Evolution of genomes, host shifts and the geographic spread of SARS-CoV and related coronaviruses

Severe acute respiratory syndrome (SARS) is a novel human illness caused by a previously unrecognized coronavirus (CoV) termed SARS‐CoV. There are conflicting reports on the animal reservoir of SARS‐CoV. Many of the groups that argue carnivores are the original reservoir of SARS‐CoV use a phylogeny to support their argument. However, the phylogenies in these studies often lack outgroup and rooting criteria necessary to determine the origins of SARS‐CoV. Recently, SARS‐CoV has been isolated from various species of Chiroptera from China (e.g., Rhinolophus sinicus) thus leading to reconsideration of the original reservoir of SARS‐CoV. We evaluated the hypothesis that SARS‐CoV isolated from Chiroptera are the original zoonotic source for SARS‐CoV by sampling SARS‐CoV and non‐SARS‐CoV from diverse hosts including Chiroptera, as well as carnivores, artiodactyls, rodents, birds and humans. Regardless of alignment parameters, optimality criteria, or isolate sampling, the resulting phylogenies clearly show that the SARS‐CoV was transmitted to small carnivores well after the epidemic of SARS in humans that began in late 2002. The SARS‐CoV isolates from small carnivores in Shenzhen markets form a terminal clade that emerged recently from within the radiation of human SARS‐CoV. There is evidence of subsequent exchange of SARS‐CoV between humans and carnivores. In addition SARS‐CoV was transmitted independently from humans to farmed pigs (Sus scrofa). The position of SARS‐CoV isolates from Chiroptera are basal to the SARS‐CoV clade isolated from humans and carnivores. Although sequence data indicate that Chiroptera are a good candidate for the original reservoir of SARS‐CoV, the structural biology of the spike protein of SARS‐CoV isolated from Chiroptera suggests that these viruses are not able to interact with the human variant of the receptor of SARS‐CoV, angiotensin‐converting enzyme 2 (ACE2). In SARS‐CoV we study, both visually and statistically, labile genomic fragments and, putative key mutations of the spike protein that may be associated with host shifts. We display host shifts and candidate mutations on trees projected in virtual globes depicting the spread of SARS‐CoV. These results suggest that more sampling of coronaviruses from diverse hosts, especially Chiroptera, carnivores and primates, will be required to understand the genomic and biochemical evolution of coronaviruses, including SARS‐CoV. © The Willi Hennig Society 2008.     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.     

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus

Coronaviruses (CoV) are enveloped positive‐strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on these structures, we have performed a time‐course experiment in which the mouse hepatitis virus (MHV)‐induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)‐electron microscopy. With our approach we were able to confirm the appearance of 6, previously reported, membranous structures during the course of a complete infection cycle. These structures include the well‐characterized double‐membrane vesicles (DMVs), convoluted membranes (CMs) and virions but also the more enigmatic large virion‐containing vacuoles (LVCVs), tubular bodies (TBs) and cubic membrane structures (CMSs). We have characterized the LVCVs, TBs and CMSs, and found that the CoV‐induced structures appear in a strict order. By combining these data with quantitative analyses on viral RNA, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of CoV infection. In particular, it provides insights in the role of each CoV‐induced structure and reveals that LVCVs are ERGIC/Golgi compartments that expand to accommodate an increasing production of viral particles.     

Scientific research progress of COVID‐19/SARS‐CoV‐2 in the first five months

A cluster of pneumonia (COVID‐19) cases have been found in Wuhan China in late December, 2019, and subsequently, a novel coronavirus with a positive stranded RNA was identified to be the aetiological virus (severe acute respiratory syndrome coronavirus 2, SARS‐CoV‐2), which has a phylogenetic similarity to severe acute respiratory syndrome coronavirus (SARS‐CoV). SARS‐CoV‐2 transmits mainly through droplets and close contact and the elder or people with chronic diseases are high‐risk population. People affected by SARS‐CoV‐2 can be asymptomatic, which brings about more difficulties to control the transmission. COVID‐19 has become pandemic rapidly after onset, and so far the infected people have been above 2 000 000 and more than 130 000 died worldwide according to COVID‐19 situation dashboard of World Health Organization ( https://covid19.who.int ). Here, we summarized the current known knowledge regarding epidemiological, pathogenesis, pathology, clinical features, comorbidities and treatment of COVID‐19/ SARS‐CoV‐2 as reference for the prevention and control COVID‐19.     

The ACE2 expression in Sertoli cells and germ cells may cause male reproductive disorder after SARS‐CoV‐2 infection

The serious coronavirus disease‐2019 (COVID‐19) was first reported in December 2019 in Wuhan, China. COVID‐19 is an infectious disease caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS‐CoV‐2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single‐cell RNA‐sequencing (scRNA‐seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS‐CoV‐2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS‐CoV‐2. The expression level of ACE2 was related to the age, and the mid‐aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS‐CoV‐2 and may enable rapid deciphering male‐related reproductive disorders induced by COVID‐19.     

Architecture and self‐assembly of the SARS‐CoV‐2 nucleocapsid protein

The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.     

Influence of hydrophobic and electrostatic residues on SARS‐coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS‐coronavirus (SARS‐CoV). SARS‐CoV entry is facilitated by the spike protein (S), which consists of an N‐terminal domain (S1) responsible for cellular attachment and a C‐terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS‐CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site‐directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH‐independent retroviral fusion proteins, SARS‐CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS‐CoV S2 analysis showed that specific hydrophobic positions at the C‐terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C‐terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C‐terminal hydrophobic residues led us to identify a 42‐residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.     

Susceptibility to COVID‐19 in populations with health disparities: Posited involvement of mitochondrial disorder,socioeconomic stress,and pollutants

SARS‐CoV‐2 is a novel betacoronavirus that has caused the global health crisis known as COVID‐19. The implications of mitochondrial dysfunction with COVID‐19 are discussed as well as deregulated mitochondria and inter‐organelle functions as a posited comorbidity enhancing detrimental outcomes. Many environmental chemicals (ECs) and endocrine‐disrupting chemicals can do damage to mitochondria and cause mitochondrial dysfunction. During infection, SARS‐CoV‐2 via its binding target ACE2 and TMPRSS2 can disrupt mitochondrial function. Viral genomic RNA and structural proteins may also affect the normal function of the mitochondria‐endoplasmic reticulum‐Golgi apparatus. Drugs considered for treatment of COVID‐19 should consider effects on organelles including mitochondria functions. Mitochondrial self‐balance and clearance via mitophagy are important in SARS‐CoV‐2 infection, which indicate monitoring and protection of mitochondria against SARS‐CoV‐2 are important. Mitochondrial metabolomic analysis may provide new indicators of COVID‐19 prognosis. A better understanding of the role of mitochondria during SARS‐CoV‐2 infection may help to improve intervention therapies and better protect mitochondrial disease patients from pathogens as well as people living with poor nutrition and elevated levels of socioeconomic stress and ECs.     

Predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals using ACE2 protein sequence homology

The article is presenting a bioinformatics based method predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals. Recently, there were reports of cats and ferrets, dogs, minks, golden hamster, rhesus monkeys, tigers, and lions testing for SARS‐CoV‐2 RNA which indicated for the possible interspecies viral transmission. Our method successfully predicted the susceptibility of these animals for contracting SARS‐CoV‐2 infection. This method can be used as a screening tool for guiding viral RNA testing for domestic and wildlife animals at risk of getting COVID‐19. We provide a list of the animals at risk of developing COVID‐19 based on the susceptibility score.     

A five‐step risk management process for geriatric dental practice during SARS‐CoV‐2 pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an RNA virus that causes coronavirus infection (COVID‐19). COVID‐19 is a highly contagious disease transmitted through respiratory droplets, saliva and other contact routes. Within 10 months of its outbreak, SARS‐CoV‐2 has infected more than 23 million people around the world. Evidence suggests that older adults are the most vulnerable to infection and have an increased risk of mortality. Reduced immunity and underlying medical conditions make them risk‐prone and vulnerable to critical care. Older adults affected with the SARS‐CoV‐2 virus present with distinct clinical manifestations necessitating specific treatment needs and management protocols. While it is crucial to prevent the spread of novel coronavirus (2019‐nCoV), the role of oral healthcare workers in addressing the specific needs of ageing adult patients by adopting specific guidelines and appropriate infection control protocols is timely. This paper aims to develop specific guidelines and protocols for the dental management of geriatric patients during the COVID‐19 pandemic.     

恒河猴感染SARS病毒致病模型的建立

为建立恒河猴严重急性呼吸道综合征(SARS)的模型并对其致病特点进行观察,采用病毒分离、免疫荧光、光镜及RT-PCR方法对病毒感染组和非感染组恒河猴不同时间、不同组织或分泌物进行检测。结果显示从恒河猴不同组织中分离到病毒,而且在病毒感染后第2d和第5d的血液、第7、9d的鼻咽分泌物、第3d的粪、第5d的粪尿中均检测到SARS-CoV RNA。光镜观察到病毒感染组肺组织肺泡问隔增宽,有大量淋巴细胞、单核细胞浸润,肺泡腔有渗出,甚至形成透明膜样物;多个肺泡形成机化性肺炎的表现。感染组肝组织可见较大的坏死灶,并伴有大量炎性细胞浸润。结论认为已成功建立了恒河猴SARS模型,可用于评价抗SARS药物和疫苗的研究。     

逆转录套式PCR检测粪便样本中的SARS冠状病毒

为了观察SARS冠状病毒在SARS患者粪便中的存在规律,建立了检测SARS冠状病毒RNA的逆转录-聚合酶链反应(RT-PCR)方法,并应用该方法检测了241份SARS患者粪便样本。部分PCR产物应用测序技术进行验证。RT-PCR的灵敏度为10^-10稀释度的病毒原液(原液为10^8TCID50／ml)。241份粪便样本的总体检出率为24．1％(58／241),其中发病后的前10d和20d的检出率均为50．0％。随着发病时间的延长,阳性检出率呈下降趋势。应用RT-PCR从粪便中检测SARS冠状病毒是可行的,在发病50d以后仍有17．0％左右的阳性检出率,提示SARS恢复期患者具有排毒的可能性,给后续的卫生防疫措施提供了一定的参考数据。     

The potential effects of DPP‐4 inhibitors on cardiovascular system in COVID‐19 patients

With the outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the public healthcare systems are facing great challenges. Coronavirus disease 2019 (COVID‐19) could develop into severe pneumonia, acute respiratory distress syndrome and multi‐organ failure. Remarkably, in addition to the respiratory symptoms, some COVID‐19 patients also suffer from cardiovascular injuries. Dipeptidyl peptidase‐4 (DPP‐4) is a ubiquitous glycoprotein which could act both as a cell membrane‐bound protein and a soluble enzymatic protein after cleavage and release into the circulation. Despite angiotensin‐converting enzyme 2 (ACE2), the recently recognized receptor of SARS‐CoV and SARS‐CoV‐2, which facilitated their entries into the host, DPP‐4 has been identified as the receptor of middle east respiratory syndrome coronavirus (MERS‐CoV). In the current review, we discussed the potential roles of DPP‐4 in COVID‐19 and the possible effects of DPP‐4 inhibitors on cardiovascular system in patients with COVID‐19.     

Molecular Targets for the Testing of COVID‐19

The pandemic outbreaks of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), spread all over the world in a short period of time. Efficient identification of the infection by SARS‐CoV‐2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID‐19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid‐February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID‐19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS‐CoV‐2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID‐19 virus.     

Identification of the membrane-active regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a 16/18-mer peptide scan: implications for the viral fusion mechanism

We have identified the membrane-active regions of the severe acute respiratory syndrome coronavirus (SARS CoV) spike glycoprotein by determining the effect on model membrane integrity of a 16/18-mer SARS CoV spike glycoprotein peptide library. By monitoring the effect of this peptide library on membrane leakage in model membranes, we have identified three regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities: region 1, located immediately upstream of heptad repeat 1 (HR1) and suggested to be the fusion peptide; region 2, located between HR1 and HR2, which would be analogous to the loop domain of human immunodeficiency virus type 1; and region 3, which would correspond to the pretransmembrane region. The identification of these membrane-active regions, which are capable of modifying the biophysical properties of phospholipid membranes, supports their direct role in SARS CoV-mediated membrane fusion, as well as facilitating the future development of SARS CoV entry inhibitors.     

Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus

Nitric oxide (NO) is an important signaling molecule between cells which has been shown to have an inhibitory effect on some virus infections. The purpose of this study was to examine whether NO inhibits the replication cycle of the severe acute respiratory syndrome coronavirus (SARS CoV) in vitro. We found that an organic NO donor, S-nitroso-N-acetylpenicillamine, significantly inhibited the replication cycle of SARS CoV in a concentration-dependent manner. We also show here that NO inhibits viral protein and RNA synthesis. Furthermore, we demonstrate that NO generated by inducible nitric oxide synthase, an enzyme that produces NO, inhibits the SARS CoV replication cycle.     

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.     

COVID‐19 and olfactory dysfunction: A possible associative approach towards neurodegenerative diseases

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the agent of novel coronavirus 2019 (COVID‐19), has kept the globe in disquiets due to its severe life‐threatening conditions. The most common symptoms of COVID‐19 are fever, sore throat, and shortness of breath. According to the anecdotal reports from the health care workers, it has been suggested that the virus could reach the brain and can cause anosmia, hyposmia, hypogeusia, and hypopsia. Once the SARS‐CoV‐2 has entered the central nervous system (CNS), it can either exit in an inactive form in the tissues or may lead to neuroinflammation. Here, we aim to discuss the chronic infection of the olfactory bulb region of the brain by SARS‐CoV‐2 and how this could affect the nearby residing neurons in the host. We further review the probable cellular mechanism and activation of the microglia 1 phenotype possibly leading to various neurodegenerative disorders. In conclusion, SARS‐CoV‐2 might probably infect the olfactory bulb neuron enervating the nasal epithelium accessing the CNS and might cause neurodegenerative diseases in the future.     

Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.