key: cord-1012673-0yd6evou authors: Tsoleridis, Theocharis; Ball, Jonathan K. title: Discovery of Novel Coronaviruses in Rodents date: 2020-05-11 journal: Coronaviruses DOI: 10.1007/978-1-0716-0900-2_2 sha: 5dca8beb1f3f64f9a5994cb80cedde498ec2062a doc_id: 1012673 cord_uid: 0yd6evou The recent emergence of SARS, SARS-CoV2 and MERS and the discovery of novel coronaviruses in animals and birds suggest that the Coronavirus family is far more diverse than previously thought. In the last decade, several new coronaviruses have been discovered in rodents around the globe, suggesting that they are the natural reservoirs of the virus. In this chapter we describe the process of screening rodent tissue for novel coronaviruses with PCR, a method that is easily adaptable for screening a range of animals. Rodents are known to be an important source of emerging viral infections [1] . Rodentia include approximately 2200 species such as voles, mice, and rats and is the single largest mammalian order comprising~40% of all mammals [2, 3] . Historically, virus discovery relied on relatively inefficient in vitro or in vivo virus isolation methods. These led to the identification of two rodent coronaviruses (CoVs); rat sialodacryoadenitis coronavirus (SADV) [4] and murine hepatitis virus (MHV) [5] , both of which are from the same viral species within the Betacoronavirus genus. However, the advent of degenerate primer PCR and unbiased viral metagenomics has significantly enhanced our ability to detect novel viruses, and their use has led to the discovery of numerous alpha-and betacoronaviruses in a range of rodent species, including field voles, bank voles, rats, and mice in East Asia and Europe [6] [7] [8] [9] [10] [11] . These findings are paving the way toward a better understanding of the longer-term evolution and origins of these important viral species [12] . In this chapter we describe a degenerate primer PCR method that we have used [10] to detect coronaviruses in a variety of tissues obtained from rodent species sampled postmortem. EDTA (or equivalent commercial ones such as Thermo Scientific 6Â DNA Loading Dye). 4. Tris-acetate-EDTA (TAE): 40 mM Tris-acetate and 1 mM EDTA (pH 8.3) buffer containing 0.5 μl/ml ethidium bromide (from a 10 mg/ml stock) (see Note 6). 1. Acquire ethical approval from relevant committees to trap, euthanize, and handle wild rodents. 2. Wild rodents can either be live trapped and euthanized later, or snap-killed by the trap. In either case, the carcasses should be stored as soon as possible at À80 C. 3. In a Class I biosafety cabinet, the carcasses should be dissected using disposable scalpels and collect the organs of interest (in this case liver and intestine). 4. The organs should be placed in a tube containing a preservative such as RNAlater and stored immediately at À80 C until they are processed for RNA extraction. 1. All the extractions from mammalian tissues should be performed inside a Class I biosafety cabinet to reduce any safety risk while handling the tissues and to avoid contamination. 2. Remove the tissue samples from the freezer, and once thawed remove from the preservative and place the sample in a petri dish (see Note 7). 3. Using a pair of tweezers, tease a section of tissue measuring approximately 1 mm 3 in volume from the tissue sample. Put the remaining tissue back in the tube with the preservative and store it at À80 C. 4. Proceed with the extraction following the steps of the extraction kit protocol. 4. Analyse the PCR products by electrophoresis through a 2% agarose gel in 1ÂTAE-EtBr at 90 V for 40 min. Coronavirusspecific PCR products should appear as a band of approximately 440 bp. 5 . Any PCR positive samples should be sent for Sanger sequencing using the sense and/or antisense PCR primers (see Note 13). 1. Seal the casting tray and add the combs of interest. 2. To prepare a 2% agarose gel add 2 g of agarose in 100 ml of 1Â TAE buffer (see Note 6). 10. Always be careful when handling EtBr and dispose of as per local regulations. There should be a dedicated space or room for preparing and running agarose EtBr gels. 1. For best quality RNA and virus retrieval, the rodent carcasses should be immediately frozen after their death. As soon as they are dissected, each tissue should be placed in a tube containing a preservative such as RNAlater (to preserve the RNA and prevent degradation) and stored immediately at À80 C. The tissues and the RNA should always be placed on ice when handled. Avoid repeated freeze-thaw cycles as they contribute to the rapid degradation of the nucleic acid. 2. There are several methods for RNA extraction, such as phenolchloroform and TRIzol. However, it is much easier to use a commercial extraction kit. We routinely use GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) which gives us good RNA recovery. 3. For the purpose of coronavirus discovery, cDNA synthesis with either random hexamers or a specific primer is acceptable. Random hexamers provide the flexibility of performing several PCRs directed at different targets such as different viruses or housekeeping genes for quality control. Primer-specific cDNA, on the other hand, is less flexible but is reportedly more sensitive. A PCR targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene (or any other housekeeping gene) is essential to assess the quality of the cDNA. 5. There are several published primer-sets for coronavirus screening [13] [14] [15] [16] [17] . However, in this chapter we describe the use of Woo et al. primer-set [13] . Although it was designed to detect the Human HKU-1 CoV, which is a betacoronavirus, it has been shown to detect alphacoronaviruses [10] . 6. It is easier to buy a commercially available 10Â TAE buffer and dilute to a final concentration of 1Â. 7. RNA is very sensitive and degrades at room temperature; therefore, all the RNA work should be done on ice to keep it stable. 8. The purity of the RNA sample will impact on downstream applications such as cDNA synthesis. Therefore, it is essential to aim for an RNA sample free from contaminants. 9 . All the PCR preparation should be performed in a dedicated pre-PCR room where there are no traces of amplicons or plasmids. The amplification and all the post-PCR handling should be done in separate rooms as far away from the pre-PCR room as possible. Thus, the risk of contamination is minimized. 10. The maximum amount of cDNA used in a PCR should not be more that 1/10th of the total PCR volume. If the user wants to add more cDNA template, then the reaction volume should be increased accordingly. 11 . In this chapter we describe the conventional PCR method. Quantification of the template is possible with quantitative real-time PCR by doing serial dilutions and using a housekeeping gene as a reference. 12. Sometimes virus titers in a sample can be very low and might not be detected using low levels of template. Larger-volume PCRs, utilizing increased template can be beneficial in these cases. This approach can also be applied to rescreen potential hits that have yielded low amounts of product (as indicated by the presence of a faint band of the expected size following agarose gel electrophoresis) in the initial screening PCR. 13. Nowadays, Sanger sequencing is commercially available and very affordable. 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