The genomes of sheeppox and goatpox viruses

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae, are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.     

Phylogenetic analysis of Chinese sheeppox and goatpox virus isolates

Background

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae family are causative agents of sheep pox and goat pox respectively, which are important contagious diseases and endemic in central and northern Africa, the Middle and Far East, and the Indian sub-continent. Both sheep pox and goat pox can cause wool and hide damage, and reduce the production of mutton and milk, which may result in significant economic losses and threaten the stockbreeding. In this study, three SPPVs and two GTPVs were collected from China in 2009 and 2011. We described the sequence features and phylogenetic analysis of the P32 gene, GPCR gene and RPO30 gene of the SPPVs and GTPVs to reveal their genetic relatedness.

Results

Sequence and phylogenetic analysis showed that there was a close relationship among SPPV/GanS/2/2011/China, SPPV/GanS/1/2011/China and SPPV/NingX/2009/China. They were clustered on the same SPPV clade. GTPV/HuB/2009/China and GS-V1 belonged to the GTPV lineage. GS-V1 was closely related to other GTPV vaccine strains. GTPV/HuB/2009/China and GS-V1 were clustered with GTPVs from China and some southern Asian countries.

Conclusion

This study may expand the datum for spread trend research of Chinese SPPVs and GTPVs, meanwhile provide theoretical references to improve the preventive and control strategy.

     

Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies.     

Differentiation of capripoxvirus species and strains by polymerase chain reaction

A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine strains used in Russia and countries of the former Soviet Union from epizootic isolates of sheeppox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.     

Sheeppox Virus SPPV14 Encodes a Bcl-2-Like Cell Death Inhibitor That Counters a Distinct Set of Mammalian Proapoptotic Proteins

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.     

Isolation and Characterization of Bluetongue Virus Recovered from Blood Samples by Immunoaffinity Purification

An immunoaffinity chromatography (IAC) method was optimized for the selective capture of bluetongue virus (BTV) from blood samples and isolation of the virus in cell culture. The antibody against BTV core particles (lacking the outer capsid proteins VP2 and VP5) was used for the optimization of IAC technique. The antibody against BTV core particle was conjugated with Protein A-virus complex and the complex was dissociated using elution buffer (4 M MgCl₂ with 75 mM HEPES, pH 6.5). The optimized IAC method specifically purified the BTV without capturing other commonly infecting small ruminant’s viruses like gaotpox virus (GTPV), sheeppox virus (SPPV), Peste des petits ruminants virus (PPRV) and Foot and mouth disease virus (FMDV). The blood samples (n?=?22), positive for BTV antigen in sandwich-ELISA were attempted for virus isolation in the BHK-21 cell using the optimized IAC method. A total of seven BTV were isolated by selective capturing of the virion particles. The isolated viruses were characterized by RNA-PAGE, sequence analysis and serum neutralization test (SNT). Electropherotypic analysis of viral dsRNA in the RNA-PAGE revealed the presence of ten dsRNA segments characteristic of BTV. Out of seven isolates, four isolates were identified as BTV-1 and three isolates were identified as BTV-16 based on nucleotide sequences of segment-2. Phylogenetic analysis of segment-2 nucleotide sequence segregated BTV-1 and BTV-16 isolates to monophyletic cluster at close proximity to other eastern topotype. In SNT, hyperimmune serum (HIS) against BTV-1 neutralized the four BTV-1 isolates up to a titer?>?256 and HIS against BTV-16 neutralized the three BTV-16 isolates up to a titer?>?128. The IAC technique will be useful for the selective capture of BTV from mixed infection (BTV with other small ruminant’s viruses) and isolation from blood sample having low viral load by enrichment.     

In vitro antiviral activity of plant extracts on goatpox virus replication

Four plants having known medicinal properties were screened for inhibition of goatpox virus (GTPV) replication in vitro. Of the 4 plants, extract of Acacia arabica (Babul) and Eugenia jambolana (Jamun) leaves had inhibition (%) 99.70 and 99.92 at their maximum non toxic concentrations, 99.93 +/- 0.38 and 1999.73 +/- 0.50 microg/ml, respectively in all cytopathic effect (CPE) inhibition assays. Inhibition of GTPV virus replication was further confirmed by PCR and SYBR Green based quantitative real-time QPCR assays specific for GTPV. Results indicated that the extract of Acacia arabica and Eugenia jambolana leaves inhibited GTPV replication in vitro.     

A new example of viral intein in Mimivirus

Background

Poxviruses encode a range of immunomodulatory genes to subvert or evade the challenges posed by the innate and adaptive immune responses. However, the inactivated poxviruses possessed immunostimulating capacity and were used as a prophylactic or metaphylactic application that efficiently reduced susceptibility to infectious diseases in different species. This fact is intensively studied in different genera of poxviruses. However, little is known about the basic mechanisms adopted by sheeppox virus (SPPV). SPPV causes an acute disease of sheep that recently, has been observed to reinfect its host in spite of vaccination.

Results

By injecting inactivated or attenuated sheeppox virus SPPV vaccine in adult male Swiss mice, SPPV was found to reduce macrophages' functions in a local event that occurs at the site of application 12 h after vaccine administration as indicated by increased level of IL-10 and decreased level of SOD from cultured peritoneal macrophages. In contrast increased levels of IL-12, and SOD activity from cultured splenic macrophages, lymphocyte response to PHA-P, and in-vivo response to T-dependant Ag were detected. These effects were observed in both attenuated and inactivated SPPV, but more prominent in attenuated one.

Conclusion

The results of this study help to elucidate, the phenomenon of existence natural SPPV infections in sheep instead of vaccination and the basic mechanisms responsible for the immunostimulating capacity of sheeppox virus. Locally, SPPV shows evidence for an immune escape mechanism that alleviates the host's immune response. Later and systemically, the virus protects the host from any fatal consequences of the immune system suppression.     

Comparative efficacy of live replicating sheeppox vaccine strains in Ovines

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD₅₀ was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.     

Nucleotide sequence analysis of VP7 gene of Indian isolates of bluetongue virus vis-à-vis other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, a family Reoviridae, is a non-enveloped with double shelled structure and ten segmented double stranded (ds) RNA genome. The RNA segment S7 encodes an inner capsid serogroup specific viral protein VP7. To amplify coding region of VP7 gene of BTV, new primers, forward primer (18-38 bp) and reverse primer (1156-1136 bp), were designed using VP7 gene sequences available in GenBank. This primer pair successfully amplified cell culture adapted Indian isolates of BTV belonging to two different serotypes 1 and 18. The coding sequences of two Indian isolates of BTV (BTV-1H and BTV-18B) were cloned into pPCR Script-Amp SK (+) plasmid vector and transformed into XL10-Gold Kan ultracompetent E. coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. The sequence analysis revealed that there was 93-97% nucleotide sequence identity in VP7 gene of three different Indian serotypes of BTV. The VP7 gene sequences of Indian isolates have comparatively less sequence homology (< 80%) with American (US), and French isolates compared to South African (SA), Australian (AUS) and Chinese (PRC) isolates. In silico restriction enzyme profile analysis of VP7 gene sequences revealed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates reported from SA, AUS and PRC using TaqI. Similarly the Indian isolates of BTV belonging to three different serotypes can be differentiated using EcoRI, Hae III and TaqI restriction enzymes.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

稳定携带山羊痘病毒DNA疫苗减毒沙门氏菌的构建

为构建质粒稳定型山羊痘DNA疫苗,采用PCR与限制性酶切技术去除真核表达质粒pcDNA3.1(+)的氨苄抗性bla基因启动子序列,构建改良质粒pmcDNA3.1(+),然后插入山羊痘病毒P32基因,获得重组表达质粒pmcDNA3.1-P32,通过TSS法将其转化至减毒沙门氏菌中,构建成功携带山羊痘DNA疫苗的重组减毒沙门氏菌SL7207(pmcDNA3.1-P32);体内和体外试验结果表明,重组质粒pmcDNA3.1-P32在沙门氏菌中的稳定性显著高于pcDNA3.1-P32。这为下一步减毒沙门氏菌介导的山羊痘DNA免疫研究奠定了基础。     

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.     

Molecular Cloning, Sequencing and Phylogenetic Analysis of Coat Protein Gene of a Biologically Distinct Citrus Tristeza Virus Isolate Occurring in Central India

Citrus tristeza virus (CTV) culture, collected from a fifteen-year-old wilted and declined mosambi sweet orange [Citrus sinensis (L) Osb] plant, was maintained under green house into acid lime [C. aurantifolia Swing] and mosambi seedlings. It gave positive reaction in ELISA, both with CTV specific polyclonal antibodies (G604) and monoclonal antibody MCA13, which specifically detects severe CTV strains. Coat protein gene (CPG) of the virus was amplified by RT PCR using CPG specific primers yielding an amplicon of 672 bp. Sequence analysis of the CPG amplicon showed 97% nucleotide sequence homology with Chinese isolate CTV-0002 (Acc. No. AJ518841) and isolate S4 (Acc. No. EF063109). In phylogenetic analysis, the present CTV isolate was displayed in different clade than other reported Indian CTV isolates, but it shared a separate clade with isolates from China, Israel, Jordan and New Zealand.     

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal β-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.     

Comparison of PCR-RFLP and PFGE for determining the clonality of Brucella isolates from human and livestock specimens

Brucellosis is an important zoonotic disease caused by different species of genus Brucella that are pathogenic for humans and a variety of animals. Accurate detection of Brucella spp. infection is important for control of disease. The aim of this study was to comparison of molecular genotyping of Brucella strains by Pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) techniques.Twenty- seven Brucella spp. were isolated from human and animal samples. The isolates identified by conventional microbiological methods and confirmed using PCR for amplification of omp2a gene. Molecular typing of Brucella strains carried out by PCR-RFLP after PstI and PFGE of chromosomal DNA after XbaI enzyme digestion. The omp2a gene PCR Products with different patterns of PCR-RFLP were sequenced.The omp2a gene amplification of all human and animal Brucella isolates were positive for 1100 bp fragment. By PCR-RFLP analysis two genotypes/patterns for human isolates and four genotypes for animal isolates were obtained. In PFGE analysis totally, 7 common clones/clusters and 3 single clones were obtained.The results of this study showed the PFGE method is the more reliable and useful assay for molecular typing of Brucella strains and is more preferred to PCR-RFLP in determination of genetic similarity among human and animal Brucella isolates. The presented data showed PCR-RFLP analysis was not able to differentiate between B. melitensis biovars and vaccine strain.     

Genetic Variability of Antigen B among Echinococcus granulosus Egyptian Isolates

Genetic polymorphisms of encoding antigen B2 gene (AgB2) in Echinococcus granulosus were studied using PCR-RFLP and DNA sequencing among 20 Egyptian isolates. Five isolates from different host origins (humans, camels, pigs, and sheep) were collected and used. All examined isolates of each host group gave very similar patterns of PCR-RFLP after restriction enzyme digestion with AluI, with the gene size of approximately 140 bp and 240 bp for sheep and human isolates, and approximately 150 bp and 250 bp for pig and camel isolates. No digestion pattern was obtained after incubation of all studied isolates with EcoRI. These results reveal high intra-group homogeneity. DNA sequence analysis highlighted that human infecting strain showed 100% identity with respect to sheep infecting isolate, 96% and 99% with pig and camel infecting isolates, respectively.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.     

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and plasmid profile of soil and clinical isolates of Nocardia.

The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for generic and species-specific differentiation of Nocardia from other morphologically similar bacterial pathogens. To examine the utility of the PCR-RFLP approach in species identification, genomic DNA was prepared from 40 soil isolates, 10 clinical isolates and 8 reference strains of Nocardia. A set of oligonucleotide primers was designed from the consensus sequence of the highly conserved groEL gene that encodes the 65-kDa heat shock protein (hsp 65). The primers selectively amplified 422 bp DNA from the genomic DNA of all Nocardia species and isolates. The digestion of the amplicons with the restriction enzyme MspI produced DNA fragments that could differentiate between different Nocardia species regardless of their origin. Additionally, the RFLP patterns obtained with restriction enzymes MspI and BsaHI resulted in the differentiation of six Nocardia species which were earlier identified by biochemical tests. Apart from soil isolates of N. asteroides, which had shown some degree of genotypic polymorphism with BsaHI, the remaining taxa yielded more consistent results. Our results on the isolation of plasmids indicated that their occurrence is not a consistent feature in Nocardia species. It is neither related to the source of origin (clinical versus saprobic), nor to virulence, anti-microbial resistance or species specificity.     

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.