Exploring the virus infection mechanisms is significant for defending against virus infection and providing a basis for studying endocytosis mechanisms. Single-particle tracking technique is a powerful tool to monitor virus infection in real time for obtaining dynamic information. In this study, we reported a quantum-dot-based single-particle tracking technique to efficiently and globally research the virus infection behaviors in individual cells. It was observed that many influenza viruses were moving rapidly, converging to the microtubule organizing center (MTOC), interacting with acidic endosomes, and finally entering the target endosomes for genome release, which provides a vivid portrayal of the five-stage virus infection process. This report settles a long-pending question of how viruses move and interact with acidic endosomes before genome release in the perinuclear region and also finds that influenza virus infection is likely to be a "MTOC rescue" model for genome release. The systemic technique developed in this report is expected to be widely used for studying the mechanisms of virus infection and uncovering the secrets of endocytosis.

BackgroundMany viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo.MethodsMice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo.ResultsZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased.ConclusionFindings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1311-5) contains supplementary material, which is available to authorized users.

In this work, we reported a simple rapid and point-of-care magnetic immunofluorescence assay for avian influenza virus (AIV) and developed a portable experimental setup equipped with an optical fiber spectrometer and a microfluidic device. We achieved the integration of immunomagnetic target capture, concentration, and fluorescence detection in the microfluidic chip. By optimizing flow rate and incubation time, we could get a limit of detection low up to 3.7 × 10(4) copy/μL with a sample consumption of 2 μL and a total assay time of less than 55 min. This approach had proved to possess high portability, fast analysis, high specificity, high precision, and reproducibility with an intra-assay variability of 2.87% and an interassay variability of 4.36%. As a whole, this microfluidic system may provide a powerful platform for the rapid detection of AIV and may be extended for detection of other viral pathogens; in addition, this portable experimental setup enables the development of point-of-care diagnostic systems while retaining adequate sensitivity.

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.