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Post by Admin on May 14, 2020 8:27:57 GMT
In most mammals, there are certain genes that can be likened to an "alarm system", informing an organism when foreign material such as a virus enters the body and triggering an immune response. A new study published in the journal Frontiers in Immunology suggests that pangolins, despite being mammals, lack two of the genes involved in such an alarm system. "Our work shows that pangolins have survived through millions of years of evolution without a type of antiviral defense that is used by all other mammals," Co-author Leopold Eckhart, PhD, from the Medical University of Vienna in Austria, says. This research is timely as pangolins can be carriers of coronaviruses, yet they appear to be able to withstand the virus. If they lack the antiviral defense system used by all other mammals, what alternative protective mechanism is at play? According to Eckhart, this question warrants further investigation: "Further studies of pangolins will uncover how they manage to survive viral infections, and this might help to devise new treatment strategies for people with viral infections." Technology Networks interviewed Eckhart to learn more about RNA sensor genes, the significance of the new research study and the challenges in this research field. Molly Campbell (MC): You conducted a genomics analysis study of three species of pangolins. What are these species, and why did you choose them? How many species of pangolins exist? Leopold Eckhart (LE): There are 8 species of pangolins and the genome sequences of three species have been determined and made publicly available. We investigated the species available: Malayan pangolin (Manis javanica), Chinese pangolin (Manis pentadactyla) and the tree pangolin (Manis tricuspis). MC: For our readers that may be unfamiliar, please can you discuss what RNA sensor genes are? LE: RNA viruses such as coronaviruses and influenza viruses depend on genetic information in the form of RNA. This viral RNA or RNA molecules that appear during viral replication are sensed by infected cells in humans and other mammals. Genes like IFIH1 and ZBP1 control the formation of particular RNA sensors. MC: Please can you provide an overview of your experimental design? LE: The SARS-CoV2 pandemic started by spill over of the virus to humans from another species. As pangolins were discussed as possible intermediate hosts of this virus, we investigated if something is special about the interaction of pangolins and viruses. We did not have access to pangolins but the complete genome information of three species of pangolins had been determined and made publicly available by other researchers. Our study was done by comparing gene sequences without any experiments on live animals. MC: Your research found that IFIH1, a sensor of intracellular double-stranded RNA, has been inactivated by mutations in pangolins. Likewise, Z-DNA-binding protein (ZBP1), which senses both Z-DNA and Z-RNA, has been lost during the evolution of pangolins – please can you expand on the significance of these findings? LE: IFIH1 and ZBP1 belong to an antiviral defense system that was previously considered essential for mammals. It is surprising that these genes have been lost during the evolution of any mammalian species. Survival of pangolins without IFIH1 and ZBP1 suggests that there are other mechanisms of antiviral defense. MC: In the press release you say: "Our work shows that pangolins have survived through millions of years of evolution without a type of antiviral defense that is used by all other mammals" – meaning it's possible that they survive viral pathogens via an alternative mechanism. Although this wasn't explored in this study, do you have any hypotheses on this? LE: Our data suggest that pangolins can either respond to RNA viruses through another sensor (for example, RIG-I) or that they tolerate infections. Tolerance would mean that the body allows the virus to grow to some extent without initiating an immediate and strong response that would potentially also damage its own tissues. It is possible that changes in the metabolism of infected animals reduce the growth of the virus but currently we do not know how pangolins control RNA virus infections. MC: Why is advancing our understanding of the immune system mechanisms of pangolins relevant in regard to the current COVID-19 pandemic? LE: Many severe cases of COVID-19 are characterized by an overreaction of the immune defense, known as a cytokine storm. A better understanding of the antiviral response of pangolins may help to find therapies that avoid inappropriate defense reactions in patients with COVID-19. MC: What challenges exist in this field of research? LE: If this research should be translated into new therapies, studies of live animals are necessary and only very few researchers are able to study exotic species such as pangolins. We do not know what the next emerging infectious disease will be – therefore, we should try to improve our understanding of the interactions of many different host species and their viruses and bacteria. We are doing basic research and we hope that support for comparative biology will increase. Leopold Eckhart was speaking to Molly Campbell, Science Writer, Technology Networks. Reference: Heinz Fischer, Erwin Tschachler and Leopold Eckhart. (2020). Pangolins lack IFIH1/MDA5, a cytoplasmic RNA sensor that initiates innate immune defense upon coronavirus infection. Frontiers in Immunology. DOI: 10.3389/fimmu.2020.00939.
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Post by Admin on May 15, 2020 7:15:45 GMT
The current hypothesis goes something like this: SARS-CoV-2 passed through a mystery animal host in its suspected evolutionary journey from bats to humans. Critically endangered pangolins have been a favoured candidate for this intermediary host, but now a genomic analysis led by geneticist Ping Liu from Guangdong Academy of Science in China has provided evidence this may not be the case. SARS-CoV-2 belongs to the Betacoronavirus genus of coronaviruses; this group of coronaviruses primarily infects mammals, and the new study suggests that pangolins are indeed natural hosts for them. The team pieced together almost an entire genome of the coronaviruses found in two sick Malayan pangolins (Manis javanica). They called the coronavirus isolated from these critically endangered animals pangolin-CoV-2020. Its final sequence had 29,521 base pairs, only slightly shorter than the 30,000-odd base pairs making up SARS-CoV-2. The resulting genome displayed a 90.32 percent sequence similarity to SARS-CoV-2 and 90.24 percent to the Rhinolophus affinis bat coronavirus BatCoV-RaTG13, which still remains the closest known relative to SARS-CoV-2, with a match of 96.18 percent. But the sequence similarities don't reflect the full story. The genetic instructions for the all-important protein spike of the SARS-CoV-2 virus matched more between the bat and human coronavirus than the pangolin one. However, the pangolin virus essentially shares the same ACE2 binding receptor as that used by the COVID-19 virus - the part of the spike that allows the virus to enter and infect human cells. This was also found in another study that is still undergoing review, and led to suggestions that the human coronavirus may be a type of hybrid (a chimera) between a bat and a pangolin virus. Liu's team also thinks these similarities may indicate that a recombination event occurred somewhere in the evolution of these different viruses - where the viral genomes exchanged pieces of their genetic materials with each other. However, their analysis of the evolutionary relationship between the three viruses did not support the idea that the human version evolved directly from the pangolin one. "At the genomic level, SARS-CoV-2 was also genetically closer to Bat-CoV-RaTG13 than pangolin-CoV-2020," they wrote in their paper. There are clearly still a lot of unknowns. With well over 4 million confirmed cases around the world, and a death toll still increasing sharply, the need to understand as much as we can about this virus just continues to intensify. However, one thing all these genetics studies have firmly ruled out is the idea that the virus was lab made. As for the pangolins, they had been rescued by the Guangdong Wildlife Rescue Center after being smuggled for black market trade, and sadly succumbed to their illness. Liu's team could not determine if their deaths were linked to the coronavirus they found. But perhaps a little good can arise from all this, at least for the world's most trafficked mammal, with the researchers concluding: "Minimising the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans." The new research was published in PLOS Pathogens: journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1008421
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Post by Admin on May 16, 2020 7:02:52 GMT
Pangolins Lack IFIH1/MDA5, a Cytoplasmic RNA Sensor That Initiates Innate Immune Defense Upon Coronavirus Infection Heinz Fischer1, Erwin Tschachler2 and Leopold Eckhart2* 1Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria 2Department of Dermatology, Medical University of Vienna, Vienna, Austria Zoonotic infections are an imminent threat to human health. Pangolins were recently identified as carriers and intermediate hosts of coronaviruses. Previous research has shown that infection with coronaviruses activates an innate immune response upon sensing of viral RNA by interferon-induced with helicase C domain 1 (IFIH1), also known as MDA5. Here, we performed a comparative genomics study of RNA sensor genes in three species of pangolins. DDX58/RIG-I, a sensor of cytoplasmic viral RNA and toll-like receptors (TLR) 3, 7, and 8, which bind RNA in endosomes, are conserved in pangolins. By contrast, IFIH1 a sensor of intracellular double-stranded RNA, has been inactivated by mutations in pangolins. Likewise, Z-DNA-binding protein (ZBP1), which senses both Z-DNA and Z-RNA, has been lost during the evolution of pangolins. These results suggest that the innate immune response to viruses differs significantly between pangolins and other mammals, including humans. We put forward the hypothesis that loss of IFIH1 and ZBP1 provided an evolutionary advantage by reducing inflammation-induced damage to host tissues and thereby contributed to a switch from resistance to tolerance of viral infections in pangolins. Introduction Emerging infectious diseases represent a major challenge to public health. The transmission of pathogens from other vertebrate animals to humans is of particular concern because the resulting diseases, known as zoonoses, have caused major epidemics in the past and continue to pose enormous threats to the human population, as exemplified by the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak (1, 2). In a broader sense, viral and bacterial pathogens are among the strongest drivers of evolutionary change and the genomes of vertebrate species have been shaped, to a large extent, by adaptations to pathogens. To cope with viral infections, vertebrate species have evolved response strategies which can be classified into resistance and tolerance (3). Resistance depends on the efficient sensing of the infection and mounting of antiviral responses that involve programmed death of infected cells, suppression of viral replication, inflammation and the establishment of adaptive immunity. However, pathogens can also trigger overreactions of the immune system which cause more harm to the individual than the infectious agent itself (4, 5). Therefore, tolerance to infections has evolved as an alternative response of many hosts to specific pathogens (6, 7). In this scenario, the pathogens are not efficiently eliminated but the pathogen or defense-induced damage to the host is reduced. Tolerance does not depend on, or is even impeded by, the early sensing of pathogen-associated patterns (PAMPs) and its mechanisms of protection are not yet fully understood (6, 8, 9). Species that tolerate infections can carry a high burden of infectious agents, and therefore may be important reservoirs for transmissions to other species. This notion is supported by the finding that bats tolerate many viral infections some of which have spread to humans causing zoonoses such as Ebola, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) (7). Pangolins have been identified, besides bats, as a possible source of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19) (10–14). Eight species of pangolins form the mammalian order Pholidota which is most closely related to Carnivora (cat-like and dog-like carnivorans). They are insectivorous and toothless animals whose body is largely covered by keratinous scales. The immune defense of pangolins has not been characterized yet except for reports on the deficiencies of TLR5, a receptor of bacterial flagellin (15) and interferon-ε, an antiviral cytokine of epithelia (16, 17). The receptor of SARS-CoV-2, i.e., angiotensin I converting enzyme 2 (ACE2) is conserved in pangolins (18) and coronaviruses isolated from pangolins have a receptor binding domain in their spike protein that is uniquely similar to that of SARS-CoV-2 (10, 19). Antiviral defense of vertebrates is initiated by sensors of viral nucleic acids. Infections with RNA viruses, such as coronaviruses, influenza viruses and Ebolavirus activate sensors of extracellular or endosomal RNA, such as TLR3, TLR7, and TLR8 (20), and sensors of intracellular RNA, such as IFIH1/MDA5, ZBP1, and DDX58/RIG-I (21–28). These sensors are specific for different subtypes of RNAs that constitute the viral genome or appear during viral replication or gene expression and they activate distinct cellular and organismal responses, such as necroptotic cell death, interferon signaling and inflammation (27, 29). Here we report a unique degeneration of the innate immune response against RNA viruses in pangolins. Materials and Methods The following genome sequences of pangolin species were analyzed: Malayan pangolin (Manis javanica), Assembly: ManJav1.0 (GCA_001685135.1), submitted by The International Pangolin Research Consortium (16); Chinese pangolin (Manis pentadactyla), Assembly: M_pentadactyla-1.1.1 (GCA_000738955.1), submitted by Washington University; Tree pangolin (Manis tricuspis), Assembly: ManTri_v1_BIUU (GCA_004765945.1), submitted by Broad Institute. Gene annotations were available in GenBank only for M. javanica (NCBI Manis javanica Annotation Release 100). Shared order of gene arrangement (synteny) in the Malayan pangolin (M. javanica), cat, dog, cattle, mouse, and human was assessed by comparison of gene loci that were downloaded from GenBank at www.ncbi.nlm.nih.gov/gene/ (last accessed on 27 March, 2020). In addition, Basic Local Alignment Search Tool (BLAST) was used to find regions of local similarity between sequences (30). Amino acid and nucleotide sequence were aligned with the Multalin software (31). Divergence times of evolutionary lineages were obtained from the Timetree website (www.timetree.org) (32). IFIH1 Is a Pseudogene in Pangolins IFIH1, also known as melanoma differentiation-associated protein 5 (MDA5), binds to double-stranded RNA in the cytosol and signals through mitochondrial antiviral-signaling protein (MAVS) to activate expression of interferons and to induce inflammation (33). IFIH1 senses cytoplasmic RNA of coronaviruses and other viruses (27, 34, 35). Comparison of the IFIH1 gene locus showed conservation of the arrangement of IFIH1 relative to the neighboring genes in mammals (Figure 1A). In the Malayan pangolin, IFIH1 is inactivated by more than 10 frameshift and in-frame stop mutations. In silico translation of the pangolin IFIH1 pseudogene (GenBank gene ID: 108398082) and alignment of the resulting amino acid sequence to that of human IFIH1 showed numerous disruptive mutations (Figure S1A). An open reading frame in exon 1 of the Malayan pangolin encodes a theoretical protein that lacks essential domains and has only 100 amino acid residues whereas functional IFIH1 proteins consist of more than 1,000 amino acid residues (Figure S2). Detailed comparative analysis of exon 1 showed the presence of multiple frameshift mutations and in-frame stop codons in the IFIH1 genes of Malayan, Chinese and tree pangolins (Figure 1B). One of the frameshift mutations and one premature stop mutation are shared by all three species, suggesting that these mutations have already been present in their last common ancestor that lived more than 20 million years ago (32). FIGURE 1 Figure 1. IFIH1 is a pseudogene in pangolins. (A) Gene locus of IFIH1 in the pangolin (M. javanica), cat, and human. Genes are represented by arrows pointing in the direction of transcription. A sequence gap is located between FAP and IFIH1 in the pangolin. (B) Inactivating mutations in exon 1 of IFIH1 in three species of pangolins. Nucleotide sequences of pangolins, cat and human were aligned. The coding sequence of human IFIH1 was translated and the amino acid sequence is shown below the nucleotide sequences. Frameshift mutations and in-frame stop codons are highlighted by red shading. Nucleotides conserved in more than 50% of the sequences are indicated by blue fonts. Nucleotides in the flanking region of the first intron are shown with gray shading. Nucleotide sequence accession numbers (GenBank): Human (NC_000002.12, nucl. 162317845-162318307, compl.), cat (NC_018730.3, nucl. 154125204-154125666, compl.), Malayan pangolin (NW_016533891.1, nucl. 53417-53871, compl.), Chinese pangolin (JPTV01003556.1, nucl. 39028-39476, compl.), tree pangolin (SOZM010146646.1, nucl. 741-1188, compl.). Abbreviations: compl., complementary; nucl., nucleotide numbers; Mj, Manis javanica; Mp, Manis pentadactyla; Mt, Manis tricuspis.
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Post by Admin on May 16, 2020 20:30:10 GMT
ZBP1 Is a Pseudogene in Pangolins ZBP1 binds to left-handed double helix structures of DNA and RNA (Z-DNA and Z-RNA) and thereupon triggers necroptosis and inflammation through interactions with receptor-interacting serine/threonine-protein kinase 3 (RIPK3) (36). Influenza virus and other viruses induce ZBP1-mediated innate immune responses in humans and mice (24, 25). Comparison of the ZBP1 gene locus showed conservation of the arrangement of ZBP1 relative to the neighboring genes in mammals (Figure 2A). In the Malayan pangolin, ZBP1 is inactivated by multiple in-frame stop codons. In silico translation of the pangolin ZBP1 pseudogene (GenBank gene ID: 108390931) and alignment of the resulting amino acid sequence to that of human ZBP1 showed premature termination of the translation product and lack of the carboxy-terminal half of the protein (Figure S1B). Mutations that prevent the production of a functional protein were found in all segments of the ZBP1 pseudogene of the Malayan pangolin. The nucleotide sequence alignment of ZBP1 exon 4 shows the presence of in-frame stop codons in three species of pangolins (M. javanica, M. pentadactyla, M. tricuspis) (Figure 2B). FIGURE 2 Figure 2. ZBP1 is a pseudogene in pangolins. (A) Gene locus of ZBP1 in the pangolin (M. javanica), cat, and human. Genes are represented by arrows pointing in the direction of transcription. (B) Inactivating mutations in exon 4 of ZBP1 in three species of pangolins. Nucleotide sequences of pangolins, cat and human were aligned. The coding sequence of human ZBP1 was translated and the amino acid sequence is shown below the nucleotide sequences. In-frame stop codons are highlighted by red shading. Nucleotides conserved in more than 50% of the sequences are indicated by blue fonts. Nucleotides in the flanking region of the introns are shown with gray shading. Nucleotide sequence accession numbers (GenBank): Human (NC_000020.11, nucl. 57614878.0.57615077, compl.), cat (NC_018725.3, nucl. 5721658-5721857), Malayan pangolin (NW_016529116.1, nucl. 156452-156651, compl.), Chinese pangolin (JPTV01006633.1, nucl. 23295.0.23494), tree pangolin (SOZM010101098.1, nucl. 532-731). Abbreviations: compl., complementary; nucl., nucleotide numbers; Mj, Manis javanica; Mp, Manis pentadactyla; Mt, Manis tricuspis. In contrast to IFIH1 and ZBP1, the genes encoding the intracellular RNA sensor RIG-I, i.e., DExD/H-box helicase 58 (DDX58), and TLR3, TLR7, and TLR8 which control the sensing of RNA in endosomes and a series of other genes involved in antiviral signaling and defense, such as MAVS, RIPK3, MLKL, SKIV2L, OAS2, RNASEL, and EIF2AK2 (PKR) do not contain disruptive mutations and therefore appear to be intact in the Malayan pangolin (M. javanica) (Table S1). DDX58 contains in-frame stop codons and frameshift mutations in the tree pangolin (M. tricuspis) but not in the Chinese pangolin (M. pentadactyla) (Figure S3), suggesting that the tree pangolin lacks functional DDX58/RIG-I in addition to the two intracellular RNA sensors (IFIH1 and ZBP1) absent in all pangolins. Pangolins Have Lost IFIH1 and ZBP1 After Their Evolutionary Divergence From Other Mammalian Lineages We screened the genomes of mammals from diverse phylogenetic lineages for functional copies (devoid of frameshift mutations and premature in-frame stop codons) of ZBP1, IFIH1 and other RNA sensor genes. Mapping the presence or absence of these genes onto the phylogenetic tree suggested that loss of both ZBP1 and IFIH1 occurred in the pangolin lineages soon after divergence from the lineage leading to Carnivora (represented by cat, dog and bear in Figure 3A). Other genes implicated in anti-RNA-viral defense are conserved in the selected set of species (Figure 3A; Table S2). FIGURE 3 Figure 3. Evolution of RNA sensor genes and possible implications on antiviral responses in pangolins. (A) Phylogenetic tree of mammals and comparison of presence (+) or absence (–) of RNA sensor genes. Evolutionary gene loss (indicated by lightning bolt symbols) was inferred from the species distribution of the genes. Species: Malayan pangolin (Manis javanica), Chinese pangolin (Manis pentadactyla), tree pangolin (Manis tricuspis), cat (Felis catus), dog (Canis lupus familiaris), bear (Ursus arctos horribilis), cattle (Bos taurus), mouse (Mus musculus), human (Homo sapiens). (B) Schematic overview of innate immune sensors of viral RNA and signaling in mammals. Only RNA sensors investigated in this study are shown. The schematic includes the hypothesis about IFIH1 and ZBP1-dependent differences in the antiviral activity and defense-induced damage to the host. The directions of the colored arrows indicate the effects of the presence or absence of RNA sensors. 5'PPP, triphosphorylated at the 5'-end; ds, double-stranded; ss, single-stranded. Discussion Based on the known target specificities of mammalian RNA sensors (Figure 3B), the loss of ZBP1 and IFIH1 suggests that the response to Z-RNA and long double-stranded RNA is diminished in pangolins. Accordingly, the resistance to RNA viruses that depend on cytoplasmic Z-RNA and long double-stranded RNA for replication has likely decreased in the evolution of pangolins. We put forward the hypothesis that strong antiviral defense was harmful and loss of ZBP1 and IFIH1 provided an evolutionary advantage by increasing tolerance to infections by certain RNA viruses, including coronaviruses. Viruses are potent drivers of evolutionary adaptations in their hosts. Both insufficient and overshooting responses to viral infections have deleterious effects, leading to strong selection for resistant or tolerant host genotypes (37, 38). Bats have retained functional RNA sensor genes (Table S3) but exert only dampened antiviral responses, indicating that they have adapted to the evolutionary pressure from viruses by decreasing inflammatory responses and by enhancing tolerance to viral replication (39–42). The results of the present study suggest that pangolins are another group of mammals with evolutionarily downregulated defense against a subset of viruses, namely those sensed by IFIH1/MDA5 or ZBP1 in other species. Our data urge to study the virus burden of pangolins, their antiviral immune response and their ability to act as reservoirs for viruses with zoonotic potential, especially coronaviruses. While genetic suppression of IFIH1/MDA5 and ZBP1-dependent pathways had neutral or beneficial effects in the evolution of pangolins, pharmaceutical suppression of IFIH1/MDA5 and ZBP1-dependent signaling may be beneficial for human patients with overreactions to viral nucleic acids.
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Post by Admin on May 18, 2020 20:35:16 GMT
Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? Ping Liu ,Jing-Zhe Jiang ,Xiu-Feng Wan,Yan Hua,Linmiao Li,Jiabin Zhou,Xiaohu Wang,Fanghui Hou,Jing Chen,Jiejian Zou,Jinping Chen Published: May 14, 2020 doi.org/10.1371/journal.ppat.1008421Abstract The outbreak of a novel corona Virus Disease 2019 (COVID-19) in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans. Author summary Recently, a novel coronavirus, SARS-CoV-2, caused a still ongoing pandemic. Epidemiological study suggested this virus was associated with a wet market in Wuhan, China. However, the exact source of this virus is still unknown. In this study, we attempted to assemble the complete genome of a coronavirus identified from two groups of sick Malayan pangolins, which were likely to be smuggled for black market trade. The molecular and evolutionary analyses showed that this pangolin coronavirus we assembled was genetically associated with the SARS-CoV-2 but was not likely its precursor. This study suggested that pangolins are natural hosts of coronaviruses. Determining the spectrum of coronaviruses in pangolins can help understand the natural history of coronaviruses in wildlife and at the animal-human interface, and facilitate the prevention and control of coronavirus-associated emerging diseases. Introduction In December 2019, there was an outbreak of pneumonia with an unknown cause in Wuhan, Hubei province, China, with an epidemiological link to the Huanan Seafood Wholesale Market, a local live animal and seafood market. Clinical presentations of this disease greatly resembled viral pneumonia. Through deep sequencing on the lower respiratory tract samples of patients, a novel coronavirus named the 2019 novel coronavirus was identified [1], the name of which was then determined as SARS-CoV-2. This virus has spread to all provinces across China and more than 200 additional countries. As of April 11, 2020, the epidemic has resulted in 83,400 laboratory confirmed cases, 3,349 of which were fatal in China, while there were 1,643,047 laboratory confirmed cases and 101,507 deaths in other countries. The global toll of new cases and deaths is still increasing sharply. To effectively control the disease and prevent new spillovers, it is critical to identify the animal origin of this newly emerging coronavirus. In the Wuhan wet market, high viral loads were reported in environmental samples. However, a variety of animals, including wildlife, were sold in this market, and the daily number and species of animals were very dynamic. Therefore, it remains unclear which animals initiated the first infections. Coronaviruses usually cause respiratory and gastrointestinal tract infections and are genetically classified into four major genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The former two genera primarily infect mammals, whereas the latter two predominantly infect birds [2]. In addition to SARS-CoV-2, other members of the Betacoronavirus genus caused the 2003 SARS (severe acute respiratory syndrome) outbreaks and the 2012 MERS (Middle East respiratory syndrome) outbreaks in humans [3, 4]. SARS-CoV and MERS-CoV are of bat origin, but both coronaviruses had an intermediate host: palm civets for SARS-CoV [5] and dromedary camels for MERS-CoV [6]. Approximately 30,000 base pairs in the coronavirus genome code for up to 11 proteins, including the surface glycoprotein Spike (S) protein binds to receptors on the host cell, which initiates virus infection. Different coronaviruses can use distinct host receptors due to structural variations in the receptor binding domains of their virus S protein. SARS-CoV uses angiotensin-converting enzyme 2 (ACE2) as one of the main receptors [7] with CD209L as an alternative receptor [8], whereas MERS-CoV uses dipeptidyl peptidase 4 (DPP4, also known as CD26) as the primary receptor. A recent study demonstrated that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming [9]. Soon after the release of the SARS-CoV-2 genome, a scientist released a full genome of a coronavirus, Bat-CoV-RaTG13, from the bat species Rhinolophus affinis, which was colonized in Yunan province, nearly 2,000 km away from Wuhan. Bat-CoV-RaTG13 was 96% identical at the whole genome level to the SARS-CoV-2, suggesting the SARS-CoV-2 could be of bat origin [1]. However, because direct human-bat contact is rare, it seems to be more likely that the spillover of SARS-CoV-2 to humans from an intermediate host rather than directly from bats, as was the cases with both SARS-CoV and MERS-CoV. The goal of this study was to determine the genetic relationship between a coronavirus from two groups of sick pangolins and SARS-CoV-2, and to assess whether pangolins could be potential intermediate hosts of SARS-CoV-2.
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