Intracellular Growth and Morphological Characteristics of Legionella pneumophila during Invasion and Proliferation in Different Cells.

Various studies have been made to attempt to study the interaction between Legionella pneumophila and the host cells. In this research, we successfully constructed a L. pneumophila mutant strain that stably expressed high levels of green fluorescent protein and used this strain to evaluate the adherence, invasion and proliferation of L. pneumophila in association with several cell lines, including seven cell lines [human macrophage-like cell lines (U937, THP-1), murine macrophage-like cell lines (J774.1A, Raw264.7), human bronchial epithelial cell lines (16HBE, Beas-2B) and human cerrical cancer cell line (HeLa)] which have been used as the host models of L. pneumophila, and two breast carcinoma cell lines (MCF-7 and MDA-MB-231). Our results showed that the two newly tested cell lines are able to support the intracellular proliferation of L. pneumophila, and there were some morphological variations during the invasion and intracellular replication of L. pneumophila in different cell lines. These results can help us find out the common and special patterns of invasion and proliferation of L. pneumophila within different hosts. This is conducive to our knowledge on the relationship and interaction between bacteria and host.

Antibiotic susceptibility of Legionella strains isolated from public water sources in Macau and Guangzhou.

The purpose of this study was to investigate the susceptibility of waterborne strains of Legionella to eight antimicrobials commonly used in legionellosis therapy. The minimum inhibitory concentrations (MICs) of 66 environmental Legionella strains, isolated from fountains and cooling towers of public facilities (hotels, schools, and shopping malls) in Macau and Guangzhou, were tested using the microdilution method in buffered yeast extract broth. The MIC50/MIC90 values for erythromycin, cefotaxime (CTX), doxycycline (DOC), minocycline (MIN), azithromycin, ciprofloxacin, levofloxacin (LEV), and moxifloxacin were 0.125/0.5 mg/L, 4/8 mg/L, 8/16 mg/L, 4/8 mg/L, 0.125/0.5 mg/L, 0.031/0.031 mg/L, 0.031/0.031 mg/L, and 0.031/0.062 mg/L, respectively. Legionella isolates were inhibited by either low concentrations of macrolides and fluoroquinolones, or high concentrations of CTX and tetracycline drugs. LEV was the most effective drug against different Legionella species and serogroups of L. pneumophila isolates. The latter were inhibited in decreasing order by MIN > CTX >DOC, while non-L. pneumophila isolates were inhibited by CTX> MIN >DOC. In this study, we evaluated drug resistance of pathogenic bacteria from the environment. This may help predict the emergence of drug resistance, improve patient outcomes, and reduce hospitalization costs.

Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice.

BACKGROUND: Nanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored. METHODS: Acute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration. RESULTS: Some PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury. CONCLUSIONS: Our data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials.

Chikungunya emergency in China: microevolution and genetic analysis for a local outbreak.

A small-scale local chikungunya outbreak occurred in a Guangdong village of southern China in October 2010. The five chikungunya viruses (CHIKV) isolated from the epidemic and three other imported cases obtained from the same period were sequenced and analyzed for phylogenesis. The results demonstrated that all of the eight sequences were clustered in the Eastern, Central, Southern, and African group. However, the local strains and imported isolates showed different sequence variations. A226V in E1 gene and V264A in E2 gene were detected in all three imported isolates, the unique substitutions S250P in E1 gene and H313Y in E2 genes could be observed in four of the five local strains. These significant variations might be some of the causes for the outbreak. It would be an important event for CHIKV to have mutated adaption to the local mosquitoes in China, Aedes albopictus and Aedes aegypti.

Preparation of lung-targeting, emodin-loaded polylactic acid microspheres and their properties.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) has been identified to have the potential to improve lung fibrosis and lung cancer. To avoid the liver and kidney toxicities and the fast metabolism of emodin, emodin-loaded polylactic acid microspheres (ED-PLA-MS) were prepared and their characteristics were studied. ED-PLA-MS were prepared by the organic phase dispersion-solvent diffusion method. By applying an orthogonal design, our results indicated that the optimal formulation was 12 mg/mL PLA, 0.5% gelatin, and an organic phase:glycerol ratio of 1:20. Using the optimal experimental conditions, the drug loading and encapsulation efficiencies were (19.0±1.8)% and (62.2±2.6)%, respectively. The average particle size was 9.7±0.7 µm. In vitro studies indicated that the ED-PLA-MS demonstrated a well-sustained release efficacy. The microspheres delivered emodin, primarily to the lungs of mice, upon intravenous injection. It was also detected by microscopy that partial lung inflammation was observed in lung tissues and no pathological changes were found in other tissues of the ED-PLA-MS-treated animals. These results suggested that ED-PLA-MS are of potential value in treating lung diseases in animals.

Smart and Sustainable Crop Protection: Design and Evaluation of a Novel α-Amylase-Responsive Nanopesticide for Effective Pest Control.

In this study, an α-amylase-responsive controlled-release formulation was developed by capping polydopamine onto ß-cyclodextrin-modified abamectin-loaded hollow mesoporous silica nanoparticles. The prepared Aba@HMS@CD@PDA were subjected to characterization using various analytical techniques. The findings revealed that Aba@HMS@CD@PDA, featuring a loading rate of 18.8 wt %, displayed noteworthy release behavior of abamectin in the presence of α-amylase. In comparison to abamectin EC, Aba@HMS@CD@PDA displayed a significantly foliar affinity and improved rainfastness on lotus leaves. The results of field trail demonstrated a significantly higher control efficacy against Spodoptera litura Fabricius compared to abamectin EC at all concentrations after 7, 14, and 21 days of spaying, showcasing the remarkable persistence of Aba@HMS@CD@PDA. These results underscore the potential of Aba@HMS@CD@PDA as a novel and persistently effective strategy for sustainable on-demand crop protection. The application of nanopesticides can enhance the effectiveness and efficiency of pesticide utilization, contributing to more sustainable agricultural practices.

Redox and Near-Infrared Light-Responsive Nanoplatform for Enhanced Pesticide Delivery and Pest Control in Rice: Construction, Efficacy, and Potential Mechanisms.

The brown planthopper, Nilaparvata lugens (Stål), is a major rice pest in various Asian countries, causing significant negative impacts on rice yield and quality. In this study, we developed a novel nanoplatform (NIT@MON@CuS) for pesticide delivery that responds to redox and near-infrared light stimuli. The nanoplatform consisted of CuS nanoparticles with mesoporous organic silica (MON), loaded with nitenpyram (NIT). With an average size of 190 nm and a loading efficiency of 22%, NIT@MON@CuS exhibited remarkable thermal response in the near-infrared region, demonstrating excellent photothermal conversion ability and stability. In vitro release kinetics demonstrated the rapid release of nitenpyram under near-infrared light and glutathione conditions, facilitating a satisfactory temperature increase and accelerated drug release. The NIT@MON@CuS-treated group exhibited a higher mortality of N. lugens, increasing from 62 to 88% compared to the group treated with nitenpyram technical after 96 h. Bioassay revealed that NIT@MON@CuS significantly enhanced nitenpyram toxicity by more than 1.4-fold against both laboratory insecticide-resistant and field strains of N. lugens. Furthermore, RT-qPCR results demonstrated that MON@CuS had the capability to reduce P450 gene expression, thereby improving the sensitivity of N. lugens to insecticides. These findings suggest that MON@CuS holds great potential as an intelligent pest control platform, offering a sustainable and efficient approach to protect crops against pests.

Metal-Organic Framework-Based Insecticide and dsRNA Codelivery System for Insecticide Resistance Management.

Targeted silencing of resistance-associated genes by specific double-stranded RNA (dsRNA) is an attractive strategy for overcoming insecticide resistance in insect pests. However, silencing target genes of insect pests by feeding on dsRNA transported via plants remains challenging. Herein, a codelivery system of insecticide and dsRNA is designed by encapsulating imidacloprid and dsNlCYP6ER1 within zeolitic imidazolate framework-8 (ZIF-8) nanoparticles to improve the susceptibility of Nilaparvata lugens (Stål) to imidacloprid. With an average particle size of 195 nm and a positive surface charge, the derived imidacloprid/dsNlCYP6ER1@ZIF-8 demonstrates good monodispersity. Survival curve results showed that the survival rates of N. lugens treated with imidacloprid and imidacloprid@ZIF-8 were 82 and 62%, respectively, whereas, in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group, the survival rate of N. lugens is only 8%. Pot experiments demonstrate that the survival rate in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group was much lower than that in the imidacloprid treatment group, decreasing from 54 to 24%. The identification of NlCYP6ER1 expression and the fluorescence tracking of ZIF-8 demonstrate that ZIF-8 can codeliver dsRNA and insecticide to insects via rice. Safety evaluation results showed that the dsNlCYP6ER1@ZIF-8 nanoparticle had desirable biocompatibility and biosafety to silkworm. This dsRNA and insecticide codelivery system may be extended to additional insecticides with potential resistance problems in the future, greatly enhancing the development of pest resistance management.

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila.

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.

[Adamantane resistance among seasonal influenza A viruses between January to October in Guangzhou, 2009].

OBJECTIVE: To study the prevalence of adamantane-resistance among influenza A viruses isolated from Guangzhou between January and October in 2009, and to provide more information for clinical usage of adamantane drugs. METHODS: Totally 311 influenza A strains isolated from 6 hospitals in Guangzhou between January and October in 2009 were selected, and the MP gene of all 311 strains (159 strains of H1 subtype, 152 strains of H3 subtype) was sequenced. The susceptibility of viruses to rimantadine was assayed by biological methods in cells. RESULT: A hundred and forty-eight strains of influenza A (H1) viruses (93.1%, 148/159) were resistant to the adamantanes, and all the 152 influenza A (H3) viruses were resistant to the adamantanes. An amino acid substitution S31N was found in most of the strains except 1 strain with double mutation V27A/S31N. Furthermore, the M gene of influenza A (H1) viruses was divided into genotype B (human) (97/159) and genotype F (European and Australian birds, 62/159), while the M gene of influenza A (H3) viruses was genotype B (human) (152/152). CONCLUSION: Resistance rate of seasonal influenza A viruses isolated from Guangzhou was high. The MP gene of influenza A (H1) may be replaced by a gene from European and Australian birds through a reassortment event.

[A pathogenic and clinical study of 882 cases of adult influenza-like illness in Guangzhou].

OBJECTIVE: This study was undertaken to describe the viral etiology and clinical features in patients with influenza-like illness (ILI) in Guangzhou. METHODS: The nasopharyngeal and throat swabs were collected from 882 patients presenting with ILI between January and September, 2009. Viral pathogens were cultured and identified by immunofluorescence technique using the Shell-Vial method. The clinical data were statistically analyzed. RESULTS: (1) Viral etiology. Of the 882 samples, 385 (43.7%) were confirmed to have at least one of the 9 different respiratory viruses detected. Among these viral isolates, 67.3% (259/385) were seasonal influenza A virus, 27.8% (107/385) were influenza B virus, and 1.3% (5/385) were human parainfluenza virus (PHIV) 1, 2, or 3. In addition, 2 cases (0.5%) of each adenovirus, HSV-1, enterovirus and respiratory syncytial virus (RSV) were also found in the samples. Co-infections with more than one virus were revealed in 8 (2.1%) of 385 samples tested, among them 6 samples were mixture of influenza A and influenza B, 1 sample was positive for both influenza B virus and HPIV-3, and 1 was for both adenovirus and RSV. Seasonal influenza B virus appeared endemic between March and May, and seasonal influenza A virus became dominant between June and August. (2) Clinical features. The percentage of patients aged from 18-30 years was much higher than that of other age groups. The most common symptoms were moderate fever and sore throat, followed by cough. The percentage of upper respiratory infection and pneumonia was 88.4% (727/882) and 10.7% (95/882) respectively. Clinical features did not discriminate between patients with seasonal influenza A and those with influenza B virus infection. The average numbers of leukocytes and lymphocytes were lower in the group positive for influenza viruses than in virus negative group. The patients with adenovirus, HPIV and RSV infection were significantly younger. No rash was observed in patients with enterovirus or HSV infection. CONCLUSIONS: (1) Seasonal influenza virus was the major viral etiologic agent of ILI in Guangzhou during the first 9 months in 2009. Influenza B and A viruses seasonally prevailed in spring and summer, respectively, while other viral etiologic agents appeared to be sporadic. (2) The analysis of clinical features in patients with ILI indicated that fever was the most common symptom, with body temperature varying greatly, and may be associated with evident respiratory and occasionally systemic symptoms. Among the cases with viral infection, the upper respiratory presentation was universal, and pneumonia was frequently noticed.

Inhibitory effect of emodin on bleomycin-induced pulmonary fibrosis in mice.

1. Currently, there is no satisfactory treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = 16 in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or 20 mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for 21 days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and 20 mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming growth factor-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and 20 mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased fibroblast proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by fibroblasts. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

[Molecular subtyping of Staphylococcus aureus isolated from a severe food-poisoning].

OBJECTIVE: To study the molecular types of Staphylococcus aureus isolated from a severe food-poisoning and to trace the possible strains. METHODS: Real-time PCR was applied to detect nuc gene as a specific marker for S. aureus, mecA gene encoding methicillin resistance and 5 other genes encoding staphylococcal enterotoxins (sea, seb, see, sed, see). Isolates were also performed with 16S rRNA oligonucleotide sequence analyzing by DNAStar MegAlign 5.0 software and pulse-field gel electrophoresis (PFGE) by BioNumerics Version 4.0 software. RESULTS: The nuc gene was detected from the 10 isolated strains, sea and seb genes were detected from 7 strains. There were 4 16 S rRNA types and 5 PFGE types found from all the strains. CONCLUSIONS: Three relative S. aureus strains were involved in the severe food-poisoning at least. Molecular subtyping might give a molecular epidemiological evidence and support the source tracing of an outbreak.

[Application of pulse-field gel electrophoresis analysis in source-tracking of food-borne disease caused by Vibrio parahaemolyticus].

OBJECTIVE: To apply pulse-field gel electrophoresis analysis(PFGE) in analysing a case of food poisoning caused by Vibrio parahaemolyticus. METHODS: PFGE using restriction enzyme Not I was employed in molecular subtyping of thirty strains of V. parahaemolyticus isolated from a case of food poisoning in Guangzhou city and PFGE patterns were analyzed by using BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA). RESULTS: Thirty strains were of the same type of pulsotype. CONCLUSIONS: Molecular subtyping by PFGE might disclose the epidemiological relationships of the strains from humans, food and the environment, giving a strong molecular epidemiological evidence and a support for the source-tracking of outbreak events.

Proteomic analysis reveals that a global response is induced by subinhibitory concentrations of ampicillin.

In this study, a recipient-donor co-culture system was used to research the effect of subinhibitory concentrations of antibiotics on horizontal transmission in bacteria and the influence of antibiotics on protein expression. We employed two-dimensional gel electrophoresis combined with mass spectrometry to compare the protein expression profiles in systems with or without 0.5 × the minimum inhibitory concentration of ampicillin. RT-PCR was used to assess the transcriptional levels of the differentially expressed genes. Fifty-seven different proteins were induced or suppressed. The upregulated proteins were involved in transcription and translation, cell wall synthesis, bacterial SOS response, and detoxifying functions, and the downregulated proteins were involved in metabolism. These results indicated that a global response was induced in the recipient-donor co-culture system by the subinhibitory concentration of ampicillin. Further analysis revealed that a global regulatory network based on key pathways was induced in the system in response to the antibiotic pressure. These findings provide a new, more comprehensive view for research on antibiotic-resistance mechanisms in recipient-donor co-culture.

Potential molecular mechanisms for fruiting body formation of Cordyceps illustrated in the case of Cordyceps sinensis.

The fruiting body formation mechanisms of Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.

[Analysis of characteristics of major pathogenicity-related genes of Vibrio cholerae isolated in Guangzhou area from 2001 to 2005].

OBJECTIVE: To apply multiplex polymerase chain reaction (MPCR) assay and sequencing in study of the carrying status of four pathogenicity-related genes of Vibrio cholerae (V.cholerae) and the variation of ctxA. METHODS: Primers targeting cholera toxin sub-unit A gene (ctxA), toxin-coregulated pilus gene (tcpA), accessory cholera enterotoxin gene (ace), zonula occludens toxin gene (zot) were designed and the MPCR method was applied to detect the pathogenicity-related genes of 276 strains of V.cholerae isolates. The amplified fragments of ctxA gene were sequenced and the genetic homology of the amplified fragments of ctxA was analyzed. RESULTS: Of the 276 strains of V.cholerae, 93.9% strains from human sources belong to the pathogenicity-related genes type A (ctxA(+)tcpA(+)ace(+)zot(+) type) and 6.1% belong to pathogenicity-related genes type C (ctxA(-)tcpA(-)ace(-)zot(-) type). Type A strains from clinical sources were isolated from patients with mild to severe symptom and carriers, among which 68.5% were isolated from patients with mild symptom and 21.9% from carriers. All 63.6% of type C strains from clinical sources were isolated from patients with mild symptom and 36.4% from carriers. The proportion of type C strains that caused mild symptom was higher than that of type A strains. Of the 78 strains isolated from the environment, 9.0% strains belong to pathogenicity-related type A and 35.9% belong to the pathogenicity-related genes type B (ctxA(-)tcpA(-)ace(+)zot(+) type), while 55.1% belong to pathogenicity-related genes type C. The sequencing results showed little genetic variation among the amplified fragments for ctxA. CONCLUSION: MPCR disclosed the polymorphic status of pathogenicity-related gene patterns in V.cholerae isolates of Guangzhou, providing effective means for further study on evolution of pathogenicity-related genes among V.cholerae isolates from human and environmental sources. This study also offers significant guidance for effective prevention, control and warning against cholera epidemic in local area.

Prevalence of 7 virulence genes of Legionella strains isolated from environmental water sources of public facilities and sequence types diversity of L. pneumopila strains in Macau.

In this study, we analyzed 7 virulence genes in 55 Legionella species (including 29 L. pneumophila and 26 non-L. pneumophila strains) which isolated from environmental water sources of the public facilities in Macau by using PCR and real-time PCR. In addition, 29 Legionella pneumophila isolates were subjected to genotyping by sequence-based typing scheme and compared with the data reported. The detection rate of flaA, pilE, asd, mip, mompS, proA and neuA genes in the L. pneumophila were 100.0%, respectively. The neuA gene was not detected in the non-L. pneumophila strains, but flaA, pilE, asd, mip, mompS, and proA genes could be amplified with a positive rate of 15.4%, 15.4%, 53.8%, 38.5%, 15.4%, and 38.5%, respectively. The results from real-time PCR were generally consistent with that of PCR. Those L. pneumophila strains were assigned into 10 sequence types (STs) and ST1 (9/29) was the dominant STs. Four new STs were found to be unique in Macau. The analysis of population structure of L. pneumophila strains which isolated from Macau, Guangzhou and Shenzhen indicated that the similar clones were existed and ST1 was the most prevalent STs. However, the distribution of the subtypes isolated from Macau was not the same extensive as those from Guangzhou and Shenzhen. The different detection rates of the 7 virulence genes in different species of Legionella might reflect their own potential for environmental adaptability and pathogenesis. And the data analyzed from STs diversity indicated the Macau L. pneumophila possessed obvious regional specificity and high genetic diversity.

Rapid identification of Chikungunya and Dengue virus by a real-time reverse transcription-loop-mediated isothermal amplification method.

Both Chikungunya and Dengue virus belong to the acute arthropod-borne viruses. Because of the lack of specific symptoms, it is difficult to distinguish the two infections based on clinical manifestations. To identify and quantitatively detect Chikungunya and Dengue viruses, a real-time accelerated reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) platform was developed, and 26-confirmed RNA samples, 42 suspects, and 18 healthy serum samples were evaluated by the method. The RT-polymerase chain reaction (PCR) and cDNA sequencing were used as references. The results showed that it could identify the Chikungunya and Dengue virus RNA correctly in all antibody-positive samples within 1 hour, without any cross-reactions. The virus load of the positive samples was quantitatively detected with a turbidimeter. The sensitivity was 100% and specificity was 95.25%. The findings indicate that the RT-LAMP is an effective method for rapid quantity detection of Chikungunya virus and Dengue virus in serum samples with convenient operation, high specificity, and high sensitivity.

In vitro inhibition of influenza virus infection by a crude extract from Isatis indigotica root resulting in the prevention of viral attachment.

Isatis indigotica root (IIR) has been widely used as a Chinese medicinal herb to treat regular seasonal influenza over the long history of traditional Chinese medicinal practice. However, its inhibitory activities against influenza virus infections along with the associated mechanisms have not been investigated comprehensively. In this study, the chemical nature, mode of action and in vitro anti-influenza activities of a crude extract (G2) of IIR were characterized. The extract was found to inhibit different subtypes of human or avian influenza viruses at various magnitudes of activity (IC50 0.39­4.3 mg/ml) in vitro, including A/PR/8/34 (H1N1), A/FM/1/47 (H1N1), A/Aichi/2/68 (H3N2), seasonal influenza (A/Guangzhou/GIRD/02/09 H1N1, B/Guangzhou/GIRD/08/09), novel swine-originating influenza (A/Guangzhou/GIRD/07/09, H1N1), A/Duck/Guangdong/09 (H6N2), A/Duck/Guangdong/94 (H7N3) and A/Chicken/Guangdong/96 (H9N2), while G2 was inactive against respiratory syncytial virus (RSV), adenovirus 3 (ADV3), parainfluenza virus 3 (PIV3) and enterovirus 71 (EV71). An apparent virus titer reduction was detected when the influenza viruses were pretreated with G2, and it was also shown that G2 exhibited inhibitory effects on influenza virus hemagglutination. In addition, G2 played a role in the early stages of infection, which did not easily result in the emergence of virus drug resistance. Thus, G2 may affect the attachment of influenza virus by interfering with the viral particles, thereby preventing the binding of influenza virus to the host cell surface.