Cloning and Phylogenetic Analysis of Nsp2 Gene of PRRSV Tianjin Isolates
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To study the variation of procine reproductive and respiratory syndrome virus(PRRSV),three PRRSV isolates(TJ-S1,TJ-S2 and TJ-S3) were obtained from Tianjin province's pig farms.A pair of primers were designed according to the published sequence of Nsp2 gene of BJ-4 strain and used to amplify the partial Nsp2 gene by RT-PCR,the PCR products were cloned into pGEM-T vector and then sequenced.The sequence was analyzed by DNAstar software.Sequence and phylogenetic analysis showed that all these 3 isolates belong to the North American genotype,TJ-S1 strain and TJ-S2 strain shared a common feature of distinctive deletions in 221 and 272-300 amino acids.The phylogenetic tree revealed that TJ-S1 strain,TJ-S2 strain had close relationship with HuN strain and SD-14 strain,TJ-S3 was close to CH-1a.Keywords:
Strain (injury)
Cloning (programming)
Sequence (biology)
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In order to realize the molecular biological characteristics and variation rule of porcine reproductive and respiratory syndrome virus(PRRSV) strains in some provinces of China during 2010-2011,15 strains of PRRSV were isolated with Marc-145 cells.15 PRRSV ORF5 genes were amplified by RT-PCR and sequences were analysized.A pair of primers were designed according to the standard sequence downloaded in NCBI,and the amplify ORF5 gene full-length(603 bp)was amplified.The result showed that the nucleotide homology of 15 isolates is 98.8%-99.9%,the amino acid homology derived by nucleotide is 95.5%-99.5%.The homology is high between 15 PRRSV isolates and American type PRRSV,so the 15 PRRSV isolates are American type PRRSV.The ORF5(151)amino acid sites of 9 PRRSV isolates in 15 PRRSV isolates have the same variation with HB-1,occured in R151 → K151.And the rest of 15 PRRSV isolates have the same ORF5(151)amino acid sites with JAX1,the ORF5(151)is R.Phylogenetic tree showed that BFLN3-2010,BFSD1-2011,BFAH2-2011 and CH-1a are in the same branch belonging to subset 2,and the rest 12 isolates and JAX1 are in the same branch belonged to subset 4.
Homology
Sequence homology
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According to the ORF5 gene sequence of Jiangxi and CH-1a strains of porcine reproductive and respiratory syndrome virus(PRRSV) available in GenBank,one pair of primers was designed and synthesized for RT-PCR amplification ORF gene from 18 samples isolated from Ningxia Hui Autonomous Region.All of the amplified fragments of ORF5 genes were 750 bp.The corresponding DNA fragments were respectively cloned into pMD18-T vector and sequenced.The sequences of ORF5 genes were analysed using DNAStar software.Nuclear acid analysis indicated that the amplified ORF genes shared 85.4%-89.0% homology with ATCC VR-2332,BJ-4 and RespPRRS MLV strains,90.0%-98.2% homology with CH-1a and 55.9%-56.6% homology with LV,respectively.The ORF5 gene phylogenetic tree showed that the PRRSV isolates from Ningxia Region were PRRSV American genotype and the same subtype with CH-1a strain.
Homology
Cloning (programming)
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To investigate the variation of porcine reproductive and respiratory syndrome virus(PRRSV) in Hebei Province,samples collected from 12 intensive pig farms suffered with PRRSV from 2004 to 2007 were tested by using RT-PCR.The ORF5 and Nsp2 genes were subjected to PCR amplification,cloning and sequencing by using DNAStar and DNAMAN softwares.The results showed that the amplified ORF5 genes shared from 98.7% to 100% amino acid homology among them,and shared from 88.9% to 100% and from 62.9% to 63.5% amino acid homology with reference strains of North American and Europe genotypes.The amplified Nsp2 genes shared from 82.8% to 100% amino acid homology among them,and shared from 76.5% to 100% amino acid homology with reference strains,respectively.The 6 Nsp2 genes amplified from samples collected from 2006 to 2007 exhibited 30 amino acid deletion(amino acid 482 and amino acid 533 to 561).Phylogenetic analyses showed the Hebei PRRSVs represented North American genotype and belonged to 2 different subgroups,of which,HB0503 was closely related to BJ4,NJ1,HN1,LP1 and VR-2332 strains,and the others were closely related to JXA1,SX and BJ strains.The results suggested that the PRRSVs from the samples collected from 2004 to 2005 were low pathogenic,and the samples collected from 2006 to 2007 were highly pathogenic.
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Cloning (programming)
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This study aimed to analyze the genetic variation of the ORF5 and Nsp2 gene in two PRRSV isolates from ShenYang and GanSu province.The ORF5 sequence and partial Nsp2 sequence were amplified by RT-PCR and the PCR products were cloned and sequenced.Multiple sequences of ORF5 and Nsp2 gene nucleotides as well as amino acids were compared with those of 10 reported PRRSV strains and analyzed using DNA Star software.The results showed that homology between ORF5 gene of two PRRSV isolates and other American genotype strains was 88.6%-98.7% in nucleotide sequence and 86.6%-98.0% in amino acids sequence;As to Nsp2 gene,the homology was 73.4%-99.8% in nucleotide sequence and 68.6%-99.5% in amino acids sequence.At the same time this gene had the same deletion compared with other variation strains in China.The data indicated that the two PRRSV isolates might be the mutant isolates of PRRSV American genotype.And our study will lay a theoretical basis for the disease prevention and vaccine design.
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To study the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV)in Sh and ong,Henan and Hebei province of China,42 samples from pig farms in three provinces were isolated and identified during 2006 to 2012.15 PRRSV isolates were successfully isolated,identified by PCR and biological characteristics were studied.The complete ORF5 and partial Nsp2 genes of 15 isolates were amplified and sequenced.The deduced amino acid sequences were blasted with deduced sequences of 68 ORF5 genes and 40 Nsp2 genes in GenBank.The analysis showed that all 15 isolates belonged to American groups,13 of them presented discontinuous amino acid deletions in Nsp2, and the homology of ORF5 deduced amino acid sequences with JXA1 were from 95.5% to 97.5% .Besides,isolate SDDY2007 showed higher similarity to vaccine strain RespPRRSV MLV(98.5% ),which was located in America subgroupⅡ.A more interesting thing was that,though Nsp2sequence of HN25-2009 contained 30 amino acids deletion,its deduced amino acid sequences showed higher similarity to VR2332(97.5% ).In short,highly pathogenic PRRSV was the dominant PRRSV in Sh and ong,Henan and Hebei during 2006 to 2012.Although there were some genetic variations among PRRSV isolates in three provinces,no obvious regional characteristics could be found.
Homology
Strain (injury)
Genetic Analysis
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ORF2a-ORF7 genes of porcine reproductive and respiratory syndrome virus(PRRSV) HS strain were obtained by RT-PCR and sequenced after being cloned directly into the pMD 18-T.The sequences of ORF2a-ORF7 genes of the isolate were compared with the sequences of other PRRSV strains in GenBank by DNAMAN software.The results showed that the ORF2a-ORF7 of the PRRSV HS strain shared 83.6%-99.7% of nucleotide homology and 86.6%-99.6% of amino acid homology with that of North American genotype(VR-2332,Resp MLV,16244B,HN1,BJ-4,CH1-a,HB-1,HB-2),respectively.And they shared only 38.9%-49% of nucleotide homology and 54.2%-78.2% of amino acid homology with that of LV,respectively.The phylogenetic trees revealed the PRRSV HS strain was clustered within North American genotype and had close relationship with HN1,VR-2332,RespMLV,16244B,BJ-4.
Homology
Cloning (programming)
Arterivirus
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【Objective】Research To research genetic evolution of Xinjiang PRRS strains ORF2-7 amino acid and provide a theoretical basis for the effective perventionprevention and control of the virus. 【Method】Four strains were treated from pig lung of clinical tissue samples originated from the suspected cases were treated with porcine reproductive and respiratory syndrome virus( PRRSV) in Xinjiang Province,RNA was extracted,and genes of PRRSV ORF2-7 were amplificated by RT-PCR and sequenced. 【Result】Nucleotide of PRRSV ORF2-7 was 3206 bp. Sequence analysis of PRRSV ORF2-7 indicated that XJTK-02 strain,XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 90. 3% ~ 98. 6% nucleotide; XJTK-02 strain shared 93% nucleotide identities to with CH-1a; XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 95. 1% ~ 98. 9% nucleotide identities to with JXA1,HuN4,JXwn06,pJX143 and HPBEDV;Tthe four strains shared 88. 9% ~ 90. 5% nucleotide identities to with North American genotype VR-2332,54. 2% ~ 54. 6% nucleotide identities with the Dutch Lelystad strain LV4. 2. 1. The results showed that the four strains PRRSV of Xinjiang belongs to North American genotype。.
Strain (injury)
clone (Java method)
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In order to offer the theoretical data for prevent and control PRRS that unparalleled outbreak in Hebei.According to the reported complete nucleotide sequence of PRRSV CH-1a and JXA1 in GeneBank,3 pairs of primers were designed to amplify ORF5-7 gene of HB-4(hs) strain with Primer 5.0 software.The ORF5-7 gene nucleotide sequences were obtained by RT-PCR and sequenced after being cloned directly into the pGM-T vector.The obtained sequences were analyzed and compared with the published complete nucleotide sequences of other strains in GeneBank by DNAstar software.The results showed that the PRRSV HB-4(hs) strain was clustered within North American genotopy.The ORF5-7 gene of HB-4(hs) strain showed highest homology of 98.8%-99.5% to the strains were isolated in recent years,showed 91.9%-92.6% identity with the strains were isolated in last years.The phylogenetic trees revealed the strains were isolated in recent years had close relationship with HB-1(sh) strain.
Primer (cosmetics)
Homology
Strain (injury)
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A strain of porcine reproductive and respiratory syndrome virus(PRRSV)was isolated from a pig herd with high fever syndrome in Shandong and it was designated as WF-1 strain.The NSP2 gene and ORF5 gene of WF-1 strain were amplified by RT-PCR and sequenced thereafter.The results showed that WF-1 strain was classified into the North American genotype and it had discontinuous 30 amino acids deletions in NSP2.The ORF5 gene of WF-1 strain shared 97.5 %,94.0 % and 87.5 % in nucleotide level,and 97.5 %,94.0 %,87.5 % in amino acid level,with those of JXA1,CH-1a and VR2332,respectively.Phylogenetic tree analysis revealed that WF-1 had closer relationship with JXA1 and SX-1 strains than classical strains,such as CH-1a,HN1,VR2332,CC-1.
Strain (injury)
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Molecular diversity of porcine reproductive and respiratory syndrome virus was analyzed by sequence comparison of E gene of the 15 virus isolates from three southern provinces (regions). A pair of primers was designed according to the published nucleotide sequences of VR-2332 and CH-1a and used to amplify complete E gene sequence by RT-PCR from virus RNA template. The PCR product of approximately 668 bp was cloned into pMD18-T vector. Nucleotide sequences comparison revealed a sequence homology of 84.1 %-100 % among all 15 local isolates, with a similarity of 81.1 %-100 % in deduced amino acid sequences. These local isolates shared 85.4 %-98.5 %, 87.1 %-96.2 % and 62.5 %-63.8 % sequence homology with VR-2332, CH-1a and LV strain respectively, which represents 83.1 %-95.5 %?￠86.1 %-95.0 %?￠51.7 %-57.2 % similarity in deduced amino acid sequences. The results showed significant genetic diversity among PRRSV epidemic strains. All 15 isolates be1onged to North American genotype. Phylogenetic tree analysis showed that the 15 strains belonged to three subgroups, with diversities evident on potential glycosylation sites, antigenic epitope regions and the amino acids associated with PRRSV virulence based on deduced amino acids of E gene.
Homology
Molecular Epidemiology
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