RESUMEN
Rapid identification of the infections of specific subtypes of influenza viruses is critical for patient treatment and pandemic control. Here we report the application of multiplex reverse transcription polymerase chain reaction (RT-PCR) coupled with membrane-based DNA biochip to the detection and discrimination of the type (A and B) and subtype (human H1N1, human H3N2, avian H5N1 and avian H7N9) of influenza viruses in circulation in China. A multiplex one-step RT-PCR assay was designed to simultaneously amplify the HA and NA genes of the four subtypes of influenza A viruses and NS genes to discriminate type A and B viruses. PCR products were analyzed by a membrane-based biochip. The analytical sensitivity of the assay was determined at a range of 2-100 copies/reactions for each of the gene transcripts. Eighty one clinical samples, containing 66 positive samples with evident seasonal influenza virus infections, were tested, which gives the clinical sensitivity and specificity of 95.5% and 100% respectively. For the avian influenza samples, 3 out of 4 H5N1 samples and 2 out of 2 H7N9 avian samples were correctly identified. We argue this method could allow a rapid, reliable and inexpensive detection and differentiation of human-infecting influenza viruses.
Asunto(s)
Gripe Humana/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Orthomyxoviridae/clasificación , Orthomyxoviridae/aislamiento & purificación , Animales , Aves/virología , China , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H3N2 del Virus de la Influenza A/clasificación , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/clasificación , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H7N9 del Virus de la Influenza A/clasificación , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Gripe Humana/diagnóstico , Tipificación Molecular/métodos , Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/virología , ARN Viral/genética , Sensibilidad y Especificidad , Proteínas Virales/genéticaRESUMEN
Efficient assembly of the influenza virus RNA-dependent RNA polymerase, a heterotrimeric complex formed by three subunits (PA, PB1 and PB2) is critical for virus replication and pathogenicity. Therefore, interfering with the assembly of the RNA-dependent RNA polymerase complex could offer novel and effective anti-influenza therapeutics. In the present study, we show that a short peptide derived from amino acids 731-757 of PB1 (PB1(731-757)) can disrupt the interaction between the C-terminal part of PB1 (denoted as PB1c corresponding to PB1(676-757)) and the N-terminal part of PB2 (denoted as PB2n corresponding to PB2(1-40) ). We further show that PB1(731-757) is capable of inhibiting viral polymerase activity and viral replication. Interestingly, we find that PB1(731-757) interacts with PB1c rather than PB2n. Furthermore, mutational analyses show that the hydrophobic sites of PB1c play an essential role in the PB1c-PB1(731-757) interaction. The characterization of the inhibitory effect of PB1(731-757) on viral polymerase activity and viral replication could offer a potential target for anti-influenza drug development.