key: cord-0033701-l3vwb2v7
authors: Tian, Hong; Wu, Jing-yan; Yin, Shuang-hui; Shang, You-jun; Man, Zi-ping; Zhao, Na; Jin, Ye; Liu, Xiang-tao
title: Genetic variation analyses of nsp2 gene of PRRSV in Ningxia Hui Autonomous Region of China
date: 2009-05-28
journal: Virol Sin
DOI: 10.1007/s12250-009-3017-4
sha: da8079c68bf14059a87f120532ea86754721885f
doc_id: 33701
cord_uid: l3vwb2v7

To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region (Ningxia) of China, the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain (ch-1a) and two other epidemic strains SD (3) and SD2006. Comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen Ningxia isolates (NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-1a was 60.3%–79.9% in the nucleotide sequence, and homology between the NX strains and SD strains was 80.3%–98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%–100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.

seven ORFs located at the 3`-terminus of the genome are postulated to encode viral structural proteins.

Among the nonstructural proteins, the multidomain protein nsp2 is the largest PRRSV replicative protein.

Genetic analysis of both Coronaviridae and Arteriviridae (1) originally identified the protein to be spanning amino acids (aa) 384 to 1 363 (nsp2 aa 1 to 981); this was subsequently projected to comprise aa Each sequence of the PCR products was determined by both enzyme analysis and sequencing.

The viral RNA of all clinical samples were extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Germany), following the manufacturer's instructions. In brief, after lysis of the specimens, the mixture was applied to a spin column as described by the manufacturer's protocol. Reverse transcription was performed at 42℃ for 1.5 h with 5μL total RNA, and then PCR was carried out in a reaction volume of 50 μL containing 1μL of each primer (S1 and R1, 50 pmol/μL), 5 μL of 10×PCR Buffer, 4 μL 2.5 mmol/L dNTPs, 3 μL of 25 mmol/L MgCl 2 , 1μL of RNase Inhibitor (40U/μL), 0.5 μL of Taq (5U/μL), 10 μL of reverse transcription production and 24.5 μL ddH 2 O.

Amplification conditions were 94℃ for 4 min, then 30 cycles that each consisted of denaturing at 94℃ 1min, annealing at 57℃ 40 s, and extension at 72℃ for 1 min, the sample was then heated at 72℃ for 10 min, and a final stage of 4℃ for 5 min. The amplified PCR products were analyzed by agarose gel electrophoresis and purified using the QIAquick gel extraction kit (Qiagen) per the manufacturer's protocol. All nucleotide sequences were obtained from clinical sample RNA by direct sequencing of PCR products.

Sequence data were analyzed using the method 

Relative to strain ch-1a, the nsp2 genes of seventeen isolates have 87 nucleotides deleted at positions 2 937 to 2 948, 2 952 to 2 978, 2 986 to 3 003 and 3 007 to 3 036, respectively; these deletions are the same as those for the SD (3) and SD2006 strains (Fig.2) .

Ningxia and ch-1a was 60.3%-79.9% in nucleotide sequence, and homology between Ningxia and the common SD strain was 80.3%-98.8% in nucleotide sequence. The nsp2 genes of seventeen isolates had 74.9%-100% nucleotide sequence identity with each other (Fig.3) . 

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