Journal of Microbiology

Volume 51(2); April 2013

Review

Minireview] The Unique Metabolism of SAR11 Aquatic Bacteria: H. James Tripp; J. Microbiol. 2013;51(2):147-153. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2671-2

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38 Citations

Abstract: The deeply branching clade of abundant, globally distributed aquatic α-Proteobacteria known as “SAR11”, are adapted to nutrient-poor environments such as the surface waters of the open ocean. Unknown prior to 1990, uncultured until 2002, members of the SAR11 clade can now be cultured in artificial, defined media to densities three orders of magnitude higher than in unamended natural media. Cultivation in natural and defined media has confirmed genomic and metagenomic predictions such as an inability to reduce sulfate to sulfide, a requirement for pyruvate, an ability to oxidize a wide variety of methylated and one-carbon compounds for energy, and an unusual form of conditional glycine auxotrophy. Here we describe the metabolism of the SAR11 type strain Candidatus “Pelagibacter ubique” str. HTCC1062, as revealed by genomeassisted studies of laboratory cultures. We also describe the discovery of SAR11 and field studies that have been done on natural populations.

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

The α-Barrel Tip Region of Escherichia coli TolC Homologs of Vibrio vulnificus Interacts with the MacA Protein to Form the Functional Macrolide-Specific Efflux Pump MacAB-TolC: Minho Lee , Hyun-Lee Kim , Saemee Song , Minju Joo , Seunghwa Lee , Daeyoung Kim , Yoonsoo Hahn , Nam-Chul Ha , Kangseok Lee; J. Microbiol. 2013;51(2):154-159. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2699-3

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18 Citations

Abstract: TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.

Prevalence of Amino Acid Changes in the yvqF, vraSR, graSR, and tcaRAB Genes from Vancomycin Intermediate Resistant Staphylococcus aureus: Jae Il Yoo , Jung Wook Kim , Gi Su Kang , Hwa Su Kim , Jung Sik Yoo , Yeong Seon Lee; J. Microbiol. 2013;51(2):160-165. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-3088-7

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26 Citations

Abstract: Vancomycin intermediate Staphylococcus aureus (VISA) strains are increasingly prevalent in the hospital setting, and are of major concern in the treatment of methicillin-resistant S. aureus infections. Multiple mutations in vancomycinsusceptible S. aureus (VSSA) strains likely led to the emergence of VISA, and point mutations in the agr, orf1, yvqF, vraSR, graSR, and tcaRAB genes of VISA strains have been shown to contribute to glycopeptide resistance. Therefore, we investigated point mutations in these genes from 87 VISA and 27 VSSA clinical strains isolated from Korean hospitals. All strains were assigned an agr type (I, II, or III) on the basis of multiplex PCR, with the majority of VISA strains belonging to agr groups I and II. Sequencing revealed amino acid changes in vraS from VISA strains which were not present in the VSSA strains. The E59D substitution in the vraR gene occurred in 36.3% of VSSA/agrI and 92.7% of VISA/agrI strains, suggesting that this mutation associated with emergence of VISA/agrI strains. VISA strains were classified into 31 mutation patterns according to mutations in the yvqF, vraSR, graSR, and tcaRAB genes. In addition, the mutation patterns were correlated with agr and sequence type (ST). The most prevalent pattern included agr type I (ST 72) strains with E59D (vraR), L26F and T224I (graS), D148Q (graR), and L218P, R283H and G312D (tcaA) amino acid substitutions. The minimum inhibitory concentration (MIC) range of mutation pattern 5 toward oxacillin and imipenem was much lower than that of patterns 6 and 24. These results improve our understanding of emergence of VISA strains.

Construction of a Streptomyces lydicus A01 Transformant with a chit42 Gene from Trichoderma harzianum P1 and Evaluation of Its Biocontrol Activity against Botrytis cinerea: Qiong Wu , Linquan Bai , Weicheng Liu , Yingying Li , Caige Lu , Yaqian Li , Kehe Fu , Chuanjin Yu , Jie Chen; J. Microbiol. 2013;51(2):166-173. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2321-8

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16 Citations

Abstract: Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.

Cloning, Annotation and Expression Analysis of Mycoparasitism-Related Genes in Trichoderma harzianum 88: Lin Yao , Qian Yang , Jinzhu Song , Chong Tan , Changhong Guo , Li Wang , Lianhai Qu , Yun Wang; J. Microbiol. 2013;51(2):174-182. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2545-7

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9 Citations

Abstract: Trichoderma harzianum 88, a filamentous soil fungus, is an effective biocontrol agent against several plant pathogens. High-throughput sequencing was used here to study the mycoparasitism mechanisms of T. harzianum 88. Plate confrontation tests of T. harzianum 88 against plant pathogens were conducted, and a cDNA library was constructed from T. harzianum 88 mycelia in the presence of plant pathogen cell walls. Randomly selected transcripts from the cDNA library were compared with eukaryotic plant and fungal genomes. Of the 1,386 transcripts sequenced, the most abundant Gene Ontology (GO) classification group was “physiological process”. Differential expression of 19 genes was confirmed by real-time RT-PCR at different mycoparasitism stages against plant pathogens. Gene expression analysis revealed the transcription of various genes involved in mycoparasitism of T. harzianum 88. Our study provides helpful insights into the mechanisms of T. harzianum 88-plant pathogen interactions.

Lactobacillus salivarius Strain FDB89 Induced Longevity in Caenorhabditis elegans by Dietary Restriction: Yang Zhao , Liang Zhao , Xiaonan Zheng , Tianjiao Fu , Huiyuan Guo , Fazheng Ren; J. Microbiol. 2013;51(2):183-188. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2076-2

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46 Citations

Abstract: In this study, we utilized the nematode Caenorhabditis elegans to assess potential life-expanding effect of Lactobacillus salivarius strain FDB89 (FDB89) isolated from feces of centenarians in Bama County (Guangxi, China). This study showed that feeding FDB89 extended the mean life span in C. elegans by up to 11.9% compared to that of control nematodes. The reduced reproductive capacities, pharyngeal pumping rate, growth, and increased superoxide dismutase (SOD) activity and XTT reduction capacity were also observed in FDB89 feeding worms. To probe the anti-aging mechanism further, we incorporated a food gradient feeding assay and assayed the life span of eat-2 mutant. The results demonstrated that the maximal life span of C. elegans fed on FDB89 was achieved at the concentration of 1.0 mg bacterial cells/plate, which was 10-fold greater than that of C. elegans fed on E. coli OP50 (0.1 mg bacterial cells/plate). However, feeding FDB89 could not further extend the life span of eat-2 mutant. These results indicated that FDB89 modulated the longevity of C. elegans in a dietary restriction-dependent manner and expanded the understanding of anti-aging effect of probiotics.

Journal Article

Biochemical Characterization of Chitinase 2 Expressed during the Autolytic Phase of the Inky Cap, Coprinellus congregatus: Yuri Kang , Hyewon Kim , Hyoung T. Choi; J. Microbiol. 2013;51(2):189-193. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2535-9

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18 Citations

Abstract: Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside was used as the substrate. The Km value and Vmax for the substrate A, 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min-1unit-1, respectively.

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

Pneumolysin-Mediated Expression of β-Defensin 2 Is Coordinated by p38 MAP Kinase-MKP1 in Human Airway Cells: Yong-Jae Kim , Hee-Sung Shin , Jung-Hoon Lee , Yong Woo Jung , Hyong-Bai Kim , Un-Hwan Ha; J. Microbiol. 2013;51(2):194-199. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2579-x

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5 Citations

Abstract: Antimicrobial peptides act as important innate immune defense mediators against invading microbes such as Streptococcus pneumoniae. Among a number of antimicrobial peptides, β-defensin 2 (BD2) has strong antimicrobial activity against S. pneumoniae. However, little is known about the molecular signaling mechanisms leading to the BD2 expression. Here, we report that BD2 is strongly induced by S. pneumoniae in human airway cells including human middle-ear cells. Among diverse pneumococcal virulence factors, pneumolysin is required for inducing BD2 whose expression is under the control of p38 mitogen-activated protein kinase (MAPK). Pneumolysin also selectively regulates the expression of MAPK phosphatase 1 (MKP1), which inhibits the p38 signaling pathway, thereby leading to upregulation of BD2 to mount an effective defense against S. pneumoniae infection. These results provide novel insights into the molecular mechanisms underlying the coordinative regulation of BD2 expression via p38-MKP1 in the pathogenesis of airway infectious diseases.

The Intracellular Mechanism of Action on Escherichia coli of BF2-A/C, Two Analogues of the Antimicrobial Peptide Buforin 2: Gang Hao , Yong-Hui Shi , Ya-Li Tang , Guo-Wei Le; J. Microbiol. 2013;51(2):200-206. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2441-1

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32 Citations

Abstract: In the present study, the antimicrobial peptides BF2-A and BF2-C, two analogues of Buforin 2, were chemically synthesized and the activities were assayed. To elucidate the bactericidal mechanism of BF2-A/C and their different antimicrobial activities, the influence of peptides to E. coli cell membrane and targets of intracellular action were researched. Obviously, BF2-A and BF2-C did not induce the influx of PI into the E. coli cells, indicating nonmemebrane permeabilizing killing action. The FITC-labeled BF2-A/C could penetrate the E. coli cell membrane and BF2-C penetrated the cells more efficiently. Furthermore, BF2-A/C could bind to DNA and RNA respectively, and the affinity of BF2-C to DNA was powerful at least over 4 times than that of BF2-A. The present results implied that BF2-A and BF2-C inhibited the cellular functions by binding to DNA and RNA of cells after penetrating the cell membranes, resulting in the rapid cell death. The structure-activity relationship analysis of BF2-A/C revealed that the cell-penetrating efficiency and the affinity ability to DNA were critical factors for determining the antimicrobial potency of both peptides. The more efficient cellpenetrating and stronger affinity to DNA caused that BF2-C displayed more excellent antimicrobial activity and rapid killing kinetics than BF2-A.

An Aqueous Extract of Yunnan Baiyao Inhibits the Quorum-Sensing-Related Virulence of Pseudomonas aeruginosa: Zu-Guo Zhao , Shuang-Shuang Yan , Yun-Mei Yu , Na Mi , La-Xi Zhang , Jun Liu , Xiao-Ling Li , Fang Liu , Jun-Fa Xu , Wei-Qing Yang , Guo-Ming Li; J. Microbiol. 2013;51(2):207-212. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2595-x

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20 Citations

Abstract: Yunnan Baiyao is a famous Chinese medicine that has long been directly applied to wounds to reduce bleeding, pain, and swelling without causing infection. However, little is known about its ability to prevent infection. The present study aimed to assess in vitro the anti-virulence activity of an aqueous extract of Yunnan Baiyao (YBX) using Pseudomonas aeruginosa as a pathogenic model. We found that a sub-MIC (2.5 mg/ml) of YBX can efficiently interfere with the quorum-sensing (QS) signaling circuit. Real-time polymerase chain reaction analysis showed that a sub-MIC of YBX downregulated the transcriptions of lasR, lasI, rhlR, and rhlI, which resulted in global attenuation of QS-regulated virulence activities, such as biofilm formation, and secretion of LasA protease, LasB elastase and pyocyanin. Further, YBX reduced the motility of P. aeruginosa related to QS, and impaired the formation of biofilms. These results suggest that YBX may possess global inhibitory activity against the virulence of P. aeruginosa and that YBX may also exhibit antimicrobial activity in vivo. The present study suggests that Yunnan Baiyao represents a potential source for isolating novel, safe, and efficacious antimicrobial agents.

Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12: Edwin David Morales-Álvarez , Claudia Marcela Rivera-Hoyos , Angélica María Baena-Moncada , Patricia Landázuri , Raúl A. Poutou-Piñales , Homero Sáenz-Suárez , Luis A. Barrera , Olga Y. Echeverri-Peña; J. Microbiol. 2013;51(2):213-221. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2416-2

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15 Citations

Abstract: The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/ pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Expression, Purification, and Biochemical Properties of Arginase from Bacillus subtilis 168: Jin-Ju Yu , Ki-Bum Park , Su-Gon Kim , Suk-Heung Oh; J. Microbiol. 2013;51(2):222-228. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2669-9

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25 Citations

Abstract: The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The Km and Vmax values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.

A Real-Time qPCR Assay to Quantify Ophiocordyceps sinensis Biomass in Thitarodes Larvae: Wei Lei , Shaosong Li , Qingyun Peng , Guren Zhang , Xin Liu; J. Microbiol. 2013;51(2):229-233. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2241-7

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11 Citations

Abstract: Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the CT values (R2=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came bodywall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these
results
show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.

Secretion of Truncated Recombinant Rabies Virus Glycoprotein with Preserved Antigenic Properties Using a Co-Expression System in Hansenula polymorpha: Weidong Qian , Frank Aguilar , Ting Wang , Bingsheng Qiu; J. Microbiol. 2013;51(2):234-240. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2337-0

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11 Citations

Abstract: Rabies virus infection remains a serious public health threat in the developing world, where cost-concerns make widescale public health interventions impractical. The development of novel and inexpensive ELISA diagnostic antigens is critical in early detection and prevention of complications. The transmembrane glycoprotein (G) of rabies virus (RV) contains an external domain capable of inducing the synthesis of anti-rabies, virus-neutralizing antibodies, in infected or immunized hosts. In our study, the external G domain was synthesized and fused in-frame with a polyhistidine-tag coding sequence present in the expression plasmid. Soluble truncated recombinant G was secreted in Hansenula polymorpha (H. polymorpha) using H. polymorpha-derived calnexin (HpCNE1) overproduction and found to be correctly N-glycosylated. The truncated recombinant G was purified from cell culture supernatant by Ni-agarose affinity chromatography and when compared with the full-length glycoprotein, found to be similarly immunogenic in vaccinated rabbits. These results subsequently led us to explore the potential of truncated recombinant G as a diagnostic antigen in ELISA. Our results show that the truncated recombinant G can detect antibodies directed to both whole virion and native glycoprotein. More sophisticated applications of truncated recombinant G would profit from the correctly N-glycosylated and soluble monomer.

Allelic MHC Class I Chain Related B (MICB) Molecules Affect the Binding to the Human Cytomegalovirus (HCMV) Unique Long 16 (UL16) Protein: Implications for Immune Surveillance: Kanya Klumkrathok , Amonrat Jumnainsong , Chanvit Leelayuwat; J. Microbiol. 2013;51(2):241-246. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2514-1

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Abstract: Unique long 16 (UL16) is a viral glycoprotein produced in a host cell infected with human cytomegalovirus (HCMV). It down regulates surface expression of MICB, one of the NKG2D ligands, by forming stable intracellular complexes and retained in the endoplasmic reticulum. Down expression of MICB renders cells less susceptible to NK cell lysis via the NKG2D receptor. Diverse UL16 sequences were identified from different strains of HCMV. MICB is known to be polymorphic. It is not known whether these polymorphisms affect the interactions between these molecules leading to alteration of the immune surveillance of HCMV. The soluble Fc fusion variant UL16 proteins from four laboratory and clinical isolates (AD169, Toledo, PH, and TR) were produced. Four allelic MICB alleles (008, 003, 004, and 00502) were cloned and stable cell lines expressing these MICB alleles were produced. The binding activities of variant UL16 to allelic MICB proteins were determined by flow cytometry. The variants of UL16 proteins did not affect the binding activities to allelic MICB proteins. However, diverse MICB alleles differentially bound UL16. We found that MICB*008 which contains methionine and asparagine at the amino acid positions 98 and 113, respectively, in the alpha 2 domain showed decreased binding activities to UL16 when compared to MICB*003, 004, and MICB*00502 containing isoleucine and aspartic acid, respectively. This finding may imply that MICB*008 is a protective allele and involved in the immune surveillance of HCMV infected patients.

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