Syndecan-4, a PRRSV attachment factor, mediates PRRSV entry through its interaction with EGFR.

The causative agent of porcine reproductive and respiratory syndrome is the PRRS virus (PRRSV), an enveloped, single-stranded and positive-sense RNA virus. The host factors and mechanisms that are involved in PRRSV entry are still largely unknown. In our present studies, we found that syndecan-4, one of the heparan sulfate proteoglycans, plays a critical role in PRRSV entry, especially in PRRSV attachment. Moreover, EGFR interacts with syndecan-4 in MACR-145 cells and disruption of their interaction impaired PRRSV entry. Furthermore, EGFR inhibitor AG1478 or syndecan-4 derived peptide SSTN87-131 inhibited syndecan-4 endocytosis induced by PRRSV entry. Altogether, syndecan-4, a PRRSV attachment factor, mediated PRRSV entry by interacting with EGFR.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant truncated Cap protein for the diagnosis of porcine circovirus-like virus P1.

BACKGROUND: Porcine circovirus-like virus P1 is a newly discovered virus. To date, there has been no specific serological assay for use in the diagnosis of P1 infection. RESULTS: Because P1 has high homology to porcine circovirus type 2 (PCV2) at the nucleotide level, the C-terminal portion of the capsid protein (amino acids 73-114), a discriminative antigen, was expressed in a prokaryotic expression system. The recombinant product (rctCap), composed of three identical repeated domains, was shown to be strongly immunoreactive to P1-specific serum. This assay was validated by comparison with an indirect immunofluorescence assay (IFA). The diagnostic sensitivity and specificity of the rctCap enzyme-linked immunosorbent assay (ELISA) developed in this study are 93.6% and 98.3%, respectively, compared with the results from IFAs on 450 sera samples from pigs. CONCLUSIONS: The indirect ELISA that we developed with rctCap, the recombinant capsid fragment containing the 217-342 nt repeat domain, was sensitive, specific, and suitable for the large-scale detection of P1 infections in swine.

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

BACKGROUND: The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1. RESULTS: A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 10(0) ~ 10(9) copies/µL. This method doesn't detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5%. The serum virus titers of P1 positive in this study were measured by this protocol to be 10(3) to 10(7) copies/mL. CONCLUSIONS: The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

LiCl inhibits PRRSV infection by enhancing Wnt/ß-catenin pathway and suppressing inflammatory responses.

Lithium chloride (LiCl) has been used as a mood stabilizer in the manic depressive disorders treatment. Recent studies show that LiCl is also a potent inhibitor for some DNA and RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen in modern pig industry. In this study, we assessed the inhibitory effect of LiCl on PRRSV infection using plaque-formation assay, Q-PCR and Western blot analysis. Our results showed that LiCl could inhibit PRRSV infection in MARC-145 and PAM-CD163 cells. Previous reports have shown that LiCl could induce the Wnt pathway in the absence of Wnt ligands. In our studies, we demonstrated that LiCl activates the Wnt pathway in PRRSV infected cells. Additionally, the knockdown of ß-catenin or the Wnt/ß-catenin pathway inhibitor PNU74654 was able to reverse the antiviral effect of LiCl, which suggested that the inhibitory effect of LiCl against PRRSV replication might be associated with the activation of the Wnt/ß-catenin pathway. We also found that lower viral replication after LiCl treatment was associated with the reduced mRNA levels of pro-inflammatory IL-8, IL-6, IL-1 ß, tumor necrosis factor α and decreased NF-κB nuclear translocation. Collectively, our data demonstrated that LiCl inhibited PRRSV infection by enhancing Wnt/ß-catenin pathway and suppressing pro-inflammatory responses.

The involvement of FAK-PI3K-AKT-Rac1 pathway in porcine reproductive and respiratory syndrome virus entry.

CD163 and sialoadhesin had been reported as the two receptors for porcine reproductive and respiratory syndrome virus (PRRSV) infection. The signaling pathway activated by PRRSV entry was seldom reported. In our studies, we demonstrated that PRRSV entry triggers FAK, PI3K, AKT and Rac1 activation. The signaling pathway FAK-PI3K-AKT-Rac1 is essential for PRRSV entry. Blocking FAK by PF573228 attenuates the activation of PI3K, AKT, Rac1 and the cytoskeleton remodeling induced by virus entry. Inhibitors to FAK, PI3K, AKT and Rac1 can significantly inhibit the virus entry. In conclusion, our observations reveal that PRRSV triggers the activation of FAK-PI3K-AKT-Rac1 signaling pathway to facilitate its entry into cells.

Transcriptional analysis of porcine circovirus-like virus P1.

BACKGROUND: Recently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts. RESULTS: The RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses. Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (-) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids). CONCLUSIONS: We show a very complex viral transcription pattern in P1-infected cells.

Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.

Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.

The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.