RESUMO
Wood decomposition is a key step of the terrestrial carbon cycle and is of economic importance. It is essentially a microbiological process performed by fungi and to an unknown extent by bacteria. To gain access to the genes expressed by the diverse microbial communities participating in wood decay, we developed an RNA extraction protocol from this recalcitrant material rich in polysaccharides and phenolic compounds. This protocol was implemented on 22 wood samples representing as many tree species from 11 plant families in the Angiosperms and Gymnosperms. RNA was successfully extracted from all samples and converted into cDNAs from which were amplified both fungal and bacterial protein coding genes, including genes encoding hydrolytic enzymes participating in lignocellulose hydrolysis. This protocol applicable to a wide range of decomposing wood types represents a first step towards a metatranscriptomic analysis of wood degradation under natural conditions.
Assuntos
Fungos/enzimologia , Perfilação da Expressão Gênica , Lignina/metabolismo , RNA/isolamento & purificação , Árvores/classificação , DNA Complementar/química , DNA Complementar/genética , Fungos/genética , Hidrólise , RNA/genética , Análise de Sequência de DNA , Árvores/enzimologia , Árvores/genética , Madeira/classificação , Madeira/enzimologia , Madeira/genéticaRESUMO
INTRODUCTION: Viral infections are a frequent cause of disseminated non-suppurative encephalitis in dogs. However, using routine diagnostic methods, the specific virus may remain unknown due to extensive or complete viral clearance or because the virus is unexpected or new. A metatranscriptomics-based approach of combining high-throughput sequencing (HTS) and bioinformatics analysis was used to investigate the viral etiology in archival cases of dogs with non-suppurative encephalitis. In formalin-fixed paraffin embedded (FFPE) brain material from the years 1976 to 2021 a high incidence of tick-borne encephalitis virus (TBEV) was detected. Moreover, canine distemper virus (CDV) was identified without typical demyelinating lesions and canine vesivirus (CaVV) was detected as an unexpected virus associated with non-suppurative encephalitis. We demonstrated the viral presence in brain tissues at the sites of inflammation by immunohistochemistry (IHC) and in situ hybridization (ISH). These results highlight the value of emerging sequencing technologies in veterinary diagnostics and expand our knowledge on the etiologies of encephalitis in dogs.
INTRODUCTION: Les infections virales sont une cause fréquente d'encéphalite non suppurée disséminée chez le chien. Cependant, en utilisant les méthodes de diagnostic de routine, le virus spécifique peut rester inconnu en raison d'une clairance virale importante ou complète ou parce que le virus est inattendu ou nouveau. Une approche métatranscriptomique combinant le séquençage à haut débit et l'analyse bioinformatique a été utilisée pour étudier l'étiologie virale dans des cas archivés de chiens atteints d'encéphalite non suppurée. Une incidence élevée du virus de l'encéphalite à tiques (TBEV) a été détectée dans le matériel cérébral fixé au formol et inclus dans la paraffine (FFPE) des années 1976 à 2021. En outre, le virus de la maladie de Carré (CDV) a été identifié sans lésions démyélinisantes typiques et le vésivirus canin (CaVV) a été détecté comme un virus inattendu associé à une encéphalite non suppurative. Nous avons démontré la présence virale dans les tissus cérébraux au niveau des sites d'inflammation par immunohistochimie (IHC) et hybridation in situ (ISH). Ces résultats soulignent la valeur des technologies de séquençage émergentes dans le diagnostic vétérinaire et élargissent nos connaissances sur les étiologies de l'encéphalite chez les chiens.