Vírus vaccínia isolados de equinos: patogenia em modelos animais e análise de genes de virulência

Abstract

Two vaccinia viruses (VACV) genetically and phenotypically divergent were isolated, in a mixed infection, from a horse lesion during an outbreak of vesicular and exanthematous disease in horses in Southern Brazil and termed Pelotas 1 (P1V) and Pelotas 2 (P2V). This thesis describes studies performed to investigate the pathogenesis of P1V and P2V infection in rabbits and guinea pigs, and to analyze the sequence of genes potentially involved in their phenotype. Chapter 1 investigated the dose-dependent susceptibility of rabbits to P1V and P2V after intranasal (IN) inoculation. Groups of weaning rabbits were inoculated with three doses of each VACV isolate (102.5 TCID50, 104.5 TCID50 e 106.5 TCID50/rabbit). The inoculation resulted in severe respiratory distress and death of most inoculated rabbits regardless the viral strain. Clinical signs started three to six days post-inoculation (pi) and culminated in death or euthanasia at days 5 to 10 pi. Viremia was detected in animals of all groups. All rabbits surviving the infection beyond day 9 pi developed neutralizing antibodies. Interstitial pneumonia, necrossupurative bronchopneumonia and diarrhea were observed in animals which died or were euthanized in extremis. These results demonstrate that P1V and P2V are virulent for rabbits and show no apparent differences in phenotype in this species. Chapter 2 describes the investigation of the susceptibility of rabbits to intradermal (ID) inoculation to VACV, in single or mixed infection. All inoculated animals developed skin lesions characterized by hyperemia, papules, vesicles pustules and ulcers. Infectious virus was detected in cutaneous lesions, lungs and intestine of animals that died during acute infection. These results demonstrate that rabbits develop cutaneous disease and systemic infection after P1V and P2V ID inoculation. Apparently, co-infected animals developed lesions more severe than those submitted to single virus infection. In chapter 3, the susceptibility and the potential of transmission of P1V and P2V by guinea pigs were investigated. For that, guinea pigs were inoculated IN with both P1V and P2V (106 TCID50/ml). The guinea pigs did not showed clinical signs but developed viremia, shed virus in secretions and seroconverted to VACV. Nevertheless, the virus was not transmitted to guinea pig sentinels maintained in close contact or when exposed to food and feces contaminated with VACV. In Chapter 4, four genes involved in virus phenotype/virulence (C7L, K2L, N1L e B1R) were submitted to nucleotide sequencing and analysis. A 15 nucleotide (nt) deletion in K2L gene was identified in P2V. The same pattern of nucleotide deletion was also detected in other genogroup 1 Brazilian VACV isolates. Point mutations were identified in K2L, C7L and N1R genes from P2V isolates when compared to P1V and to a standard VACV strain. The molecular analysis of these genes would not allow the establishment of association between the sequences/genotype and phenotype. However, this analysis indicate that the 15 nt deletion in K2L gene may be used as a molecular marker for genogroup 1 Brazilian VACV isolates. In summary, the results obtained in these studies demonstrate: i. P1V and P2V produce systemic and cutaneous disease in rabbits but they do not exhibit evident differences in virulence for rabbits; ii. Guinea pigs are susceptible to mixed P1V an P2V infection but apparently do not effectively transmit the virus; iii. P1V and P2V present some sequence differences in virulence genes and that a 15 nt deletion in K2L gene may be used as a molecular marker to distinguish between VACV genogroups.