Meng Ge

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.

Identification of new defective interfering RNA species associated with porcine reproductive and respiratory syndrome virus infection.

In the present study, seven new defective interfering (DI) RNA species of porcine reproductive and respiratory syndrome virus (PRRSV) were identified. RT-PCR, Northern blot and sequence analyses indicated that these DI RNA specie have deletions of 8513-9176 nucleotides located between Nsp1/Nsp2 and Nsp10. Compared with the previous DI RNAs of PRRSV reported, they have three distinct characteristics: much smaller deletion sizes; different nucleotide repeats (2-12nt) used in the junction sites and in-frame deletions. The results further suggested that the similarity-assisted RNA recombination may be the main cause of generation of DI RNAs in PRRSV and probably in other arteriviruses.

First isolation and genetic characteristics of porcine sapeloviruses in Hunan, China.

Outbreaks of diarrhea in piglets cause serious economic consequences in China. Diarrhetic fecal samples from 20 Hunan farm piglets were tested and found to be positive for porcine epidemic diarrhea virus (PEDV) by RT-PCR, although incubation with porcine kidney (PK-15) cells failed to produce infectious PEDV. Four porcine sapelovirus (PSV) strains (designated as PSV-HuNs) were isolated from four of the samples. Genomic sequence analysis revealed open reading frames encoding polyproteins of 2,331 (HuN1, 2 and 3) and 2,332 (HuN4) amino acids. Homology comparisons of the VP1 gene of the four Hunan strains with previously reported PSV strains revealed nucleotide sequence identities ranging from 74.2 to 98.6%, and deduced amino acid sequence identities from 79.5 to 98%. Phylogenetic analyses based on full-length and partial VP1 gene sequences showed that 3 of the PSV-HuN strains (HuN2, 3 and 4) clustered within a clade distinct from HuN1 as well as from all PSVs previously isolated in China, thereby showing that genetic diversity exists within Chinese PSVs. In addition, recombination analysis among PSVs indicates that a recombinant (HuN2 strain) exist in China.

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.

Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species.

Prevalence of Porcine teschovirus genotypes in Hunan, China: identification of novel viral species and genotypes

Porcine teschovirus (PTV) comprises at least 13 genotypes (PTV 1-13). Here, the genotypes of field strains prevalent among pig populations in Hunan Province, China, were identified. Multiple PTV genotypes, including all genotypes except PTV 7 and 8, were found co-circulating in the pig populations, reflecting a high genetic diversity. Moreover, we identified nine novel PTV genotypes, provisionally designated as PTV 14-22. PTV 21-HuN41 and PTV 21-HuN42 were successfully isolated, and their nearly complete genomes were sequenced. Homology comparison of the polyprotein genes of PTV 21-HuN41-42 to those of other known PTVs revealed low identities, ranging from 70.1 to 71.9 % (nucleotide identity) and 75.4 to 77.6 % (amino acid identity). Moreover, PTV 21-HuN41-42 were identified as a novel teschovirus species (tentatively Teschovirus B), based on the analyses of phylogenetics and evolutionary divergence. The findings of this study are expected to greatly enrich our knowledge of PTV genotypes.