A case of severe hookworm infection / 中南大学学报(医学版)

With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.

Estandarización de una técnica de RT-PCR anidada para detección de alfavirus / Standardization of a nested RT-PCR technique for alphavirus detection

El género Alphavirus está constituido por virus de ARN de los cuales, varias especies son causantes de enfermedades humanas y animales como los virus chikungunya, Mayaro y los virus de encefalitis equinas, por lo que son considerados un problema de salud pública a nivel regional. En Paraguay han sido reportadas infecciones humanas por chikungunya pero son necesarios más estudios para ampliar conocimientos sobre circulación y ecoepidemiología de los alfavirus. La transcripción reversa de ARN seguida de una reacción en cadena de la polimerasa (RT-PCR) anidada es de gran utilidad como herramienta diagnóstica y en la vigilancia epidemiológica. El objetivo de este estudio fue definir las condiciones óptimas de reacción y determinar el límite de detección para una RT-PCR anidada para la detección genérica de alfavirus. El límite de detección obtenido, de 0,47 UFP/mL, indica una alta sensibilidad, pudiéndose aplicar la técnica a muestras humanas y animales de suero, líquido cefalorraquídeo, órganos y a pooles de mosquitos. Este trabajo servirá de base a otros estudios de detección e identificación de especies de alfavirus circulantes en nuestro país, lo que contribuiría a fortalecer su vigilancia y prevención.

The genus Alphavirus consists of RNA viruses of which several species areresponsible for human and animal diseases, such as chikungunya, Mayaro and equineencephalitis viruses, and are therefore considered a regional public health problem. InParaguay, human infections have been reported by chikungunya, but more studies areneeded to increase knowledge on the circulation and ecoepidemiology of alphaviruses.Reverse RNA transcription followed by a nested polymerase chain reaction (RT-PCR) isvery useful as a diagnostic tool and in epidemiological surveillance. The objective ofthis study was to define optimal reaction conditions and to determine the limit ofdetection for a nested RT-PCR for generic alphavirus detection. The detection limitobtained, of 0,47 PFU/mL, indicate high sensitivity, and the possibility of applying thetechnique to human and animals samples of serum, cerebrospinal fluid, organs andmosquito pools. This work will serve as a basis for other studies of detection andidentification of alphavirus species circulating in our country, which would helpstrengthen the surveillance and prevention.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCID₅₀/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Characterizing PML/RARα Isoforms of Acute Promyelocytic Leukemia (APL) in Malay patients.

Aim: Reciprocal translocation between retinoic acid receptor alpha (RARα) gene on chromo- some 17 and promyelocytic leukemia (PML) gene on chromosome 15 is the hallmark for acute promyelocytic leukemia (APL). Three different PML/RARα isoforms have been described; S-form, L-form and V-form. Our aims were to characterize the different types of PML/RARα iso- forms in Malay patients with APL and to determine the outcome of these different types of iso- forms. Materials and methods: RT-PCR analysis was performed on 20 patients recruited from hematology-oncology ward. RT-PCR detected fusion transcript of PML/RARα in all patients. Results and Discussion: Of these patients, 65% (13 patients) exhibited L/V-form, and 35% (7 patients) S-form. Total white blood cell count (TWBC) was higher in L/V-form (25 x 109/l) compared to S-form (2.1 x 109/l) (p < 0.05). Five years survival rate was 100% and 33.3% for L/V-forms and S-forms respectively (p<0.005). Conclusion: We conclude that L/V- forms is the commonest isoform among Malays. They presented at younger age with higher TWBC counts. Although the sample size is small, our preliminary data showed an interestingly longer survival outcome among L/V-forms compared to S-form. PML/RARα isoforms could be used in future as risk stratification feature in patients diagnosed as APL. Further study with more number of patients is required.

Detection of dengue virus RNA in blood clots by multiplex nested reverse transcription-PCR / 中国人兽共患病学报

Dengue is the most common vector borne viral disease of humans globally.Detection of viral RNA from suspected patient specimens is rapid,specific and confirmative in laboratory diagnosis of dengue infections during the acute phase.In this study,a multiplex nested reverse transcription PCR (RT-PCR) system was established for clinical specimens.While other nucleic acid amplification tests showed relatively low sensitivity,the multiplex nested RT PCR assay detected 4 cases among blood clots from 8 serologically confirmed dengue patients.These results suggested that blood clots of dengue patients could be used in laboratory diagnosis,and that the multiplex nested RT PCR assay,which simplified the detection procedure,could facilitate viral RNA detection of specimens in clinical laboratories.

The correlation between micrometastasis and hMAM in the peripheral blood of the patients with breast cancer / 国际外科学杂志

Objective To study the correlation between micrometastasis and human manmmaglobin mRNA(hMAM-mRNA)in tlle peripheral blood of the patients with breast cancer.Methods Peripheral blood samples were obtained from 92 patients with breast carcinoma,and 20 patients were with benign breast diseases,and 10 patients with other system malignant tumor,and 10 normal healthy volunteers.To identify breast carcinoma cells in periphersl blood,hMAM-mRNA Was amplified from total RNA extracted from whole blood by nested reverse transcription-polymerase chain reaction(Nested-RT-PCR).The expression of hMAM-mRNA wag contracted to MCF-7 breast cancer cell.Results The hMAM-mRNA wag only detectable in the blood from breast disease patients.Among 92 breast carcinoma patients,40 were nested-RT-PCR positive for hMAM-mRNA.The positive rate was 43.5%(40/92).The expression of hMAM-mRNA in pe-ripheral blood was not correlated with patient's estrogen or pregnant receptor;menstruation state;primarytumour size.The expression of hMAM-mRNA in peripheral blood wag correlated with patient's age:the state of c-erbB-2 and carcino embryonic antigen and carbohydrate antigen 15-3.Conclusion The hMAM-mRNA in peripheral blood may be indicator of the micmmetastasis of breast cancer.

Epidemiological investigation of Borna disease virus infection in horses and donkeys in Yili, Xinjiang / 中华微生物学和免疫学杂志

Objective To investigate the epidemiology of BDV infection in Yili horses and Yili donkeys and to analyze phylogenetic source of BDV in Yili area, Xinjiang. Methods We established fluo- rescence quantitative nested RT-PCR to detect BDV p24 segment in peripheral blood mononuclear cells (PBMCs) of 518 Yili horses and 206 Yili donkeys in Yili area, Xinjiang. Positive products were validated by detecting BDV p40 segment and plasmid to preclude the contamination, and were sequenced to analyze the homology of gene sequence, amino acid sequence and phylogenetic tree. Results The positive rates of BDV infection in PBMCs of 518 Yili horses and 206 Yili donkeys were 0.97% and 1.94%, respectively. The results of BDV p40 segment verification were positive in all of the samples of BDV p24 positive. All the samples tested were not contaminated by plasmid. There was a homology of the gene sequence of positive PCR samples with strain He/80. And the gene sequence revealed more than 93% identical to H1766 and strain V. Conclusion Our study suggested BDV natural infection in Yili horses and Yili donkeys. The en- demic BDV had a high degree of identity to strain He/80.

Detection of MLL gene rearrangement in acute leukemia and its clinical significance / 白血病·淋巴瘤

Objective To study the frequency of mixed-lineage leukemia(MEL)gene rearrangement and frequent types of fusion genes and the clinical characteristics in acute leukemia(AL)with MLL gene rearrangement.Methods MLL gene rearrangement was detected by multiplex nested RT-PCR in 109 cases of AL.Immunophenotypes were screened by flow cytometry and the chnical features of MLL gene rearranged were analyzed.Results MLL gene rearrangement were identified in 6.4%(7/109)of the patients,4 cases of AML,1 case of M2,1 case of M4,2 cases of M5,3 cases of.B-ALL.MLL-AF4,MLL-AF6,MLL-AF9,MLL-AF10.MLL-ENL were deleted by multiplex nested RT-PCR.Patients with MLL gene rearrangement presented higher initial white blood cell count(WBC)(P=0.019).Conclusion Multiplex nested RT-PCR is a fast and effective method to detect gene rearrangement of MLL in AL Patients with MLL gene rearrangement presented higher WBC and poor prognosis.

Epidemiological investigation on natural infection to Borna disease virus (BDV) among horses in Yili,Xinjiang / 中华流行病学杂志

Objective To investigate the epidemiological pattern of Borna disease virus (BDV)infection in horses and to analyze the phylogenetic tree of derived BDV in Yili, Xinjiang. Methods We established a modified nested RT-PCR (nRT-PCR) to detect BDV p24 segment in peripheral blood mononuclear cells (PBMCs) and brain tissues of 120 horses in Yili, Xinjiang. Positive products were analyzed by sequencing and homology analysis. Results The positive rate of BDV infection was 2.5% in both PMBCs and brain tissues at the same time. The gene sequence revealed in positive PCR samples was more than 93 % ,identical to that of BDV derived from horses in other countries. We also noticed a high degree of identity ( >98 % ) to standard strain He/80 in gene sequence of positive PCR samples. Conclusion Our study found the presence of BDV natural infection in horses in Yili. The endemic BDV had a high degree of identity to standard strain He/80.

Apply Nested-RT-PCR technique to identify bcr/abl fusion gene in the chronic myelogenous leukemia

Background: Bcr/abl fusion gene plays an important role in diagnosing and treating chronic myelogenous leukemia. Objective: to detect fusion genes: b3a2, b2a2, b3a3, b2a3 and e1a2 in patients with chronic myelogenous leukemia by using Nested RT - PCR technique. Subjects and methods: Peripheral blood samples were analyzed by Nested RT - PCR assay from 30 adult patients. Results: 28/30 patients showed bcr/abl fusions gene; among them 20/30 patients showed b3a2 fusions gene, 5/30 patients showed b2a2 fusions gene, 2/30 patients showed co-expression of the b3a2 and b2a2. 1/30 showed e1a2; 2/30 patients showed negative fusion gene. Count of leukocytes and platelets of patients with b3a2 fusion genes were 311.3 G/l and 597.5 G/l, respectively and of patients with b2a2 fusion genes were 136.7 G/l and 333 G/l, respectively. Conclusion: Most of patients showed b3a2 fusion gene, while remaining showed b2a2 transcripts or the co-expression of the b3a2, only one case showed e1a2 fusion gene, two patients showed negative fusion gene. There was no case which showed b3a3 or b2a3 fusion gene. Nested RT assay should be used to determine bcr/abl fusion genes for patients with chronic myelogenous leukemia\r\n', u'\r\n', u'

Research on correlation between E-cadherin mRNA expression and lung cancer / 临床检验杂志

Objective To explore the correlation of E-cadherin(E-cad) mRNA expression with tumorigenesis and development of lung cancer.Methods The relative quantitative nested RT-PCR was used to detect the expression of E-cad mRNA in cancerous tissues,adjacent and distal tissues of tumor from 50 lung cancer patients,and non-cancerous benign lung tissues from 5 lung disorder patients.Results The means of expression rate of E-cad mRNA in cancerous tissue,adjacent and distal tissues of tumor,and non-cancerous tissues were 1.33?0.53,1.65?0.60,2.01?0.46 and 2.09?0.09 respectively.The data were evaluated by analysis of variance.There were significant difference among cancerous tissue,adjacent and distal tissue(F=18.810,P0.05).Also,there were no significant differences among squamous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of cancerous,adjacent and distal tissue.Conclusion The reduced expression of E-cadherin mRNA may play important role for tumorigenesis and development of lung cancer,and there was no evident relationship of E-cad mRNA with different pathological types of lung cancer.

Prognostic Value of Bone Marrow Micrometastasis Detected by Nested RT-PCR for Cytokeratin 19 and Mammaglobin in Breast Cancer

PURPOSE: Breast cancers frequently undergo distant metastasis during the early phase, on which the survival of patients is greatly dependent. It has been suggested that the occurrence of micrometastasis relates with other prognostic features of breast cancer, such as lymph node metastasis and the presence of vascular invasion. The aim of this study was to examine the presence of keratin-19 and mammaglobin mRNA in bone marrow aspirates obtained from breast cancer patients, and their possible correlation with tumor staging and disease free survival. METHODS: Bone marrow samples were obtained from 254 breast cancer patients at the time of surgery. We separated the mononuclear fraction from the samples and carried out nested reverse transcriptase polymerase chain reaction for the detection of keratin-19 and mammaglobin mRNA using two different pairs of primers. We also studied the possible correlations between the tumor size, nodal involvement, stage, and distant metastasis. RESULTS: Seventy-five of the 254 samples were studied for cytokeratin 19 and the others for cytokeratin and mammaglobin. The median follow-up time was 21.1 months. Sixty-five (26%) of the 254 samples were cytokeratin 19 positive and 25 (14.3%) of the 175 were mammaglobin positive. Eight cases (12.3%) in the cytokeratin positive group showed a recurrent disease in distant organs. Whereas, six (3.2%) out of 185 cytokeratin negative patients had distant recurrences. Mammaglobin positivity was not correlated with distant metastasis. The stage, nodal status, and estrogen receptor were independent of bone marrow micrometastasis. CONCLUSION: Bone marrow micrometastasis, detected by nested RT-PCR for cytokeratin 19, could be a useful predictive marker for the distant metastasis of breast cancer.

Expression of Maspin is associated with the Intestinal Type of Gastric Adenocarcinoma / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Maspin is known as a tumor suppressor gene, but its significance has been questioned in various human cancers. The aim of this study was to investigate the expression pattern of Maspin in human gastric adenocarcinomas and its possible correlation with clinicopathological findings. MATERIALS AND METHODS: The expression of Maspin mRNA was measured by nested RT-PCR using 60 frozen adenocarcinomas of the stomach and 31 noncancerous tissues from the proximal resection margin. Immunohistochemical study for Maspin protein expression was carried out using 62 paraffin-embedded tissues, composed of both cancer and noncancerous tissues. RESULTS: Maspin mRNA expression was detected in 80.0% (48 of 60) of the gastric adenocarcinomas, but in only 22.6% (7 of 31) of the normal gastric mucosa (p<0.001). The positive rate of Maspin protein expression was higher in the adenocarcinomas than the normal tissues (62.9% vs. 27.4%, p<0.05). In addition, the intestinal type of tumors showed significantly higher expression levels compared to the diffuse type of tumors (81.5% vs. 48.6%, p<0.05). CONCLUSION: Our results suggest that Maspin is frequently expressed in human gastric cancers, and its expression might be associated with tumorigenesis of the intestinal type of gastric cancer.

Molecular detection of the mammaglobin expression in the peripheral blood of Thai breast cancer patients.

Mammaglobin (hMAM) is expressed exclusively in the mammary glands of adult woman and mammary tumour cell lines. Thus, we examined hMAM expression as a market for the detection of carcinoma cells in the peripheral blood of patients with breast cancer in Thailand. In addition, we studied the correlation between hMAM expression in circulation mammary carcinoma cells and clinicopathological prognostic factors of breast cancer. Blood sample obtained from two hundred breast cancer patients at various stages of their disease and from sixty females without breast cancer (thirty healty individuals and thirty patients with various malignancies other than breast cancer) were screened for hMAM mRNA by a nested reverse transcriptase polymerase chain reaction (RT-PCR) assay. We found significant differences between patients with breast cancer and those with other malignancies or healthy controls. None of the samples from the peripheral blood of sixty females without breast cancer was positive, whereas sixty four (32%) of the two hundred sample from breast cancer patients tested positive for hMAM mRNA. While our hMAM nested RT-PCR approach has 100% specificity, its sensitivity is only 32%. The presence or absence of hMAM expression in these breast cancer patients was not associated with clinicopathological prognostic factors including stage, oestrogen and progesterone status, lymph node metastases, histological type, tumour size, differentiation, lymphatic invasion, vascular invasion, menopausal status or age. We summarized that the hMAM nested RT-PCR assay may be an effective tool for the detection of circulating mammary carcinoma cells of breast cancer patients. Nevertheless, the clinical relevance hMAM RT-PCR based tumour cell detections should be further evaluated in prospective studies.

Measurement of tumor cells in the peripheral blood of patients with breast cancer by the MAGE genes as specific markers / 医学研究生学报

Objective: To detect tumor cells in the peripheral blood of patients with breast cancer by usingthe MAGE-A1 and MAGE-A3 genes as specific tumor markers. Methods: Peripheral blood was obtained from 40 patients with breast cancer and 20 patients with benign diseases. The mRNA of the MAGE-A1 and MAGE-A3 genes in the peripheral blood mononuclear cells (PBMC) was detected by nested RT-PCR. The MAGE-A1 and MAGE-A3 transcripts in breast cancer were detected by RT-PCR. Results: Of the 40 breast cancer patients, MAGE-A1 and MAGE-A3 mRNA were positive in 12.5% (5/40)and 17.5%(7/40)of PBMC, respectively, and in 15.0 %(6/40)and 22.5 %(9/40)of breast cancer tissues, respectively. In the PBMC of the 40 breast cancer patients, 10(25.0%)samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMC from the patients whose tumors did not express the MAGE genes, nor in the PBMC from the 20 patients with benign diseases. The positive rate of MAGE mRNA in the PBMC was closely correlated with the TNM stages. Conclusion: MAGE-A1 and MAGE-A3 mRNA could be specifically detected in the PBMCof breast cancer patients by our methods. They may be used as specific tumor markers for the detection of the circulating breast cancer cells.

Influence of Bone Marrow Micrometastasis in Recurrence and Survival of Breast Cancer

PURPOSE: Breast cancers frequently develop distant metastasis in the early phase. The survival rate of patients depends on a distant metastasis. The occurrence of a micrometastasis has been related to the prognostic features of breast cancer, such as a lymph node metastasis and the presence of a vascular invasion. The aim of this study was to examine the presence of RNA from epithelial tumors in bone marrow from a series of breast cancer patients and its correlation with the tumor staging and disease free survival. METHODS: Bone marrow samples were obtained from 59 patients with breast cancer at the time of surgery. The mononuclear fraction was separated and a nested reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for the detection of keratin-19 with different two pairs of primers. After surgery, the patients were followed up for a 3-month interval. Its correlation with the tumor size, nodal involvement, stage, and recurrence was investigated. RESULTS: A bone marrow micrometastasis was detected by nested RT-PCR for Keratin-19 mRNA in one case in 4 DCIS, 13 in 30 patients with T1, 11 in 20 patients with T2, and all 4 cases in patients with a T3 lesion. Recurrence was observed in 7 cases and all of them tested positive for a micrometastasis in the bone marrow. CONCLUSION: The nested RT-PCR for keratin-19 mRNA from the bone marrow in patients with breast cancer is sensitive and reliable. Moreover, early recurrence has been observed in patients with tumor mRNA present in the bone marrow. Additional studies with a large number of patients and a long term follow up are needed.

Use of cancer-testis antigen mRNA as specific markers to evaluate the prognosis of patients with hepatocellular carcinoma undergoing liver transplantation / 中国普通外科杂志

Objective To investigate the relationship between the expression of MAGE-1,SSX-1,and CTp11 mRNA in the peripheral blood of patients with hepatocellular carcinoma (HCC) receiving orthotopic liver transplantation (OLT) and the prognosis of these patients. [WT5”HZ]Methods The expression of the three genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR,and a follow-up was carried out in 26 patients. Results Before OLT,the positive rate of MAGE-1,SSX-1 and CTp11 transcripts was 40.0% (14/35),37.1% (13 /34),and 31.4% (11/35),respectively in the PBMC samples of HCC patients;Sixty percent (21/35) of the PBMC samples were positive for at least one of the three gene transcripts. The follow-up of 26 patients showed that,the tumor recurrence/metastasis rate after OLT (75.0%) in 16 patients with positive preoperative PBMC samples of at least one gene transcripts was significantly higher than that of the other 10 patients who was negative preoperatively for gene transcripts (30.0%)( P = 0.043).Postoperative tumor recurrence or metastasis developed in 15 out of 20(75%) patients with persistently perioperative positive MAGE-1,SSX-1 and /or CTp11 transcripts in PBMC samples or swithing from negative to positive perioperatively,in contrast to no recurrence nor metastasis in the other 6 patients with the gene transcripts switching from positive to negative postoperatively,or negative perioperatively ( P =0.002). [WT5”HZ]Conclusions MAGE-1,SSX-1 and CTp11 transcripts may act as combined tumor-specific makers to detect tumor cells in the peripheral circulation of HCC patients,and the detection is valuable for selecting OLT candidates and predicting the postoperative prognosis.

The correlation between micrometastasis and SBEN expression in the peripheral blood of patients with breast cancer / 中国普通外科杂志

Objective To study the expression of small breast epithelial mucin(SBEM) in the peripheral blood of the patients with breast cancer,and explore the correlation between micrometastasis and SBEM.Methods Peripheral blood samples were obtained from 92 patients with breast carcinoma,20 patients with benign breast diseases,10 patients with malignant tumor not originating from breast,and 10 normal healthy volunteers.SBEM-mRNA expression was detected by nested reverse transcription-polymerase chain reaction(Nested-RT PCR).Results Among 92 breast carcinoma patients,43(46.7%) were Nested-RT-PCR positive for SBEM-mRNA,but it was negative in all the other groups.The expression of SBEM-mRNA in peripheral blood did not correlate with ER or PR status,menstruation state or primary tumour size.but correlated with patient′s age,the state of Her-2,CEA,CA15-3 and axillary lymph node metastasis of breast cancer.Conclusions The expression of SBEM-mRNA in peripheral blood of breast cancer can be of potential use to monitor the micrometastasis of breast cancer.

THE DETECTION OF AFP mRNA IN PERIPHERAL BLOOD AND ITS CLINICAL SIGNIFICANCE / 肿瘤研究与临床

Objective:To search the marker of micrometastatic hepatocellular carcinoma (HCC).Methods:Peripheral blood samples were obtained from 65 patients with hepatocellular carcinoma,21 non HCC malignant tumors,22 chronic hepatitis B or cirrhosis,and 21 normal healthy volunteers.To identify hepatocellular carcinoma cells in peripheral blood, liver specific alpha fetoprotein(AFP)mRNA was amplified from total RNA extracted from whole blood by nested reverse transcription polymerase chain reaction (Nested RT PCR).Results:AFP mRNA was not detected in the normal healthy volunteers and patients with non HCC malignant tumors.The presence of AFP mRNA in patients with HCC(44/65,67.7%)was higher than those with chronic hepatitis B or cirrhosis(2/22,9.1%, P

Partial Sequence Analysis of Puumala Virus M Segment from Bats in Korea

Hantavirus is a genus of the Bunyaviridae family causing two serious diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Puumala virus is a member of hantavirus originally found in Europe, and its natural reservoir is Clethrionomys glareolus. It is also associated with the hurnan disease nephropathia epidemica, a milder form of HFRS. To identify the hantaviruses in bats, bats were collected from Jeong-Sun, Won-Joo, Chung-Ju and Hwa-Cheon area in Korea, and nested RT-PCR was performed with serotype specific primer from M segment. Interestingly, Puumala virus was detected in bats (Rhinolophus ferrum-equinum) only from Won-Joo. The 327 bp nested RT-PCR product, was sequenced. The sequence database search indicates that the sequence is homologous to the published sequence of Puumala viruses. The sequence similarities were ranged from 71% to 97%. The highest sequence similarity was 97% with Puumala virus Vranicam strain, and the lowest was 71% with Puumala virus K27 isolate. Puumala virus Vranicam strain was isolated from a bank vole (Clethrionomys glareolus) in Bosnia-Hercegovina. Puumala virus K27 was isolated from human in Russia. This analysis confirms that bats (Rhinolophus ferrum-equinum) in Korea are natural reservoir of Puumala virus.