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:
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Cloning (programming)
Sequence (biology)
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Objective The objective of this study was to analyze the genetic variation of the nucleocapsid protein(N protein) in Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) in China by cloning and sequencing the ORF7 gene of 5 PPRSV strains and comparing it to other PRRSV epidemic strains in GenBank.Methods One pair of primers was designed according to the published full genome sequence of the ATCC VR-2332 strain in GenBank;the ORF7 full length gene of three Gansu epidemic strains(named GSZY,GSBY,and GSLZ strains),one Shanxi epidemic strain(named the Shanxi strain),and one Jiangxi epidemic strain(named the Jiangxi strain) were amplified by RT-PCR.The PCR products were cloned into a pGEM-T Easy vector,and recombinant vectors were constructed and then sequenced.Multiple sequences of ORF7 gene nucleotides and amino acids were compared with those of 13 PRRSV strains and analyzed using DNA Star software.Results Results indicated that the ORF7 gene shared 91.9%-94.3% homology of nucleotides with an American virus(ATCC VR-2332 strain),which caused the variation of PRRS in the early stages,while shared 98.7%-99.7% homology with other epidemic strains in China,and 57.7%-58.2% homology with a European strain(LV strain).Conclusion Results showed that there is variation in the N protein(amino) acid(sequence) of the American strain but that it is relatively conserved among strains in China.
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A pair of the special primers were designed and synthesized according to the ORF5 sequence of porcine reproductive and respiratory syndrome virus(PRRSV) AF494042 strain published in GenBank.The ORF5 gene of field virulent strain that was collected from Henan province was amplified by RT-PCR and cloned into pGEM-T Easy vector.The nucleotide sequence was determined by sequencing and compared with other ORF5 genes in GenBank,and the structure of ORF5 gene was analyzed too.The results showed that the amplified fragment length was 721 bp.Compared with the corresponding region with other PRRSV strains,the results showed that the nucleotide sequence homology was 93.5% with AF494042 straine.There have high homologies when compared with Shanxi and Shandong isolates,from 97.8% to 99.2 %;and the nucleotide sequence homologies were from 93.4% to 98.7% when compared with Hebei,Anhui and Heilongjiang isolates;while there has the lowest homology when compared with the Henan 2004 isolates,just 88.7%.The further analysis of the field strain was done by the phylogenetic tree.In all of the PRRSV isolates,the HN-09 sequenced strain has closer relation with Shanxi and Shandong isolates,but it showed distant relationship with the Henan isolates in 2004.The composition of amino acid and percentage of secondary structure of GP5 protein were compared analyse between HN-09 and AF494042 isolate using the ANTHEPROT software.The results showed that the composition of amino acid and secondary structure of HN-09 isolate have some difference when compared with AF494042 isolate,but the distribution of secondary structure unit did not change too much.
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Cloning (programming)
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Sequence homology
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To investigate the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV),the full-length genome of PRRSV GX1003 strain from Guangxi province were amplified by RT-PCR,sequenced and analyzed.The results showed that,excluding the poly(A) tail,the genomic sequence of GX1003 strain was 15 320 nucleotides(nt) in length.Sequence analysis revealed that GX1003 strain shared 85.0%-99.4% nucleotide identity and 82.1%-99.1% amino acid identity with North American(NA) type isolates,and shared 60.6% nucleotide identity and 51.3% amino acid identity with European(EU) prototype LV strain.Sequence comparison demonstrated that GX1003 strain had discontinuous 30 aa deletion in 481 aa and 533-561 aa of NSP2 region compared with that of the NA prototype VR-2332,which was considered as the genetic marker of highly pathogenic PRRSV(HP-PRRSV),and had new multiple genomic variations in different regions.Phylogenetic analysis showed that all the NA type isolates could be divided into four subgroups,while GX1003 strain distributed in subgroup 4 represented by the highly pathogenic JXA1 strain.The results indicated that GX1003 strain from Guangxi province belonged to HP-PRRSV with new genetic variations in different regions.
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Genetic Analysis
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To fully understand the knowledge of highly pathogenic porcine reproductive and respiratory syndrome(PRRSV) molecular epidemiology and genetic variation in Shandong area,a total of 14 PRRSV isolates were isolated in 2007~2009,and Nsp2 gene and ORF5 gene of these isolates were amplified by RT-PCR and sequenced.Comparing with JXA1 strain,Nsp2 gene shared 96.1%～99.8% of nucleotide homologies and 92.9%~100% of the deduced amino acid homologies,while the ORF5 gene shared 98.0%~99.8% of nucleotide homologies and 97.5%~99.5% of the deduced amino acid homologies.Sequence results showed that all the 14 PRRSV isolates had discontinuous 30 amino acids deletions in Nsp2;the GP5 protein encoded by ORF5 gene had a few mutation but no deletion.The phylogenetic tree revealed JQ strain isolated in 2007 had closer relationship with JXA1 strain.Among the 12 PRRSV strains isolated in 2009,two strains still locate in the same branch with the domestic strains isolated between 2006 and 2007,nine strains constitute an independent branch,and 1 strain(DLS-1) forms in a separate branch,indicating that those PRRSV isolates have no obvious geographical characteristics but have an annual feature in Shandong area.
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The ORF2a-ORF7 genes of porcine reproductive and respiratory syndrome virus(PRRSV) TS strain isolated in our laboratory from Tangshan Hebei were obtained by RT-PCR and sequenced after being cloned directly into the pMD19-T.The sequences of ORF2a-ORF7 genes of the isolate were compared with the sequences of other PRRSV strains in GenBank by DNAStar software.The results showed that the ORF2a-ORF7 of the PRRSV TS strain shared 88.9%-94.7%of nucleotide homology with North American genotype(VR-2332) and highly homology with the strains isolated from Hebei province in recent years.But,they shared only 61.2%-69.0%of nucleotide homology with LV strain,and belong to American genotype.The phylogenetic trees revealed the domestic strains could be divided into two subgroups,the Hebei strains were clustered within one subgroup and had close relationship with highly pathogenic representative strain JXAl.This study will provide reference of PRRS prevention and control in Hebei province.
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Sequence homology
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Amplifying the NSP2 and GP5 genes sequences of nine strains PRRSV isolated from different areas of Henan between 2006 and 2009,these genes of NSP2 exhibited different sizes in length ranging from 2 850 bp to 2 937 bp and coded 950-979 aa,and the length of GP5 were 603 bp and coded 200 aa.Nucleotide and deduced amino acid sequences analysis revealed that 7 out of 9 Nsp2 sequences contained the same 30 aa deletion and the results indicated that 9 strains were American.In order to compare the consistency between Nsp2 and GP5 variations,the Nsp2 and GP5 genes sequence of nine strains PRRSV genome isolated from Henan were analyzed,then exploring the genetic variation characteristics of epidemic PRRSV strains.
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Five pairs of specific primer of Nsp2 and ORF5-7 genes were designed according to the gene sequence of ATCC VR-2322 of American standard strain.The cDNA fragment of PRRSV Henan strains were amplied by RT-PCR,then cloned into pMD-18T vector respectively and sequenced.After sequencing,the Nsp2 and ORF5-7 genes were blasted with PRRSV genome sequence downloaded from NCBI.Compared with other American isolated,the homology rate of the Nsp2 and ORF5-7 genes and amino acid were 89.1% to 98.1% and 81.4% to 97.1% respectively.Compared with other Europe isolated,the homology rate of the Nsp2 and ORF5-7 genes and amino acid were 46.9% to 61.2% and 41.4% to 45.1% respectively.The result showed that the PRRSV isolated from Henan were mutant compare with American genetype.
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Molecular Epidemiology
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Samples were collected from two farms in Anhui province with suspected clinical signs of porcine reproductive and respiratory syndrome in 2008,and two porcine reproductive and respiratory syndrome virus isolates(HS08 and ZJ08) were isolated after 4-5 passages in Marc-145 cells.The NSP2 and ORF7 genes of the isolates were amplified using RT-PCR which was followed with cloning and sequencing.The two isolates were similar to each other(nucleotide homology 99.0%),and they contained a discontinous 30 amino acid deletion in the NSP2 gene.The two isolates had 80.3% and 80.2% nucleotide homology with strains CH-1a,respectively,in the NSP2 gene.They had 98.4% and 99.5% nucleotide homology with the highly pathogenic strain HuN4 isolated in 2007 in the ORF7 gene,and they had mutations of K46→R46,H109→Q109,V117→A117 in the ORF7 gene.Phylogenetic analysis also revealed that the two isolates belonged to the cluster of highly pathogenic strains emerging in China in recent years.
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Porcine reproductive and respiratory syndrome (PRRS) has leaded to an enormous loss per year to the swine industry, its etiology porcine reproductive and respiratory syndrome virus (PRRSV) is a highly mutated virus in pigs. To fully understand the genetic characteristics of PRRSV genome in South China, this study collected the lung samples infected with PRRSV in Guangdong and Hainan province from 2014 to 2015 and tried to isolate the PRRSV. Finally, the complete genomes of isolated strains were sequenced and analyzed.Virus isolation was performed in MARC-145 cells. The 13 fragments of PRRSV genome were amplified by RT-PCR and the complete PRRSV genome sequence was obtained by SeqMan program of DNASTAR7.0 software. Nucleotide and deduced amino acid (AA) sequences of NSP2 and ORF5 were aligned using the MegAlign program of DNASTAR7.0 software to determine sequence homology. A phylogenetic tree was constructed using MEGA5.2 software with the neighbor-joining method to analyze the evolutionary relationship.11 PRRSV strains were isolated in South China from 2014 to 2015. All the isolated strains clustered into subgenotype V along with the HP-PRRSV representative strains JXA1, HuN4 and JXwn06. The subgenotype V was furtherly divided into two groups. AA sequence alignment analysis indicated that all the isolated strains had 1 AA deletion and 29 AA continuous deletion at position 481 and 533-561. Notably, GDHY strain had another 120 AA continuous deletion at position 629-748. All the isolated strains had an A137S mutation in the residue A137 of GP5 which was considered to differentiate vaccine strains. All the isolated strains had a L39I mutation in the primary neutralizing epitope (PNE) of GP5. Except GDHZ had a N34T mutation, all the other isolated strains had conserved N30, N44 and N51 glycosylation sites in the four potential N-glycosylation sites (N30, N34, N44 and N51) of GP5.Our study showed that the prevalent strains in this region were highly pathogenic PRRS virus-like. Moreover, one new strain having another 120 amino acids continuous deletion except the discontinuous 30 (29+1) amino acids deletion in NSP2 region had emerged. Besides, the isolated strains had extensive amino acids substitutions in the putative signal, extravirion and intravirion regions of GP5. These results showed that PRRSV has undergone extensive variation in South China, providing some theoretical basis for researching effective vaccince to better controling the PRRSV in this area.
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Isolation, identification and genomic analysis of HP-PRRSV obtained from one province in east China.
【Objective】PRRSV strains from pig IFArms suspected to have broken out PRRS in 3 different regions of one province in middle east China were isolated.The genome of the obtained PRRSV isolates was determined and then molecular characteristics of dominant strains was analyzed,the evolutionary law of PRRSVs isolated in China recent years according detaild sequences analysis was observed.【Method】 Samples of lymph nodes and lungs from dead or sick pigs were collected,treated samples by PRRSV RTPCR were determined,then the strains from positive samples were isolated.IFA was conducted after the i-solation and TCID50 of IFA-positive isolates was measured.6 pairs of primers were designed based on PRRSV VR-2332,CH-1a,HB2 and JXA1 and other PRRSV strains available on NCBI Genebank.The genome of obtained PRRSV isolates were amplified and determined by these primers.Detailed sequences anal-ysis and evolutionary relationship research among 3 obtained isolates and LV,VR-2332,JXA1 and other 13 domestic and foreign representative strains or isolates were carried out.【Result】3PRRSVstrains were obtained and were named as SDCXA/2008,SDWF and SDLY respectively.The Blast result of complete genome indicated that all 3 isolates were Amercian-type strains.According to the sequences analysis,the genome sequences of 3 isolates showed a very low identity of 61.6%-61.8% with Europe-type PRRSV,a relatively low identity of 86.6%-97.1% with American-type strains isolated before 2006,a high identity of 98.3%-99.7% with American-type strains isolated after 2006.Nsp2gene was the most variable labile gene compared with ORF5 and ORF3 gene.ORF5 gene was more mutable than ORF3 gene.The deduced aa of Nsp2 gene was most variable too.The sequences analysis showed that all 3 isolates were high pathogenic PRRSV like representative HP-PRRSV strains of JXA1and HUN4 having 30 discontinuous aa deletions at the position of 481and 532-560aa site in Nsp2 deduced aa sequence.The sequences analysis with and phylogenetic tree was combined and 3obtained HP-PRRSV strains were clossified into JXA1-like HPPRRSV group.【Conclusion】3HP-PRRSV strains were obtained,the genetic characteristics of 3 strains showed to be comparatively stable,but some mutations appeared in the genomes suggested that the PRRSVs would continually evolve with variation in future.The result also showed that there were no apparent relations between the distribution features of PRRSVs and the places.
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