Rongli GUO ,Jichun WANG ,Aihua MAO ,Libin WEN ,Bin LI ,Yanxiu NI ,Kongwang HE *

1.Institute of Veterinary Medicine，Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;

2.Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture,Nanjing 210014,China;

3.National Research Center of Veterinary Biologicals Engineering and Technology,Nanjing 210014,China

Responsible editor:Xiaohui FAN Responsible proofreader:Xiaoyan WU

Porcine pseudorabies(PR),also known as Aujeszky＇s disease,is a highly infectious disease that is mainly characterized by livestock fever,itching (except pigs) and encephalomyelitis,which is caused by porcine pseudorabies virus (PRV) or porcine herpesvirus Type I[1].PRV can infect pigs of different ages.Specifically,PRV causes abortion,mummification or embryonic death;the mortality rate of newborn piglets infected with PRV reaches 100%;adult pigs usually exhibit silent infection without any clinical symptoms but carry and release viruses for a long term.So far,the occurrence of pseudorabies has been reported in more than 40 countries around the world.In China,Deng et al.[2]reported that pseudorabies occurred in 31 provinces and cities including Hong Kong and Taiwan.Many countries in Europe and America have successfully eradicated PRV from pig farms by immunization with genedeleted Bartha vaccines and other comprehensive prevention and control measures[3].The outbreak of pseu dorabies has been effectively controlled with similar methods in many pig farms in China.However,since 2012,it has been reported that pseudorabies occurred in several pig farms immunized with Bartha vaccines[4-6],leading to weak fetuses,stillbirth,abortion,as well as neurological symptoms,respiratory symptoms and other clinical symptoms in piglets with relatively high mortality rate in Heilongjiang,Jilin,Tianjin,Hebei,Hubei,Sichuan,Jiangsu,Guangzhou and other regions.In addition,PRV mutants were isolated from infected pig farms[4,7-8].At present,porcine pseudorabies has caused great economic losses to the pig industry in China.

PRV genome encodes 11 types of envelope proteins,including gB,gC,gD,gE,gH,gI,gK,gL,gM,gN and gG glycoproteins[9-10].To be specific,gE glycoprotein is a major virulence factor of PRV,which not only plays an important role in the mediation of cell fusion and infection,virus spread among cells and virus release,but also play a certain role with gI in the invasion and spread of PRV in the nervous system[11-14].

In this study,a virus strain was isolated from the brain tissues of stillborn fetuses delivered by sows with suspected PRV infection in a pig farm in Jiangsu Province,which was identified as PRV and named PRV N5B strain;gE gene in PRV N5B strain was sequenced and compared with the published sequences in GenBank;subsequently,PRV N5B strain was used to infect piglets,aiming at laying foundation for the investigation of characteristics of current epidemic pseudorabies virus and improvement of prevention and control measures.

Materials and Methods

Cells and experimental animals

Vero cells,preserved by Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture; ten 6-week-old healthy piglets without PRV vaccination; detection results of gE and gB antibodies by using PRV gE antibody detection kit(CIVTEST SUIS ADVgE batch number:CAE.62HT) and gB antibody detection kit (CIVTEST SUIS ADVgB batch number:CAB.47GC) were both negative.

Reagents and medium

Agarose Gel DNA Purification Kit,purchased from Axygen Corporation;DNA Marker DL 2000,2×GC Buffer Ⅰ(II),LA Taq DNA polymerase (5 U/μl),purchased from TaKaRa Corporation;DMEM medium,purchased from Gibco Inc.

Isolation and incubation of PRV

Virus and disease material processingThe positive control virus PRV SD strain was isolated and preserved by Key Laboratory of Animal Diseases Diagnostic and Immunology in 2004.Stillborn fetuses delivered by sows with suspected PRV infection were collected from a pig farm in Jiangsu Province,where all the pigs were immunized with PRV Bartha attenuated vaccine.Brain tissues were collected from stillborn fetuses,added with PBS solution,ground into paste,prepared into 1:5 suspension,frozen and thawed three times,and centrifuged at 12 000 r/min for 10 min.The supernatant was collected and sterilized by filtration with a 0.22 μm disposable filter.The filtrate was preserved at-80 ℃before use.

Virus isolation and determination of 50% tissue culture infectious dose TCID50After filter-sterilization,the above virus liquid was inoculated to single-layered Vero cells,adsorbed at 37 ℃for 1 h,added with cell maintenance medium (DMEM 2%FBS),and incubated at 37 ℃in a 5% CO2incubator.Cytopathic effects (CPE)were observed every day.Cell cultures without typical lesions were collected for three blind passages.When typical lesions were observed,the virus was purified by three rounds of colony selection.Subsequently,the purified virus was propagated to detect 50% tissue culture infectious dose (TCID50)[1].

PCR identification and sequencing Primer design and synthesisAccording to PRV gene sequence(JF797218.0) in GenBank,specific primers were designed for the amplification of full-length gE gene sequence.gE F:5’-AAGATGACGTTGGCCGAGCT-3’; gE R:5’-TTGTCGCTCTCGCTGTAGTA-3’.The designed primers were synthesized by Shanghai Invitrogen Biotechnology Co.,Ltd.

Viral DNA extraction and PCR amplificationViral DNA was extracted in accordance with the instructions of viral DNA extraction kit(Beijing Tiandz Inc.).The total PCR reaction volume was 50 μl,containing 25.0 μl of 2× GC Buffer Ⅰ,8.0 μl of dNTPs(2.5 mmol/L),0.5 μl of LA Taq DNA polymerase (5 U/μl),2.0 μl of template DNA,1.0 μl of upstream primer,1.0 μl of downstream primer,and 12.5 μl of ddH2O.The PCR amplification was started with initial denaturation at 95 ℃for 5 min,followed by 38 cycles of denaturation at 94 ℃for 30 s,annealing at 48 ℃for 30 s,and extension at 72℃for 150 s;the amplification was completed by holding the reaction mixture at 72 ℃for 10 min to allow complete extension of PCR products.PCR products were detected by using electrophoresis on 1% agarose gel,purified using gel DNA recovery kit,and sequenced by Shanghai Invitrogen Biotechnology Co.,Ltd.

Phylogenetic analysis of gE gene

gE gene sequences of PRV N5B 2013 strain isolated in this study and PRV SD2004 strain (positive control)were compared with those of 19 representative PRV strains in GenBank.Multiple sequence alignment and phylogenetic analysis were performed by Clastal Method using sequence analysis software DNASTAR.Lasergene.

Infection experiment of piglets Animal groupingPRV antibodynegative piglets were randomly divided into two groups.Five piglets in infection group were inoculated intranasally with 2 ml of 15thgeneration virus liquid of PRV N5B strain(106TCID50/0.1 ml);five piglets in control group were inoculated intranasally with 2 ml of DMEM medium.

Clinical observationAfter inoculation,piglets were observed twice a day.The clinical symptoms were recorded,and the rectal temperature was measured once a day.

Pathological observationIn the experimental period,piglets died of the infection were anatomized to observe the pathological lesions.

Virus detectionNasal swabs and rectal swabs were collected every day and inoculated to single-layered Vero cells for virus isolation.Moreover,brain tissues and viscera with severe lesions of piglets died of the infection were collected and inoculated to single-layered Vero cells for virus isolation.Cell cultures without typical lesions were collected for three blind passages.

Results and Analysis

Virus isolation,purification and TCID50 determination

The virus liquid was inoculated to single-layered Vero cells.CPE could be observed at 36 h post-inoculation(Fig.1).The diseased cells were gridlike in the early period; as time went by,the lesion area was expanded,and cells began to fuse with the formation of obvious syncytia,which turned round and were exfoliated finally.As the pathological status were stabilized,the virus was purified by three rounds of colony selection.The incubated virus in different generations was inoculated to Vero cells for propagation and TCID50determination.The results showed that TCID50of virus incubated for 4,8,10 and 15 generations reached 103.75/0.1 ml,105.5/0.1 ml,105.875/0.1 ml and 107.125/0.1 ml,respectively.

PCR identification of the virus

PCR amplification products (containing full-length gE gene sequence)of the isolated virus strain were detected by 1% agarose electrophoresis and observed under an ultraviolet light.Results showed that clear specific bands were amplified,which were in accordance with the the expected size(Fig.2).

Phylogenetic analysis of gE gene

Sequencing results showed that gE gene sequences of PRV N5B strain and PRV SD strain were consistent,both 1 740 bp in length.Subsequently,gE gene sequences of PRV N5B strain,PRV SD strain and 19 representative PRV strains in GenBank were obtained for multiple sequence alignment and phylogenetic analysis.According to the phylogenetic tree of gE genes (Fig.3),gE genes were remarkably divided into two categories.To be specific,one category consisted of Chinese isolates and was divided into two groups,one of which (boxed)consisted of strains isolated since 2012,including PRV N5B strain isolated in this study;AY173124 2002 strain and SD strain were clustered into the other category,which indicated that PRV N5B strain and strains newly isolated in 2012 (boxed) were clustered into a relatively independent branch,exhibiting a distant genetic relationship with previous isolates and foreign isolates.

According to multiple sequence alignment results,PRV strains isolated since 2012 (boxed) and strains in the other category exhibited multiple base substitutions,especially for G-A and T-C substitutions,which was mainly characterized by the insertions of GAC and CGA consecutive bases at loci 142-144 and 1 488-1 490,respectively(Fig.4).Individual strains (KF0104-99.1) only exhibited GAC insertion at loci 142-144.Although Shandong isolate (AY173124 2002) and SD strain(2004) both exhibited insertions at two loci,there were many base substitutions compared with PRV strains isolated since 2012.

Homology analysis of gE genes showed that gE nucleotide sequence of PRV N5B2013 strain shared 96.4% -100% homology with other PRV isolates.gE sequence of PRV N5B strain shared the highest homology (99.7%-100%) with PRV strains isolated since 2012 (boxed),which also shared 99.0%-99.2% homology with PRV strains isolated previously in China (AY173124 2002 strain and SD strain) and 96.8%-97.2% homology with PRV isolates in the other category.

Animal experiments

Clinical symptomsThe body temperature of piglets increased significantly at 24 h post-infection and was maintained at about 42 ℃at 3-5 d post-infection.Diseased piglets exhibited anorexia and lassitude;respiratory symptoms were aggravated significantly with the exacerbation of disease,and diseased piglets mainly exhibited asthma,sneezing,expiratory dyspnea,as well as some neurological symptoms such as hindlimb standing instability,dementia,or even apastia,recumbency and death in serious cases.The mortality rate of experimental piglets reached 100%.All infected piglets exhibited no obvious symptom of itching.In control group,piglets had normal temperature and clinical performance with no abnormalities during 1-14 d.

Autopsy,PCR assay and virus de

tection In infection group,four out of five piglets exhibited severe symptoms and died.Autopsy results showed that dead piglets exhibited significantly increased cerebrospinal fluid,vascular dilatation and hemorrhage,obvious pulmonary edema,acute hemorrhage necrotizing,inflammations in the the entire respiratory tract,obvious inguinal lymph node swelling and hyperaemia,splenic marginal congestion and swelling,and blutene chloaide in the kidneys; in control group,no significant pathological changes were observed in the organs of piglets.Therefore,various organs of infected piglets were damaged by the virus,especially leading to significant pathological changes in respiratory tract and cerebrospinal fluid.In infection group,the target fragment could be detected in the brain tissues and visceral tissues of five piglets by PCR.After inoculation to Vero cells,typical PRV CPE was produced.Nasal swabs and anal swabs were collected every day,diluted,centrifuged and inoculated to Vero cells; at 4,5 and 6 d post-inoculation,CPE was produced by samples,indicating that viruses were released remarkably by piglets at 4,5 and 6 d post-inoculation.

Discussions

Pigs are the main hosts of pseudorabies virus.Nonpregnant adult gilts infected with pseudorabies virus generally exhibit no obvious clinical symptoms; however,pseudorabies virus infection in pregnant sows leads to stillbirth and abortion; pseudorabies virus infection in newborn piglets leads to relatively high mortality rates and neurological symptoms[1].Since the end of 2011,typical pseudorabies symptoms occurred successively in several pig farms in China,although all these farms were immunized with Bartha vaccines.Infected pigs exhibited high fever (40-42 ℃),depression,loss of appetite,coughing,asthma and neurological symptoms; newborn piglets exhibited high mortality rates with the symptom of itching that was not found in previous cases[15].According to PRV detection reports in Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture,6-week-old piglets in pig farms without immunization exhibited higher mortality rates after PRV infection.

In this study,by serial passage in Vero cells,PRV N5B strain was isolated from the brain tissues of stillborn fetuses delivered by sows with suspected PRV infection in a pig farm in Jiangsu Province.gE gene of PRV N5B strain was amplified and sequenced for phylogenetic analysis.The results show that gE gene of PRV N5B strain shares vary high homology(99.7% -100% ) with epidemic PRV strains but differs significantly from PRV strains isolated previously in China and foreign isolates.PRV N5B strain and PRV strains newly isolated in 2012 were clustered into an independent category,exhibiting a distant genetic relationship with previous isolates and foreign isolates.Based on multiple sequence alignment analysis,PRV strains isolated since 2012 exhibit multiple base substitutions with PRV strains in the other category,which is mainly characterized by the insertions of consecutive bases GAC and CGA at loci 142-144 and 1 488-1 490,respectively.This is consistent with related reports about the genetic variations of gE gene of PRV strains isolated in recent years[4,7-8].The results confirm that PRV N5B strain isolated in this study is an epidemic PRV strain,and its antigenicity exhibits certain variations compared with previous isolates.

Infection experiments were conducted with PRV N5B strain isolated by Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture.The results of animal experiments show that PRV N5B strain can infect 6-week-old piglets and causes typical symptoms of pseudorabies.Compared with previous cases of PRV infection,piglets infected with PRV N5B strain exhibit continuously increasing temperature.Body temperature of piglets increases significantly at 24 h post-infection and remains at about 42 ℃at 3-5 d postinfection,with obvious respiratory symptoms such as asthma,coughing,expiratory dyspnea and secretion increase,slight neurological symptoms,and unobvious itching symptoms.Tong et al.[8],Wang et al.[15]reported that 15-day-old and 35-day-old piglets inoculated with epidemic PRV strains had obvious itching symptoms and neurological symptoms such as ataxia.An et al.[4]found that 3-month-old pigs inoculated with epidemic PRV strains exhibited no obvious neurological symptoms or itching symptoms.Therefore,piglets of different ages show different clinical symptoms after inoculation with epidemic PRV strains.PRV N5B strain is highly lethal to 6-week-old piglets,and the piglets will die within 3-7 d after intranasal inoculation.All inoculated piglets release virus at 4-6 d.He et al.[16]reported that 20-day-old piglets inoculated with classical strain E A exhibited typical PRV symptoms,with a mortality rate of 75%.Thus,PRV N5B strain isolated in this study exhibits higher pathogenicity to piglets,indicating that current epidemic PRV strains with high pathogenicity may be the main cause of the outbreak of porcine pseudorabies in Bartha-immunized herd[4,6].This study can provide a theoretical basis for the development of new vaccines to prevent and control porcine pseudorabies induced by epidemic PRV strains.

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