[Analysis on transmission chain of a cluster epidemic of COVID-19, Nanchang].

Objective: Based on an investigation of an outbreak of COVID-19 in Nanchang, to understand the transmission process, analyze the infectivity of the cases in incubation period and asymptomatic carrier, and evaluate the transmission risks in different exposures. Methods: Case investigation was based on the traditional epidemiological survey, combined with analysis based on big data about population movement trajectories. Transmission chain was identified to indicate transmission relationship. Results: A total of 27 cases were found in this cluster epidemic, including 25 confirmed cases, 1 suspected case (index case) and 1 asymptomatic carrier. A total of 347 close contacts were found. The secondary attack rate was 7.2% (25/347). The infection rates in close contacts of the first, second, third and fourth generation cases were 52.6% (10/19), 6.1% (13/213), 2.3% (2/88) and 0.0% (0/27), respectively. Asymptomatic carrier caused household transmission. The infection rates in close contacts after having meals, sharing rooms/beds, having work contacts, having neighbor contacts, having same time medical services or sharing wards and sharing vehicles with the patients were 10.6%(17/160), 10.0%(20/201), 5.3%(5/94), 0.0%(0/30), 0.0%(0/18) and 0.0%(0/17), respectively. Conclusions: The infection source of this cluster epidemic was a suspected case from Wuhan. Analysis based on big data about population movement trajectories can help to search the cases and close contacts accurately. The proposed epidemic prevention and control measures based on this investigation were effective.

Beneficial effect of Rhodopseudomonas palustris on in vitro rumen digestion and fermentation.

As a member of photosynthetic bacteria, Rhodopseudomonas palustris, which has extraordinary metabolic versatility, has been applied as one of potential probiotics in feed industry. To explore whether R. palustris can increase rumen microbial viability and thus improve microbial fermentation, a 2×5 factorial experiment was conducted to evaluate the effect of R. palustris at dose rates of 0, 1.3, 2.6, 3.9, 5.2×106 cfu/ml on ruminal fermentation of two representative total mixed rations (HY, a ration for high-yield (>32 kg/d) lactating cows; LY, a ration for low-yield (<25 kg/d) lactating cows). After a 48 h in vitro rumen incubation, both rations resulted in different fermentation characteristics. The HY in comparison with LY group presented greater in vitro dry matter disappearance (IVDMD), cumulative gas production (GP48) and total volatile fatty acids (VFA, P<0.01). Increasing R. palustris addition linearly increased IVDMD (P<0.01) and GP48 (P<0.05), and the IVDMD increment in response to R. palustris addition was greater in LY than HY group (6.4% vs 1.4%). Meanwhile, increasing R. palustris addition also linearly enhanced microbial protein synthesis and increased total VFA production (P<0.01), especially in LY group (up to 21.5% and 24.5% respectively). Unchanged acetate and declined propionate in molar percentage were observed in response to the R. palustris addition. Furthermore, increasing R. palustris addition altered fermentation gas composition in which molar O2 proportion in headspace of fermentation system was linearly reduced by 46.1% in LY and 32.9% in HY group, respectively (P<0.01), and methane production in both ration groups was enhanced by 1.9-4.1% (P=0.02). In summary, the R. palustris addition exhibited high potential for promoting the growth of rumen microorganism and enhancing microbial fermentation towards non-glucogenic energy supply by maintaining an anaerobic environment to microbe equilibrium.

[Clinical experiences of laparoscopic radical cystectomy and urinary diversion in the elderly patients with bladder cancer].

Objective: To optimize the surgical procedures of laparoscopic radical cystectomy and urinary diversion for the elderly patients with bladder cancer, generalize operating technique, summarize clinical experiences. Methods: From July 2004 to October 2016, laparoscopic radical cystectomy (LRC) and urinary diversion was performed in 68 elderly patients (≥75 years old) diagnosed with bladder cancer in urology department of Beijing Chaoyang Hospital, Capital Medical University, and the relevant clinical and follow-up data were retrospectively reviewed. All the patients were pathologically diagnosed and their perioperative data, postoperative pathological results, postoperative complications and follow-up outcomes were recorded and analyzed. Results: Among 68 elderly patients with bladder cancer, fifty patients were male and 18 were female, the age of whom were (79±4) (range 75 to 91) years old. The Charlson comorbidity index (CCI) score was 6±1 (range 5 to 7). All the 68 operations were successfully performed without conversion to open surgery. There were 26 cases receiving cutaneous ureterostomy, 34 cases receiving ileal conduit (intracorporeal for 16 cases and extracorporeal for 18 cases) and 8 cases receiving orthotopic ileal neobladder (intracorporeal for 4 cases and extracorporeal for 4 cases; Xing's technique for 4 cases, T-Pouch for 2 cases and Studer-Pouch for 2 cases) respectively, and the operation time of these three groups were (221±47) min, (315±70) min and (358±90) min respectively, the estimated blood loss were 100 (87, 200)ml, 300 (250, 500) ml and 250 (113, 725) ml respectively, the time of intake of liquid diet were 3 (2, 4) d, 6 (5, 7) d and 9 (5, 12) d respectively, and the postoperative hospital stay were (12±6) d, (24±11) d, and (27±11) d respectively. Postoperative pathological results showed urothelial carcinoma in 64 patients, squamous cell carcinoma in 2 patients and adenocarcinoma in 2 patients. Sixty patients received laparoscopic pelvic lymphadenectomy and the number of dissected lymph nodes was 17.1±7.0. There were 46 cases with T stage greater than or equal to T2 (46/68, 67.6%), 4 cases of low grade (4/68, 5.9%) and 60 cases of high grade (60/68, 88.2%). All the early postoperative (within 30 days after the operation) complications were grade Ⅰ-Ⅱ mainly manifested as fever, pain and infection that could get better with symptomatic treatment, and the incidence rate were 30.8% for cutaneous ureterostomy, 29.4% for ileal conduit and 37.5% for orthotopic ileal neobladder. The patients were followed up for a median period of 93.5 months without obvious hydronephrosis and impaired renal function. The 5-year cancer specific survival rate and overall survival rate were 57% and 50% respectively. There was significant difference between the preoperative and postoperative QOL (quality of life) score (56.0±10.0 and 47.4±5.8 respectively, P<0.05) which indicated that the patients' postoperative quality of life was greatly improved. Conclusions: Laparoscopic radical cystectomy and urinary diversion for the elderly patients with bladder cancer is safe and feasible, and owns great therapeutic value.

Growth under elevated air temperature alters secondary metabolites in Robinia pseudoacacia L. seedlings in Cd- and Pb-contaminated soils.

Plant secondary metabolites play a pivotal role in growth regulation, antioxidant activity, pigment development, and other processes. As the global climate changes, increasing atmospheric temperatures and contamination of soil by heavy metals co-occur in natural ecosystems, which alters the pH of rhizosphere soil and influences the bioavailability and mobility of metals. Elevated temperatures in combination with heavy metals are expected to affect plant secondary metabolites, but this issue has not been extensively examined. Here, we investigated secondary metabolites in Robiniapseudoacacia seedlings exposed to elevated temperatures using a passive warming device in combination with Cd- and Pb-contaminated soils. Heavy metals significantly stimulated the accumulation of saponins, phenolic compounds, and flavonoids in leaves and stems; alkaloid compounds increased in leaves and decreased in stems, and condensed tannins fluctuated. Elevated temperatures, alone and in combination with Cd and Pb, caused increases in secondary metabolites in the plant tissues. Phenolic compounds showed the greatest changes among the secondary metabolites and significant interactive effects of temperature and metals were observed. These results suggest that slightly elevated temperature could enhance protective and defense mechanisms of Robinia pseudoacacia seedlings exposed to heavy metals by stimulating the production of secondary metabolites.

Salmonella induce autophagy in melanoma by the downregulation of AKT/mTOR pathway.

Salmonella have been demonstrated to inhibit tumor growth. However, the mechanism of Salmonella-induced tumor cell death is less defined. Autophagy is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol. Tumor cells frequently display lower levels of basal autophagic activity than their normal counterparts and fail to increase autophagic activity in response to stresses. Autophagy is involved in the cell defense elimination of bacteria. The signaling pathways leading to activation of Salmonella-induced autophagy in tumor cells remain to be elucidated. We used autophagy inhibitor (3-Methyladenine) and apoptosis inhibitor (Z-VAD-FMK) to demonstrate that Salmonella may induce cell death via apoptosis and autophagic pathway. Meanwhile, we suggested that Salmonella induce autophagy in a dose- and time-dependent manner. The autophagic markers were increased after tumor cell infected with Salmonella. In addition, the protein express levels of phosph-protein kinase B (P-AKT), phosph-mammalian targets of rapamycin (P-mTOR), phosph-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased by western analysis after Salmonella infection. In conclusion, our results point out that Salmonella induce the autophagic signaling pathway via downregulation of AKT/mTOR pathway. Herein, our findings that Salmonella in controlling tumor growth may induce autophagic signal pathway.

Light-sensitive intelligent drug delivery systems of coumarin-modified mesoporous bioactive glass.

Functionalized mesoporous bioactive glasses (MBG) with photoactive coumarin demonstrates photo-responsive dimerization resulting in reversible gate operation. Coumarin-modified MBG was used as a drug delivery carrier to investigate drug storage/release characteristics using phenanthrene as a model drug. Irradiation with UV light (>310 nm) induced photo-dimerization of the coumarin-modified MBG, which led to the pores' closing with cyclobutane dimers and trapping of the guest phenanthrene in the mesopores. However, irradiating the dimerized-coumarin-modified MBG with shorter wavelength UV light (approximately 250 nm) regenerates the coumarin monomer derivative by the photo-cleavage of cyclobutane dimers, such that trapped guest molecules are released from the mesopores. The structural, morphological, textural and optical properties are well characterized by X-ray diffraction, transmission electron microscopy, N(2) adsorption/desorption, and UV-visible spectroscopy. The results reveal that the MBG exhibits the typical ordered characteristics of the hexagonal mesostructure. The system demonstrates great potential in light-sensitive intelligent drug delivery systems and disease therapy fields.

The possible role of arterial radial vibration in heart rate and blood pressure matching.

MOTIVATION: This study tests if the 'oscillating spring' analogy for the radial vibration of the arterial wall can help to describe the relationship between the heart rate (HR), the blood pressure (BP), and properties of the arterial wall when different types of stimulation are applied on the cardiovascular system. It may help to suggest a possible role for arterial radial vibration in the association between the arterial stiffening and hypertension. METHODS: Either mechanical stimulation was applied (0.5-mmHg pressure variation) to Wistar rats by at near-HR frequency (group A) or administered Propranolol (2mg/kg i.p.; group B), and measured HR and BP simultaneously. RESULTS: In both groups, HR and BP were noted to change in the same direction (r2 = 0.72 and 0.62, respectively; p both < 0.05 by F-test). ANCOVA was performed on these two regression lines, and it was found that there was no significant difference between them (p > 0.3). CONCLUSION: In both groups, changes in haemodynamic parameters can be explained by the 'oscillating spring' analogy for the radial vibration of the arterial wall. This illustrates that, when facing various stimulations, it may be an important regulatory mechanism for the heart and the arteries to restore their frequency-matching condition in order to improve the arterial transmission efficiency. Paying more attention to the radial movement of the wall may therefore help to suggest a novel explanation of the mechanism that underlies the bidirectional relation between hypertension and arterial stiffening.

Evaluating microcirculation by pulsatile laser Doppler signal.

Laser Doppler flowmetry (LDF) is a popular method for monitoring the microcirculation, but it does not provide absolute measurements. Instead, the mean flux response or energy distribution in the frequency domain is generally compared before and after stimulus. Using the heartbeat as a trigger, we investigated whether the relation between pressure and flux can be used to discriminate different microcirculatory conditions. We propose the following three pulsatile indices for evaluating the microcirculation condition from the normalized pressure and flux segment with a synchronized-averaging method: peak delay time (PDT), pressure rise time and flux rise time (FRT). The abdominal aortic blood pressure and renal cortex flux (RCF) signals were measured in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The mean value of the RCF did not differ between SHR and WKY. However, the PDT was longer in SHR (87.14 +/- 5.54 ms, mean +/- SD) than in WKY (76.92 +/- 2.62 ms; p < 0.001). The FRT was also longer in SHR (66.56 +/- 1.98 ms) than in WKY (58.02 +/- 1.77 ms; p < 0.001). We propose that a new dimension for comparing the LDF signals, which the results from the present study show, can be used to discriminate RCF signals that cannot be discriminated using traditional methods.

Effects of whole-body mechanical stimulation at double the heart rate on the blood pressure waveform in rats.

The effects of mechanical stimulation on hemodynamics, such as due to mechanotransduction in vascular endothelial cells, have been widely discussed recently. We previously proposed a resonance model in which the arterial system is treated as a pressure-transmitting system, and suggested that the application of external mechanical stimulation with frequencies near the heart rate (HR) or harmonics thereof can be sensed by the arterial system and induce hemodynamic changes. In this study, we monitored the effects of external mechanical stimulation at a frequency of double the HR on BPW (blood pressure waveform), HRV (HR variability) and BPHV (blood-pressure-harmonics variability) in rats. A motor beating a waterbed mattress was used to generate pressure variations of 0.5 mmHg to apply onto the rats. The experiments were performed on three groups of rats with different beating frequencies: (A) double the HR, (B) 5% deviation from double the HR and (C) 1.5 times the HR. The experimental procedure was a 15 min control period followed by application of the mechanical stimulation for 15 min and further recording for 15 min (OFF period). During the OFF period, the amplitude of the second harmonic in the BPW significantly increased by >5% in group A with decreased HRV and BPHV. The second harmonic increased less in group B, and decreased in group C. The increase in the second-harmonic amplitude in group A may be due to the filtering properties of the renal arterial structure. This mechanism could be used to improve the local blood supply into the kidneys, and hence provide a new treatment modality for some important diseases, such as renal hypertension or nephrosis.

Raising harmonic variation of arterial pulse in dying rats.

Our previous study revealed that the coefficient of variation of harmonic magnitude (HCV) of radial arterial pulse was significantly raised before the death of cancer patients. In this study, we recorded the caudate arterial pulse of 24 Sprague-Dawley rats that had a fatal dose of urethane injected into their abdomens. Twenty rats were dead within 3 hours after the injection and four survived. We defined the last 100 minutes of each rat's life as the dying process. During the dying process, we found that both the systolic blood pressure and diastolic blood pressure dropped steeply during the last 5 minutes. However, all HCVs, except HCV1, climbed steeply before the last 5 minutes. The HCV1 of the dying rats was significantly higher than that of rats that survived, starting from the first minute (P < 0.01). The HCV2 of the dying rats was significantly higher than that of the survived rats starting from the 52nd minute (P < 0.05). The HCV3 and HCV4 of the dying rats were significantly higher than those of the survived rats until the 70th minute and the 80th minute, respectively (P < 0.05). Furthermore, HCV2-HCV4 proceeded with the dying process and increased gradually. We concluded that HCVs, which failed first in the high-frequency components and then in the low-frequency components, could provide physicians with earlier information to prevent the coming failure of circulatory system, and could reflect quantitatively pathological severity and predict patient outcome. The specific Fourier components in the pulse provide more physiological information than systolic and diastolic blood pressures.

Population structure of wild bananas, Musa balbisiana, in China determined by SSR fingerprinting and cpDNA PCR-RFLP.

Both demographic history and dispersal mechanisms influence the apportionment of genetic diversity among plant populations across geographical regions. In this study, phylogeography and population structure of wild banana, Musa balbisiana, one of the progenitors of cultivated bananas and plantains in China were investigated by an analysis of genetic diversity of simple sequence repeat (SSR) fingerprint markers and cpDNA PCR-RFLP. A chloroplast DNA (cpDNA) genealogy of 21 haplotypes identified two major clades, which correspond to two geographical regions separated by the Beijiang and Xijiang rivers, suggesting a history of vicariance. Significant genetic differentiation was detected among populations with cpDNA markers, a result consistent with limited seed dispersal in wild banana mediated by foraging of rodents. Nuclear SSR data also revealed significant geographical structuring in banana populations. In western China, however, there was no detected phylogeograpahical pattern, possibly due to frequent pollen flow via fruit bats. In contrast, populations east of the Beijiang River and the population of Hainan Island, where long-range soaring pollinators are absent, are genetically distinct. Colonization-extinction processes may have influenced the evolution of Musa populations, which have a metapopulation structure and are connected by migrating individuals. Effective gene flow via pollen, estimated from the nuclear SSR data, is 3.65 times greater than gene flow via seed, estimated from cpDNA data. Chloroplast and nuclear DNAs provide different insights into phylogeographical patterns of wild banana populations and, taken together, can inform conservation practices.

Validation of fermentation processes.

The ability to prepare consistent biopharmaceutical products depends extensively on possession of banked and characterized cell substrates and on development of production processes which can be validated. While the attributes that define cell characterization have been extensively detailed by ICH and the regulatory agencies in the past decade, little has been specified regarding process validation for biological processes. The extent to which validation concepts can be applied to biological processes varies depending on the nature of the process, the nature of the product, and the level of knowledge regarding the relationship between process parameters and product quality. Expectations concerning the rigour of the validation programme should be adjusted accordingly. There is no single approach that is appropriate for all processes and products. At a minimum, there should be an attempt to define which process parameters are critical, and to focus the attention of validation efforts on these parameters.

Influencing the heart rate of rats with weak external mechanical stimulation.

The ventricular-arterial coupling is assumed to minimize the expenditure of cardiac energy. From the conjecture of the resonance theory, the arterial system transmits pressure waves and resonates with the heartbeat, therefore, the arterial system is similar to a mechanical resonator. Theoretically, the heart rate can be paced with weak external mechanical stimulation and corresponding blood pressure changes can be observed. A waterbed was activated to generate 0.5-mmHg pressure vibrations as a stimulus and the rate was set to deviate 5% from the control heart rate. Among 13 studies on seven rats, the linear regression between X (stimulation frequency--control heart rate) and Y (actual changes of the heart rate) is Y = 0.992X = 0.062 (Hz) with a correlation coefficient of 0.97 (Y = X implies complete steering). The intercorrelation coefficient between the change in mean blood pressure and the heart rate was 0.79. The study showed that this weak mechanical stimulation influences the heart rate, and the blood pressure changes according to the heart rate. Cardiovascular optimization and the resonance theory may explain the way one may regulate the heart rate and the blood pressure of humans noninvasively in the future.

Crystallization and preliminary crystallographic studies of a ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer.

Ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer is a good model for the study of the thermostability of proteins. It has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as precipitant at 290 K. The crystal belongs to the hexagonal space group P6(1)/P6(5), with unit-cell parameters a = b = 48.32, c = 86.42 A. The asymmetric unit contains a single molecule of L30e, with a corresponding crystal volume per protein mass (V(M)) of 2.68 A(3) Da(-1) and a solvent content of 54%. A complete data set diffracting to 1.96 A resolution was collected from a single crystal at 100 K.

Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha.

p73 is a homologue of the tumour suppressor p53 and contains all three functional domains of p53. The alpha-splice variant of p73 (p73 alpha) contains near its C-terminus an additional structural domain known as the sterile alpha-motif (SAM) that is probably responsible for regulating p53-like functions of p73. Here, the 2.54 A resolution crystal structure of this protein domain is reported. The crystal structure and the published solution structure have the same five-helix bundle fold that is characteristic of all SAM-domain structures, with an overall r.m.s.d. of 1.5 A for main-chain atoms. The hydrophobic core residues are well conserved, yet some large local differences are observed. The crystal structure reveals a dimeric organization, with the interface residues forming a mini four-helix bundle. However, analysis of solvation free energies and the surface area buried upon dimer formation indicated that this arrangement is more likely to be an effect of crystal packing rather than reflecting a physiological state. This is consistent with the solution structure being a monomer. The p73 alpha SAM domain also contains several interesting structural features: a Cys-X-X-Cys motif, a 3(10)-helix and a loop that have elevated B factors, and short tight inter-helical loops including two beta-turns; these elements are probably important in the normal function of this domain.

[Studies of FISH and karyotype of Gossypium barbadense].

Based on the fluorescent in situ hybridization (FISH) of somatic chromosome of Gossypium barbadense with the probe of genomic DNA (gDNA) of Gossypium arboreum, two sets of chromosomes were easily distinguished by signals hybridized or not. The FISH directly proved that G. barbadense originated from two different diploid species, but was not in concordance with the former point that every chromosome of A sub-genome of tetraploid species was bigger than that of other sub-genome (D genome). The karyotype formula of G. barbadense based on its FISH was 2n = 4x = 52 = 38 m + 14sm(6sat). There were three pairs of satellite chromosomes which were all sm types. Their satellites located in short arms but originated differently from their chromosomes of sub-genome. Fragment translocations occured in the long arms of homologous chromosomes of number 5, 6 and 9 of A sub-genome. It was suggested that the translated fragments come from D sub-genome. The fragments are fairly large with the relative lengths of 19.21%, 17.69% and 12.88% of their whole chromosomes, respectively. At least five pairs of chromosomes in D sub-genome show some hybridized signals of gDNA probe of G. arboreum in their centromere regions, which indicated that there would be chromatin introgressions from A sub-genome.

Interaction between HIV type 1 glycoprotein 120 and CXCR4 coreceptor involves a highly conserved arginine residue in hypervariable region 3.

Several seven-transmembrane chemokine receptors are known to function as entry coreceptors for human immunodeficiency virus type 1. CCR5 and CXCR4 are the major coreceptors for non-syncytium-inducing (NSI) and syncytium-inducing (SI) viruses, respectively. During the natural course of infection, the emergence of variants with a phenotypic transition from NSI to SI and rapid disease progression is associated with expanded coreceptor usage to CXCR4. Characteristic amino acids at several positions in the hypervariable region 3 (V3) of gp120 have been linked to CXCR4 utilization. Previously, we reported that a highly conserved arginine residue of V3 played an important role in CCR5 utilization. In this study, the possible involvement of the same arginine residue in CXCR4 utilization was investigated. Amino acid substitutions introduced to this arginine on R5X4 viruses were found to have a significant effect on their utilization of CXCR4. These results, taken together with those reported previously, suggest that this highly conserved arginine may contribute to the functional convergence of chemokine coreceptor utilization by human immunodeficiency viruses and may represent a unique target for future antiviral design.

Molecular biology of human immunodeficiency virus type 1.

Since the discovery of human immunodeficiency virus (HIV) as the etiologic agent of acquired immune deficiency syndrome (AIDS) more than a decade ago, tremendous progress has been made in various aspects of this virus and its interplay with the host immune system. The advent of potent combination therapy has made it possible to achieve effective and durable control of HIV-1 replication in vivo, yet the persistence of the latent reservoirs pose a new challenge. The recent identifications of several cellular proteins interacting with different viral gene products have not only shed new insights into our understanding of the HIV-1 and the host cell biology, but also provided the bases for developing novel strategies to block HIV-1 replication. It is from this perspective that we review the current understanding of the molecular mechanisms governing the HIV-1 life cycle.

Influence of spleen meridian herbs on the harmonic spectrum of the arterial pulse.

Pulse analysis is a powerful method in Chinese medicine. We suggest that the effect of herbal medicine is to redistribute the blood to organs and meridians. In this report, by injecting extracts into rats and then analyzing the blood pressure wave measured at the caudate arteries, we studied eight important spleen meridian related herbs: They were Semen Lablab, Fructus Amomi Globosi, Rhizoma Atractylodis Macrocephalae, Rhizoma Atractylodis, Tuber Pinelliae, Radix Codonopsitis, Pericarpium Aurantii and Rhizoma Polygonati. All eight herbs increased the intensity of the 3rd harmonic (C3) of the pressure pulse which is correlated to the spleen and spleen meridian, as described in traditional Chinese medical literature. All of them also increased the 2nd harmonic (which is correlated to the kidney and the kidney meridian) as well as decreased the heart load (DC term of pressure wave, C0). Tuber Pinelliae, Radix Codonopsitis, Pericarpium Aurantii and Rhizoma Polygonati decreased the 1st harmonic (correlated to the liver meridian) significantly, while Rhizoma Atractylodis Macrocephalae only decreased C1 slightly. Except for Semen Lablab, all the others decreased the intensity of the 5th (stomach meridian) and the 7th harmonics. The effects of kidney herbs: Cortex Eucommiae and Radix Achyranthis were also shown for comparison.

Quantitative competitive reverse transcription-PCR for quantification of dengue virus RNA.

A quantitative competitive reverse transcription-PCR assay was developed to quantify dengue virus RNA in this study. The main features include a primer pair targeting a highly conserved region in the capsid and the addition of competing RNA that contains an internal deletion to provide a stringent internal control for quantification. It can be utilized to quantify RNA isolated from the four dengue virus serotypes but not RNA isolated from other flaviviruses, including Japanese encephalitis virus and hepatitis C virus, both prevalent in Asia. It can also be used to quantify dengue virus RNA isolated from the plasma of infected individuals. The sensitivity of the assay was estimated to be 10 to 50 copies of RNA per reaction, and twofold differences in virus titer are distinguishable. This assay is a convenient, sensitive, and accurate method for quantification and can be used to further understanding of the pathogenesis of dengue virus infection.