Songlin Qiao

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.

Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China

Porcine epidemic diarrhea virus (PEDV) has caused devastating impact on pig-rearing industry in China and current vaccine is not effective against the circulating PEDV variants. In the present study, the full-length genome sequence from a PEDV isolate (CH/HNQX-3/14) was determined. The complete genome sequence analysis showed that the CH/HNQX-3/14 possessed unique deletion regions in the S and ORF3 genes. It was identified as a recombinant strain using phylogenetic analysis and recombination detection program. Further analyses of the full-length sequence suggest that CH/HNQX-3/14 is a natural recombinant between the attenuated vaccine strains (CV777 and DR13) and circulating wild-type strain (CH/ZMDZY/11). The recombination occurred not only in structural protein-coding region (S1 and N genes) but also in non-structural protein-coding region (replicases 1a and ORF3 genes). These results provided new evidence that PEDV strains circulating in China underwent recombination between vaccine and field strains, suggesting that recombination contributes to the genetic diversity of PEDV. Our findings provide valuable information on PEDV evolution and underscore the need for ongoing surveillance of this economically important swine disease.

Phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in central China based on the ORF3 gene and the main neutralization epitopes.

Since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central China. To investigate the epidemic characteristics of PEDV, the complete ORF3 genes of 14 PEDV field strains from central China during 2012 to 2013 were cloned, sequenced and compared with reference strains. Phylogenetic analysis based on the complete ORF3 gene showed that the PEDVs in central China and the reference strains could be divided into three groups: G1, G2, and G3. The 14 PEDV isolates were classified as G1 and showed a close relationship to some Chinese strains isolated previously in central China and differed genetically from recent isolates from southern China, Korean strains (SM98 and DB1865, 2012), the Chinese LZC strain (2007), and the vaccine strain (CV777) being used in China. Our findings suggested that the PEDVs circulating between 2012 and 2013 in central China might have evolved from earlier strains in the local region. To determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the S gene. The results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. We speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure. Together, these findings may be useful for understanding the epidemiology of PEDV and may be relevant for designing of new and more efficacious vaccines.

Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O

An immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine. In the strip, the expressed protein of VP1, the main protective antigen of FMDV, labeled with colloidal gold was used as the detector, the staphylococcal protein A (SPA) and swine anti-foot-and-mouth disease virus (FMDV) antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. 296 swine serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial liquid-phage blocking ELISA (LPB ELISA) kit and peptide ELISA kit. The strip was shown to be of high specificity and sensitivity. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents nor equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.

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.

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.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Identification of the linear epitope for Fc‐binding on the bovine IgG2 Fc receptor (boFcγ2R) using synthetic peptides

To identify the linear epitope for Fc‐binding on the bovine IgG2 Fc receptor (boFcγ2R), peptides derived from the membrane‐distal extracellular domain (EC1) of boFcγ2R corresponding to the homologous region of human FcαRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot‐blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F–G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2‐binding. Such functional epitope peptide should be very useful for understanding the IgG‐Fcγ interaction and development of FcR‐targeting drugs.

Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in china

Recently, NADC30-like porcine reproductive and respiratory syndrome viruses (PRRSVs), which are genetically similar to the NADC30 strain isolated in the United States of America in 2008, have become prevalent in China. Here, a novel variant PRRSV strain named HNhx was successfully isolated on porcine alveolar macrophages from Henan province and the full-length genome sequence was determined. Phylogenetic analysis indicated that HNhx strain was classified into the NADC30-like PRRSV subgroup, in which all the strains had the unique discontinuous 131-amino acid deletion relative to that of the nonstructural protein 2 (Nsp2) of the VR2332 strain. Genetically, HNhx shared 92.9% nucleotide similarity to NADC30. Furthermore, HNhx strain contained extensive amino acid mutations in GP5. In particular, the S32H, N33D, D34N, and S36G variations resulted in that HNhx lost all the putative N-linked glycosylation sites at amino acid positions 30, 32, 33, 34, and 35. Recombination analysis revealed that HNhx was the result of recombination between the NADC30 strain and the highly pathogenic PRRSV vaccine strain circulating in China in Nsp4 (nt 5261) to Nsp9 (nt 7911). The novel genome data of HNhx will be helpful for understanding the evolution and epidemiology of PRRSV in China.

Development of a Peptide-Based Immunochromatographic Strip for Differentiation of Serotype O Foot-and-Mouth Disease Virus—Infected Pigs from Vaccinated Pigs

An immunochromatographic strip for discriminating Foot-and-mouth disease virus (FMDV) infected from vaccinated pigs was developed based on synthetic peptide. Five peptides designed from the amino acid sequences of nonstructural proteins (NSP) of FMDV were synthesized, and pep5 located in NSP 3B reacted strongly with serum from FMDV-infected pigs but did not react with serum samples from healthy vaccinated pigs. An immunochromatographic strip was developed by using colloidal gold labeled with pep5 as the detector. Staphylococcal protein A and rabbit against peptide-conjugated ovalbumin antibody immunoglobulin G were blotted on the nitrocellulose membrane for the test and control lines. In comparison with 2 commercial NSP enzyme-linked immunosorbent assays, the peptide-based strip showed good specificity and sensitivity. The apparent agreements of this new assay with Ceditest® ELISA and UBI® ELISA were 98.59% and 96.63%, respectively. These results indicate that the strip can be adequately used to discriminate FMDV-infected animals from vaccinated animals.