RESUMO
Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((R)) RT-PCR assay, based on a conserved region in the HA9 gene. The assay was validated with viruses representing different genetic subtypes and other common avian pathogens, and was found specific to H9N2. The real-time RT-PCR assay was compared to RT-PCR, which is in routine diagnostic use. Real-time RT-PCR was found to be more sensitive than RT-PCR by 1.5-2.5 orders of magnitude when testing tracheal swabs directly and by 2-3 orders of magnitude allantoic fluid after AIV propagation in embryonated eggs. Sensitivity was quantified by using 10-fold dilutions of the H9-gene amplification fragment, and real-time RT-PCR was found to be 10(4)-fold more sensitive than RT-PCR. Clinical samples, which included tracheal and cloacal swabs, as well as allantoic fluid, were tested by both methods. By real-time RT-PCR 20% more positive H9N2 samples were detected than by RT-PCR. The real-time RT-PCR assay was found suitable for detection and epidemiological survey not only of Israeli H9N2 viruses, but also for isolates from other parts of the world.
Assuntos
Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Alantoide/virologia , Animais , Galinhas , Cloaca/virologia , Hemaglutininas Virais/genética , Israel , Sensibilidade e Especificidade , Traqueia/virologiaAssuntos
Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/química , Neurotoxinas/toxicidade , Fosfitos/toxicidade , Animais , Neoplasias Encefálicas/patologia , Cromatografia em Gel , Meios de Cultura , Humanos , Masculino , Neuroblastoma/patologia , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
The restriction cleavage sites of the BamHI-B and BamHI-E DNA fragments of several Herpes simplex virus type 1 (HVS-1) strains were mapped. These fragments are situated at the ends of the long unique regions and share homologous sequences in the repeat components (TRL and IRL) of the genome. All the strains analyzed were found to have deletions in the Hpal-P fragment, situated in the BamHI-B fragment. Five strains were further analyzed and the deletions were located in the Smal-A fragment (within the Hpal-P fragment). The BamHI-E fragment of four recombinants (obtained by recombination between the HFEM genome and the BamHI-B fragment or part of it from the HSV-1 F strain) were almost identical but differed from another strain [NIH(LP)]. Comparison of the BamHI-B and the BamHI-E fragments of the same strain revealed that the fragments were not identical in all cases.
Assuntos
DNA Recombinante/análise , DNA Viral/análise , Mapeamento por Restrição , Simplexvirus/genética , Clonagem Molecular , Desoxirribonuclease BamHI , Eletroforese em Gel de Ágar , Genes Virais , Plasmídeos , Polimorfismo de Fragmento de RestriçãoRESUMO
The virulence of herpes simplex virus-1 (HSV-1) strains by the intraperitoneal (ip) route of injection in mice depends on the presence of an intact sequence in the HpaI DNA fragment P within coordinates 0.762 to 0.787. Deletion of the HpaI-P region (e.g., strain HFEM) abrogates the ability of the virus to infect mice by the ip route without affecting pathogenicity by the intracerebral (ic) route. A recombinant virus (M1C1) derived from DNA of the HSV-1 HFEM strain and the MLUIDNA fragment (coordinates 0.761 to 0.796) spanning the HpaI-P sequence of the pathogenic strain F regained pathogenicity for mice by the ip route.