Cloning and Sequence Analysis of the M gene of Porcine Epidemic Diarrhea Virus LJB/03

Porcine epidemic diarrhea virus (PEDV) LJB/03 was isolated from the fece of piglets infected with PEDV on a pig farm, Heilongjiang province, China. The M gene of LJB/03 was amplified from the RNA extracted directly from the fece samples by RT-PCR and cloned into pMD18-T vector. The M gene cDNA was sequenced and encompasses an open reading frame of 681 nucleotides, encoding a 226-amino acid protein. The LJB/03 M gene has a base composition of 152 adenines (22%), 153 cytosines (23%), 161 guanines (24%), and 214 thymines (31%). Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 M gene has a high sequence homology to those of other PEDV isolates, 97.80% with JMe2, 96.92% with KPEDV-9 (Korean field isolate), 97.36% with KPEDV-9 (Korean), 97.80% with Br1/87, and 97.94% with CV777. The encoded protein shared 97.79% amino acid identities compared with CV777, 97.35% with Br1/87, 97.79% with JMe2, 96.90% with KPEDV-9 (Korean field isolate), 96.46% with KPEDV-9 (Korean). Sequence analysis of the M gene, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, showed that all other PED viruses analyzed fell into three groups, and the LJB/03 itself branched in an independent group. These data revealed that the M gene nucleotide sequence of LJB/03 has some mutations in comparison with the other PED viruses.     

Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered diagnostic reagents. Also, sequences of the nucleotides and deduced amino acids of the Chinju99 N gene were analyzed by alignment with those of CV777 and Br1/87. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of Chinju99 was 1326 bases long and encoded a protein of 441 amino acids with predicted M _r of 49 kDa. It consisted of 405 adenine (30.5%), 293 cytosine (22.1%), 334 guanines (25.2%) and 294 thymines (22.2%) residues. The Chinju99 N ORF nucleotide sequence was 96.5% and 96.4% homologous with that of the CV777 and Br1/87, respectively. The Chinju99 N protein revealed 96.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained seven potential sites for threonine (T)- or serine (S)-linked phosphorylation by each protein kinase C and casein kinase II.     

Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration with high mortality rates in swine. It has become increasingly problematic in China. Since the nucleocapsid (N) protein is highly conserved, it is a candidate protein for early diagnosis and vaccine development. In this study, the N genes of 15 PEDV strains were amplified by RT-PCR and cloned into the pMT-19T vector, sequenced, and compared to each other as well as to PEDV reference strains. The nucleotide sequences of the N gene of the Chinese PEDV strains consist of 1326 nucleotides and encode a 441-aa-long peptide. The nucleotide sequences of the fifteen PEDV strains in our study were 96.1-100 % identical to each other, and the deduced amino acid sequences were 94.8-100 % identical. Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.2-99.7 % identical to those of the Chinese PEDV strains, and their deduced amino acid sequences were 94.1-99.5 % identical. In addition, the fifteen strains showed a high degree of nucleotide sequence identity to the early domestic strains (98.4-99.7 %) except the LZC strain, but less sequence identity to the vaccine strain (CV777) used in China (94.7-97.7 %). Phylogenetic analysis showed that the Chinese PEDV strains are composed of a separate cluster including three early domestic strains (JS-2004-02, LJB/03 and DX) but differ genetically from the vaccine strain (CV777) and the early Korean strains (Chinju99 and SM98).     

Cloning and Sequence Analysis of the Spike Gene of Porcine Epidemic Diarrhea Virus Chinju99

The spike (S) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to aid in the development of genetically engineered vaccines and diagnostic reagents against PEDV. The nucleotide sequence encoding the entire S gene open reading frame (ORF) of Chinju99 was 4152 bases long encoding 1383 amino acids. It consisted of 1001 adenine (24.1%), 849 cytosine (20.4%), 877 guanine (21.1%) and 1425 thymine (34.3%) residues. The Chinju99 S ORF nucleotide sequence was 94.5% homologous with that of the Br1/87 and CV777 strains, respectively. The Chinju99 S protein had 92.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained 27 potential sites for asparagine (N)-linked glycosylation and there was a stretch of highly hydrophobic residues at position 1325–1350.     

Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13

The spike (S) gene of the attenuated porcine epidemic diarrhea virus (PEDV) DR13 was cloned and sequenced to further explore the functions of wild type PEDV and attenuated PEDV. Sequencing revealed a single large ORF of 4,149 nucleotides encoding a protein of 1,382 amino acids with predicted M _r of 151 kDa. The coding region of the S gene of attenuated PEDV DR13 had 20 nucleotide changes that appeared to be significant determinants of function in that they produced changes in its predicted amino acid sequence. Notably, attenuated PEDV DR13 has previously been found to exhibit reduced pathogenicity in pigs. The regions containing these 20 nucleotide changes may therefore be crucial for PEDV pathogenicity. The attenuated PEDV DR13 S protein contains 28 Asn-Xaa-Ser/Thr sequons, 21 asparagines that are predicted to be N-glycosylated and a stretch of highly hydrophobic residues at positions 1,327–1,347, which is predicted to form an α-helix and to function as a membrane anchor. One (from N to K at 378) of the changes in the deduced amino acid sequence destroyed N-linked glycosylation sites, while another change (from N to S at 114) created a new one at a different location. These alterations in N-linked glycosylation sites reflected 3 nucleotide changes, which were related to the above-mentioned nucleotide changes and are suggested to influence the pathogenicity of attenuated PEDV DR13. Attenuated PEDV DR13 has 96.5, 96.4, 96.1, 93.9, 93.5 and 96.6% DNA sequence identities with CV777, Br1/87, JS-2004-2, Spk1, Chinju99 and parent DR13, respectively. Likewise, it shares 95.7, 95.4, 95.6, 92.0, 91.6 and 95.7% identity with those genes at the deduced amino acid sequence level. Phylogenetic analysis suggested that attenuated PEDV DR13 is closely related to CV777, Br1/87, JS-2004-2 and parent DR13, rather than to Spk1 and Chinju99 and is especially close to the Chinese PEDV strain JS-2004-2. Nucleotide sequence data reported is available in the GenBank database under the Accession Nos. DQ462404 and DQ862099.     

Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea

Porcine epidemic diarrhea virus (PEDV) causes a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine, thereby incurring heavy economic losses in Korea. Spike (S) glycoprotein has been suggested as an important determinant for PEDV biological properties. In this study, the nucleotide and deduced amino acid sequences of the partial S glycoprotein genes of Korean PEDV isolates, including epitope region that is capable of inducing PEDV-neutralizing antibodies, were determined. The partial S glycoprotein genes were amplified by RT-PCR, cloned, sequenced, and compared with each other as well as with reference PEDV strains. By phylogenetic analysis, the Korean PEDV isolates were divided into three groups (G1, G2, G3), which had three subgroups (G1-1, G1-2, G1-3). Group1 (G1) Korean PEDV isolates were highly homologous to CV777, Br1/87, JS-2004-2, KPED-9, P-5V, SM98-1, parent DR13, and attenuated DR13, group2 (G2) Korean PEDV isolates were highly homologous to Spk1, and group3 (G3) was Chinju99 at the nucleotide and deduced amino acid sequence levels. In addition, the G1 Korean PEDV isolates didn’t had several specific nucleotides and amino acids which were found in the G2 and G3 Korean PEDV isolates, and especially the G1-1 Korean PEDV isolates had specific nucleotides and amino acids which were not found in the G1-2, G1-3, G2, and G3 Korean PEDV isolates. It was suggested that many Korean PEDV isolates are closely related to the G1 including CV777, Br1/87, JS-2004-2, KPED-9, P-5 V, SM98-1, parent DR13, and attenuated DR13 rather than to the G2 and G3 including Spk1 and Chinju99, and notably more prevalent PEDVs isolated in Korea are especially close to the Chinese PEDV strain JS-2004-2 rather than Korean PEDV strains Spk1, Chinju99, KPED-9, SM98-1, parent DR13, and attenuated DR13.     

Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China

Since late 2010, porcine epidemic diarrhea virus (PEDV) has been re-emerging in immunized swine herds with devastating impact in the Hebei province of China. Seven prevailing strains of PEDV were isolated from fecal samples out of piglets suffering from severe diarrhea. The M gene of the seven PEDV isolates encompasses an open reading frame of 681 nucleotides, encoding a protein of 226 amino acids. The seven PEDV isolates showed 99.4-99.9 % nucleotide sequence identity and 98.2-99.1 % deduced amino acid identity. When compared with other Chinese isolates and foreign isolates, the seven isolates showed high nucleotide identity with the Thailand isolate M-NIAH1005 (99.6-99.9 %) and Korea isolate PFF188 (99.7-100 %), but low identity with other Chinese isolates (96.6-99.1 %) and with the vaccine strain CV777 used in China (97.8-98.2 %). Phylogenetic analyses showed that all seven Chinese field isolates were grouped together in the same cluster. Although CV777 was also separated into the same cluster with the seven isolates, they were belonged to different sub-cluster. These results showed that the seven prevailing isolates in China are closely related phylogenetically to each other and have close relationships with the Korean strain PFF188 and Thailand strain M_NIAH1005. However, they differ genetically from other Chinese isolates and the vaccine strain CV777. Therefore, a more efficient vaccine strain should be chosen to prevent outbreaks of PEDV in China.     

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China

A total of 127 porcine samples were collected from 48 farms in six provinces in south China. The positive rate of porcine epidemic diarrhea virus (PEDV) was 43.0 % (55/127), and the co-infection rate of PEDV and transmissible gastroenteritis virus (TGEV) was 12.0 % (15/127). The partial S gene and complete M gene were amplified from PEDV-positive strains by RT-PCR, cloned, sequenced and compared with each other, as well as with the reference strains in GenBank. Sequence homology results of the partial S gene and complete M gene showed that all south China field PEDV strains had nucleotide (deduced amino acid) sequence identities of 86.7-98.7 % (83.2-99.3 %) and 96.1-100 % (95.0-100%), respectively, with the foreign reference strains reported in GenBank. Phylogenetic analysis of the partial S gene showed that all the south China PEDV strains and two Thailand strains (08UB01 and 08RB07) belong to the same group and differ genetically from European strains and early domestic strains. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777).     

Completion of the Porcine Epidemic Diarrhoea Coronavirus (PEDV) Genome Sequence

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5 end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.     

粉尘螨变应原第5组分基因克隆及分子特征分析

目的 获得粉尘螨变应原第5组分的编码基因(Der f5)并了解其分子特征.方法 用RNAiso试剂盒提取粉尘螨总RNA,根据GenBank已公布的Der f5核酸序列设计引物,用RT-PCR扩增获得其编码基因,插入pMD19-T Simple载体进行序列测定和分析.结果 获得的Der f5基因与参考序列(GenBank AY283283)同源性达97.8%,含1个完整的开放阅读框架(ORF),由132个氨基酸组成,信号肽位于1～19AA、跨膜区域位于1～19AA,为细胞外疏水性蛋白.二级结构由延伸主链(1.52%)、无规则卷曲(7.58%)和α螺旋(90.91%)组成.具有酪蛋白激酶Ⅱ磷酸化位点2个.其氨基酸序列与屋尘螨变应原第5组分相似率为78%.结论 成功克隆了 Der f5,并初步预测得其分子特征.     

小鼠凋亡相关新基因TFAR15的克隆和序列分析

目的克隆人白血病细胞凋亡相关新基因TFAR15的小鼠同源序列 ,并比较其在不同种属间的序列同源性。方法利用EST(expressedsequencetag)拼排、RT PCR、DNA序列测定和计算机分析技术 ,对小鼠TFAR15进行研究。结果首次成功地进行了小鼠TFAR15全长cDNA的克隆和序列分析 ,发现小鼠TFAR15与小鼠其它cDNA没有明显的同源性 ,因此提交GenBank并被收录 ,登录号为AF159368。小鼠TFAR15和人TFAR15在核苷酸水平上有92.3 %的同源性 ,在氨基酸水平上有高达98.6 %的同源性 ;同时发现小鼠TFAR15在氨基酸水平上与线虫C14A4.11蛋白质有38 %的同源性。功能区分析发现 ,小鼠TFAR15cDNA序列含编码212个氨基酸的开放读码框架 ,有2个可能的N 糖基化位点 ,3个可能的caseinkinaseII磷酸化位点 ,4个可能的PKC磷酸化位点 ,并且可能是一种胞浆蛋白(cytoplasmicprotein)。结论小鼠TFAR15是进化上高度保守的新基因 ,可能具有重要的功能。     

甲型流感病毒（N1N2）亚型毒株血凝素和神经氨酸酶基 …

目的 研究新分离到的Ｈ１Ｎ２亚型毒株血凝素（ＨＡ）和神经氨酸酶（ＮＡ）基因的来源。方法 病毒通过鸡胚增殖后提取其ＲＮＡ,通过逆转录合成ｃＤＮＡ,经ＰＣＲ扩增和产物纯化,用双脱氧链终止法进行核苷酸序列测定,并用ＭｅｇＡｌｉｇｎ（１．０３版）和Ｅｄｉｔｓｅｑ（３．６９版）软件进行种系发生学分析。结果 新分离到Ｈ１Ｎ２毒株ＨＡ１区氨基酸序列与Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）和Ａ／Ｇｕａｍｇｄｏｎｇ／６／９     

Molecular epidemiology of porcine epidemic diarrhea virus in China

Since early 2006, porcine epidemic diarrhea virus (PEDV) has been reemerging in immunized swine herds. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. The entire ORF3 genes of 12 PEDV field strains and one vaccine strain were sequenced. The ORF3 genes of Chinese PEDV field strains (excluding CH/GSJIII/07) contain a single 672- or 675-nucleotide (nt) ORF, which encodes a 223- or 224-aa-long peptide. However, the CV777 vaccine strain and CH/GSJIII/07 contain a 276-nt ORF because of a 49-nt deletion at nt 245–293. The Chinese PEDV field strains and PEDV reference strains are divided into three groups based on the phylogenetic relationship of their ORF3 genes. Chinese PEDV field strains (excluding CH/GSJIII/07) have a close phylogenetic relationship to Korean strains and are genetically different from the PEDV vaccine strains. However, CH/GSJIII/07 has a close phylogenetic relationship to two vaccine strains, suggesting that it might have evolved from a live vaccine strain. Chinese PEDV field strains (excluding CH/GSJIII/07) can be differentiated from PEDV vaccine strains by a nested RT-PCR method.     

Bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011–2013 in Guangdong,China

Three strains of porcine epidemic diarrhea virus (PEDV) were isolated from dead or diseased pigs at different swine farms in Guangdong during 2011–2013, and their S genes were sequenced. In the same period, seven PEDV strains were also isolated in Guangdong by other laboratories. The spike sequences of 10 Guangdong isolates were compared with vaccine strains and reference pathogenic isolates using six bioinformatics tools. The results revealed that 10 Guangdong strains, excluding strain GDS03, had distinct characteristics in terms of primary structure, secondary structure, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites. Phylogenetic analysis also confirmed these findings and revealed that all PEDV strains were clustered into three distinct groups. Ten Guangdong strains, not including GDS03, belong to Group 1, whereas four vaccine strains and GDS03 belong to Group 3, which is evolutionarily distant from Group 1. Alignment analysis of the neutralizing region amino acid sequences indicated that the amino acid substitutions of Y/D766S, T549S, and G594S that are present in the Guangdong strains, not including GDS03, were a sign of predominant genetic changes among the isolated strains. GDS03 is closely related to the 83P-5 vaccine strain, which suggests that it might represent re-isolation of the vaccine strain or vaccine variants. Taken together, these results indicate that there have been predominant new strains circulating in Guangdong from 2011 to 2013, and the circulating PEDV strains have a genetic composition that is distant from reference strains, especially the vaccine strains; however, the vaccinations might also provide some level of cross-protection, as there have been no changes in the neutralizing epitopes of SS2 and 2C10. This explains why there have been constant but infrequent outbreaks recently in comparison to late 2010 in which PEDV outbreaks were more frequent and severe. In addition, the USA-Colorado-2013 strain had the same amino acid substitutions in the neutralizing regions as the Guangdong strains except GDS03, which suggests that the information and strategies in this study may play role in PEDV variant research in other countries.     

Phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in central China based on the ORF3 gene and the main neutralization epitopes

Since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central China. To investigate the epidemic characteristics of PEDV, the complete ORF3 genes of 14 PEDV field strains from central China during 2012 to 2013 were cloned, sequenced and compared with reference strains. Phylogenetic analysis based on the complete ORF3 gene showed that the PEDVs in central China and the reference strains could be divided into three groups: G1, G2, and G3. The 14 PEDV isolates were classified as G1 and showed a close relationship to some Chinese strains isolated previously in central China and differed genetically from recent isolates from southern China, Korean strains (SM98 and DB1865, 2012), the Chinese LZC strain (2007), and the vaccine strain (CV777) being used in China. Our findings suggested that the PEDVs circulating between 2012 and 2013 in central China might have evolved from earlier strains in the local region. To determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the S gene. The results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. We speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure. Together, these findings may be useful for understanding the epidemiology of PEDV and may be relevant for designing of new and more efficacious vaccines.     

Sequence analysis of aMolluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization

The nucleotide sequence of a near left-terminal region from the genome ofMolluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacentBamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb).BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N), which had been mapped 30–50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.The contribution to this paper by Antonia Martin-Gallardo and Marta Moratilla is equal, and the order of authorship is arbitrary.     

Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes

The 3' end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3' end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3'-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.