Gaiping Zhang

Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages

In 2006 China experienced outbreaks of a severe form of porcine reproductive and respiratory syndrome (PRRS) characterized by high fever, morbidity and mortality in swine irrespective of age. It is thought that secondary bacterial infections may contribute to the generation of this severe form of the disease. To determine the mechanisms by which a highly pathogenic PRRSV strain causes high fever we used an in vitro model to investigate the production of the pro-inflammatory cytokines IL-1β and TNF-α by macrophages in response to inoculation with PRRSV with or without LPS. Firstly we demonstrated, through an animal inoculation trial, that the isolate HN07-1 was a highly pathogenic strain and sequencing showed that the virus had the same genomic characteristics as previously described isolates. Porcine alveolar macrophage (PAM) cultures infected with PRRSV strains showed increased cytokine secretion and this was greater in the more virulent strain. Addition of LPS further increased cytokine secretion and again the effect was greater with the more virulent strain. Incubation of PAMs with PRRSV strain HN07-1 resulted in a significant increase in surface CD14 expression. This may explain the synergistic action between PRRSV and LPS in the induction of inflammatory cytokine secretion seen in the PAMs and so offer an explanation for the high fever that is characteristic of infections by the highly pathogenic PRRSV.

Marek’s disease virus-encoded microRNAs: genomics, expression and function

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek’s disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek’s disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

Molecular characteristics and evolutionary analysis of field Marek's disease virus prevalent in vaccinated chicken flocks in recent years in China

Marek’s disease is a highly contagious, oncogenic, and immunosuppressive avian viral disease. Surveillance of newly registered Marek’s disease virus (MDV) isolates is meaningful for revealing the potential factors involved in increased virulence. Presently, we have focused on the molecular characteristics of all available MDVs from China, including 17 new Henan isolates. Based on Meq, gE, and gI genes, we found that most Chinese isolates contain conserved amino acid point mutations in Meq, such as E77, A115, A139, R176, and A217, compared to USA virulent MDVs. However, the 59-aa or 60-aa insertions are only found in a few mild MDVs rather than virulent MDVs in China. Further phylogenetic analysis has demonstrated that a different genotype of MDV has been prevalent in China, and for virulent MDVs, their recent evolution has possibly been geographically restricted. Our study has provided more detailed information regarding the field MDVs circulating in China.

Molecular epidemiology of outbreak-associated pseudorabies virus (PRV) strains in central China.

In several parts of China, there have been a large number of pseudorabies (PR) outbreaks which have devastated many swine farms even though the herds had been previously immunized with gE-deleted vaccines (Bartha-K61). The emergence of these outbreak-associated PRV strains might indicate that Bartha-K61 vaccine could not provide effective protection and poses challenges for current serologic diagnostics of anti-PRV antibodies. Here, we performed phylogenetic analyses based on partial gE, gB, and gC genes to provide information about the molecular epidemiology, diagnostics, and immune protection in these outbreak-associated PRV strains. Our results indicated that the maximal nucleotide sequence divergence for gE, gB, and gC genes are 1.7, 0.4, and 2.7xa0% within the cluster where outbreak-associated PRV strains were located, and are 2.3, 2.7, and 7.6xa0% with other clusters in the phylogenetic trees, respectively. Phylogenetic analyses revealed that gE, gB, and gC genes of the twelve outbreak-associated PRV strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor with strains Ea-China-1999, Fa-China-2001, and BJ-China-2008. The genetic relationship between these outbreak-associated PRV strains and strain Bartha is not close which may genetically explain the emergence of PR outbreaks in Bartha-K61-vaccinated swine farms. We suggest that these outbreak-associated PRV strains originate from earlier strains in local regions in China.

Porcine FcγRIIb Mediates Enhancement of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection

Antibody-dependent enhancement (ADE) of virus infection caused by the uptake of virus-antibody complexes by FcγRs is a significant obstacle to the development of effective vaccines to control certain human and animal viral diseases. The activation FcγRs, including FcγRI and FcγRIIa have been shown to mediate ADE infection of virus. In the present paper, we showed that pocine FcγRIIb, an inhibitory FcγR, mediates ADE of PRRSV infection. Stable Marc-145 cell lines expressing poFcγRIIb (Marc-poFcγRII) were established. The relative yield of progeny virus was significantly increased in the presence of sub-neutralization anti-PRRSV antibody. The Fab fragment and normal porcine sera had no effect. Anti-poFcγRII antibody inhibited the enhancement of infection when cells were infected in the presence of anti-PRRSV antibody, but not when cells were infected in the absence of antibody. These results indicate that enhancement of infection in these cells by anti-PRRSV virus antibody is FcγRII-mediated. Identification of the inhibitory FcγR mediating ADE infection should expand our understanding of the mechanisms of pathogenesis for a broad range of infectious diseases and may open many approaches for improvements to the treatment and prevention of such diseases.

Genetic analysis of strains of Japanese Encephalitis Virus isolated from swine in central China.

In this study, four strains of Japanese Encephalitis Virus (JEV) were isolated from the cerebrospinal fluid of aborted fetuses or stillborn piglets collected randomly from a number of piggeries in central China. The E genes were cloned by RT–PCR and sequenced. Phylogenetic analysis was performed with 48 JEV isolates previously reported in China and other countries, and showed that all four isolates can be classified into the subcluster of genotype III. The results strongly suggest that the genotype III of JEV is the major variant currently circulating in the swinery of central China.

Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus.

To investigate the host–pathogen interactions between infectious bursal disease virus (IBDV) and target B-lymphocytic cells, a cDNA T7 phage display library from the chicken bursa of Fabricius was constructed and screened for virus binding. Surface immunoglobulin M (sIgM) was isolated as a putative candidate binding site and its interactions with IBDV were further investigated using a chicken bursal lymphoma-derived cell line DT40. The results showed that the λ light chain of sIgM specifically interacted with IBDV in a virulence-independent manner in vitro, and most of the binding of IBDV to DT40 cells was inhibited by sIgM-specific monoclonal antibodies. Further, the infectivity of IBDV in vitro was reduced by sIgM-specific monoclonal antibodies. Our data provided evidence that sIgM may participate as one of the putative membrane binding sites responsible for IBDV infection.

Antibody-dependent enhancement of PRRSV infection down-modulates TNF-α and IFN-β transcription in macrophages

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Previous studies have shown that Fcγ receptor (FcγR)-mediated entry of infectious PRRSV immune complexes into macrophages plays a pivotal role in the pathogenesis of the disease. This study demonstrates that PRRSV was able to suppress the transcription of key antiviral genes tumor necrosis factor-α (TNF-α) and interferon-β (IFN-β), when infection was via the ADE pathway. Investigation of this infection pathway found that PRRSV suppresses the antiviral genes by disrupting the transcription of the genes coding for the associated transcription factors interferon regulatory factor-1 (IRF-1), interferon regulatory factor-3 (IRF-3) and nuclear factor kappa B (NF-κB). The ADE pathway of infection allows PRRSV to specifically target antiviral genes and alters the innate intracellular immune responses in macrophages. The ADE mechanism described in this study furthers our understanding of pathogenesis following PRRSV infection and is of general relevance to virally induced disease and in relation to antiviral vaccination strategies.

Identification of host cell binding peptide from an overlapping peptide library for inhibition of classical swine fever virus infection

The envelope proteins of classical swine fever virus (CSFV) mediate the binding of CSFV to cell surface molecules and allow CSFV subsequent to enter host cells. However, the proteins binding to host cells and their binding sequences are uncertain. The results showed that the protein E1, E2, and Erns were displayed on the surfaces of T7 phages. The E2 and Erns phage clones showed high binding affinity to host cells, in which the E2 phage clone interacted more specifically with host cells than with other cells, while the Erns phage clone interacted with all tested cells. A 30-mer phage displaying peptide library was constructed and screened against immobilized host cells, in which each peptide was overlapped 10aa to another peptide and spanned all amino acid sequences of Erns and E2. Fifty-eight clones with specific binding to host cells were isolated. Amino acid sequence analyses for two phage clones (P2 and P6) demonstrated the strongest binding positions were at 101–130 (S2) in Erns, and 141–170 (S6) in E2, respectively. The synthetic peptides (S2 and S6) could inhibit the binding of phage clones (P2 and P6) and CSFV to cell. About 86.74 and 74.24% inhibition rates of CSFV infection were achieved at 55xa0μM of the synthetic peptides S2 and S6. The results also indicated that the S2 (LAEGPPVKECAVTCRYDKDADINVVTQARN) and S6 (AVSPTTLRTEVVKTFRRDKPFPHRMDCVTT) from CSFV were host cell binding peptides, and both of them had potential for research of CSFV entering host cells.

A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells

An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken β-actin/rabbit β-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3′-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.