Background: Gastroenteritis by rotavirus is responsible for approximately 810 annual deaths/year in children under 5 years in Peru and emerging rotavirus genotypes have led to concerns regarding cross-protection by the vaccines available. Moreover, there are no reports on the molecular-epidemiology of rotavirus diarrhea in Peru Methodology: A total of 131 stool samples were obtained from children under 5 years old hospitalized from January 2010 to December 2012 in the Hospital Regional de Cajamarca, Peru. ELISA and RT-PCR techniques were performed for rotavirus detection. G and P typing of rotavirus-positive samples were obtained by semi-nested multiplex RT-PCR and sequencing was performed to confirm the PCR results. Results: Of the 117 samples available, 18.80% (22/117) tested positive for rotavirus by ELISA and 35.90% (42/117) by RT-PCR. Among the G-genotype identified, G9 in 35.71% (15/42) and G12 in 33.33% (14/42) were the most prevalent. With the most common combination being G12/P6 in 23.81% (10/42). Conclusions: A high prevalence of the G12/P6 genotype was detected. It is know that this genotype is not covered by the current vaccines available. More in depth studies are needed to know the current rotavirus genotypes presents in Peru. / Tesis

<p>Porcine reproductive and respiratory syndrome virus (PRRSV) is estimated to cost the US swine industry $664 million in annual production losses. Therefore, the objective of this project was to evaluate the effect of MCFA on PRRSV replication using in vitro and in-vivo studies. The overarching hypothesis was that MCFA would inhibit or reduce viral replication of PRRSV infection in vitro and reduce viral load in-vivo. In the first experiment (Chapter 2), MARC-145 cells were used to determine the effects of individual MCFA (C6, C8, C10, and C12) exposure at concentrations ranging from 1-1000 µg/mL prior to and following inoculation of North American Type II (P-129) or European Type I (Lelystad) PRRSV. Viral replication was determined using FITC labeled IgG anti-PRRSV monoclonal antibody and TCID₅₀was calculated for each concentration. Data were analyzed using the Proc Mixed procedure of SAS. Incubation of MARC-145 cells with caproic acid (C6) at concentrations of 1-1000 µg/mL prior to and after inoculation with Type II North American (P129) or Type I European (Lelystad) PRRSV did not alter viral replication (<i>P</i> > 0.10). However, incubation of MARC-145 cells with caprylic (C8), capric (C10), and lauric (C12) acid prior to and after inoculation with Type I and Type II PRRSV did reduce viral replication at concentrations ranging from 100-1000 µg/mL. In general, the effective dose required to reduce (<i>P </i>< 0.05) viral replication (Log₁₀TCID₅₀/mL)decreased as MCFA chain length increased. In experiment 2 (Chapter 3), the use of MCFA combinations (C8:C10; C8:C12; C10:C12; and C8:C10:C12) to reduce viral replication of PRRSV in MARC-145 cells was investigated. The MCFA combinations were analyzed at six different concentrations ranging from 50-500 µg/mL with North American Type II (P-129) and European Type I (Lelystad) PRRSV. Viral replication was determined as described in experiment 1 (Chapter 2) using FITC labeled IgG anti-PRRSV monoclonal antibody and Log₁₀TCID₅₀/mL was calculated for each concentration. Data were analyzed using the Proc Mixed procedure of SAS. Incubation of MARC-145 cells with MCFA combinations prior to and after inoculation with Type II North American (P129) and Type I European (Lelystad) PRRSV resulted in reduced viral replication at MCFA concentrations of 200-500 µg/mL and was concentration dependent. Reduction of viral replication with MCFA was further evaluated by independently incubating MARC-145 cells or PRRSV. Results indicated that viral replication was reduced when MARC-145 cells were incubated with MCFA and not when PRRSV was incubated with MCFA. In experiment 3 (Chapter 4), 112 mixed sex pigs (PIC 1050 females x PIC 359 sire), weaned at 21 d of age, weighing 7.5 ± 0.68 kg, were used in a 33d PRRSV challenge study. Pigs were blocked by body weight and sex and randomly assigned to one of four treatments in a 2x2 factorial design with pigs receiving 0 or 0.30% MCFA in the diet and placebo or PRRSV inoculation. Following a 5 d adjustment to diets and rooms, pigs were inoculated with either a placebo (sterile PBS) or Type II North American (P129) PRRSV (1 x 10⁵,TCID₅₀/mL) given in 1 mL each intranasal and IM injection. Each room contained 4 pens with 7 pigs per pen and an equal ratio of barrows to gilts within treatment. Diets were formulated to meet or exceed all nutritional requirements (NRC, 2012) and were fed in 4 nursery phases. Feed budgets by phase were 1.13 kg/pig in phase 1, 2.72 kg/pig in phase 2, 6.35 kg/pig in phase 3, and phase 4 fed until the end of the experiment. MCFA (C8:C12) were mixed in a 1:1 ratio (wt:wt), and then mixed with finely ground corn to prepare a premix added to diets at 0.60% to provide 0.30% total MCFA. Control diets used soybean oil mixed with finely ground corn at the same 0.60% inclusion to keep ME levels constant across treatments. Body weights, feed intakes, blood samples, and temperatures were determined or collected on d 0, 3, 7, 10, 14, 21, and 28 post inoculation. Sections of tonsil, lung, and intestines were collected at d 10 post-inoculation from 1 pig per pen and at d 28 from all remaining pigs. Data were analyzed using the PROC Mixed procedure of SAS with pen as the experimental unit for growth and performance measurements and pig as the experimental unit for viral load analysis. Serum viral load confirmed PRRSV was only detectable in challenged pigs. Body weights were not different (<i>P</i> > 0.05) between treatments prior to d 14 post inoculation. Body weights from d 14 to 28 post inoculation were reduced (<i>P</i> < 0.05) in PRRSV infected pigs compared to non-infected pigs. Overall ADG and ADFI were reduced (<i>P </i>< 0.05) for PRRSV infected pigs compared to non-infected pigs by an average of 18 and 28%, respectively. Body temperatures were not different between treatments. Viral load measured in the lung was not different (<i>P </i>> 0.05) between PRRSV infected treatments. Tonsil viral load was not different (<i>P</i> > 0.10) between PRRSV treatments. However, there was a trend (<i>P</i> ≤ 0.10) for an effect of day post inoculation with control-fed, PRRSV-infected pigs having higher viral loads at d 10 post inoculation compared to d 28 post inoculation. Overall, no effects of MCFA on PRRSV viral load or performance were observed during the in-vivo trial. MCFA was effective at reducing viral replication of PRRSV in MARC-145 cells in vitro. However, the results could not be confirmed in the in-vivo experiment. Porcine alveolar macrophages should be used to confirm the in vitro inhibition of PRRSV replication observed in MARC-145 cells. In order to fully understand the application of MCFA to inhibit PRRSV infection in pigs, more studies should be conducted to evaluate the form of MCFA as well as viral inoculation with field strains of PRRSV. </p>

Diarrhoeal disease is considered the second most common cause of morbidity and fourth most common cause of mortality, worldwide. Low-income countries such as those in Africa and Asia bear the greatest burden of gastroenteritis. Diarrhoeal disease affects individuals of all ages, however, children <5 years of age, the immunocompromised and the elderly population ≥65 years of age are most severely affected. The elderly population, particularly immunocompromised patients residing in long-term care facilities represent high-risk groups for gastroenteritis and surveillance of these individuals in South Africa is under-represented. It has been observed that an individual’s fucosyltransferase 2 (FUT2) secretor status has been associated with different degrees of infection by rotaviruses and noroviruses. A total of 1 012 stool specimens from elderly patients were collected over an 18-month surveillance period, of which 340 specimens met the inclusion criteria and were tested. Virus screening was performed using a lyophilised real-time multiplex RT-PCR/PCR screening iv assay testing for norovirus GI and GII, rotavirus, human adenovirus, human astrovirus and sapovirus. Careful analysis of the real-time (RT)-PCR amplification plots and export data identified 50 viruses in 40 patient specimens. Seven norovirus GI/GII dual-infections were observed and three co-infections were identified, each with an astrovirus accompanying infection by a rotavirus, sapovirus and human adenovirus. FUT2 genotyping was performed to acquire the secretor status for all the rotavirus- and norovirus-positive individuals. The real-time TaqMan® SNP Genotyping Assay was inconsistent in amplifying the SNP in the FUT2 gene from stool-extracted DNA of elderly patients, and therefore an alternative, conventional genotyping PCR approach was performed. This approach was successful in acquiring the secretor status of 14/21 patient specimens. A total of 10 homozygous secretors, three heterozygous secretors and one homozygous non-secretor were identified. Virus-positive specimens identified in this study were genotyped and subjected to phylogenetic analysis. Overall, 14 norovirus- GI, 12 norovirus- GII, 10 sapovirus-, six human adenovirus-, six human astrovirus- and two rotavirus-positives were identified. From the 14 norovirus GI positives, three polymerase regions and two capsid regions were successfully genotyped. The polymerase strains all belonged to genotype GI.P1 and the capsid sequences were all GI.1 genotypes. Only one virus was successfully dual-genotyped as GI.1[P1]. For norovirus GII, a total of nine polymerase and nine capsid strains were genotyped successfully. All the polymerase sequences belonged to the GII.P31 genotype and eight capsid sequences identified as GII.4 Sydney 2012 strains, with a single GII.6 genotype identified. Three of the six adenovirus positives were genotyped, of which one strain grouped into species C and two strains grouped into species D, and shared a clade with a type 17 reference strain. Human astrovirus dual-genotyping was successful for three strains, which identified as type 2 for both the serine protease and capsid types. A single rotavirus strain was genotyped for VP4 and VP7 and identified as G9P[6]. Only two sapovirus-positives were successfully genotyped as GI.2 and GIV.1, respectively. This study highlights the epidemiological importance of clinical surveillance in the geriatric population, acting as a cornerstone for future studies in South Africa. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2019. / The dissertation is under embargo until September 2022. / National Research Foundation / Poliomyelitis Research Foundation / Medical Virology / MSc (Medical Virology) / Restricted

La pandemia por COVID-19 representa un reto para los servicios de salud y ha producido un colapso de estos en muchas partes del mundo y el cirujano dentista no ha estado al margen de esta situación y ha jugado un rol importante, produciéndose una serie de cambios y reforzando otros, como las medidas de bioseguridad, para una atención estomatológica segura. En la siguiente revisión de la literatura, se muestra la preponderancia que tiene la cavidad bucal en la enfermedad del COVID-19 tanto en la infectividad y severidad en la presentación de los síntomas. así mismo se discute el riesgo de la labor que cumple el cirujano dentista y cómo nuestra profesión puede ayudar a mitigar los contagios a través de la disminución de la carga viral con el uso de determinados productos tienen una sólida base científica con investigaciones realizadas para demostrar esta acción.

An investigation was undertaken in some detail of three virus strains of insect pests of agricultural importance, viz. a nuclear-polyhedrosis virus of the lucerne caterpillar, Colias electo, and a granulosis virus of the bollworm, Heliothis armigera, both found in South Africa, and a nuclear-polyhedrosis virus of the bollworm, Heliothis zea, isolated in America, with a view to ascertaining a knowledge of some of the fundamental properties and basic biology of these infective agents. On the basis of the information gained the viruses could be differentiated and their broad classification was established. The morphology of the polyhedra, capsules and virus particles observed by light and electron microscopy has been completed and measurements of the viral elements have been made. Some biophysical properties of the virus particles and their inclusion bodies were recorded, i.e. their resistance to chemical and physical treatments and their relative mobility in an electric field under standard conditions. Observations were made on procedures which brought about varying degrees of purification and concentration of the virus particles from putrefying larvae and the most successful of these were found to be reproducible. They involved the purification of the inclusion bodies and their digestion by weak alkali to release the virus particles. Both preparations of the viral elements were further purified by zone electrophoresis in sucrose density gradients. Some information was gathered on the mode of transmission of the infection from insect to insect by contact or cannibalism, from one generation to the next through the eggs, and particularly from one area to another by virus survival in avian faeces. The incidence and rate of the infection in the larvae was increased by environmental changes such as raising the temperature and also to some extent by spraying with a suspension of endospores of Bacillus thuringiensis. Exposure to other stress conditions was not successful in initiating a fatal infection in the insects. Of particular interest, however, was the observation that by injecting a 'foreign' virus a fatal infection was induced by activation of a native occult virus in the larvae of the silkworm, Bornbyx mori. In the context of the possible application of these infective agents to future methods of biological control of economically disastrous pests, these preliminary experiments were not unrewarding.

Se realizó un estudio epidemiológico de Diarrea viral bovina (DVB) y neosporosis en 425 vacas en producción de 37 hatos del Valle del Mantaro-Región Junín, Perú, mediante ELISA e identificando sus factores de riesgo aplicando una encuesta. Para DVB se encontraron prevalencias: muestral 66.33%, predial 97.3% (36/37) y promedio/hato 64.78%, Concepción tuvo las prevalencias promedio/hato y muestral más altas, luego Jauja, Chupaca y Huancayo; la prevalencia de animales persistentemente infectados (PI) con DVB fue 5.8%. Las prevalencias de neosporosis fueron: muestral 15.29%, predial 56.76% (21/37) y promedio/hato 12.76%; Huancayo registró la más alta prevalencia muestral y Concepción la prevalencia promedio/hato más alta, Jauja registro los menores valores para ambas. Fueron factores de riesgo para DVB: número servicios/preñez (2-3 OR: 3.9 y >3 OR: 32); vacas repetidoras (OR: 5.5); crianza con cuyes (OR: 5.8); animales silvestres (OR: 3.75); hato abierto (OR: 2.58); ubicación en Concepción (OR: 3.5) y Jauja (OR: 3); siendo factores de protección la crianza con alpacas (OR: 0.08) y aves (OR: 0.2), existiendo asociación positiva entre altas prevalencias DVB y presencia de vacas repetidoras, abortos y nacimientos anómalos y el uso de agua de acequias para bebida. Factores de riesgo para neosporosis fueron: retención de placenta/metritis (> 3) (OR: 11.7); reproducción artificial (IA o IA/MN) (OR:5.07); crianza con aves (OR: 4.7); alta presencia de ratas (OR: 6.8); uso de puquiales (OR: 3.7) y colindancia con centros poblados (OR: 2.8); fue factor de protección el uso de instalaciones adecuadas (OR: 0.26); hubo asociación negativa entre valores altos de seroprevalencia de neosporosis y presencia de vacas repetidoras, y asociación positiva entre casos de abortos y nacimientos anómalos con uso de agua de acequias. La coinfección (DVB y neosporosis) fue del 8.9% y el riesgo epidemiológico exógeno fue más importante para neosporosis mientras el riesgo epidemiológico endógeno para DVB. Palabras clave: Diarrea viral bovina, neosporosis, seroprevalencia, persistentemente infectado, factores de riesgo / --- It was conducted an epidemiological study of bovine viral diarrhea (BVD) and neosporosis in 425 cows in production of 37 herds of Mantaro’s Valley-Junín Region, Peru, by ELISA and identifying their risk factors by applying an epidemiological survey. The values of DVB prevalences were: sample 66.33%, praedial 97.3% (36/37) and average/herd 64.78%, Concepción had prevalences average/herd and sample higher, then Jauja, Chupaca and Huancayo; the prevalence of animals persistently infected (PI) with DVB was 5.8%. The values of neosporosis prevalences were: sample 15.29%, praedial 56.76% (21/37) and average/herd 12.76%; Huancayo had the highest sample prevalence and Concepción the highest average/herd prevalence, Jauja registered lower values for both. Were risk factors for DVB: number services/pregnancy (2-3 OR: 3.9 and > 3 OR: 32); repeater cows (OR: 5.5); breeding with guinea pigs (OR: 5.8); wild animals (OR: 3.75); open herd (OR: 2.58); location in Conception (OR: 3.5) and Jauja (OR: 3); being protective factors the breeding with alpaca (OR: 0.08) and birds (OR: 0.2), existing positive association between high prevalences DVB and the presence of repetitive cows, abortions and abnormal births as well as the use of irrigation ditch as drinking water. Were risk factors for neosporosis: placenta retention/metritis (> 3) (OR: 11.7); artificial reproduction (IA or IA/MN) (07 OR:5.), breeding birds (OR: 4.7); high presence of rats (OR: 6.8); use of springs (OR: 3.7) and close to the herd to the towns (OR: 2.8); was protection factor the use of adecuate cattle facilities (OR: 0.26); there was a negative association between seroprevalence of neosporosis and presence of cows repeaters high values , and positive association between cases of abortions and abnormal births with use of water from irrigation ditches. (DVB and neosporosis) co-infection was 8.9% and the exogenous epidemiological risk was most important for neosporosis as endogenous epidemiological risk for DVB. Key words: bovine viral diarrhea, neosporosis, seroprevalence, persistently infected, risk factors / Tesis

Cell wall is the major barrier in the delivery of biomolecules such as nucleic acids into the plant cell. Biological (bacteria or viruses) and biolistic (particle-based) methods are used to deliver nucleic acids into the plant cell. However, these methods have significant limitations when it comes to species range, scalability, and field assays. In this work, we report the use of layered double hydroxide (LDH) topically applied to deliver RNA molecules into the plant cell. LDH were assembled by methanol-based co-precipitation of magnesium and aluminum nitrate solution with sodium hydroxide and finally dispersed in deionized water. The assembled LDH were physically characterized by AFM, zeta-sizer and their binding to RNA was confirmed by gel electrophoresis. LDH complexed with double stranded RNA (dsRNA) was topically applied to Nicotiana benthamiana leaves. As a model system, virus specific dsRNA-LDH complexes were used to activate cellular RNAi machinery against Tomato Yellow leaf Curl Virus (TYLCV) in N. benthamiana plants. Our results demonstrated that topical application of the TYLCV specific dsRNA-LDH complexes reduce viral genome accumulation and viral symptoms development. Similarly, dsRNA-LDH protected plants produce typical leaves, flowers, and seeds, confirming efficient virus resistance compared unprotected TYLCV infected plants. Topical application and noninvasive delivery of nucleic acid has several advantages, as these methods are specie independent, easy to scale up, applied with low-pressure spray, requires no tissue culture and no sophisticated equipment. The LDH based noninvasive delivery of nucleic acids has the capability to overcome the cell wall barrier limitations and will open new opportunities to exploit the full potential of cellular machinery to produce resilient plants and insure sustainable food production.

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