The antibacterial effect of silver nanoparticle-treated silk fabrics was tested against E. coli and S. aureus by using a shaking flask method according to the antibacterial standard of knitted products (FZ/T 73023-2006, China). This standard specified the requirements of the antibacterial fabric, test methods, and inspection rules, which are applicable to the https://www.selleckchem.com/products/mk-5108-vx-689.html antibacterial fabrics made by natural fiber, chemical fiber, and blended fiber. A sample fabric with a weight of 0.75 g was cut into small pieces with a size around 0.5 × 0.5 cm2 and was immersed into a flask containing 70 ml of 0.3 mM PBS (monopotassium phosphate,

pH ≈ 7.2) culturing solution with a bacterium concentration of 1 × 105 to 4 × 105 colony-forming units (CFU)/ml. The flask was then shaken at 150 rpm on a rotary shaker at 24°C for 18 h. From each incubated sample, 1 ml of solution was taken and diluted to 10, 100, and 1,000 ml and then distributed onto an agar plate. All plates were incubated at 37°C for 24 h, and the colonies formed were counted by eyes. The percentage reduction was determined as follows (FZ/T 73023-2006,

China): where A and B are the bacterial colonies of the original silk fabrics and the silver-treated silk fabrics, respectively. To evaluate the durability of the nanoparticle-treated silk fabrics against repeated launderings, AATCC Test Method 61-1996 was applied. An AATCC standard wash machine (Atlas Launder-Ometer) selleckchem and detergent (AATCC Standard Detergent WOB) were used. Samples were cut into several

5 × 15 cm2 swatches and put into a stainless steel container with 150 ml of 0.15% (w/v) WOB detergent solution and 50 steel balls (0.25 in. in diameter) at 49°C for various washing times to simulate 5, 10, 20, and 50 wash cycles of home/commercial launderings. Results and discussion Synthesis of silver nanoparticles in solution Figure  2 shows the FTIR spectra of RSD-NH2 and the resulting silver colloid. (-)-p-Bromotetramisole Oxalate Comparing the spectra of the pure polymer and the silver/RSD-NH2 nanohybrid, the band positions of RSD-NH2 show an apparent shift. The band position at 3,068.9 cm−1, corresponding to amide B (NH stretching vibration modes) of RSD-NH2, shifted to a lower region (3,066 cm−1) after the formation of silver nanoparticles. The band position of CH2 symmetric stretching at 2,819.7 cm−1 shifted to 2,821.4 cm−1. The band position of amide I of RSD-NH2 at 1,652.3 cm−1 moved to a lower region (1,651.9 cm−1). It indicated that there are some interactions between the silver nanoparticles and RSD-NH2. The principle is illustrated in Figure  3: the molecule of RSD-NH2 contains numerous secondary and tertiary amine groups, as well as some primary amine groups at the peripheral region. These amine groups are able to attract silver ions and provide an electron source for the reduction process.

As to more specific lifestyle factors related to diet, the potential adverse skeletal Blebbistatin order effects of low calcium intake, high sodium intake and excessive caffeine consumption have been addressed in the section on nutrition. The use of carbonated soda drinks and more in particular of colas has been associated

with lower bone mass. Besides displacement of more nutrient- and calcium-rich beverages, caffeine, and phosphoric acid content in colas have also been implicated as contributing to the adverse skeletal effects [13, 91]. Excessive alcohol consumption is generally recognized as a secondary cause of osteoporosis and as a risk factor for fracture [79]. Alcohol may interfere with bone metabolism through direct toxic effects on osteoblasts and indirectly www.selleckchem.com/products/ABT-888.html through adverse skeletal effects of nutritional deficiencies in calcium, vitamin D, and proteins that are prevalent in heavy drinkers. However, increased fracture risk is explained only for a minor part by

increased bone fragility and other factors, perhaps resulting in an increased risk for falls, are involved. In a meta-analysis of three prospective studies in a total of 5,939 men and 11,032 women, followed for 75,433 person-years [92], alcohol consumption was non-linearly associated with an increased fracture risk. Consumption of 2 units or less (1 unit = 10 g ethanol) per day was not associated with an increased fracture rate, whereas higher SDHB alcohol

intake was associated both in men and women with an increased risk of any fracture (risk ratio (RR) = 1.23; 95% CI, 1.06–1.43), any osteoporotic fracture (RR = 1.38; 95% CI, 1.16–1.65), or hip fracture (RR = 1.68; 95% CI, 1.19–2.36). A similar threshold of around 2 units per day for the association of alcohol intake and fracture risk was reported in earlier studies [93, 94]. At variance with the findings in some other studies, there were no significant difference between gender for either the risk ratios or threshold; above the threshold, there was a dose–effect. Also at variance with some other studies reporting a J-shaped association between alcohol consumption and fracture risk, fracture risk was not higher in subjects abstaining from alcohol use as compared with those consuming 1or 2 units per day [79, 92]. However, it should be noted that a number of both cross-sectional and prospective studies failed to detect an increased fracture risk associated with alcohol intake (see reference [1] for review). Smoking has adverse skeletal effects and current smoking is associated with an increased fracture risk [79]. Albeit it has been reported that the adverse effects on BMD are apparent after the age of 50 and increase with age [95], smoking has been shown to also adversely affect bone health in young individuals during bone maturation [96].

Purified mouse IgG1, mouse anti-DNAM-1, NKp46, NKp44, NKp30 or all four together (all at 10 μg/ml) were added to defined wells during 4 hours of cytotoxicity in order to assess specific activating NK cell receptor-tumor ligand interactions. Reduction in cytotoxicity was calculated based on

percentage cytotoxicity in the presence of indicate blocking mAb(s) versus percentage cytotoxicity in the presence of mouse control mAb. The % reduction in ADCC was calculated with percentage cytotoxicity in the presence of human IgG1 set at 100%. To minimize changes that may occur when cell lines are established from primary tumors, the gastric cell lines used in these studies were cultured for less than 10 passages after isolation from the primary tumor tissue. Statistics Paired two-tailed Student’s t tests were used to calculate p values. P < 0.05 was considered to be significant. Results

Cytotoxic Evofosfamide NK cells are efficiently expanded from PBMC from normal individuals and patients with various solid tumors without the need of primary enrichment protocols To achieve large-scale expansion of human NK cells, PBMC were co-cultured in a 1 to 1.5 ratio with lethally irradiated K562 cells expressing membrane-bound IL-15 and 4-1BBLigand (K562-mbIL15-4-1BBL) in culture media containing 200 units IL2/ml. After 14 days of culture, NK cells (CD56+CD3- as defined by flow cytometry) expanded greater than 2 orders of magnitude from PBMC (mean 165 fold; range 4-567 fold, n = 6) and cell products became significantly enriched in NK cells (day 0 with mean 7%, range 3.2%-12.6% versus day 14 with mean 45.6%, range 7.4%-76.4%; P = 0.0140). Staurosporine At the same time, NKT cells (CD56+CD3+ as defined by flow cytometry) expanded at an average Metformin supplier of 57 fold (range 7-234), although no significant enrichment (day 0 with mean

3.8%, range 0.8%-8.1% versus day 14 with mean 11.4%, range 2.3%-17.9%; P = 0.1907) was observed. In contrast, a significant decrease in T cells (CD3+ as defined by flow cytometry) was noted after 14 days of expansion (day 0 with mean 54.5%, range 39.9%-71.2% versus day 14 with mean 30.0%, range 4.2%-58.4%; P = 0.0436) with an absolute expansion of 7 fold (range 2-19). The distribution of NK cells and NKT cells in PBMC after expansion is shown in Figure 1A. Figure 1 Cytolytic NK cells are efficiently expanded from PBMC. In the presence of K562-IL15-41BBL (A) expanded cells become significantly enriched (P = 0.0307) in NK cells (defined by CD56+CD3- cells) after 14 days of culture. Expanded cells were evaluated for cytolytic activity using 4 hour51Cr release assays. Ex-vivo expanded cells from PBMC (■ donor 1 and △ donor 2), but not freshly purified non-expanded NK cells (◇), efficiently lysed allogeneic tumor cell lines derived from breast (MCF-7) and prostate (LNCaP) cancers but not allogeneic or autologous PBMC derived from donor 1 (B).

PubMedCentralPubMed selleck chemicals 8. Nataro JP, Kaper JB: Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998, 11:142–201.PubMedCentralPubMed 9. Girón JA, Jones T, Millán-Velasco F, Castro-Muñoz E, Zárate L, Fry J, Frankel G, Moseley SL, Baudry B, Kaper JB: Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico. J Infect Dis 1991, 163:507–513.PubMedCrossRef 10. Nataro JP, Kaper JB,

Robins-Browne R, Prado V, Vial P, Levine MM: Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J 1987, 6:829–831.PubMedCrossRef 11. Johnson JR, Murray AC, Gajewski A, Sullivan M, Snippes P, Kuskowski MA, Smith KE: Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken STI571 nmr products. Antimicrob Agents Chemother 2003, 47:2161–2168.PubMedCentralPubMedCrossRef

12. Braun V, Pilsl H, Gross P: Colicins: structures, modes of action, transfer through membranes, and evolution. Arch Microbiol 1994, 161:199–206.PubMedCrossRef 13. Gillor O, Nigro LM, Riley MA: Genetically engineered bacteriocins and their potential as the next generation of antimicrobials. Curr Pharm Des 2005, 11:1067–1075.PubMedCrossRef 14. Moreno F, San Millán JL, Hernández-Chico C, Kolter R: Microcins. Biotechnology 1995, 28:307–321.PubMed 15. Šmarda J, Šmajs D: Colicins–exocellular lethal proteins of Escherichia coli. Folia Microbiol (Praha) 1998, 43:563–582.CrossRef 16. Šmajs D, Weinstock GM: Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes. J Bacteriol 2001, 183:3949–3957.PubMedCentralPubMedCrossRef 17. Šmajs D, Weinstock GM: The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake. J Bacteriol 2001, 183:3958–3966.PubMedCentralPubMedCrossRef 18. Riley MA, Wertz JE: Bacteriocin diversity: ecological and evolutionary perspectives. Biochimie 2002, 84:357–364.PubMedCrossRef 19. Patzer OSBPL9 SI, Baquero MR, Bravo D, Moreno F, Hantke K: The colicin

G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology (Reading, Engl) 2003,149(9):2557–2570.CrossRef 20. Azpiroz MF, Poey ME, Laviña M: Microcins and urovirulence in Escherichia coli. Microb Pathog 2009, 47:274–280.PubMedCrossRef 21. Šmajs D, Micenková L, Šmarda J, Vrba M, Ševčíková A, Vališová Z, Woznicová V: Bacteriocin synthesis in uropathogenic and commensal Escherichia coli: colicin E1 is a potential virulence factor. BMC Microbiol 2010, 10:288.PubMedCentralPubMedCrossRef 22. Budič M, Rijavec M, Petkovšek Z, Zgur-Bertok D: Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia. PLoS ONE 2011, 6:e28769.PubMedCentralPubMedCrossRef 23.

Table 2 Photocurrent density-voltage characteristics of TiO 2 nanofiber cells Cell ZnO thickness (nm) J sc(mA/cm2) V oc(V) FF η (%) τ d(ms) τ n(ms) L n(μm) II 0 14.5 0.825 0.53 6.34 1.88 107.7 138.3 IV 4 15.0 0.828 0.54 6.71 1.43 119.5 166.9 V 10 16.5 0.833 0.54 7.42 1.21 154.3 206.4 VI 15 17.3 0.842 0.55 8.01 1.08 179.7 235.7 VII 20 14.8 0.825 0.53 6.47 4.62 354.5 159.9 TiO2 nanofiber cells with ZnO layer of different thicknesses, the transit time (τ d) and electron lifetime (τ n), and diffusion length (L n). The schematic view of electron transfer with ZnO layer is shown in Figure  8. The interfacial processes involved

in charge transportation in the cell are depicted in Figure  8b. As exciton dissociation occurs, SB202190 concentration electrons injected into the TiO2 conduction band will transport to the FTO by diffusion [33]. Because the conduction band edge of ZnO is a little more negative than that of TiO2, an energy barrier is introduced at the interface of FTO/TiO2, in which ultrathin ZnO layer can effectively suppress the back electron transfer from FTO to electrolytes or may block injected electron transfer from TiO2

to FTO. The back reaction was studied using IMVS measurements. The electron lifetime τ n obtained from IMVS (as shown in Table  2) is 107.7 ms for the cell without ZnO layer but is significantly increased from 119.5 to 354.5 ms with ZnO layer thickness increasing from 4 to 20 nm. The striking increase in the lifetime shows direct evidence that ultrathin ZnO layers prepared by ALD method dipyridamole successfully suppress the charge recombination between electrons emanating from the FTO substrate and I3 − ions in

the electrolyte. The transit times of electrons calculated learn more from IMPS measurements reflect charge transport and back reaction. Although an energy barrier is induced by introduction of ZnO layer between the TiO2 and FTO, the electron transit time estimated from IMPS measurement is decreased from 1.88 to 1.08 ms for cells with ZnO layer thickness increasing from 0 to 15 nm. However, when the thickness of ZnO layer further increases, the change trend is reverse, and electron transit time for the cell with 20-nm-thick ZnO layer is markedly increased to 4.62 ms. It is put forward that relative to the cell without ZnO blocking layer, the electron transport in the cells with ZnO layers is determined by the two competition roles of the suppression effect of recombination with I3 − and potential barrier blocking effect. The increased electron lifetime has verified that ultrathin ZnO layer effectively slows the back recombination of electrons at the interface of FTO/electrolyte, so the decreased electron transit time reveals that the suppression effect is stronger than the potential barrier effect when the ZnO layer thickness is smaller than 20 nm. The obtained values of L n/d of cells IV to VII are shown in Table  2, which are all larger than that of the reference cell without ZnO layer, with the largest value of 8.

The high levels of secretion and the degree of conservation within the genus are congruent SC79 supplier with Pam modulating these important activities. Very little is known about Photorhabdus infections in humans, but a recent study has found that, unlike the extracellular growth of P. luminescens in insects [27], a clinical isolate of P. asymbiotica is a facultative intracellular pathogen when incubated with human

macrophage-like cells [28]. Future studies may investigate what role if any Pam has in the infection of mammalian cells. Conclusions In this study we show that the highly abundant Pam protein is able to bind to exopolysaccharides and change the attachment properties of Photorhabdus. Deletion of pam altered bacterial adhesion to surfaces but did not cause a decrease in virulence towards Galleria mellonella larvae. However, Pam is produced during insect infection

suggesting a role for this protein in the insect cadaver, possibly in the colonization of the insect body. Sequence analysis of pam in multiple isolates showed that it is ancestral and conserved in the genus Photorhabdus and thus deserves further investigations to clarify its role in the complex cycle of Photorhabdus biology. Methods Bacterial strains, plasmids and culture conditions. DNA amplification and cloning The strains used in this study are: P. asymbiotica strain ATCC43949 [29], P. luminescens subspecies laumondii strain TT01 [30] and a wild-type spontaneous rifampicin-resistant JAK inhibitor selleck inhibitor P. luminescens TT01rif (this study). A knock-out strain in the pam gene was constructed from TT01rif and named TT01pam. The pam gene was deleted from the chromosome by allelic exchange using the suicide vector pDS132 [31] and correct chromosomal

deletion was confirmed by PCR and DNA sequencing of the region near the deleted gene. The pam knock-out strain grew similarly to the wild-type strain in rich and minimal media and insect plasma (filtered hemolymph). Escherichia coli EC100 (Epicentre Biotechnology, USA) was used for heterologous production of Pam. The pam gene was PCR amplified from P. asymbiotica ATCC43949 genomic DNA using the primers PamF: 5′ TTAATCTTGGAATTCATTAAACACATT 3′ and PamR: 5′ TTAAAGCTTAGGTTACAATAGTATATTCT 3′. Using EcoRI and HinDIII restriction sites incorporated in the primers, the product was directionally cloned downstream of an arabinose-inducible promoter in the pBAD30 plasmid [32] to create the pBADpam expression construct. Pam expression in E. coli EC100 containing pBADpam was induced by addition of 0.2% (w/v) L-arabinose overnight, and E. coli EC100 carrying pBAD30 empty vector was used as control. Cloned P. asymbiotica ATCC43949 pam in pET-28α (Novagen, USA) and expressed in E. coli BL21 (DE3) (Novagen, USA) was used for the feeding assays, and compared to E. coli EC100 carrying pET-28α as control.

In this study, a Sb-doped ZnO microrod array was successfully grown on an Al-doped n-type ZnO thin film by electrodeposition. Strong violet luminescence, originated from free electron-to-acceptor level transitions, was identified by temperature-dependent learn more photoluminescence measurements. This acceptor-related transition was attributed to substitution of Sb dopants for Zn sites, instead of O sites, to form a complex with two Zn vacancies (VZn), the SbZn-2VZn complex. This

SbZn-2VZn complex has a lower formation energy and acts as a shallow acceptor which can induce a strong violet luminescence. ZnO homojunction diode made with Sb-doped ZnO and Al-doped ZnO exhibits the expected p-n diode characteristic on the current-voltage (I-V) measurement and confirms that the Sb-doped ZnO microrod array is p-type and can be

fabricated successfully using the electrodeposition method. Finally, the photoresponse of the ZnO p-n diode operating at an increased PD-1/PD-L1 inhibitor reverse bias shows a good optical response and high photocurrent gain, indicating that it can be a good candidate for use as an ultraviolet photodetector. Methods A Sb-doped ZnO microrod array was electrodeposited on a patterned Al-doped n-type ZnO thin film at 98°C for 1 h [15]. The array pattern was fabricated on the ZnO thin film by optical lithography method. The reaction solution for the growth of the microrod

array was a mixture of 0.05 M zinc nitrate (Zn(NO3)2·6H2O), 0.05 M hexamethylenetetramine (C6H12N4), and 0.05 g antimony acetate (Sb(CH3COO)3). The conditions for electrodeposition were optimized at I = 10 mA and V = 3.1 V. Ohmic contacts on the n-type ZnO thin film and on the Sb-doped ZnO microrod array were fabricated by depositing aluminum and gold antimony (AuSb), respectively, for the electrical characterization. The surface morphology and the crystalline structure of the sample were examined using a 5-FU scanning electron microscope (SEM; HITACHI S-2400, Chiyoda-ku, Japan) and by X-ray diffraction (XRD; PANalytical X’Pert PRO, Almelo, The Netherlands), respectively. The chemical and elemental identification on the surface was carried out by X-ray photoelectron spectroscopy (XPS). A He-Cd laser at the wavelength of 325 nm was used for the photoluminescence (PL) measurement. Keithley 236 and 4200-SCS (Cleveland, OH, USA) were used for the characteristic I-V measurement. To study the photoresponse of our ZnO homojunction device, we employed a xenon arc lamp (LHX150 08002, Glasgow, UK) as a variable-wavelength light source. The monochromatic light was selected using an iHR-320 monochromator (HORIBA Scientific, Albany, NY, USA) and irradiated on the sample. The beam line of 365 nm was selected in the photocurrent measurement.

Of the 163 genes that encode for various parts of the amino acid transport and metabolism, the PM upregulated a significant number of genes (20 and 37 genes) compared to the WT in standard and Populus hydrolysate media. Most significantly, the PM increased the expression of 10 of the 15 genes along the histidine metabolism pathway compared to the WT

in standard medium (Table 4). Cthe_2880-Cthe_2889 is a single operon and is among the most highly differentially expressed genes in the PM versus WT comparison, with an Luminespib in vivo average 23-fold to 31-fold increase in expression in standard and Populus hydrolysate media. The PM decreases the expression of one gene in this pathway, Cthe_3028 which converts histidine to histamine (Figure 3). De novo biosynthesis of histidine during fermentation may be constrained by the high NADH/NAD+ ratio during anaerobic growth and the requirement for further

reduction of NAD+ in Citarinostat datasheet the two terminal steps of biosynthesis [17]. Histidine may be limited by the addition of furfural [17]. The PM has two mutations involved with glutamate catabolism; a possible gain in function in argD (Cthe_1866, E55G) and a possible loss in function in proB (Cthe_1766, A149T) [17]. These two mutations seem to be a beneficial shift from proline production to glutamate and arginine production in PM [17,18,32]. The shift in amino acid production may also assist in the increased expression in the histidine pathway since glutamate is utilized in the pathway. The PM also significantly increases the expression of 6 of the 18 genes belonging to valine, leucine and isoleucine biosynthesis, which may help balance carbon and electron flow. An increase in amino acid production can also help overcome weak acid stress [17,18,33]. Table

4 Fold change in gene expression in histidine metabolism pathways Gene Product PM vs. WT 0 PM vs. WT 10 PM 0 vs. 10 PM 0 vs. 17.5 WT 0 vs. 10     ML LL ML LL ML LL ML LL ML LL Cthe_2880 ATP phosphoribosyltransferase regulatory subunit 98.42 29.12 98.72 80.51 1.25 1.01 1.12 −1.06 1.25 −2.73 Cthe_2881 ATP phosphoribosyltransferase 78.48 23.79 85.06 Montelukast Sodium 100.15 1.64 1.24 1.35 −1.01 1.52 −3.40 Cthe_2882 histidinol dehydrogenase 35.86 18.44 28.45 44.69 1.49 1.33 1.37 1.40 1.88 −1.83 Cthe_2883 histidinol-phosphate aminotransferase 38.12 19.61 23.12 40.22 1.15 1.22 1.19 1.42 1.89 −1.69 Cthe_2884 Imidazoleglycerol-phosphate dehydratase 7.45 7.71 17.31 17.09 1.23 1.25 1.18 1.27 −1.89 −1.77 Cthe_2886 Imidazole glycerol phosphate synthase subunit hisH 11.99 12.29 14.84 15.87 1.19 1.12 1.09 −1.01 −1.04 −1.16 Cthe_2887 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase 13.46 11.01 10.02 14.54 1.44 1.20 1.29 1.13 1.93 −1.10 Cthe_2888 Imidazole glycerol phosphate synthase subunit hisF 12.46 14.23 10.04 18.19 1.61 1.30 1.54 1.24 1.99 1.

Wiley-Interscience,

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BSR-T7/5 cells (a cell line derived from BHK-21, which constitutively expresses T7

RNA polymerase [44]) were maintained in Glasgow minimal essential medium (GMEM) supplemented with 4% tryptose phosphate broth, 10% fetal bovine serum (FBS) and were additionally provided with G418 (1 mg mL-1) on every second passage to ensure maintenance of the T7 polymerase gene. BHK-21 cells were grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% FBS. RNA extraction, RT-PCR and nucleotide sequencing RNA was extracted from virus stock of Asia1/JSp1c8, Asia1/JSM4, and Asia1/JS/China/2005 using RNeasy mini kit (Qiagen, Valencia, CA) according to the VRT752271 purchase manufacturer’s instructions. Viral cDNAs were synthesized from the viral RNAs, as previously described [45]. Briefly, viral cDNAs were synthesized using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA)

with NK61 primer (5′-GACATGTCCTCCTGCATCTG-3′) and the VP1 coding regions were amplified by PCRs with the primer pair NK61/VP31 (5′-TAGTGCTGGYAARGACTTTG-3′). The PCRs were performed using PrimeSTAR HS DNA Polymerase (Takara, Dalian, China). PCR amplifications were carried out for 30 cycles of denaturation at 98°C for 20 s, annealing at 68°C for 1 min, and extension at 72°C for 8 min. Following amplification, the cDNA fragments were purified from agarose gels using a kit (Qiagen) and sequenced YH25448 nmr by Sunny Biotech (Shanghai, China). In order to detect heterogeneity of the VP1 gene,

the amplicons were cloned into a pGEM-T vector (Promega, Madison, WI, USA) using standard molecular cloning techniques [46] and plasmids derived from 10 positive clones for each sample were sequenced. Additionally, the capsid-encoding regions of Asia1/JSp1c8, Asia1/JSM4, and Asia1/JS/CHA/05 were also amplified and sequenced. Construction of genome-length Tyrosine-protein kinase BLK cDNA clone of Asia1/JSp1c8 and derivation of G-H loop VP1 mutants Recombinant DNA techniques were used according to standard procedures [46]. The viral RNA of Asia1/JSp1c8 was used as a template for first-strand cDNA synthesis with M-MLV reverse transcriptase by using specific oligonucleotide primers (E1′, E2′, E3′, E4′, and E5′). A total of five fragments (E1-E5; Figure 5), covering the complete virus genome, were subsequently amplified by PCR. Two fragments (E1 and E2 corresponding to nucleotide 1-390, 362-700) were amplified with the E1/E1′ and E2/E2′ primer pairs by PCR. T7 RNA polymerase promoter was introduced in the E1 primer. Cycling conditions for both PCRs were as follows: initial denaturation at 94°C for 1 min, 30 cycles of 98°C for 20 s, 68°C for 40 s, and then 72°C for 8 min. E12 fragments were generated by overlap PCR fusion E1 and E2 fragments with primer pair E1/E2′. PCR amplifications involved initial denaturation at 94°C for 1 min, followed by 30 cycles of 98°C for 20 s, 68°C for 1 min, then 72°C for 8 min.