Molecular Characterization of the <i>ORF3</i> and <i>S1</i> Genes of Porcine Epidemic Diarrhea Virus Non S-INDEL Strains in Seven Regions of China, 2015

<div><p>In an effort to trace the evolution of porcine epidemic diarrhea virus (PEDV), <i>S1</i> and <i>ORF3</i> genes of viruses identified in 41 pig farms from seven regions (North, Northeast, Northwest, Central, East, South West, and South, respectively) of China in 2015 were sequenced and analyzed. Sequence analysis revealed that the 41 <i>ORF3</i> genes and 29 <i>S1</i> genes identified in our study exhibited nucleotide homologies of 98.2%–100% and 96.6%–100%, respectively; these two genes exhibited low nucleotide sequence similarities with classical CV777 strain and early Chinese strain LZC. Phylogenetic analysis indicated that the identified PEDV strains belonged to global non S-INDEL strains, and exhibited genetic diversity; <i>S1</i> gene of the HLJ2015/DP1-1 strain harbored an unique deletion of 12 nucleotides (A¹¹³⁰CAACTCCACTG¹¹⁴¹); while the Chinese PEDV S-INDEL reference strains included two types of the “CV777” S-INDEL as well as the “US” S-INDEL, and all co-circulated with Chinese non S-INDEL strains. Of 29 identified <i>S1</i> genes, the SS2 epitope (Y⁷⁴⁸SNIGVCK⁷⁵⁵) was highly conserved, while the SS6 epitope (L⁷⁶⁴QDGQVKI⁷⁷¹) and pAPN receptor-binding region (aa 490–615) exhibited amino substitutions. Nine possible recombination events were identified between the 29 identifed <i>S1</i> genes and the 3 <i>S1</i> reference genes from early Chinese PEDV strains. The complete <i>S</i> genes of selected Chinese PEDV field strains (2011–2015) showed 5.18%–6.07% nucleotide divergence, which is far higher than the divergence observed in early Chinese PEDV strains (3.1%) (<i>P</i><0.05). Our data provide evidence that PEDV non S-INDEL strains with genetic diversities and potential recombination circulate in seven regions of China in 2015; Chinese PEDV S-INDEL strains exhibit genetic diversity and co-circulate with non S-INDEL strains.</p></div

Potential recombination analysis between the 29 identified <i>S1</i> genes and the 3 <i>S1</i> genes from early Chinese PEDV strains.

<p>Potential recombination analysis between the 29 identified <i>S1</i> genes and the 3 <i>S1</i> genes from early Chinese PEDV strains.</p

Information about PEDV-positive samples identified in our study.

<p>Information about PEDV-positive samples identified in our study.</p

<i>P. gingivalis</i> strain W83 morphology and 16S rRNA gene analyzing.

<p>A, The colonial morphology of <i>P. gingivalis</i> strain W83 on the BHI agar plate. B, The PCR amplification result of 16s rRNA for <i>P. gingivalis</i>. Lane 1: The PCR amplification product; Lane 2: DL 2 000 DNA marker. C, The PCR amplification result of ragB from <i>P. gingivalis</i> strain W83. Lane 1: DL 2 000 DNA marker; Lane 2:The PCR amplification product. D, The oligonucleotide sequences of PCR primers for the genes of 16s rRNA and ragB from <i>P. gingivalis</i> strain W83.</p

The anti-RagB IgG antibody levels in serum and RagB-specific antibody-forming cells in spleens and bone marrow.

<p>A, The levels of RagB-specific antibody in pIRES, pIRES-<i>ragB</i> and pIRES<i>-ragB-mGITRL</i> groups; The sum of RagB-specific antibody-forming cells of spleen (B) or bone marrow (D) in three group respectively. The comparison of RagB-specific antibody-forming cell level in spleen (C) or bone marrow (E) between pIRES-ragB-mGITRL group and pIRES-ragB group. The results are expressed as means ± SD obtained for five mice per group. *P<0.05 vs. pIRES-<i>ragB</i> group.</p

Divergence analysis of complete <i>S</i> gene of the selected Chinese PEDV strains.

<p><i>Note</i>. The values (red) represent “average value ± standard deviation” of the nucleotide divergence (%) of each group; the different letters represent statistically significantly difference (<i>P</i><0.05) among six groups, and the same letters (one or more) represent no significant difference (<i>P</i>>0.05) among six groups.</p

Construction of recombinant plasmid pIRES<i>-ragB-mGITRL</i> for immunization.

<p>Amplified <i>ragB</i> and <i>mGITRL</i> genes were inserted into the eukaryotic expression vectors pIRES, respectively. CMV, human cytomegalovirus immediate-early promoter; IRES, internal ribosome entry site; SV40, simian virus 40; Amp^r, ampicillin resistance gene; Neo^r, neomycin resistance gene.</p

Phylogenetic analysis of the identified PEDV strains on the basis of <i>S1</i> gene sequences.

<p><i>Note</i>. The “CV777” S-INDEL strain, North American (NA) prototype strain USA/Colorado/2013 (non S-INDEL), US S-INDEL strain OH851, Chinese S-INDEL strains (LZC, JS2008, and CH/S), Japanese S-INDEL strain ZK-O, South Korea S-INDEL strains (SM98 and virulent DR13), European S-INDEL strains (FR/001/2014, 15V010/BEL/2015, and GER/L00721/2014), Chinese PEDV field strains, and other countries non S-INDEL strains were choose for genotyping and phylogenetic analysis of the 29 <i>S1</i> genes identified in our study. Light purple dot represents PEDV strains identified in North China; red dot represents PEDV strains identified in East China; pink dot represents PEDV strains identified in Northeast China; grey dot represents PEDV strains identified in Northwest China; green dot represents PEDV strains identified in South China; yellow dot represents PEDV strains identified in Central China; blue green dot represents PEDV strains identified in Southwest China.</p

Expression of <i>RagB</i> or <i>mGITRL</i> in COS-7 cells or muscle cells of mice.

<p>These data were representative of three independent experiments with similar results.</p

Phylogenetic analysis of the identified PEDV strains on the basis of <i>ORF3</i> gene sequences.

<p><i>Note</i>. The classical PEDV strain CV777, North American (NA) prototype strain USA/Colorado/2013 (non S-INDEL), US S-INDEL strain OH851, Chinese early PEDV strains (CH/S), Chinese PEDV field strains (2006–2015), and other countries PEDV field strains were choose for genotyping and phylogenetic analysis of the 41 <i>ORF3</i> genes identified in our study. Light purple dot represents PEDV strains identified in North China; red dot represents PEDV strains identified in East China; pink dot represents PEDV strains identified in Northeast China; grey dot represents PEDV strains identified in Northwest China; green dot represents PEDV strains identified in South China; yellow dot represents PEDV strains identified in Central China; blue green dot represents PEDV strains identified in Southwest China.</p