Optimizing Methods To Extract RNA From Murine Fecal Pellets To Quantify And Compare Shedding And Transmission Of MNV Strains
Abstract
Norovirus being one of the leading causes of foodborne gastroenteritis and it is critical to understand the transmission, so the knowledge can be applied in protection and prevention in the spread of disease. Here a comparative analysis of fecal shedding of MNV-1, MNV-3 and CR-6, three murine norovirus strains was carried out to analyze amount of virus in stool. Being a virus that infects the gut exclusively, the transmission is by the fecal oral route, so the study facilitates understanding infection rates in mice. The aim of this study was to optimize a method of extracting RNA from murine fecal pellets a lesser invasive way of analyzing viral titers than gut tissue analysis. The findings indicate that RNA extraction of the viral genome from the Zymo kit yields purer results in comparison to the Qiagen kit as viral multiplication titers show a disparity in the control and the samples of infected mice.
Introduction
Noroviruses are one of the leading causes of non-bacterial gastrointestinal disorders around the world that are estimated to be extremely significant emerging pathogens. They are the leading cause of outbreaks of childhood diarrhea globally. It is an RNA Virus of the Caliciviridae family and is an enteric single stranded RNA (+ strand) virus. 2 They result in about 685 million cases of diseases every year and result in 200, 000 deaths globally each year. Those affected in the group include mostly children below the age of 5 and result in around 50, 000 deaths each year. They consist of a group of single stranded positive sense RNA non- enveloped virus. They are generally classified into 5 different genetic clusters or genogroups out of which I, II and IV affect humans and V after being isolated in mice, is being used as a study model. It was observed that MNV-3 attenuation is maintained as per studies carried wherein he infection was by oral gavage. The results showed from the figures cited from the study that MNV-1 as a strain is more virulent than MNV-3, based on the survival kinetics, weight loss in mice and fecal consistencies. Hence understanding the transmission kinetics becomes imperative.
There are 7 major genotypes of Noroviruses containing different strains. Group I, II and IV cause disease in humans. Group V are Murine Noroviruses. Murine Noroviruses Strains MNV-1, MNV-3, CR-6. Murine Noroviruses as model organisms for understanding pathogenesis of human NorovirusesHuman norovirus previously known as the Norwalk virus was first identified in a gastroenteritis outbreak in the region of Norwalk during an outbreak.
Important characteristics of MNV is the analysis of pathogenicity of the viral strain. There are specific properties that MNV-1, MNV-3 and CR-6 share with Caliciviridae in general and then specific3 coat protein and structure lead to comparing the different strains.
Materials and methods
RAW Cells RNA extraction and RAW 264.
RAW 264. 7 are cells that are mediums for multiplying virus MNV-1 were seeded with MNV-1 and then viral genome was extracted, and viral titers were quantified using qPCR.
Fecal Sample Collection
The collection of feces was from MNV1 infected mice at days 1 and 2 post infection. The mice under ACS guidelines were handled in the mouse room designated for Norovirus work. The method included, collection of fresh fecal pellets by anal stimulation by abdominal hand motion. Approximately two fecal pellets were collected per animal, with two fecal pellets obtained from two mice in two cages that had not been infected and two pellets obtained from 2 infected mice in cages.
Fecal Sample Preparation
The fecal samples were collected in sterile tubes with lot number- that contained sterile Zirconia beads. The tubes were weighed after adding the Zirconia beads and then autoclaved to ensure sterility. Addition of the fecal pellets was done in a laboratory biosafety cabinet following ACS rules and procedures. After placing the feces into individual zirconia bead carrying tubes they were transported to the Biosafety level-2 cabinets for processing them further as per requirements of the individual Qiagen and Zymo protocol. Viral RNA extraction by feces using the Qiagen kitFecal pellets were collected from mice that were infected with MNV-1. The fecal pellets tubes were labelled as infected and mock tubes. They were placed on ice right after being excreted by the animal by methods that are stated above. Pellets from uninfected mice were placed as negative controls each in the tubes. They were weighed after placing the fecal pellets in the tubes. The weights were noted for calculating the log viral genome titers per gram. Bead beating was carried out twice for a 1-minute time span the tubes in the bead beater machine for 1 minute and cool on ice for 2 minutes and repeat the beating for one more time. After this centrifugation was carried out for 30 seconds at 5000rpm. Buffer AVL containing carrier RNA with concentrations defined by manufacturer are defined ahead is placed on ice after preparation. 560 μl of prepared Buffer AVL containing carrier RNA was added into a 1. 5 ml microcentrifuge tube. There was addition of 140 μl of fecal supernatant fluid in Buffer AVL–carrier RNA in the microcentrifuge tube that was mixed by pulse-vortexing for 15s. To ensure efficient lysis, it was essential that the sample was mixed thoroughly with Buffer AVL to yield a homogeneous solution. This was incubated at room temperature (15–25°C) for 10 minutes. Viral particle lysis is completed after lysis for 10 min at room temperature. Longer incubation times usually have no effect on the yield or quality of the purified RNA. There was brief centrifugation of the tube to remove drops from the inside of the lid. After which 560 μl ethanol (96–100%) was added to the sample and mixed by pulse-vortexing again. Only ethanol is used in the process since other alcohols may result in reduced RNA yield and purity. Carefully 630μl of the solution was pipetted to the QIAamp Mini column that is commercially provided by the manufacturer without wetting the rim. After which centrifugation was carried out at 6000 x g (8000 rpm) for 1 min. The QIAamp Mini column was placed into a clean 2 ml collection tube twice. The QIAamp Mini column, which now had RNA bound, was now washed with 500 μl Buffer AW1 with centrifuge at 6000 x g (8000 rpm) for 1 min. The QIAamp Mini column was placed in a clean 2 ml collection tube that was provided. After which 500 μl Buffer AW2 was added after which a full speed elution was carried out. There was elimination of AW2 carryover. After this step water was added to the column and centrifugation was carried out to elute the RNA.
Viral RNA extraction by feces using the Zymo kit
The 1mm sterile bead tubes were taken and labelled as infected and mock. The weights were recorded. The fecal pellets were kept on ice and placed in the hood. After collection a pellet was placed in each of the bead tubes. Decontamination was carried out and tubes were brought out of the hood. After which weighing was carried out. Viral RNA Buffer was added to each fecal pellet, and all the tubes were bead beated for 2 minutes at maximum speed. The tubes were brought to the RNA processing area and the mixture was transferred into a Zymo-Spin™ IC Column along with a Collection Tube and were centrifuged for 2 minutes. Then this was transferred into a new collection tube. 500μl of Viral Wash Buffer was added to the column, this was centrifuged for 30 seconds and the flow-through was discarded. 500 μl of ethanol (95-100%) was added to the column and centrifugation for 1 minute was carried out to ensure complete removal of the wash buffer. After careful transfer into a centrifugation tube 50μl of DNase/RNase-Free Water was added directly to the column matrix and centrifuged for 30 seconds.
Determination of Nucleic Acid Purity
The A260/A280 ratios were determined using the Nano drop software which are available via Nanodrop ND-1000 Spectrophotometer (Nano- drop Technologies) was used to measure the level of contaminating substances present in the RNA extracted that included other nucleic acids that may be contaminating agents as well as proteins that absorb light at 260 nm and 280 nm. 10Quantification of the viral Genome by qPCRIn the CFX Manager file The X axis represents the cycle number and the Y axis represents the fluorescence in RFU. It is always a sigmoid curve that is a highly quantitative measurement. The faster the RFU reaches the threshold the more target RNA was there to begin with. A lower Ct value means a higher expression. If the RFU curve is more on the left of the graph, then that means that there was a large copy number of target RNA present. It’s the value that will be used for the analysis. The Ct value was set as 42 for the purpose of this study because more cycles of amplification were needed to detect the fluorescence. The primers taken for this study were specific for the MNV genome as seen by the karst lab. The qPCR machine measures the intensity of fluorescence emitted by intercalated SYBR dye at each cycle. During the first cycles, there is not enough fluorescence to be detected, but the reaction rapidly produces more and more amplicons and the fluorescence builds up. The Ct measure is a determined PCR cycle and represents the basic result of a qPCR experience. It’s taken in the exponential phase, where the curve is linear. The threshold (red line) is placed in the linear phase, and the Ct is measured where the PCR curve crosses the threshold.
Statistical Analysis
Statistical Analysis was carried out by means of the PRISM software that indicates the comparative analysis for all purity samples of MNV-1 and MNV-3. All the data plotted was normalized based on the minimum level of background that was detected. Interpretation of qPCR valuesOn the PCR Machine the total cycle number was set to be 42. The amplification that is happening in 42 cycles was estimated to show up in PCR results. As a result, the cDNA amplified in the process was the target. The Ct values was a measure to determine the SYBR dye intercalation in the DNA strands with every successive run and provided fluorescence that the PCR machine measured. The principle was greater the cycle number for the cDNA means smaller the amount was present as it took that many cycles to multiply. We want to see a difference between the values in the RT+ and RT-. As RT- is a control the number of repeats should be very high (the 40s) or N/A that means that no amplification detected in the multiplication of the 42 cycles set. If the RT+ sample has a Ct value equal to or higher than that of its corresponding RT- sample, it must be considered negative for the target gene. Water was added as providing a baseline to show basic impurities. The copy number of viral genomes calculated in the water standard was considered the limit of detection for the plate. Any samples with a Ct value equal to or higher than that of water are considered negative for the target gene.
Results and Discussion
Optimization of a method of purification for viral RNA from feces was done using the two commercially available methods11 that were available. As proof of concept the first result determined was of RAW Cells to visualize if there is any degradation of RNA inherently by the kits Qiagen and Zymo where two bands for RAW 264. 7 cells can be visualized at 4000bp and 1500 base pairs respectively. As shown in the figure there is no degradation of cellular RNA.
It is observed that the two kits provide comparable quantification of viral genome in samples known to contain virus after multiplication in RAW cells. Zymo yields a result for A260/A280 which is in the range of 1. 8 to 2. 0 and shows a concentration of viral genome between 0 and 50 ng/ul and Qiagen shows a range of values between 1. 8 to 3. 1 for the A260/A280 ratio whereas yields values ranging from between 65 to 250 ng/ul of the RNA concentrations. Zymo showing tighter results closer to two indicates better quality levels whereas the Qiagen indicates better quantification values.
Comparing the quantification of log viral genome/ gm of stool for both MNV-1 and CR6-1 we see that after baseline elimination of the minimum value of signal that we see in our PCR plates for MNV-1 the viral titers obtained from stool were very low. As a positive control the CR-6 strain infected IFNAR -/- mice stool samples were collected wherein, reproducing the experiment one day post infection we wanted to see a 6 fold increase 12 in the amount of viral RNA for the Zymo kit which was observed. The study indicated that These are the same strain of mouse we are getting stool from to test qPCR results from fecal RNA extracted using Zymo or Qiagen. The study from which this was taken showed mice infected with CR6 and these feces were collected at 14dpi. There should be consistently high shedding in these mice, so if the results were replicated that would validate that our fecal RNA extraction/qPCR protocol was working.
The data indicates can facilitate in testing important experimental questions, of how much virus is shed comparatively from mice infected with different MNV strains, or how soon does virus begin to be shed and which can give us comparative results.
The conclusion being that the amount of fecal shedding for MNV-1, 1dpi is less as the quantification is low as indicated. CR-6 4dpi shows a 6-fold increase in the amount of viral RNA/ gm of stool and shows a multi fold increase in the viral titers. As a result, there is no degradation being caused of the Viral RNA by the kit itself on extraction from the stool 1dpi. This could indicate variability in viral transmission kinetics that play a key role in understanding the transmission efficiency of the viral RNA.
