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Tick-borne encephalitis virus (TBEV) is a leading cause of human neurological infection in many parts of Europe and Asia. Although several TBEV isolates have been reported, current understanding of the biological characteristics of a Chinese strain is limited. In this study, a Far-Eastern strain of TBEV designated WH2012 was isolated in northern China. Its genome has been sequenced and found to be closely related to other Chinese TBEV isolates. Human cell lines of neural origin exposed to WH2012 showed cytopathic effects and WH2012 replicated most efficiently in human neuroblastoma cells SK-N-SH. In addition, WH2012 possessed a pathogenic potential in the mouse model, characterized by inducing a complete paralysis in the hindlimbs with a fatal outcome. We herein describe the first data regarding biological properties of TBEV from China. This study may help future research on pathogenic mechanisms of the neurological disease induced by TBEV infection in China.

Background Tick-borne encephalitis virus (TBEV) is one of the most important flaviviruses that targets the central nervous system (CNS) and causes encephalitides in humans. Although neuroinflammatory mechanisms may contribute to brain tissue destruction, the induction pathways and potential roles of specific chemokines in TBEV-mediated neurological disease are poorly understood. Methods BALB/c mice were intracerebrally injected with TBEV, followed by evaluation of chemokine and cytokine profiles using protein array analysis. The virus-infected mice were treated with the CC chemokine antagonist Met-RANTES or anti-RANTES mAb to determine the role of RANTES in affecting TBEV-induced neurological disease. The underlying signaling mechanisms were delineated using RANTES promoter luciferase reporter assay, siRNA-mediated knockdown, and pharmacological inhibitors in human brain-derived cell culture models. Results In a mouse model, pathological features including marked inflammatory cell infiltrates were observed in brain sections, which correlated with a robust up-regulation of RANTES within the brain but not in peripheral tissues and sera. Antagonizing RANTES within CNS extended the survival of mice and reduced accumulation of infiltrating cells in the brain after TBEV infection. Through in vitro studies, we show that virus infection up-regulated RANTES production at both mRNA and protein levels in human brain-derived cell lines and primary progenitor-derived astrocytes. Furthermore, IRF-3 pathway appeared to be essential for TBEV-induced RANTES production. Site mutation of an IRF-3-binding motif abrogated the RANTES promoter activity in virus-infected brain cells. Moreover, IRF-3 was activated upon TBEV infection as evidenced by phosphorylation of TBK1 and IRF-3, while blockade of IRF-3 activation drastically reduced virus-induced RANTES expression. Conclusions Our findings together provide insights into the molecular mechanism underlying RANTES production induced by TBEV, highlighting its potential importance in the process of neuroinflammatory responses to TBEV infection. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0665-9) contains supplementary material, which is available to authorized users.

Human bocavirus (HBoV) mainly infects young children. Although many infected children suffer from respiratory or gastroenteric tract diseases, an association between HBoV and these diseases is not definite. Because modulation of type I IFN is crucial for viruses to establish efficient replication, in this study, we tested whether HBoV modulates type I IFN production. We observed that a nearly full-length HBoV clone significantly reduced both Sendai virus (SeV)- and poly(deoxyadenylic-thymidylic) acid-induced IFN-β production. Further study showed that NP1 blocked IFN-β activation in response to SeV, poly(deoxyadenylic-thymidylic) acid, and IFN-β pathway inducers, including retinoic acid-inducible protein I, mitochondrial antiviral signaling protein, inhibitor of κB kinase ε, and TANK-binding kinase 1. In addition, NP1 interfered with IRF-3–responsive PRD(III-I) promoter activated by SeV and a constitutively active mutant of IRF-3 (IRF-3/5D). Although NP1 suppressed the IRF-3 pathway, it did not affect IRF-3 activation processes, including phosphorylation, dimerization, and nuclear translocation. Coimmunoprecipitation assays confirmed the interaction between NP1 and IRF-3. Additional deletion mutagenesis and coimmunoprecipitation assays revealed that NP1 bound to the DNA-binding domain of IRF-3, resulting in the interruption of an association between IRF-3 and IFNB promoter. Altogether, our results indicate that HBoV NP1 blocks IFN production through a unique mechanism. To our knowledge, this is the first study to investigate the modulation of innate immunity by HBoV. Our findings suggest a potential immune-evasion mechanism used by HBoV and provide a basis for better understanding HBoV pathogenesis.

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α–mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α–mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α–mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.

Continuous efforts have been made to develop a prophylactic vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, two recombinant baculoviruses, vAc-N and vAc-S, were constructed, which contained the mammalian-cell activate promoter element, human elongation factor 1alpha-subunit (EF-1alpha), the human cytomegalovirus (CMV) immediate-early promoter, and the nucleocapsid (N) or spike (S) gene of bat SARS-like CoV (SL-CoV) under the control of the CMV promoter. Mice were subcutaneously and intraperitoneally injected with recombinant baculovirus, and both humoral and cellular immune responses were induced in the vaccinated groups. The secretion level of IFN-gamma was much higher than that of IL-4 in vAc-N or vAc-S immunized groups, suggesting a strong Th1 bias towards cellular immune responses. Additionally, a marked increase of CD4 T cell immune responses and high levels of anti-SARS-CoV humoral responses were also detected in the vAc-N or vAc-S immunized groups. In contrast, there were significantly weaker cellular immune responses, as well as less antibody production than in the control groups. Our data demonstrates that the recombinant baculovirus can serve as an effective vaccine strategy. In addition, because effective SARS vaccines should act to not only prevent the reemergence of SARS-CoV, but also to provide cross-protection against SL-CoV, findings in this study may have implications for developing such cross-protective vaccines.

Virus-like particles (VLPs) represent a promising vaccine against severe acute respiratory syndrome coronavirus (SARS CoV). In this study, recombinant baculovirus vAcS and vAcME were constructed to express the S protein and the M and E proteins of SARS CoV, respectively. Electron microscope analysis demonstrated the formation of VLPs in vAcME and vAcS coinfected insect cells. Mice immunized four times with VLPs developed high antibody titres against SARS CoV. In addition, VLPs elicited cell-mediated immunity as demonstrated by enhanced interferon-gamma and interleukin-4 production. VLPs also conferred protective immunity against the infection of Spike protein pseudotyped murine leukaemia virus. Our findings demonstrate that SARS CoV VLPs are immunogenic and can elicit strong SARS CoV-specific humoral and cellular immune responses in mice. This is the first study describing the immunogenicity of SARS CoV VLPs, providing valuable data for developing a protective vaccine against SARS CoV infection.

DNA vaccines induce humoral and cellular immune responses in animal models and humans. To analyze the immunogenicity of the severe acute respiratory syndrome (SARS) coronavirus (CoV), SARS-CoV, spike DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2), DNA vaccine plasmids pcDNA-S and pcDNA-IL-2 were constructed and inoculated into BALB/c mice with or without pcDNA-IL-2 by using three different immunization routes (the intramuscular route, electroporation, or the oral route with live attenuatedSalmonella entericaserovar Typhimurium). The cellular and humoral immune responses were assessed by enzyme-linked immunosorbent assays, lymphocyte proliferation assays, enzyme-linked immunospot assays, and fluorescence-activated cell sorter analyses. The results showed that specific humoral and cellular immunities could be induced in mice by inoculating them with SARS-CoV spike DNA vaccine alone or by coinoculation with IL-2-expressing plasmids. In addition, the immune response levels in the coinoculation groups were significantly higher than those in groups receiving the spike DNA vaccine alone. The comparison between the three vaccination routes indicated that oral vaccination evoked a vigorous T-cell response and a weak response predominantly with subclass immunoglobulin G2a (IgG2a) antibody. However, intramuscular immunization evoked a vigorous antibody response and a weak T-cell response, and vaccination by electroporation evoked a vigorous response with a predominant subclass IgG1 antibody response and a moderate T-cell response. Our findings show that the spike DNA vaccine has good immunogenicity and can induce specific humoral and cellular immunities in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances the humoral and cellular immune responses. Different vaccination routes also evoke distinct immune responses. This study provides basic information for the design of DNA vaccines against SARS-CoV.

Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS. It is believed that SARS-CoV originates from wild animals. We have developed a multitarget real-time Taqman reverse transcription-PCR (RT-PCR) assay for the quantitative detection of SARS-CoV. The sequences of the Taqman probes with a minor groove binder and the corresponding primers were based on the sequences of the N gene, open reading frame (ORF) 3, and ORF 8. The overall linear range of this assay was from at least 10 1 to 10 6 copies per reaction, and the detection limit could reach less than 10 copies per reaction. The quantification results for SARS-CoV from cell culture correlated well with those of the RT-PCR by using any two of the three sets of primer and probe used in this assay. However, the results of quantification of SARS-CoV obtained by using a few available throat swab specimens from SARS patients and the N gene as the target were almost 10 times higher than those obtained by using ORF 3 and ORF 8. Using this assay, we also detected an apparently SARS-CoV-related coronavirus in the throat swab specimens from masked palm civets in the west part of Hubei Province, People's Republic of China.

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro . Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.

The pathogenesis of SARS coronavirus (CoV) remains poorly understood. In the current study, two recombinant baculovirus were generated to express the spike (S) protein of SARS-like coronavirus (SL-CoV) isolated from bats (vAcBS) and the envelope (E) and membrane (M) proteins of SARS-CoV, respectively. Co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (BVLPs) as demonstrated by electron microscopy. Incorporation of S protein of vAcBS (BS) into VLPs was confirmed by western blot and immunogold labeling. Such BVLPs up-regulated the level of CD40, CD80, CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-alpha in immature dendritic cells (DCs). Immune responses were compared in immature DCs inoculated with BVLPs or with VLPs formed by S, E and M proteins of human SARS-CoV. BVLPs showed a stronger ability to stimulate DCs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of IL-6 and TNF-alpha. Further study indicated that IFN-gamma+ and IL-4+ populations in CD4+ T cells increased upon co-cultivation with DCs pre-exposed with BVLPs or SARS-CoV VLPs. The observed difference in DC-stimulating activity between BVLPs and SARS CoV VLPs was very likely due to the S protein. In agreement, SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than SARS-CoV S DNA in mice. Our data have demonstrated for the first time that SL-CoV VLPs formed by membrane proteins of different origins, one from SL-CoV isolated from bats (BS) and the other two from human SARS-CoV (E and M), activated immature DCs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. Finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of SARS-like CoV.