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Purpose: To establish a highly sensitive and specific approach for the detection of circulating PIK3CA H1047R mutation in breast cancer (BC) patients and to investigate the association between the prevalence of PIK3CA H1047R mutation and clinical presentations., Methods: A proper blocker was designed in an allele-specific manner and optimized for PCR-based identification of the PIK3CA H1047R mutation. The established technique was validated in cell-free DNA samples from 196 recruited BC patients., Results: The allele-specific PCR assay with a properly designed blocker was able to detect the H1047R mutant variant with 0.01%. By applying the newly established assay, 62 cases (31.6% of the total recruited cases) were found to carry a blood-circulating H1047R mutant. Wherein, the detected mutant rates increased with disease stages from 2/18 (11.1%) of stage I to 17/71 (23.9%) of stage II, 20/53 (37.7%) of stage III, and 23/31 (42.6%) of stage IV (p = 0.025), respectively. Higher frequencies of H1047R mutation were associated with late-stage (p = 0.033) or recurrence (p = 0.045) or metastatic patients (p = 0.049) as well as radiation-treated human epidermal growth factor receptor 2 (HER2) positive BC (p = 0.004). PIK3CA mutant carriers were frequently observed in patients under the age of 50 who had liver-metastasized or brain metastases or lymph node-invaded (p < 0.05)., Conclusion: A novel allele-specific PCR assay with high sensitivity was established successfully for the detection of the PIK3CA H1047R mutation in clinical practice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Thao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke., Methods: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary., Results: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains., Conclusions: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Honey is a nutritious food made by bees from nectar and sweet deposits of flowering plants and has been used for centuries as a natural remedy for wound healing and other bacterial infections due to its antibacterial properties. Honey contains a diverse community of bacteria, especially probiotic bacteria, that greatly affect the health of bees and their consumers. Therefore, understanding the microorganisms in honey can help to ensure the quality of honey and lead to the identification of potential probiotic bacteria., Methods: Herein, the bacteria community in honey produced by Apis cerana was investigated by applying the next-generation sequencing (NGS) method for the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. In addition, lactic acid bacteria (LAB) in the honey sample were also isolated and screened for in vitro antimicrobial activity., Results: The results showed that the microbiota of A. cerana honey consisted of two major bacterial phyla, Firmicutes (50%; Clostridia , 48.2%) and Proteobacteria (49%; Gammaproteobacteria , 47.7%). Among the 67 identified bacterial genera, the three most predominant genera were beneficial obligate anaerobic bacteria, Lachnospiraceae (48.14%), followed by Gilliamella (26.80%), and Enterobacter (10.16%). Remarkably, among the identified LAB, Lactobacillus kunkeei was found to be the most abundant species. Interestingly, the isolated L. kunkeei strains exhibited antimicrobial activity against some pathogenic bacteria in honeybees, including Klebsiella spp ., Escherichia coli , Enterococcus faecalis , Pseudomonas aeruginosa and Staphylococcus aureus . This underscores the potential candidacy of L. kunkeei for developing probiotics for medical use. Taken together, our results provided new insights into the microbiota community in the A. cerana honey in Hanoi, Vietnam, highlighting evidence that honey can be an unexplored source for isolating bacterial strains with potential probiotic applications in honeybees and humans., Competing Interests: The authors declare there are no competing interests., (©2024 Lanh et al.)

Introduction: In the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates., Methods: The profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination., Results: The number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the bla KPC-2 , bla NDM-5 , bla OXA-1 , bla OXA-48 , and bla OXA-181 β-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the β-lactamase gene clusters in plasmid contigs that carried the same AMR genes., Discussion: Our study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nguyen, Le, Nguyen, Nguyen, Dong, Hayer, Bañuls and Tran.)

Honeybees play a vital role in the ecological environment and agricultural economy. Increasing evidence shows that the gut microbiome greatly influences the host's health. Therefore, a thorough understanding of gut bacteria composition can lead to the development of probiotics specific for each development stage of honeybees. In this study, the gut microbiota at different developmental stages (larvae, pupae, and adults) of the honeybees Apis cerana in Hanoi, Vietnam, was assessed by sequencing the V3-V4 region in the 16S rRNA gene using the Illumina Miseq platform. The results indicated that the richness and diversity of the gut microbiota varied over the investigated stages of A. cenara . All three bee groups showed relative abundance at both phylum and family levels. In larvae, Firmicutes were the most predominant (81.55%); however, they decreased significantly along with the bee development (33.7% in pupae and 10.3% in adults) in favor of Proteobacteria . In the gut of adult bees, four of five core bacteria were found, including Gilliamella apicola group (34.01%) Bifidobacterium asteroides group (10.3%), Lactobacillus Firm-4 (2%), and Lactobacillus Firm-5 (1%). In contrast, pupae and larvae lacked almost all core bacteria except G. apicola (4.13%) in pupae and Lactobacillus Firm-5 (4.04%) in larvae. This is the first report on the gut microbiota community at different developmental stages of A. cerana in Vietnam and provides potential probiotic species for beekeeping., Competing Interests: The authors declare no conflict of interest.

Background: Porcine epidemic diarrhea virus (PEDV) is a dangerous virus causing large piglet losses. PEDV spread rapidly between pig farms and caused the death of up to 90% of infected piglets. Current vaccines are only partially effective in providing immunity to suckling due to the rapid dissemination and ongoing evolution of PEDV., Methods: In this study, the complete genome of a PEDV strain in Vietnam 2018 (IBT/VN/2018 strain) has been sequenced. The nucleotide sequence of each fragment was assembled to build a continuous complete sequence using the DNASTAR program. The complete nucleotide sequences and amino acid sequences of S, N, and ORF3 genes were aligned and analyzed to detect the mutations., Results: The full-length genome was determined with 28,031 nucleotides in length which consisted of the 5'UTR, ORF1ab, S protein, ORF3, E protein, M protein, N protein, and 3'UTR region. The phylogenetic analysis showed that the IBT/VN/2018 strain was highly virulent belonged to the G2b subgroup along with the Northern American and Asian S-INDEL strains. Multiple sequence alignment of deduced amino acids revealed numerous mutations in the S, N, and ORF3 regions including one substitution 766 P > L 766 in the epitope SS6; two in the S 0 subdomain ( 135 DN 136 > 135 SI 136 and N 144 > D 144 ); two in subdomain S HR1 at aa 1009 L > M 1009 and 1089 S > L 1089 ; one at aa 1279 P > S 1279 in subdomain S HR2 of the S protein; two at aa 364 N > I 364 and 378 N > S 378 in the N protein; four at aa 25 L > S 25 , 70 I > V 70 , 107 C > F 107 , and 168 D > N 168 in the ORF3 protein. We identified two insertions (at aa 59 NQGV 62 and aa 145 N) and one deletion (at aa 168 DI 169 ) in S protein. Remarkable, eight amino acid substitutions ( 294 I > M 294 , 318 A > S 318 , 335 V > I 335 , 361 A > T 361 , 497 R > T 497 , 501 SH 502 > 501 IY 502 , 506 I > T 506 , 682 V > I 682 , and 777 P > L 777 ) were found in S A subdomain. Besides, N- and O-glycosylation analysis of S, N, and ORF3 protein reveals three known sites (25 G+ , 123 N+ , and 62 V+ ) and three novel sites (144 D+ , 1009 M+ , and 1279 L+ ) in the IBT/VN/2018 strain compared with the vaccine strains. Taken together, the results showed that mutations in the S, N, and ORF3 genes can affect receptor specificity, viral pathogenicity, and the ability to evade the host immune system of the IBT/VN/2018 strain. Our results highlight the importance of molecular characterization of field strains of PEDV for the development of an effective vaccine to control PEDV infections in Vietnam., Competing Interests: The authors declare there are no competing interests., (©2021 Tran et al.)

Vibriosis in farmed animals is a serious threat to aquaculture worldwide. Using probiotics and anti- Vibrio antimicrobial substances in aquaculture systems can be a means of preventing Vibrio infections. Therefore, we aimed to characterize and compare 16 potential anti- Vibrio probiotics (Vi+) isolated from marine sponges and fish intestines collected from the Vietnam Sea, as well as an anti- Vibrio bacteriocin to fully explore their application potentials. 16S rRNA sequencing confirmed all Vi+ to be Bacillus species with different strain variants across two sample types. An obvious antimicrobial spectrum toward Gram-negative bacteria was observed from intestinal Vi+ compared to sponge-associated Vi+. The reason was the higher gene frequency of two antimicrobial compounds, non-ribosomal peptides (NRPS) and polyketide type-I (PKS-I) from intestinal Vi+ (66.7%) than sponge-associated Vi+ (14.3% and 0%, respectively). Additionally, a three-step procedure was performed to purify an anti- Vibrio bacteriocin produced by B. methylotrophicus NTBD1, including (i) solvent extraction of bacteriocin from cells, (ii) hydrophobic interaction chromatography, and (iii) reverse-phase HPLC. The bacteriocin had a molecular weight of ~2-5 kDa, was sensitive to proteolysis and thermally stable, and showed a broad antimicrobial spectrum, all of which are essential properties for promising feed additives. This study provides necessary information of the potential of probiotic Bacillus species with anti- Vibrio antimicrobial properties to study their further use in sustainable aquaculture.

Background: Deformed wing virus (DWV) is a virulent virus that causes honeybee disease. DWV can exist as a latent infection in honeybees, outbreak into epidemics, and cause serious damage to beekeeping cross the world, including Vietnam., Methods: The two DWV strains circulating in Vietnamese honeybee, Apis cerana, were first isolated from adult honeybees in North Vietnam (DWV-NVN) and South Vietnam (DWV-SVN). Their complete nucleotide sequences were determined, aligned, and compared with other DWV strains ., Results: The two Vietnamese DWV strains comprised 10,113 bp and contained a large single open reading frame (ORF) of 2,893 amino acids, initiating at nucleotide 1,130 and terminating at nucleotide 9,812. Multiple nucleotide sequence alignment between these two DWV-VN strains and DWV strains in A. mellifera was performed. The DWV-VN strains showed a low genetic identity (from 91.4% to 92.0%) with almost of these strains, but lower identities (89.2% and 89.4%) with UK2 and (89.6%) with the China2 strain. Low identities (91.7% and 91.9%) were also observed between the China3 strain (in A. cerana ) and the DWV-VN strains, respectively. The deduced amino acid sequence alignment showed high genetic similarities (97.0%-97.9%) when the USA1, Chile, Italy1, France, UK1, UK2, Japan, Korea2, China1, China2 and China3 strains were compared to the DWV-VN strains. This ratio was 96.7% and 96.8% when the Korea1 strain was compared to the DWV-SVN and DWV-NVN strains, respectively. Numerous amino acid substitutions were identified in the L, VP3, and RdRp sequences. Notably, we observed six substitutions positioned at amino acids 27 (E > I), 98 (S > T), 120 (A > V), 153 (M > T), 170 (D > F), and 174 (Y > F) in the L protein, two amino acid changes at positions 980 (S > A) and 1032 (E > T) in VP3, and one amino acid change at position 2627 (R > C) unique to the DWV-VN strains. Phylogenetic analysis based on complete genome sequences, RdRp sequences and Simplot analysis indicated that there was a significant difference between DWV-VN strains in A. cerana and DWV strains in A. mellifera . The results suggested that the genetic variations of the DWV-VN strains in A. cerana help them to adapt geographical conditions and may lead to change the viral pathogenicity of DWV-VN strains., Competing Interests: The authors declare there are no competing interests., (©2020 Thu et al.)

Introduction: Left ventricular dysfunction is quite common in septic shock. Speckle-tracking echocardiography (STE) is a novel, highly sensitive method for assessing left ventricular function, capable of detecting subclinical myocardial dysfunction, which is not identified with conventional echocardiography. We sought to evaluate subclinical left ventricular systolic function in patients with septic shock using speckle-tracking echocardiography., Methods: From May 2017 to December 2018, patients aged ≥18 years admitted to the intensive care unit with the diagnosis of sepsis and septic shock based on the sepsis-3 definition were included. Patients with other causes of cardiac dysfunction were excluded. Transthoracic echocardiography was performed for all the patients within 24 hours of diagnosis. Left ventricular systolic function was assessed using conventional echocardiography and speckle-tracking echocardiography., Results: Patients with septic shock ( n  = 90) (study group) and 37 matched patients with sepsis but no septic shock (control group) were included. Left ventricular ejection fraction (LVEF) by conventional echocardiography showed no significant difference between two groups (58.2 ± 9.9 vs. 58.6 ± 8.3, p =0.804). The global longitudinal strain (GLS) by STE was significantly reduced in patients with septic shock compared with that in the control (-14.6 ± 3.3 vs. -17.1 ± 3.3, p < 0.001). Based on the cutoff value of GLS ≥ -15% for the definition of subclinical left ventricular systolic dysfunction, this dysfunction was detected in 50 patients with septic shock (55.6%) and in 6 patients in the control group (16.2%) ( p < 0.05)., Conclusions: Speckle-tracking echocardiography can detect early subclinical left ventricular systolic dysfunction via the left ventricular global longitudinal strain compared with conventional echocardiographic parameters in patients with septic shock., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Pham Dang Hai et al.)

Background: Neisseria meningitidis remains the main cause of sporadic meningitis and sepsis in military camps in Vietnam. Yet, very limited molecular data of their genotypic and epidemiological characteristics are available from Vietnam, and particularly the military environment. Whole genome sequencing (WGS) has proven useful for meningococcal disease surveillance and guiding preventative vaccination programs. Previously, we characterized key genetic and epidemiological features of an invasive N. meningitidis B isolate from a military unit in Vietnam. Here, we extend these findings by sequencing two additional invasive N. meningitidis B isolated from cerebrospinal fluid (CSF) of two meningitis cases at another military unit and compared their genomic sequences and features. We also report the sequence types and antigenic profiles of 25 historical and more recently emerged N. meningitidis isolates from these units and other units in proximity., Methods: Strains were sequenced using the Illumina HiSeq platform, de novo assembled and annotated. Genomes were compared within and between military units, as well as against the global N. meningitidis collection and other isolates from the Southeast Asia region using PubMLST. Variations at the nucleotide level were determined, and phylogenetic relationships were estimated. Antigenic genotypes and vaccine coverage were analyzed using gMATS and PubMLST. Susceptibility of isolates against commonly used antibiotic agents was examined using E-test., Results: Genome comparison revealed a high level of similarity among isolates both within and between units. All isolates showed resistance to chloramphenicol and carried identical catP gene with other Southeast Asian isolates, suggesting a common lineage. Their antigenic genotypes predicted no coverage by either Bexsero ® or Trumenba ® , and nucleotide variation analysis revealed diverse new, unassigned alleles at multiple virulence loci of all strains. Groups of singleton and unique novel sequence types extending beyond individual camps were found from epidemiological data of 25 other isolates. Our results add to the sparse published molecular data of N. meningitidis in the military units in Vietnam, highlight their diversity, distinct genetic features and antibiotic resistance pattern, and emphasize the need for further studies on the molecular characteristics of N. meningitidis in Vietnam., Competing Interests: The authors declare there are no competing interests., (©2020 Le et al.)

The isolation, identification and characterization of a novel huperzine A (HupA)-producing fungal strain Rsp5.2 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in Vietnam. The fifty-eight endophytic fungi were recovered from roots of natural H. serrata in Lao Cai province of northern Vietnam and screened for HupA-producing by thin-layer chromatography (TLC). The only one of the 58 strains, Rsp5.2, could produce HupA. The amount of HupA produced by Rsp5.2 was quantified to be 19.45 µg g -1 dried mycelium by high-performance liquid chromatography (HPLC). Acetylcholine esterase (AchE) inhibition (IC 50 ) of the crude HupA extract from Rsp5.2 fermentation broth was 2.849 ± 0.0026 µg mL -1 . The fungus was identified as Fusarium sp. Rsp5.2 by morphological characteristics and Internal Transcribed Spacer (ITS) sequences. This is the first report of Fusarium sp. as a HupA-producing endophyte isolated from H. serrata.

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig-fertilization medium was superior medium to Tyrode's albumin lactate pyruvate-polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 10 6  sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus-oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig-fertilization medium from 2 to 5 mmol/L and the prolongation of the co-culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig-fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility., (© 2018 Japanese Society of Animal Science.)

Sacbrood virus (SBV) is one of the most common viral infections of honeybees. The entire genome sequence for nine SBV infecting honeybees, Apis cerana and Apis mellifera, in Vietnam, namely AcSBV-Viet1, AcSBV-Viet2, AcSBV-Viet3, AmSBV-Viet4, AcSBV-Viet5, AmSBV-Viet6, AcSBV-Viet7, AcSBV-Viet8, and AcSBV-Viet9, was determined. These sequences were aligned with seven previously reported complete genome sequences of SBV from other countries, and various genomic regions were compared. The Vietnamese SBVs (VN-SBVs) shared 91-99% identity with each other, and shared 89-94% identity with strains from other countries. The open reading frames (ORFs) of the VN-SBV genomes differed greatly from those of SBVs from other countries, especially in their VP1 sequences. The AmSBV-Viet6 and AcSBV-Viet9 genome encodes 17 more amino acids within this region than the other VN-SBVs. In a phylogenetic analysis, the strains AmSBV-Viet4, AcSBV-Viet2, and AcSBV-Viet3 were clustered in group with AmSBV-UK, AmSBV-Kor21, and AmSBV-Kor19 strains. Whereas, the strains AmSBV-Viet6 and AcSBV-Viet7 clustered separately with the AcSBV strains from Korea and AcSBV-VietSBM2. And the strains AcSBV-Viet8, AcSBV-Viet1, AcSBV-Viet5, and AcSBV-Viet9 clustered with the AcSBV-India, AcSBV-Kor and AcSBV-VietSBM2. In a Simplot graph, the VN-SBVs diverged stronger in their ORF regions than in their 5' or 3' untranslated regions. The VN-SBVs possess genetic characteristics which are more similar to the Asian AcSBV strains than to AmSBV-UK strain. Taken together, our data indicate that host specificity, geographic distance, and viral cross-infections between different bee species may explain the genetic diversity among the VN-SBVs in A. cerana and A. mellifera and other SBV strains., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

This article reports on the ability of yeast Trichosporon asahii B1 biofilm-associated cells, compared with that of planktonic cells, to transform sec-hexylbenzene and its metabolites. This B1 strain was isolated from a petroleum-polluted sediment collected in the QuangNinh coastal zones in Vietnam, and it can transform the branched aromatic hydrocarbons into a type of forming biofilm (pellicle) more efficiency than that the planktonic forms can. In the biofilm cultivation, seven metabolites, including acetophenone, benzoic acid, 2,3-dihydroxybenzoic acid, β-methylcinnamic acid, 2-phenylpropionic acid, 3-phenylbutyric acid, and 5-phenylhexanoic acid were extracted by ethyl acetate and analyzed by HPLC and GC-MS. In contrast, in the planktonic cultivation, only three of these intermediates were found. An individual metabolite was independently used as an initial substrate to prove its degradation by biofilm and planktonic types. The degradation of these products indicated that their inoculation with B1 biofilms was indeed higher than that observed in their inoculation with B1 planktonic cells. This is the first report on the degradation of sec-hexylbenzene and its metabolites by a biofilm-forming Trichosporon asahii strain. These results enhance our understanding of the degradation of branched-side-chain alkylbenzenes by T. asahii B1 biofilms and give a new insight into the potential role of biofilms formed by such species in the bioremediation of other recalcitrant aromatic compounds.

Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNA-based PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4-100%. Two of the four genotypes had high similarity (99.8-100%) with known sequences. The other two genotypes have never been identified., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.

To understand the role of human 15-lipoxygenase 1 (15-LOX1) in vascular wall remodeling, we have studied the effect of the major 15-LOX1 metabolite of arachidonic acid, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), on vascular smooth muscle cell (VSMC) migration both in vitro and in vivo. Among 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 15(S)-HETE potentially stimulated more vascular smooth muscle cell (VSMC) migration. In addition, 15(S)-HETE-induced VSMC migration was dependent on Src-mediated activation of signal transducer and activator of transcription-3 (STAT-3). 15(S)-HETE also induced monocyte chemoattractant protein-1 (MCP-1) expression via Src-STAT-3 signaling, and neutralizing anti-MCP-1 antibodies completely negated 15(S)-HETE-induced VSMC migration. Cloning and characterization of a 2.6-kb MCP-1 promoter revealed the presence of four putative STAT-binding sites, and the site that is proximal to the transcription start site was found to be essential for 15(S)-HETE-induced Src-STAT-3-mediated MCP-1 expression. Rat carotid arteries that were subjected to balloon injury and transduced with Ad-15-LOX1 upon exposure to [(3)H]arachidonic acid ex vivo produced 15-HETE as a major eicosanoid and enhanced balloon injury-induced expression of MCP-1 in smooth muscle cells in Src and STAT-3-dependent manner in vivo. Adenovirus-mediated delivery of 15-LOX1 into rat carotid artery also led to recruitment and homing of macrophages to medial region in response to injury. In addition, transduction of Ad-15-LOX1 into arteries enhanced balloon injury-induced smooth muscle cell migration from media to intima and neointima formation. These results show for the first time that 15-LOX1-15(S)-HETE axis plays a major role in vascular wall remodeling after balloon angioplasty.

Gerbils (Meriones unguiculatus) were inoculated intraperitoneally (i.p.) with Neospora caninum tachyzoites to examine parasite distribution and histological lesions at different time points over a 9-day period of infection. Gerbils were sacrificed 12 h post-infection (PI), then daily intervals up to day 9 PI. The parasite was detected by PCR assay targeting the Nc5 sequence of N. caninum. The parasite was not found in any organs until day 5 PI, however, from day 8 PI onwards, they were detected in all the organs examined as demonstrated by PCR. The first target organs in acute N. caninum infection were liver, spleen, and kidney, but not the blood as was expected. Histologic lesions were detected in the liver and spleen only, no lesions were found in other organs examined until the end of the experiment. Notably, the focal miliary hepatitis was observed in the liver of infected gerbils just after 1 day post-inoculation, whereas splenic lesions were not found until day 5 PI. These results reinforce the applicability of gerbils as a suitable model of acute neosporosis and provide new insights into the response of gerbils to N. caninum intraperitoneal infection.

Interconversion between tachyzoites and bradyzoites of Neospora caninum plays a pivotal role in transmission of the parasite. Although significant efforts have been made toward understanding the mechanisms that trigger and control stage conversion of the parasite, little is known about this process. We used annealing control primer (ACP)-based polymerase chain reaction (PCR) technique to characterize the differences in transcription between tachyzoites and bradyzoites of N. caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by treatment of infected cultures with 70 microM sodium nitroprusside. Subsequently, the gene expression profiles of the tachyzoites and bradyzoites were analyzed through comparison of the level of messenger RNA expression. ACP-based PCR revealed 85 amplicons that were consistently differentially expressed between tachyzoite and bradyzoite stages. Of the 85 differentially expressed transcripts identified, ten were cloned into Topo TA cloning vector, sequenced, and further analyzed by the Basic Local Alignment Search Tool. These differentially expressed transcripts include a combination of known genes and as yet unidentified genes. The present work provides candidate genes for further investigation on molecular basis of stage conversion from tachyzoites to bradyzoites of N. caninum.

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Z alpha domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Z alpha domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Z alpha domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Z alpha domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Z alpha domains bind Z-DNA tightly. Ability of Z alpha(E3L) converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Z alphas can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the beta-wing play an important role in this stabilization. Notably the Z alpha domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zalpha proteins.