Chen Huan-chun

Quantitative and Qualitative Study of Enzyme-linked Immunosorbent Assay to Detect IgG against Japanese Encephalitis Virus in Swine Sera

This study was designed to investigate the application of indirect enzyme-linked immunoassay (ELISA) in detecting IgG against Japanese encephalitis virus in swine sera and the qualitative nature of this test. The attenuated strain SA14-14-2 of Japanese encephalitis virus (JEV) was inoculated into 9-day-old chicken embryos and virus was harvested, purified and suspended in 0.9% saline as JEV antigen. The control antigen was prepared by the same method as for the antigen. In the ELISA, the optimal concentrations of antigen coated and dilution factor were selected using χ2 test. Ninety-two swine sera negative to haemagglutination inhibition (HI) were tested by this assay and the positive threshold was determined. The results of this study indicate that indirect ELISA has high specificity, sensitivity and reproducability. Simultaneous testing of 74 serum samples from nine pig farms was carried out to compare the existing HI test and the indirect ELISA. The coincidence rate of the two assays was 85.1% (63/74) and no significant difference was observed between them (p>0.05). This ELISA test can detect 46 swine serum samples qualitatively and the titre of eight swine serum samples through endpoint dilution quantitatively within one 96-well plate.

The development and application of the latex agglutination test to detect serum antibodies against Japanese encephalitis virus

The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20 000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.

Development of a latex agglutination test using the major epitope domain of glycoprotein E of pseudorabies virus expressed in E. coli to differentiate between immune responses in pigs naturally infected or vaccinated with pseudorabies virus.

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.

Microarray-Based Detection and Differentiation of Virulent and Attenuated Pseudorabies Virus

DNA microchip used in this study was formed from miniature arrays of pseudorabies virus (PrV) gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip (microarrays) was used for differentiation between virulent and attenuated PrV. The presence of four gene segments (gB, gD, gE+, and gE-) encoding conservative glycoprotein B (gB), D (gD), and E (gE) of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE+ gene fragments was shown in virulent (gE+ genotype) and attenuated PrV (gE- genotype), whereas gE- gene (deleted domain in gE gene) was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV-2). The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK−/gE−/GP5+ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK−/gE−/LacZ+, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK−/gE−/LacZ+, resulting in a PRV recombinant named TK−/gE−/GP5m+, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK−/gE−/GP5m+ and TK−/gE−/GP5+ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK−/gE−/GP5m+ against PRRSV were higher than that induced by TK−/gE−/GP5+. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.

Cloning, expression and immunogenic characterization of the apxIIA gene of Actinobacillus pleuropneumoniae

The structural gene encoding ApxⅡ toxin (apxIIA) was amplified from the genomic DNA of Actinobacillus pleuropneumoniae serotype 2 (APP-2) strain HB08 and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blotting analysis showed that the apxIIA gene was expressed in Escherichia coli BL21 (DE3) and the expression products could react with ApxⅡ antibodies. The recombinant ApxⅡ was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice with an interval of two weeks using unfolded and refolded recombinant proteins, the native ApxⅡ extracted from the cultural supernatant of APP strain of serotype 7 (APP-7) and PBS, respectively. Serum antibody was examined by ApxⅡ-specific ELISA two weeks post every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08×108CFU (colony form unit) per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the misfolded recombinant protein group, while all the mice in the PBS group died in 36 h post challenge. The data revealed that the refolded recombinant ApxⅡ had excellent immunogenicity and could elicit protection from the lethal challenge of APP.

Development and preliminary application of gE enzyme-linked immunosorbent assay for detection of the antibody to gE protein of Pseudorabies virus in pigs

Abstract To differentiate pigs infected with Pseudorabies virus (PrV) from pigs vaccinated with gE-PrV, aglycoprotein E enzyme-linked immunosorbent assay (gE-ELISA) based on recombinantglycoprotein E (gE) (which was expressed by Escherichia coli, purified, denatured andrenatured) was developed. By testing 115 serum samples, the diagnostic specificity andsensitivity of the developed gE-ELISA were evaluated to be 94.5% and 96.7%, respectively. Fiveserum samples were tested with plates from five lots, and the results had a coefficient ofvariation of less than 10%, showing good reproducibility of gE-ELISA. This gE-ELISA wascompared with a commercial blocking ELISA by testing 356 serum samples. The agreement rateof the two assays was 92.13% (328/356). These results suggested that the gE-ELISA developedin our laboratory could be used in differentiating PrV-infected and gE-PrV-vaccinated pigs. Keywords: Pseudorabies virus; glycoprotein E; enzyme-linked immunosorbent assay (ELISA) Introduction Aujeszky’s disease is one of the most serious infectiousdiseases of several domestic and wild animals. It is causedby Pseudorabies virus (PrV). Swine are its natural hostand reservoir. Once piglets and other animals are infectedby PrV, the mortality is generally 100%. Reproductionrates will be low if boars and sows are infected by PrV.Pseudorabies can easily spread between pigs. It causeshigh economic losses in the pig industry (Mettenleiter,2000). Control of pseudorabies depends mainly onvaccination, as there are no effective therapeutic drugs.In many developed countries, pseudorabies has beencontrolled or eradicated by using marker vaccines anddifferentiation diagnostic assays (Stegemam, 1997). But inChina, pseudorabies still breaks out frequently and badlyaffects the Chinese pig industry. Therefore, studying themarker vaccine and differentiation diagnostic assays, andputting eradication campaigns into practice, are essentialin China.The genome of PrV codes for more than 100 proteins,and there are about 50 proteins on the mature virus. Atleast 11 glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL,gM and gN) were found in the membrane of PrV (Hampl,1984). The gE gene is a non-essential glycoprotein genefor PrV replication, but is associated with its virulence.Thus deletion of the gE gene does not influencereplication, but decreases the virulence of the virus.