Studies on genetic properties of porcine parvoviruses

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses