Meilin Jin

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.

Complete Genome Sequence of Haemophilus parasuis SH0165

Haemophilus parasuis is the causative agent of Glässers disease, which produces big losses in swine populations worldwide. H. parasuis SH0165, belonging to the dominant serovar 5 in China, is a clinically isolated strain with high-level virulence. Here, we report the first completed genome sequence of this species.

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.

A pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with H5N1 avian influenza virus in mice and chickens

Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was used as a vector to express the hemagglutinin (HA) protein of highly pathogenic H5N1 avian influenza virus, A/Chicken/Hubei/327/2004 (HB/327). The resultant recombinant baculovirus (BV-G-HA) mediated gene delivery and HA expression efficiently in mammalian cells. Mice immunized with 1 x 10(9)PFU of BV-G-HA developed significantly higher levels of H5-specific antibodies and cellular immunity than those that received 100 microg of DNA vaccines expressing HA, and were completely protected from lethal challenge with HB/327. Different vaccination doses were further tested in chickens, and these experiments demonstrated that 1 x 10(8)PFU of BV-G-HA offered complete protection from challenge with 100 LD(50) of HB/327. These data indicate that the pseudotype baculovirus-mediated vaccine could be utilized as an alternative strategy against the pandemic spread of H5N1 influenza virus.

A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip

A novel fluorescence immunoassay method for fast and ultrasensitive detection of avian influenza virus (AIV) was developed. The immunoassay method which integrated lateral flow test strip technique with fluorescence immunoassay used the label-free and high luminescent quantum dots (QDs) as signal output. By the sandwich immunoreaction performed on lateral flow test strip, the gold nanoparticle (NP) labels were captured in the test zone and further dissolved to release a large number of gold ions as a signal transduction bridge that was detected by the QDs-based fluorescence quenching method. Under the optimal conditions, the relative fluorescence intensity of QDs was linear over the range of 0.27-12 ng mL(-1) AIV, and the limit of detection was estimated to be 0.09 ng mL(-1) which was 100-fold greater than enzyme-linked immunosorbent assay (ELISA). The sensitive and specific response was also coupled with high reproducibility in the proposed method. A series of six parallel measurements produced reproducible fluorescent signals with a relative standard deviation of 4.7%. The proposed method can be used to directly detect clinical sample without any pretreatment, and showed high efficiency (90.0%), sensitivity (100.0%) and specificity (88.2%) compared with virus isolation (gold method). The new method shows great promise for rapid, sensitive, and quantitative detection of AIV in-field or point-of-care diagnosis.