| The Porcine Epidemic Diarrhea Virus(PEDV)is the causative agent of porcine epidemic diarrhea,a highly contagious disease that affects farm animals.The virus can cause diarrhea,vomiting,anorexia,dehydration and other clinical symptoms in pigs.The mortality rate of piglets infected with this disease is very high;In addition,the disease has the characteristics of contact transmission,which can be transmitted through contaminated water,feed and other channels,resulting in restrictions on the import and export trade of pigs and by-products.At present,vaccine immunization is the most important method to prevent and treat PED,and it has achieved obvious results in the early stage of PED outbreak.However,after 2010,due to the constant variation and recombination of the gene structure of PEDV,many new variant strains emerged,resulting in the poor match of the antigenicity of the commercial vaccines sold on the market and the current epidemic strains,resulting in the greatly reduced or even ineffective protection of the original vaccines.The PED epidemic broke out in most parts of China,and then also broke out in various parts of the world,This has caused huge economic losses to the global pig industry.Since 2010,the PEDV variant strain has shown the characteristics of stronger pathogenicity and wider epidemic range,which has brought greater pressure to the prevention and control of PED.In view of the lack of clear understanding of the genetic structure,biological characteristics and epidemiological investigation of the current PEDV epidemic strain,this experiment will carry out research from the following aspects to further understand the molecular characteristics and pathogenicity of the current PEDV epidemic strain in Hebei Province,and provide theoretical basis for the development and development of new and efficient vaccines and antiviral drugs.1.Epidemiological investigation was conducted on piglet diarrhea in six different regions of Hebei Province(Baoding,Handan,Xingtai,Cangzhou,Zhangjiakou,Cangzhou)from 2021-2022,and 134 suspected PED samples were collected for detection.In addition,this study successfully designed a pair of specific fluorescent primers for the conserved M gene,and established a general TaqMan qPCR detection method for screening PEDV in pig farms.The results showed that the method was highly specific and had no cross reaction with other common gastrointestinal viruses such as porcine delta virus(PDCoV),porcine rotavirus(PoRV)and infectious gastroenteritis virus(TGEV);The minimum detection limit of the established detection method for pMD19T-PEDV standard is 1.09×101 Copy/μL.It is about 100 times more sensitive than ordinary PCR,with high sensitivity;The coefficient of variation within and between groups is less than 1%,with good repeatability.The qPCR detection method established in this study was used to detect the suspected diarrhea samples collected above.It was found that 80 samples were positive for PEDV,with a positive rate of 59.70%.The detection rate of PEDV was 75.00%in Baoding and 45.45%in Cangzhou.The results showed that the epidemic situation of PED in the sampling area of this study was relatively serious,and the prevention and control of the disease was urgent2.In this study,a total of 10 PEDV epidemic strains were isolated from the 80 selected PED samples,and the full amplification of PEDV S gene was carried out,and the homology and genetic evolution of gene sequence were analyzed with reference to the S gene of 30 classical strains published from NCBI.The study found that 10 strains of PEDV S gene isolated in this study were located in two different groups,G Ⅱ-a and G Ⅱ-b,and were closely related to the epidemic strains in China in recent years,but far from the vaccine strains and early epidemic strains.The nucleotide similarity and amino acid homology with classical strain CV777 were 92.6%～94.4%and 91.3%～93.8%,respectively.The results showed that the current epidemic strain of PEDV has mutated,which may cause the original commercial vaccine to lose its protective effect.3.Inoculate 10 PEDV samples collected in the region with Vero cells for continuous blind transmission to the third generation,and add appropriate amount of trypsin(10μg/mL)to promote the combination of PEDV and Vero cells,and detect the isolation of virus by PCR.The results showed that when one of the PEDV samples was transferred to the second generation,Vero cells formed syncytium,and RT-PCR was positive for PEDV.It was confirmed by indirect immunofluorescence test and transmission electron microscope test that a cell-adapted virus strain CH/HB/CZ01 was successfully isolated in this study.The virus strain caused typical cytopathic changes such as obvious shrinkage and exfoliation in Vero cells,and the viral titer of the virus increased with the increase of generation and finally stabilized at 106.5 TCID50/mL.Then the green fluorescent PEDV virus particles can be observed by indirect immunofluorescence test.In order to further verify the virus isolated in this experimental study,the morphology of the isolated virus was observed through the electron microscope test.It was found that in the electron microscope field of vision,the virus particles with typical coronavirus morphological characteristics and a nearly circular shape of about 100 nm could be clearly seen on the surface of the virus.In order to understand the variation characteristics of the isolate,the whole genome of the isolate CH/HB/CZ01 was amplified and sequenced,and the S,ORF3 and M genes were selected for gene homology and genetic evolution analysis.The results showed that the nucleotide homology of the S,M,ORF3 genes of the strain and the reference strain were 93.3%～98.3%,94.9%,99.3%,94.7%～98.8%,respectively.The strain belonged to the GⅡ-b subgroup in the same branch as the epidemic strain in China in recent years,of which the S gene had the greatest variability,and the CH/JLDH/2016(MF346935)strain with the highest amino acid homology had 25 amino acid point mutations.Based on the above analysis,the isolated strain CH/HB/CZ01 is far from the vaccine strain,not in the same branch,and is close to the epidemic virus strain in recent years.Through animal regression test,the isolated PEDV virus solution was inoculated into healthy piglets at 2.0 mL/head,and all of them had symptoms such as watery diarrhea,and the rectal swabs were identified as PEDV positive by RT-PCR after 2 days of inoculation.The results showed that the isolate had strong pathogenicity. |
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