Epidemiological Investigation Of Classical Swine Fever In Shandong Province And Analysis Of Genetic Variation Of E2 Gene

Classical Swine Fever（CSF）is an acute,pyretic,and contact infectious disease caused by Classical Swine Fever Virus（CSFV）.It is highly contagious and lethal.Pig is the only natural host in this disease.CSF is one of the most harmful pig diseases in the world pig industry which has caused huge economic losses.The CSFV first reported in Ohio in 1833and spread rapidly to all continents.In 1954,Chinese scientists developed a highly effective c-strain vaccine,which quickly controlled the CSF epidemic in China and helped many countries eradicate the threat of CSFV.In the late 1990s,Changchun Tu and other scholars conducted a systematic survey of CSFV in various regions of China and classified the CSF strains.According to the survey,there are gene type I（1.1 subtypes）,gene type II（2.1,2.2,2.3 subtypes）and gene type III（3.4 subtypes）of CSFV in China.At present,the mainly subtype in China is 2.1 subtype,and the gene type III exists only in Taiwan.This study conducted a systematic epidemiological investigation of the CSF epidemic in Shandong Province,based on the analysis of CSF clinical samples received from various cities in Shandong from 2016 to 2018.It included background investigation of CSF clinical cases,histopathological examinations and pathogen examinations,separation and identification of CSFV,and detection of CSFV-E2 antibodies.The major protective antigen gene（E2 gene）was sequenced and analyzed for genetic variation.The results showed that the symptoms of the affected pigs were not the same.The acute cases were characterized by high fever,neurological symptoms,skin spotted hemorrhage and high mortality.Symptoms of pregnant sows were abortion and stillbirth;boars showed symptoms such as acroposthitis and genital spine urinary retention;piglets had many neurological symptoms,such as bruxism,retropulsion,spin,tonic reflex,unable to stand,and even coma.Chronic cases were characterized by persistent fever,depression,diarrhea,marasmus,and it might become complex due to secondary or concurrent bacterial or other viral infections.The disease has no obvious seasonality,and it has changed from occurrence in winter-spring to the current four seasons.The necropsy lesions in acute cases were mainly characterized by extensive hemorrhage of the whole-body tissues and organs such as lymph nodes,kidneys,and gastrointestinal mucosa.There was hemorrhagic infarction at the edge of the spleen.In addition to hemorrhagic lesions and intestinal mucosal ulcer were shown in chronic cases.The pathological features of CSF were hemorrhagic necrosis of lymphoid tissue,viral encephalitis,and extensive hemorrhage of tissues and organs.Among the 1,716 samples submitted for testing,207 samples were detected for CSFV-positive by RT-PCR,and the positive rate was 12.06%（207/1716）.The results of CSFV-E2 antibody test showed that in12,709 serum samples,the positive rate was 76.85%（9767/12709）.According to the subcluster statistics,the antibody positive rates of boars,pregnant sows,Reserve sows,piglets and growing-finishing pigs were 89.34%,84.44%,83.25%,70.94%and 67.66%separately.The antibody positive rate and antibody level were relatively high,but the immune effect of piglets and growing-finishing pigs was limited.The probable reason was the current occurrence of CSF.The lack of biosafety measures is also an important reason for the frequent occurrence of infectious diseases.The E2 genes of 41 isolates were sequenced.Using the methods of homology analysis,genetic evolution analysis and mutation site analysis,the isolates were compared with reference strains.The results showed that most of the isolates were in 2.1d subtype of CSFV,and only one strain was 2.1b subtype.The nucleotide homology and amino acid homology between them were 81.1%-96.2%and 88.2%-98.4%,and the isolates had the highest homology with the 2.1 subtype.TA mutation was found in the glycosylation site（986NYAK-NYTE989）by the mutation site analysis.It was speculated that the E2 protein produced the same mutation as the vaccine strain HCLV under the pressure of vaccine immunization.The epitope"⁷¹⁷TTWKEYSH⁷²⁴","⁷⁷¹LLFDGTNP⁷⁷⁸","⁸²⁹TAVSPTTLR⁸³⁷"were also mutated,but the effects of the mutations need to be further studied,and the epitope"⁹⁹⁵YYEP⁹⁹⁸"has not changed.Based on the above research results,the current infection pressure of CSFV in Shandong Province is still very heavy.Although the use of CSF vaccine can effectively protect the herd of swine,it does not completely eliminate the risk of epidemics.All scale pig farms should increase the monitoring of vaccine immune effects,adjust and optimize vaccine immunization procedures,and improve biosafety measures.On the basis of comprehensive prevention and control,they would achieve the detection and purification of core pigs.This study further enriches the epidemiological data of CSF in Shandong Province,and has certain theoretical guiding significance for the development of new vaccines.