Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCT␥ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCT␥ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCT␥ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCT␥ to NBs and identify the chaperonin CCT␥ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.

Cross-species transmissions of swine influenza viruses (SIVs) raise great public health concerns. In this study, subcellular proteomic profiles of human A549 cells inoculated with H3N2 subtype SIV were used to characterize dynamic cellular responses to infection. By 2DE and MS, 27 differentially expressed (13 upregulated, 14 downregulated) cytoplasmic proteins and 20 differentially expressed (13 upregulated, 7 downregulated) nuclear proteins were identified. Gene ontology analysis suggested that these differentially expressed proteins were mainly involved in cell death, stress response, lipid metabolism, cell signaling, and RNA PTMs. Moreover, 25 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between mock- and virus-infected A549 cells. Western blot analysis confirmed that changes in abundance of identified cellular proteins heterogeneous nuclear ribonucleoprotein (hnRNP) U, hnRNP C, ALDH1A1, tryptophanyl-tRNA synthetase, IFI35, and HSPB1 in H3N2 SIV-infected cells were consistent with results of 2DE analysis. By confocal microscopy, nucleus-to-cytoplasm translocation of hnRNP C and colocalization between the viral nonstructural protein 1 and hnRNP C as well as N-myc (and STAT) interactor were observed upon infection. Ingenuity Pathway Analysis revealed that cellular proteins altered during infection were grouped mainly into NFκB and interferon signaling networks. Collectively, these identified subcellular constituents provide an important framework for understanding host/SIV interactions and underlying mechanisms of SIV cross-species infection and pathogenesis.

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The rabies virus is a neurotropic virus that causes fatal disease in humans and animals. However, not all commercial antibodies against rabies virus (RABV) structural proteins are generally available, and production of high-quality monoclonal antibodies (MAbs) requires high purification of virus particles and special facilities and is time-consuming. By using RABV-infected suckling mouse brain as antigens in this study, 11 hybridoma cells secreting MAbs against RABV were obtained, which showed strong reactivity with RABV-infected Vero cells in immunofluorescence assay. Among the 11 MAbs, three MAbs (1B11, 1C8, and 8H12) showed a neutralizing effect to RABV, while MAb 4B7 recognized the recombinant nucleoprotein (N) of RABV expressed in Vero cells; seven MAbs (1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3) reacted specifically with phosphoprotein (P) of RABV. The MAbs developed in this study will be useful in establishing a diagnostic test and study on the interactions between RABV and its host.

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Klebsiella pneumoniae belongs to Enterobacteriaceae, which is the commonest bacterium causing nosocomial respiratory tract infection. It ranks second in bacteremia and urinary tract infection in gram-negative bacteria. Therefore, the rapid and accurate identification of K. pneumoniae was of great significance for the guide of clinical medication, and timely treatment of patients. The purpose of this study was to establish a rapid and sensitive molecular detection method for K. pneumoniae based on loopmediated isothermal amplification (LAMP) technology. Firstly, local BLAST and NCBI BLAST were used to analyze the genome of K. pneumoniae. According to the principle of interspecific and intraspecific specificity, CelB (GenBank ID 11847805) was selected as the specific gene. Then, the LAMP and PCR identification systems were established with this target gene. Thirty-six clinical isolates of K. pneumoniae and 50 non-K. pneumoniae were used for the specific evaluation, and both LAMP and PCR could specifically distinguish K. pneumoniae from non-K. pneumoniae. A 10-fold series diluted positive plasmids and simulated infected blood samples were used as the templates in the sensitivity assay, and the results showed that the sensitivity could reach 1 copy/reaction. In summary, a rapid, specific, and sensitive LAMP method was established to detect K. pneumoniae in clinics.