Multiple genes expression and rotavirus-VLPs production in silkworm.

<p>(a) The larvae of six days post injection were observed using the fluorescence detection device. 1: the mock injected larvae; 2&3: the larvae injected with invasive and DAP auxotrophic <i>E. coli</i> carrying BmBacmid with six genes including <i>egfp</i>, <i>dsRed</i>, <i>eyfp</i>, <i>vp2</i>, <i>vp6</i> and <i>vp7</i> at a dose of 8.0×10⁸ cells per larva. (b) The hemolymph from a red larva was observed using laser confocal microscope. The images of hemocytes were taken at the bright (trans) channel (I), GFP (515 nm) detection channel (II), DsRed (590 nm) channel (III) and YFP (530 nm) channel (IV). (c) Western blot analysis of the hemolymph from the red larvae using anti-VP2, anti-VP6 and anti-VP7 rabbit antiserums. (d) The EM image of hemocytes collected from red larvae. R: the round Rotavirus-VLPs; B: the rod shape baculovirus particle, bar = 100 nm.</p

The flowchart of producing infective BmNPV expressing ten heterologous genes in silkworm larvae or pupae.

<p>The target genes are cloned into the three donor vector (pCTdual, pRADM and pUCDMIG) using usual method. The first two genes carried by pCTdual are inserted into BmBacmid through <i>I-Sce</i> I linearization and <i>red-gam</i> homologous recombination. Four genes in pRADM and the other four genes in pUCDMIG are then introduced into BmBacmid via Tn7 transposition and cre-loxp recombination, respectively. As a result, ten foreign expression cassettes and three antibiotic screening markers, as well as a GFP illumination marker are introduced into BmBacmid. The invasive and DAP auxotrophic <i>E. coli</i> carrying recombinant BmBacmid are injected into silkworm larvae at an appropriate dose. Consequently, recombinant BmNPV will be produced and multiple foreign genes will be expressed in green <i>B. mori</i> larvae or pupae.</p

SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.

<p>5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.</p

Additional file 1: of Evolutionary and genetic analysis of the VP2 gene of canine parvovirus

Table S1. The detail numbers of the three discovered mutation sites of the 424 sequences. Table S2. The nucleotide contents of the selected sequences and the mean ± SD values of the A%,T%,G%,C%,respectively. Table S3. The correlation analysis of codon usage indices. *Signifies 0.05 > p > 0.01; **signifies p < 0.01. Table S4. The abundance of the 16 dinucleotides. Table S5. The detail information of the 424 sequences. (DOCX 96 kb

Additional file 1: of Evolutionary and genetic analysis of the VP2 gene of canine parvovirus

Table S1. The detail numbers of the three discovered mutation sites of the 424 sequences. Table S2. The nucleotide contents of the selected sequences and the mean ± SD values of the A%,T%,G%,C%,respectively. Table S3. The correlation analysis of codon usage indices. *Signifies 0.05 > p > 0.01; **signifies p < 0.01. Table S4. The abundance of the 16 dinucleotides. Table S5. The detail information of the 424 sequences. (DOCX 96 kb

The prevalence of HEV RNA in pig bile samples collected from the Pearl River Delta. ^b

b<p>Pig bile samples have been collected from 9 districts of the Pearl River Delta since 2011.</p

The information of swine farms and samples.

<p>Bile samples were collected from 22 different swine farms including large-scale farms and family-scale farms in 9 districts of Pearl River Delta. Swine sera were sampled from 12 large-scale swine farm in Guangdong. N, nursery pig; G, growing pig; S, sow; B, boar.</p

The anti-HEV rates of the population in Guangdong province, China, according to age, gender, and pig-exposure status.

<p>a: OR, odds ratio; b: 95%CI, 95%confidence interval; c: X², Chi-Square Test; d: ref, reference; UF, urban female; UM, urban male; FF, swine farm female; FM, swine farm male; **, significant difference; *, different.</p

The estimated seroprevalence of the hepatitis E virus in Guangdong Province.

<p>The best fit trendline for the positive rate data was selected based on the R-squared value of the curves drawn using the Microsoft Office Excel 2007 program (Microsoft, Redmond, WA). 1. Overall gender-specific seroprevalence; 2. Gender-specific seroprevalence of swine farm; 3. Gender-specific seroprevalence of the general population; 4. Overall population-specific seroprevalence; 5. Population-specific seroprevalence of females; 6. Population-specific seroprevalence of males.</p

A phylogenetic tree based on the full nucleotide sequence of HEV.

<p>Total RNA positive bile samples are 48, from 9 different districts in Guangdong. We have chosen at least one sample from each district by random sampling. The amplification products of ORF2 (509 nucleotides, primer sequences were HEV-INF and HEV-INR as described above) from 10 positive bile samples were sequenced and compared. The nucleotide sequence identity among the 10 swine HEV isolates obtained from pigs from different farms in three years ranged between 94.3 and 99.8%. Phylogenetic trees were constructed by the neighbor-joining method based on the partial nucleotide sequence of the ORF2 region (509 nucleotides). The bootstrap values (expressed as percentages) were determined on 1000 re-samplings of the data sets. △were the isolates in this study.</p