Jiyong Zhou

Proteomics Analysis of Host Cells Infected with Infectious Bursal Disease Virus

The effect of infectious bursal disease virus (IBDV) infection on cellular protein expression is essential for viral pathogenesis. To characterize the cellular response to IBDV infection, the differential proteomes of chicken embryo fibroblasts, with and without IBDV infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared at 48 and 96 h after IBDV infection. Mass spectrometry identified 51 altered cellular proteins, including 13 up-regulated proteins and 38 down-regulated proteins 12–96 h after infection. Notably 2-DE analysis revealed that IBDV infection induced the increased expression of polyubiquitin, apolipoprotein A-I, heat shock 27-kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and ribosomal protein SA isoform 2. In addition, IBDV infection considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover 38 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between infected and uninfected chicken embryo fibroblasts. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor expression and the induction of polyubiquitin during IBDV infection. Subcellular distribution analysis of the cytoskeletal proteins vimentin and β-tubulin clearly demonstrated that IBDV infection induced the disruption of the vimentin network and microtubules late in IBDV infection. Thus, this work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis.

Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus type 2.

Type 2 porcine circovirus (PCV2) is associated with post-weaning multisystemic wasting syndrome in pigs. In this study, three monoclonal antibodies (mAbs) against the capsid protein (Cap) of PCV2, eight mAbs to Cap of type 1 porcine circovirus (PCV1) and five mAbs specific for Cap of both PCV1 and PCV2, were generated and used to finely map the antigenic sites of PCV1 and PCV2, and to identify the antigenic phenotype of PCV2 with different length of genome. Five linear B-cell epitopes, including the residues 231-233 and 195-202 specific for PCV2, residues 92-103 specific for PCV1, and residues 156-162 and 175-192 shared between PCV1 and PCV2, were finely defined with synthetic peptides, and the critical residue in epitope 231-233 and 156-162 was located at residues 233 ((233)Proline) and 156 ((156)Tyrosine), respectively. The conformational epitopes recognized by mAbs with neutralizing activity against both PCV1 and PCV2 were detected in transfected PK-15 and the residues 231-233 also participated in the formation of conformational epitopes. Analysis of antigenic diversity on these epitopes exhibited three antigenic phenotypes of PCV2, (1766)PCV2, (1767)PCV2 and (1768)PCV2 using mAbs. The results from this study first demonstrated the different antigenic phenotype between PCV2 isolates.

Expression of Immunogenic S1 Glycoprotein of Infectious Bronchitis Virus in Transgenic Potatoes

ABSTRACT The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

ABSTRACT The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.

Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2

ABSTRACT Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To date, viral proteins Cap, Rep, Rep′, and ORF3, encoded by the PCV2 genome, have been described. Here, transcription and translation of a novel viral gene within the PCV2 genome (designated ORF4) was determined and functionally analyzed in vitro and in vivo. Northern blot analysis indicated that the RNA transcribed from the ORF4 gene is about 180 bp in length and overlaps ORF3 in the same direction. Site-directed mutagenesis confirmed that the viral ORF4 protein is not essential for virus replication in PK-15 cells and in mice infected with an ORF4-deficient PCV2 (PCV2Δ). PCV2Δ triggered higher activity levels of caspase-3 and -8 than wild-type PCV2 (wPCV2) in PK-15 cells. The antigenic epitopes of two mouse monoclonal antibodies (MAbs) raised against the viral ORF4 protein were mapped to the same 19KSSASPR25 peptide. Expression of ORF4 was confirmed using the specific MAbs in wPCV2-infected PK-15 cells and mice. Mice infected with PCV2Δ had a higher serum viral load (genomic copies) and more severe lymphoid tissue damage in the spleen than those infected with wPCV2. Meanwhile, flow-cytometric analysis indicated that the PCV2Δ infection caused a significant decrease of CD4+ and CD8+ T lymphocytes. Our results demonstrate that ORF4 is a newly discovered viral protein that is not essential for PCV2 replication but plays a role in suppressing caspase activity and regulating CD4+ and CD8+ T lymphocytes during PCV2 infection.

Differential proteome analysis of host cells infected with porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which is an emerging swine immunosuppressive disease. To uncover cellular protein responses in PCV2-infected PK-15 cells, the comprehensive proteome profiles were analyzed utilizing two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification. Multiple comparisons of 2-DE revealed that the majority of changes in protein expression occurred at 48-96 h after PCV2 infection. A total of 34 host-encoded proteins, including 15 up-regulated and 19 down-regulated proteins, were identified by MALDI-TOF/TOF analysis. According to cellular function, the differential expression proteins could be sorted into several groups: cytoskeleton proteins, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation. Western blot analysis demonstrated the changes of alpha tubulin, beta actin, and cytokeratin 8 during infection. Colocalization and coimmunoprecipitation analyses confirmed that the cellular alpha tubulin interacts with the Cap protein of PCV2 in the infected PK-15 cells. These identified cellular constituents have important implications for understanding the host interactions with PCV2 and brings us a step closer to defining the cellular requirements for the underlying mechanism of PCV2 replication and pathogenesis.

Epidemiology, Evolution, and Pathogenesis of H7N9 Influenza Viruses in Five Epidemic Waves since 2013 in China

H7N9 influenza viruses were first isolated in 2013 and continue to cause human infections. H7N9 infections represent an ongoing public health threat that has resulted in 1344 cases with 511 deaths as of April 9, 2017. This highlights the continued threat posed by the current poultry trade and live poultry market system in China. Until now, there have been five H7N9 influenza epidemic waves in China; however, the steep increase in the number of humans infected with H7N9 viruses observed in the fifth wave, beginning in October 2016, the spread into western provinces, and the emergence of highly pathogenic (HP) H7N9 influenza outbreaks in chickens and infection in humans have caused domestic and international concern. In this review, we summarize and compare the different waves of H7N9 regarding their epidemiology, pathogenesis, evolution, and characteristic features, and speculate on factors behind the recent increase in the number of human cases and sudden outbreaks in chickens. The continuous evolution of the virus poses a long-term threat to public health and the poultry industry, and thus it is imperative to strengthen prevention and control strategies.

Serological investigation and genomic characterization of PCV2 isolates from different geographic regions of Zhejiang province in China.

Sera collected from 46 swine farms in Zhejiang province were evaluated for the presence of antibodies to PCV2 using an indirect-fluorescent antibody procedure. In addition PCV2 isolated from superficial inguinal lymph node samples collected from 40-to 90-day-old pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) using the PK-15 cell line were sequenced and compared. Overall seroprevalence of PCV2 antibody averaged 58.34% for all samples. Breakdown of serology by groups was as follows: 59.38% for sows, 57.41% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalence of Landrace sows was higher than that of Yorkshire and Duroc sows, but non-significant (p > 0.05). Serological analysis also showed that seroprevalence of PCV2 antibody was a negative correlation to that of PRRSV antibody. The complete genomes of five PCV2 isolates identified in the herds with PMWS consisted of 1767nt, containing the 11 potential ORFs. Genome of the virus isolates shared 93.8% to 99.8% identity with PCV2 reference strains from GenBank, 76.6% to 77.9% identity with PCV1. Phylogenetic analysis indicated that there were two subgenotypes within PCV2: subgenotype I (1767 nt) and subgenotype II (1768 nt).

Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice

The protective immune response against porcine circovirus 2 (PCV2) infection in mice was characterized using flow cytometric analysis (FCM), assays of antibody (of different IgG isotypes) and viraemia, and histopathological examination. An open reading frame 2 plasmid (pORF2) and the capsid protein (Cap) of PCV2 were used as DNA and subunit vaccines, respectively. In FCM analysis, although pORF2 and Cap alone showed comparable efficacy in eliciting lymphoproliferative responses and Cap-specific CD4(+) T cells, pORF2 was superior to the Cap protein in triggering CD8(+) T cells. A virus neutralization assay showed that pORF2 evoked stronger recall virus-neutralizing (VN) antibody responses than the Cap protein on PCV2 challenge. Correspondingly, VN antibody kinetics coincided with those of Cap-specific IgG2a, but not with the kinetics of IgG and IgG1. Following virus challenge, real-time PCR and histopathological analysis confirmed that only low viral DNA loads and mild microscopic lesions appeared in pORF2-immunized mice. These findings indicate that CD8(+) T cells and VN antibody responses correlating mainly with Cap-specific IgG2a play crucial roles in protecting against PCV2 infection, and that the protective immunity induced by the pORF2 plasmid is superior to that induced by the PCV2 Cap protein.

Characterization of chicken interleukin 2 receptor α chain, a homolog to mammalian CD25

To identify chicken IL‐2R α chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL‐2‐dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL‐2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.