Genetic characterization of picobirnaviruses detected in fecal samples from different hosts

Author(s)

Maria Clara Duarte Fregolente Alves

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2010-07-16

Examining board members

Maria Silvia Viccari Gatti; Fabiana Fantinatti Garboggini; Rovilson Gilioli; Dolors Ursula Mehnert; Vera de Souza Gouvêa

• Advisor: Maria Silvia Viccari Gatti

Abstract

Picobirnaviruses (PBV) belong to the Picobirnaviridae family, genus Picobirnavirus, and Human picobirnavirus and Rabbit picobirnavirus are the type species. These small non-enveloped viruses, with two genetic segments of double-stranded RNA, can be found in diarrheic or nondiarrheic fecal samples from different hosts like mammals, including humans, birds and reptiles. PBV infection and its association with gastroenteritis are still unknown, but they are considered opportunistic and emergent pathogens, and their zoonotic potential has also been suggested. Techniques for PBV identification include: polyacrylamide gel electrophoresis (PAGE) and RTPCR. The first one allows characterization of PBV according to the migration pattern of their genomic segments. In the RT-PCR, seven primers' pairs have been designed, including one that allows classification of PBV into genogroups I or II. The aim of this project was the genetic and phylogenetic characterization of PBV identified in fecal samples from different natural hosts by complete and partial sequencing of PBV genomic segments and set up of a conserved region for designing primers able to detect all PBV by RT-PCR. Fecal samples from pigs, rabbits, rats, dogs, snakes, wild rats, capybaras, horses and cattle were analyzed for PBV occurrence. PBV were identified in all studied hosts by PAGE and genogroup I was identified in the majority of them by RT-PCR, except in capybaras and cattle. Genogroup II was not identified. Genogroup I circulation in different hosts suggests that there is no genogroup-host species' specificity. Partial sequencing of small PBV's genomic segment identified in fecal samples from dogs, snake and rats showed homology mainly to human PBV sequences. Coexistence of two or more PBV population in the same host could be determined in fecal samples from horses, pigs, rat, wild rat and rabbit by partial sequencing of small PBV's genomic segment after cloning, suggesting that reassortment may occur in nature, allowing host species jump. These results support the emergent and zoonotic potential of PBV. The heterogeneity found in PBV's nucleotide sequences after cloning suggests the existence of PBV quasispecies in these hosts. The little variation in nucleotide sequences of PBV identified in hosts living in an open environment could be justified by a tendency of less contact among these animals, allowing less viral spread. The classification system used nowadays is cannot be considered appropriated as it doesn't consider the heterogeneity found in PBV's genogroup I sequences. Also, PBV that don't belong to any of the described genogroups, remain with no classification. Therefore, a new standard nomenclature for PBV, based on its host, country and year of identification was proposed. Unfortunately, it was not possible to sequence the complete genome of PBV found in this study. Also, no conserved sequence could be identified for primers' design, which would be capable of standardized PBV detection. Additional studies are ongoing to try to define nucleotide sequences identity percentages for genogroups and/or genotypes classification (AU)