Browsing by Author "Lamothe Reyes, Yaneysis"

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    Evaluación de la capacidad de protección temprana de la variante Thiverval del virus de la Peste Porcina clásica y del rol de CD46 durante su entrada a la célula.
    (Universidad de Concepción., 2023) Lamothe Reyes, Yaneysis; Sánchez Ramos, Oliberto; Figueroa Yévenes, Maximiliano
    Classical Swine Fever is a multisystemic viral disease that exclusively affects pigs and generates significant economic losses. Its causative agent, the Classical Swine Fever virus, is a virus that exposes in its envelope the Erns, E1 and E2 proteins, which are involved in the initial union and entry into the cell. However, it is the interaction of E2 with one or more receptors on the cell membrane that mediates endocytosis of the virus. According to the evidence, the CD46 membrane protein is involved in this process, although it has been observed that some viral variants, after adaptation to cell culture, develop infection mechanisms independent of this protein. It is unknown if these variants use different mechanisms to enter the cell and if there are modifications in the E2 protein that could account for this phenomenon. Vaccination is a viable alternative to contain the spread of the virus and eventually eradicate it from those regions of the world where it remains endemic. In these contexts, modified live vaccines are widely used, based on virus variants that have been attenuated after numerous passages in culture. However, the appearance of variants of low or moderate virulence has been reported in areas where an active vaccination policy is carried out. These new variants favor the permanence of the virus in the swine population and the generation of chronic and persistent forms of the disease. It is believed that the immunological pressure exerted against vaccines used for many years, such as those based on the China-strain, has contributed to this phenomenon. Therefore, there is a need to evaluate new vaccine alternatives that are equally safe and effective in these scenarios. Thiverval is an attenuated variant of the Classical Swine Fever virus approved by the World Organization for Animal Health for vaccination against the disease. However, the safety and efficacy reports after in vivo studies with this variant date back to the 1970s and were obtained with techniques that are outdated to date. In addition, it is unknown whether the variant can be transmitted to non-vaccinated animals, its replication rate in tissues and organs, and its ability to confer clinical and virological protection in less than 7 days after a single dose. Updating the information regarding this variant and evaluating its ability to confer early protection may favor the use of vaccines based on this strain in settings where disease control has been difficult. On the other hand, it is unknown if the mutations that led to the attenuation of this variant generate changes in the interaction with CD46. This knowledge constitutes a useful tool for the potential development of viral variants that can be used as vaccines, using methods that directly modify the virus entry mechanism with possible cross-sectional use in other veterinary diseases. Therefore, the general objective of this research was to evaluate the early protection capacity of the Thiverval variant of the Classical Swine Fever virus and the role of CD46 during its entry into the cell. To assess the protective effect of the vaccine, two groups of animals were immunized with the Thiverval variant 16 days apart, and a contact group and a control group were also included in the study. All the animals were challenged with Margarita, a highly virulent CSFV variant, at 21- or 5-days post immunization (dpi). The results of this study show that the Thiverval variant has a low replication rate in the organism and that it is not transmitted to the contact group. It also induces the expression of IFN-α and the humoral response against E2 and Erns, the main proteins of the virus envelope. Neutralizing antibodies are detected from day 14 post immunization and clinical protection against exacerbated symptoms of the disease is achieved with only 5 dpi. In addition, the formulation controls viral replication after challenge, virologically protecting the animals. The role of CD46 during viral infection was assessed in vitro by pre-incubating cells with dilutions of an anti-CD46 polyclonal serum prior to the addition of Thiverval. A 1/20 dilution did not generate complete inhibition of Thiverval infection, however, when the Margarita variant was used, the infection rate was practically nil. This suggests the existence of CD46-independent entry mechanisms after attenuation. An in-silico analysis of the E2 structure of the Thiverval variant showed how the presence of mutations in the region previously defined as important for the interaction with CD46 could be affecting the interaction with the receptor. The results obtained in this work demonstrate the early protection capacity of the Thiverval variant against infection by the virus, which makes it an attractive alternative for the control of the disease in endemic areas. In addition, this research lays the groundwork for further investigation of whether mutations that affect a virus's initial interaction with its cell-entry receptors could be responsible for its attenuation. This would open the doors to the possibility of artificially creating attenuated variants for vaccine purposes using an approach based on protein structure and altering the interaction of the virus with its natural receptor.
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