Jason C. Huang

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

Abstract DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing α-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.

Detection of swine-origin influenza A (H1N1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Abstract Swine-origin influenza A (H1N1) virus (S-OIV) was identified as a new reassortant strain of influenza A virus in April 2009 and led to an influenza pandemic. Accurate and timely diagnoses are crucial for the control of influenza disease. We developed a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB) which combines a sandwich immunoassay with the LSP technique using antibodies against the hemagglutinin (HA) proteins of S-OIVs. The detection limit of the LSPCF-FOB for recombinant S-OIV H1 protein detection was estimated at 13.9pg/mL, which is 103-fold better than that of conventional capture ELISA when using the same capture antibodies. For clinical S-OIV isolates measurement, meanwhile, the detection limit of the LSPCF-FOB platform was calculated to be 8.25×104 copies/mL, compared with 2.06×106 copies/mL using conventional capture ELISA. Furthermore, in comparison with the influenza A/B rapid test, the detection limit of the LSPCF-FOB for S-OIV was almost 50-fold in PBS solution and 25-fold lower in mimic solution, which used nasal mucosa from healthy donors as the diluent. The findings of this study therefore indicate that the high detection sensitivity and specificity of the LSPCF-FOB make it a potentially effective diagnostic tool for clinical S-OIV infection and this technique has the potential to be applied to the development of other clinical microbe detection platforms.

Arginines in the CDR of anti-dsDNA autoantibodies facilitate cell internalization via electrostatic interactions

Internalization of autoantibodies against double‐stranded DNA (anti‐dsDNA) is crucial to the pathogenesis of systemic lupus erythematosus. Anti‐dsDNA may bind to cell‐surface targets in order to facilitate the subsequent cell penetration of the anti‐dsDNA. In this study, we observed that the 9D7 monoclonal anti‐dsDNA autoantibody (9D7 mAb) penetrates into Jurkat cells via a novel alternative pathway. Endocytosis inhibitors or a lipid‐raft inhibitor did not significantly change the penetration of 9D7 mAb into the Jurkat cells. However, heparin sulfate, chondroitin sulfate B, decaarginine and chondroitinase ABC significantly suppressed the internalization and the 9D7 mAb inhibited the internalization of Tat‐GFP. Moreover, the penetration of the 9D7 mAb was significantly reduced in proteoglycan‐deficient cells (pgs A‐745). Positively charged amino acids including arginine are commonly found in the CDR of the 9D7 mAb. Point mutations to the arginine residues in the CDR of the H chain of the recombinant 9D7 mAb significantly attenuated its DNA‐binding and cell‐penetration abilities. These findings indicate that cell penetration of anti‐dsDNA is due to the electrostatic interactions of arginine residues in the CDR with the negatively charged sulfated polysaccharides on the cell surface.

Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Abstract In order to enhance the sensitivity of conventional immunoassay technology for the detection of SARS coronavirus (SARS-CoV) nucleocapsid protein (N protein), we developed a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor that combines sandwich immunoassay with the LSP technique. Experimentally, a linear relationship between the fluorescence signal and the concentration of recombinant SARS-CoV N (GST-N) protein in buffer solution could be observed from 0.1pg/mL to 1ng/mL. In addition, the concentration of GST-N protein in diluted serum across a similar range could also be measured. The correlation coefficients (linear scale) for these two measurements were 0.9469 and 0.9624, respectively. In comparison with conventional enzyme linked immunosorbent assay (ELISA), the detection limit of the LSPCF fiber-optic biosensor for the GST-N protein was improved at least 104-fold using the same monoclonal antibodies. Therefore, the LSPCF fiber-optic biosensor shows an ability to detect very low concentration (∼1pg/mL) of SARS-CoV N protein in serum. The biosensor should help with the early diagnosis of SARS infection.

Diverse origin of P[19] rotaviruses in children with acute diarrhea in Taiwan: Detection of novel lineages of the G3, G5, and G9 VP7 genes†

We previously reported the detection of genotype P[19] rotavirus strains from children hospitalized with acute dehydrating diarrhea during a 5‐year surveillance period in Taiwan. The characterization of five P[19] strains (0.4% of all typed), including three G3P[19], a novel G5P[19], and a unique G9P[19] genotype is described in this study. Phylogenetic analysis of the VP4, VP7, VP6, and NSP4 genes was performed, which demonstrated novel lineages for respective genotypes of the VP4 and the VP7 genes. The sequence similarities of the P[19] VP4 gene among Taiwanese human strains was higher (nt, 91.5–96.2%; aa, 93.7–97.6%) than to other P[19] strains (nt, 83.5–86.6%; aa, 89.4–94.1%) from different regions of the world. The VP7 gene of the three G3P[19] Taiwanese strains shared up to 93.4% nt and 97.5% aa identity to each other but had lower similarity to reference strain sequences available in GenBank (nt, <90.1%; aa, <95.6%). Similarly, the VP7 gene of the novel G5P[19] strain was only moderately related to the VP7 gene of reference G5 strains (nt, 82.2–87.3%; aa, 87.0–93.1%), while the VP7 gene of the single G9P[19] strain was genetically distinct from other known human and animal G9 rotavirus strains (nt, ≤92.0%; aa, ≤95.7%). Together, these findings suggest that the Taiwanese P[19] strains originated by independent interspecies transmission events. Synchronized surveillance of human and animal rotaviruses in Taiwan should identify possible hosts of these uncommon human rotavirus strains. J. Med. Virol. 83:1279–1287, 2011.

Identification of porcine rotavirus-like genotype P[6] strains in Taiwanese children.

The molecular characterization of genotype P[6] rotavirus strains collected from children admitted to hospital with acute dehydrating diarrhoea during a 6-year surveillance period in Taiwan is described in this study. In total, three G4P[6] strains, one G5P[6] and one G12P[6] were characterized by sequencing and phylogenetic analysis of the VP4, VP7, VP6 and NSP4 genes. Whilst all four genes of the single Taiwanese G12P[6] strain clustered with the respective genes of globally common human rotavirus strains, the G4 and G5 strains showed remarkable similarities to porcine rotavirus strains and putative porcine-origin human P[19] strains reported previously from Taiwan. The overall proportion of porcine rotavirus-like strains in Taiwan remains around 1 % among hospitalized children; however, the circulation and sporadic transmission of these heterotypic strains from pigs to humans could pose a public-health concern. Therefore, continuation of strain monitoring is needed in the vaccine era to detect any possible vaccine breakthrough events associated with the introduction of such heterologous rotavirus strains.

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein.

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure -- a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.

Influenza A virus in Taiwan, 1980–2006: Phylogenetic and antigenic characteristics of the hemagglutinin gene

Limited amount of information is available in Taiwan on the genetic or antigenic characteristics of influenza A virus prior to the establishment of a Taiwan surveillance network in 2000. Isolates of H1N1 and H3N2 viruses in Taiwan between 1980 and 2006 were studied, and part of the hemagglutinin gene was analyzed due to its importance in terms of viral infection and antibody neutralization. Results from a phylogenetic analysis indicate continuous evolutionary topology in H3N2 isolates, and two distinct H1N1 lineages. Many genetic relationships between vaccine strains and epidemic isolates appearing in Taiwan before other global locations were also observed and recorded in addition to a gradual increase in the number of N‐linked glycosylation sites on partial HA1 proteins since 1980. The results from pairwise comparisons of HA1 nucleotide and deduced amino acid sequences indicate shared identities within groups organized according to their bootstrap and P‐values of approximately 95.5–100% and 95.7–100% in H1N1 and 94.5–100% and 93.2–100% in H3N2 viruses, respectively. Comparisons of amino acid substitutions in the five antigenic regions reveal highly non‐synonymous changes occurring in the Sb region of H1N1 and in the B region of H3N2. The results of an antigenic analysis using a hemagglutinin inhibition (HI) test indicate the presence of some epidemic strains 1–2 years earlier in Taiwan than in other parts of the world, as well as higher vaccine mismatch rates. This information supports the need for continuous surveillance of emerging influenza viruses in Taiwan, which will be useful for making global vaccine decisions. J. Med. Virol. 81:1457–1470, 2009.

Anti-ribosomal phosphoprotein autoantibody triggers interleukin-10 overproduction via phosphatidylinositol 3-kinase-dependent signalling pathways in lipopolysaccharide-activated macrophages.

Anti‐ribosomal phosphoprotein autoantibodies have been shown to be significantly associated with multiple manifestations of systemic lupus erythematosus (SLE). High levels of interleukin‐10 (IL‐10) have been demonstrated to contribute to lupus susceptibility and severity. In this study, we investigated the molecular mechanisms of anti‐ribosomal phosphoprotein monoclonal antibody (anti‐P mAb)‐induced autoimmune responses. Anti‐P mAb promoted IL‐10 overproduction in a dose‐ and time‐dependent manner in both lipopolysaccharide (LPS)‐activated RAW 264.7 cells and primary human macrophages. Anti‐P mAb enhanced phosphorylation of Akt (PKB; protein kinase B), extracellular signal regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase 1/2 (JNK1/2), while phosphorylation of p38 remained unaltered. Furthermore, anti‐P mAb decreased glycogen synthase kinase 3 (GSK3) activity and reduced the phosphorylation of IκBα in LPS‐activated macrophages. The Syk, phosphatidylinositol 3‐kinase (PI3K), protein kinase C (PKC), JNK and ERK signalling pathways involved in anti‐P mAb‐triggered IL‐10 secretion were also confirmed using various pharmacological inhibitors. In addition, nuclear factor (NF)‐κB had negative regulatory effects on anti‐P mAb‐triggered IL‐10 secretion. Using reporter plasmids containing the nuclear factor binding sites of NF‐κB, cAMP‐enhanced activation protein 1 (AP‐1), serum response element (SRE) or cyclic AMP response element (CRE), treatment of anti‐P mAb led to activation of the corresponding factors that bind to the AP‐1 site, SRE and CRE in the LPS‐activated macrophages. Furthermore, by transfection with reporter plasmids bearing various lengths of the IL‐10 promoter, the AP‐1 binding site, SRE and CRE were shown to be required for anti‐P mAb‐induced effects. Collectively, our results provide a molecular model for anti‐P mAb‐induced IL‐10 overproduction in LPS‐activated macrophages, which may play a role in the pathogenesis of SLE.

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

Abstract The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.