A Rapid Way To Titrating Hantaan Virus With Flow Cytometry Method

As a member of genus Hantavirus in the family of Bunyaniridae, Hantaan virus （HTNV） posses a negative-sense RNA genome which consists of three segments, designated the large （L）, medium （M）, and small （S） segments encoding the viral RNA polymerase, two surface glycoproteins （G1 and G2）, and the nucleocapsid protein （NP） respectively. GP contains neutralizing epitopes and type specific epitopes which play an important role in the process of entering the target cells and are major sites sensed and recognized by immune cells, while NP contains group specific epitopes which induce strong humoral and cellular immune response in the body. In their natural hosts （rodents of the families Muridae and Cricetidae）, hantaviruses establish a persistent and subclinical infection, which causes no apparent harm. Hantavirus can be transmitted via aerosolized excreta to humans and causes two acute infectious diseases: hemorrhagic fever with renal syndrome （HFRS） in Eurasia and hantavirus pulmonary syndrome （HPS） or hantavirus cardiopulmonary syndrome （HCPS） in South and North America. Until now, 7 sero/genotypes of hantaviruses have been identified in China. Among them, only HTNV and Seoul virus （SEOV） cause HFRS. Every year, 60 000 to 100 000 HFRS cases have been reported worldwide, mostly from China. During the 58-year period of 1950-2007, a total of 1 557 622 cases of HFRS and 46 427 deaths （3%） were reported in China. Today, HFRS remains a major public health problem in China.The most widely used cell line to propagate HTNV is Vero and Vero-E6 cell which both are cloned African green monkey kidney cells. HTNV grows slowly in Vero-E6 cells where it does not cause any cytopathic effect （CPE）, such as altered shape, cell lysis, inclusion bodies, altered membrane permeability, apoptosis, etc. The infectious titer of the virus usually reaches its peak after 7-14 days of incubation. Another useful way to obtain high titers of HTNV is to culture virus in the brain of suckling mouse.The infectious titer of virus is one of the most important issues in virology research which is usually expressed by plaque forming unit （PFU） or 50% tissue culture infective dose (TCID₅₀). Plaque assay is a classical method to measure the virus titer in which the plaques caused by infectious viruses are calculated. To perform a plaque assay, 10-fold serial dilutions of a virus stock are prepared, and aliquots are inoculated onto confluent cell monolayers. After an incubation period to allow virus to infect cells, the monolayers are covered with a nutrient medium containing a substance, usually agar or methyl cellulose. When cell plates are incubated, the original infected cells release viral progeny which are restricted to neighboring area by the substance. Consequently, each infectious particle produces a circular zone of infected cells named a plaque. And the dyes, usually the neutral red or the crystal violet, are used to stain living cells in order to enhance the contrast between the living cells and plaques which can not be stained. The plaques are counted by naked eyes or under microscope, and the titer of a virus stock can be calculated in PFU per milliliter. However, the plaque assay is usually time-consuming which usually requires 7-10 days or longer time to complete and is strict to experimental conditions, such as cell conditions, the even distribution of cells in the plates, the staining method, etc. Due to many influential factors, it is hard to get the stable and reliable results of plaque assay in different laboratories and at different times.TCID₅₀ assay is a relative stable and statistical way of measuring viral titers. It is also useful for viruses which are not cytopathic or do not produce plaques. To conduct TCID₅₀ assay, host cells are plated and serial dilutions of virus are added. After incubation, the percentage of cell death or positive cells is observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID₅₀ result. There are several statistical methods for analyzing the results, such as Reed-Muench method or Spearman-Karber method. Compared with plaque assay, the TCID₅₀ assay may take up shorter time and the results can be predicted.Flow cytometry （FCM） can be defined as the measurement of physical properties of single particle at a high speed which can obtain a number of different multi-parameters of thousands of cells, including cell number, cell size, cellular granularity, and mean fluorescent intensity （MFI）, etc. FCM has been extended to the field of virology in which FCM has allowed rapid and sensitive detection and quantitation of virus infection. Recently, FCM-based methods have been developed to determine the infectious titers of viruses such as human immunodeficiency virus type 1 （HIV-1）, herpes simplex virus （HSV）, dengue virus （DENV）, influenza virus （IV）, rabies virus （RV）, rotavirus, vaccinia virus （VACV）, adenovirus （AV）, and baculovirus （BV）. However, the use of FCM for the rapid measurement of Hantaviruses infectious titer has not been reported. In the present study, a rapid and sensitive way to titer HTNV 76-118 strain cultured in Vero-E6 cells with FCM-based method was developed and compared with indirect immunofluorescence assay （IFA）.Results: 1. The propagation of HTNVHTNV 76-118 strain was inoculated onto Vero-E6 cells for propagation. After 8-10 days of culture and several rounds of passages, the cells were frozen and thawed three times. The supernatant was centrifuged to pellet cellular debris, and was aliquoted and stored at -80℃.2. Plaque assayThe virus stock was serially diluted using 10-fold dilutions and added to Vero-E6 cells grown in six-well plates. After adsorption, methyl cellulose overlays were added to the Vero-E6 cells, and the plates were incubated at 37℃in the presence of 5% CO2 for 10 days. Then, the cells were fixed by methanal and stained by crystal violet solution. The plaques were observed and counted by naked eyes.3. Titration of HTNV by IFAVero-E6 cells were inoculated with 10-fold of serial dilution of virus stock. After incubation for 12 h, 24 h, 36 h, 48 h and 72 h at 37℃in 5% CO₂, IFA was performed. The positive cells were observed using an inverted fluorescence microscope. The Reed-Muench method was used to calculate the TCID₅₀. We found that the optimal time point to perform IFA was at 36 h after incubation, and the titer of HTNV 76-118 strain was 1.12×10⁶ TCID₅₀/mL.4. Titration of HTNV by FCMVero-E6 cells were infected by 10-fold dilution of HTNV 76-118 strain and incubated for 12 h, 24 h, 36 h, 48 h and 72 h at 37℃in the presence of 5% CO₂. Then cells were harvested using typsin and resuspended in monoclonal antibody 3G1 against HTNV NP, then in fluorescein isothiocyanate （FITC）-labeled goat anti-mouse secondary antibody. The samples were analyzed on a FACScan flow cytometer to measure the percentage of positive cells. We found that the optimal time point to measure virus titer was at 36 h after infection, and the percentage of the positive cells was （10.06±0.42） %. The infectious unit （IU） determined by FCM was 1.9×10⁶/mL, and the lower limit of FCM method was 47.5 IU/mL.Conclusion: Compared with the classical plaque assay and TCID₅₀ method, FCM is a rapid and sensitive way to detect and titrate HTNV.