RESUMO
Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vírus da Influenza A/isolamento & purificaçãoRESUMO
The origin and evolution of the classical swine fever (CSF) epizootic that occurred in Cuba from 1993 to 1997 has been investigated by the analysis of E2 gene sequences from 15 representative viral isolates as well as the vaccine and the challenge strains used in this country. In the phylogenetic tree derived from these sequences, the Cuban isolates were located in a defined cluster within the previously reported genomic subgroup 1.2. This cluster was related, although distinguishable, from the live vaccine used in Cuba since 1965. Two further groups were identified. One of them included the early viruses isolated in the western part of Cuba until 1996 and the strain Margarita, used for vaccine potency tests since 1965. These results are consistent with the strain Margarita being the origin of the western outbreaks. The viruses isolated from 1996 in eastern Cuba defined a related, but independent group. The level of sequence variation observed in this group does not exclude an independent origin for the eastern isolates.
Assuntos
Vírus da Febre Suína Clássica/genética , Peste Suína Clássica/epidemiologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Peste Suína Clássica/fisiopatologia , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/classificação , Vírus da Febre Suína Clássica/isolamento & purificação , Cuba/epidemiologia , DNA Viral/química , DNA Viral/genética , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Suínos , Proteínas do Envelope Viral/químicaRESUMO
Two murine monoclonal antibodies (mAbs) directed against different epitopes on recombinant porcine interferon-alpha (IFN-alpha) were selected and used to construct a two-site ELISA. This ELISA, when performed in a one-step version, detected about 0.5 units ml-1 of IFN-alpha and showed similar sensitivity but better precision than a cytopathic effect inhibition bioassay. Estimates of IFN-alpha in tissue culture medium by the two assays correlated well. In contrast, one or several factors in porcine serum reduced the sensitivity of the ELISA. Measurements of IFN-alpha in porcine serum was, however, possible in a two-step version of the ELISA, with a sensitivity of about 1 unit IFN-alpha ml-1. Results of ELISA and bioassay agreed, except that the ELISA possibly produced false positive results in two out of a total of 91 sera negative in the bioassay. In addition, one of 23 sera positive in the bioassay was negative in the ELISA.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Interferon-alfa/sangue , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Bioensaio , Meios de Cultura , Epitopos/imunologia , Interferon Tipo I/imunologia , Camundongos , Proteínas Recombinantes , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SuínosRESUMO
A simple reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed for the specific amplification of DNA after reverse transcription of RNA from the classical swine fever virus (CSFV). A pair of oligonucleotides was selected from an area of high homology in the genome of CSFV strains, but which differed from the corresponding sequences in the genome of bovine viral diarrhea virus (BVDV) strains. Using these primers (CSFV1-CSFV2), a CSFV specific DNA band of 174 bp was amplified from the CSFV RNA extracted from four reference strains and 14 field isolates, as well as from 25 organ extracts and eight buffy coats and serum samples of experimentally infected animals. No amplification was observed with the RNA from four BVDV reference and vaccine strains and seven field isolates. This RT-PCR assay made it possible, in a one-step reaction, to detect CSFV rapidly, sensitively and specifically in cell culture supernatants and in clinical specimens.