Puyan Chen

Serologic differences among nondefective reticuloendotheliosis viruses

SummaryAntigenic relationships among 26 isolates of reticuloendotheliosis virus (REV) obtained from several avian species were compared by cross neutralization tests with polyclonal chicken sera and by immunofluorescent assays with monoclonal antibodies to REV strain T. The isolates were all strongly related by neutralization assays and thus probably constitute a single serotype. However, 3 antigenic subtypes were suggested by minor but distinct differences in neutralization titers. The validity of these 3 subtype designations was confirmed by differential reactivity of viral isolates to selected monoclonal antibodies. Subtype-associated differences in serum antibody titers were noted following the inoculation of chickens with the REV isolates.

Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII

Japanese encephalitis virus (JEV) infection is a major cause of acute viral encephalitis both in humans and animals. The domain III of virus envelope protein (EDIII) plays important roles in interacting with host cell receptors to facilitate virus entry. In this study, recombinant JEV EDIII was expressed and purified. The protein showed the ability to inhibit JEV infection in BHK-21 cells with 50% inhibition at a concentration of 25μg/ml. Based on NMR structure of JEV EDIII, we chose several loop peptides that were reported to be related to receptor binding to test their possible inhibitory activities on virus infection. Our in vitro experiments demonstrated that one of the loop peptides (loop3) can prevent JEV infection with 50% inhibition at concentration of 10μM by interfering in virus attachment to the cells. Our in vivo experiments on mice showed the loop3 was the most protective peptide when administered before virus challenge. Therefore, the loop3 peptide may be served as basis for the development of novel antiviral agents against Japanese encephalitis virus or other flaviviruses infection.

In vitro inhibition of the replication of classical swine fever virus by porcine Mx1 protein.

Classical swine fever virus (CSFV) is the causative pathogen of classical swine fever (CSF), a highly contagious disease of swine. Mx proteins are interferon-induced dynamin-like GTPases present in all vertebrates with a wide range of antiviral activities. Although Zhao et al. (2011) have reported that human MxA can inhibit CSFV replication, whether porcine Mx1 (poMx1) has anti-CSFV activity remains unknown. In this study, we generated a cell line designated PK-15/EGFP-poMx1 which expressed porcine Mx1 protein constitutively, and we observed that the proliferation of progeny virus in this cell line was significantly inhibited as measured by virus titration, indirect immune fluorescence assay, Q-PCR and Western blot. Furthermore, when PTD-poMx1 fusion protein expressed in Escherichia coli (Zhang et al., 2013) was used to treat CSFV-infected PK-15 cells, the results showed that PTD-poMx1 inhibited CSFV replication in a dose-dependent manner. Additionally, the proliferation of progeny virus was inhibited as measured by virus titration and Q-PCR. Overall, the results demonstrated that poMx1 effectively inhibited CSFV replication, suggesting that poMx1 may be a valuable therapeutic agent against CSFV infection.

Isolation and potential immunological characterization of TPSGLVY, a novel bursal septpeptide isolated from the bursa of Fabricius

The bursa of Fabricius is central immune organ unique to birds, and the extract is immunocompetent in stimulating B cell differentiation and enhancing antibody production. However, except for bursin, the active peptides from the bursa of Fabricius are little reported. In the paper, a novel bursal septpeptide (BSP-II) with the amino acids sequence of TPSGLVY was identified and similar to the MGC53864 protein of Gallus gallus. We investigated the effects of BSP-II on the immune response in terms of the antibodies titers (IgG1 and IgG2alpha), the levels of interferon-gamma and interleukin-4 cytokines, spleen cell lymphocyte proliferation, and the T-lymphocyte subtype composition. It was noteworthy that BSP-II potentiates the Th1 and Th2-type immune responses in dose-dependent manner. BSP-II had specific enhancing effects on the hybridoma SP2/0 cell proliferation at two different serum concentrations (20% and 5%), but had no connection with the dose of BSP-II. The antibody secreting level of hybridoma SP2/0 cells rose in 5% and 20% serum when the concentrations of BSP-II increased. Also, BSP-II had effect on the viabilities of tumor cells (Hela and SP2/0). All the results indicated that BSP-II was able to significantly induce various immune responses and involved in the cell viability of different tumor cell lines. Our observations implied that BSP-II might be a novel biological active factor from the bursa of Fabricius with immunomodulatory activities.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012

Since October 2010, clinical outbreaks of diarrhea in suckling piglets have reemerged in pig-producing areas of China, causing an acute increase in the morbidity and mortality in young piglets. Four viruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (GAR), and porcine circovirus 2 (PCV2), are the major causative agents of enteric disease in piglets. A novel multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of the four viruses in field samples from piglets. A mixture of four previously published pairs of primers were used for amplification of viral gene, yielding four different amplicons with sizes of 481 bp for PCV2, 651 bp for PEDV, 859 bp for TGEV, and 309 bp for GAR, respectively. The sensitivity of the mRT-PCR using plasmids containing the specific viral target fragments was 2.17 × 10(3), 2.1 × 10(3), 1.74 × 10(4) and 1.26 × 10(4)copies for the four viruses, respectively. A total of 378 field samples were collected from suckling piglets with diarrhea in East China from October 2010 to December 2012, and detected by mRT-PCR. The PEDV-positive rates of the three years were 69.2%, 62.8% and 54.9%, respectively, suggesting that PEDV was a major pathogen in these diarrheal outbreaks. Taken together, all data indicated that this mRT-PCR assay was a simple, rapid, sensitive, and cost-effective detection method for clinical diagnosis of mixed infections of porcine diarrhea associated viruses.

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene.

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.

Isolation, antiproliferation on tumor cell and immunomodulatory activity of BSP-I, a novel bursal peptide from chicken humoral immune system

The bursa of Fabricius (BF) is acknowledged as central humoral immune organ unique to birds. Our purpose was to identify the potential function of a novel bursal-derived bioactive peptide. A bursal septpeptide (BSP-I), EPASGMM, first isolated from BF, reduced MCF and Hela tumor cells proliferation, and enhanced antitumor factor p53 luciferase activity and protein expression. Further, we found the significantly immune inducing function of BSP-I on antigen-specific immune response in BALB/c mice intraperitoneally immunized with inactivated avian influence virus (AIV, H(9)N(2) subtype) vaccine, including of enhancing the antibody (IgG, the isotypes IgG1 and IgG2a) production, and stimulating cytokines IL-4 and IFN-γ level, and inducing T cell immunophenotyping and lymphocyte proliferation. These results suggested that as the bioactive peptide from avian humoral immune system, various biological function of BSP-I may have far-reaching implication on immune system significance, which might provide novel insight on linking between humoral immune system and development of effective immunotherapeutic strategies for treating human cancers diseases.

Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus.

To elucidate the role of microRNAs (miRNA) in the regulation of gene expression in Japanese encephalitis virus (JEV) infected swine testis (ST) cells, we analyzed miRNA and mRNA expression profiles of JEV infected ST cells by high-throughput sequencing technology as compared to uninfected controls. The results showed that 104 known miRNAs and 9 new miRNA candidates were differentially expressed in ST cells after JEV infection. We identified 396 differentially expressed mRNAs. Bioinformatics analysis identified 435 known miRNA-mRNA interaction pairs and 94 novel miRNA-mRNA interaction pairs involving miRNAs inversely correlated with the expression of their predicted target mRNAs. The known miRNAs inversely correlated with their target genes were involved in the biological processes of immunity, cytokine production, inflammation, and apoptosis. Selected miRNA-mRNA interactions were validated by luciferase reporter assay. Overall, our findings indicate that miRNAs may play critical roles in the pathogenesis of JEV infection.

Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV). Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865) and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716), were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine) than that of mono-epitope peptide(rE2-a or rE2-b). Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals) vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.