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Background: Encephalomyocarditis virus (EMCV) can infect a variety of animal species and humans. Although the EMCV infection is known to induce autophagy to promote its replication in host cells, the viral proteins that are responsible for inducing autophagy are unknown. Methods: The recombinant plasmids that were expressing the EMCV proteins were constructed to analyze the role of each protein in the induction of autophagy. Autophagy inductions by the EMCV proteins in BHK-21 cells were investigated by confocal microscopy, Western blotting and transmission electron microscopy. ER stress in BHK-21 cells was examined by detecting the marker molecules using western blotting and luciferase assays. Results: This study presents the first demonstration that the nonstructural proteins 2C or 3D of EMCV were involved in inducing autophagy in BHK-21 cells that were expressing 2C or 3D, and we found that inhibiting Beclin1 expression influenced this autophagy induction process. Next, 2C and 3D were shown to be involved in inducing autophagy by activating the ER stress pathway. Finally, EMCV 2C or 3D were demonstrated to regulate the proteins associated with PERK and ATF6alpha pathway. Conclusions: Our findings indicate that 2C and 3D are involved in EMCV-induced autophagy by activating ER stress molecules and regulating the proteins expression associated with UPR pathway, helping to better understand the EMCV-induced autophagy process.
Background: The influenza RNA dependent RNA polymerase synthesizes viral RNA in the nucleus as functional viral ribonucleoprotein (vRNP) complexes with RNA and nucleoprotein (NP). The N-terminus of NP contains an unconventional nuclear localization signal (NLS) important for initial vRNP nuclear localization but which also interacts with various host factors. Methods: To study the role of the N-terminus of NP aside from NLS function, we generated an N-terminal NP deletion mutant, del20NLS-NP, encoding the conventional SV40 T-antigen NLS in place of the first 20 amino acids of NP. We characterized expression, location, and activity of del20NLS-NP compared to wild type NP using reconstituted vRNP assays, cellular fractionation, Western blotting, and reverse transcription-PCR. We assessed NP nucleotide binding with gel-shift assays and analyzed NP complexes using 1D blue native gel electrophoresis. Results: del20NLS-NP is expressed, localized in the nucleus and cytoplasm, and maintains ability to bind nucleic acids. Despite this, del20NLS-NP exhibits a defect in viral RNA expression exacerbated by increasing vRNA template length. We find diminished del20NLS-NP high molecular weight complexes in protein extracts; evidence the defect is with functional vRNP formation. Interestingly, the shortest template, NS vRNA, exhibits a limited defect. However, this is not due to short template size, but rather activity of the NS protein(s). Expression of NS1 rescues the gene expression defect primarily at the protein level, a finding consistent with the known role of NS1 as a viral mRNA translational enhancer. NS1 mutant analysis confirms NS1-RNA binding is not required for the translational enhancement and reveals the NS1-CPSF30 interaction surface is essential. Conclusions: del20NLS-NP is a nuclear localized NP mutant able to bind nucleic acids but inefficient for assembly of functional vRNPs inside the host cell. Our results add to growing evidence the N-terminus of NP plays important roles aside from vRNP nuclear localization. We demonstrate the utility of this partially functional NP mutant to characterize the influence of additional proteins on viral gene expression. Our studies reveal the NS1-CPSF30 interaction surface is required for the ability of NS1 to enhance viral protein translation, supporting a function for this NS1 domain in the cytoplasm.
Background: Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Methods: Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3H2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription - quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Results: Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. Conclusions: These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.
Background: Herpes viruses infection transmitted through healthy but infected blood donors pose a danger to herpes-naive immunocompromised recipients. The risk of transfusion-related HHV-8 transmission is different in endemic and not endemic areas. HHV-6 and HHV-8 seroprevalence and viral load among blood donors have been reported from different countries. The aim of our study was to assess the seroprevalence of HHV-8 and HHV-6 in volunteer blood donors from Greece which is unknown.FindingsSerum samples from 179 healthy blood donors were tested for the presence of IgG antibodies against HHV-6 and HHV-8 with ELISA. None of the 179 donors of Greek origin tested was positive for HHV-8. HHV-6 seropositivity was assessed in 160 blood donorsaapos; samples and was found to be 78.75% (126/160). The HHV-6 seroprevalence did not differ either between males and females or among different decade age groups. Conclusions: The fact, that no blood donor was positive for HHV-8 IgG antibodies indicates that the risk for transfusion related HHV-8 transmission in Greece, if any, is negligible and does not warrant broad testing for HHV-8. Definitely further studies are needed, in order to clarify the potential risk of HHV-6 transmission.
Background: The deciphering of cellular networks to determine susceptibility to infection by HIV or the related simian immunodeficiency virus (SIV) is a major challenge in infection biology. Results: Here, we have compared gene expression profiles of a human CD4+ T cell line at 24 h after infection with a cell line of the same origin permanently releasing SIVmac. A new knowledge-based-network approach (Inter-Chain-Finder, ICF) has been used to identify sub-networks associated with cell survival of a chronically SIV-infected T cell line. Notably, the method can identify not only differentially expressed key hub genes but also non-differentially expressed, critical, aapos;hiddenaapos; regulators. Six out of the 13 predicted major hidden key regulators were among the landscape of proteins known to interact with HIV. Several sub-networks were dysregulated upon chronic infection with SIV. Most prominently, factors reported to be engaged in early stages of acute viral infection were affected, e.g. entry, integration and provirus transcription and other cellular responses such as apoptosis and proliferation were modulated. For experimental validation of the gene expression analyses and computational predictions, individual pathways/sub-networks and significantly altered key regulators were investigated further. We showed that the expression of caveolin-1 (Cav-1), the top hub in the affected protein-protein interaction network, was significantly upregulated in chronically SIV-infected CD4+ T cells. Cav-1 is the main determinant of caveolae and a central component of several signal transduction pathways. Furthermore, CD4 downregulation and modulation of the expression of alternate and co-receptors as well as pathways associated with viral integration into the genome were also observed in these cells. Putatively, these modifications interfere with re-infection and the early replication cycle and inhibit cell death provoked by syncytia formation and bystander apoptosis. Conclusions: Thus, by using the novel approach for network analysis, ICF, we predict that in the T cell line chronically infected with SIV, cellular processes that are known to be crucial for early phases of HIV /SIV replication are altered and cellular responses that result in cell death are modulated. These modifications presumably contribute to cell survival despite chronic infection.
Background: Tetherin is an interferon-inducible host cell factor that blocks the viral particle release of the enveloped viruses. Most knowledge regarding the interaction between tetherin and viruses has been obtained using the primate lentiviral system. However, much less is known about the functional roles of tetherin on other lentiviruses. Equine infectious anemia virus (EIAV) is an important macrophage-tropic lentivirus that has been widely used as a practical model for investigating the evolution of the host-virus relationship. The host range of EIAV is reported to include all members of the Equidae family. However, EIAV has different clinical responses in horse and donkey. It’s intriguing to investigate the similarities and differences between the tetherin orthologues encoded by horse and donkey. Results: We report here that there are two equine tetherin orthologues. Compared to horse tetherin, there are three valine amino acid deletions within the transmembrane domain and three distinct mutations within the ectodomain of donkey tetherin. However, the antiviral activity of donkey tetherin was not affected by amino acid deletion or substitution. In addition, both tetherin orthologues encoded by horse and donkey are similarly sensitive to EIAV Env protein, and equally activate NF-κB signaling. Conclusion: Our data suggest that both tetherin orthologues encoded by horse and donkey showed similar antiviral activities and abilities to induce NF-κB signaling. In addition, the phenomenon about the differential responses of horses and donkeys to infection with EIAV was not related with the differences in the structure of the corresponding tetherin orthologues.
Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.
Background: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to improved management and husbandry practices, the availability of several anti-PCV2 vaccines provides an efficient immunological option for reducing the impact of these diseases. Most anti-PCV2 vaccines are marketed as injectable formulations. Although these are effective, there are problems associated with the use of injectable products, including laborious and time-consuming procedures, the induction of inflammatory responses at the injection site, and treatment-associated stress to the animals. Oral vaccines represent an improvement in antigen delivery technology; they overcome the problems associated with injection management and facilitate antigen boosting when an animals’ immunity falls outside the protective window. Methods: Chitosan microparticles were used as both a vehicle and mucosal adjuvant to deliver yeast-derived PCV2 virus-like particles (VLPs) in an attempt to develop an oral vaccine. The physical characteristics of the microparticles, including size, Zeta potential, and polydispersity, were examined along with the potential to induce PCV2-specific cellular immune responses in mice after oral delivery. Results: Feeding mice with PCV2 VLP-loaded, positively-charged chitosan microparticles with an average size of 2.5 μm induced the proliferation of PCV2-specific splenic CD4+/CD8+ lymphocytes and the subsequent production of IFN-γ to levels comparable with those induced by an injectable commercial formulation. Conclusion: Chitosan microparticles appear to be a safe, simple system on which to base PCV2 oral vaccines. Oral chitosan-mediated antigen delivery is a novel strategy that efficiently induces anti-PCV2 cellular responses in a mouse model. Further studies in swine are warranted.
Background: Substitutions of amino acid (aa) 70 in the core region of hepatitis C virus genotype 1b (HCV 1b) are a predictor of the non-virological response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy. The aim of our study was to develop quantitative real-time reverse transcription polymerase chain reaction (qPCR) assays to quantify wild-type (70 W) and mutant (70 M) strains of HCV 1b. Methods: We used the TaqMan system to quantify strains 70 W and 70 M. Codon 70 in the HCV 1b core region can be either CGN or CAN, therefore degenerate TaqMan minor groove binder (MGB) probes with inosine were used. We determined detection limits, sensitivity and specificity of the methods developed. Direct sequencing and cloning of the HCV core region were used to confirm the reliability of our new system. Serum samples from 138 Chinese patients infected with HCV 1b were examined with the system we developed and compared with results obtained from the Roche TaqMan RT-PCR HCV RNA quantitation system. Results: Degenerate MGB probes were able to clearly distinguish 70 W from 70 M. The detection limit was 103 copies/mL. Cross-reactivity tests confirmed the specificity of our method. Our system can effectively quantify 70 W and 70 M for 99.6% of patients with HCV 1b. Further tests involving cloning and sequencing confirmed the reliability of our system. Conclusions: We developed an assay system using degenerate TaqMan MGB probes with inosine to quantify wild-type and mutant viral RNAs of the HCV 1b core region at aa 70. Our developed assay system had high levels of sensitivity and accuracy, and could prove useful in investigating dynamic changes during PEG-IFN/RBV therapy to assess virological responses.
Background: Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections. Methods: In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010. Results: F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was (112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese. Conclusion: The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.