| Porcine epidemic diarrhea is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus, which is characterized by diarrhea, vomiting, dehydration and high mortality rate of piglets. Porcine epidemic diarrhea virus(PEDV) is an enveloped, single-stranded RNA virus belonging to the genus Alphacoronavirus, subfamily Coronavirinae, family Coronaviridae.In China, PED has been reported in many swine-producing provinces, with mortality rates up to 90-100%, mainly involving breeding piglets up to 7 days of age, usually 2-3 days after birth. As one of the major swine-producing provinces in China, Shandong has suffered significant economic losses because of these new PED outbreaks, despite the availability of a vaccine. Since 2012, a remarkable increase in PED outbreaks occurred in many pig farms in Shandong province, which had brought a heavy blow to the pig industry. In order to investigate the molecular diversity and epidemiology of the virus, and develop new type effective oral vaccines for the current PEDV, following researches were conducted.We present the first molecular epidemiological analysis of PEDVs isolated from Shandong Province during the period from January 2012 to July 2015. 146 samples were tested as PEDV positve from collected 205 PED suspected samples, and the detection rate of PEDV was 71.2%(146/205) determined by reverse transcription-polymerase chain reaction(RT-PCR). Sequence analysis of the S genes of 38 strains which obtained from 13 different cities revealed high levels of sequence homology(95.0–99.9% nt, 94.3–99.8% aa), and differences in gene length differentiated these strains into the G2 and G3 clades of S gene phylogentic tree. This tree was drawn by analysis of 41 representative classical PEDV strains from the NCBI database and the above-mentioned 38 local strains, which was divided into three clades: G1, G2 and G3. G1 contained mainly the classical strains and vaccine strains, G2 contained 7 of the 38 strains origanted from 4 different cities in Shandong province. The G3 group included 56 epidemic strains, 31 of which were derived from this study, the other 25 including 17 Chinese epidemic strains, 3 Korean epidemic strains and 5 American epidemic strains. The length of G3 in S gene was mostly 4161 bp, but the Gd branch was an exception, with a length of 4158 bp. CH-SDLY-2-2014 and CH-SDLY-3-2014, isolated from Linyi in South Shandong in 2014, belonged to the Gd subclade and appeared to represent a novel genotype, which was the first discovery in North China. Multiple sequence alignments revealed many insertions or deletions in the S genes and mutations relating to virulence were detected in the antigenic regions, which may be one of the main reasons for the frequent outbreaks of swine epidemic diarrhea recently. Then we concluded that strains harboring two predominant S gene genotypes G2 and G3(including Gd) had been circulating in Shandong from 2012 to 2015.The complete genome of CH-SDLY-2014 was 28,035 nucleotides(nt) in length without the poly(A) tail. Its genomic organization was similar to that of other reported PEDVs with the characteristic gene order. On the basis of CLUSTAL method, strain CH-SDLY-2014 showed 96.6-99.0% and 93.5-98.5% nucleotide homology and amino acid homology to PEDVs currently circulating in the world for the whole genome and the full-length spike(S) gene, respectively. By phylogenetic analysis of the complete genome, the phylogenetic tree was divided into three groups(G1, G2 and G3), among them, the G3 including 2 subgroups(3a and 3b). The novel CH-SDLY-2014 strain was clustered in the subgroup 3a including 6 PEDV strains from South China, but distantly related to other PEDV strains circulating in North China or other countries. The same topology was also found in the phylogenetic analysis of the full-length S gene. Interestingly, results showed that subgroup 3b strains predominantly displayed a 4161-nt S gene sequence, but subgroup 3a displayed a 4158-nt gene sequence. Subgroup 3a had 14 field strains collected after 2011, the novel CH-SDLY-2014 strain in this study located in North China, 12 strains located in South China and one strain from South Asia. They all shared a typical molecular characteristic displaying a 3-nt deletion(nt 3580-3582 compared with CV777) in the part of the S gene encoding the C-terminal region. This feature may be used as the basis for differentiation 3a strains from other PEDV strains.S protein is the surface protein of PEDV particle, in addition to the role of mediating immune, this protein has other biological functions such as promotion the fusion of the viral and cell membranes, recognition of target cells, which has been selected as target protein for reseach of new type effective PEDV vaccine. In this study, the recombinant plasmid was built by connecting the SPEDV gene with p ET-30 a, and imported into the E.coli BL21 for induced expression in vitro. The SPEDV protein was expressed efficiently through optimization of various expression conditions such as expression time, temperatureand dosage of inducer(The ideal expression conditions: the recombinant E.coli were expressed for 4 hours at 37℃ induced by 1 m M IPTG). Then using the purified expression protein, we established an basic indirect ELISA used for detesting antibody of SPEDV protein.As a gene engineering vaccine recipient bacterium, lactobacillus could combine its biological functions with specific immune function of exogenous gene, so as to enhance the nonspecific immunity and specific immunity in animals. In this study, we transfered recominant p Rc/CMV2-S1-Rep.8014 and p Rc/CMV2-S2-Rep.8014 to L. acidophilus 1C1 to develope recombinant lactobacillus vaccine expressing PEDV SPEDV protein. Subsequently, multiple groups of BALB/c mouses were oral immunized by this vaccine, humoral and cellular immune parameters were detection for exploring the immune effect of the oral vaccine.BALB/c mouses were vaccinated by recombinant L.acidophilus 1C1: p Rc/CMV2-S1(2)-Rep.8014, p Rc/CMV2-S1-Rep.8014 and p Rc/CMV2-S2-Rep.8014, at the same time, control groups were immuned by PBS, p Rc/CMV2 and commodity vaccine respectvely. Blood and small intestine and feces samples were collected, and sera were isolated to detect the specific Ig G antibody while small intestine and feces samples were to be tested for the SIg A antibody. Results showed that oral vaccine groups were lower than commodity vaccine group in the degrees of Ig G antibody(p <0.05), but the specific SIg A antibody in oral vaccine group was much higher than that in commodity vaccine group(p <0.01). The percentages of both CD4+ and CD8+ T lymphocytes in splenocytes of mice immunized with p Rc/CMV2-S1(2)-Rep.8014 were significantly increased compared with the other groups(p <0.05). The levels of Th1-type cytokines IFN-γand Th2-type cytokines IL-4 in the sera of immunized mice were measured by ELISA. Results showed that the oral vaccine had a certain immune effect, and can stimulate the body to produce higher humoral immune antibody.With the development of molecular biology, the NGS technology has a great influence on the structure and function of bacteria in the environment. Rarefaction curve reflected the amount of data in the sample was sufficient, and the biodiversity in S1 and S2 groups in the early or late period were lower than that in the middle period. The intestinal microbiota composition of S1 and S2 group were most similar. Community structure analysis showed that Lactobacillus and Firmicutes were highly abundant in two treatment groups. We speculated that the third Lactobacillus oral vaccine should be added to the immune system which might improve the maintenance of the antibody level. |
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