Preparation and characterization of emamectin benzoate nanocapsules based on the dual role of polydopamine.

BACKGROUND: Developing pesticide-controlled release formulations with foliage adhesion has become the focus of current research in the field of crop protection. In this study, an excellent adhesive nanocapsule loaded with emamectin benzoate (Eb@PDA) was prepared via emulsion interfacial polymerization based on the self-polymerization ability and adhesion properties of polydopamine (PDA). RESULTS: The physicochemical properties of the Eb@PDA were characterized by scanning electron microscopy, transmission electron microscopy, particle size statistics, Fourier transform infrared spectroscopy and X-ray diffraction. The Eb@PDA presented a regular spherical shape, with an average particle size of 163.8 nm. Compared with conventional formulations, it had higher pesticide-loading content (34%) and excellent adhesion onto corn leaf. In addition, Eb@PDA showed sustained-release characteristics, facilitating the release of Eb at low pH and high temperature. Eb@PDA could effectively protect Eb against photodegradation and had a longer effective period for controlling Spodoptera frugiperda and Spodoptera exigua. Furthermore, acute toxicity tests showed that the 50% lethal concentration (LC50 ) was 80.91 and 57.91 mg kg-1 at 7 and 14 days, respectively, indicating a lower toxicity of the Eb@PDA to earthworms. The cells (L02) treated with Eb@PDA showed a higher cell viability but a lower apoptosis rate (only 5.75%), demonstrating the lower cytotoxicity of the Eb@PDA. CONCLUSION: The self-prepared Eb@PDA could be used as a formulation with the advantages of slow release, UV shielding, strong leaf adhesion, superior insecticidal properties, sustained effectiveness and biosafety. It will also facilitate the development of an efficient and safe pesticide delivery system. © 2022 Society of Chemical Industry.

Preparation of eugenol nanoemulsions for antibacterial activities.

Eugenol is a versatile plant essential oil, but its high volatility and low water solubility greatly limit its application. Accordingly, this study prepared eugenol nanoemulsions by a high-speed shearing technique. Through visual inspection and a series of characterizations, including dynamic light scattering, and confocal laser scanning microscopy, the optimized formula was determined to be 5% (w/w) oil phase (eugenol) and 8% (w/w) surfactant (Tween-80), and the optimized shearing time was 5 min. The optimized nanoemulsion had good stability, small droplets (85 nm), and uniform distribution. At a concentration of 0.02 mg µL-1, the nanoemulsion showed strong inhibition against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Scanning electron microscopy (SEM) images showed severe deformation and membrane rupture of both bacteria treated by the nanoemulsion. This result was further confirmed by the leakage of proteins in both bacteria after treatment. The results of reactive oxygen species (ROS) and malondialdehyde (MDA) measurements indicated that the increased levels of ROS in both bacteria treated by the nanoemulsion triggered lipid peroxidation, thus increasing the MDA levels, ultimately causing changes in cell membrane permeability and disruption of the membrane structure. In addition, the nanoemulsion had a small effect on the proliferation and apoptosis of hepatocytes (L02) and lung cells (BEAS-2B), indicating its good biocompatibility. In this study, we developed a novel eugenol nanoemulsion with high stability and good biological activity, which may provide a promising and effective method for wound treatment in the healthcare area.

Synthesis, antibacterial evaluation, and safety assessment of CuS NPs against Pectobacterium carotovorum subsp. carotovorum.

BACKGROUND: Copper agents have been widely used in crop protection because of their unique mechanism against resistant pathogenic bacteria; however, their application brings environmental pollution and biosafety problems. Therefore, environmentally friendly copper agents have attracted attention. In this study, copper sulfide nanoparticles (CuS NPs) were prepared, characterized, analyzed for antibacterial activity and safety. RESULTS: Characterization results showed that the prepared pure CuS NPs have flake nanostructures, hexagonal crystal system, and size range from 40 to 60 nm. These CuS NPs exerted excellent antibacterial effects [median effective concentration (EC50 ) = 17 mg L-1 ] against Pectobacterium carotovorum subsp. carotovorum (Pcc) in vitro and can effectively delay and reduce bacterial infection in vivo. Antibacterial mechanism analysis revealed that CuS NPs can increase the levels of reactive oxygen species (ROS) and lipid peroxidation and destroy the structure of bacterial cells as observed through scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) spectroscopy. These NPs can also inhibit the motility of Pcc. At 7 and 14 days, the 50% lethal concentrations (LC50 ) of CuS NPs against earthworms were 1136 and 783 mg kg-1 , respectively, indicating their low acute toxicity to earthworms and environmental friendliness. Furthermore, the cells (L02) treated by CuS NPs showed relatively high cell viability (> 96%) and low apoptosis rate (only 5.2%), proving that CuS NPs had low cytotoxicity. CONCLUSION: Compared with commercial dicopper chloride trihydroxide (Cu2 (OH)3 Cl), CuS NPs could be used as a highly effective, lowly toxic, and environmentally friendly antibacterial agent. © 2021 Society of Chemical Industry.

Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70.

BACKGROUND: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS: We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA. RESULTS: Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-ß and TNF-α in transfected cells. CONCLUSIONS: Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.

[PreC/C gene-targeting RNA interference suppresses hepatitis B virus replication and expression in human hepatoma cells].

OBJECTIVE: To explore the antiviral efficacy of small interfering RNAs (siRNAs)/shRNA targeting preC/C of HBV in human hepatoma cells Huh-7 and HepG2.2.15 cells. METHODS: Three 21 nucleotide(nt) siRNAs for treating HBV preC/C gene were designed and synthesized according to the HBV genome in GenBank accession numbers (U95551); simultaneously, one 21-nt-long non-homologous siRNA was also designed randomly for negative control. They were cloned into vector pU6 for constructing shRNA-expressing plasmids pU6-C1, pU6-C2, pU6-C3 and control pU6-C4. To assess the function of siRNAs, a reporter gene system was constructed. The HBV preC/C gene was synthesized by PCR with pT-HBV1.3 as the template. The preC/C gene was then inserted into the enhanced green fluorescent protein expression vector (EGFP-N1) in order to construct the recombinant plasmid pEGFP-preC/C (E-C), which carries the EGFP reporter gene. The three shRNA-expressing plasmids-pU6-C1, pU6-C2, or pU6-C3-was each then cotransfected into Huh-7 cells along with either reporter gene expression vector E-C or the controls; or these three plasmids-pU6-C1, pU6-C2, or pU6-C3-was each cotransfected into HepG2.2.15 cells along with the controls. First, upon determination of the number of cells exhibiting EGFP expression in Huh-7cells as detected by an BH-2 fluorescence microscope and FACS-440 flow cytometry at different times after cotransfection, the investigators evaluated the inhibitory efficiency of the three shRNA-expressing plasmids by an EGFP reporter system in cultured cells. Subsequently, the expression amount of HBsAg and HBeAg in HepG2.2.15 cell supernatant at 24, 48, 72 and 96 h post-cotransfection was detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the expression of HBsAg and HBcAg at 72 h post-cotransfection in HepG2.2.15 cells. The copy level of HBV mRNA transcripts cDNA in HepG2.2.15 cells was further investigated through quantitative real-time polymerase chain reaction (real-time PCR). RESULTS: In comparison with single plasmid transfection pEGFP-N1 or E-C, fluorescence microscope examination and flow cytometry detection at 48 hours after cotransfection indicated that the expression of the reporter gene EGFP in cotransfected group Huh-7 cell involving pU6-C1, pU6-C2 or pU6-C3 resulted in an 80% reduction in EGFP signal relative to the controls (P < 0.01). It was also found through immunofluorescence that the expression of HBsAg and HBcAg in HepG2.2.15 cells was reduced markedly (P < 0.01), that the copy level of HBV mRNA transcripts cDNA as detected at 48 hours after cotransfection by quantitative real-time PCR was reduced respectively by 73.9% ± 1.2% (P = 0.029), 48.2% ± 1.8% and 35.8% ± 1.4% (P = 0.037, 0.040) relative to the control, that it conformed with that detected by fluorescence microscope/flow cytometry, ELISA, and immunofluorescence (P < 0.01). Thereby further corroborating the antiviral efficacy of RNAi. The efficacy was obvious at 48 h, reaching a peak at 72 h. CONCLUSION: For the first time it has been found that RNAi induced by siRNA/shRNA targeting HBV preC/C gene is effective and specific in inhibiting HBV replication and expression in human hepatoma cells Huh-7 and HepG2.2.15 cells. Our data suggest that RNAi may provide an effective, viable approach in gene therapy to treating major infectious diseases such as HBV/HCV/HIV infection.

Cloning and characterization of porcine interferon-δ-related genes identified by genomic database screening.

Interferon-δ (IFN-δ) belongs to type I IFN. It was first identified by Lefevre in 1993. The porcine interferon-δ (PoIFN-δ)-related sequences were reported in 2009. The IFN-δ-related sequences have not been studied as closely as IFN-α and IFN-ß. In this study, we identified 11 functional genes and 7 pseudogenes of the PoIFN-δ-related subtypes from the porcine genome database. Three of these genes were cloned, expressed, and functionally studied. PoIFN-δ-related subtypes can bind to the type I IFN receptors, activate the Janus kinase-signal transduce and activator of transcription (JAK-STAT) signaling pathway, induce IFN stimulate genes, and other cytokines. The antiviral activity of PoIFN-δ-related subtypes varies a lot in different subtypes. Among all the PoIFN-δ subtypes detected, PoIFN-δ8 has the strongest antiviral and immunomodulatory activities.

[C gene-targeting short hairpin RNA suppresses the replication and expression of hepatitis B virus in BHK-21 cells].

OBJECTIVE: The vaccines currently developed against infectious diseases fail to induce effective antiviral immune responses to control an abrupt outbreak of viral diseases epidemic in a short space of time. Hence there is an urgent need to develop emergency vaccines capable of producing prophylactic effects against infectious diseases. RNA interference (RNAi) is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes. The rapidity and uniqueness of RNAi responses can make up for the current inadequacy of antiviral preventive regimes. Here we evaluate the antiviral potential of short hairpin RNA (shRNA) targeting C (core) gene of hepatitis B virus (HBV). It plays essential roles in HBcAg encoding and viral attachment to susceptible cells during its life cycle. The present study was intended to investigate the inhibitory effect of C-specific shRNAs on HBV replication and expression in BHK-21 cells. METHODS: The reporter gene expression vector of pC-EGFP-N1 was constructed by cloning the DNA (PCR product) of HBV C into the EcoRI-HindIII sites of pEGFP-N1 to fuse C to enhanced green fluorescent protein (EGFP) for providing a reporting system for monitoring siRNA function. Plasmid pC was constructed by cloning the DNA of HBV C into the EcoRI-HindIII sites of pCDNA3.1B(-) directly under the control of cytomegalovirus promoter. Two plasmids (S1 & S2) were constructed to express shRNAs targeting C of HBV with the length of 24 nucleotide (nt) homologous in sequence to the HBV C gene. Plasmids were designed and synthesized according to the HBV genome (HBV genotype B, ayw1 subtype) of chronic hepatic B patients from 56 ethnic minorities in China. After cloning and sequencing, the investigators registered them with the GenBank accession numbers of AY517488 (CYN/2002) and AY517489 (CYN/2000), etc. Simultaneously, one of nonspecific shRNA-S3 with a length of 24 nt was also designed randomly for negative control. After cloning into vector pU6 for constructing shRNA-expressing plasmids, they were then cotransfected into BHK-21 cells along with reporter gene expression vector of pC-EGFP-N1. First, upon a determination of the number of cells exhibiting EGFP expression in BHK-21 cells as detected by an Olympus BH-2 fluorescence microscope and FACS-440 flow cytometry (Becton-Dickinson, USA) at different times after cotransfection, the investigators evaluated the gene inhibitory efficiency of both plasmids by an EGFP reporter system in BHK-21 cells. Subsequently, the antiviral efficacy of both plasmids in BHK-21 cells was further investigated by real-time quantitative polymerase chain reaction. RESULTS: In comparison with single plasmid transfection pC-EGFP-N1 or pEGFP-N1, fluorescence microscope and flow cytometry detection at 24 h post-cotransfection revealed that the expression of reporter gene EGFP in cotransfection group involving S1 or S2 and S1 + S2 cotransfection group was reduced significantly by about 90% in EGFP signal versus the control. And the EGFP expression in control plasmid cotransfected S3 or pU6 was not significantly reduced in BHK-21 cells (P < 0.01). It was found that the expression of mRNAs of HBV C and EGFP gene as detected by real-time quantitative PCR was the same as that detected by fluorescence microscope and flow cytometry (P < 0.01), thereby further corroborating the antiviral efficacy of RNAi. The antiviral efficacy extended to almost 48 hours. The results indicted that a DNA vector-based RNAi technology could effectively and specifically inhibit the replication and expression of HBV in BHK-21 cells. CONCLUSION: For the first time it has been found that RNAi induced by shRNA targeting C gene exerts an effective and unique inhibition of HBV replication and expression in BHK-21 cells. Thus RNAi may provide an effective emergency vaccine against major infectious diseases such as HBV infection.

Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice.

Foot-and-mouth disease (FMD) is a highly contagious disease that afflicts cloven-hoofed animals. The etiological agent of FMD is foot-and-mouth disease virus (FMDV). The VP1 gene of FMDV is essential during the life cycle of the virus and plays a key role in the attachment of the virus to susceptible cells. We constructed a plasmid, pCWN11, that expresses siRNAs multiple-targeting the VP1 genes of FMDV. We evaluated the gene silencing efficiency of the plasmid using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. The antiviral potential of the plasmid in BHK-21 cells and suckling mice were investigated. The results indicate that cotransfection of pCWN11 with any one of three serotypes VP1-EGFP plasmids resulted in a reduction in the EGFP signal relative to the control. Moreover, the antiviral potential induced by pCWN11 was evident during challenge with one FMDV isolate of either serotype O (HKN/2002) or serotype Asia I (YNBS/58), and the inhibition extended to almost 40 h. Furthermore, subcutaneous injection of pCWN11 in the neck made suckling mice significantly less susceptible to FMDV serotype O and Asia I.

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine.

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10(9) CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID(50) of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 x 10(9) CFU of p3D-NT56/S. cho were protected in 9 days.

Characterization and virus-induced expression profiles of the porcine interferon-omega multigene family.

Interferon-omega is a member of the type I interferon family. In this work, 8 functional porcine interferon-omega genes and 4 pseudogenes present on porcine chromosome 1 were identified in the porcine genome database by BLAST scanning. Their genetic and genomic characteristics were investigated using bioinformatics tools. Then the PoIFN-omega functional subtype genes were isolated and expressed in BHK-21 cells. The PoIFN-omega subtypes possessed about 10(4) to 10(5) units of antiviral activity per milliliter. PoIFN-omega 7 had the highest antiviral activity, about 20 times that of PoIFN-omega 4, which had the lowest antiviral activity. Differential expression of the subtypes was detected in PK15 cells and porcine peripheral blood mononuclear cells (PBMCs) in response to pseudorabies virus and poly(I).poly(C). Expression of PoIFN-omega 2/-omega 6 was up-regulated to the greatest extent by virus infection.

[Transfection of siRNA expressing plasmids targeting S gene inhibits replication and expression of hepatitis B virus in hepatic cancer cells].

OBJECTIVE: To study the inhibitive effects of transfection of siRNA expressing plasmids targeting S gene, one of the 4 open reading frames of hepatitis B virus (HBV), on the replication and expression of HBV. METHODS: Two plasmids expressing 2 siRNAs targeting S gene, one of the 4 open reading frames of HBV (S1 and S2) and one nonspecific plasmid (siRNA-S3), as negative control, with the length of 21 nt heterologous to the HBV/U95551 genome were constructed, and then transfected into the hepatic cancer cells of the line HepG2.2.15. 48 hours later, real-time PCR was used to evaluate the gene silencing efficiency and ELISA was used to detect the expression of HBsAg and hepatitis B e antigen (HBeAg), protein markers of HBV, in the supernatants. RESULTS: The inhibition rates of HBsAg and HBeAg expression of the HepG2.2.15 cells transfected with S1 were 60% and 56% respectively, those of the HepG2.2.15 cells transfected with S2 were 73% and 70% respectively, those of the HepG2.2.15 cells transfected with S1+S2 were 82% and 78% respectively, and those of the HepG2.2.15 cells transfected with S3 were not significantly different from those of the blank control group. RT-PCR showed that the mRNA expression rates of HBsAg and HBeAg in the HepG2.2.15 cells transfected with S1, S2, and S1+S2 were inhibited by 64%-88% t respectively. CONCLUSION: Transfection of the vector plasmids expressing the siRNAs targeting S gene inhibits the expression of HBsAg and HBeAg in hepatic cancer cells. RNAi may provide a viable strategy against viruses for the prevention and treatment of HBV infection.

Identification of a putative invertebrate helical cytokine similar to the ciliary neurotrophic factor/leukemia inhibitory factor family by PSI-BLAST-based approach.

Most of our knowledge of helical cytokine-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. It is hard to predict helical cytokines in invertebrates based on sequences from mammals and vertebrates, because of their long evolutionary divergence. In this article, we collected 12 kinds of fish cytokines and constructed their respective consensus sequences using hidden Markov models; then, the conserved domains region of each consensus sequence were further extracted by the SMART tool, and used as the query sequence for PSI-BLAST analysis in Drosophila melanogaster. After two filtering processes based on the properties of helical cytokines, we obtained one protein named CG14629, which shares 25% identities/46% positives to fish M17 cytokine in the half length of the N-terminus. Considering the homology between M17 and LIF/CNTF (leukemia inhibitory factor/ciliary neurotrophic factor), and the close relationship between Dome, the putative cytokine receptor in Drosophila cells, and LIFR/CNTFR (LIF receptor/CNTF receptor), the results suggest that CG14629 is a good candidate for the helical cytokine ortholog in D. melanogaster.

A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide.

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.

Detection of differential expression of porcine IFN-alpha subtypes by reverse transcription polymerase chain reaction.

The porcine interferon-alpha (IFN-alpha) multigene family is a new IFN-alpha system in recent research. Characterization of the PoIFN-alpha multigene family has been described in our previous work, and 14 functional PoIFN-alpha genes were detected in the porcine genome. In this study, we designed subtype-specific primers and consensus primers for PoIFN-alpha. The expression of PoIFN-alpha was detected using the two PCR strategies in three systems, namely, poly(I).poly(C)-DEAE-dextran-induced PK15 cells, pseudorabies virus-infected PK15 cells, and infected PK15 cells with an attenuated strain of swine fever virus, respectively. In poly(I).poly(C)-DEAE-dextran-induced PK15 cells, the expression of IFN-alpha2, -alpha3, -alpha4, -alpha8, and -alpha9 after 6-h/24-h inducement in PK15 cells were observed. In pseudorabies virus-infected PK15 cells, the expression of PoIFN-alpha2, -alpha3, -alpha8, -alpha9, -alpha10, and -alpha13 was observed after 6-h/24-h infection, and in the attenuated strain of swine fever virus-infected PK15 cells, upregulation of PoIFN-alpha2, -alpha3, -alpha4, -alpha8, -alpha9, and -alpha10 was detected. The results of realtime quantitative PCR analysis suggested that the expression was time-dependent in pseudorabies virus/poly(I).poly(C)-DEAE-dextran-induced PK15 cells, but in the attenuated swine fever virus-infected PK15 system, the expression level of IFN-alpha subtypes was not obviously time dependent.

Alpha interferon is a powerful adjuvant for a recombinant protein vaccine against foot-and-mouth disease virus in swine, and an effective stimulus of in vivo immune response.

The adjuvant effect of porcine interferon-alpha (PoIFN-alpha) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV. The PoIFN-alpha gene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant, injected together with low-dose IgG-FMDV. This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T-cell-mediated immune responses, whereas only a modest humoral and cellular response was observed with low-dose vaccine alone. As an adjuvant for the protein vaccine, PoIFN-alpha induced strong inflammatory cytokines production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses. The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo. In response to viral challenge, all control animals developed viremia and lesions, whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge (dpc). Our studies indicate that porcine IFN-alpha is a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.

Characterization of the porcine alpha interferon multigene family.

The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-alpha (PoIFN-alpha) multigene family. Fourteen functional PoIFN-alpha genes and two PoIFN-alpha pseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes. A phylogenetic tree of the porcine IFN-alpha gene family defined the evolutionary relationship of the various subtypes. In addition, analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome. Eight PoIFN-alpha subtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line. We detected the level of transcription by real-time quantitative RT-PCR analysis. The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus (VSV) and PK 15 cells/Pseudorabies Virus (PRV) respectively. We found the antiviral activities of intact PoIFN-alpha genes are approximately 2-50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15-55 times higher in PK 15 cells. There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility.

[Expression in Escherichia coli of hepatitis B virus genes from minority nationality patients in Yunnan province with chronic hepatitis B and their antigenicity].

OBJECTIVE: To investigate the expression in Escherichia coli (E. coli) of hepatitis B virus (HBV) genes from minority nationality patients in Yunnan province with chronic hepatitis B (CHB) and their antigenicity. METHODS: Peripheral blood samples were collected from 25 minority nationality patients with CHB in Yunnan province. Twenty-five CHB patients of Han nationality in Yunnan were used as controls. The full length HBV preS2/S and C genes were amplified by PCR, cloned, sequenced, and inserted into the prokaryotic expression vector p lambda PR. The recombinant plasmids p lambda PR-S2S and p lambda PR-C were transfected into E. coli of the line TOP10. The expression of the non-fusion proteins encoded by the HBV preS2S and C genes was determined by sodium dodecyl sulphate polyacrlamide gel electrophoresis (SDS-PAGE) and Western blotting. The antigenicity of the non-fusion proteins was examined by ELISA. Fifty samples of serum of patients with hepatitis A, 50 samples of serum of patients with hepatitis C, and 50 samples of serum of healthy blood donors were used as controls in the study of the antigenicity of non-fusion proteins. RESULTS: SDS-PAGE showed that the recombinant plasmids p lambda PR-S2S and p lambda PR-C were both stably and highly expressed in the E. coli for all 50 CHB patients. The molecular weights of the expressed non-fusion proteins, with a concentration of 16% and a purity of 50%, were between 31,000 and 21,000. Western blotting and ELISA showed that the purified recombinant non-fusion proteins reacted strongly with the antibodies HBpreS2S/SAb and HBcAb and the serum of CHB patients, but there was no cross-activity between the non-fusion proteins and all the serum samples of controls with HA and HC, and normal controls. CONCLUSION: The HBV preS2S and C genes from the minority nationality patients with CHB can be stably and highly expression in E. coli. The non-fusion proteins encoded by the HBV preS2S and C genes have high antigenicity. These findings have potential values in development of HB vaccines.

[Identification of hepatitis B virus genotypes in patients with chronic hepatitis B from different nationalities in ethnic minority areas in Yunnan Province, China].

OBJECTIVE: To determine whether there is evolutionary difference in hepatitis B virus (HBV) genotypes among the patients with chronic hepatitis B (CHB) of different nationalities and its clinical significance. METHODS: Peripheral blood samples were collected from 50 CHB patients, 25 of diverse nationalities and 25 of Han nationality from the ethnic minority regions in Yunnan Province, China, The HBV preS2/S (pre S2/S) and C genes were amplified by PCR. The PCR products were inserted into the vector pBluescriptIISK (pBS). The cloned preS2/S and C genes were sequenced. RESULTS: The sequences of HBV preS2/S and C genes from the 50 patients were 846 (with 49.1% of GC) and 552 (with 46.1% of GC) nucleotides (nt) in length, and encoded 281 and 183 amino acids (aa) respectively. These findings were registered in GenBank Accession Numbers: AY517619, AY517620, AY517488, AY517489, AY517598, AY517599. Compared with the HBV and subtype sequences in the GenBank database, the HBV preS2/S and C genes among all the subjects were homologous to ayw1 in sequence by 97.5% - 98.6% and 94.5% - 97.8% respectively. The "a" determinant region of S genes in all cases were found to be Arginine (AGA) and Lysine (AAA) at corresponding aa 122nd and 160th respectively. HBV genotype B was identified in all patients with CHB (ayw1 subtype). Genotypes A, C, D, E, F, G, and H were not detected in any of them. The quasi-species nature of the HBV in the sera was observed in 2 of the 50 samples examined (4%). There was not a significant difference in the prevalence of HBV genotype B between the 25 diverse nationality patients and the 25 control Han nationality patients (P > 0.05). In the 50 CHB patients, the preS2/S genes were identified to have aa substitutions at the positions R124K (1.1%), L172P (1.3%), M306T (1.5%), and I361M (1.6%), with a frequency of more than 1%. In all subjects, the frequency of aa G145R (0.4%) substitutions was less than 1%. In all subjects, nt variations of C genes caused aa substitutions among aa 27 - 63, 80 - 110, and 135 - 153 involved in T and B cell epitopes. In 45 CHB patients, C genes was identified to have aa substitutions at the positions V 27, N38, V63, Q135, and A147, due to nt variations of 1979A to G, 2012T to A, 2088G to T, 2304C to A, and 2339A to G transitions respectively. The frequency of aa substitutions of C genes was more than 1%. Whereas as for the other 5 severe CHB patients, the C gene variations of A to G, and A to C transitions at nt positions 2159 and 2189 led to aa substitutions of S to G and I to L at positions G87 and L97. No insertion or deletion was found in preS2/S and C regions. HBV genotype B was not relevant to different nationalities (all P > 0.05). CONCLUSION: It is the first time that the genotype of the HBV epidemic strains in the ethnic minority areas of Yunnan Province has been identified as genotype B subtype ayw1. The HBV genotype B is not related with nationality. A novel genotyping method by using PCR, gene cloning, followed by DNA sequencing that can identify all major genotypes has been developed. HBV genotype B is the geographic original strain in this area and is correlated with the severity of liver diseases and curative effect. HBV viral is the only significant variable associated with the CHB patients' prognosis.

Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.