Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Relationship between Penicillin-Binding Proteins Alterations and ß-Lactams Non-Susceptibility of Diseased Pig-Isolated Streptococcus suis.

Streptococcus suis is a zoonotic pathogen causing disease in both animals and humans, and the emergence of increasingly resistant bacteria to antimicrobial agents has become a significant challenge globally. The objective of this study was to investigate the genetic basis for declining susceptibility to penicillin and other ß-lactams among S. suis. Antimicrobial susceptibility testing and penicillin-binding proteins (PBP1a, PBP2a, PBP2b, and PBP2x) sequence analysis were performed on 225 S. suis isolated from diseased pigs. This study found that a growing trend of isolates displayed reduced susceptibility to ß-lactams including penicillin, ampicillin, amoxicillin/clavulanic acid, and cephalosporins. A total of 342 substitutions within the transpeptidase domain of four PBPs were identified, of which 18 substitutions were most statistically associated with reduced ß-lactams susceptibility. Almost all the S. suis isolates which exhibited penicillin-non-susceptible phenotype (71.9%) had single nucleotide polymorphisms, leading to alterations of PBP1a (P409T) and PBP2a (T584A and H588Y). The isolates may manifest a higher level of penicillin resistance by additional mutation of M341I in the 339STMK active site motif of PBP2x. The ampicillin-non-susceptible isolates shared the mutations in PBP1a (P409T) and PBP2a (T584A and H588Y) with additional alterations of PBP2b (T625R) and PBP2x (T467S). The substitutions, including PBP1a (M587S/T), PBP2a (M433T), PBP2b (I428L), and PBP2x (Q405E/K/L), appeared to play significant roles in mediating the reduction in amoxicillin/clavulanic acid susceptibility. Among the cephalosporins, specific mutations strongly associated with the decrease in cephalosporins susceptibility were observed for ceftiofur: PBP1a (S477D/G), PBP2a (E549Q and A568S), PBP2b (T625R), and PBP2x (Q453H). It is concluded that there was genetically widespread presence of PBPs substitutions associated with reduced susceptibility to ß-lactam antibiotics.

Differential distribution of eicosanoids and polyunsaturated fatty acids in the Penaeus monodon male reproductive tract and their effects on total sperm counts.

Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E2 (PGE2) and prostaglandin F2α in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ2, (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE2 in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE2 in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp.

Antimicrobial Susceptibility of Streptococcus suis Isolated from Diseased Pigs in Thailand, 2018-2020.

Streptococcus suis is a porcine and zoonotic pathogen that causes severe systemic infection in humans and pigs. The treatment of S. suis infection relies on antibiotics; however, antimicrobial resistance (AMR) is an urgent global problem, pushing research attention on the surveillance of antibiotic-resistant S. suis to the fore. This study investigated the antimicrobial susceptibility of 246 S. suis strains isolated from diseased pigs in Thailand from 2018-2020. The major sources of S. suis strains were lung and brain tissues. PCR-based serotyping demonstrated that the most abundant serotype was serotype 2 or ½, followed by serotypes 29, 8, 9, and 21. To the best of our knowledge, this is the first report describing the distribution of AMR S. suis serotype 29 in diseased pigs. The antimicrobial susceptibility test was performed to determine the minimum inhibitory concentrations of 35 antimicrobial agents. The results showed that important antimicrobial agents for human use, amoxicillin/clavulanic acid, daptomycin, ertapenem, meropenem, and vancomycin, were the most effective drugs. However, a slight decrease in the number of S. suis strains susceptible to amoxicillin/clavulanic acid and vancomycin raised awareness of the AMR problem in the future. The data indicated a tendency of reduced efficacy of available veterinary medicines, including ampicillin, cefepime, cefotaxime, ceftiofur, ceftriaxone, chloramphenicol, florfenicol, gentamicin, penicillin, and tiamulin, for the treatment of S. suis infection, thus emphasizing the importance of the prudent use of antibiotics. The widespread of multidrug-resistant S. suis strains was identified in all serotypes and from different time periods and different regions of the country, confirming the emergence of the AMR problem in the diseased pig-isolated S. suis population.

Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

A novel bicyclic 2,4-diaminopyrimidine inhibitor of Streptococcus suis dihydrofolate reductase.

Streptococcus suis is a Gram-positive bacterial pathogen of pigs and an emerging zoonotic pathogen. It has become increasingly resistant to multiple classes of antibiotics. New drug candidates and knowledge of their targets are needed to combat antibiotic-resistant S. suis. In this study, the open-source Pathogen Box compound library was screened. Thirty hits that effectively inhibited S. suis growth at 10 µM were identified. Among the most potent hits, MMV675968 (a diaminoquinazoline analog) was shown to target S. suis dihydrofolate reductase (SsDHFR) via (1) growth inhibition of an E. coli surrogate whose growth is dependent on exogenously expressed SsDHFR and (2) inhibition of in vitro SsDHFR activity. Thymidine supplement is able to reverse growth inhibition by MMV675968 in both E. coli surrogate and S. suis, indicating that a thymidine-related pathway is a major target of MMV675968. Comparison of MMV675968 with seven DHFR inhibitors representing different core structures revealed that bicyclic 2,4-diaminopyrimidines with long and flexible side chains are highly effective in inhibiting SsDHFR and S. suis growth. MMV675968 and related compounds thus may serve as starting points for developing antibiotics against drug resistant S. suis.

Genome sequences of antibiotic-resistant Streptococcus suis strains isolated from human patients and diseased and asymptomatic pigs in Thailand.

Streptococcus suis, a zoonotic bacterial pathogen, has negative economic impacts on both intensive swine production and human health worldwide. Whole-genome sequencing and comparative genomic analysis have been widely used for comprehensive classification and investigation of the genetic basis of several S. suis strains obtained from distinct hosts in different geographic areas, revealing great genetic diversity of this zoonotic pathogen. In this study, whole-genome sequences of antibiotic-resistant S. suis strains isolated from human patients (2 strains), diseased pigs (4 strains), and asymptomatic pigs (3 strains) in Thailand were compared with known genomes of 1186 S. suis strains. Single-nucleotide polymorphism-based phylogenetic analysis indicated that the Thai-isolated S. suis strains have close genetic relatedness to S. suis strains isolated from Canada, China, Denmark, Netherlands, United Kingdom, and United States of America. The genome analysis revealed genes conferring antibiotic resistance (aad(6), ant(6)-Ia, ermB, tet(O), patB, and sat4) and gene clusters (aph(3')-IIIa and aac(6')-Ie-aph(2″)-Ia) associated with aminoglycoside, macrolide, and fluoroquinolone resistance in S. suis in Thailand. This work provides additional resources for future genomic epidemiology investigation of S. suis.

Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum.

The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.

KITSUNE: A Tool for Identifying Empirically Optimal K-mer Length for Alignment-Free Phylogenomic Analysis.

Genomic DNA is the best "unique identifier" for organisms. Alignment-free phylogenomic analysis, simple, fast, and efficient method to compare genome sequences, relies on looking at the distribution of small DNA sequence of a particular length, referred to as k-mer. The k-mer approach has been explored as a basis for sequence analysis applications, including assembly, phylogenetic tree inference, and classification. Although this approach is not novel, selecting the appropriate k-mer length to obtain the optimal resolution is rather arbitrary. However, it is a very important parameter for achieving the appropriate resolution for genome/sequence distances to infer biologically meaningful phylogenetic relationships. Thus, there is a need for a systematic approach to identify the appropriate k-mer from whole-genome sequences. We present K-mer-length Iterative Selection for UNbiased Ecophylogenomics (KITSUNE), a tool for assessing the empirically optimal k-mer length of any given set of genomes of interest for phylogenomic analysis via a three-step approach based on (1) cumulative relative entropy (CRE), (2) average number of common features (ACF), and (3) observed common features (OCF). Using KITSUNE, we demonstrated the feasibility and reliability of these measurements to obtain empirically optimal k-mer lengths of 11, 17, and ∼34 from large genome datasets of viruses, bacteria, and fungi, respectively. Moreover, we demonstrated a feature of KITSUNE for accurate species identification for the two de novo assembled bacterial genomes derived from error-prone long-reads sequences, and for a published yeast genome. In addition, KITSUNE was used to identify the shortest species-specific k-mer accurately identifying viruses. KITSUNE is freely available at https://github.com/natapol/kitsune.

Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase.

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6-2.7 µM, IC90 of 0.7-3.9 µM, and MIC of 1.6-10 µM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.

Antimicrobial susceptibility of Streptococcus suis isolated from diseased pigs, asymptomatic pigs, and human patients in Thailand.

BACKGROUND: Prophylaxis and treatment of emerging zoonotic Streptococcus suis infection in agricultural and healthcare settings mainly rely on antibiotics. However, continued use of antibiotics contributing to emergence and widespread of antibiotic resistant S. suis becomes a significant challenge in many endemic countries, including Thailand. Meanwhile, the knowledge of antibiotic susceptibility patterns of bacterial pathogens is required for overcoming the antimicrobial resistance problem, the information of antibiotic susceptibility of S. suis strains isolated in Thailand remains limited. This study aims to assess the susceptibility of Thai-isolated S. suis strains to different antibiotic classes in order to gain an insight into the distribution of antibiotic-resistant patterns of S. suis strains in different regions of Thailand. RESULTS: This study revealed the antimicrobial resistance and multidrug resistance of 262 S. suis strains isolated in different regions of Thailand. Susceptibility testing indicated widespread resistance to macrolides and tetracyclines of S. suis strains in the country. Beta-lactam antibiotic drugs (including cefotaxime and ceftiofur), vancomycin, chloramphenicol, as well as florfenicol were potentially the most effective therapeutic drugs for the treatment of S. suis infection in both pigs and humans. High prevalence of intermediate susceptibility of S. suis isolated from asymptomatic pigs for penicillin G, gentamicin, enrofloxacin, and norfloxacin could be the premise of the emergence of S. suis antibiotic resistance. Resistance was also found in S. suis strains isolated from asymptomatic pigs indicating that they could act as reservoirs of antibiotic resistance genes. CONCLUSIONS: To the best of our knowledge, this is the first report on antimicrobial resistance of a large collection of S. suis strains isolated from pigs and humans in Thailand. It revealed the multidrug resistance of S. suis strains in pigs and humans. The information gained from this study raises an awareness and encourage best practices of appropriate antibiotic drug prescribing and use among human health and agriculture sectors.

Expression and Characterization of Serotype 2 Streptococcus suis Arginine Deiminase.

BACKGROUND: Arginine deiminase (ArcA) has been speculated to facilitate the intracellular survival of Streptococcus suis under acidic conditions. However, the physical and biological properties and function of SS2-ArcA have not yet been elucidated. METHODS: Recombinant SS2-ArcA (rSS2-ArcA) was expressed and purified using Ni-NTA affinity chromatography. Under various pH and temperature conditions, the enzymatic properties of purified rSS2-ArcA and crude native SS2-ArcA were determined. RESULTS: The SS2-arcA-deduced amino acid sequence contained a conserved catalytic triad (Cys399-His273-Glu218). The optimum temperature and pH of 47-kDa rSS2-ArcA and crude native SS2-ArcA were 42°C and pH 7.2. The rSS2-ArcA and crude native SS2-ArcA were stable for 3 h at 4 and 25°C, respectively. The pH stability and dependency tests suggested that rSS2-ArcA and crude native SS2-ArcA were functionally active in acidic conditions. The L-arginine substrate binding affinity (Km) values of rSS2-ArcA (specific activity 16.00 U/mg) and crude native SS2-ArcA (specific activity 0.23 U/mg) were 0.058 and 0.157 mM, respectively. rSS2-ArcA exhibited a weak binding affinity with the common ArcA inhibitors L-canavanine and L-NIO. Furthermore, the partial inactivation of SS2-ArcA significantly impaired the viability and growth of SS2 at pH 4.0, 6.0, and 7.5. CONCLUSIONS: This study profoundly demonstrated the involvement of ArcA enzymatic activity in S. suis survival under acidic conditions.

Loop-Mediated Isothermal Amplification and LFD Combination for Detection of Plasmodium falciparum and Plasmodium vivax.

Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (µL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777-784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.

Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick.

The significant strides made in reducing global malaria burden over the past decades are being threatened by the emergence of multi-drug resistant malaria. Mechanisms of resistance to several classes of antimalarial drugs have been linked to key mutations in the Plasmodium falciparum genes. Pyrimethamine targets the dihydrofolate reductase of the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS), and specific point mutations in the dhfr-ts gene have been assigned to resistant phenotypes. Several molecular methods are available to detect the mutant genotypes including DNA sequencing and PCR-based methods. In this study, we report the development of PfSNP-LAMP to detect nucleotide polymorphism in the dhfr gene associated with N51I mutation and antifolate resistance. The PfSNP-LAMP method was validated with genomic DNA samples and parasite lysates prepared from sensitive and pyrimethamine resistant strains of P. falciparum.

Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax.

Malaria is largely a preventable and curable disease. However, a delay or an inappropriate treatment can result in serious adverse outcomes for patient. Rapid, simple and cost-effective diagnostic tests that can be easily adapted and rapidly scaled-up at the field or community levels are needed. In this study, accelerated detection methods for the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) dihydrofolate reductase-thymidylate synthase were developed based on the loop-mediated isothermal amplification (LAMP) method. The developed methods included the use of species-specific biotinylated primers to amplify LAMP amplicons, which were then hybridized to specific FITC-labeled DNA probes and visualized on a chromatographic lateral flow dipstick (LFD). The total LAMP-LFD assay time was approximately 1.5h. The LAMP-LFD assays showed similar detection limit to conventional PCR assay when performed on plasmid DNA carrying the malaria dhfr-ts genes. The LAMP-LFD showed 10 folds higher detection limit than PCR when performed on genomic DNA samples from Pf and Pv parasites. The dhfr-ts LAMP-LFD assays also have the advantages of reduced assay time and easy format for interpretation of results.

Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases.

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus.

Neuraminidase amino acids 149 and 347 determine the infectivity and oseltamivir sensitivity of pandemic influenza A/H1N1 (2009) and avian influenza A/H5N1.

Pandemic influenza A/H1N1 (2009) and avian influenza A/H5N1 neuraminidase (NA) differ at two critical residues, positions 149 and 347. Recombinant influenza A viruses were constructed in which these two residues in pandemic influenza A/H1N1 (2009) NA were changed to the corresponding amino acids of avian influenza A/H5N1 NA, and vice versa. Recombinant viruses bearing N1 NA with the oseltamivir resistance mutation H274Y in combination with mutations at residues 149 and 347 were also constructed. Recombinant viruses grew normally in allantoic fluid and were subsequently studied for viral infectivity (TCID50), substrate binding (Km) and sensitivity to oseltamivir (Ki). The data demonstrated that infectivity of mutant viruses in Madin Darby canine kidney cells was comparable to, or even greater than, the infectivity of the parental viruses harboring wild-type N1 NA. Furthermore, mutations at NA residues 149 and 347 altered Km and Ki values, and thus modulated oseltamivir sensitivity. Although these mutants have yet to be observed among natural isolates, the minimal costs to the growth of recombinant viruses indicate their possible viability. Reassortment between pandemic influenza A/H1N1 (2009) and avian influenza A/H5N1 viruses may therefore generate new influenza A viruses with increased infectivity and oseltamivir resistance, and continued surveillance will be crucial for public health preparedness.

Enhanced expression of secretable influenza virus neuraminidase in suspension mammalian cells by influenza virus nonstructural protein 1.

Influenza neuraminidase (NA) is a major target for anti-influenza drugs. With an increasing number of viruses resistant to the anti-NA drug oseltamivir, functionally active recombinant NA is needed for screening novel anti-NA compounds. In this study, the secretable NA (sNA) head domain of influenza A/Vietnam/DT-036/05 (H5N1) virus was expressed successfully in human embryonic kidney (HEK-293T) cells and shown to be enzymatically active. The inclusion of a plasmid encoding nonstructural protein 1 (NS1) of influenza A/Puerto Rico/8/34 virus with the sNA plasmid in the cotransfection demonstrated an increase in H5N1 sNA expression by 7.4 fold. Subsequently, the sNA/NS1 cotransfection protocol in serum-free 293-F suspension cell culture was optimized to develop a rapid transient gene expression (TGE) system for expression of large amounts of H5N1 sNA. Under optimized conditions, NS1 enhanced H5N1 sNA expression by 4.2 fold. The resulting H5N1 sNA displayed comparable molecular weight, glycosylation, K(m) for MUNANA, and K(i) for oseltamivir carboxylate to those of H5N1 NA on the virus surface. Taken together, the NS1-enhancing sNA expression strategy presented in this study could be used for rapid high-level expression of enzymatically active H5N1 sNA in suspension mammalian cells. This strategy may be applied for expression of sNA of other strains of influenza virus as well as the other recombinant proteins.

Characterization of band 3-ankyrin-Protein 4.2 complex by biochemical and mass spectrometry approaches.

The elastic property of red blood cell is supported by interaction between red cell membrane and the intricate cytoskeleton network underlying the membrane bilayer cytoplasmic face. One of the major scaffold protein linkers is band 3-ankyrin complex. Defects occurring in this complex have been found in many inherited diseases, causing red blood cell abnormalities. Here we combined the power of mass spectrometry with conventional biochemical purification methods in order to study the native interactions among band 3, ankyrin and Protein 4.2. This approach provided in vivo evidence for the association between band 3 and N-terminal ankyrin purified directly from the cell membrane. The C-terminal regions of ankyrin were not found to be a stable partner of the band 3 complex. Protein 4.2 was shown here to be an integral part of the complex. Its association to the band 3-ankyrin complex could withstand harsh purification conditions. Our findings lend additional support to the interaction between band 3 and ankyrin N-terminal domain previously shown by in vitro binding assays and provide evidence for a band 3 core complex comprising of band 3, ankyrin and Protein 4.2.