Da-Liang Jiang

Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2-94.9% and 89.9-93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1-95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.

Epitope screening of the PCV2 Cap protein by use of a random peptide-displayed library and polyclonal antibody.

Capsid protein (Cap), the only structural protein of porcine circovirus 2 (PCV2), is involved in the host protective response and is a target for vaccine development. To find a rapid and easy way to fully map the antigenic epitopes of Cap, purified Cap-specific polyclonal antibodies were used to screen a random heptapeptide phage display library. After three rounds of screening, twenty phage clones that had binding activity to Cap-specific antibodies (tested by phage ELISA) were sequenced. When the inserted amino acid sequences were aligned with the Cap protein sequence, eight core regions in Cap ((50)SRTFGYT(56), (62)VRTPSW(67), (68)AVDMMR(73), (79)FLPPGG(84), (86)SNPRSVPF(93), (102)KVEFWP(107), (119)GSSXXXLDDN(128) and (229)PPLNP(233)) were identified, three of which ((50)SRTFGYT(56), (86)SNPRSVPF(93) and (102)KVEFWP(107)) for the first time. Nine phagetopes representing the eight regions were chosen to immunize Kunming mice. All except minotopes (50)SRTFGYT(56) and (229)PPLNP(233) induced antibodies against PCV2 when injected into Kunming mice.

Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

ABSTRACT A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

Small-Sized Circular Genomes Similar to Genome of Porcine Circovirus 2

ABSTRACT Circular genomes smaller than and similar to the genome of porcine circovirus 2 were obtained from pig tissues along with the full-length genome of porcine circovirus 2. The 922-, 839-, and 617-nucleotide-long genomes exhibit high homology to the rep gene plus the origin of replication sequence of porcine circovirus 2.

Seroprevalence of porcine cytomegalovirus and sapovirus infection in pigs in Hunan province, China

The seroprevalence of porcine cytomegalovirus (PCMV) and sapovirus (SaV) infections in pigs was investigated in Hunan province, China, between May 2005 and October 2010. A total of 500 pig serum samples collected from 10 representative administrative regions in Hunan province were evaluated for antibodies against PCMV and SaV using enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of porcine cytomegalovirus and sapovirus in pigs was 96.40% (482/500) and 63.40% (317/500), and the seropositivity of 10 herds we surveyed varied, ranging from 94.74% to 98.48% and 56.36% to 72.50%, respectively. The highest prevalence was found in breeding sows (96.67% for PCMV and 83.33% for SaVs). The results of the present survey indicated that infections with porcine cytomegalovirus and sapovirus are highly prevalent in pigs in Hunan province, China.