cDNA. These results demonstrate HBV replication in the baboon liver. Transmission of the virus was shown by the detection of HBV DNA in the sera of the four inoculated baboons at various times throughout the 26 week follow-up period. These baboons also showed transient seroconversion for HBsAg and HBeAg during this period with intermittent fluctuations in ALT levels. Moreover, using DNA extracted from liver tissue obtained at necropsy from one of the injected baboons, the sequence of the HBV surface gene amplified was found to be identical to the sequence of the isolate from inoculum. The finding of subgenotype A2 in the baboon is paradoxical because subgenotypes A1 and A3 as well as genotypes D and E predominate in Africa. The possibility exists that subgenotype A2 is an older strain that has been overtaken by these other strains. There is however a scarcity of subgenotype A2 sequencing data from Africa and a higher circulation of this subgenotype could be uncovered with more extensive molecular epidemiological studies in more remote areas. Alternatively, a recent discovery of alternative compartmentalization of subgenotype A2 infections in the peripheral blood lymphocyte population of individuals from India, where subgenotype A1 also predominates, could explain the lack of detection of this subgenotype in Africa. Occult hepatitis B infection is defined as the presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for HBsAg by currently available assays. The detection of HBV DNA in the baboon liver and serum in the absence of serological markers therefore classifies this infection as occult. To our knowledge, this is the first study to demonstrate a naturally occurring occult HBV infection in non-human primates.