Identification of Vibrio parahaemolyticus based on the concatenated DNA sequencing of 16S rRNA and gyrB gene / 上海预防医学

Objective:To establish the concatenated DNA sequencing of 16S ribosomal RNA （16S rRNA） and DNA gyrase subunit B （gyrB） gene， and provide evidence for the identification and classification of Vibrio parahaemolyticus （V. parahaemolyticus）. Methods:Typical strains in the genus of Vibrio spp. was selected， such as V. parahaemolyticus， V. alginolyticus and other species for examination of 16S rRNA and gyrB gene as target. Primers were separately designed to amplify these two nucleotide fragments. Phylogenetic analysis was performed using the concatenated sequences. Results:The concatenated 16S rRNA+gyrB nucleotide sequence of V. parahaemolyticus formed a single cluster in the phylogenetic analysis， which identified the typical strains of Vibro spp. at the species level. Conclusion:In our study， an identification method of V. parahaemolyticus is established based on concatenated 16S rRNA+gyrB nucleotide sequencing. It can identify the strains of V. parahaemolyticus at the species level， which may be applied in phylogenetic analysis and contamination tracing of V. parahaemolyticus in food and drug control.

Successive subculture and genetic stability of hepatitis A virus vaccine strain H2 / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic stability of virus seed H2M20K7 (K7) of live attenuated Hepatitis A virus H2 strain (HAV, H2 strain) for production of hepatitis A (Live) vaccine, lyophilized after continuous passages.@*Methods@#The virus seed K7 of H2 strain was proliferated and passaged in KMB17 cells in cell factories. Viruses of different passages were harvested after continuous passages. Virus RNA was extracted and the complete genomes of different virus passages (K7, K10, K11, K13, K15, K18) were sequenced by using next-generation deep sequencing. The mutation rates of different passages were compared. The infectivity titers of different virus passages of H2 strain were tested by ELISA.@*Results@#The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed. The structure of gene was stable and non-synonymous mutation rate was lower than 0.57%. The mutation rate of 5 ’non-coding regions was lower than 0.1%. There was no significant mutation in VP1/2 A and 2C virulence site. The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID50/ml. No statistically significant difference was found in this study.@*Conclusions@#The gene structure of the master virus seed, working seed and different passages of H2M20K7 after subculture was stable and the mutation rate was low. No significant mutation was found in 5’non-coding regions, and the critical virulence sites such as VP1/2 A, 2B and 2C showed attenuated characteristics with low mutation rate. Virulence of the virus did not changed. The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.

Characteristics of an avian influenza A (H9N2) virus isolated from an infant / 中华实验和临床病毒学杂志

Objective@#To analyze the genome characteristics of an avian influenza A (H9N2) virus isolated from an 11-month-old infant, and to look for possible sources of infection.@*Methods@#Throat swabs were collected from an infant with influenza-like illness in influenza sentinel surveillance hospitals and isolated for influenza viruses using cells. The isolates were identified for influenza virus types and subtypes by the method of hemagglutination assay, hemagglutination inhibition assay and fluorescence PCR. Whole genome sequencing of the isolated virus was carried out. The genome nucleic acid sequences and the deduced amino acid sequences were analyzed by comparing the phylogenetic trees which were constructed by bioinformatics software.@*Results@#A seasonal un-typed influenza virus was isolated from the infant with influenza like illness. With fluorescent PCR method , it was identified as H9N2 subtype of avian influenza virus and the case was confirmed as a human infected with an avian influenza A(H9N2) virus. Epidemiological studies revealed that the case had no clear history of poultry contact and exposure. Blast analysis shows that eight segments of the viral genome are avian origin, and 97.5%-99.8% homology with that of viruses isolated from the live-poultry markets. The virus belongs to G57 genotype, deduced amino acid sequence analysis shows that the virus has typical low pathogenic avian influenza characteristics.@*Conclusions@#Although the case does not have a clear history of contact or exposure to poultry, molecular traceability suggests that possible sources of infection may be still from poultry.

First isolation and identification of Getah virus SC1210 in Sichuan / 中华实验和临床病毒学杂志

Objective@#To study the genome molecular characteristics of Getah virus (SC1210) which isolated in Sichuan province in 2012.@*Methods@#Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the isolate and the genome was sequenced by the second Ion Torrent PGM. Computer softwares, including Mega Align and Mega 6, were used to analyze the nucleotide and deduced amino acid sequence, and draw phylogenetic trees.@*Results@#SC1210 was identified as Getah virus. The full genome sequence was 11 690nt, the nucleotide and amino acid homology of the full sequence with other strains were 99.2%-99.7% and 96.5%-99.4%.The capsid protein of SC1210 consisting of 804 nucleotides, encoding 268 amino acids and the full-length of E2 protein, had 1 266 nucleotides, encoding 422 amino acids. The nucleotide homology of the capsid protein and the E2 protein with other strains were 94.9%-99.2% and 94.6%-99.6%, and the amino acid were 97%-99.6% and 97.1%-99.5%. The 3′ UTR of the virus included 402 nucleotides and there were three repeat sequence elements and 19 nucleotides conservation sequence.@*Conclusions@#The first GETV isolate SC1210 in Sichuan province has a closer relationship with Yunnan strain YN040 and a far genetic relationship with MM2021.

Clinical efficacy of a combination of Percoll continuous density gradient and swim-up techniques for semen processing in HIV-1 serodiscordant couples / 亚洲男科学杂志(英文版)

To evaluate the clinical efficacy of a procedure comprising a combination of Percoll continuous density gradient and modified swim-up techniques for the removal of human immunodeficiency virus type 1 (HIV-1) from the semen of HIV-1 infected males, a total of 129 couples with an HIV-1 positive male partner and an HIV-1 negative female partner (serodiscordant couples) who were treated at Keio University Hospital between January 2002 and April 2012 were examined. A total of 183 ejaculates from 129 HIV-1 infected males were processed. After swim-up, we successfully collected motile sperms at a recovery rate as high as 100.0% in cases of normozoospermia (126/126 ejaculates), oligozoospermia (6/6), and asthenozoospermia (36/36). The recovery rate of oligoasthenozoospermia was 86.7% (13/15). In processed semen only four ejaculates (4/181:2.2%) showed viral nucleotide sequences consistent with those in the blood of the infected males. After using these sperms, no horizontal infections of the female patients and no vertical infections of the newborns were observed. Furthermore, no obvious adverse effects were observed in the offspring. This protocol allowed us to collect HIV-1 negative motile sperms at a high rate, even in male factor cases. We concluded that our protocol is clinically effective both for decreasing HIV-1 infections and for yielding a healthy child.

Identificación de microorganismos asociados a residuos de higuerilla (Ricinus communis) / Identification of microorganisms associated with castor (Ricinus communis) waste / Identificação de microorganismos associados resíduos de castor mamona (Ricinus communis)

El objetivo de este artículo fue aislar e identificar los microorganismos presentes en los residuos de fruto y torta de higuerilla (Ricinus communis). Se utilizaron medios de cultivo selectivos para la caracterización morfológica y bioquímica y para la identificación molecular se usó la técnica de PCR con oligonucleótidos universales RM y RB del gen 16S para bacterias y secuencias intergénicas ITS1 e ITS4 para hongos y levaduras. Las secuencias fueron analizadas identificándose nueve especies de hongos, siendo Penicillium brevicompactum predominante; 12 especies de bacterias, donde el género más recurrente fue Bacillus sp.; y dos especies de levaduras, Rhodosporidium paludigenum y Pichia burtonni. La identificación de la microbiota nativa presente en los residuos de higuerilla es muy promisoria, aportando un amplio conocimiento sobre la versatilidad metabólica de cada una de las cepas aisladas. El mayor número de aislamientos se obtuvieron de la torta por el alto contenido de nutrientes presentes en este residuo.

The aim of this article is to isolate and to identify microorganisms present in the fruit and cake waste of castor (Ricinus communis). Selective culture media for morphological and biochemical characterization were used. For molecular identification, PCR technique was performed with universal primers RM and RB from the bacterial 16S gene and the ITS5 and ITS4 intergenic sequences from molds and yeasts. Sequences were analyzed identifying 9 fungal species being predominant Penicillium brevicompactum; 12 species of bacteria, where genus Bacillus sp. was the most recurrent; and two species of yeast Pichia burtonni and Rhodosporidium paludigenum. The identification of this native microbiota in the waste of castor is very promising, providing a broad knowledge of the metabolic versatility of each of the isolates. The majority of isolates were obtained from the cake by the high content of nutrients in the waste.

O objetivo da pesquisa foi isolar e identificar os microrganismos presentes nos resíduos da fruta e bolo de mamona (Ricinus communis). Foram utilizados meios de cultura seletivos para caracterização morfológica e bioquímica. Para a identificação molecular empleou-se a técnica de PCR com primers gerais RM do gen RB 16S para bactérias e sequências intergênicas ITS1 e ITS4 para fungos e leveduras. As sequências foram analisadas e identificaram-se nove espécies de fungos, sendo o predominante Penicillium brevicompactum; 12 espécies de bactérias, nas quais o gênero mais recorrente foi o Bacillus sp.; e duas espécies de levedura Rhodosporidium paludigenum e Pichia burtonni. A identificação da microbiota nativa presente nos resíduos de rícino é muito promissoria, proporcionando um amplo conhecimento da versatilidade metabólica de cada uma das cepas isoladas. A maioria das amostras foram obtidas a partir do bolo pelo alto conteúdo de nutrientes nestos resíduos.

Effect of Next-Generation Exome Sequencing Depth for Discovery of Diagnostic Variants

Sequencing depth, which is directly related to the cost and time required for the generation, processing, and maintenance of next-generation sequencing data, is an important factor in the practical utilization of such data in clinical fields. Unfortunately, identifying an exome sequencing depth adequate for clinical use is a challenge that has not been addressed extensively. Here, we investigate the effect of exome sequencing depth on the discovery of sequence variants for clinical use. Toward this, we sequenced ten germ-line blood samples from breast cancer patients on the Illumina platform GAII(x) at a high depth of ~200x. We observed that most function-related diverse variants in the human exonic regions could be detected at a sequencing depth of 120x. Furthermore, investigation using a diagnostic gene set showed that the number of clinical variants identified using exome sequencing reached a plateau at an average sequencing depth of about 120x. Moreover, the phenomena were consistent across the breast cancer samples.

Antigenic variations of human influenza virus in Shiraz, Iran.

Purpose: Influenza virus is a major cause of human respiratory infections and responsible for pandemics and regional outbreaks around the world. This investigation aims to determine the prevalent influenza genotypes during 2005-2007 outbreaks in Shiraz, the capital city of Fars province, southern Iran and compare the results obtained with those of previous study. Materials and Method: Of the 300 pharyngeal swabs collected from influenza patients, 26 were found to be positive by culture and hemagglutination (HA) assays. Typing and subtyping of the isolates carried out by using multiplex RT-PCR and phylogenetic analysis performed on isolated HA genes using neighbour-joining method. Result: Out of 26 positive isolates 12 and 14 were H1N1 and H3N2 respectively. The phylogenetic and amino acid sequence analyses of our H1N1 isolates showed 99-100% genetic resemblance to A/NewCaledonia/20/99 (H1N1) vaccine strain. Most of the Iranian H3N2 isolates varied form A/California/7/2004 vaccine strain in 20 amino acids of which positions 189,226 and 227 were located in antigenic sites of HA1 molecule. These substitutions were not observed in any of the H3N2 subtypes from the same region reported previously. Conclusion: The H3N2 subtype strains prevalent during the 2005/7 influenza outbreak in southern Iran demonstrated a drastic antigenic variation and differed from A/California/7/2004 vaccine strain. The H1N1 subtypes showed a notable resemblance to A/NewCaledonia/20/99 vaccine strain and therefore were predicted to be capable of conferring sufficient immunity against H1N1 subtypes.

Identification of Bacterial and Fungal Isolates by Sequence Analysis of 16S rRNA and Internal Transcribed Spacer / 대한임상미생물학회지

BACKGROUND: Accurate and rapid identification of pathogens is one of the most important tasks of the clinical microbiology laboratory, and, in cases of rare pathogens, the identification is difficult and time-consuming upon the use of conventional methods alone. Herein, we will report our molecular work involving the identification of bacteria and fungi. METHODS: Sixty bacterial isolates had been collected from November 2004 to May 2007, and 15 fungal isolates had been collected from September 2005 to May 2007. Species identifications were performed using sequence analyses of the 16S rRNA region of bacteria and the internal transcribed spacer (ITS) region of fungi. The data were compared with those of GenBank (http://www.ncbi.nlm.nih.gov/) or EMBL (http://www.ebi.ac.uk/embl/). RESULTS: Sixty bacterial isolates included: 23 isolates with genus information (group 1), 17 isolates (group 2) that were too fastidious for genus or species identification, 16 isolates (group 3) with results from identification kits having low confidence, and 4 isolates (group 4) with odd antibiograms according to the species. In 58 of 60 isolates, identification of the genus or species could be obtained using molecular genetic methods. Thirty-eight isolates (63%) and 20 (33%) of 58 isolates could be identified at the species and genus levels, repectively. Among the total of 15 fungal isolates, 11 (73%) and 4 (27%) isolates were identified at the species and genus levels, respectively. CONCLUSION: 16S rRNA and ITS sequencing analyses are very useful for identifying the species or genus of a pathogenic microorganism in the clinical microbiology laboratory.

Partial genome molecular characteristics of Getah virus newly isolated in China / 中华微生物学和免疫学杂志

Objective To study the genome molecular characteristics of Getah virus(DY0824)which isolated in Shandong province,2008 by molecular biology methods.Methods Reverse transcriptasepolymerase chain reaction(RT-PCR)was used to amplify the structural gene and 3'UTR fragments then the RT-PCR products were inserted into PGEM-T easy to be sequenced.Computer software was used to analyze the nucleotide and deduced amino acid sequence,and draw phylogenetic trees,including Clustal X1.83 and MegaAlign and Mega4.Results The capsid protein of DY0824 consists of 804 nucleotides,encoding 268 amino acids and the full-length of E2 protein is 1266 nucleotides,encoding 422 amino acids.The nucleotide homology of the capsid protein and the E2 protein with other strains were 95.4%-99.9%and 94.8%-99.5%,and the amino acid were 97.4%-100%and 97.6%-100%.The 3'UTR of the virus include 401 nucleotides and there are three repeat sequence elements.Conclusion Compared with the prototype virus,the Getah virus isolated in Shandong province had 7 amino acid differences in capsid protein genes and 10 amino acid differences in E protein genes.The 3'UTR region had multi-nucleotide changes.

Isolation and Complete Nucleotide Sequence of the Measles Virus IMB-1 Strain in China / 中国病毒学·英文版

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4% -5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H 1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.

Research on genetic characteristics in the HA1 of influenza A(H1N1) viruses isolated in Taian City / 中华疾病控制杂志

Objective To analyze the data of influenza A(H1N1) viruses surveillance and genetic characteristics from Taian City during 2005-2008,so a scientific basis can be provided for the prevention and treatment of influenza.Methods The specimens from Influenza-Like Illness(ILI) were collected.The viruses were isolated with MDCK cell and identified with HAI and RT-PCR.The product of PCR were sequenced.Then the sequences were analyzed through biometric software.Results A total of 121 influenza strains were obtained from 615 specimens,and 4 of them were identified as A(H1N1) subtype.There were 3 strains mutated on several sites.Compared with strains isolated in 2005,there were 5 and 8 mutations in the amino acid sequences of virus strains isolated in 2007 and 2008 respectively.And there were a total of 22 amino acid mutations compared with A/Brisbane/59/2007(H1N1).Conclusions Influenza type A(H1N1) are detected in Taian City.There are several mutations in the amino acid sequences of virus strains isolated in Taian. The antigenic drift of virus strains is due to accumulation of amino acid substitutions

Comparison of Cj1136,Cj1138 and Cj1139 genes among Campylobacter jejuni strains / 中华流行病学杂志

Objective By sequenceing the Cj1136,Cj1138 and Cj1139 gene of Campylobacter jejuni(C. Jejuni) strains associated with Guillain-Barre Syndrome(GBS),features of Cj1136,Cj1138 and Cj1139 gene were studied.Results were compared with the C.jejuni strain NCTC11168, to find the mutations in sequence of C.jejuni which inducing GBS and their polygenetic relationship was analyzed.Methotis Three GBS-associated C.jejuni strains were isolated from stools of GBS patients from Hebei province who had been diagnosed as clinical AMAN pattern and electrophysiological tests were performed.After distilling and sequencing Cj1136,Cj1138 and Cj1139 genes,results were spliced and assembled into a complete sequence by the terminals overlapped with each other.Sequences of Cj1136,Cj1138 and Cj1139 genes were compared with NCTC11168,to find the mutations and gene feature.Results The Cj1136,Cj1138 and Cj1139 gene of the three GBS-associated C.jejuni strains were composed by 1173 base pairs,1170 base pairs,912 base pairs respectively. The alignment with the related sequence of NCTC11168 showed that there were two same mutations in the Cj1138 gene of the three C.jejuni stains.Data from phylogenetic analysis demonstrated that the three C.jejuni strains were genetically closed to NCTC11168,with the biggest phylogenetic distance between the three of them as 2.1%.Conclusion When compared with NCTC11168 the Cj1138 gene of the three GBS-associated C.jejuni strains had the same mutations which might be related to the development of GBS.Relation between the variation and GBS-pathogenesis remained to be confirmed.The mutations found in the three C.jejuni strains established the foundation for exploring the biological characteristics of GBS-associated C.jejuni strains and demonstrated that the GBS-associated C.jejuni strains of Hebei province having its regional features.

Complete Nucleotide Sequence of a Mumps Virus SP Strain Isolated in China / 中国病毒学·英文版

The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4%-6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.

Quantitative evaluation of viral fitness by nucleotide sequence determination / 重庆医科大学学报

Objective:To evaluate reproductivity of nucleotide sequencing for quantitative determination of viral fitness. Methods:F gene fragments from respiratory syncytial virus (RSV) A2 strain,palivizumab escape mutants,F212 and MP4,were amplified by RT-PCR,gel purified,quantified by spectrophotometry and adjusted to same concentration. A2,F212 and MP4 RT-PCR products were mixed at different ratios and determined for nucleotide sequence. Electropherograms at sites with ambiguity were visually measured by using ABI EDIT software. Proportion of RT-PCR products from prototype virus or escape mutants represented viral fitness level. Molecular cloning was utilized to validate input ratios of RT-PCR products. Results:Dual peaks were seen at 816 and 828 positions in the F gene for A2/F212 and A2/MP4mixtures,respectively. Proportion of prototype virus and escape mutants were gained by measuring the heights of dual peaks. Measured ratios were similar to premixed ratios which were confirmed by molecular cloning. Conclusion:Nucleotide sequencing is reliable,easy assay for evaluation of viral fitness and practical for screening escape mutants and viral fitness in viral infections,such as HIV infection.

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species.

Sequencing of WLAX gene in Guillain-Barr? Syndrome-associated Campylobacter jejuni strains / 基础医学与临床

Objective To investigate the characteristic of sequences of WLAX gene in Campylobacter jejuni(C.jejuni)strains.Methods WLAX gene and the neighbouring sequences were amplified by polymerase chain reaction(PCR).The PCR products were cloned into the vectors of plasmid.The positive recombinants were sequenced and the results were processed by software DNAstar.Results The variation frequency of WLAX sequences in GBS-related C.jejuni was higher than that in non-GBSrelated C.jejuni.The nucleotide sequences of WLAX gene in all the strains in the present study differed from that in genome sequencing strain NCTC11168.The phylogenic tree reflected the regional feature of C.jejuni.Conclusions The probability of sequence variation of WLAX in GBS-related C.jejuni is significantly higher than non-GBS-associated C.jejuni strains,the relation between the variation and GBS-pathogenesis remains to be further confirmed.

Partial Nucleotide Sequence of Porcine Parvovirus (VRI-1 Strain): Identification of the Putative Defective Genomes

A porcine parvovirus, designated as VRI-1, was isolated from a 30-day-old piglet. Replicative form of viral DNA from ST cells infected with VRI-1 was directly cloned into pUC19. The cloned DNA fragment contained the entire nonstructural and structural protein genes, covering approximately 85% of the viral genome. The nucleotide sequence of VRI-1 showed 99.4~99.5% identity in the nonstructural protein (NS) and 99.0~99.2% identity in the structural protein with previously reported PPV strains, respectively. Among the cloned genes, two types of defective genomes with deletion of 100 and 247 nucleotides at almost similar location of 3' region within NS gene were also identified in this study.

Production, Characterization, and Variable Region Analysis of Monoclonal Antibodies Specific for Hepatitis B Virus S Antigen

BACKGROUND: Hepatitis B virus (HBV) infection is one of the worldwide public health problem affecting about 300 million people. The envelope protein of HBV consists of three components known as preS1, preS2, and S antigen. According to the recent study, anti-HBs Ab showed effective neutralization ability against HBV from chronic hepatitis B and liver transplant patients, suggesting the possible development of therapeutic antibody. METHODS: Spleen cells immunized with S antigen of HBV were fused with myeloma cell line to obtain HBsAg specific monoclonal antibodies. High affinity antibodies against HBsAg (adr, ad and ay type) were selected by competitive ELISA method. Nucleotide sequence of the variable regions of monoclonal antibodies was analyzed by RT-PCR followed by conventional sequencing method. RESULTS: We produced 14 murine monoclonal antibodies which recognize S antigen of HBV. Two of them, A9-11 and C6-9 showed the highest affinity. The sequence analysis of A9-11 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain I (B) and light chain lambda 1, respectively. Likewise, the sequence analysis of C6-9 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain II (B) and light chain kappa 1, respectively. Neutralization assay showed that A9-11 and C6-9 effectively neutralize the HBV infection. CONCLUSION: These results suggest that A9-11 and C6-9 mouse monoclonal antibodies can be used for the development of therapeutic antibody for HBV infection.

Nucleotide Sequencing and Expression of Type 2 Porcine Circoviruses Isolated in Korea

Porcine circovirus (PCV) is a small, nonenveloped virus that contains a single-stranded circular DNA genome of about 1.76 Kb and belongs to the family Circoviridae. The PCV-2 was thought to be one of the causative agents for postweaning multisystemic wasting syndrome (PMWS) in pigs. In this study, the complete genome of two PCV-2 Korean isolates (KSY-1 and KSY-2) were sequenced and characterized. Also, the ORF2 gene of KSY-1 isolate was expressed in baculovirus expression system and the expressed protein was characterized. The sequence data indicated that the PCV-2 genome of two Korean isolates were 1,768 bases in length and encoded 2 major proteins, Rep (ORF1, 314 amino acids, 37 kDa) and a capsid (ORF2, 233 amino acids, 28~30 kDa) protein. There were 5 glycosylation sites and stem-loop structures with the nonanucleotide (5-AAGTATTAC-3), typically seen in PCV-2. Compared to nucleotide sequences of PCV-1 and PCV-2 reference strains, two Korean isolates were closely related; that is, they showed 98% homology in nucleotide sequence each other. Also, they showed 95~99% homology in nucleotide sequences with those of PCV-2 isolates but 76% similarity with those of PCV-1 reference strains. A phylogenetic analysis revealed that nucleotide sequences of Korean isolates were close to those of PCV-2 (AF055392) isolated in Canada. The baculovirusexpressed ORF2 migrated at 30 kDa and reacted with PCV-2 specific antiserum by indiect fluorescent antibody and Western blot analyses. It is concluded that our results could be valuable to understand the molecular characteristics of PCV-2 and to develop diagnostic methods for PCV-2 infections.