| Senecavirus A(SVA)belongs to genus Senecavirus of family Picornaviridae,which is a new pathogen causing porcine idiopathic vesicular disease in recent years.Senecavirus A mainly infects pigs and characteristic symptoms of sick pigs are are blisters and ulcers in multiple organs of the whole body,such as the mouth and hoof crown.The mortality of piglets is as high as 30%-70%.Since the first outbreak of SVA infection in Guangdong,China in 2015,there have been cases of SVA infection in Shandong,Henan,Heilongjiang,Hubei,Sichuan,Fujian,Guangdong and other provinces.The research on its epidemiology and pathogenesis is limited at home and abroad,due to senecavirus A disease is a new infectious disease.In view of this,two SVA strains GX84/2020 and GX94/2020 were isolated and sequenced.Phylogenetic analysis based on the full-length sequences of two isolates and 210 SVA strains show that SVA can be roughly divided into two genotypes,GX84/2020 and GX94/2020 belong to genotype ?,and all SVA strains show the characteristics of evolution over time,which can be divided into three stages:relatively stable period(1986-2010),rising period(2010-2017)and falling period(after 2017).In addition,the results of pigs challenged showed that GX84/2020 and GX94/2020 could successfully infect pigs and cause typical clinical symptoms of SVA,such as elevated body temperature,claudication,blisters and ulcers in nose,lip and hoof.Finally,SVA replicons combined with site directed mutation was used to find that(H42A and Q79A)mutations and(?5H,?20G,?34N,?42H,?56R)deletions of L protein can significantly inhibit the expression of reporter gene,indicating that SVA L protein has an important impact on viral transcription and translation.This study reveales for the first time that SVA L protein has the function of regulating viral transcription and translation.1.Isolation,identification and genetic evolution analysis of Senecavirus AThere were some pigs had clinical symptoms such as depression,loss of appetite,blisters and erosion on lips and feet in a pig farm in Guangxi in 2020,which was suspected to be SVA infection.In order to determine the pathogen of the disease,two blister scabs at the lesion were detected by RT-PCR,and both of them were SVA positive.After grinding and aseptic treatment,the positive disease material was inoculated with well-growing ST-R cells for virus isolation,and identified by IFA and WB.Meanwhile,the full-length genome of the SVA isolated strains was amplified,sequenced and spliced.The results strains were named GX84/2020(GenBank No:MW117126)and GX94/2020(GenBank No:MW117127)and uploaded to GenBank.Subsequently,we study the phylogenetic analysis and evolutionary dynamics analysis of GX84/2020 and GX94/2020 strains and 218 SVA strains that have full of complete genome originate from GenBank.The results of phylogenetic analysis showed that all SVA strains were mainly divided into two genotypes,Type ? strains were from 19892006,type ? strains were from 2007-2020,and GX84/2020 and GX94/2020 belongs to genotype ?.Further evolutionary dynamics analysis showed that SVA has the characteristics of evolution over time.On the whole,the historical scale of the population can be divided into three stages:from 1986 to 2010,it remained relatively stable;From 2010 to 2017,there was an obvious upward trend;After 2017,there was a decline.GX84/2020 and GX94/2020 strains belong to the third stage.2.Pathogenicity analysis of Senecavirus AIn order to determine the pathogenicity of Guangxi isolates,12 healthy piglets aged 8-10 weeks were divided into three groups for pig challenge test,namely GX84/2020 group,GX94/2020 group and PBS control group,with 4 pigs in each group.The results showed that there were differences in the degree of clinical lesions in the infected groups,but all pigs showed the typical symptoms of SVA dieases.Among them,two pigs in GX84/2020 group had relatively serious clinical symptoms,including elevated body temperature,blisters on nose and lip,blisters and ulcers on hoof,rupture of hoof shell and severe claudication.But the other two pigs had mild symptoms,only fever and hoof swelling were observed.In contrast,the clinical symptoms of GX94/2020 infected group were mild,in addition to fever and loss of appetite,one pig had swelling and blisters on its snout;Swelling between the toes of 1 pig's hoof;The other 2 pigs had ulceration and scab on their hoofs.In addition,the changes of detoxification amount of pig nose swab and serum neutralizing antibody level from 0 to 14 days were also monitored.The results of nasal swab detoxification showed that virus shedding was detected in the nasal swab on the 3rd day after virus attack,and reached the peak level on the 7th day.The neutralizing antibody results showed that the neutralizing antibody was detected on the 5th day after infected,the neutralizing antibody titer increased rapidly after the 5th day,and began to rise slowly on the 7th day until the neutralizing antibody still existed on the 14th day.It should be noted that the neutralizing antibody titer showed an inflection point on the 7th day,which was exactly the same as the time when the nasal swab virus disappeared in the body.3.Function study of L protein of Senecavirus AL protein(lead protein)is the first protein encoded by Senecavirus A.Because L protein sequence is highly variable in small RNA virus and its function is not conservative,its function in the whole life activity of SVA virus is not clear at present.,The L gene was mutated with H5A?H42A?R56A?Q79A amino acids and deleted with ?5H??20G??34N??42H??56R??69Q amino acids based on the infectious clone pC3-SVA-GD05.Then the virus was rescued and the virus titer was determined.The results showed that ?42H,?56R and Q79A were fatal to the virus.The titer of H42A and ?20G virus decreased by 5 orders of magnitude,which seriously weakened the virus;H5A,A5H,?34N,?69Q decreased by two orders of magnitude;R56A decreased by one order of magnitude.In order to further determine the role of L gene,we established the subgenomic replicator of SVA virus,mutated and deleted L gene,and then studied the effect of L gene on virus transcription and translation.The results showed that the level of RNA produced by intracellular replication of SVA replicator decreased after H42A and Q79A mutations,indicating that L protein mutations affected virus replication.but Rep-?5H,Rep-?42A,Rep-?42H,Rep-?56R,Rep-?20G,Rep?34N and Rep-Q79A seriously(300-700 times)inhibited the transcription and translation of the virus,and Rep-H42A and rep-?69Q reduced the transcription and translation activity of the virus.These results suggest that SVA L protein plays a regulatory role in viral transcription and translation.In general,L protein is essential for the survival of SVA virus.It plays an important role in the process of virus replication,transcription and translation. |
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