Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 103 pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10–100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.

Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

Mongolian gerbils (Meriones unguiculatus) are widely used as animal models for a variety of infectious diseases. However, immunological reagents such as cytokines have not been characterized. Two heterohybridomas, D9(E6)C2B3 and D9(E4), obtained by fusion of gerbil splenocytes with mouse myeloma cells (P3-X63-Ag8.653), expressed gerbil CD3G mRNA. These cells were suggested to be T cell heterohybridomas. They also expressed gerbil IL6 [D9(E6)C2B3] and TGFB [D9(E4) and D9(E6)C2B3] mRNAs. The addition of conditioned medium (CM) obtained from the culture of D9(E6)C2B3 significantly enhanced antibody secretion and expression of gerbil Cγ1 and Cε IGHC mRNAs in the B11D2(C2) heterohybridoma, which secretes gerbil IgG1. However, the addition of CM from both heterohybridomas did not improve in proliferation of B11D2(C2) cells. These results indicate that CM from D9(E6)C2B3 improved the culture of gerbil–mouse heterohybridomas, possibly by secreting gerbil IL6.

The expression level of transcription factor c-Myb oscillates during hematopoiesis. Fbw7 promotes ubiquitin-mediated degradation of c-Myb, which is dependent on phosphorylation of Thr572. To investigate the physiological relevance of Fbw7-mediated c-Myb degradation, we generated mutant mice carrying c-Myb-T572A (TA). Homozygous mutant (TA/TA) mice exhibited a reduction in the number of peripheral red blood cells and diminished erythroblasts in bone marrow, presumably as a result of failure during erythroblast differentiation. We found that c-Myb high-expressing cells converged in the Lin − CD71 + fraction, and the expression of c-Myb was higher in TA/TA mice than in wild-type mice. Moreover, TA/TA mice had an increased proportion of the CD71 + subset in Lin − cells. The c-Myb level in the Lin − CD71 + subset showed three peaks, and the individual c-Myb level was positively correlated with that of c-Kit, a marker of undifferentiated cells. Ultimately, the proportion of c-Myb hi subgroup was significantly increased in TA/TA mice compared with wild-type mice. These results indicate that a delay in reduction of c-Myb protein during an early stage of erythroid differentiation creates its obstacle in TA/TA mice. In this study, we showed the T572-dependent downregulation of c-Myb protein is required for proper differentiation in early-stage erythroblasts, suggesting the in vivo significance of Fbw7-mediated c-Myb degradation. Ablation of c-Myb expression by gene targeting indicates that c-Myb, a member of the hematopoietic transcription factor family, is essential for fetal liver hematopoiesis and erythroid development 1,2. c-Myb is consistently expressed at high levels in immature progenitors of all hematopoietic lineages and is associated with the regulation of proliferation, differentiation, and survival of progenitors 3. Levels of c-Myb decrease during terminal differentiation to mature blood cells 4 ; tetracycline-regulated expression of c-Myb in c-Myb −/− embryonic stem (ES) cells prevents the terminal differentiation of erythrocytes and megakaryocytes 5. These observations prove that appropriate levels of c-Myb protein are strictly defined in distinct stages of differentiation of the hematopoietic cell lineage to maintain normal proliferation and differentiation. Consistent with this role, several c-Myb target genes linked to proliferation and differentiation, such as c-Myc and c-Kit, have been identified in myeloid cells 2. The cellular abundance of c-Myb is controlled not only by transcription but also by post-transcriptional mechanisms. c-Myb is highly expressed in immature erythroid progenitors with the level of expression decreasing during maturation. It is suggested that the decrease in c-Myb is due to a block in transcription elongation 6,7. It is thought that c-Myb undergoes post-translational modifications, including phosphorylation and ubiquitylation,