RESUMEN
Major themes in pathogen evolution are emergence, evolution of virulence, host adaptation and the processes that underlie them. RNA viruses are of particular interest due to their rapid evolution. The in vivo molecular evolution of an RNA plant virus was demonstrated here using a necrotic isolate of cowpea mild mottle virus (CPMMV) and a susceptible soybean genotype submitted to serial inoculations. We show that the virus lost the capacity to cause necrosis after six passages through the host plant. When a severe bottleneck was imposed, virulence reduction occurred in the second passage. The change to milder symptoms had fitness benefits for the virus (higher RNA accumulation) and for its vector, the whitefly Bemisia tabaci. Genetic polymorphisms were highest in ORF1 (viral replicase) and were independent of the symptom pattern. Recombination was a major contributor to this diversity - even with the strong genetic bottleneck, recombination events and hot spots were detected within ORF1. Virulence reduction was associated with different sites in ORF1 associated to recombination events in both experiments. Overall, the results demonstrate that the reduction in virulence was a consequence of the emergence of new variants, driven by recombination. Besides providing details of the evolutionary mechanisms behind a reduction in virulence and its effect under viral and vector fitness, we propose that this recombination-driven switch in virulence allows the pathogen to rapidly adapt to a new host and, potentially, switch back.
Asunto(s)
Carlavirus , Hemípteros , Virus ARN , Vigna , Animales , Carlavirus/genética , Virus ADN/genética , Hemípteros/genética , ARN , Virus ARN/genética , Recombinación Genética , Virulencia/genéticaRESUMEN
Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.
Asunto(s)
Carlavirus/genética , ADN Complementario , Genoma Viral , Clonación Molecular , Orden Génico , Sistemas de Lectura Abierta , Enfermedades de las Plantas/virología , Glycine max/virologíaRESUMEN
The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.
Asunto(s)
Bovinos , Sustitutos de la Leche Humana , Técnicas y Procedimientos Diagnósticos , Técnicas In Vitro , Infecciones por Mycobacterium , Mycobacterium avium/aislamiento & purificación , Paratuberculosis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Bovinos , Muestras de Alimentos , MétodosRESUMEN
The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.
RESUMEN
In the current study, the virulence factors in Escherichia coli isolates from bovine mastitis were investigated, and the connection between these factors and infection was evaluated using phenotypic and genotypic analyses. Twenty-seven E. coli isolates were analyzed, and 2 were shown to produce verotoxin. All isolates had the ability to produce biofilms, although at different levels. One isolate was found to be sensitive to the bactericidal activity of bovine serum, 11 were intermediate, and 15 were resistant. Some isolates showed resistance to trimethoprim sulfa (9) and ampicillin (4), intermediate resistance to neomycin (1) and trimethoprim sulfa (5), and simultaneous resistance to ampicillin and trimethoprim sulfa (4). The fimH gene was found in all isolates and was associated with other virulence markers: pap (1), stb (8), cs31a (3), stb and vt2 (2), cs31a and stb (3), east1 and kps (1), stb and east1 (1), cs31a and east1 (1), and cs31a, stb, pap, and iucD (1). Serogroups were determined for 3 isolates: O93:H4, O83:H19, and O15:H11. Phylogenetic analysis showed that 23 isolates belonged to group A and 4 belonged to B1. The findings revealed that these E. coli isolates are opportunistic pathogens with different virulence factors. The results indicate that the pathogenicity route of E. coli in bovine mastitis is not a consequence of 1 specific virulence factor.