Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity: Implication for epidemiological surveillance in endemic areas.

Classical swine fever (CSF), caused by CSF virus (CSFV), is considered one of the most important infectious diseases with devasting consequences for the pig industry. Recent reports describe the emergence of new CSFV strains resulting from the action of positive selection pressure, due mainly to the bottleneck effect generated by ineffective vaccination. Even though a decrease in the genetic diversity of the positively selected CSFV strains has been observed by several research groups, there is little information about the effect of this selective force on the virulence degree, antigenicity and pathogenicity of this type of strains. Hence, the aim of the current study was to determine the effect of the positive selection pressure on these three parameters of CSFV strains, emerged as result of the bottleneck effects induced by improper vaccination in a CSF-endemic area. Moreover, the effect of the positively selected strains on the epidemiological surveillance system was assessed. By the combination of in vitro, in vivo and immunoinformatic approaches, we revealed that the action of the positive selection pressure induces a decrease in virulence and alteration in pathogenicity and antigenicity. However, we also noted that the evolutionary process of CSFV, especially in segregated microenvironments, could contribute to the gain-fitness event, restoring the highly virulent pattern of the circulating strains. Besides, we denoted that the presence of low virulent strains selected by bottleneck effect after inefficient vaccination can lead to a relevant challenge for the epidemiological surveillance of CSF, contributing to under-reports of the disease, favouring the perpetuation of the virus in the field. In this study, B-cell and CTL epitopes on the E2 3D-structure model were also identified. Thus, the current study provides novel and significant insights into variation in virulence, pathogenesis and antigenicity experienced by CSFV strains after the positive selection pressure effect.

Positive selection pressure on the B/C domains of the E2-gene of classical swine fever virus in endemic areas under C-strain vaccination.

In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. Interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. This study aimed to assess positive selection pressure acting on partial E2 gene of CSF viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (CSFV) evolution in swine populations under regular vaccination. Selection pressure analysis were performed to detect positive selection acting on a particular lineage as well as among sites of the E2-B/C-domain of CSFV nucleotide sequences, reported in a previous study and in the present work, several models, available in the CODEML module of PAML 4.3, were used. In addition, a representative Cuban CSF isolate was assessed in an experimental infection trial for their clinical virulence in order to expand the knowledge regarding CSF viruses circulating in pig populations. The viral genomes sequenced in this study were grouped in a defined cluster within the genotype 1.2, as it has been reported previously for Cuban CSF viruses. The selection pressure analysis didn't find evidence of positive selection (dN/dS of>1) along any branch. The positive selective pressure analysis estimated six new sites under positive selection on E2 partial gene analysed. Besides, the clinical manifestations of the CSF-disease were related mainly to a mild course of the illness. The high number of positively selected sites suggests that these changes could be associated to viral evasion of the host-immune response. These observations highlight a possible association between escape viral variants and the alterations observed in the virulence and pathogenesis of the virus. Therefore, while the vaccination programs have not led to a genotype change, alterations in virulence were suggested to arise.

Classical swine fever virus E2 glycoprotein antigen produced in adenovirally transduced PK-15 cells confers complete protection in pigs upon viral challenge.

E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.

Situation of classical swine fever and the epidemiologic and ecologic aspects affecting its distribution in the American continent.

Classical swine fever (CSF) is a viral transboundary animal disease that is highly contagious among domestic and wild pigs, such as boars and peccaries. Today, far from being what was classically described historically, the disease is characterized as having a varied clinical picture, and its diagnosis depends on resorting to proper sample collection and prompt dispatch to a laboratory that can employ several techniques to obtain a definitive diagnosis. Laboratory findings should be complemented with a field analysis of the occurrence of disease to have a better understanding of its epidemiology. The disease is still present in various regions and countries of Latin America and the Caribbean, thus hindering production, trade, and the livestock economy in the region. Consequently, it is among the diseases included in List A of the Office International des Epizooties (OIE). Currently, there are epidemiologic and ecologic aspects that characterize its geographical distribution in the region such as: continued trends in the demand for pork and pork products; an increase in swine investment with low production costs which are able to compete advantageously in international markets; the convention of associating CSF in the syndrome of "swine hemorrhagic diseases" owing to the historical description of its acute presentation and not to the new and more frequent subacute presentations or the diseases with which it may be confused (notably, porcine reproductive and respiratory syndrome and porcine dermopathic nephropathy syndrome, among others); dissemination of the virus through asymptomatic hosts such as piglets infected in utero; frequent lack of quality control and registration of vaccines and vaccinations; feeding of swine with contaminated food waste (swill); the common practice of smuggling animals and by-products across borders; the backyard family production system or extensive open field methods of swine rearing with minimal input in care and feeding; poor understanding of the epidemiologic role that boars and peccaries could have in the transmission and maintenance of the disease in the Americas; and new procedures in animal welfare that some countries are adopting for the production, transport, and slaughter of domestic animals. Consequently, many countries (i.e., Canada, USA, Chile, Belize, Costa Rica, Panama, and Mexico, where 13 of 32 States are disease free) have given priority to the control and progressive eradication of CSF. In other parts of the Americas, the disease appears under control, as is the case of the five countries of the Andean Region and the 12 northern States of Brazil. In South America, Chile, Uruguay and 13 States in Brazil are disease free. Argentina has mounted a national campaign and is in the process of eradicating the disease. No recent information on its presence or distribution in Paraguay is available. With no master strategy to harmoniously progress in the control and eradication of the disease, 17 countries of the region, jointly led by the Food and Agriculture Organization of the United Nations, developed the Continental Plan for the Eradication of CSF whose objective is expected to be reached by 2020.

Inactivation of classical swine fever virus: association of hydrostatic pressure and ultraviolet irradiation.

Reversible pressure-induced disassembly of several viruses has suggested the idea of using hydrostatic pressure to suppress virus infectivity. In this study, the effects of high hydrostatic pressure and ultraviolet (UV) irradiation were investigated on classical swine fever virus (CSFV) in an attempt to eliminate residual infectivity. The structural modifications were followed by intrinsic fluorescence and biological activity assays. The kinetics of CSFV inactivation showed that pressure-induced inactivation was not enough to eliminate viral infectivity. However, when pressure was applied in association with UV irradiation no infectious focus was observed. The application of these two methods against CSFV can be an attractive inactivation strategy for the development of a vaccine.

Characterization of lesions caused by a South American virulent isolate ('Quillota') of the hog cholera virus.

In this study, macroscopic and histopathological lesions produced by a virulent South American isolate ('Quillota') of hog cholera virus were studied. The virus was inoculated in doses of 10(5)TCID50 in each of 35 pigs of 20 kg live weight. The animals were slaughtered from 4 to 18 days post-inoculation. The presence of virus antigens in lymphatic tissue was confirmed by both direct immunofluorescence and Avidin-Biotin-Peroxidase techniques in formalin-embedded tissue samples. Histological sections were stained with haematoxylin-eosin and Mallory's phosphotungstic acid haematoxylin methods. The 'Quillota' isolate used in this study caused a disease characterized by vascular lesions (splenic infarcts, haemorrhages in the lymph nodes and the urinary system and disseminated microthrombosis), and necrosis of lymphocytes, particularly in the B-areas of the lymphoid organs, lesions that are characteristic of the acute form of the disease. Other lesions observed were a non-purulent meningoencephalitis, the necrosis of the epithelial cells of tonsils, the presence of fibrin nets in the red pulp and a marked thickening of the alveolar septa.

Seguridad de la vacuna Cerdovirac como preventiva de la peste porcina clásica / Safety of the Cerdovirac vaccine as preventive of classical hog swine plague

La peste porcina clásica es una enfermedad viral, específica de los cerdos, la cual sólo puede ser prevenida por vacunación. Este trabajo reporta sobre la seguridad de administrar virus vivo modificado por lapinización como profiláctico contra infecciones experimentales homólogas. De las mediciones de temperatura, antes o después de la vacunación y confrontación, en combinación con los resultados histopatológicos, se reporta la incapacidad de los virus modificados, a diferencia de la cepa virulenta de confrontación, de ocasionar infección patológica visualizada por el aparecimiento de fiebre y microcoagulaciones sistémicas con daños endoteliales de los vasos sanguíneos e infiltración linfoplasmocitaria en los tejidos. A esto se suma la habilidad de estos virus para proteger totalmente contra dosis letales cuando se administran a individuos sanos con pesos alrededor de 9.5 kg o más. La resistencia humoral parece ser importante según los resultados de la administración de suero realizada temprano a los 3 días de la infección; caso contrario, los efectos del virus de confrontación resultaron ser mortalmente irreversibles