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Eucalyptus is a large genus of the Myrtaceae family with high value in various fields of industry. Recently, attention has been focused on the functional properties of Eucalyptus extracts. These extracts have been traditionally used to combat various infectious diseases, and volatile oils are usually considered to play a major role. But the positive effects of non-volatile acylphloroglucinols, a class of specialized metabolites with relatively high content in Eucalyptus, should not be neglected. Herein, non-volatile acylphloroglucinols from leaves of Eucalyptus robusta were evaluated for their abilities to inhibit Zika virus (ZIKV) which is associated with severe neurological damage and complications. The results showed eucalyprobusone G, a new symmetrical acylphloroglucinol dimer, possessed the significant ability to inhibit ZIKV without inducing cytotoxicity. The EC50 values of eucalyprobusone G against the African lineage (MR766) and Asian lineage (SZ-WIV01) of ZIKV were 0.43 ± 0.08 and 10.10 ± 3.84 μM which were 110 times and 5.8 times better than those of the reference compound ribavirin, respectively. Further action mode research showed that eucalyprobusone G impairs the viral binding and RdRp activity of NS5. The results broaden the functional properties of Eucalyptus robusta and indicate acylphloroglucinol dimers could be developed as anti-ZIKV agents.

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.

P-selectin glycoprotein ligand-1 (PSGL-1) has been proved to serve as the functional receptor for enterovirus 71 (EV71). We found the abundant expression of PSGL-1 on monocyte-derived dendritic cells (MDDCs). However, we have previously demonstrated that MDDCs did not support efficient replication of EV71. Dendritic cells (DCs) have been described to be subverted by various viruses including EV71 for viral dissemination, we thus explore the potential contribution of PSGL-1 on DC-mediated EV71 transmission. We found that the cell-surface-expressing PSGL-1 on MDDCs mediated EV71 binding, and intriguingly, these loaded-viruses on MDDCs could be transferred to encountered target cells; Prior-treatment with PSGL-1 antibodies or interference with PSGL-1 expression diminished MDDC-mediated EV71 transfer and rescued virus-induced cell death. Our data uncover a novel role of PSGL-1 in DC-mediated EV71 spread, and provide an insight into blocking primary EV71 infection.

To obtain full-length FKN nucleotide sequences of homonids including human, chimpanzee, gorilla, orangutan and gibbon, and Old World Monkeys including macaque and leaf monkey and make phylogenetic analysis, three exons of FKN were amplified by degenerated PCR using obtained peripheral blood cells DNA as template which was extracted from homonids and Old World Monkeys. After extracting and purifying from agarose gels, PCR products were sequenced and then spliced by using BioEdit. All the FKN sequences were aligned and compared the percent identity by using DNAStar. The phylogenetic tree was constructed using maximum evolution approach in Mega. The negative selection sites were analyzed by using Datamonkey. There is an apparent 30 bp nucleotides deletion mutation in homonids FKN comparing to that of Old World Monkeys besides other point mutations. Homology of nucleotide sequence between human and chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey is 99.2%, 98.4%, 98.1%, 96.5%, 95.9% and 93.8%, respectively. Homology of amino acid sequence of them is 98.5%, 98.0%, 97.7%, 94.7%, 93.7% and 90.5%, respectively. In the same time, the genealogical relationship of human is a lot closer to chimpanzee than it is to gorilla and other apes. It is generally agreed that the evolution rule of FKN gene is in accord with that of the higher primates. In addition, Datamonkey shows that there are 3 negative selection sites 53Q, 84D and 239N in FKN. The full-length FKN gene of human, chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey were sequenced successfully, and the FKN sequences analysis lays the foundation for further studying its evolution in immunological function in higher primates and the relation between its structure and function.

Four hundred forty-six serum samples from HBsAg-positive chronic hepatitis B patients were collected from five areas in China (eastern coastal city, Shanghai; southwestern inland city, Chengdu; mid-inland city, Wuhan; southern island city, Haikou; and northeastern city, Changchun). Precore stop codon variants (e-minus mutants) were screened using a rapid method of polymerase chain reaction (PCR) amplification of a precore and partial core gene fragment (nucleotides 1785–2172), followed by dot-blot hybridization with specific oligonucleotide probes (M0, and M1 + M2). The sequence of the M0 probe covered the distal precore region of wild-type virus (nucleotides 1887–1908), and the sequences of the M1 and M2 probes were from sequences mutated at nt. 1898, (TGGTAG) with or without additional change at nt. 1901. A significantly lower incidence of the precore stop codon was found in anti-HBe-positive serum samples from Haikou (17.6%), whereas in other areas the percentages of this mutation in anti-HBe positive sera ranged from 47.4% to 78.9%. In HBeAg-positive samples, the rate of e-minus mutant in coexistence with wild-type virus was low in specimens from Haikou (9.5%) and Changchun (2.9%) compared to other areas in China. In contrast, coexistence of mutant and wild-type virus was frequently detected in samples from Wuhan (50.0%). J Med Virol 51:85–89, 1997. © 1997 Wiley-Liss, Inc.

One-day-old ducklings experimentally infected with duck hepatitis B virus (DHBV) were found to be immunologically tolerant to virus antigens (DHBsAg, DHBcAg), with no humoral or cellular immune responses being detected. When immunized with virus antigens in Freund's complete adjuvant, no immune responses could be induced. Rabbit anti-DHBs sera were complexed to a solid matrix (Staphylococcus aureus Cowan A strain) and purified DHBsAg was bound to this complex to form a solid matrix antibody-antigen (SMAA) complex. This SMAA was used as an immunogen to immunize the tolerant ducks. After three injections, in 12 of 17 ducks serum DHBV DNA became absent and serum DHBsAg was cleared. In eight of 16 ducks, low titres of anti-DHBs could be detected. The SMAA approach shows potential for application in immunoregulatory treatment for chronically infected hepatitis B patients.

Several examples have shown that C3d3, when fused to a corresponding antigen, had a strong adjuvant effect on certain specific antibody production. In a previous study, we attempted to prove that this was the case of the human chorionic gonadotrophin beta chain (hCGbeta)-induced immunity following DNA vaccination. However, we found that C3d3 when fused to hCGbeta inhibited rather than enhanced the antigen-specific immune response. In the present study, using hCGbeta DNA vaccine preparations, we demonstrated that C3d3 inhibited the antigen-specific humoral antibody response and several other immune responses, such as the hCGbeta specific lymphoproliferation. Such inhibitory effects of C3d3 were not related to the expression level of the target protein, the gender of the test mice, or the vector used. Contrastingly, C3d3 fused with the envelope protein of hepatitis B virus (PreS2/S) used as a control system resulted in the enhancement of both humoral and cell-mediated antigen-specific immune responses against HBV-preS2/S, which was consistent with other groups' adjuvant-effect findings. We further showed that the mechanisms involved in the inhibitory effect of C3d3 might be possible due to impairing the function of antigen presenting B lymphocytes and reducing the expression of transcription factors (T-bet and GATA-3) and cytokine IL-4. Collectively, unlike its usual expected adjuvant function, the fusion of C3d3 with the tumor-associated antigen hCGbeta was found to inhibit both humoral and cell-mediated antigen-specific immune responses. These findings indicate that research concerning tumor immune escapes and vaccine designs require further extensive attention.

To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form.One to four copies of C3d-P28 coding gene, amplified by PCR and modified by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]). HBV-preS2/S coding sequence was then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 microg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 microg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA, respectively.HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the groups primed with pVAON33-S2/S-P28.4 (P0.01). After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2/S-P28 fusions than that with DNA expressing preS2/S only (P0.05). Interestingly, mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals. The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P0.01).Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.

Recently, increasing attention is being paid to the viral etiology of atherosclerosis (AS). Human cytomegalovirus (HCMV) is thought to be the most possible etiological factor of AS. In our study, artery vascular tissue specimens derived from 75 patients with AS were studied for detection of HCMV immediately early (IE) and later (L) gene fragments by polymerase chain reaction (PCR) and in situ hybridization; sera collected from the same patients were examined for HCMV specific IgG and IgM by enzyme-linked immunosorbent assay (ELISA). Twenty two normal arterial tissues and sera were used as controls. We found that the positive rate of HCMV L and IE gene fragments were significantly higher in AS patients than those in controls. HCMV DNA were mainly observed in nucleus of endothelial cells and muscularis under intima as well as smooth muscle of media of AS area, rarely found in controls. These results suggested that HCMV infection may relate to the pathogenesis of AS; and the artery itself may be the site of HCMV latency. In addition, higher levels of HCMV IgG and IgM were found to be associated with virus persistence, indicating that a periodically active latent infection or a continuously active infection is presented in AS patients.

Background: Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71–host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. Methods: EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. Results: MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. Conclusion: MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection.

Recently, more and more studies suggested a new, possible role of markers of infection and inflammation beyond traditional cardiovascular risk factors, in the development and progression of atherosclerosis. Our aim was to study the mechanism of MCMV infection contributing to atherogenesis. ApoE knockout C57BL/6 mice were fed with hypercholesterol diet and inoculated ip with 3×10 pfu/100 μl MCMV. The plasma concentrations of cholesterol and the expression of MCP-1, GRO-α, FKN, RANTES, IL-4 and IFN-γ were detected in the atherosclerosis lesion. Results showed that the concentration of plasma cholesterol increased over 500 mg/dl and distinct atherosclerotic plaque could be observed in aorta after 12 weeks with hypercholesterol diet. In MCMV infection, impressive atherosclerotic plaques were observed in the aorta and the infiltration of inflammatory cells in endothelium were significantly enhanced. RT-PCR results showed that MCMV infection could induce the expression of MCP-1, GRO-α, FKN and RANTES on the atherosclerotic lesion and correlated with the atherosclerotic plaque. The local expression of cytokine IFN-γ was enhanced in MCMV infection. Our findings showed that MCMV infection contributed to atherogenesis probably by inducing expression of chemokines and immune response deviation to Th1 response. Surely, there will be other pathways involved the atherogenesis.