ABSTRACT
Avian influenza A(H7N9) virus, which emerged in China in the spring of 2013, has infected hundreds of people and resulted in many deaths. Herein, a rapid and quantitative assay is proposed for the one-step detection of H7N9 virions. Immunomagnetic nanospheres (IMNs) and antibody-conjugated quantum dots (Ab-QDs) are simultaneously employed to capture and identify the target virus, leading to a high efficiency, good specificity, and strong anti-interference ability. Moreover, this reliable detection assay, which combines the efficient magnetic enrichment and the unique photophysical properties of QDs, can achieve a high sensitivity for a low detection limit. At the same time, this detection strategy shows great flexibility for employment in a variety of fluorescence detectors, including fluorescence spectrometry, microscope assays, and handheld UV lamp tests. Furthermore, our one-step detection strategy induces very little change in the integrity of the vulnerable virions, which enables additional genotyping testing following the fluorescence detection. The present study, thus, reports a rapid and quantitative approach for the detection of H7N9 virions based on simultaneous magnetic capture and QD labeling, thereby providing a higher probability for detection and therefore faster diagnosis of H7N9-infected patients.
Subject(s)
Immunomagnetic Separation/methods , Influenza A Virus, H7N9 Subtype/ultrastructure , Microscopy, Fluorescence/methods , Quantum Dots , Viral Load/methods , Virion/ultrastructure , Influenza A Virus, H7N9 Subtype/isolation & purification , Reproducibility of Results , Sensitivity and Specificity , Staining and Labeling/methods , Virion/isolation & purificationABSTRACT
Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300 µg aluminum hydroxide, 1.5 µg HA, and 1.5 µg HA plus 300 µg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.
Subject(s)
Ferrets , Influenza A Virus, H7N9 Subtype/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , Adaptation, Biological , Animals , Antigens, Viral/immunology , Dogs , Female , Immunization , Influenza A Virus, H7N9 Subtype/ultrastructure , Lung/immunology , Lung/pathology , Lung/virology , Madin Darby Canine Kidney Cells , Reassortant VirusesABSTRACT
In early spring 2013, the emergence of the influenza A (H7N9) virus in humans in Eastern China raised concerns of a new influenza pandemic. Development of a safe and effective H7N9 influenza vaccine is urgently needed. To this end, we first synthesized the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A (H7N9) virus A/AnHui/1/2013. Using reverse genetics, we rescued a reassortant virus (H7N9/PR8) that contained the HA and NA genes from wild-type H7N9 and six genes encoding internal proteins from the A/Puerto Rico/8/34 (PR8) virus. Next, the pathogenicity of the reassortant virus was evaluated both in vivo and in vitro. We found that the virus was non-pathogenic in mice and was stable after serial passaging in eggs. Furthermore, we found that a monovalent influenza A (H7N9) split vaccine prepared from the virus was immunogenic in mice and ferrets. When given intramuscularly, the vaccine (two doses of at least 15-µg) completely protected mice from normally lethal wild-type H7N9 virus challenge. In summary, our H7N9 vaccine, developed over a short time, is a potential candidate for further clinical evaluation and human use.
Subject(s)
Influenza A Virus, H7N9 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Animals , Capsid/ultrastructure , Chickens , Chlorocebus aethiops , Female , Ferrets , Humans , Influenza A Virus, H7N9 Subtype/genetics , Influenza A Virus, H7N9 Subtype/ultrastructure , Influenza, Human/immunology , Influenza, Human/virology , Mice, Inbred BALB C , Reverse Genetics , Vaccination , Vaccines, Attenuated/immunology , Vero Cells , Virus ReplicationABSTRACT
In this study, we investigated the ultrastructural modifications induced by influenza A (H7N9) virus in human lung epithelial cells. One particular characteristic of H7N9 viral infection is the formation of numerous M1-associated striated tubular structures within the nucleus and the cytoplasm, which have only previously been observed for a limited number of influenza A viruses, notably the 2009 pandemic (H1N1) virus.
