Epidemiologia molecular de amostras brasileiras do vírus da doença de Aujeszky

Abstract

Aujeszky´s disease is a disease caused by Suid Herpesvirus 1 (SuHV-1) and responsible for considerable economic losses in the swine industry. The illness is marked by severe clinical signs with high letality in young animals and can establish latent infection in the neuronal ganglia. The first diagnostic in Brazil was done in 1912 in São Paulo (SP). New outbreaks occurred since then with an impact of one million reais per year in Santa Catarina State (SC). It is a notifiable disease since 1934 and an eradication program was approved in 2007. The objective of this work was to use molecular epidemiology as a tool to assist in the study of the outbreaks. Twenty five isolates from south and southeast regions, the standard strain Shope and the vaccine strain Bartha were multiplied in PK-15 cells and submitted to PCR for gE and gC genes. The strain Bartha (sB) only amplified for gC as expected. The amplicons were purified, quantified and sequenced using Big Dye Terminator Kit. Sequences were edited and aligned with Bioedit (v.7.0.5.3) and the phylogenetic trees constructed in MEGA 4.0 using the Neighbor-Joining Method. Sequences for gE were highly conserved and grouped in two major groups (A and B, divided in B1 and B2). A group were formed by Shope, isolates from 1983 and 1984 from Paraná (PR), Minas Gerais(MG) and two strains from SP. Isolates from SC and Rio Grande do Sul (RS) clustered in B1 group. The B2 group had three isolates, one from each southern state from outbreaks occurred in 1983 (SC), 2000 (PR) and 2003 (RS). Phylogenetic tree for gC had higher bootstraps values with almost all isolates in the same groups as in gE tree. Only two strains from RS (years 1954 and 2003) clustered in B2. All other strains from SC and two from SP clustered in B1 group. Strains Shope, Bartha and an isolate from SP (1990) clustered in a fourth and new group. Results showed that Brazilian isolates clustered in four different groups. The most recent outbreaks in PR and RS were caused by strains similar to those occurred in SC since 1984. Two alleles for gE were found and could be related to periods of outbreaks in southern states. The nonsynonymous mutations had no influence in the functional sites for N-glycosilation (N-g) in both genes. gC sequences had more variability, some triplets deletions and additions. None of the six N-g sites or the three heparin-binding domains were affected by the mutations. A hot spot region between nucleotides 535 and 559 resulted in a change from hydrophobic to a hydrophilic region in isolates related to Santa Catarina,Rio Grande do Sul and sB. Despite the many mutations in sB, these changes did not occur in 22-amino-acid N-terminal hydrophic domain. This domain functions as a signal sequence and was previous described that mutations in this region in sB were responsible for the reduction in N-g and maturation of gC, which would affect virulence. All Brazilian isolates, except for one sample from São Paulo, clustered in different clades from the international isolates. Results prove that Brazilian isolates diverge genetically from international strains but do not have significant mutations in amino acid compositon. It also showed a different result for the signal previously described for sB