| Avian influenza viruses (AIVs) belong to the family of Orthomyxoviridae. The H5N1 AIVs was first detected in the sick geese in Guangdong province in 1996. H5N1 AIVs has evolved into a number of different clades based on the HA gene. Recently, different NA subtypes (including N2, N3, N5, N6, and N8) have emerged in the novel H5 reassortants among Clade 2.3.4.4. In addition, in early 2013, a novel recombinant H7N9 virus emerged in the Eastern China and caused death of human with mortality rate as high as 39%. Phylogenetic analysis showed that the H7N9 avian influenza virus is a natural reassortant of three avian influenza viruses:H7N3, H10N9 and H9N2. These novel recombinant viruses pose a great threat to poultry industry and public health. More importantly, surveillance of the influenza viruses revealed that these novel recombinants are currently widely circulating in poultry in China, and are still in evolution. Currently, many expermantal methods are available for the detection and surveillance of AIVs, including virus isolation, hemagglutination (HA) assay and hemagglutination inhibition (HI), enzyme linked immunoabsorbent assay (ELISA) and RT-PCR. However, theie methods can not satisify the need of simple operation and quick diagonosis. Therefore, in this study, we used three H5 subtype strains, A/duck/Eastern China/031/2009 (031), A/duck/Shandong/Q5/2013 (QD5) and H7N9 A/Chinken/jiaxin/JX148/2013(JX148) to development the monoclonal antibodies (McAbs) against the N5, N8 and N9 NA proteins. Based on the qualified NA monoclonal antibodies, we developed the N5 subtype colloidal gold test strip to provide a technical foundation for the avian influenza surveillance and diagnostic methods in the future.1. Construction and identification of the N5, N8 and N9 NA eukaryotic expression plasmidsThe H5N5 (031), H5N8 (QD5) and H7N9 (JX148) three different NA subtypes of strains were inoculated 9-10 days old SPF chicken embryos, incubation at 35℃. On the one hand, by viral allantoic fluid sucrose density gradient centrifugation were collected and concentrated with formaldehyde inactivated, purified whole virus immunogen. On the other hand, by recombinant DNA technology, gene fragments of different NA subtypes are connected to the eukaryotic expression vector pcDNA3.1 (+) eukaryotic expression plasmid pcDNA3.1-N5, pcDNA3.1-N8, pcDNA3.1-N9. After digestion and sequencing are correct, the eukaryotic expression plasmids were transiently transfected MDCK and 293T cells were mixed, can successfully expressed NA protein identification of eukaryotic expression plasmid by indirect immunofluorescence.2. Preparation and identification of the N5, N8 and N9 NA monoclonal antibodiesWe first immunized the eight-week-old BALB/c mice with the purified whole virus and the immune adjuvant, then the cell fusion, IFA and limiting dilution methods were chosen to select the positive clones. The resμlts showed that a number of subclones were obtained for all the three strains of the H5N8 and H5N5 virusesand for two strains of H7N9 viruses. At the same time, these 8 monoclonal antibodies were further identified for the antibody subclasses, specificity and titers. The resμlts showed that three subclones from H5N8 virus (QD5) namely B8C10-5, E8 and C10-3, have a titer of 1:105,1:106,1:106 respectively. In addition, their antibody subclasses were IgM, IgM, IgA, respectively. Three subclones from H5N5 (031) namely 24,10-5 and C6 have a titer of 1:105,1:106,1:106 respectively, and their antibody subclasses were IgG2b, IgG1, IgG2b. As for the H7N9 (JX148) virus, the titers of the 4-B3-C5 andCl-B3-4 were 1:105 and 1:107, respectively, and the antibody subclasses were IgM and IgGA. The indirect immunofluorescence assay resμlts showed that all the ascites have the specific fluorescence reaction except the C6 of the H5N5 (QD5). The specificity experiment resμlts showed that all the monoclonal antibodies except for the C10-3 (H5N8) and 10-5 (H5N5) had no cross reactivity with the H5N2, H6N2, H3N2, H11N2, H10N3, H5N1and H1N1 viruses. The superposable test showed that the antigen recognition sites of the B8C10-5 and E8 monoclonal antibody are identical, Rest C 10-3,24,10-5, C6,4-B3-C5, C1-B3-4 six monoclonal antibody are recognize different antigenic site.3. Development of the N5 subtype monoclonal antibody immunochromatographic stripThen, two highly efficient paired ascites 24 (H5N5) and C6 (H5N5) were then selected for further purified. The immunochromatographic strip was developed for rapid detection of the N5 subtype of avian influenza viruses with sandwich method. The monoclonal antibody 24 for the N5-AIVs was labeled with colloidal gold, C6 for N5-AIV was used as the detection reagent, and the anti-mouse secondary antibody was blotted on the test line. We then determined the specificity of the immunochromatographic strip. Our resμlts demonstrated that only the standard N5-AIVs antigen displayed positive lines appreared as a purple strip on the inmmuochromatographic strips, while other subtypes of avian influenza viruses, including the H5N1, H3N2, H10N3, and H7N9 were negative. The sensitivity test resμlts also showed that the inmmuochromatographic strips have higher sensitivity than the ELISA assay and easy to operate. The developed N5 subtype colloidal gold strip has important value of clinical applications. |
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