Characterization of Gut Microbiota Compositions along the Intestinal Tract in CD163/pAPN Double Knockout Piglets and Their Potential Roles in Iron Absorption.

The gut microbiota is known to play a role in regulating host metabolism, yet the mechanisms underlying this regulation are not well elucidated. Our study aimed to characterize the differences in gut microbiota compositions and their roles in iron absorption between wild-type (WT) and CD163/pAPN double-gene-knockout (DKO) weaned piglets. A total of 58 samples along the entire digestive tract were analyzed for microbial community using 16S rRNA gene sequencing. The colonic microbiota and their metabolites were determined by metagenomic sequencing and untargeted liquid chromatography-mass spectrometry (LC-MS), respectively. Our results showed that no alterations in microbial community structure and composition were observed between DKO and WT weaned piglets, with the exception of colonic microbiota. Interestingly, the DKO piglets had selectively increased the relative abundance of the Leeia genus belonging to the Neisseriaceae family and decreased the Ruminococcaceae_UCG_014 genus abundance. Functional capacity analysis showed that organic acid metabolism was enriched in the colon in DKO piglets. In addition, the DKO piglets showed increased iron levels in important tissues compared with WT piglets without any pathological changes. Pearson's correlation coefficient indicated that the specific bacteria such as Leeia and Ruminococcaceae_UCG_014 genus played a key role in host iron absorption. Moreover, the iron levels had significantly (P < 0.05) positive correlation with microbial metabolites, particularly carboxylic acids and their derivatives, which might increase iron absorption by preventing iron precipitation. Overall, this study reveals an interaction between colonic microbiota and host metabolism and has potential significance for alleviating piglet iron deficiency. IMPORTANCE Iron deficiency is a major risk factor for iron deficiency anemia, which is among the most common nutritional disorders in piglets. However, it remains unclear how the gut microbiota interacts with host iron absorption. The current report provides the first insight into iron absorption-microbiome connection in CD163/pAPN double knockout piglets. The present results showed that carboxylic acids and their derivatives contributed to the absorption of nonheme iron by preventing ferric iron precipitation.

Recombinant DNA vaccine of Hantavirus Gn and LAMP1 induced long-term immune protection in mice.

BACKGROUND: Prophylaxis is widely adopted the best choice against Hemorrhagic fever with renal syndrome (HFRS) caused by Hantavirus. However, loss of memory immune response maintenance remains as major shortcoming in current HFRS vaccine. A recombinant DNA vaccine, pVAX-LAMP/Gn was previously proved efficient, requiring long-term evaluations. METHODS & RESULTS: Immune responses of Balb/c mice were assessed by specific and neutralizing antibodies, interferon-γ ELISpot assay, and cytotoxic T-lymphocyte cytotoxicity assay. HTNV-challenge assay identified long-term protection. Safety was confirmed by histological and behavioral analysis. Epitope-spreading phenomenon was noted, revealing two sets of dominant T-cell epitopes cross-species. CONCLUSION: pVAX-LAMP/Gn established memory responses within a long-term protection. Lysosome-targeted strategy showed promise on Gn-based DNA vaccine and further investigations are warranted in other immunogenic Hantaviral antigens.

Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection.

Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.

The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms.

miR-365, a novel negative regulator of interleukin-6 gene expression, is cooperatively regulated by Sp1 and NF-kappaB.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in host defense. IL-6 expression can be regulated at both a transcriptional and a post-transcriptional level. We used a combination of bioinformatics and experimental techniques to demonstrate that the miR-365 is a direct negative regulator of IL-6. Overexpression of miR-365 mimics decreased activity of a luciferase reporter containing the IL-6 3'-UTR and led to repression of IL-6 protein. In contrast, ectopic expression of a miR-365 inhibitor elevated IL-6 expression. The negative regulation of miR-365 was strictly dependent on a microRNA binding element in the 3'-UTR of IL-6 mRNA. Deletion mutant analysis of the miR-365 promoter showed that two transcription factors, Sp1 and NF-κB, are essential for the transcriptional regulation of miR-365. We also demonstrate that the MAPK/ERK pathway contributes to the regulation of miR-365. Furthermore, miR-365 exhibited a greater negative regulatory effect on IL-6 than hsa-let-7a, a previously identified microRNA negatively regulating IL-6. Taken together, our results show that miR-365 is a novel negative regulator of IL-6.

[Construction of herpes simplex virus-1 virion protein 22-mediated microdystrophin gene recombinant adenovirus and study on the property of protein transduction].

OBJECTIVE: To construct the recombinant adenovirus containing herpes simplex virus-1 virion protein (VP) 22 and human microdystrophin gene, then the adenovirus was transfected into C2C12 myoblast and studied on the property of protein transduction with VP22-mediated microdystrophin in C2C12 myoblast. METHODS: The full-length VP22 cDNA was obtained from recombinant plasmid pSINrep5-VP22 with PCR, and the product was directionally inserted into pShuttle-CMV to acquire the plasmid pCMV-VP22. Microdystrophin cDNA was obtained from recombinant plasmid pBSK-micro digested with restrictive endonuclease NotI, and the product was directionally inserted into pCMV-VP22 to acquire the plasmid pCMV-VP22-MICDYS. The plasmid of pCMV-VP22-MICDYS was lined with Pme I, and the fragment containing VP22-microdystrophin was reclaimed and transfected into E1 coli BJ5183 with plasmid pAdeasy-1. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the cytopathic effect of cells and by PCR method to acquire the recombinant adenovirus Ad-VP22-MICDYS. Finally, the C2C12 myoblast were transfected with the recombinant adenovirus Ad-VP22-MICDYS and Ad-MICDYS, and the expression of microdystrophin was detected by RT-PCR, Western blot and immunocytochemistry. RESULTS: The recombinant adenovirus including VP22 and microdystrophin gene was successfully constructed. VP22 transferred VP22-microdystrophin fused protein from infected C2C12 myoblast into uninfected cells and enhance the expression of microdystrophin in myoblast. CONCLUSIONS: Recombinant adenovirus containing VP22 and microdystrophin gene was constructed successfully. VP22 can enhance the expression with microdystrophin in myoblast. It lays the foundation for further studying on VP22-mediated recombinant including microdystrophin gene to cure Duchenne muscular dystrophy.

[Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice].

Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.

[Co-expressed GP5 and M proteins of porcine reproductive and respiratory syndrome virus can form heterodimers].

In order to investigate the characterization of in vitro co-expressed GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV), eukaryotic expression plasmids pCI-ORF5 (expressing GP5 protein alone), pCI-ORF6 (expressing M protein alone), and pCI-ORF5/ORF6 (co-expressing GP5 and M proteins) were constructed. After transient transfection, Western blot analysis under nonreducing condition demonstrated that co-expressed GP5 and M proteins could form disulfide-linked heterodimers (GP5-M) in transiently transfected BHK-21 cells. To further study the influence of GP5-M heterodimers formation on the subcellular localizations of GP5 or M proteins, green fluorescence protein (EGFP) and red fluorescence protein (RFP) were used as markers. The results of fluorescence distribution showed that co-expressed GP5-EGFP chimera and M-RFP chimera boosted the transport of GP5 from the endoplasmic reticulum (ER) to the Golgi complex, indicating that the formation of GP5-M heterodimers may be involved in posttranslational modification, transport, and subcellular localization of GP5. These results presented here lay foundation to further study the molecular mechanism of GP5-M heterodimer formation and its role in protective immunity of PRRSV.

[Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1].

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.

[Bias of base composition and codon usage in pseudorabies virus genes].

The complete sequence of the Pseudorabies Virus (PRV) genomic DNA has not yet been determined, primarily because of the high content of G + C nucleotides of about 74%. We examined the base composition and codon usage of the 68 known PRV genes. As a result, we found a strong bias towards GC-rich codons especially NNC or NNG (N represents any one of four nucleotides) in PRV genes. This demonstrated that the usage bias of synonymous codon and amino acid is the main cause of the high G + C content of PRV. The results showed that the genome regions adjacent UL48, UL40, UL14, IE180 genes where the G + C content occurs as pronounced waves are corresponding to the replication origins. It was also found that the codon usage patterns of regulatory genes are apparently different from other PRV genes. A corresponding analysis of amino acid compositions indicated that the bias of codon usage could be related to the differences of gene function.

[Expression of GP5-M fusion protein of porcine reproductive and respiratory syndrone virus (PRRSV) and establishment of ELISA diagnose based on the recombinant fusion protein].

The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.

[High expression of the foot-and-mouth disease's structural protein P1 in Escherichia coli and analysis of its biology activity].

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.

[Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus].

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.

DNAskew: statistical analysis of base compositional asymmetry and prediction of replication boundaries in the genome sequences.

Sueoka and Lobry declared respectively that, in the absence of bias between the two DNA strands for mutation and selection, the base composition within each strand should be A=T and C=G (this state is called Parity Rule type 2, PR2). However, the genome sequences of many bacteria, vertebrates and viruses showed asymmetries in base composition and gene direction. To determine the relationship of base composition skews with replication orientation, gene function, codon usage biases and phylogenetic evolution, in this paper a program called DNAskew was developed for the statistical analysis of strand asymmetry and codon composition bias in the DNA sequence. In addition, the program can also be used to predict the replication boundaries of genome sequences. The method builds on the fact that there are compositional asymmetries between the leading and the lagging strand for replication. DNAskew was written in Perl script language and implemented on the LINUX operating system. It works quickly with annotated or unannotated sequences in GBFF (GenBank flatfile) or fasta format. The source code is freely available for academic use at http://www.epizooty.com/pub/stat/DNAskew.

[Construction and growth properties of recombinant pseudorabies virus expressing modified enhanced green fluorescent protein].

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence w as amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of P RV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/LacZ(+), was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p.i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24-36 h p.i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG(-)/M1(+) in tissue cultures were further examined and the titer of gG(-)/M1(+) was similar to that of PRV Ea wild strain and the parental strain gG(-)/LacZ(+). The above results indicated that the recombinant virus expressing the modified EGFP can be used as an in vivo marker to monitor replication and spread, as well as t o study the molecular pathogenesis of PRV.