Journal of Microbiology

Volume 48(5); October 2010

Research Support, Non-U.S. Gov'ts

Genomic Diversity of Legionella pneumophila Serogroup 1 from Environmental Water Sources and Clinical Specimens Using Pulsed-Field Gel Electrophoresis (PFGE) from 1985 to 2007, Korea: Hae Kyung Lee , Yeon Ho Kang , Jae Yon Yu; J. Microbiol. 2010;48(5):547-553. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0031-z

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Abstract: The molecular typing of 202 Legionella pneumophila sg 1 isolates obtained from environmental water sources and clinical specimens from 1985 to 2007 was conducted using pulsed-field gel electrophoresis (PFGE). In this study, a total of 212 isolates were grouped into 35 different PFGE types and Type 1 was the predominant type, accounting for 28.7% in PFGE types. Type 1 and Type 8 were observed continuously from 1985 to 2007. In the analysis of the distribution of PFGE types in six geographic regions (Seoul-Incheon, Gangwon, Chungcheong, Gyeongsang, Jeolla, and Jeju), Type 1 was predominant throughout four regions except for Jeolla and Jeju, and Type 6 was observed in four regions except two regions (Gangwon and Jeju). Six clinical isolates belonged to PFGE Type 1, Type 6, Type 9, and Type 15. Type 1 among these types, was isolated from 3 patients with confirmed nosocomial infection at the hospital and Type 6, Type 9, and Type 15 were isolated 3 patients with suspected community-acquired infection. Type R, PFGE pattern of L. pneumophila sg 1 (ATCC 33152, Philadelphia-1), was not observed in the isolates evaluated in this study. Therefore, our results suggest that PFGE Type 1 was very prevalent in the environmental and clinical isolates in Korea. Type 1 was distributed continuously for many years throughout Korea.

Detection of Xanthomonas arboricola pv. pruni by PCR Using Primers Based on DNA Sequences Related to the hrp Genes: So Yeon Park , Young Sun Lee , Young Jin Koh , Jae-Sun Hur , Jae Sung Jung; J. Microbiol. 2010;48(5):554-558. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0072-3

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Abstract: Efficient control of Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, requires a sensitive and reliable diagnostic tool. A PCR detection method that utilizes primers to target the hrp gene cluster region was developed in this study. The nucleotide sequence of the PCR product amplified with primers specific for the hrp region of the xanthomonads and genomic DNA of X. arboricola pv. pruni was determined, and the sequence was aligned with that of X. campestris pv. campestris, which was obtained from the GenBank database. On the basis of the sequence of the amplified hrp region, a PCR primer set of XapF/R specific to X. arboricola pv. pruni was designed. This primer set yielded a 243-bp product from the genomic DNA of X. aboricola pv. pruni strains, but no products from other 21 strains of Xanthomonas or from two epiphytic bacterial species. Southern blot hybridization with the probe derived from the PCR product with the primer set and X. aboricola pv. pruni DNA confirmed the PCR results. The Xap primer system was successfully applied to detect the pathogen from infected peach fruits. When it was applied in field samples, the primer set was proved as a reliable diagnostic tool for specific detection of X. aboricola pv. pruni from peach orchards.

Diversity of Endophytic Bacteria in Ginseng and Their Potential for Plant Growth Promotion: Regupathy Thamizh Vendan , Young Joon Yu , Sun Hee Lee , Young Ha Rhee; J. Microbiol. 2010;48(5):559-565. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0082-1

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Abstract: Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different beneficial relationships. The diversity of bacterial endophytes associated with ginseng plants of varying age levels in Korea was investigated. Fifty-one colonies were isolated from the interior of ginseng stems. Although a mixed composition of endophyte communities was recovered from ginseng based on the results of 16S rDNA analysis, bacteria of the genus Bacillus and Staphylococcus dominated in 1-year-old and 4-year-old plants, respectively. Phylogenetic analysis revealed four clusters: Firmicutes, Actinobacteria, α-Proteobacteria, and γ-Proteobacteria, with Firmicutes being predominant. To evaluate the plant growth promoting activities, 18 representative isolates were selected. Amplification of nifH gene confirmed the presence of diazotrophy in only two isolates. Half of the isolates solubilized mineral phosphate. Except four, all the other endophytic isolates produced significant amounts of indole acetic acid in nutrient broth. Iron sequestering siderophore production was detected in seven isolates. Isolates E-I-3 (Bacillus megaterium), E-I-4 (Micrococcus luteus), E-I-8 (B. cereus), and E-I-20 (Lysinibacillus fusiformis) were positive for most of the plant growth promoting traits, indicating their role in growth promotion of ginseng.

Application of Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Monitor Effect of Biocontrol Agents on Rhizosphere Microbial Community of Hot Pepper (Capsicum annuum L.): Young Tae Kim , Myoungho Cho , Je Yong Jeong , Hyang Burm Lee , Seung Bum Kim; J. Microbiol. 2010;48(5):566-572. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0126-6

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Abstract: Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.

Bacterial Diversity in the Sediment from Polymetallic Nodule Fields of the Clarion-Clipperton Fracture Zone: Chun-Sheng Wang , Li Liao , Hong-Xiang Xu , Xue-Wei Xu , Min Wu , Li-Zhong Zhu; J. Microbiol. 2010;48(5):573-585. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0151-5

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Abstract: The Clarion-Clipperton Fracture Zone (CCFZ) is located in the northeastern equatorial Pacific and contains abundant polymetallic nodules. To investigate its bacterial diversity, four libraries of 16S rRNA genes were constructed from sediments of four stations in different areas of the CCFZ. In total, 313 clones sequenced from the 4 libraries were assigned into 14 phylogenetic groups and 1 group of 28 unclassified bacteria. High bacterial diversity was predicted by the rarefaction analysis. The most dominant group overall was Proteobacteria, but there was variation in each library: Gammaproteobacteria was the most dominant group in two libraries, E2005-01 and ES0502, while Alphaproteobacteria and Deltaproteobacteria were the most dominant groups in libraries EP2005-03 and WS0505, respectively. Seven groups, including Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Betaproteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, were common to all four libraries. The remaining minor groups were distributed in libraries with different patterns. Most clones sequenced in this study were clustered with uncultured bacteria obtained from the environment, such as the ocean crust and marine sediment, but only distantly related to isolates. Bacteria involved in the cycling of metals, sulfur and nitrogen were detected, and their relationship with their habitat was discussed. This study sheds light on the bacterial communities associated with polymetallic nodules in the CCFZ and provides primary data on the bacterial diversity of this area.

Evaluation of the Sensitivity and Specificity of Primer Pairs and the Efficiency of RNA Extraction Procedures to Improve Noroviral Detection from Oysters by Nested Reverse Transcription-Polymerase Chain Reaction: Cheonghoon Lee , Sooryun Cheong , Hee-Jung Lee , Miye Kwon , Ilnam Kang , Eun-Gyoung Oh , Hong-Sik Yu , Soon-Bum Shin , Sang-Jong Kim; J. Microbiol. 2010;48(5):586-593. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0047-4

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Abstract: Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoVpolluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoVseeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp? Viral RNA Mini kit with a QIAshredderTM Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.

Effects of Crude Oil on Marine Microbial Communities in Short Term Outdoor Microcosms: Seung Won Jung , Joon Sang Park , Oh Youn Kown , Jung-Hoon Kang , Won Joon Shim , Young-Ok Kim; J. Microbiol. 2010;48(5):594-600. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0199-2

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Abstract: To assess the effects of crude oil spills on marine microbial communities, 10 L outdoor microcosms were manipulated over an exposure period of 8 days. The responses of microbial organisms exposed to five crude oil concentrations in 10 to 10,000 ppm (v/v) were monitored in the microcosms. The abundance of microalgae and copepods decreased rapidly upon the addition of crude oil at concentrations over 1,000 ppm, whereas the total density of heterotrophic bacteria increased dramatically at the higher concentrations. Bacterial diversity, determined by denaturing gradient gel electrophoresis, was increased at higher concentrations. In particular, the intensity of the bands representing Jannaschia sp. and Sulfitobacter brevis increased with the addition of oil. These results indicate that crude oil spills with concentrations over 1,000 ppm seriously affected the structure of the microbial communities.

Pseudoxanthomonas icgebensis sp. nov., Isolated from the Midgut of Anopheles stephensi Field-Collected Larvae§: Asha Rani , Anil Sharma , Tridibes Adak , Raj K. Bhatnagar; J. Microbiol. 2010;48(5):601-606. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0125-7

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Abstract: A Gram-negative, aerobic, golden yellow, rod-shaped bacterium, a strain designated ICGEB-L15T, was isolated from the larval midgut of Anopheles stephensi captured in District Jhajjar, Haryana, India. The strain ICGEB-L15T grows at 30-50°C (optimum 30-37°C), pH 6.5-8.5 (optimum 7.0-8.0) and in the presence of 2% NaCl. The major fatty acids were iso-C15:0 (22.5% of total fatty acid), anteiso-C15:0 (16.5%), iso-C17:1ω9c (10.3%), iso-C16:0 (7.3%), C16:0 (6.1%), and iso-C11:0 (5.3%). The strain showed the highest 16S rRNA gene sequence similarities with the type strains Pseudoxanthomonas daejeonensis KCTC 12207T (97.4%), Pseudoxanthomonas kaohsiungensis J36T (97.17%), and Pseudoxanthomonas mexicana AMX 26BT (97.11%). The DNA relatedness between ICGEB-L15T and Pseudoxanthomonas daejeonensis KCTC 12207T, Pseudoxanthomonas kaohsiungensis J36T and Pseudoxanthomonas mexicana AMX 26BT was 24.5%, 28.2%, and 33.6%, respectively. The G+C content of genomic DNA was 69.9 mol%. The major isoprenoid quinone of strain ICGEB-L15T was Q-8. The strain ICGEB-L15T represents a novel species of the genus Pseudoxanthomonas based on physiological, biochemical and phylogenetic properties; therefore, the name Pseudoxanthomonas icgebensis sp. nov. is proposed. The type strain is ICGEB-L15T (=KACC 14090T =DSM 22536T).

Journal Article

Re-identification of Aspergillus fumigatus sensu lato Based on a New Concept of Species Delimitation: Seung-Beom Hong , Dae-Ho Kim , In-Cheol Park , Young-Joon Choi , Hyeon-Dong Shin , Robert Samson; J. Microbiol. 2010;48(5):607-615. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0084-z

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Abstract: The species concept of Aspergillus fumigatus sensu stricto has recently been defined by polyphasic taxonomy. Based on the new concept of species delimitations, 146 worldwide strains of Aspergillus fumigatus sensu lato were re-identified. Of those 146 strains, 140 (95.8%) could be identified as A. fumigatus sensu stricto, 3 (2.1%) as A. lentulus, and the remaining 3 strains as A. viridinutans complex, Neosartorya udagawae, and N. cf. nishimurae. Of 98 clinical strains, only 1 from dolphin nostril was identified as A. lentulus and not A. fumigatus sensu stricto. Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers PELF and URP1F produced nearly the same band patterns among 136 strains of A. fumigatus sensu stricto while discriminated the species from its related species. We also discussed about identification of several atypical A. fumigatus strains from clinical environments.

Research Support, Non-U.S. Gov'ts

New Taxa in Alphaproteobacteria: Brevundimonas olei sp. nov., an Esterase-Producing Bacterium: Myungjin Lee , Sathiyaraj Srinivasan , Myung Kyum Kim; J. Microbiol. 2010;48(5):616-622. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-9367-7

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Abstract: A polyphasic taxonomic approach was used to characterize a Gram-negative, non-motile bacterium, designated MJ15T, that was isolated from soil of a GS-Caltex Oil reservoir in Korea. As shown by comparative 16S rRNA gene sequence analysis, strain MJ15T belongs to genus Brevundimonas. The 16S rRNA gene sequence similarities ranged from 95.6-99.2% between strain MJ15T and validated representatives of the genus Brevundimonas. With respect to Brevundimonas species, strain MJ15T exhibited DNA-DNA relatedness values below 40.7%. The G+C content of the genomic DNA was 61.7 mol%. Strain MJ15T contained ubiquinone Q-10. The major fatty acids were C16:0 (27.7%), C19:0 cyclo ω8c (23.2%), summed feature 8 (containing C18:1 ω7c/C18:1 ω6c) (28.5%), and major hydroxyl fatty acid was C12:0 3OH (3.7%). Based upon its phenotypic and genotypic properties, as well as its phylogenetic distinctiveness, strain MJ15T (KCTC 22461T; JCM 16237T) should be classified in the genus Brevundimonas as the type strain of a novel species. The name Brevundimonas olei sp. nov. is proposed for this new species.

Nocardioides ginsengisegetis sp. nov., Isolated from Soil of a Ginseng Field: Wan-Taek Im , Se-Young Kim , Qing-Mei Liu , Jung-Eun Yang , Sung-Taik Lee , Tae-Hoo Yi; J. Microbiol. 2010;48(5):623-628. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0001-5

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Abstract: A Gram-positive, rod-shaped, non-spore-forming bacterium (Gsoil 485T) was isolated from the soil of a ginseng field located in Pocheon province in South Korea. This bacterium was characterized in order to determine its taxonomic position by using the polyphasic approach. On the basis of 16S rRNA gene sequence similarity, strain Gsoil 485T was shown to belong to the family Nocardioidaceae and related to Nocardioides koreensis (96.8% 16S rRNA gene sequence similarity), Nocardioides basaltis (96.7%), Nocardioides salarius (96.7%), and Nocardioides sediminis (96.5%). The sequence similarity with other species that had validly published names within the genus Nocardioides was less than 96.4%. Strain Gsoil 485T was characterized chemotaxonomically as having LL-2,6-diaminopimelic acid in a cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone, and iso-C16:0, C18:1 ω9c as the major fatty acids. The G+C content of genomic DNA was 71.6 mol%. The chemotaxonomic properties and phenotypic characteristics supported the affiliation of strain Gsoil 485T to the genus Nocardioides. The results of both physiological and biochemical tests allowed for genotypic differentiation of strain Gsoil 485T from the recognized Nocardioides species. Therefore, strain Gsoil 485T is considered to represent the novel species, for which the name Nocardioides ginsengisegetis sp. nov. is proposed, with the type strain Gsoil 485T (KACC 14269T =KCTC 19469T =DSM 21349T).

Characterization of Escherichia coli EutD: a Phosphotransacetylase of the Ethanolamine Operon: Federico P. Bologna , Valeria A. Campos-Bermudez , Damián D. Saavedra , Carlos S. Andreo , María F. Drincovich; J. Microbiol. 2010;48(5):629-636. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0091-0

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Abstract: The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism.

Characterization of Deinococcus radiophilus Thioredoxin Reductase Active with Both NADH and NADPH: Hee-Jeong Seo , Young Nam Lee; J. Microbiol. 2010;48(5):637-643. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0283-7

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Abstract: Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2'5' ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 μM and 25 μM/min, whereas those for NADH are 30.2 μM and 192 μ M/min. The Km and Vmax for 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 μM and 756 μM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu2+, Zn2+, Hg2+, and Cd2+, but moderately reduced (ca. 50%) by Ag+. A significant inhibition of TrxR by N ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H2N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.

Identification of Vibrio natriegens uvrA and uvrB Genes and Analysis of Gene Regulation Using Transcriptional Reporter Plasmids: Keryn L. Simons , Susan M. Thomas , Peter A. Anderson; J. Microbiol. 2010;48(5):644-656. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-9370-z

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Abstract: Nucleotide excision repair (NER) rectifies a variety of chemically and structurally distinct DNA lesions. The current model of NER is based upon the enteric bacterium Escherichia coli and there is scarce information about how other bacterial species respond to, and correct, DNA damage. Here we report the isolation and functional analysis of the uvrA and uvrB genes from Vibrio natriegens, a naturally occurring marine bacterium. Genetic studies were completed to assess the repair capabilities of V. natriegens uvrA and uvrB in E. coli uvrA and uvrB mutants. In addition to the genetic studies, transcriptional fusions between the luciferase gene and the 5′ regulatory regions of uvrA and uvrB gene of V. natriegens and E. coli were constructed. Luminescent measurements from E. coli transformed with these constructs showed that whilst the response to UV irradiation of either E. coli or V. natriegens uvrA regulatory sequences was similar, both the rate and induction of luminescence detected from the uvrB regulatory regions differed.

Replication and Pathogenesis of the Pandemic (H1N1) 2009 Influenza Virus in Mammalian Models: Donghyok Kwon , Kyeongcheol Shin , Seungtae Kim , Yooncheol Ha , Jang-Hoon Choi , Jeong Seon Yang , Joo-Yeon Lee , Chanhee Chae , Hee-Bok Oh , Chun Kang; J. Microbiol. 2010;48(5):657-662. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0120-z

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Abstract: This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.

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