ABSTRACT
Objective:To establish and validate a fluorescence focus assay (FFA) for rapid titration of Japanese encephalitis virus (JEV) and to evaluate its application in the production of Japanese encephalitis vaccine.Methods:Recombinant JEV non-structural protein 1 (NS1) was expressed in a prokaryotic expression system. After purification, JEV-NS1 was used to immunize rabbits to induce polyclonal antibody. FFA was established with the polyclonal antibody to titer JEV. The accuracy of FFA was validated by comparing with plaque assay, and the specificity, precision, linearity, range and robustness of FFA were also validated. Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results:FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV, and there was no cross reaction with other viruses (tick-borne encephalitis virus, yellow fever virus, coxsackievirus A2, coxsackievirus A4). Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine. FFA was more consistency than plaque assay.Conclusions:The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.
ABSTRACT
Avicel is made of a mixture of microcrystalline cellulose (MCC) and carboxymethyl cellulose (CMC), and used for virus plaque assay. The avicel in common use is produced by FMC Biopolymer. Due to the relatively fixed proportion of MCC and CMC, avicel in common use is not suitable for plaque determination experiment of all types of viruses. In this study, we evaluated the effect of avicel made of different proportions of MCC and CMC on virus plaque assay, and developed an improved avicel virus plaque assay featured with simple and convenient operation, good practicability and high stability. To generate avicel overlays with different proportions of MCC and CMC, twelve different 2×avicel solutions were prepared. Their overall viscosity and bottom viscosity were measured to evaluate the ease of operation. The results showed that most of the 2×avicel solutions (except the 4.8% MCC+1.4% CMC and 4.8% MCC+1.0% CMC group) were easy to absorb and prepare nutrient overlap than 2×CMC solution. In order to find the best scheme to detect the titer of porcine epidemic diarrhea virus (PEDV), these avicel overlay solutions with different proportion of MCC and CMC were used as a replacement in the standard plaque assay. By comparing the size, clarity, stability and titer accuracy of virus plaque, we identified that 0.6% MCC and 0.7% CMC was the most preferable composition of avicel overlay for PEDV plaque assay. In conclusion, we developed an improved virus plaque assay based on avicel, which may facilitate the research of virus etiology, antiviral drugs and vaccines.
Subject(s)
Animals , Carboxymethylcellulose Sodium/chemistry , Cellulose/chemistry , SwineABSTRACT
ABSTRACT: Equid alphaherpesvirus type 1 (EHV-1) is distributed worldwide and is a major agent of abortion, respiratory and neurological disease in horses. No specific treatment is available for EHV-1 infection, yet the potential of antiviral therapy has been explored. In this study we investigated the in vitro activity of Acyclovir, Ganciclovir, Foscarnet, Famciclovir, Vidarabina and Cidofovir against EHV-1. For this, the MTT test was performed, in which all the tested drugs showed no toxicity up to 200μg/mL. Subsequently, different drug concentrations were submitted to viral plaque reduction assays in cell culture. The selectivity index (SI) of the compounds was determined using the cytotoxic concentration for 50% of cells (CC50), obtained by MTT, and effective drug concentration to inhibit by 50% the number of viral plaques (EC50). Ganciclovir (SI: 490; EC50: 1.9 μg/mL) was the most efficient and safest drug against EHV-1, followed by Cidofovir (SI: 150, EC50: 5.7μg/mL), Acyclovir (SI: 37.4, EC50: 22.2μg/mL), Famciclovir (SI: 25.1, EC50: 24.5μg/mL), Vidarabine (SI: 12.2, EC50: 40.9μg/mL) and Foscarnet (SI: 6.9, EC50: 49.5 μg/mL), respectively. These results indicated that Ganciclovir (followed by Cidofovir), is a promising candidate for use in in vivo experiments.
RESUMO: O alfaherpesvírus equino tipo 1 (EHV-1) está amplamente distribuído nos rebanhos equinos de todo o mundo e é um dos principais agentes causadores de abortos, doença respiratória e neurológica em equinos. Ainda não há tratamento específico para a infecção pelo EHV-1 em equinos, mas o potencial da terapia antiviral tem sido investigado. Neste trabalho, foi investigada a atividade anti-herpética in vitro dos fármacos Aciclovir, Ganciclovir, Foscanet, Famciclovir, Vidarabina e Cidofovir frente ao EHV-1. Para isso, foi realizado o teste de MTT, em que todas as drogas não apresentaram citotoxicidade até a dose de 200μg/mL. A seguir, diferentes concentrações dos fármacos foram submetidas ao teste de redução de placas virais em cultivo celular. O índice de seletividade (IS) dos compostos foi determinado usando a concentração citotóxica para 50% dos cultivos celulares (CC50), obtida pelo MTT, e pela concentração dos fármacos efetiva para inibir em 50% o número de placas virais (EC50). O Ganciclovir (IS: 490; EC50: 1,9μg/mL) foi o mais eficiente e seguro frente ao EHV-1, seguido pelo Cidofovir (IS: 150; EC50: 5,7 μg/mL), Aciclovir (IS: 37,4; EC50: 22,2μg/mL), Famciclovir (IS: 25,1; EC50: 24,5μg/mL), Vidarabina (IS: 12,2; EC50: 40,9μg/mL) e Foscarnet (IS: 6,9; EC50: 49,5μg/mL). Estes resultados indicam que o Ganciclovir constitui-se em um candidato para uso em experimentos in vivo.
ABSTRACT
Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.
Subject(s)
Animals , Humans , Mice , Antibodies , Flow Cytometry , Lymphocytic choriomeningitis virus , Lymphocytic Choriomeningitis , Methods , Nucleoproteins , Polymerase Chain ReactionABSTRACT
Objective To establish the crystal violet plaque assay for detection of virus titer of recombined Tiantan vaccinia AIDS vaccine,and provide more stable method of virus titration for rTV AIDS Vaccine.Methods Optimized the concentration of Vero cells,the time and temperature of virus adsorption,and the time of determination for CPE,then established the crystal violet plaque assay for virus titer of rTV.Counting and analysis the plaques by BioSpot Reader,then analyzed the relativity of plaques counted with BioSpot Reader and manual; Several lots rTV AIDS Vaccine and Tiantan vaccinia were titrated by the method of plaque formation-hemadsorption assay,neutral red and crystal violet plaque assay,then analyzed the relativity of the results of three methods ; meanwhile,the virus titer of samples were determine repeatedly by the crystal violet plaque assay,then calculated the coefficient of variation( CV),and verified the precision of the method; SPSS17.0 was used in statistical analysis of the experimental results.Results When the concentration of Vero cells was 5.0×105-9.O×105 cells/ml,virus been adsorbesd 2 h at 37℃,then cultivated 72 h after adding the culture medium containing methyl cellulose.Plaques counted by BioSpot Reader was highly related with counted by manual (r =0.985),so BioSpot Reader counting can objectively reflect the virus plaques with various size,and reduce the error by manual counting; compared the virus titration for different lots of rTV AIDS vaccine and Tiantan vaccinia with three methods,the crystal violet plaque assay was highly related with plaque formation-hemadsorption assay (r =0.997,P＜0.01 ) and neutral red plaque assay(r=0.980,P＜0.01 ).Conclusion Crystal violet plaque assay was established for virus titration of rTV AIDS Vaccine.
ABSTRACT
Objective To establish the plaque assay for the titration of GFP-labeled recombinant human metapneumovims(rhMPV). Methods Vero-E6 cells were selected as host cells for titration. GFP-labeled hMPV was serially diluted and added to each well to infect the cells. The plates were covered with low melting agarose overlay and incubated for different days in incubator. The plates were then observed under fluorescence microscope for plaques with green flourecence, at the same time, the number of plaques was counted by blue plaque-forming. Results Under the low melting agarose overlay, Vero-E6 cells grew well until the CPE caused by hMPV was seen. Clear green flourescence could be observed the first day post infection, much clearer on the third day post infection but showed somewhat fusion between plaques later on.Blue plaques on the fifth day after infection were large and easy to observe. The recombinant GFP-labeled hMPV could replicate up to 1 × 106 PFU in the Vero-E6 cells. Conclusion Plaque assay for titration of recombinant GFP-rhMPV has been sucessfully established. This methodology will offer a solid base for further studies on pathogenesis and vaccine development of this virus.
ABSTRACT
Objective To establish plaque reduction assay and evaluate the activities of oseltamivir (tamiflu),amantadine,ribavirin and herb radix isatidis against influenza virus in vitro.Methods Plaque reduction assay was used to determine IC_(50) values of four studied drugs above in this susceptibility testing in which 8 clinical isolates(three influenza A virus isolates and five influenza B virus isolateds)were inoculated and tested.Results By testing of 8 clinical isolates of influenza virus A and B isolated between the year 2001 to 2008,oseltamivir and amantadine were found to be sensitive to influenza A virus with IC_(50) of 0.064 -0.128 mg/L and 0.5 mg/L,respectively.However,ribavirin(IC_(50)>8 mg/L)was not found to be sensitive,and herb radix isatidis had totally no activities.Unfortunately.all four studied drugs were not found to have activities against influenza B virus in vitro.Conclusions It Was indicated that oseltamivir and amantadine.but not ribavirin and herb radix isatidis.are sensitive to influenza A virus.All four studied drugs were not found to have activities against influenza B virus in vitro.
ABSTRACT
The effective inactivation of microorganisms in drinking water by Ultraviolet (UV) irradiation is regarded as a new low-cost water treatment method shoeing high removal rate of relatively stable infectious virus particles including poliovirus. In the present study, we examined virus inactivation by UV in various water environments. Samples were collected from finished water and surface water, and tested for turbidity. UV dose of 18, 22, 30, 36 and 40 milli-Joule (mJ)/cm2 were used by combination of 2 mW/cm2 UV intensity and time of 9, 11, 15, 18 and 20 second. Depths of water were fixed at 0.37 cm and 8 cm, and virus titers were shown by plaque forming unit (PFU). Poliovirus was inactivated to 99.0% by 18 mJ/cm2 of UV dose in the condition of 0.08 Nephelometry Turbidity Unit (NTU) and 8 cm depth of water. Poliovirus at 30 mJ/cm2 of UV dose under the same condition was inactivated to 99.7%. Furthermore, Poliovirus at 56.60 NTU and 8 cm depth of water was inactivated to 92.0% and 98.5% by 18 mJ/cm2 and 30 mJ/cm2 of UV dose, respectively. The degrees of virus inactivation were dependent upon the UV dose, the turbidit, y and the depth of water. In conclusion, introduction of UV disinfections can be considered in drinking water purification systems in case reasonable engineering support is possible.
Subject(s)
Drinking Water , Nephelometry and Turbidimetry , Poliovirus , Shoes , Virion , Virus Inactivation , Water Purification , WaterABSTRACT
The effective inactivation of microorganisms in drinking water by Ultraviolet (UV) irradiation is regarded as a new low-cost water treatment method shoeing high removal rate of relatively stable infectious virus particles including poliovirus. In the present study, we examined virus inactivation by UV in various water environments. Samples were collected from finished water and surface water, and tested for turbidity. UV dose of 18, 22, 30, 36 and 40 milli-Joule (mJ)/cm2 were used by combination of 2 mW/cm2 UV intensity and time of 9, 11, 15, 18 and 20 second. Depths of water were fixed at 0.37 cm and 8 cm, and virus titers were shown by plaque forming unit (PFU). Poliovirus was inactivated to 99.0% by 18 mJ/cm2 of UV dose in the condition of 0.08 Nephelometry Turbidity Unit (NTU) and 8 cm depth of water. Poliovirus at 30 mJ/cm2 of UV dose under the same condition was inactivated to 99.7%. Furthermore, Poliovirus at 56.60 NTU and 8 cm depth of water was inactivated to 92.0% and 98.5% by 18 mJ/cm2 and 30 mJ/cm2 of UV dose, respectively. The degrees of virus inactivation were dependent upon the UV dose, the turbidit, y and the depth of water. In conclusion, introduction of UV disinfections can be considered in drinking water purification systems in case reasonable engineering support is possible.
Subject(s)
Drinking Water , Nephelometry and Turbidimetry , Poliovirus , Shoes , Virion , Virus Inactivation , Water Purification , WaterABSTRACT
Hantaviruses are negative-strand RNA viruses that contain three segmented (L/M/S) genome and belong to the genus hantavirus of the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, hantaviruses have a possibility to produce reassortants that containing genomic sets mixed with different segments originated from both parental viruses during the genetic interaction. To investigate whether this phenomenon occurs in vitro, Hantaan (HTN) and Seoul (SEO) viruses were co-infected into Vero-E6 cells and virulent Maaji (MAA) virus was superinfected into avirulent Prospect Hill (PH) virus-infected Vero-E6 cells, respectively. To select only reassortants among progeny viruses, well separated plaque clones were analyzed by multiplex RT-PCR. The putative reassortant viruses detected by 1st multiplex RT-PCR were plaque-purified three times and confirmed by 2nd multiplex RT-PCR. Only 3 reassortants like HTN/HTN/SEO, SEO/HTN/HTN and SEO/HTN/SEO and only 2 reassortants like PH/MAA/MAA, MAA/MAA/PH as designated in order of L/M/S of genomic segments have been identified so far. These results indicate that genetic reassortment can be induced by mixed-infection of two more distantly related serotypes of hantavirus. Interestingly, reassortant SEO/HTN/SEO containing HTN viral M RNA segment is isolated more frequently. This implies that preferential selection of M genome segments occurred when RNA genomes were packaged into virion and also the process of packaging of RNA segments into virion is not random phenomenon. These reassortants would be helpful to know whether genetic reassortment is dependent on genetic distance between hantaviruses and which viral RNA segment plays an important role in coding for virulence marker. Therefore, genetic reassortment can be useful genetic tool to understand genetical, and biological function of hantavirus.
Subject(s)
Humans , Bunyaviridae , Clinical Coding , Clone Cells , Genome , Orthohantavirus , Parents , Product Packaging , Reassortant Viruses , RNA , RNA Viruses , RNA, Viral , Seoul , Virion , VirulenceABSTRACT
Objective To develop plaque assay for SARS virus, in order to provide a reliable means for SARS research. Methods SARS virus BJ 01 strain in various dilutions was inoculated to Vero E6 cells, the cells were then covered with nutritious agar. The titers of the plaque were measured by Dullbecco R method. Results The plaque of SARS virus appeared on the third day after the cell cultures infected with virus. The plaque was round in shape, 2.5-3 mm in diameter. Conclusion The plaque assay developed in present study was stable and regular, and it could be used in SARS research.