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The mechanism of rabies virus (RABV) infection still needs to be further characterized. RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells. Herein, based on a reverse genetic system of the Evelyn-Rokitnicki-Abelseth (rERA) strain, we generated a recombinant RABV rERA-N/mCherry strain that stably expresses an additional ERA nucleoprotein that fuses with the red fluorescent protein mCherry (N/mCherry). The rERA-N/mCherry strain retained growth property similar to the parent strain rERA in vitro. The N/mCherry expression showed genetic stability during passage into mouse neuroblastoma (NA) cells and did not change the virulence of the vector. The rERA-N/mCherry strain was then utilized as a visual viral model to study the RABV-cell binding and internalization. We directly observed the red self-fluorescence of rERA-N/mCherry particles binding to the cell surface, and further co-localizing with clathrin in the early stage of infection in NA cells by fluorescence microscopy. Our results showed that the rERA-N/mCherry strain uses clathrin-dependent endocytosis to enter cells, which is consistent with the well-known mechanism of RABV invasion. The recombinant RABV rERA-N/mCherry thus appears to have the potential to be an effective viral model to further explore the fundamental molecular mechanism of rabies neuropathogenesis.

Newcastle disease virus (NDV) infects poultry and antagonizes host immunity via several mechanisms. Dendritic cells (DCs) are characterized as specialized antigen presenting cells, bridging innate and adaptive immunity and regulating host resistance to viral invasion. However, there is little specific knowledge of the role of DCs in NDV infection. In this study, the representative NDV lentogenic strain LaSota was used to explore whether murine bone marrow derived DCs mature following infection. We examined surface molecule expression and cytokine release from DCs as well as proliferation and activation of T cells in vivo and in vitro in the context of NDV. The results demonstrated that infection with lentogenic strain LaSota induced a phenotypic maturation of immature DCs (imDCs), which actually led to curtailed T cell responses. Upon infection, the phenotypic maturation of DCs was reflected by markedly enhanced MHC and costimulatory molecule expression and secretion of proinflammatory cytokines. Nevertheless, NDV-infected DCs produced the anti-inflammatory cytokine IL-10 and attenuated T cell proliferation, inducing Th2-biased responses. Therefore, our study reveals a novel understanding that DCs are phenotypically mature but dysfunctional in priming T cell responses during NDV infection.

The V protein of Newcastle disease virus (NDV) has been shown to inhibit the secretion of interferon (IFN) during infection, which is responsible for the promotion of NDV pathogenicity. However, the ability of the V protein to suppress host innate immunity is not well understood. In this study, we explored the function of V protein and its relationship with virulence by generating V protein-inserted recombinant (r) NDVs. Using rNDVs as a model, we examined the efficiency of infection, IFN responses, and apoptosis of host cells during infection. We found that viral propagation occurred smoothly when V protein from lentogenic NDV is inserted instead of the V protein from the velogenic strain. The infection of lentogenic V protein-inserted rNDV induced less expression of IFNs and downstream antiviral proteins via efficient degradation of p-STAT1 and MDA5. Moreover, velogenic V protein triggered a higher apoptosis rate during infection thereby restricting the replication of NDV. Conversely, lentogenic V protein inhibits IFN responses efficiently and induces less apoptosis compared to the velogenic strain. Our findings provide a novel understanding of the role of V protein in NDV pathogenicity.

Middle East respiratory syndrome coronavirus (MERS-CoV), a member of the Coronaviridae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome (ARDS), as well as extrapulmonary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here, we generated recombinant nonvirulent Newcastle disease virus (NDV) LaSota strain expressing MERS-CoV S protein (designated as rLa-MERS-S), and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa-MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

The envelope attachment glycoprotein (G) and fusion protein (F′) of Nipah virus (NiV) play a key role in viral entry and induction of neutralization antibody. In this study, recombinant baculoviruses, rBac-NF and rBac-NG, were generated to express F and G proteins of NiV. The expressions of recombinant G (rNG) and F (rNF) proteins in rBac-NF and rBac-NG-infected cells were confirmed by Western blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG-infected insect cells elicited G and F protein-specific antibody responses in mice. Furthermore, the G and F protein-specific antibodies could neutralize the infectivity of the VSVΔG*F/G, the NiV F and G envelope glycoproteins of pseudotype recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated that the F and G proteins expressed by the recombinant baculoviruses could be safe diagnostic antigens for the surveillance and monitoring of NiV and could also be promising subunit vaccines for the prevention of NiV.

To identify glucocorticoid induced cataract (GIC)-specific modified crystallins and related changes, we analyzed rat crystallins and related changes in lenses exposed to dexamethasone (Dex). To carry out proteomics analyses, we separated soluble lens proteins with two-dimensional electrophoresis (2-DE) and modified crystallins were analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Related changes in mRNA, protein levels and morphological and functional changes of modified crystallins were also determined. Measured masses (except for γD-crystallin as the larger and cross-link form), the isoelectric points (PIs; except for βB3-crystallin as the alkalinization form) and amino acid sequences of all known rat crystallins matched previously reported data. Analysis by 2-DE indicated that αA, αB, βB3 and γD increased when lenses were exposed to 5 μM Dex; βA4 increased when lenses were exposed to 1 μM Dex and the five proteins that had the highest expressional trend were identical with the results of Q-PCR. βA3/A1 crystallin (expressional trend identical with results of Q-PCR) and the serum albumin precursor gradually disappeared when exposed to 1–50 μM Dex. Results of Western blotting, immunohistochemistry or fluorescence analysis showed that αA and αB increased most when exposed to 5 μM Dex and βA1/A3 and KI-67 decreased obviously when exposed to 1–50 μM Dex. Electron microscopy showed that the condition of the lens was better when lenses were exposed to 5 μM Dex than at other levels and cracks between the fiber cells became larger when lenses were exposed to 1–50 μM Dex. A chaperone role of α-crystallin protecting heated catalase (CAT) and the activity of superoxide dismutase (SOD), glutathione (GSH), and caspase-3 were highest when exposed to 5 μM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased). In conclusion, the proteomics analysis and related changes of rat crystallins when lenses were exposed to Dex in this study will be useful for comparison with normal lens proteins and GIC. We also provided a mechanism for GIC from a proteomics aspect based on the in vitro model.

We investigated the antibody response of DNA immunization with two mammalian codon optimized envelope glycoprotein genes, F and G, of Nipah virus in a mouse model. The results indicated that G gene immunization elicited more significant specific serum IgG response and neutralization antibody response than F gene did, suggesting that the G gene DNA immunization is a potential vaccine strategy against Nipah virus.

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.