Genomic characteristics of an avipoxvirus 282E4 strain.

Avipoxvirus 282E4 strain was extensively applied into recombinant vaccine vector to prevent other infectious diseases. However, little information on the genomic background, functional and genetic evolutionary of the isolate 282E4 strain was clarified. The results showed that the linear genome of avipoxvirus 282E4 was 308,826 bp, containing 313 open reading frames (ORFs) and 12 new predicted ORFs. The 282E4 strain appears to encode two novel thymidine kinase proteins and two TGF-beta-like proteins that may be associated with the suppression of the host's antiviral response. Avipoxvirus 282E4 also encodes 57 ankyrin repeat proteins and 5 variola B22R-like proteins, which composed 7% of the avipoxvirus 282E4 genome. GO and KEGG analysis further revealed that 12 ORFs participate in viral transcription process, 7 ORFs may function during DNA repair, replication and biological synthesis, and ORF 208 is involved in the process of virus life cycle. Interestingly, phylogenetic analysis based on concatenated sequences p4b and DNA polymerase of avipoxviruses gene demonstrates that avipoxvirus 282E4 strain is divergent from known FWPV isolates and is similar to shearwater poxvirus (SWPV-1) that belongs to the CNPV-like virus. Sequencing avipoxvirus 282E4 is a significant step to judge the genetic position of avipoxviruses within the larger Poxviridae phylogenetic tree and provide a new insight into the genetic background of avipoxvirus 282E4 and interspecies transmission of poxviruses, meanwhile, explanation of gene function provides theoretical foundation for vaccine design with 282E4 strain as skeleton.

Complete genomic analysis of rabbit rotavirus G3P[22] in China.

A rabbit rotavirus Z3171 isolate from diarrheic rabbits was identified and sequenced. The genotype constellation of Z3171 is G3-P[22]-I2-R3-C3-M3-A9-N2-T1-E3-H3, which is different from the constellation observed in previously characterized LRV strains. However, the genome of Z3171 differed substantially from those of the rabbit rotavirus strains N5 and Rab1404 in terms of both gene content and gene sequence. Our study suggests that either a reassortment event occurred between human and rabbit rotavirus strains or there are undetected genotypes circulating in the rabbit population. This is the first report of detection of a G3P[22] RVA strain in rabbits in China.

Generation of duck Tembusu virus using a simple reverse genetic system in duck embryo fibroblast cells.

Outbreaks of duck Tembusu virus (DTMUV) have caused serious economic losses in China since 2010. In this study, an infectious clone of the DTMUV BZ-2010strain, isolated from layer cherry duck in China, was constructed using the bacterium-free infectious subgenomic-amplicons method. The subgenomic-amplicons of the human cytomegalovirus promoter (pCMV) at the 5' terminus of the first DNA fragment, the entire genome of DTMUV, and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) at the 3' terminus of the last DNA fragment were synthesized and amplified by PCR in three DNA fragments. The pCMV and HDR/SV40pA were used to drive the viral RNA transcription and generate a full-length RNA transcript of the virus, and were found to be effective in reassembling DTMUV in duck embryo fibroblast cells. The RNA transcripts from the infection clone were infectious in duck embryo fibroblast cells, generating the reconstituted DTMUV. This study provided a valuable reverse genetic tool for the further study DTMUV pathogenesis.

Novel duck reovirus exhibits pathogenicity to specific pathogen-free chickens by the subcutaneous route.

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 µL (10-5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 µL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

Proteome analysis of reticuloendotheliosis-virus-infected chicken embryo fibroblast cells through iTRAQ-based quantitative proteomics.

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.

An epidemiological investigation of porcine circovirus 3 infection in cattle in Shandong province, China.

BACKGROUND: Porcine circovirus type 3 (PCV3) is a single-stranded, closed circular DNA virus, which causes porcine dermatitis and nephropathy syndrome (PDNS), multisystemic inflammation, and reproductive failure. The present study aimed to investigate the seroprevalence of PCV3 in cattle (Bos taurus) in Shandong province, China, and examine its genome diversity. RESULTS: PCR amplification and sequencing showed that 74 of 213 bovine samples (34.7%) were positive for PCV3. Among them, the capsid gene (n = 12) and the complete genome (n = 4) were sequenced. These sequences had high identities to the reference capsid gene (98.0-100%) and the complete genome (97.5-99.8%). The PCV3 strains were classified into two different genotypes (PCV3a and PCV3b), according to phylogenetic analysis based on the complete genome and capsid gene sequences. Specifically, the bovine-origin strains in this study were grouped into PCV3a, showing a close relationship with PCV3-US/SD2016 (American strain; GenBank: KX966193.1). Notably, a comparison of the inferred amino acid sequences revealed a mutation from D124 to Y124. CONCLUSION: This was the first seroprevalence and genetic investigation of PCV3 in cattle in Shandong province, China. The results could provide insights into the epidemiology and pathogenesis of this important virus.

ITRAQ-Based Quantitative Proteomics Reveals the Proteome Profiles of Primary Duck Embryo Fibroblast Cells Infected with Duck Tembusu Virus.

Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.

SIV-Specific Antibodies are Elicited by a Recombinant Fowlpox Virus Co-expressing SIV Gag and envT.

Given the failures of past HIV-1 vaccine clinical trials, potential HIV-1 vaccine candidates should be rigorously screened in preclinical models including simian immunodeficiency virus (SIV) primate models and small animal models. In this study, we tested the immunogenicity of a recombinant fowlpox virus (rFPV) expressing the SIV gag and SIV envT (rFPVsg-se) proteins in BALB/c mice, to establish a foundation for further development. rFPVsg-se was constructed through homologous recombination techniques and purified through plaque screening assays using enhanced green fluorescent protein as the reporter gene. The integration, transcription, and translation of the SIV genes were measured by PCR (genomic DNA), RT-PCR (RNA), Western-blot, respectively. The levels of SIV-specific antibodies were assessed by ELISA following a single immunization (n = 18/group) or a prime-boost strategy (n = 24/group) with rFPVsg-se and compared to FPV and PBS controls. Residual virus was measured in distant organs following immunization using PCR. SIV-specific IgG titers against gag and gp120 were detected following single vaccination and the prime-boost. As expected the titers were higher following the prime-boost approach. The levels of Gag- and gp120-specific antibodies were significantly higher than controls (p < 0.01) 14 days after the booster immunization. Residual rFPVSg-Se was detected in the muscle at the site of injection, but not in distant organs, from day 1-7 post immunization. In summary, rFPVsg-se induced high levels of SIV-specific antibodies suggesting it may be a viable candidate for further development.

Construction, Selection and Immunogenicity of Recombinant Fowlpox Candidate Vaccine Co-expressing HIV-1 gag and gp145.

An HIV candidate vaccine for the Chinese population was designed by constructing a recombinant fowlpox virus expressing HIV-1 gag and HIV gp145 proteins via homologous recombination and plaque screening using enhanced green fluorescent protein (EGFP) as the reporter gene. EGFP in the recombinant was then knocked out with the Cre/Loxp system yielding rFPVHg-Hp, which was identified at the genomic, transcriptional and translational levels. The immunogenicity of rFPVHg-Hp was analyzed by measuring levels of HIV-specific antibodies and IFN-γ-secreting splenocytes by enzyme-linked immunosorbent assay and IFN enzyme-linked immune spot test in the BALB/c mouse model. Results showed that rFPV could not stimulate HIV-1 specific antibodies or IFN-γ-secreting cells by a single immunization. Meanwhile, in the prime-boost strategy, HIV-p24 antibodies (P < 0.01) and IFN-γ-secreting cells (P < 0.05) were induced strongly by the candidate vaccine after the boost immunization. Thus, both humoral and cellular immunity could be elicited by the candidate vaccine in a prime-boost immunization strategy. This study provides a foundation for future preclinical studies on the HIV rFPVHg-Hp candidate vaccine.

Identification of Goose-Origin Parvovirus as a Cause of Newly Emerging Beak Atrophy and Dwarfism Syndrome in Ducklings.

A recent epizootic outbreak, in China, of duck beak atrophy and dwarfism syndrome (BADS) was investigated using electron microscopic, genetic, and virological studies, which identified a parvovirus with a greater similarity to goose parvovirus (GPV) (97% protein homology) than to Muscovy duck parvovirus (MDPV) (90% protein homology). The new virus, provisionally designated GPV-QH15, was found to be antigenically more closely related to GPV than to MDPV in a virus neutralization assay. These findings were further supported by phylogenetic analysis showing that GPV-QH15 evolved from goose lineage parvoviruses, rather than from Muscovy duck- or other duck species-related parvoviruses. In all, two genetic lineages (GPV I and GPV II) were identified from the GPV samples analyzed, and GPV-QH15 was found to be closely clustered with two known goose-origin parvoviruses (GPVa2006 and GPV1995), together forming a distinctive GPV IIa sublineage. Finally, structural modeling revealed that GPV-QH15 and the closely related viruses GPVa2006 and GPV1995 possessed identical clusters of receptor-interacting amino acid residues in the VP2 protein, a major determinant of viral receptor binding and host specificity. Significantly, these three viruses differed from MDPVs and other GPVs at these positions. Taken together, these results suggest that GPV-QH15 represents a new variant of goose-origin parvovirus that currently circulates in ducklings and causes BADS, a syndrome reported previously in Europe. This new finding highlights the need for future surveillance of GPV-QH15 in poultry in order to gain a better understanding of both the evolution and the biology of this emerging parvovirus.

Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability.

The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

OBJECTIVE: To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. METHODS: We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. RESULTS: DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. CONCLUSION: The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus.

Construction and characterization of novel fowlpox virus shuttle vectors.

Viral vectors are important vehicles in vaccine research. Avipoxviruses including fowlpox virus (FPV) play major roles in viral vaccine vector development for the prevention and therapy of human and other veterinary diseases due to their immunomodulatory effects and safety profile. Recently, we analyzed the genomic and proteomic backgrounds of the Chinese FPV282E4 strain. Based on analysis of the whole genome of FPV282E4, the FPV150 and FPV193 loci were chosen as insertion sites for foreign genes, and two shuttle vectors with a triple-gene expression cassette were designed and constructed. Homologous recombination between the FPV virus genome and sequences within the shuttle plasmids in infected cells was confirmed. The recombinants were obtained through several rounds of plaque purification using enhanced green fluorescent protein as a reporter and evaluated for the correct expression of foreign genes in vitro using RT-PCR, real-time PCR and Western blotting. Morphogenesis and growth kinetics were assayed via transmission electron microscopy and viral titering, respectively. Results showed that recombinant viruses were generated and correctly expressed foreign genes in CEF, BHK-21 and 293T cells. At least three different exogenous genes could be expressed simultaneously and stably over multiple passages. Additionally, the FPV150 mutation, FPV193 deletion and insertion of foreign genes did not affect the morphogenesis, replication and proliferation of recombinant viruses in cells. Our study contributes to the improvement of FPV vectors for multivalent vaccines.

Complete genome sequence of an avian reovirus isolated from wild mallard ducks in china.

We report here the complete sequence of novel duck reovirus (N-DRV) strain SD12 isolated from diseased wild mallard ducklings in the Shandong Province of China in 2012. The complete genome consists of 23,420 nucleotide base pairs (bp), including 10 segments ranging from 1,191 bp (S4) to 3,959 bp (L1).

In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine.

This study aims to evaluate the functional and probiotic characteristics of eight indigenous Lactobacillus strains in vitro. The selected lactobacilli include strains of Lactobacillus casei subsp. casei, Lactobacillus salivarius subsp. salicinius, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus rhamnosus. All strains tolerated both pH 2 for 3 h and 1% bile salt for 24 h. The strains CICC 23174 and CGMCC 1.557 were the most adhesive strains producing the highest quantity of EPS. Although a wide variation in the ability of the eight strains to deplete cholesterol and nitrite, antagonize pathogens, scavenge free radical, and stimulate innate immune response were observed, the strains CICC 23174 and CGMCC 1.557 showed the widest range of these useful traits. Taken together, the strains CICC 23174 and CGMCC 1.557 exhibited the best probiotic properties with the potential for use in the production of probiotic fermented foods.

Enhanced immune responses in pigs by DNA vaccine coexpressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus.

The European (EU) type of porcine reproductive and respiratory syndrome virus (PRRSV) has recently emerged in China. In this study, three recombinant DNA vaccines, pVAX1-EU-ORF3-ORF5 (coexpressing EU type PRRSV GP3 and GP5), pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed and evaluated for their abilities to induce humoral and cellular responses as well as to protect piglets against homologous virus challenge. All piglets were given booster vaccinations at 21 days after the initial inoculation and then challenged 14 days later. Pigs inoculated with pVAX1-EU-ORF3-ORF5 developed significantly higher (P<0.05) PRRSV-specific antibody responses, neutralizing antibodies and levels of IL-4 and IL-10 than those given pVAX1-EU-ORF3, pVAX1-EU-ORF5 or pVAX1. Moreover, pigs immunized with pVAX1-EU-ORF3-ORF5 had markedly increased levels of IFN-γ and IL-2 in serum and T-lymphocytes (CD3(+)CD4(+) and CD3(+)CD8(+) T cells) in peripheral blood. Thus, EU-type PRRSV GP3 and GP5 proteins demonstrated good immunogenicity and reactogenicity and could induce cellular immunity in pigs. Following challenge with the Lelystad virus (LV) strain, piglets inoculated with pVAX1-EU-ORF3-ORF5 showed viremia and virus load distributed in organ tissues that were significantly lower (P<0.05) than those in the pVAX1-EU-ORF3 group and control group, and slightly lower than those in the pVAX1-EU-ORF5 group (P>0.05). As GP3 could enhance humoral- and cell-mediated immune responses to GP5, the results of this study suggested that these two proteins delivered by a vaccine can synergistically induce immunity against PRRSV.

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus.

BACKGROUND: The European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV. RESULTS: To evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN-γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group. CONCLUSIONS: Using Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More importantly, better PRRSV-specific neutralizing antibody titers and cell-mediated immune responses were observed in mice immunized with the DNA vaccine co-expressing GP3 and GP5 proteins than in mice immunized with a DNA vaccine expressing either protein singly. The results of this study demonstrated that co-immunization with GP3 and GP5 produced a better immune response in mice.

Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization.

This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA (pCCMp24) and recombinant attenuated vaccinia virus Tian Tan (rddVTT-CCMp24). Intramuscular immunization was performed on days 0 (prime) and 21 (boost). The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus (HIV)-specific binding antibody levels and cytokine (interleukin-2 and interleukin-4) concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4(+)/CD8(+) cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8(+) T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-γ response, providing a basis for further studies.

Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment.

Staphylococcus aureus is an opportunistic pathogen that can colonize human and animal intestinal tracts, causing certain gastrointestinal diseases. The adherence of enteric pathogens to host intestinal epithelial cells is important for their pathogenesis. In the present study, Lactobacillus salivarius and Lactobacillus plantarum were investigated in vitro to examine their ability to competitively exclude S. aureus. Various factors involved in attachment, including bacterial status and cell concentration, growth phase, competition patterns, and surface-layer protein extracts, were also investigated. Live lactobacilli in the mid-log growth phase exhibited maximum inhibitory activity when lactobacilli were pre- or co-incubated with S. aureus. However, the inhibitory activity was significantly reduced when the lactobacilli were inactivated by heating or treated with LiCl. Furthermore, both lactobacilli possessed certain cell surface properties, such as hydrophobicity, autoaggregation, and coaggregation ability. L. salivarius and L. plantarum strongly inhibited S. aureus adherence to Caco-2 cells and their inhibition activity was significantly influenced by several factors that affect adhesion inhibition.