Anding Zhang

Identification of immunogenic cell wall-associated proteins of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. An immunoproteome‐based approach was developed to identify candidate antigens of SS2 for vaccine development. Hyperimmune sera, convalescent sera, and control sera were analyzed for reactivity by Western Blot against SS2 cell wall‐associated proteins (WAPs) separated by 2‐DE. A total of 34 proteins were identified by immunoproteomic analysis, of which 15 were recognized by both hyperimmune sera and convalescent sera, including most WAPs currently characterized as SS2 vaccine candidate antigens: muramidase‐released protein (MRP), surface protein SP1 (Sao), and glyceraldehyde‐3‐phosphate dehydrogenase (GapdH). The novel immunogenic proteins may be developed as alternative antigens for further study of SS2 vaccine and diagnostics.

The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus.

The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49th to 68th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007

The aim of this study was to illustrate the characteristics of 407 strains of Streptococcus suis (S. suis) isolated from diseased pigs in China. The results revealed that S. suis, with 56.6% of the Streptococci isolates, had replaced Streptococcus equi subsp. zooepidemicus as the predominant agent. Among the strains investigated, serotype 2 (43.2%) was most prevalent, followed by serotypes 3 (14.7%) and 4, 8, 5, 7, 1/2 (3.2-6.4%). Serotype 2 was more frequently isolated from swine with systemic infection, while serotype 3 was significantly associated with pneumonia. A high percentage of S. suis serotype 2 strains (54%) belonged to the genotype sly+ mrp+ epf+, which is a highly virulent strain, as confirmed by mice infection test. Biofilm producers only had lowly virulence compared to strains with the same genotype, indicating that biofilm could be associated with virulence but be not the character of virulent strains. The present study contributes to understanding the characteristics of Streptococcus suis and controlling Streptococcal disease in China.

Identification and characterization of a novel protective antigen, Enolase of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. In the present study, a novel immunogenic Enolase identified in the previous study was inducibly overexpressed in Escherichia coli, and the purified recombinant protein could elicit a significant humoral antibody response and confer efficient immunity against challenge with lethal dose of SS2 or SS7 infection in mouse model. The roles Enolase plays in pathogenicity of SS2 were also explored as reasons for which Enolase could be a protective antigen. The Enolase was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the indirect immunofluorescent assay and the inhibition assay. These suggested that Enolase could play important roles in pathogenicity and may serve as a novel vaccine candidate against SS2 infection.

Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5

Haemophilus parasuis is the aetiological agent of Glässers disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis in young pigs. To develop more effective vaccines, an immunoproteome-based approach was used to analyze the outer membrane proteins of H. parasuis serovar 5. A total of 15 proteins with high immunogenicity were identified and all were showed to be immunogens for the first time in H. parasuis. Further analyses of 8 selected proteins revealed that (1) significantly higher level of serum antibodies against 6 proteins was detected with convalescent sera and immunized sera; (2) antisera against 5 of the selected proteins could effectively inhibit H. parasuis growth in mouse blood; and (3) 4 proteins could induce protective response of the vaccinated mice against H. parasuis. The results suggest these 4 proteins (PalA, Omp2, D15 and HPS_06257) have strong potential to be vaccine candidates.

Isolation and molecular characterization of equine H3N8 influenza viruses from pigs in China.

During 2004–2006 swine influenza virus surveillance, two strains of H3N8 influenza viruses were isolated from pigs in central China. Sequence and phylogenetic analyses of eight gene segments revealed that the two swine isolates were of equine origin and most closely related to European equine H3N8 influenza viruses from the early 1990s. Comparison of hemagglutinin (HA) amino acid sequences showed several important substitutions. One substitution caused the loss of a potential glycosylation site, and two substitutions, located at the cleavage site and adjacent to the receptor-binding pocket, respectively, had been reported previously in canine H3 HAs. This expansion of host range of equine H3N8 influenza viruses with mutations in the HA protein might raise the possibility of transmission of these viruses to humans.

Quantum-dots-based fluoroimmunoassay for the rapid and sensitive detection of avian influenza virus subtype H5N1

The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Rapid and sensitive diagnostic methods are required for the H5N1 surveillance. In this study, the fluorescent (FL) probe of CdTe quantum dots (QDs) was designed using covalently linked rabbit anti-AIV H5N1 antibody. Based on these QD-antibody conjugates, a novel sandwich FL-linked immunosorbent assay (sFLISA) was developed for H5N1 viral antigen detection. The sFLISA allowed for H5N1 viral antigen determination in a linear range of 8.0 × 10(-3) to 5.1 × 10(-1)  μg mL(-1) with the limit of detection (LOD) of 1.5 × 10(-4)  μg mL(-1) . In comparison with virus isolation for 103 clinic samples, the sensitivity and specificity of sFLISA were found to be 93.6 and 91.1% respectively. The sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1 and showed great potential for biological applications in immunoassays.

Comparative genomic analysis of Streptococcus suis reveals significant genomic diversity among different serotypes

BackgroundStreptococcus suis (S. suis) is a major swine pathogen and an emerging zoonotic agent. Serotypes 1, 2, 3, 7, 9, 14 and 1/2 are the most prevalent serotypes of this pathogen. However, almost all studies were carried out on serotype 2 strains. Therefore, characterization of genomic features of other serotypes will be required to better understand their virulence potential and phylogenetic relationships among different serotypes.ResultsFour Chinese S. suis strains belonging to serotypes 1, 7, 9 and 1/2 were sequenced using a rapid, high-throughput approach. Based on the 13 corresponding serotype strains, including 9 previously completed genomes of this bacterium, a full comparative genomic analysis was performed. The results provide evidence that (i) the pan-genome of this species is open and the size increases with addition of new sequenced genomes, (ii) strains of serotypes 1, 3, 7 and 9 are phylogenetically distinct from serotype 2 strains, but all serotype 2 strains, plus the serotype 1/2 and 14 strains, are very closely related. (iii) all these strains, except for the serotype 1 strain, could harbor a recombinant site for a pathogenic island (89 K) mediated by conjugal transfer, and may have the ability to gain the 89 K sequence.ConclusionsThere is significant genomic diversity among different strains in S. suis, and the gain and loss of large amount of genes are involved in shaping their genomes. This is indicated by (i) pairwise gene content comparisons between every pair of these strains, (ii) the open pan-genome of this species, (iii) the observed indels, invertions and rearrangements in the collinearity analysis. Phylogenetic relationships may be associated with serotype, as serotype 2 strains are closely related and distinct from other serotypes like 1, 3, 7 and 9, but more strains need to be sequenced to confirm this.

Development and evaluation of a DAS-ELISA for rapid detection of avian influenza viruses.

Abstract Rapid detection of avian influenza virus (AIV) infection is critical for control of avian influenza (AI) and for reducing the risk of pandemic human influenza. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for this purpose. The method employed a monoclonal antibody (MAb) as the capture antibody and rabbit polyclonal IgG labeled with horseradish peroxidase as the detector antibody, and both antibodies were against type-specific influenza A nucleoprotein (NP). The DAS-ELISA could detect minimally 2.5 ng of influenza viral protein in virus preparations treated with Triton X-100, which is equvilent to 2.5 × 102 EID50 virus particles. This DAS-ELISA could detect all 15n AIV subtypes (H1–H15) and did not cross react with other avian pathogens tested. The DAS-ELISA were directly compared with virus isolation (VI) in embryonated chicken eggs, the current standard of influenza virus detection, for 805 chicken samples. The DAS-ELISA results correlated with VI results for 98.6% of these samples, indicating a sensitivity of 97.4% and specificity of 100%. The method was further tested with H5N1 and H9N2 AIV experimentally infected chickens, ducks, and pigeons, as well as field samples obtained from central China in 2005. The DAS-ELISA method has demonstrated application potential as an AIV screening tool and as a supplement for virus isolation in Asia.

Complete Genome Sequence of Streptococcus suis Serotype 14 Strain JS14

Streptococcus suis is an important zoonotic agent leading to a variety of diseases in swine and can be transmitted to human beings upon close contact. Here, we report the complete genome sequence of S. suis serotype 14 strain JS14 which was isolated from a diseased pig in Jiangsu Province, China.