Reed-Muench Method Research Articles

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Berbamine, a bioactive alkaloid, suppresses equine herpesvirus type 1 in vitro and in vivo.

Equine herpesvirus type 1 (EHV-1) poses a global threat to equines. The anticancer agent berbamine (BBM), a bioactive alkaloid, has been shown to inhibit viral infection. However, whether BBM can inhibit EHV-1 infection remains unclear. This study investigated the effect of BBM treatment on EHV-1 infection. Quantitative PCR (qPCR), immunoblotting, the Reed-Muench method, and pathological examination were employed to study the ability of BBM to inhibit EHV-1 infection, viral DNA replication, viral protein production, virion secretion, and cytopathogenesis in vitro and in vivo. The in vitro studies revealed that 10 μM BBM effectively suppressed EHV-1 viral entry into cells, viral DNA replication, and virion secretion, while the in vivo studies verified the ability of BBM to suppress EHV-1-induced damage of brain and lung tissues and animal mortality. These findings strongly suggest that BBM could be a serious contender in the therapeutic control of EHV-1 infection of equines.

Both Maternal and Newborn IgMs Inhibit Transmissible Gastroenteritis Virus Internalization in LLC-PK1 Cells

Immunoglobulins M (IgMs) are the evolutionally oldest class of antibodies in higher eukaryotes. This pool of antibodies is one of the first to appear in humans and begins to be-synthesized at the early stages of the neonatal period. Most of the repertoire of IgMs of the newborns consists of the so-called «natural», or «naive», antibodies synthesized by the body without external antigenic stimulation. In addition to the classical functions of human immunoglobulins M (such as antigen recognition and initiation of innate immune responses), antibodies of this class exhibit a variety of non-canonical functions. The non-canonical functions are the action of antibodies as agonists/antagonists of various receptors, cleavage of antigen due to the catalytic activity of IgM, direct inactivation of pathogens in the absence of effector cells and molecules, etc. The aim of this work was to study and compare the antiviral activity of total preparations of immunoglobulin M of newborns and adults, obtained from umbilical cord and venous blood sera, on the LLC-PK1 cell line model infected with the transmissible gastroenteritis virus (TGEV). In addition, in the course of the studies, a decision was made to investigate the effect of combined preparations of immunoglobulin M on the change in signal transduction in the epidermal growth factor receptor as one of the mechanisms of TGEV internalization during infection of target cells. Methods. Highly purified preparations of total IgM of adults or newborns were obtained using the methodologies of sequential salt fractionation and affinity chromatography. This work used the model of the interaction of the transmissible gastroenteritis virus with cells of the LLC-PK1 line and the monitoring of changes in the phosphorylation state of the epidermal growth factor receptor of these cells during virus infection to study the effect of human IgM on the internalization of the virus and its interaction with the receptor system of the host cell. The degree of cytopathogenic effect of the virus was determined visually by changes in cell morphology. The mean infectious dose for transmissible gastroenteritis virus in the cell culture of the LLC-PK1 line was determined by the Reed-Muench method. Analysis of changes in the phosphorylation of the epidermal growth factor receptor was performed using the Western-blot analysis method. Results. The addition of a total high-purified sample of human IgM reduces the degree of efficiency of TGEV infection of the LLC-PK1 cell line and modulates the phosphorylation levels of these cells. Conclusions. The total preparations of IgM obtained from human venous blood of adults and from umbilical cord blood of newborns can affect the internalization of the transmissible gastroenteritis virus in the LLC-PK1 cell line. The original model of virus (TGEV) — cell line (LLC-PK1) was applied and tested to study the effect of native total preparations of immunoglobulin M on the internalization of the virus into the cell. The obtained data can be useful in further studies for a better understanding of the process of development and functioning of the immune system of newborns.

Improved assay for detecting SARS-CoV-2 from nonporous hospital surfaces using surrogate human coronavirus OC43

Background: Understanding SARS-CoV-2 persistence on surfaces can help inform transmission risk from surfaces in healthcare and community settings. A sensitive viral infectivity assay is crucial for the detection of infective virus in environmental investigations. The conventional cell culture-based infectivity assay is limited by the time dependence, subjectivity, and insensitivity of cytopathic effect (CPE) scoring. We validated an integrated cell-culture and reverse-transcription quantitative RT-PCR method (cc-RT-qPCR) to improve SARS-CoV-2 detection and reduce detection time. We compared cc-RT-qPCR with CPE-scored cell culture to evaluate assay sensitivity of recovered virus from stainless-steel coupons simulating nonporous healthcare surfaces. Method: Human β-coronavirus OC43, a model strain for SARS-CoV-2, was propagated on HRT-18G cells in growth medium at 33°C in a 5% CO2 incubator. The OC43 infectivity was determined by cell culture with a 10-fold dilution series of viral samples in 96-well plates, and incubation for 7 days at 33°C to confirm CPE. Plates were CPE-scored and TCID50 was calculated using the Reed-Muench method. For the cc-RT-qPCR assay, CPE-negative wells were interrogated for viral intracellular replication using RT-PCR; infectivity was based on a titer increase of ≥ 2 logs 7 days after inoculation using RT-qPCR. CPE-positive or replicative virus-harboring cells were enumerated to determine TCID50. The sensitivity of both CPE-scored cell culture and cc-RT-qPCR assays were evaluated by inoculating 105 TCID50/mL OC43 in infection media and artificial saliva matrices onto coupons and dried in an environmental chamber at 26°C and 57% relative humidity for 6 hours. Viral eluates from coupons served as test samples. Results: Low-titer infectious OC43 (0.75 log10) was detected by both methods 7 days after incubation; however, infectivity confirmation required 4 and 6 days after incubation, respectively, for cc-RT-qPCR and CPE-scored cell culture methods. When cells were inoculated with OC43 at titer range 1.75–4.75 log10, CPE presented at 4–5 days after incubation, while viral replication was already detected at 3 days after incubation via RT-PCR. Upon virus titration, cc-RT-qPCR demonstrated greater sensitivity, detecting up to 1 log10 higher of infectious OC43 than cell culture alone at 0 and 6 hours (P ≤ .05) dried in infection medium and 0 hours (P ≤ .05) in saliva. Conclusions: Our data demonstrated greater sensitivity and shorter times to detect viral replication by cc-RT-qPCR, minimizing potential for false-negative results with cell culture alone. This sensitive assay may provide investigators with quicker results for informing infection control practices to reduce risk of transmission from deposited bodily fluids on surfaces, eg, coughing and sneezing.Funding: NoneDisclosures: None

Acute Venom Toxicity Determinations for Five Iranian Vipers and a Scorpion

Background: Poisoning due to the bites and stings of venomous snakes and scorpions is a neglected public health problem, particularly in rural areas. Poor health facilities and inadequate knowledge of health care personnel are the major factors that result in envenomated human victims not receiving adequate care and medical attention. There is a great need for up-to-date and effective healthcare knowledge and awareness of the potency and lethality of venomous creatures in Iran. Assessment of the potency, acute toxicity, and lethal effects of venomous creatures come from a variety of specific tests, such as the 50% median lethal dose (LD50) and ample animal experimentations. Methods: In the present study, using modified Reed-Muench method, the LD0, LD50, and LD100 values of the venoms from five Iranian vipers and one scorpion were determined. The studied venomous creatures were: Macrovipera lebetina, Vipera albicornuta, Vipera raddei, Caucasicus intemedius agkistrodon, Montivipera latifii, and one scorpion Hemiscorpius lepturus. The venoms were injected in Albino mice (n=204) intraperitoneally, and their toxicities determined. Results: The results revealed that the LD50 values of the above-mentioned creatures were 3.87, 2.05, 1.63, 1.45, 0.84, and 6.33 mg/kg, respectively. Among the vipers, M. latifii had the most potent venom while M. lebetina’s venom had the lowest toxicity. Conclusion: Theoretically, the determined LD50 values provide for objective comparisons of the toxicity among of the venoms. However, comparison becomes complicated due to variations in the venoms’ LD50. Further, based on the venoms’ toxicity levels, H. lepturus’ venom caused the lowest toxicity in the Albino mice.

Chemical composition and larvicidal activity of the essential oil of Pimenta dioica leaves

In this study, we investigated the main constituent, the predominant class and biological activity of the essential oil extracted from the leaves of Pimenta dioica and the pattern of the major constituent against larvae in the third stage of Aedes aegypti. For this reason, we extracted the oil by hydrodistillation, identified its components by gas chromatography coupled with mass spectrometry (GC/MS) and calculated the lethal concentration (LC50) of the larvicidal activity using the Reed-Muench method. The results show that the oil consists mainly of eugenol, in which the phenylpropanoid class predominated and the lethal concentration, LC50, was 38.86 μg mL-1at a confidence level of 2.25 μg mL-1, while the eugenol standard presented LC5079.75 μg mL-1at a confidence level of 2.10 μg mL-1. Given the facts, we conclude that the oil is more active than the standard and that it has the potential to replace chemical larvicides.

In-vitro Assessment of Antiviral Activity of the Herbo Mineral Capsule, Fema SakthiTM, against Human Coronavirus (HCoV) on VERO Cells

Introduction: With the spread of COVID-19 pandemic, healthcare workers and patients look for alternate medicines including Siddha, Ayurveda, Unani and other forms of traditional medicines as we still do not have promising antiviral drugs for COVID-19. Objective: To evaluate the in-vitro antiviral activity of Fema Sakthi™ (FS) by eliciting the inhibition of cytopathic effect of Human Coronavirus (HCoV) on African Green Monkey kidney cells (VERO cells). Materials and Method:The cytopathic effect (CPE) was performed on Vero cells with Human Coronavirus 229E, a type of Coronavirus associated with respiratory infections. The Median Tissue Culture Infectious Dose (TCID50) was evaluated using Reed-Muench method. 100 TCID50 of HCoV 229E viral suspensions were added to VERO cell culture to induce the cytopathic effect. Uninfected and untreated cells were used as control and five concentrations (62.5, 125, 250, 500 & 1000 μg/mL) of Fema Sakthi™ (FS) were used to study the anti-viral activity. After incubation for 72 hours, the cell viability was observed under the inverted microscope after staining with 0.1% crystal violet. Results: Fema Sakthi™ (FS) was found to exhibit inhibition of cytopathic effect at lower concentrations (62.5, 125 and 250 μg/mL) but at higher concentrations (500 and 1000 μg/mL), the formulation itself was cytotoxic to the cells. Conclusion: This preliminary study showed that FS has antiviral activity at lower concentrations 62.5, 125 and 250 μg/mL on the VERO cells. However, further specific studies have to be carried out to confirm the anti-viral activity and clinical efficacy using other preclinical and clinical models of Human Coronavirus (HCoV) including COVID-19.

Low Neutralizing Antibody Titers against the Mu Variant of SARS-CoV-2 in 31 BNT162b2 Vaccinated Individuals in Colombia.

Global surveillance programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are showing the emergence of variants with mutations in the spike protein. Genomic and laboratory surveillance are important to determine if these variants may be more infectious or less susceptible to antiviral treatments and vaccine-induced antibodies. Three of the most predominant SARS-CoV-2 variants in Colombia during the epidemiological peaks of 2021 were isolated: Mu, a variant of interest; Gamma, a variant of concern; B.1.111, which lacks genetic markers associated with greater virulence. Microneutralization assays were performed by incubating 120 mean tissue culture infectious doses (TCID50) of each SARS-CoV-2 isolate with five two-fold serial dilutions of sera from 31 BNT162b2-vaccinated volunteers. The mean neutralization titer (MN50) was calculated by the Reed–Muench method. At the end of August, Mu represented 49% of coronavirus disease 2019 (COVID-19) cases in Colombia, followed by 25% of Gamma. In contrast, B.1.111 became almost undetectable. The evaluation of neutralizing antibodies suggests that patients vaccinated with BNT162b2 generate neutralizing antibody titers against the Mu variant at significantly lower concentrations relative to B.1.111 and Gamma. This study shows the importance of continuing surveillance programs of emerging variants, as well as the need to evaluate the neutralizing antibody response induced by other vaccines.

Способность экстрактов некоторых съедобных грибов подавлять репродукцию вируса гриппа

In this work, extracts from the mycelium of some xylophytic fungi: Pleurotus eryngii, Auricularia auriculajudae, Tremella fuciformis were obtained, the toxicity of the obtained extracts was evaluated, and the antiviral activity was researched. The influenza virus was grown in the allantois cavity of chicken embryos. The infectious titer of the influenza virus was determined by titration on chicken embryos. The presence of the virus was judged by the reaction of hemagglutinating activity. The virus infectivity titer was calculated using the Reed-Muench method. The hemagglutinating activity of viruses was determined by a standard method using 0.75 % suspension of chicken red blood cells. The main criterion for studying the specific antiviral effect of compounds was the CTI (chemical-therapeutic index), that is determined by the ratio of the average toxic concentration of the substance (TC50) to the average effective viral inhibitory concentration (EC50). The antiviral activity of the obtained extracts was studied on a model of human, animal and bird influenza viruses, in the dose range from 0.025 mcg/ml to 1.25 mcg/ml using the methodological recommendations of the “Guidelines for conducting Preclinical studies of medicines”. While studying the acute toxicity of 3 obtained fungi extracts, it was found that all studied fungi compounds with a single intragastric injection to white mongrel mice, did not show a toxic effect in the studied dose range. During the study of the ability to suppress the reproduction of various strains of the influenza virus, it was found that extracts of the fungi Auricularia auricula-judae and Tremella fuciformis have possessed antiviral properties, and extracts exceed in terms of CTI to commercial anti-influenza drugs.

Sensitivity of infl uenza virus strains isolated from various regions of Kazakhstan in 2018–2019 to antiviral drugs

The purpose of the study was to examine susceptibility of the Kazakhstan strains of infl uenza A/H1N1 and type B viruses, isolated from various regions of Kazakhstan in 2018–2019, to antiviral drugs. Materials and methods. The susceptibility analysis of 20 strains of infl uenza A/H1N1 and B viruses was carried out with chemotherapeutic agents including Remantadine, Tamifl u, Arbidol, and Ingavirin. Viruses were cultured in the allantoic cavity of developing 10-day-old chicken embryos for 48 hours at 36 °C. The hemagglutinating activity was determined according to the conventional method on 96-well plates using 0.75% chicken red blood cell suspension; the infectivity was calculated by the Reed-Muench method. The sensitivity of virus strains to diff erent concentrations of antiviral drugs was evaluated by the level of reproductive suppression of 100 lg EID50/0.2 ml of virus in chicken embryos. Statistical analysis was performed with the use of Microsoft Offi ce Excel 2010 software. Results. A study of sensitivity to chemotherapeutic agents demonstrated heterogeneity of Kazakhstan 2018–2019 infl uenza A and B viruses population on this feature. The sensitivity to Tamifl u was found in all Kazakhstan strains of infl uenza A/H1N1 virus and three type B strains (inhibitory concentration was 0.44–25.38 μg/mL). The reproduction of most viruses was eff ectively inhibited by tamifl u at a concentration of 0.68–3.23 μg/mL. The inhibitory concentration for the three strains of A/H1N1 virus was 7.23–25.38 μg/mL. Remantadin inhibited the reproduction of viruses at higher doses (12.60–25.55 μg/mL). All viruses under study were resistant to Arbidol and Ingavirin. One type B infl uenza virus was found to be weakly sensitive to Ingavirin. Conclusion. The heterogeneity of the infl uenza virus population in their sensitivity to antiviral drugs indicates the need for constant epidemiological surveillance in order to identify drug-resistant variants.

Neutralization antibody titers against SARS-CoV-2 in an infant born to a mother with COVID-19

Neutralization antibody titers against SARS-CoV-2 in an infant born to a mother with COVID-19

2018–2019 antiviral drug sensitivity of the influenza virus strains isolated from various regions of Kazakhstan

Influenza is a serious public health problem. The ability of influenza virus to change upon replication is the most serious issue for practical medicine and virology, which can fundamentally alter virus biological properties, such as infectivity and virulence. The high mutational variability of influenza viruses can contribute to rapidly emerging drug resistance. Therefore, the study of antiviral drug sensitivity among influenza viruses is necessary to justify proper drug use for treatment and prevention of influenza infection. The aim of the study was to examine antiviral drug susceptibility of influenza A/H1N1 and B virus strains isolated from various regions of Kazakhstan in the years 2018–2019. Materials and methods. The susceptibility analysis of 20 strains of influenza A/H1N1 and B viruses was carried out by using chemotherapeutic agents including Remantadine, Tamiflu, Arbidol, and Ingavirin. Viruses were cultured in the allantoic cavity of developing 10-day-old chicken embryos for 48 hours at 36оC. The hemagglutinating activity was determined according to the standard method on 96-well plates using 0.75% chicken red blood cell suspension; the infectivity was calculated by the Reed–Muench method. The susceptibility of virus strains to different concentrations of antiviral drugs was evaluated by the level of virus reproductive suppression of 100 lg EID50/0.2 ml in chicken embryos. Statistical analysis was performed using Microsoft Office Excel 2010 software. Results. A study of susceptibility to chemotherapeutic agents demonstrated heterogeneity of influenza A and B virus population isolated in Kazakhstan during the 2018–2019 period. The susceptibility to tamiflu was found in all Kazakhstan strains of influenza A/H1N1 virus and three type B strains (inhibitory concentration was 0.44–25.38 μg/mL). The reproduction of most viruses was effectively inhibited by Tamiflu at a concentration of 0.68–3.23 μg/mL. The inhibitory concentration for three strains of A/H1N1 virus was 7.23–25.38 μg/mL. Remantadine inhibited reproduction of viruses at higher doses (12.60–25.55 μg /mL). All investigated viruses were resistant to Arbidol and Ingavirin. A single type B influenza virus strain was found to be weakly susceptible to Ingavirin. Conclusion. The heterogeneity of influenza virus population in susceptibility to antiviral drugs suggest a need for constant epidemiological surveillance in order to identify drug-resistant variants.

Salvia plebeia R. Br. polysaccharides (SPP) against RSV (respiratory syncytial virus) infection: Antiviral effect and mechanisms of action

ObjectiveTo investigate the antiviral effect of Salvia plebeia R. Br. polysaccharides (SPP) against RSV and underlying mechanisms. MethodsSPP was extracted via alcohol-precipitation method and extract was separated into various fractions using ultrafiltration method. The polysaccharide content was determined using UV-Vis. Antiviral effect of SPP and fractions was measured using MTT method and Reed-Muench method. Sixty Balb/c mice were randomly divided into 6 groups, and received either Ribavirin or SPP. Their body weight and food intake were recorded every day throughout the experiment period. The lung index inhibition ratio and pulmonary virus titer were determined followed by the histological analysis of lungs. Furthermore, time-of-addition and effective stage analysis were carried out to determine the mechanism of action. The TLR-3 and TLR-4 levels in the lungs were determined using qRT-PCR. The levels of IFN-γ, IL-2 and TNF-α in serum were determined using ELISA. ResultsThe SPP content is 4.396%. SPP has shown a good anti-RSV effect both in vitro (TI = 123.041) and in vivo models. The antiviral activity of fractions with molecular weight ≥ 10,000 is found to possess more potent antiviral activity than other fractions. SPP inhibits the RSV proliferation and reduces the lung lesions induced by RSV. The mechanism of action involves the inhibition of TLR-3 and TLR-4 in lungs, up-regulation of IFN-γ and IL-2, and down-regulation of TNF-α in serum. It is also shown to improve the body’s immune function. ConclusionSPP has a potential to treat diseases caused by RSV.

Chemical composition and larvicidal activity of essential oil of the bark of Citrus sinensis (L.) Osbeck

Aim: We determined the chemical composition and larvicidal activity of the essential oil distilled from the Citrus sinensis (L.) Osbeck husks and the pattern of the major constituent. Materials and methods: For this, we distill the oil by hydrodistillation, identify the components by gas chromatography coupled with mass spectrometry (GC-MS), test the larvicidal activity against Aedes aegypti and calculate the lethal concentration at 50% (LC50) by the Reed-Muench method and the confidence interval by the Pizzi method for both oil and standard. Results: it showed that the oil consists mostly of limonene and showed larvicidal activity (LC50 of 199.01(± 2.10) μg·mL-1) greater than the lemonade standard (126.03 (± 2.09) μg·mL-1). Conclusion: Therefore, we conclude that distilled oil has the potential to replace chemical larvicides.

Assessment of Antiviral Activity of Curcuma longa on Two RNA Viruses

Major pandemics and seasonal epidemics that have ravaged the world in the past and even at present, are mostly caused by RNA viruses. This has necessitated the need for continuous research to identify important natural products, with antiviral potentials, which can be harnessed for use in the prevention and treatment of viral infections. This study therefore, evaluated the antiviral property of Curcuma longa on two important RNA viruses of public health importance, namely polio and measles viruses. Extraction of active ingredients from turmeric rhizomes was done with the use of Analar grade methanol and concentrated using rotary evaporator. Polio and measles viruses were isolated from their respective vaccines using Reed-Muench method. Infective doses of the viruses and toxicity profile of extract were determined. Confluent Vero cells were inoculated with the viruses at different dilutions of the extract, incubated and observed for 7 days. Methanol extract of Curcuma longa inhibited polio virus at the maximum non-toxic concentration (MNTC) of 0.031μg μL-1 and inhibitory concentration (IC50) of 0.067 μg μL-1 with selectivity index of 2.16. Inhibition by the extract was observed prior to infection with the viruses. Phytochemical analysis of the extract showed presence of terpenes, saponins, alkaloids, flavonoids, tannins, cardiac glycosides and phenol as the bioactive phytochemicals. This study has shown that curcuma longa has potent inhibitory activity, hence can be harnessed in the development of an effective antiviral agent against polio and measles viruses.

Atividade larvicida do óleo essencial de Alpinia zerumbetfrente as larvas do mosquito Aedes aegypti

O presente estudo teve por objetivo avaliar a toxicidade e a atividade larvicida do óleo essencial (OE) das folhas de Alpinia zerumbet. O (OE) foi extraído por hidrodestilação a 100°C por 3h e determinaram-se os parâmetros físico-químicos mediante a Farmacopeia Brasileira. A toxicidade foi realizada através do bioensaio de Artemia salina Leach, em seguida, avaliou-se a atividade larvicida frente ao Aedes aegypti.Calculou-se a CL50 para a toxicidade e a atividade larvicida frente as larvas do mosquito com base no método de Reed-Muench. Classificou-se o OE quanto à toxicidade e utilizou-se o critério de Dolabela. No bioensaio de toxicidade, o CL50321,15 mg L-1, sendo classificado como atóxico. A atividade larvicida frente ao Aedes aegyptiapresentou 50,70 mg L-1 segundo o OE. Os fenólicos totais apresentaram resultado de 513,574 mg EAT g-1. O OE apresentou resultados satisfatórios contra os microrganismos testados, revelando sua eficiência no combate e controle de microrganismos patogênicos e larvicidas.

A modified Reed-Muench method of estimation in dose-effect relationship

A modified Reed-Muench method of estimation in dose-effect relationship

Assessment of replicate numbers for titrating avian influenza virus using dose-response models.

Embryonating chicken eggs (ECEs) are among the most sensitive laboratory host systems for avian influenza virus (AIV) titration, but ECEs are expensive and require space for storage and incubation. Therefore, reducing ECE use would conserve resources. We utilized statistical modeling to evaluate the accuracy and precision of AIV titration with 3 instead of 5 ECEs for each dilution by the Reed-Muench method for 50% endpoint calculation. Beta-Poisson and exponential dose-response models were used in a simulation study to evaluate observations from actual titration data from 18 AIV isolates. The reproducibility among replicates of a titration was evaluated with one AIV isolate titrated in 3 replicates with the beta-Poisson, exponential, and Weibull dose-response models. The standard deviation (SD) of the error between input and estimated virus titers was estimated with Monte Carlo simulations using the fitted dose-response models. Good fit was observed with all models that were utilized. Reducing the number of ECEs per dilution from 5 to 3 resulted in the width of the 95% confidence interval increasing from ±0.64 to ±0.75 log10 50% ECE infectious doses (EID50) and the SD of the error increased by 0.03 log10 EID50. Our study suggests that using fewer ECEs per dilution is a viable approach that will allow laboratories to reduce costs and improve efficiency.

A Collaborative Study to Establish the Second Korean National Reference Standard for Snake Venom

In 2015, a candidate for the second national reference standard (NRS) of Gloydius snake venom was produced to replace the first NRS of Gloydius snake venom. In the present study, the potencies of the candidate were determined by a collaborative study, and the qualification of the candidate was estimated. The potencies of the candidate were determined by measuring the murine lethal titers and lapine hemorrhagic titers of venom against the regional working reference standard (RWRS) for antivenom using the methods described in the previous report for the first NRS of Gloydius snake venom. Three Korean facilities contributed data from a total of 30 independent assays. Subsequently, two foreign national control research laboratories contributed to this collaborative study. The results were calculated using the Reed-Muench method for lethality and determined using a mixed-effects model for hemorrhage. The general common potencies of the lethal and hemorrhagic titers were obtained from the results of the 30 tests performed at three Korean facilities. The results are expressed in micrograms for 1 test dose (TD) with a 95% confidence interval as follows: a lethal titer of 90.13 μg/TD (95% confidence interval = 87.39∼92.86 μg) and a hemorrhagic titer of 10.80 μg/TD (95% confidence interval = 10.46∼11.14 μg). In addition, the candidate preparation showed good quality evaluation according to the results of the quality estimation of the candidate and is judged to be suitable to serve as the Korean NRS for snake venom. In conclusion, the second NRS of Gloydius snake venom was established in this study and will be used for national quality control, including a national lot release test of Korean antivenom products.

Dengue virus type 2 infection inhibits the expression of toll-like receptors 4 and 7 in murine bone marrow-derived dendritic cells

Objective To analyze the changes in the expression of Toll-like receptors 4 (TLR4) and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 (DEN2) infection. Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injection and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively. RT-PCR was performed to identify DEN2 RNA. Reed-Muench method was used to determine the 50% tissue culture infective dose (TCID50) of DEN2. Dendritic cells (DCs) were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry. The prepared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluorescence assay. Dynamic changes in the expression of CD11c, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry. Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR. Results The TCID50 of DEN2 to C6/36 cells was 10-5.8. Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro. The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1×105 TCID50 of DEN2 were statistically different from those of the negative control group. Neither of the two groups showed a significant difference in the percentages of membrane molecules over time. However, the percentages of membrane molecules on DCs increased with increasing viral load. Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load. Conclusion DEN2 infection promotes the maturation of DCs. Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection. Key words: Dengue virus type 2; DC; TLR4; TLR7