Expression Of Avian Influenza Virus Neuraminidase Protein And Development Of RT-PCR Technique For Detection Of H7N9Subtype Avian Influenza Virus

Avian influenza is an infectious disease of birds caused by the avian influenza viruses (AIV). Avian influenza viruses do not typically replicate efficiently in humans, indicating direct transmission of avian influenza virus to humans is unlikely. However, since1997, several cases of human infections with different subtypes (H5N1, H7N7, and H9N2) of AIV have been identified and raised the pandemic potential of AIV in humans. In order to differently diagnose AIV NA subtypes, N3-N9neuraminidase protein produced in insect cells by recombinant baculovirus was developed. The N3-N9DNA fragments of AIV were amplified by PCR. The genes of interest were firstly cloned into pFastBacHTA transfer vector. Then the recombinant vectors were cloned into the DH10Bac cells, named as Bacmid-NA. Insect cells sf9were transfected with Bacmid-NA and the recombinant plasmid were collected from the infected cells. The recombinant proteins N3-N9were analyzed by neuraminidase test and western blot. The results indicated that the N3-N9protein had good reactivity with antiserum and all of them were enzymatically active.Another reverse-transcription PCR assay to rapidly detect the novel H7N9subtype avian influenza virus was developed and evaluated during my experiment period. Two pairs of RT-PCR primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9virus, respectively. A one-step method was used to establish the RT-PCR system. The specificity assays were evaluated using influenza A viruses of various genetic backgrounds and other avian pathogens. The sensitivity assays were determined using viral RNA extracted from serially diluted AIV-infected allantoic fluid. In addition, a blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. The H7HA can be detected by this RT-PCR assay, while the other H1-H6and H8-H15subtype HA, as well as other avian pathogens were detected negative in specificity assay. Similarly only the N9NA related to the novel H7N9virus was detected, the other N1-N9NA were detected negative. Results of10-fold dilution series of allantoic fluid by one step RT-PCR assays showed that detection limit of the assay was approximately104.5EID50per reaction. Furthermore, the assays showed clinical specificity for identification cloacal swabs of H7N9virus. The RT-PCR assay established in this study can be used as a referee method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.