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The mechanism of interactions between natural killer (NK) cells and dendritic cells (DCs) in regulating the early stage of innate immune response and the subsequent adaptive immune response has been elucidated in recent years. NK-DC interactions result in the activation of NK cells and the maturation and/or apoptosis of DCs through direct cell-to-cell contact and releasing cytokines. NK cells can control and enhance DC-mediated antiviral immune response by inducing DCs to mature in favor of Th1 immune response and providing antigens to DCs and killing immature DCs (iDCs). DCs stimulate NK cells through soluble and cell-contact activators, thereby increasing their proliferation, survival and cytotoxicity to shape the innate immune response. Recent studies have shown that interactions between NK cells and DCs also play a critical role in several antiviral responses. Therefore, this paper reviewed the NK-DC interactions and their relationship with antiviral responses, thus providing a theoretical basis for studying the molecular mechanism of NK-DC interactions in viral infectious diseases.
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Objective To analyze how enterovirus D68 (EV-D68) protease 2A affects the anti-vi-ral interferon typeⅠ(IFN-Ⅰ) pathway in 293T cells following infection. Methods Western blot was used to detect the expression of recombinant protease 2A, IFN-α and signal transducers and activators of tran-scription 1 (STAT1) at protein level. Expression of EV-D68 viral protein (VP1) and protease 2A was ana-lyzed by immunofluorescence at different time points. Cytopathic effects were recorded to calculate 50％ cell culture infective dose ( CCID50 ) . Expression of the genes involved in the anti-viral IFN-Ⅰ pathway was measured by real-time PCR (RT-PCR). Results The recombinant plasmid pCLIPf-2A was successfully constructed and the expression of recombinant protease 2A could be detected by Western blot 24 h after transfection. The recombinant protease 2A promoted the proliferation of EV-D68 at the late stage of infection and induced the production of IFN-α. Expression of the genes involved in the anti-viral IFN-Ⅰ pathway at mRNA level was up- or down-regulated to different degrees with various trends in different groups following infection. Expression of STAT1 was enhanced in all groups. Conclusions EV-D68 protease 2A promoted the activation of anti-viral IFN-Ⅰpathway in response to viral infection and enhanced the proliferation of virus at the late stage of infection.
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Objective To investigate the role of CXC chemokine ligand 5 (CXCL5) in the patho-genesis of dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD). Methods A mouse model of IBD was established by giving 3% DSS in drinking water. Influences of CXCL5 knockout on mouse body weight, clinical symptoms, survival rate, pathological injury and the secretion of inflammatory cyto-kines were analyzed. Results CXCL5 levels in serum of mice with DSS-induced IBD were significantly higher than those of the normal control group. DSS-induced weight gain, death, pathological damages and inflammatory cytokine secretion were alleviated in mice after knocking out CXCL5. Conclusion CXCL5 might promote the secretion of inflammatory cytokines in mice with DSS-induced acute colitis and aggravate pathological damages,suggesting that CXCL5 might be a potentially important candidate target for the treat-ment of IBD.
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Objective To express and purify the recombinant UL7 protein of herpes simplex virus 1 (HSV-1), to prepare the corresponding UL7-specific polyclonal antibody and to preliminarily analyze the expression of UL7 protein during the proliferation of HSV-1. Methods The UL7 gene was amplified by PCR and then cloned into the pGEX-5X-1 vector for expression of UL7 protein in the prokaryotic expression system. The constructed expression plasmid, pGEX-5X-1-UL7, was transformed into E. coli BL21 (DE3) to induce the expression of UL7 protein by IPTG. The purified GST-UL7 fusion protein was used as antigen to inject the ICR mouse for the preparation of polyclonal antibody specific for UL7 protein. The titer and speci-ficity of the polyclonal antibody were analyzed by using indirect ELISA and Western blot assay, respectively. The UL7 protein-specific polyclonal antibody was used to detect the expression of UL7 protein at different time points after infecting Vero cells with HSV-1. Results The GST-UL7 fusion protein was efficiently ex-pressed in E. coli BL21 (DE3). The UL7 protein-specific polyclonal antibody was prepared with high titer (1 ∶ 105) and high specificity as indicated by the indirect ELISA and Western blot assay. The expression of UL7 protein was detected at different time points after infecting Vero cells with HSV-1. Conclusion The GST-UL7 fusion protein was obtained successfully and the UL7 protein-specific polyclonal antibody was pre-pared. Accompany with the proliferation of HSV-1, the expression of UL7 protein was detected at different time points by using the polyclonal antibody.
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Objective To investigate the characteristics of VP1 gene hypervariable region in hu-man enterovirus type 68(HEV68)strains isolated in China. Methods Nucleotide sequences of the VP1 gene in the Chinese strains and strains isolated in other countries were aligned by using Clustal W in the MEGA6 program. The phylogenetic trees were constructed by using Neighbor-Joining(NJ)method in the MEGA6 program. Sequence of the amino acids encoded by that region was analyzed by compared with that of the standard strain Fermon. Results A total of 80 strains of EV68 had been isolated in China by the end of 2015. Most of the mutations occurred in BC and DE loops. The mutation sites lied in the VP1 gene of Chi-nese isolates were at 83,89,91,94,96,97,98,102,109,139,141,142,143,144 and 147. Glycine was missing from most of the amino acid sequences encoded by the VP1 gene of Chinese strains. The phylo-genetic analysis indicated that 53 and 21 EV68 strains isolated in China belonged to B and C clades,respec-tively. Conclusion Compared with the standard strain Fermon,the Chinese strains changed a lot in BC-loop and DE-loop,which were associated with the antigenicity and virulence of EV68. The EV68 strains iso-lated in China belonged to B and C clades.
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Objective To investigate the epidemic patterns and the characteristics of influenza in Chi-na through a Meta-analysis based on the studies published in domestic literatures.Methods Related articles published during 2005 to 2012 were screened out from domestic databases and analyzed through a Meta-analysis with Review Manager 5.0 software.Results Twenty-two articles covering 957 901 patients with influenza-like-illness (ILI) and 148 233 pathogen samples were screened out according to the inclusion criteria.No significant difference with the ILI diagnosis rate was found between subjects at age 0-4 years and those at age 15-59 years. Higher ILI diagnosis rates were observed in those two groups as compared with subjects elder than 60 years old. Most of the pathogen samples were carried by subjects aged 25-59 years.More influenza virus strains were isola-ted in 2009 as compared with those of the seven other years (OR=2.25, 95%CI=1.27-3.70).There was sta-tistical difference between the numbers of influenza A H1N1 and seasonal influenza A strains (OR=2.25, 95%CI=1.30-3.91) .Significant difference was also observed between the numbers of influenza A and influenza B strains (OR=4.05, 95%CI=2.53-6.47).Conclusion There was significant difference with the diagnosis rate between subjects aged 0-4 years and those aged≥60 years.More attention should be paid to people at high risk of infection (0-4 years old and≥60 years old) and those at 25-29 years with high mobility and social inter-course for the timely prevention and control of pandemic influenza.The detection rate of influenza virus strains was increased during the outbreak of novel influenza A H1N1 infection in 2009.After that outbreak, the detec-tion rate of novel influenza A H1N1 strains was 2.25 times the rate of seasonal influenza strains.The detection rate of influenza A was 4.05 times the rate of influenza B virus strains.Therefore, it is necessary to strengthen the surveillance for influenza A virus and other epidemic influenza virus strains.
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Objective To establish a non-traumatic mouse model of acid aspiration-induced lung injury which al-lows longitudinal studies.Method C57BL/6 mice were anesthetized and orotracheally intubated with a 20 gauge angio-catheter guided by optical fiber.The mice were subsequently placed in the right lateral decubitus position and external com-pression to the left lung was manually applied.A polyethylene catheter was advanced into the right lung and used to instill either hydrochloric acid (2.5μL/g, 0.1 mol/L, pH 1.5) or saline as control.Then the mice were recovered with supple-mental oxygen for 4 hours.The pulmonary physiological function and survival of mice within 2 weeks after surgery were as-sessed.Results Methylene blue instillation showed that the staining fluid went into the right lung of the non-traumatically intubated mice.The survival rate of the mice with non-traumatic instillation was 80%, statistically significantly higher than those with tracheostomy instillation.Histological examination and lung function ( wet/dry ratio, elastance and arterial oxy-gen saturation) assay demonstrated that acid instillation caused a profound pathological changes and functional impairment of the lung.Besides, acid aspiration into the mouse lung caused a significant increase in neutrophil infiltration in mouse pulmonary alveoli and high concentrations of inflammatory factors (TNF-α, IL-6, CXCL1 and CXCL2) in the bronchoalve-olar lavage fluid.Conclusions We successfully established a mouse model of acid aspiration-induced lung injury, which may serve as a reliable model for longitudinally studying pulmonary immune-inflammatory mechanism in humans.
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<p><b>OBJECTIVE</b>To investigate the effect of temperature on the stability of intermediate and final products of inactivated enterovirus 71 vaccine, which was prepared in human diploid cells.</p><p><b>METHODS</b>The different batches of harvest viral cultures, the vaccine stock solutions and the final productions of inactivated enterovirus 71 vaccine were stored at different temperatures. The samples of viral culture stored at -20°C or 4°C were harvested at 0, 6, 12 and 24 months later. The samples of vaccine stock solutions stored at -20°C were harvested at 0, 6, 12 and 24 months later, and that stored at 4°C were harvested at 0, 1, 3, 6 and 12 months later. The samples of finial products were harvested at different time points (0, 6, 12 and 24 months for storing at 4°C; 0, 7, 14, 28, 42 and 60 d for storing at 25°C; 0, 3, 7, 14 and 21 d for storing at 37°C). The viral titer, antigen content, antigen purity, endotoxin content, effectiveness, pH and appearance of samples were determined, respectively. A total of 1 800 BLAB/c mice were immunized by vaccine and 150 control mice were injected by diluents without antigen via intraperitoneal. The tail vein blood (500 µl per mouse) from 1 950 mice were harvested after 4 weeks post injected. The neutralization antibody titers of the serum were tested to calculate the half effective dose (ED50) of final products. All results were analyzed using analysis of variance to compare the differences of the above indexes.</p><p><b>RESULTS</b>The viral titers of harvest viral culture of inactivated EV71 vaccine were (6.67 ± 0.13), (6.56 ± 0.09), (6.52 ± 0.04), (6.39 ± 0.16) lgCCID50/ml (CCID50, the half cell culture infective dose) after 0, 6, 12 and 24 months storage at -20°C; and (6.67 ± 0.13), (6.41 ± 0.13), (6.19 ± 0.18), (5.97 ± 0.09) lgCCID50/ml at 4°C. The viral titers reduced with time (F = 9.81 or 44.16, P < 0.05). The antigen contents of the vaccine stock solution were maintained at (3 626.67 ± 1 382.56) EU/ml within 3 months at 4°C, but were (2 080.00 ± 876.36), (951.17 ± 346.35) EU/ml at 6 and 12 months, respectively. The ED50 of the final production were (31.00 ± 2.71), (32.93 ± 3.22), (39.37 ± 3.44) and (46.04 ± 3.25) EU/ml after 0, 6, 12 and 24 months storage at 4 °C, but were (31.00 ± 2.71), (32.23 ± 2.66), (34.70 ± 1.77), (40.04 ± 2.10), (47.78 ± 1.93) and (56.97 ± 0.50) EU/ml at 0, 7, 14, 28, 42 and 60 days at 25°C, and were (31.00 ± 0.00), (36.20 ± 0.00), (41.87 ± 0.50), (53.25 ± 0.50) and (64.84 ± 0.58) EU/ml at 0, 3, 7, 14 and 21 days at 37°C, respectively. The ED50 had increased with the time by and had significantly differences compared with the beginning level (F = 28.49, 215.15 or 156.12, P < 0.05).</p><p><b>CONCLUSION</b>There is a good stability of the intermediate and final productions of inactivated enterovirus 71 (EV71) vaccines, within 24 months at -20°C or 6 months at 4°C storage for viral culture, 24 months at -20°C or 3 months at 4°C storage for stock solution and 24 months at 4°C or 28 d at 25°C or 7 d at 37°C storage for finial vaccine.</p>
Subject(s)
Animals , Humans , Mice , Drug Storage , Methods , Enterovirus A, Human , Immunization , Vaccination , Vaccine Potency , Vaccines, InactivatedABSTRACT
In light of the scarcity of reports on the interaction between HSV-1 nucleocapsid protein UL25 and its host cell proteins,the purpose of this study is to use yeast two-hybrid screening to search for cellular proteins that can interact with the UL25 protein.C9orf69,a protein of unknown function was identified.The interaction between the two proteins under physiological conditions was also confirmed by biological experiments including co-localization by fluorescence and immunoprecipitation.A preliminary study of the function of C9orf69 showed that it promotes viral proliferation.Further studies showed that C9orf69 did not influence viral multiplication efficiency by transcriptional regulation of viral genes,but indirectly promoted proliferation via interaction with UL25.
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The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.
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Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein,infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein.Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus,persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracyclinedependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of internal ribosome entry sites (IRES) on transcriptional regulation.
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The protein HTRP (human transcription regulator protein) is encoded by the differential gene htrp and induced by Herpes simplex virus type 1 (HSV-1) infection in KMB-17 cells. HTRP was found to interact with SAP30 (mSin3A Association Protein), one of the components of co-repressor complex mSin3A, which is part of the deacetylation transfer enzyme HDAC. To reveal the biological significance of the interaction between HTRP and SAP30, real- time PCR and a dual-luciferase detecting system was used. The results indicate that HTRP could inhibit the transcription of a viral promoter, whose interaction with SAP30 synergistically affects transcriptional inhibition of the viral genes, and is related to HDAC enzyme activity. ChIP experiments demonstrate that HTRP could promote HDAC activity by increasing the deacetylation level of lysine 14 and lysine 9 in histone H3.
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The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4% -5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H 1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.
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The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4%-6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.
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Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.
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The Sindbis-like virus was first discovered in China in 1986. Its complete genomic sequence consists of more than 11 000 bp encoding more than 3 700 amino acids. It contains a 5' non-transcriptional region (5'-NTR) in a non-structural region, four non-structural proteins (nsP1, nsP2, nsP3, nsP4) regions, capsids in conserved and non-conserved regions and structural E1, E2, E3, 6K regions and a 3' non-transcriptional region (3'-NTR). The Sindbis-IMB was isolated from the blood of a patient suspected to have encephalitis, and was followed by identification and passage. The virus RNA was extracted from virus supernatant in infected cells and the whole genome was divided into 12 fragments; RT-PCR was then performed to amplify the 12 fragments for complete sequencing. The results showed that the whole genomic sequence of Sindbis-IMB consists of 11 717 bp encoding 3 773 amino acids. Homology comparison with other Sindbis-like isolates demonstrated the highest similarity was the YN87448 with a variation of 1% strain isolated in Yunnan Province and the second highest to the SAAR86 strain with a variation of～1.2%.The nucleotide sequence variations were present in non-structural regions, resulting in amino acids K, E, N, R, H, and L in protein sequences in positions 230, 231, 443,781, 1 582, and 1746 in the new isolation respectively. Furthermore, three additional amino acids--glutamic acid, serine and alanine--were noted in nsp4 terminus as compared to the YN87448 isolate.
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HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.
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An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.
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The three immediate-early proteins of HSV-1, ICPO, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.