No description available.

<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>

No description available.

Productive HIV-1 transcription yields a single ∼­9.2kb RNA. From this ∼9.2kb genomic RNA, greater than 40 different subgenomic mRNAs can be produced through alternative splicing using four 5' splice sites (ss) and seven 3'ss. Splice site utilization is governed by the inherent strength of the splice sites and by several identified cis acting elements. The HIV-1 Vif protein, required to overcome the cellular antiviral factor APOBEC3G, is encoded by a singly-spliced mRNA coupling 5'ss D1 to 3'ss A1. Alternatively, mRNAs spliced at A1 can utilize a downstream 5'ss, D2, resulting in inclusion of non-coding exon 2 in a small percentage of mRNAs. Expression of vif mRNA within infected cells is required but maintained at low levels. The purpose of studies described in this thesis was to identify and characterize elements within the HIV-1 genome regulating vif mRNA splicing. We identified an exonic splicing enhancer (ESE) within the 18nt downstream of HIV-1 3'ss A1, ESEVif. Mutation of ESEVif within the HIV-1 proviral clone pNL4-3 resulted in a dramatic decrease in vif mRNA, Vif protein, and undetectable levels of non-coding exon 2 inclusion. The cellular splicing factor SRp75 was found to selectively bind ESEVif in vitro. ESEVif mutant virus replicated in APOBEC3G-deficient T-cell lines as efficiently as wild-type virus. In APOBEC3G-producing T-cell lines, ESEVif mutant virus replicated to lower levels than wild-type virus. Other studies have identified additional mRNA splicing elements regulating splicing at A1: the downstream 5'ss D2 can promote or repress splicing, a G4 motif downstream of D2 represses splicing, and two ASF/SF2 dependent splicing enhancers, ESEM1 and ESEM2, promote splicing. Mutational analysis described in this thesis determined that loss of both ESEVif and the G4 motif resulted in wild-type levels of splicing at A1. Mutations of each identified ESE influenced vif mRNA splicing in the order ESEVif>ESEM2>ESEM1. The data presented in this thesis support a model of vif mRNA splicing regulation in which exon 2 ESE act to overcome the negative G4 motif insuring sufficient levels of Vif production for efficient replication in the presence of APOBEC3G.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

Positive-strand RNAviruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Os herpesvírus são normalmente adaptados a um único grupo de hospedeiros, e esta associação parasita-hospedeiro está ligada à sua seleção e coevolução. Estes agentes podem causar infecções latentes, onde normalmente o vírus não se replica. Durante o ciclo lítico, no entanto, outras células são infectadas e liberam partículas virais capazes de infectar outros indivíduos. O CFPHV (Chelonid fibropapilloma-associated herpesvirus) tem sido apontado como principal agente infeccioso ligado a fibropapilomatose em tartarugas-marinhas. A doença caracteriza-se por uma proliferação cutânea benigna mas que, dependendo da sua severidade, pode comprometer a sobrevivência do indivíduo afetado, sendo por isso apontada como importante ameaça a conservação de tartarugas-marinhas, particularmente de tartarugas verdes (Chelonia mydas), a principal espécie acometida pela doença. Alguns aspectos da biologia do CFPHV e sua relação com as tartarugas verdes foram estudados no presente trabalho. Primeiramente, a capacidade deste agente em se disseminar pelo ambiente e infectar outros indivíduos, e as possíveis vias envolvidas nesta dispersão. Em seguida, avaliou-se os possíveis tecidos em que o herpesvírus pode estabelecer a infecção latente. Por fim, determinou-se a prevalência de indivíduos de Chelonia mydas infectados pelo CFPHV em duas áreas de alimentação (Ubatuba-SP e Vitória-ES) e em uma áreas mista - de alimentação e reprodução (Fernando de Noronha-PE). No primeiro estudo, observou-se que a prevalência de CFPHV nas amostras de secreções de Chelonia mydas variou entre 0%, no Espírito Santo, a 25%, em São Paulo. Os haplótipos afetados foram CMA-3 e CMA-8, e a variante viral encontrada não havia sido detectada anteriormente no Brasil, mas possui elevada similaridade com vírus provenientes do Golfo da Guiné e de Porto Rico. Os resultados sugerem que estes vírus podem ser transmitidos por secreções e também circular entre diferentes regiões. No segundo estudo, detectou-se a presença de CFPHV no cérebro de 5 animais necropsiados e também na pele e em lesões fibropapilomatosas. Em um dos animais foi detectada a presença de uma única variante de CFPHV no cérebro, pele e tumores. Esta variante ainda não havia sido detectada no Brasil e apresentou 100% de identidade com a variante detectada nas secreções. Para avaliar a relação entre haplótipos e variantes virais, o terceiro estudo determinou a prevalência de CFPHV em pele e tumores de 136 indivíduos - 9,56% de indivíduos sadios apresentavam o agente em tecido epitelial e 45,58% dos animais foram positivos para CFPHV, quando considerados também animais com fibropapilomatose. Duas novas variantes de herpesvírus foram encontradas: Var. 7, em Ubatuba-SP e Vitória-ES e Var. 8, em Vitória-ES. Não houve associação entre uma variante viral e um haplótipo. Os resultados observados permitem apontar que o CFPHV pode estabelecer infecções latentes; o vírus pode \"migrar\" entre diferentes regiões, junto com seus hospedeiros; partículas virais podem ser liberadas por secreções; duas novas variantes foram identificadas. Altas taxas de substituição de nucleotídeos em CFPHV podem indicar o surgimento das variantes destas áreas, mas a alta similaridade entre as variantes detectadas e àquelas de Porto Rico e Golfo da Guiné sugerem também a entrada de novas variantes na costa brasileira. / Herpesviruses are usually adapted to a single group of hosts, and this host-parasite association is linked to its selection and co-evolution. These agents can cause latent infections, where the virus usually does not replicate. During the lytic cycle, however, other cells are infected and release viral particles capable of infecting other individuals.The CFPHV (Chelonid fibropapilloma-associated herpesviru) has been indicated as the main infectious agent linked to fibropapillomatosis on sea turtles. The disease is characterized by a benign skin proliferation, but, depending on its severity, can compromise the survival of the affected individual, therefore considered an important threat to the conservation of sea turtles, especially green turtles (Chelonia mydas), the main species affected by the disease. Some aspects of the CFPHV biology and its relation to green turtles were studied in this work. Firstly, the ability of this agent to spread in the environment and infect other individuals, and the possible pathways involved in this dispersion. Then, potential tissues wherein the herpesvirus can establish latent infection were assessed. Finally, we determined the prevalence of Chelonia mydas individuals infected by CFPHV in two feeding areas (Ubatuba-SP and Vitória-ES) and in a mixed area of feeding and reproduction (Fernando de Noronha-PE). In the first study, it was observed that the prevalence of CFPHV in samples of Chelonia mydas secretions ranged from 0% in Espírito Santo, to 25% in São Paulo. Affected haplotypes were CM-A3 and CM-A8, and viral variant found had not been previously detected in Brazil, but it is significantly similar to viruses found in the Gulf of Guinea and Puerto Rico. The results suggest that these viruses can be transmitted by secretions and can also circulate among different regions. Considering the low maintenance of the agent within the environment, they are probably brought by individuals with the latent virus, being capable of releasing viral particles during the herpesvirus replication cycle. In the second study, the presence of CFPHV was detected inside the brain of 5 necropsied animals, besides the detection of the virus on the skin and fibropapillomatosis lesions. In one of the animals, it was possible to characterize the CFPHV and the presence of a single viral variant inside the brain, tumors and on the skin of the same animal was detected. This variant had not yet been detected in Brazil and showed 100% identity with the variant detected in secretions. These results indicate that the virus may establish a latent infection in nerve tissue. To evaluate the relationship between haplotypes and viral variants, the third study determined the prevalence of CFPHV on skin and tumors of 136 individuals - 9.56% of healthy individuals showed the agent in epithelial tissue and 45.58% of the animals were positive for CFPHV, when also considered animals with fibropapillomatosis. Two new variants of the herpesvirus were found, Var. 7 in Ubatuba-SP and Vitória-ES and Var. 8 only in Vitória-ES. C. mydas individuals of different haplotypes were infected, and there was no association between a viral variant and a haplotype. The observed results permitted to point that CFPHV can establish latent infections in Chelonia mydas; the virus can \"migrate\" among different regions, along with its hosts; viral particles can be released by secretion; viral variants previously detected were not found in these areas, but two new variants were detected. The high nucleotide substitution rates observed in CFPHV may indicate the emergence of these variants in these areas, but the high similarity among the detected variants and those identified in Puerto Rico and Gulf of Guinea also suggest the entry of new variants into the Brazilian coast.

Les bracovirus forment une symbiose avec les guêpes parasitoïdes, demeurant dans leur génome et produits uniquement dans leurs ovaires. Nous avons caractérisé comment les cercles d’ADNdb contenu dans les particules étaient amplifiés depuis leur forme provirale avant leur encapsidation.Nous avons montré que le site d’intégration du génome viral est conservé chez les bracovirus et organisé dans le génome de la guêpe en un macrolocus regroupant la majorité des segments proviraux et 7 loci isolés. Nous avons mis en évidence 12 unités de réplication (UR) et que les 9 gènes viraux du cluster nudiviral étaient amplifiés sur une UR sans être encapsidés. Nous avons identifié des concatémères tête-tête et queue-queue comme étant les intermédiaires de réplication des UR, caractéristiques d’une réplication linéaire du génome viral. Enfin, nous avons montré que l’ADN polymérase B2 appartenait à un élément Maverick. L’absence de gènes viraux de la réplication du génome viral semble indiquer que la machinerie réplicative cellulaire serait impliquée. Il reste maintenant à mettre en évidence les différents facteurs cellulaires participant à l’amplification du génome viral. / Bracovirus form a symbiosis with parasitoid wasp, remaining in their genome and products only in their ovaries. We characterized how packaged dsDNA circles were amplified from their proviral genome before packaging in viral particles.We showed that viral genome integration site is conserved in bracovirus and organized in the wasp genome in a macrolocus where the majority of proviral segments was found and 7 isolated loci. We showed 12 replication units (UR) and the 9 nudiviral genes from cluster were amplified on a UR without being packaged. We identified concatemers head-head and tail-tail as the replication intermediates of UR, indicating a linear replication of the viral genome. Finally, we showed that DNA polymerase B2 belonged to a Maverick element.The absence of viral gene involved in the genome replication suggests that the cellular replication machinery is involved. It remains to highlight the different cellular factors involved in the amplification of the viral genome.

Epstein-Barr virus (EBV) BKRF4 gene product is a tegument protein encoded by a gene with no sequence homology outside of the gamma subfamily of Herpesviridae. Its positional homologs are necessary for an efficient viral lytic program, in particular viral progeny egress and primary infection. To characterize BKRF4 in this regard, EBV recombinant viruses deficient for BKRF4 were developed using site-directed mutagenesis and a bacterial artificial chromosome (BAC)-based recombineering system. Stable human embryonic kidney (HEK) 293 cell lines containing these genomes were generated and the phenotypes of these mutants were analyzed following stimulation of the viral lytic cycle. During the lytic program, BKRF4-null cell lines showed decreased protein expression of various EBV lytic genes that were analyzed using immunostaining and flow cytometry. Reduced amounts of extracellular viral progeny were observed when quantified by real-time PCR and infectivity assays as compared to wild type. These findings suggest an active role of BKRF4 in EBV infection, possibly in viral egress.

Os herpesvírus são normalmente adaptados a um único grupo de hospedeiros, e esta associação parasita-hospedeiro está ligada à sua seleção e coevolução. Estes agentes podem causar infecções latentes, onde normalmente o vírus não se replica. Durante o ciclo lítico, no entanto, outras células são infectadas e liberam partículas virais capazes de infectar outros indivíduos. O CFPHV (Chelonid fibropapilloma-associated herpesvirus) tem sido apontado como principal agente infeccioso ligado a fibropapilomatose em tartarugas-marinhas. A doença caracteriza-se por uma proliferação cutânea benigna mas que, dependendo da sua severidade, pode comprometer a sobrevivência do indivíduo afetado, sendo por isso apontada como importante ameaça a conservação de tartarugas-marinhas, particularmente de tartarugas verdes (Chelonia mydas), a principal espécie acometida pela doença. Alguns aspectos da biologia do CFPHV e sua relação com as tartarugas verdes foram estudados no presente trabalho. Primeiramente, a capacidade deste agente em se disseminar pelo ambiente e infectar outros indivíduos, e as possíveis vias envolvidas nesta dispersão. Em seguida, avaliou-se os possíveis tecidos em que o herpesvírus pode estabelecer a infecção latente. Por fim, determinou-se a prevalência de indivíduos de Chelonia mydas infectados pelo CFPHV em duas áreas de alimentação (Ubatuba-SP e Vitória-ES) e em uma áreas mista - de alimentação e reprodução (Fernando de Noronha-PE). No primeiro estudo, observou-se que a prevalência de CFPHV nas amostras de secreções de Chelonia mydas variou entre 0%, no Espírito Santo, a 25%, em São Paulo. Os haplótipos afetados foram CMA-3 e CMA-8, e a variante viral encontrada não havia sido detectada anteriormente no Brasil, mas possui elevada similaridade com vírus provenientes do Golfo da Guiné e de Porto Rico. Os resultados sugerem que estes vírus podem ser transmitidos por secreções e também circular entre diferentes regiões. No segundo estudo, detectou-se a presença de CFPHV no cérebro de 5 animais necropsiados e também na pele e em lesões fibropapilomatosas. Em um dos animais foi detectada a presença de uma única variante de CFPHV no cérebro, pele e tumores. Esta variante ainda não havia sido detectada no Brasil e apresentou 100% de identidade com a variante detectada nas secreções. Para avaliar a relação entre haplótipos e variantes virais, o terceiro estudo determinou a prevalência de CFPHV em pele e tumores de 136 indivíduos - 9,56% de indivíduos sadios apresentavam o agente em tecido epitelial e 45,58% dos animais foram positivos para CFPHV, quando considerados também animais com fibropapilomatose. Duas novas variantes de herpesvírus foram encontradas: Var. 7, em Ubatuba-SP e Vitória-ES e Var. 8, em Vitória-ES. Não houve associação entre uma variante viral e um haplótipo. Os resultados observados permitem apontar que o CFPHV pode estabelecer infecções latentes; o vírus pode \"migrar\" entre diferentes regiões, junto com seus hospedeiros; partículas virais podem ser liberadas por secreções; duas novas variantes foram identificadas. Altas taxas de substituição de nucleotídeos em CFPHV podem indicar o surgimento das variantes destas áreas, mas a alta similaridade entre as variantes detectadas e àquelas de Porto Rico e Golfo da Guiné sugerem também a entrada de novas variantes na costa brasileira. / Herpesviruses are usually adapted to a single group of hosts, and this host-parasite association is linked to its selection and co-evolution. These agents can cause latent infections, where the virus usually does not replicate. During the lytic cycle, however, other cells are infected and release viral particles capable of infecting other individuals.The CFPHV (Chelonid fibropapilloma-associated herpesviru) has been indicated as the main infectious agent linked to fibropapillomatosis on sea turtles. The disease is characterized by a benign skin proliferation, but, depending on its severity, can compromise the survival of the affected individual, therefore considered an important threat to the conservation of sea turtles, especially green turtles (Chelonia mydas), the main species affected by the disease. Some aspects of the CFPHV biology and its relation to green turtles were studied in this work. Firstly, the ability of this agent to spread in the environment and infect other individuals, and the possible pathways involved in this dispersion. Then, potential tissues wherein the herpesvirus can establish latent infection were assessed. Finally, we determined the prevalence of Chelonia mydas individuals infected by CFPHV in two feeding areas (Ubatuba-SP and Vitória-ES) and in a mixed area of feeding and reproduction (Fernando de Noronha-PE). In the first study, it was observed that the prevalence of CFPHV in samples of Chelonia mydas secretions ranged from 0% in Espírito Santo, to 25% in São Paulo. Affected haplotypes were CM-A3 and CM-A8, and viral variant found had not been previously detected in Brazil, but it is significantly similar to viruses found in the Gulf of Guinea and Puerto Rico. The results suggest that these viruses can be transmitted by secretions and can also circulate among different regions. Considering the low maintenance of the agent within the environment, they are probably brought by individuals with the latent virus, being capable of releasing viral particles during the herpesvirus replication cycle. In the second study, the presence of CFPHV was detected inside the brain of 5 necropsied animals, besides the detection of the virus on the skin and fibropapillomatosis lesions. In one of the animals, it was possible to characterize the CFPHV and the presence of a single viral variant inside the brain, tumors and on the skin of the same animal was detected. This variant had not yet been detected in Brazil and showed 100% identity with the variant detected in secretions. These results indicate that the virus may establish a latent infection in nerve tissue. To evaluate the relationship between haplotypes and viral variants, the third study determined the prevalence of CFPHV on skin and tumors of 136 individuals - 9.56% of healthy individuals showed the agent in epithelial tissue and 45.58% of the animals were positive for CFPHV, when also considered animals with fibropapillomatosis. Two new variants of the herpesvirus were found, Var. 7 in Ubatuba-SP and Vitória-ES and Var. 8 only in Vitória-ES. C. mydas individuals of different haplotypes were infected, and there was no association between a viral variant and a haplotype. The observed results permitted to point that CFPHV can establish latent infections in Chelonia mydas; the virus can \"migrate\" among different regions, along with its hosts; viral particles can be released by secretion; viral variants previously detected were not found in these areas, but two new variants were detected. The high nucleotide substitution rates observed in CFPHV may indicate the emergence of these variants in these areas, but the high similarity among the detected variants and those identified in Puerto Rico and Gulf of Guinea also suggest the entry of new variants into the Brazilian coast.