What is the result of phylogenetic analysis of SARS-CoV-2 genome sequence?

Evolutionary relationships and sequence-structure determinants in human SARS coronavirus-2 spike proteins for host receptor recognition

Coronavirus disease 2019 (COVID-19) is a pandemic infectious disease caused by novel severe acute respiratory syndrome coronavirus-2 (SARS CoV-2). The SARS CoV-2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and SARS CoV-2 via their glycosylated spike proteins recognize the human angiotensin converting enzyme-2 (ACE-2) receptor. We generated multiple sequence alignments and phylogenetic trees for representative spike proteins of SARS CoV and SARS CoV-2 from various host sources in order to analyze the specificity in SARS CoV-2 spike proteins required for causing infection in humans. Our results show that among the genomes analyzed, two sequence regions in the N-terminal domain "MESEFR" and "SYLTPG" are specific to human SARS CoV-2. In the receptor-binding domain, two sequence regions "VGGNY" and "EIYQAGSTPCNGV" and a disulfide bridge connecting 480C and 488C in the extended loop are structural determinants for the recognition of human ACE-2 receptor. The complete genome analysis of representative SARS CoVs from bat, civet, human host sources, and human SARS CoV-2 identified the bat genome (GenBank code: MN996532.1) as closest to the recent novel human SARS CoV-2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933 and MG772934) are evolutionary intermediates in the mutagenesis progression toward becoming human SARS CoV-2.

Insights into molecular evolution recombination of pandemic SARS-CoV-2 using Saudi Arabian sequences

The recently emerged SARS-CoV-2 (Coronaviridae; Betacoronavirus) is the underlying cause of COVID-19 disease. Here we assessed SARS-CoV2 from the Kingdom of Saudi Arabia alongside sequences of SARS-CoV, bat SARS-like CoVs and MERS-CoV, the latter currently detected in this region. Phylogenetic analysis, natural selection investigation and genome recombination analysis were performed. Our analysis showed that all Saudi SARS-CoV-2 sequences are of the same origin and closer proximity to bat SARS-like CoVs, followed by SARS-CoVs, however quite distant to MERS-CoV. Moreover, genome recombination analysis revealed two recombination events between SARS-CoV-2 and bat SARS-like CoVs. This was further assessed by S gene recombination analysis. These recombination events may be relevant to the emergence of this novel virus. Moreover, positive selection pressure was detected between SARS-CoV-2, bat SL-CoV isolates and human SARS-CoV isolates. However, the highest positive selection occurred between SARS-CoV-2 isolates and 2 bat-SL-CoV isolates (Bat-SL-RsSHC014 and Bat-SL-CoVZC45). This further indicates that SARS-CoV-2 isolates were adaptively evolved from bat SARS-like isolates, and that a virus with originating from bats triggered this pandemic. This study thuds sheds further light on the origin of this virus. AUTHOR SUMMARY The emergence and subsequent pandemic of SARS-CoV-2 is a unique challenge to countries all over the world, including Saudi Arabia where cases of the related MERS are still being reported. Saudi SARS-CoV-2 sequences were found to be likely of the same or similar origin. In our analysis, SARS-CoV-2 were more closely related to bat SARS-like CoVs rather than to MERS-CoV (which originated in Saudi Arabia) or SARS-CoV, confirming other phylogenetic efforts on this pathogen. Recombination and positive selection analysis further suggest that bat coronaviruses may be at the origin of SARS-CoV-2 sequences. The data shown here give hints on the origin of this virus and may inform efforts on transmissibility, host adaptation and other biological aspects of this virus.

Median-joining network analysis of SARS-CoV-2 genomes is neither phylogenetic nor evolutionary

Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity

Monitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a mutation that leads to weaker receptor binding capability, which concerns a SARS-CoV-2 sample collected on 27th January 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and raises the alarm that the ongoing vaccine development may become futile in future epidemic if more mutations were identified. Highlights Based on the currently available genome sequence data, we proved that SARS-COV-2 genome has a much lower mutation rate and genetic diversity than SARS during the 2002-2003 outbreak. The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human. We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a sample collected on 27th January 2020.

The origin and underlying driving forces of the SARS-CoV-2 outbreak

The spread of SARS-CoV-2 since December 2019 has become a pandemic and impacted many aspects of human society. Here, we analyzed genetic variation of SARS-CoV-2 and its related coronavirus and found the evidence of intergenomic recombination. After correction for mutational bias, analysis of 137 SARS-CoV-2 genomes as of 2/23/2020 revealed the excess of low frequency mutations on both synonymous and nonsynonymous sites which is consistent with recent origin of the virus. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China was two time higher than those derived from the rest of the world. In addition, in network analysis, haplotypes collected from Wuhan city were at interior and have more mutational connections, both of which are consistent with the observation that the outbreak of cov-19 was originated from China. SUMMARY In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated.

Genome analysis of SARS-CoV-2 isolates occurring in India: Present scenario.

Background The origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still a debatable topic. The association of the virus spread from the market is supported by the close relation of genome sequences of environmental surface samples with virus samples from earliest patients by phylogenetic analysis. Objectives To have an insight into the SARS-CoV-2 genome sequences reported from India for better understanding on their epidemiology and virulence. Methods Genome sequences of Indian isolates of SARS-CoV-2 were analyzed to understand their phylogeny and divergence with respect to other isolates reported from other countries. Amino acid sequences of individual open reading frames (ORFs) from SARS-CoV-2 Indian isolates were aligned with sequences of isolates reported from other countries to identify the mutations occurred in Indian isolates. Results Our analysis suggests that Indian SARS-CoV-2 isolates are closely related to isolates reported from other parts of the world. Most ORFs are highly conserved; mutations were also detected in some ORFs. We found that most isolates reported from India have key mutations at 614th position of the S protein and 84th position of the ORF 8, which has been reported to be associated with high virulence and high transmission rate. Conclusion An attempt was made to understand the SARS-CoV-2 virus reported from India. SARS-CoV-2 reported from India was closely similar to other SARS-CoV-2 reported from other parts of the world, which suggests that vaccines and other therapeutic methods generated from other countries might work well in India. In addition, available sequence data suggest that majority of Indian isolates are capable of high transmission and virulence.

Molecular evolution and phylogenetic analysis of SARS-CoV-2 and hosts ACE2 protein suggest Malayan pangolin as intermediary host

An emergence of a novel coronavirus, causative agent of COVID19, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), occurred due to cross-species transmission. Coronaviruses are a large family of viruses able to infect a great number of hosts. Entrance of SARS-CoV-2 depends on the surface (S) protein interaction with host ACE2 protein and cleavage by TMPRSS2. ACE2 could be a species-specific barrier that interferes with bat-to-human coronavirus cross-species transmission. Molecular analysis supported bats as natural hosts for SARS-CoV and involved them in MERS-CoV origin. The genomic similarity between bat RaTG13 CoV strain and SARS-CoV-2 implicates bats in the origin of the new outbreak. Additionally, there is a hypothesis for the zoonotic transmission based on contact with Malayan pangolins by humans in Huanan seafood market in Wuhan, China. To investigate bats and pangolin as hosts in SARS-CoV-2 cross-species transmission, we perform an evolutionary analysis combining viral and host phylogenies and divergence of ACE2 and TMPRSS2 amino acid sequences between CoV hosts. Phylogeny showed SARS-like-CoV-2 strains that infected pangolin and bats are close to SARS-CoV-2. In contrast to TMPRSS2, pangolin ACE2 amino acid sequence has low evolutionary divergence compared with humans and is more divergent from bats. Comparing SARS-CoV with SARS-CoV-2 origins, pangolin has yet lower ACE2 evolutionary divergence with humans than civet—the main intermediary host of SARS-CoV. Thus, pangolin has become an opportune host to intermediates bat-to-human SARS-CoV-2 jump and entry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00321-1) contains supplementary material, which is available to authorized users.

Recombination and lineage-specific mutations led to the emergence of SARS-CoV-2

The recent outbreak of a new coronavirus (SARS-CoV-2) in Wuhan, China, underscores the need for understanding the evolutionary processes that drive the emergence and adaptation of zoonotic viruses in humans. Here, we show that recombination in betacoronaviruses, including human-infecting viruses like SARS-CoV and MERS-CoV, frequently encompasses the Receptor Binding Domain (RBD) in the Spike gene. We find that this common process likely led to a recombination event at least 11 years ago in an ancestor of the SARS-CoV-2 involving the RBD. As a result of this recombination event, SARS-CoV and SARS-CoV-2 share a similar genotype in RBD, including two insertions (positions 432-436 and 460-472), and alleles 427N and 436Y. Both 427N and 436Y belong to a helix that interacts with the human ACE2 receptor. Ancestral state analyses revealed that SARS-CoV-2 differentiated from its most recent common ancestor with RaTG13 by accumulating a significant number of amino acid changes in the RBD. In sum, we propose a two-hit scenario in the emergence of the SARS-CoV-2 virus whereby the SARS-CoV-2 ancestors in bats first acquired genetic characteristics of SARS-CoV by incorporation of a SARS-like RBD through recombination before 2009, and subsequently, the lineage that led to SARS-CoV-2 accumulated further, unique changes specifically in the RBD.

[Source of the COVID-19 pandemic: ecology and genetics of coronaviruses (Betacoronavirus: Coronaviridae) SARS-CoV, SARS-CoV-2 (subgenus Sarbecovirus), and MERS-CoV (subgenus Merbecovirus).]

Since the early 2000s, three novel zooanthroponous coronaviruses (Betacoronavirus) have emerged. The first outbreak of infection (SARS) caused by SARS-CoV virus occurred in the fall of 2002 in China (Guangdong Province). A second outbreak (MERS) associated with the new MERS-CoV virus appeared in Saudi Arabia in autumn 2012. The third epidemic, which turned into a COVID-19 pandemic caused by SARS-CoV-2 virus, emerged in China (Hubei Province) in the autumn 2019. This review focuses on ecological and genetic aspects that lead to the emergence of new human zoanthroponous coronaviruses. The main mechanism of adaptation of zoonotic betacoronaviruses to humans is to changes in the receptor-binding domain of surface protein (S), as a result of which it gains the ability to bind human cellular receptors of epithelial cells in respiratory and gastrointestinal tract. This process is caused by the high genetic diversity and variability combined with frequent recombination, during virus circulation in their natural reservoir - bats (Microchiroptera, Chiroptera). Appearance of SARS-CoV, SARS-CoV-2 (subgenus Sarbecovirus), and MERS (subgenus Merbecovirus) viruses is a result of evolutionary events occurring in bat populations with further transfer of viruses to the human directly or through the intermediate vertebrate hosts, ecologically connected with bats. This review is based on the report at the meeting «Coronavirus - a global challenge to science¼ of the Scientific Council «Life Science¼ of the Russian Academy of Science: Lvov D.K., Alkhovsky S.V., Burtseva E.I. COVID-19 pandemic sources: origin, biology and genetics of coronaviruses of SARS-CoV, SARS-CoV-2, MERS-CoV (Conference hall of Presidium of RAS, 14 Leninsky Prospect, Moscow, Russia. April 16, 2020).

Molecular epidemiology, evolution and phylogeny of SARS coronavirus

Abstract Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs (SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats [mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9]. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species.

Genome analysis of SARS-CoV-2 isolates occurring in India: Present scenario

Background: The origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still a debatable topic. The association of the virus spread from the market is supported by the close relation of genome sequences of environmental surface samples with virus samples from earliest patients by phylogenetic analysis. Objectives: To have an insight into the SARS-CoV-2 genome sequences reported from India for better understanding on their epidemiology and virulence. Methods: Genome sequences of Indian isolates of SARS-CoV-2 were analyzed to understand their phylogeny and divergence with respect to other isolates reported from other countries. Amino acid sequences of individual open reading frames (ORFs) from SARS-CoV-2 Indian isolates were aligned with sequences of isolates reported from other countries to identify the mutations occurred in Indian isolates. Results: Our analysis suggests that Indian SARS-CoV-2 isolates are closely related to isolates reported from other parts of the world. Most ORFs are highly conserved; mutations were also detected in some ORFs. We found that most isolates reported from India have key mutations at 614th position of the S protein and 84th position of the ORF 8, which has been reported to be associated with high virulence and high transmission rate. Conclusion: An attempt was made to understand the SARS-CoV-2 virus reported from India. SARS-CoV-2 reported from India was closely similar to other SARS-CoV-2 reported from other parts of the world, which suggests that vaccines and other therapeutic methods generated from other countries might work well in India. In addition, available sequence data suggest that majority of Indian isolates are capable of high transmission and virulence.

Molecular Mechanism of Evolution and Human Infection with SARS-CoV-2

The outbreak of a novel coronavirus, which was later formally named the severe acute respiratory coronavirus 2 (SARS-CoV-2), has caused a worldwide public health crisis. Previous studies showed that SARS-CoV-2 is highly homologous to SARS-CoV and infects humans through the binding of the spike protein to ACE2. Here, we have systematically studied the molecular mechanisms of human infection with SARS-CoV-2 and SARS-CoV by protein-protein docking and MD simulations. It was found that SARS-CoV-2 binds ACE2 with a higher affinity than SARS-CoV, which may partly explain that SARS-CoV-2 is much more infectious than SARS-CoV. In addition, the spike protein of SARS-CoV-2 has a significantly lower free energy than that of SARS-CoV, suggesting that SARS-CoV-2 is more stable and may survive a higher temperature than SARS-CoV. This provides insights into the evolution of SARS-CoV-2 because SARS-like coronaviruses have originated in bats. Our computation also suggested that the RBD-ACE2 binding for SARS-CoV-2 is much more temperature-sensitive than that for SARS-CoV. Thus, it is expected that SARS-CoV-2 would decrease its infection ability much faster than SARS-CoV when the temperature rises. These findings would be beneficial for the disease prevention and drug/vaccine development of SARS-CoV-2.

Binding Ability Prediction between Spike Protein and Human ACE2 Reveals the Adaptive Strategy of SARS-CoV-2 in Humans

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing an outbreak of COVID-19 globally in the past six months. A relatively higher divergence on the spike protein of SASR-CoV-2 enables it to transmit across species efficiently. We particularly believe that the adaptive mutations of the receptor-binding domain (RBD) of spike protein in SARS-CoV-2 might be essential to its high transmissibility among humans. Thus here we collected 2,142 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny, and also analyzed the interaction between the polymorphisms of spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 and coronavirus in other hosts show SARS-CoV-2 is highly possible originated from Bat-CoV (RaTG13) found in horseshoe bat and a recombination event may occur on the spike protein of Pangolin-CoV to imbue it the ability to infect humans. Moreover, compared to the S gene of SARS-CoV-2, it is more conserved in the direct-binding sites of RBD and we noticed that spike protein of SARS-CoV-2 may under a consensus evolution to adapt to human hosts better. 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 were simulated and their stability and affinity binding to hACE2 (S19-D615) were calculated. Our analysis indicates SARS-CoV-2 could infect humans from different populations with no preference, and a higher divergence in the spike protein of SARS-CoV-2 at the early stage of this pandemic may be a good indicator that could show the pathway of SARS-CoV-2 transmitting from the natural reservoir to human beings.

An emergence of a novel coronavirus, causative agent of COVID19, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), occurred due to cross-species transmission. Coronaviruses are a large family of viruses able to infect a great number of hosts. Entrance of SARS-CoV-2 depends on the surface (S) protein interaction with host ACE2 protein and cleavage by TMPRSS2. ACE2 could be a species-specific barrier that interferes with bat-to-human coronavirus cross-species transmission. Molecular analysis supported bats as natural hosts for SARS-CoV and involved them in MERS-CoV origin. The genomic similarity between bat RaTG13 CoV strain and SARS-CoV-2 implicates bats in the origin of the new outbreak. Additionally, there is a hypothesis for the zoonotic transmission based on contact with Malayan pangolins by humans in Huanan seafood market in Wuhan, China. To investigate bats and pangolin as hosts in SARS-CoV-2 cross-species transmission, we perform an evolutionary analysis combining viral and host phylogenies and divergence of ACE2 and TMPRSS2 amino acid sequences between CoV hosts. Phylogeny showed SARS-like-CoV-2 strains that infected pangolin and bats are close to SARS-CoV-2. In contrast to TMPRSS2, pangolin ACE2 amino acid sequence has low evolutionary divergence compared with humans and is more divergent from bats. Comparing SARS-CoV with SARS-CoV-2 origins, pangolin has yet lower ACE2 evolutionary divergence with humans than civet-the main intermediary host of SARS-CoV. Thus, pangolin has become an opportune host to intermediates bat-to-human SARS-CoV-2 jump and entry.

Since the early 2000s, three novel zooanthroponous coronaviruses (Betacoronavirus) have emerged. The first outbreak of infection (SARS) caused by SARS-CoV virus occurred in the fall of 2002 in China (Guangdong Province). A second outbreak (MERS) associated with the new MERS-CoV virus appeared in Saudi Arabia in autumn 2012. The third epidemic, which turned into a COVID-19 pandemic caused by SARS-CoV-2 virus, emerged in China (Hubei Province) in the autumn 2019. This review focuses on ecological and genetic aspects that lead to the emergence of new human zoanthroponous coronaviruses. The main mechanism of adaptation of zoonotic betacoronaviruses to humans is to changes in the receptor-binding domain of surface protein (S), as a result of which it gains the ability to bind human cellular receptors of epithelial cells in respiratory and gastrointestinal tract. This process is caused by the high genetic diversity and variability combined with frequent recombination, during virus circulation in their natural reservoir - bats (Microchiroptera, Chiroptera). Appearance of SARS-CoV, SARS-CoV-2 (subgenus Sarbecovirus), and MERS (subgenus Merbecovirus) viruses is a result of evolutionary events occurring in bat populations with further transfer of viruses to the human directly or through the intermediate vertebrate hosts, ecologically connected with bats. This review is based on the report at the meeting «Coronavirus - a global challenge to science» of the Scientific Council «Life Science» of the Russian Academy of Science: Lvov D.K., Alkhovsky S.V., Burtseva E.I. COVID-19 pandemic sources: origin, biology and genetics of coronaviruses of SARS-CoV, SARS-CoV-2, MERS-CoV (Conference hall of Presidium of RAS, 14 Leninsky Prospect, Moscow, Russia. April 16, 2020).

Evolutionary relationships and sequence‐structure determinants in human SARS coronavirus‐2 spike proteins for host receptor recognition

Coronavirus disease 2019 (COVID‐19) is a pandemic infectious disease caused by novel Severe Acute Respiratory Syndrome coronavirus‐2 (SARS CoV‐2). The SARS CoV‐2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and SARS CoV‐2 via their glycosylated spike proteins recognize the human angiotensin converting enzyme‐2 (ACE‐2) receptor. We generated multiple sequence alignments and phylogenetic trees for representative spike proteins of SARS CoV and SARS CoV‐2 from various host sources in order to analyze the specificity in SARS CoV‐2 spike proteins required for causing infection in humans. Our results show that among the genomes analysed, two sequence regions in the N‐terminal domain (NTD); "MESEFR" and "SYLTPG" are specific to human SARS CoV‐2. In the receptor binding domain (RBD), two sequence regions; "VGGNY" and "EIYQAGSTPCNGV" and a disulfide bridge connecting 480C and 488C in the extended loop are structural determinants for the recognition of human ACE‐2 receptor. The complete genome analysis of representative SARS CoVs from bat, civet, human host sources and human SARS CoV‐2 identified the bat genome (GenBank code: MN996532.1) as closest to the recent novel human SARS CoV‐2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933 and MG772934) are evolutionary intermediates in the mutagenesis progression towards becoming human SARS CoV‐2. This article is protected by copyright. All rights reserved.

[Analysis of variation and evolution of SARS-CoV-2 genome].

OBJECTIVE To analyze the evolution and variation of SARS-CoV-2 during the epidemic starting at the end of 2019. METHODS We downloaded the full-length genome sequence of SARS-CoV-2 from the databases of GISAID and NCBI. Using the software for bioinformatics including MEGA-X, BEAST, and TempEst, we constructed the genomic evolution tree, inferred the time evolution signal of the virus, calculated the tMRCA time of the virus and analyzed the selection pressure of the virus during evolution. RESULTS The phylogenetic tree showed that SARS-CoV-2 belonged to the Sarbecovirus subgenus of β Coronavirus genus together with bat coronavirus BetaCoV/bat/Yunnan/RaTG13/2013, bat-SL-CoVZC45, bat-SL-CoVZXC21 and SARS-CoV. The genomic sequences of SARS-CoV-2 isolated from the ongoing epidemic showed a weak time evolution signal with an average tMRCA time of 73 days (95% CI: 38.9-119.3 days). No positive time evolution signal was found between SARS-CoV-2 and BetaCoV/bat/Yunnan/RaTG13/2013, but the former virus had a strong positive temporal evolution relationship with bat-SL-CoVZC45 and SARS-CoV. The major cause for mutations of SARS-CoV-2 was the pressure of purification selection during the epidemic. CONCLUSIONS SARS-CoV-2 may have emerged as early as November, 2019, originating most likely from bat-associated coronavirus. This finding may provide evidence for tracing the sources and evolution of the virus.

A simple method for detection of a novel coronavirus (SARS-CoV-2) using one-step RT-PCR followed by restriction fragment length polymorphism

A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. Early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. We aimed to establish a simple method for the detection of SARS-CoV-2 in differentiating with SARS-CoV. Primers of our in-house reverse transcription polymerase chain reaction (RT-PCR) assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I, and AluI to distinguish between SARS-CoV-2 and SARS-CoV. In this report, a 396-bp fragment of the RdRp gene and 345-bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP-amplified product of SARS-CoV-2 generating three fragments of 45, 154, and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating two fragments of 248 and 97 bp without cutting to SARS-CoV. The accuracy of the assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, in the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV.

CoV-Seq: SARS-CoV-2 Genome Analysis and Visualization

Summary COVID-19 has become a global pandemic not long after its inception in late 2019. SARS-CoV-2 genomes are being sequenced and shared on public repositories at a fast pace. To keep up with these updates, scientists need to frequently refresh and reclean datasets, which is ad hoc and labor-intensive. Further, scientists with limited bioinformatics or programming knowledge may find it difficult to analyze SARS-CoV-2 genomes. In order to address these challenges, we developed CoV-Seq, a webserver to enable simple and rapid analysis of SARS-CoV-2 genomes. Given a new sequence, CoV-Seq automatically predicts gene boundaries and identifies genetic variants, which are presented in an interactive genome visualizer and are downloadable for further analysis. A command-line interface is also available for high-throughput processing. Availability and Implementation CoV-Seq is implemented in Python and Javascript. The webserver is available at http://covseq.baidu.com/ and the source code is available from https://github.com/boxiangliu/covseq. Contact jollier.liu@gmail.com Supplementary information Supplementary information are available at bioRxiv online.

Molecular Characterization and Amino Acid Homology of Nucleocapsid (N) Protein in SARS-CoV-1, SARS-CoV-2, MERS-CoV, and Bat Coronavirus

Coronavirus disease - 2019 (COVID-19) pandemic, due to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is posing a severe bio threat to the entire world Nucleocapsids of SARS-CoV-2 and the related viruses were studied for gene and amino acid sequence homologies In this study, we established similarities and differences in nucleocapsids in SARS-CoV-2, severe acute respiratory syndrome - coronavirus-1 (SARS-CoV-1), bat coronavirus (bat-CoV) and Middle East respiratory syndrome - coronavirus (MERS-CoV) We conducted a detailed analysis of the nucleocapsid protein amino acid and gene sequence encoding it, found in various coronavirus strains After thoroughly screening the different nucleocapsids, we observed a close molecular homology between SARS-CoV-1 and SARS-CoV-2 More than 95% sequence similarity was observed between the two SARS-CoV strains Bat-CoV and SARS-CoV-2 showed 92% sequence similarity MERS-CoV and SARS-CoV-2 nucleocapsid analysis indicated only 65% identity Molecular characterization of nucleocapsids from various coronaviruses revealed that SARS-CoV 2 is more related to SARS-CoV 1 and bat-CoV SARS-CoV 2 exhibited less resemblance with MERS-CoV SARS-CoV 2 showed less similarity to MERS-CoV Thus, either SARS-CoV-1 or bat-CoV may be the source of SARS-CoV-2 evolution Moreover, the existing differences in nucleocapsid molecular structures in SARS-CoV-2 make this virus more virulent and highly infectious, which means that the non-identical SARS-CoV-2 genes (which are absent in SARS-CoV-1 and bat-CoV) are responsible for COVID-19 severity We observed that SARS-CoV-2 nucleocapsid from different locations varied in amino acid sequences This revealed that there are many SARS-CoV-2 subtypes/subsets currently circulating globally This study will help to develop antiviral vaccine and drugs, study viral replication and immunopathogenesis, and synthesize monoclonal antibodies that can be used for precise COVID-19 diagnosis, without false-positive/false-negative results

Comparative transcriptome analysis reveals the intensive early-stage responses of host cells to SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a widespread outbreak of highly pathogenic COVID-19. It is therefore important and timely to characterize interactions between the virus and host cell at the molecular level to understand its disease pathogenesis. To gain insights, we performed high-throughput sequencing that generated time-series data simultaneously for bioinformatics analysis of virus genomes and host transcriptomes implicated in SARS-CoV-2 infection. Our analysis results showed that the rapid growth of the virus was accompanied by an early intensive response of host genes. We also systematically compared the molecular footprints of the host cells in response to SARS-CoV-2, SARS-CoV and MERS-CoV. Upon infection, SARS-CoV-2 induced hundreds of up-regulated host genes hallmarked by a significant cytokine production followed by virus-specific host antiviral responses. While the cytokine and antiviral responses triggered by SARS-CoV and MERS-CoV were only observed during the late stage of infection, the host antiviral responses during the SARS-CoV-2 infection were gradually enhanced lagging behind the production of cytokine. The early rapid host responses were potentially attributed to the high efficiency of SARS-CoV-2 entry into host cells, underscored by evidence of a remarkably up-regulated gene expression of TPRMSS2 soon after infection. Taken together, our findings provide novel molecular insights into the mechanisms underlying the infectivity and pathogenicity of SARS-CoV-2.

COVID-19 Variants Database: A repository for Human SARS-CoV-2 Polymorphism Data

COVID-19 is a newly communicable disease with a catastrophe outbreak that affects all over the world. We retrieved about 8,781 nucleotide fragments and complete genomes of SARS-CoV-2 reported from sixty-four countries. The CoV-2 reference genome was obtained from the National Genomics Data Center (NGDC), GISAID, and NCBI Genbank. All the sequences were aligned against reference genomes using Clustal Omega and variants were called using in-house built Python script. We intend to establish a user-friendly online resource to visualize the variants in the viral genome along with the Primer Infopedia. After analyzing and filtering the data globally, it was made available to the public. The detail of data available to the public includes mutations from 5688 SARS-CoV-2 sequences curated from 91 regions. This database incorporated 39920 mutations over 3990 unique positions. According to the translational impact, these mutations include 11829 synonymous mutations including 681 synonymous frameshifts and 21701 nonsynonymous mutations including 10 nonsynonymous frameshifts. Development of SARS-CoV-2 mutation genome browsers is a fundamental step obliging towards the virus surveillance, viral detection, and development of vaccine and therapeutic drugs. The SARS-COV-2 mutation browser is available at http://covid-19.dnageography.com.

Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop

The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) originally arose as part of a major outbreak of respiratory disease centered on Hubei province, China. It is now a global pandemic and is a major public health concern. Taxonomically, SARS-CoV-2 was shown to be a Betacoronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2). Subsequently, betacoronaviruses from pangolins were identified as close relatives to SARS-CoV-2. Here, we perform structural modeling of the SARS-CoV-2 spike glycoprotein. Our data provide support for the similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains. We suggest this loop confers fusion activation and entry properties more in line with betacoronaviruses in lineages A and C, and be a key component in the evolution of SARS-CoV-2 with this structural loop affecting virus stability and transmission.

Human Gene Sequences in SARS-CoV-2 and Other Viruses.

In a previous study, we identified a 117 base severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 Orf1b gene in non-structural protein 14 (NSP14), which is an exonuclease and NSP15, an endoribonuclease. In the current study we compared the human genome with other viral genomes to determine some of the characteristics of human sequences found in the latter. Most of the viruses had human sequences, but they were short. Hepatitis A and St Louis encephalitis had human sequences that were longer than the 117 base SARS-Cov-2 sequence, but they were in non-coding regions of the human genome. The SARS-Cov-2 sequence was the only long sequence found in a human gene (NTNG1). The related coronaviruses SARS-Cov had a 41 BP human sequence on chromosome 3 that was not part of a human gene, and MERS had no human sequence. The 117 base SARS-CoV-2 human sequence is relatively close to the viral spike sequence, separated only by NSP16, a 904 base sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 (ACE2) and internalization of the complex by the host cell. We have no explanation for the NSP14 and NSP15 SARS-Cov-2 sequences we observed here or how they might relate to infectiousness. Further studies are warranted.

On spatial molecular arrangements of SARS-CoV2 genomes of Indian patients

A pandemic caused by the SARS-CoV2 is being experienced by the whole world since December, 2019. A thorough understanding beyond just sequential similarities among the protein coding genes of SARS-CoV2 is important in order to differentiate or relate to the other known CoVs of the same genus. In this study, we compare three genomes namely MT012098 (India-Kerala), MT050493 (India-Kerala), MT358637 (India-Gujrat) from India with NC_045512 (China-Wuhan) to view the spatial as well as molecular arrangements of nucleotide bases of all the genes embedded in these four genomes. Based on different features extracted for each gene embedded in these genomes, corresponding phylogenetic relationships have been built up. Differences in phylogenetic tree arrangement with individual gene suggest that three genomes of Indian origin have come from three different origins or the evolution of viral genome is very fast process. This study would also help to understand the virulence factors, disease pathogenicity, origin and transmission of the SARS-CoV2.

Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Human Gene Sequences in SARS-CoV-2 and Other Viruses

Genomic, geographic and temporal distributions of SARS-CoV-2 mutations

The COVID-19 pandemic is the most significant public health issue in recent history. Its causal agent, SARS-CoV-2, has evolved rapidly since its first emergence in December 2019. Mutations in the viral genome have critical impacts on the adaptation of viral strains to the local environment, and may alter the characteristics of viral transmission, disease manifestation, and the efficacy of treatment and vaccination. Using the complete sequences of 1,932 SARS-CoV-2 genomes, we examined the genomic, geographic and temporal distributions of aged, new, and frequent mutations of SARS-CoV-2, and identified six phylogenetic clusters of the strains, which also exhibit a geographic preference in different continents. Mutations in the form of single nucleotide variations (SNVs) provide a direct interpretation for the six phylogenetic clusters. Linkage disequilibrium, haplotype structure, evolutionary process, global distribution of mutations unveiled a sketch of the mutational history. Additionally, we found a positive correlation between the average mutation count and case fatality, and this correlation had strengthened with time, suggesting an important role of SNVs on disease outcomes. This study suggests that SNVs may become an important consideration in virus detection, clinical treatment, drug design, and vaccine development to avoid target shifting, and that continued isolation and sequencing is a crucial component in the fight against this pandemic. Significance Statement Mutation is the driving force of evolution for viruses like SARS-CoV-2, the causal agent of COVID-19. In this study, we discovered that the genome of SARS-CoV-2 is changing rapidly from the originally isolated form. These mutations have been spreading around the world and caused more than 2.5 million of infected cases and 170 thousands of deaths. We found that fourteen frequent mutations identified in this study can characterize the six main clusters of SARS-CoV-2 strains. In addition, we found the mutation burden is positively correlated with the fatality of COVID-19 patients. Understanding mutations in the SARS-CoV-2 genome will provide useful insight for the design of treatment and vaccination.

SARS-CoV-2: Structural diversity, phylogeny, and potential animal host identification of spike glycoprotein

To investigate the evolutionary history of the current pandemic outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a total of 137 genomes of coronavirus strains with release dates between January 2019 and 25 March 2020, were analyzed. To investigate the potential intermediate host of the SARS-CoV-2, we analyzed spike glycoprotein sequences from different animals, with particular emphasis on bats. We performed phylogenetic analysis and structural reconstruction of the spike glycoproteins with subsequent alignment and comparison. Our phylogenetic results revealed that SARS-CoV-2 was more similar to the bats' betacoronavirus isolates: HKU5-related from Pipistrellus abramus and HKU4-related from Tylonycteris pachypus. We also identified a yak betacoronavirus strain, YAK/HY24/CH/2017, as the closest match in the comparison of the structural models of spike glycoproteins. Interestingly, a set of unique features has been described for this particular strain of the yak betacoronavirus. Therefore, our results suggest that the human SARS-CoV-2, responsible for the current outbreak of COVID-19, could also come from yak as an intermediate host.

Snapshot of the evolution and mutation patterns of SARS-CoV-2

The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.

Unsupervised cluster analysis of SARS-CoV-2 genomes reflects its geographic progression and identifies distinct genetic subgroups of SARS-CoV-2 virus

Over 10,000 viral genome sequences of the SARS-CoV-2 virus have been made readily available during the ongoing coronavirus pandemic since the initial genome sequence of the virus was released on the open access Virological website (http://virological.org/) early on January 11. We utilize the published data on the single stranded RNAs of 11, 132 SARS-CoV-2 patients in the GISAID (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) database, which contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Among many important research questions which are currently being investigated, one aspect pertains to the genetic characterization/classification of the virus. We analyze data on the nucleotide sequencing of the virus and geographic information of a subset of 7, 640 SARS-CoV-2 patients without missing entries that are available in the GISAID database. Instead of modelling the mutation rate, applying phylogenetic tree approaches, etc., we here utilize a model-free clustering approach that compares the viruses at a genome-wide level. We apply principal component analysis to a similarity matrix that compares all pairs of these SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index (Jaccard, 1901; Tan et al., 2005; Prokopenko et al., 2016; Schlauch et al., 2017). Our analysis results of the SARS-CoV-2 genome data illustrates the geographic and chronological progression of the virus, starting from the first cases that were observed in China to the current wave of cases in Europe and North America. We also observe that, based on their sequence data, the SARS-CoV-2 viruses cluster in distinct genetic subgroups. It is the subject of ongoing research to examine whether the genetic subgroup could be related to diseases outcome and its potential implications for vaccine development.

Molecular Epidemiology of Severe Acute Respiratory Syndrome–Associated Coronavirus Infections in Taiwan

Background In 2003, Taiwan experienced a series of outbreaks of severe acute respiratory syndrome (SARS) and 1 laboratory-contamination accident. Here we describe a new phylogenetic analytical method to study the sources and dissemination paths of SARS-associated coronavirus (SARS-CoV) infections in Taiwan Methods A phylogenetic analytical tool for combining nucleotide sequences from 6 variable regions of a SARS-CoV genome was developed by use of 20 published SARS-CoV sequences; and this method was validated by use of 80 published SARS-CoV sequences. Subsequently, this new tool was applied to provide a better understanding of the entire complement of Taiwanese SARS-CoV isolates, including 20 previously published and 19 identified in this study. The epidemiological data were integrated with the results from the phylogenetic tree and from the nucleotide-signature pattern Results The topologies of phylogenetic trees generated by the new and the conventional strategies were similar, with the former having better robustness than the latter, especially in comparison with the maximum-likelihood trees: the new strategy revealed that during 2003 there were 5 waves of epidemic SARS-CoV infection, which belonged to 3 phylogenetic clusters in Taiwan Conclusions The new strategy is more efficient than its conventional counterparts. The outbreaks of SARS in Taiwan originated from multiple sources

Evidence of Increasing Diversification of Emerging SARS-CoV-2 Strains

BACKGROUND: On January 30th, 2020, an outbreak of atypical pneumonia caused by a novel Betacoronavirus (ßCoV), named SARS-CoV-2, was declared a public health emergency of international concern by the World Health Organization. For this reason, a detailed evolutionary analysis of SARS-CoV-2 strains currently circulating in different geographic regions of the world was performed. METHODS: A compositional analysis as well as a Bayesian coalescent analysis of complete genome sequences of SARS-CoV-2 strains recently isolated in Europe, North America, South America and Asia was performed. RESULTS: The results of these studies revealed a diversification of SARS-CoV-2 strains in three different genetic clades. Co-circulation of different clades in different countries, as well as different genetic lineages within different clades were observed. The time of the most recent common ancestor (tMRCA) was established to be around November 1, 2019. A mean rate of evolution of 6.57 x 10-4 substitutions per site per year was found. A significant migration rate per genetic lineage per year from Europe to South America was also observed. CONCLUSION: The results of these studies revealed an increasing diversification of SARS-CoV-2 strains. High evolutionary rates and fast population growth characterizes the population dynamics of SARS-CoV-2 strains. This article is protected by copyright. All rights reserved.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient’s sequence clustered with sequences from Germany while the tourist’s sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients’ sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

SARS-CoV-2 host diversity: An update of natural infections and experimental evidence

Coronavirus disease-19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is now a pandemic threat. This virus is supposed to be spread by human to human transmission. Cellular angiotensin-converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2 which is identical or similar in different species of animals such as pigs, ferrets, cats, orangutans, monkeys, and humans. Moreover, a recent study predicted that dogs might be secondary hosts during the evolution of SARS-CoV-2 from bat to human. Therefore, there is a possibility of spreading SARS-CoV-2 through domestic pets. There are now many reports of SARS-CoV-2 positive cases in dogs, cats, tigers, lion, and minks. Experimental data showed ferrets and cats are highly susceptible to SARS-CoV-2 as infected by virus inoculation and can transmit the virus directly or indirectly by droplets or airborne routes. Based on these natural infection reports and experimental data, whether the pets are responsible for SARS-CoV-2 spread to humans; needs to be deeply investigated. Humans showing clinical symptoms of respiratory infections have been undergoing for the COVID-19 diagnostic test but many infected people and few pets confirmed with SARS-CoV-2 remained asymptomatic. In this review, we summarize the natural cases of SARS-CoV-2 in animals with the latest researches conducted in this field. This review will be helpful to think insights of SARS-CoV-2 transmissions, spread, and demand for seroprevalence studies, especially in companion animals.

Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak

An outbreak of coronavirus disease 2019 (COVID-19) caused by the 2019 novel coronavirus (SARS-CoV-2) began in the city of Wuhan in China and has widely spread worldwide. Currently, it is vital to explore potential intermediate hosts of SARS-CoV-2 to control COVID-19 spread. Therefore, we reinvestigated published data from pangolin lung samples from which SARS-CoV-like CoVs were detected by Liu et al. [1]. We found genomic and evolutionary evidence of the occurrence of a SARS-CoV-2-like CoV (named Pangolin-CoV) in dead Malayan pangolins. Pangolin-CoV is 91.02% and 90.55% identical to SARS-CoV-2 and BatCoV RaTG13, respectively, at the whole-genome level. Aside from RaTG13, Pangolin-CoV is the most closely related CoV to SARS-CoV-2. The S1 protein of Pangolin-CoV is much more closely related to SARS-CoV-2 than to RaTG13. Five key amino acid residues involved in the interaction with human ACE2 are completely consistent between Pangolin-CoV and SARS-CoV-2, but four amino acid mutations are present in RaTG13. Both Pangolin-CoV and RaTG13 lost the putative furin recognition sequence motif at S1/S2 cleavage site that can be observed in the SARS-CoV-2. Conclusively, this study suggests that pangolin species are a natural reservoir of SARS-CoV-2-like CoVs.

The origin and underlying driving forces of the SARS-CoV-2 outbreak

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019–12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 ‘SARS-related-conserved’ regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 ‘SARS-CoV-2-conserved-structured’ regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5’ UTR, frame-shifting element, and 3’ UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 ‘SARS-CoV-2-conserved-unstructured’ genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

Whole genome sequencing of SARS-CoV-2 isolated in Guangdong Province and factors influencing the sequencing/ 广东省新型冠状病毒全基因组序列测定及其影响因素分析

Objective: To obtain the genome sequences of SARS-CoV-2 in respiratory specimens in Guangdong Province with next-generation sequencing (NGS) and analyze the factors influencing sequencing. Methods: Eight upper and lower respiratory tract specimens were collected from patients with SARS-CoV-2 infection in Guangdong Province in January 2020. RNA library construction was used to obtain the genome sequences of SARS-CoV-2. A bio-informatics software package (CLC Genomics Workbench 12.0) was used to analyze and compare the genomic sequences. Results: Five SARS-CoV-2 genome sequences were obtained from the eight specimens and two were obtained from lower respiratory tract specimens. The nucleotide homology to SARS-CoV-2 was 97.74%-99.90%. The Ct values were lower, while the sequencing depth, coverage, relative abundance and genome integrity were higher in sequencing the SARS-CoV-2 in lower respiratory tract specimens. Conclusions: The low Ct value of SARS-CoV-2 in the samples was good for sequencing.

Phylogeny of SARS-CoV as inferred from complete genome comparison

SARS-CoV, as the pathogeny of severe acute respiratory syndrome (SARS), is a mystery that the origin of the virus is still unknown even a few isolates of the virus were completely sequenced. To explore the genesis of SARS-CoV, the FDOD method previously developed by us was applied to comparing complete genomes from 12 SARS-CoV isolates to those from 12 previously identified coronaviruses and an unrooted phylogenetic tree was constructed. Our results show that all SARS-CoV isolates were clustered into a clique and previously identified coronaviruses formed the other clique. Meanwhile, the three groups of coronaviruses depart from each other clearly in our tree that is consistent with the results of prevenient papers. Differently, from the topology of the phylogenetic tree we found that SARS-CoV is more close to group 1 within genus coronavirus. The topology map also shows that the 12 SARS-CoV isolates may be divided into two groups determined by the association with the SARS-CoV from the Hotel M in Hong Kong that may give some information about the infectious relationship of the SARS.

Genomic surveillance of SARS-CoV-2 reveals community transmission of a major lineage during the early pandemic phase in Brazil

Despite all efforts to control the COVID-19 spread, the SARS-CoV-2 reached South America within three months after its first detection in China, and Brazil became one of the hotspots of COVID-19 in the world. Several SARS-CoV-2 lineages have been identified and some local clusters have been described in this early pandemic phase in Western countries. Here we investigated the genetic diversity of SARS-CoV-2 during the early phase (late February to late April) of the epidemic in Brazil. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 in Brazil and the community transmission of a major B.1.1 lineage defined by two amino acid substitutions in the Nucleocapsid and ORF6. This SARS-CoV-2 Brazilian lineage was probably established during February 2020 and rapidly spread through the country, reaching different Brazilian regions by the middle of March 2020. Our study also supports occasional exportations of this Brazilian B.1.1 lineage to neighboring South American countries and to more distant countries before the implementation of international air travels restrictions in Brazil.

The novel coronavirus SARS-CoV-2: From a zoonotic infection to coronavirus disease 2019

The novel coronavirus (CoV), severe acute respiratory syndrome (SARS)-CoV-2 is an international public health emergency. Until now, the intermediate host and mechanisms of the interspecies jump of this virus are unknown. Phylogenetic analysis of all available bat CoV complete genomes was performed to analyze the relationships between bat CoV and SARS-CoV-2. To suggest a possible intermediate host, another phylogenetic reconstruction of CoV genomes obtained from animals that were hypothetically commercialized in the Chinese markets was also carried out. Moreover, mutation analysis was executed to suggest genomic regions that may have permitted the adaptation of SARS-CoV-2 to the human host. The phylogenetic analysis demonstrated that SARS-CoV-2 formed a cluster with the bat CoV isolate RaTG13. Possible CoV interspecies jumps among bat isolates were also observed. The phylogenetic tree reconstructed from CoV strains belonging to different animals demonstrated that SARS-CoV-2, bat RaTG13, and pangolin CoV genomes formed a monophyletic cluster, demonstrating that pangolins may be suggested as SARS-CoV-2 intermediate hosts. Three AA substitutions localized in the S1 portion of the S gene were observed, some of which have been correlated to structural modifications of the S protein which may facilitate SARS-CoV-2 tropism to human cells. Our analysis shows the tight relationship between SARS-CoV-2 and bat SARS-like strains. It also hypothesizes that pangolins might have been possible intermediate hosts of the infection. Some of the observed AA substitutions in the S-binding protein may serve as possible adaptation mutations in humans but more studies are needed to elucidate their function.

Selectomic and Evolvability Analyses of the Highly Pathogenic Betacoronaviruses SARS-CoV-2, SARS-CoV, and MERS-CoV

SARS-CoV-2, the causative agent of COVID-19, is widespread in several countries around the world following its late-2019 emergence in the human population. Rapid development of molecular diagnostic tests and subunit vaccines have been prioritized, and as such evaluating the SARS-CoV-2 genomic plasticity and evolutionary dynamics is an urgent need. We determined the SARS-CoV-2 selectome by calculating rates of pervasive and episodic diversifying selection for every amino acid coding position in the SARS-CoV-2 genome. To provide context for evolutionary dynamics of a highly pathogenic betacoronavirus following a zoonotic spillover into human hosts, we also determined the selectomes of SARS-CoV and MERS-CoV, and performed evolvability calculations for SARS-CoV-2 based on SARS-CoV. These analyses identify the amino acid sites within each coding sequence that have been subjected to pervasive diversifying selection or episodic diversifying selection, and report significantly evolvable sites in the ORF1a polyprotein, the spike protein, and the membrane protein of SARS-CoV-2. These findings provide a comprehensive view of zoonotic, highly pathogenic betacoronavirus evolutionary dynamics that can be directly applied to diagnostic assay and vaccine design for SARS-CoV-2.

A Simple Method for Detection of a Novel Coronavirus (SARS-CoV-2) using One-step RT-PCR followed by Restriction Fragment Length Polymorphism

BACKGROUND: A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. An early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. OBJECTIVES: We aimed to establish a simple method for detection of SARS-CoV-2 in differentiating with SARS-CoV. STUDY DESIGN: Primers of our in-house RT-PCR assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I and AluI to distinguish between SARS-CoV-2 and SARS-CoV. RESULTS AND DISCUSSIONS: In this report, a 396 bp fragment of the RdRp gene and 345 bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP amplified product of SARS-CoV-2 generating 3 fragments of 45, 154 and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing 2 fragments of 76 and 320 bp, whereas, the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating 2 fragments of 248 and 97 bp without cutting to SARS-CoV. Accuracy of assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV. This article is protected by copyright. All rights reserved.

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2

SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 5 2020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2

To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.

[Analysis of variation and evolution of SARS-CoV-2 genome]

OBJECTIVE: To analyze the evolution and variation of SARS-CoV-2 during the epidemic starting at the end of 2019. METHODS: We downloaded the full-length genome sequence of SARS-CoV-2 from the databases of GISAID and NCBI. Using the software for bioinformatics including MEGA-X, BEAST, and TempEst, we constructed the genomic evolution tree, inferred the time evolution signal of the virus, calculated the tMRCA time of the virus and analyzed the selection pressure of the virus during evolution. RESULTS: The phylogenetic tree showed that SARS-CoV-2 belonged to the Sarbecovirus subgenus of ß Coronavirus genus together with bat coronavirus BetaCoV/bat/Yunnan/RaTG13/2013, bat-SL-CoVZC45, bat-SL-CoVZXC21 and SARS-CoV. The genomic sequences of SARS-CoV-2 isolated from the ongoing epidemic showed a weak time evolution signal with an average tMRCA time of 73 days (95% CI: 38.9-119.3 days). No positive time evolution signal was found between SARS-CoV-2 and BetaCoV/bat/Yunnan/RaTG13/2013, but the former virus had a strong positive temporal evolution relationship with bat-SL-CoVZC45 and SARS-CoV. The major cause for mutations of SARS-CoV-2 was the pressure of purification selection during the epidemic. CONCLUSIONS: SARS-CoV-2 may have emerged as early as November, 2019, originating most likely from bat-associated coronavirus. This finding may provide evidence for tracing the sources and evolution of the virus.

Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection

Abstract SARS-CoV-2 causes the recent global COVID-19 public health emergency. ACE2 is the receptor for both SARS-CoV-2 and SARS-CoV. To predict the potential host range of SARS-CoV-2, we analyzed the key residues of ACE2 for recognizing S protein. We found that most of the selected mammals including pets (dog and cat), pangolin and Circetidae mammals remained the most of key residues for association with S protein from SARS-CoV and SARS-CoV-2. The interaction interface between cat/dog/pangolin/Chinese hamster ACE2 and SARS-CoV/SARS-CoV-2 S protein was simulated through homology modeling. We identified that N82 in ACE2 showed a closer contact with SARS-CoV-2 S protein than M82 in human ACE2. Our finding will provide important insights into the host range of SARS-CoV-2 and a new strategy to design an optimized ACE2 for SARS-CoV-2 infection.

Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CLpro targeting repurposed drug candidates

BACKGROUND: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. METHODS: For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CLpro) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. RESULTS: Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CLpro enzyme, which regulates viral replication machinery. CONCLUSIONS: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

Evolutionary Trajectory for the Emergence of Novel Coronavirus SARS-CoV-2

Over the last two decades, the world experienced three outbreaks of coronaviruses with elevated morbidity rates. Currently, the global community is facing emerging virus SARS-CoV-2 belonging to Betacoronavirus, which appears to be more transmissible but less deadly than SARS-CoV. The current study aimed to track the evolutionary ancestors and different evolutionary strategies that were genetically adapted by SARS-CoV-2. Our whole-genome analysis revealed that SARS-CoV-2 was the descendant of Bat SARS/SARS-like CoVs and bats served as a natural reservoir. SARS-CoV-2 used mutations and recombination as crucial strategies in different genomic regions including the envelop, membrane, nucleocapsid, and spike glycoproteins to become a novel infectious agent. We confirmed that mutations in different genomic regions of SARS-CoV-2 have specific influence on virus reproductive adaptability, allowing for genotype adjustment and adaptations in rapidly changing environments. Moreover, for the first time we identified nine putative recombination patterns in SARS-CoV-2, which encompass spike glycoprotein, RdRp, helicase and ORF3a. Six recombination regions were spotted in the S gene and are undoubtedly important for evolutionary survival, meanwhile this permitted the virus to modify superficial antigenicity to find a way from immune reconnaissance in animals and adapt to a human host. With these combined natural selected strategies, SARS-CoV-2 emerged as a novel virus in human society.

A High-Coverage SARS-CoV-2 Genome Sequence Acquired by Target Capture Sequencing

This manuscript is based on the method we developed urgently to deal with the research requirement in the conflict between achieving a complete genome sequence for the evolutionary history of SARS-CoV-2 study and the low viral RNA concentration. Here, in this manuscript, we developed a set of SARS-CoV-2 enrichment probes to increase the sensitivity of sequence-based virus detection and characterization via obtaining the comprehensive genome sequence. Following the CDC health and safety guidelines, we test the concept using the culturing supernatant contain SARS-CoV-2 particles, and its full-length sequence was used for further analysis. The fraction of SARS-CoV-2 endogenous DNA was 93.47% with Cluster Factor about 1.1, which demonstrate that the numbers of mapped reads to SARS-CoV-2 reference sequence significantly increased, compared to metagenomic sequencing technology, following SARS-CoV-2 probe enrichment. Moreover, based on the high-quality sequence, we discussed the heterozygosity and viral expression during replication of coronavirus, and its phylogenetic relationship with other selected high-quality samples from The Genome Variation Map (GVM) (on 2020/03/22). We believe this manuscript is valuable for all the researchers who are interested in using clinical warp samples to obtain the high coverage of SARS-CoV-2 genome sequence with a relatively low concentration of viral particles. This would allow the clinician to correlate the diagnostic data with molecular monitoring in viral evolutional, the most importantly, to track the functional mutation of SARS-CoV-2.

Could SARS-CoV-2 affect male fertility?

We performed this systematic review to evaluate the possibility of an impact of SARS-CoV-2 infection on male fertility. SARS-CoV-2 enters the cells with the help of ACE2; therefore, testicular expression of ACE2 was analysed from transcriptome sequencing studies and our unpublished data. Literature suggested that SARS-CoV-1 (2002-2004 SARS) had a significant adverse impact on testicular architecture, suggesting a high possibility of the impact of SARS-CoV-2 as well. Out of two studies on semen samples from COVID-19 affected patients, one reported the presence of SARS-CoV-2 in the semen samples while the other denied it, raising conflict about its presence in the semen samples and the possibility of sexual transmission. Our transcriptome sequencing studies on rat testicular germ cells showed ACE expression in rat testicular germ cells. We also found ACE2 expression in transcriptome sequencing data for human spermatozoa, corroborating its presence in the testicular germ cells. Transcriptome sequencing data from literature search revealed ACE2 expression in the germ, Sertoli and Leydig cells. The presence of ACE2 on almost all testicular cells and the report of a significant impact of previous SARS coronavirus on testes suggest that SARS-CoV-2 is highly likely to affect testicular tissue, semen parameters and male fertility.

Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions

The COVID-19 pandemic has spread across the globe at an alarming rate in the last four months. However, unlike any of the previous global outbreaks the availability of hundreds of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 480 full-length (>29000 nt) sequences from the 1575 SARS-CoV-2 sequences available and identified 37 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, two of the 37 substitutions occur within highly conserved secondary structures in the 5’ and 3’ untranslated regions that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define 3 mutually exclusive lineages (A1, B1 and C1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2.

Understanding evolution of SARS-CoV-2: A perspective from analysis of genetic diversity of RdRp gene

Coronavirus disease 2019 emerged as the first example of "Disease X", a hypothetical disease of humans caused by an unknown infectious agent that was named as novel coronavirus and subsequently designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The origin of the outbreak at the animal market in Wuhan, China implies it as a case of zoonotic spillover. The study was designed to understand evolution of Betacoronaviruses and in particular diversification of SARS-CoV-2 using RNA dependent RNA polymerase (RdRp) gene, a stable genetic marker. Phylogenetic and population stratification analyses were carried out using maximum likelihood and Bayesian methods, respectively. Molecular phylogeny using RdRp showed that SARS-CoV-2 isolates cluster together. Bat-CoV isolate RaTG13 and Pangolin-CoVs are observed to branch off prior to SARS-CoV-2 cluster. While SARS-CoV form a single cluster, Bat-CoVs form multiple clusters. Population-based analyses revealed that both SARS-CoV-2 and SARS-CoV form separate clusters with no admixture. Bat-CoVs were found to have single and mixed ancestry and clustered as four sub-populations. Population-based analyses of Betacoronaviruses using RdRp revealed that SARS-CoV-2 is a homogeneous population. SARS-CoV-2 appears to have evolved from Bat-CoV isolate RaTG13, which diversified from a common ancestor from which Pangolin-CoVs have also evolved. The admixed Bat-CoV sub-populations indicate that bats serve as reservoirs harboring virus ensembles that are responsible for zoonotic spillovers such as SARS-CoV and SARS-CoV-2. The extent of admixed isolates of Bat-CoVs observed in population diversification studies underline the need for periodic surveillance of bats and other animal reservoirs for potential spillovers as a measure towards preparedness for emergence of zoonosis.

The insert sequence in SARS-CoV-2 enhances spike protein cleavage by TMPRSS

At the end of 2019, the SARS-CoV-2 induces an ongoing outbreak of pneumonia in China1, even more spread than SARS-CoV infection2. The entry of SARS-CoV into host cells mainly depends on the cell receptor (ACE2) recognition and spike protein cleavage-induced cell membrane fusion3,4. The spike protein of SARS-CoV-2 also binds to ACE2 with a similar affinity, whereas its spike protein cleavage remains unclear5,6. Here we show that an insertion sequence in the spike protein of SARS-CoV-2 enhances the cleavage efficiency, and besides pulmonary alveoli, intestinal and esophagus epithelium were also the target tissues of SARS-CoV-2. Compared with SARS-CoV, we found a SPRR insertion in the S1/S2 protease cleavage sites of SARS-CoV-2 spike protein increasing the cleavage efficiency by the protein sequence aligment and furin score calculation. Additionally, the insertion sequence facilitates the formation of an extended loop which was more suitable for protease recognition by the homology modeling and molicular docking. Furthermore, the single-cell transcriptomes identified that ACE2 and TMPRSSs are highly coexpressed in AT2 cells of lung, along with esophageal upper epithelial cells and absorptive enterocytes. Our results provide the bioinformatics evidence for the increased spike protein cleavage of SARS-CoV-2 and indicate its potential target cells.

SARS-CoV-2 genome evolution exposes early human adaptations

The set of mutations observed at the outset of the SARS-CoV-2 pandemic may illuminate how the virus will adapt to humans as it continues to spread. Viruses are expected to quickly acquire beneficial mutations upon jumping to a new host species. Advantageous nucleotide substitutions can be identified by their parallel occurrence in multiple independent lineages and are likely to result in changes to protein sequences. Here we show that SARS-CoV-2 is acquiring mutations more slowly than expected for neutral evolution, suggesting purifying selection is the dominant mode of evolution during the initial phase of the pandemic. However, several parallel mutations arose in multiple independent lineages and may provide a fitness advantage over the ancestral genome. We propose plausible reasons for several of the most frequent mutations. The absence of mutations in other genome regions suggests essential components of SARS-CoV-2 that could be the target of drug development. Overall this study provides genomic insights into how SARS-CoV-2 has adapted and will continue to adapt to humans. SUMMARY In this study we sought signals of evolution to identify how the SARS-CoV-2 genome has adapted at the outset of the COVID-19 pandemic. We find that the genome is largely undergoing purifying selection that maintains its ancestral sequence. However, we identified multiple positions on the genome that appear to confer an adaptive advantage based on their repeated evolution in independent lineages. This information indicates how SARS-CoV-2 will evolve as it diversifies in an increasing number of hosts.

Unsupervised cluster analysis of SARS-CoV-2 genomes indicates that recent (June 2020) cases in Beijing are from a genetic subgroup that consists of mostly European and South(east) Asian samples, of which the latter are the most recent

Research efforts of the ongoing SARS-CoV-2 pandemic have focused on viral genome sequence analysis to understand how the virus spread across the globe. Here, we assess three recently identified SARS-CoV-2 genomes in Beijing from June 2020 and attempt to determine the origin of these genomes, made available in the GISAID database. The database contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Including the three new samples and excluding samples with missing annotations, we analyzed 7, 643 SARS-CoV-2 genomes. Using principal component analysis computed on a similarity matrix that compares all pairs of the SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index, we find that the newly discovered virus genomes from Beijing are in a genetic cluster that consists mostly of cases from Europe and South(east) Asia. The sequences of the new cases are most related to virus genomes from a small number of cases from China (March 2020), cases from Europe (February to early May 2020), and cases from South(east) Asia (May to June 2020). These findings could suggest that the original cases of this genetic cluster originated from China in March 2020 and were re-introduced to China by transmissions from samples from South(east) Asia between April and June 2020.

SARS-CoV-2 orf1b Gene Sequence in the NTNG1 Gene on Human Chromosome 1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.

The Architecture of SARS-CoV-2 Transcriptome

Summary SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3′ tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

SARS-CoV-2 sequence typing, evolution and signatures of selection using CoVa, a Python-based command-line utility

The current global pandemic COVID-19, caused by SARS-CoV-2, has resulted in millions of infections worldwide in a few months. Global efforts to tackle this situation have produced a tremendous body of genomic data, which can be used for tracing transmission routes, characterization of isolates, and monitoring variants with potential for unusual virulence. Several groups have analyzed these genomes using different approaches. However, as new data become available, the research community needs a pipeline to perform a set of routine analyses, that can quickly incorporate new genome sequences and update the analysis reports. We developed a programmatic tool, CoVa, with this objective. It is a fast, accurate and user-friendly utility to perform a variety of genome analyses on hundreds of SARS-CoV-2 sequences. Using CoVa, we define a modified sequence typing nomenclature and identify sites under positive selection. Further analysis identified some peptides and sites showing geographical patterns of selection. Specifically, we show differences in sequence type distribution between sequences from India and those from the rest of the world. We also show that several sites show signatures of positive selection uniquely in sequences from India. Preliminary evolutionary analysis, using features that will be incorporated into CoVa in the near future, show a mutation rate of 7.4 × 10−4 substitutions/site/year, confirm a temporal signal with a November 2019 origin of SARS-CoV-2, and a heterogeneity in the geographical distribution of Indian samples.

Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.

Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism

The spread of COVID-19 caused by the SARS-CoV-2 outbreak has been growing since its first identification in December 2019. The publishing of the first SARS-CoV-2 genome made a valuable source of data to study the details about its phylogeny, evolution, and interaction with the host. Protein-protein binding assays have confirmed that Angiotensin-converting enzyme 2 (ACE2) is more likely to be the cell receptor through which the virus invades the host cell. In the present work, we provide an insight into the interaction of the viral spike Receptor Binding Domain (RBD) from different coronavirus isolates with host ACE2 protein. By calculating the binding energy score between RBD and ACE2, we highlighted the putative jump in the affinity from a progenitor form of SARS-CoV-2 to the current virus responsible for COVID-19 outbreak. Our result was consistent with previously reported phylogenetic analysis and corroborates the opinion that the interface segment of the spike protein RBD might be acquired by SARS-CoV-2 via a complex evolutionary process rather than a progressive accumulation of mutations. We also highlighted the relevance of Q493 and P499 amino acid residues of SARS-CoV-2 RBD for binding to human ACE2 and maintaining the stability of the interface. Moreover, we show from the structural analysis that it is unlikely for the interface residues to be the result of genetic engineering. Finally, we studied the impact of eight different variants located at the interaction surface of ACE2, on the complex formation with SARS-CoV-2 RBD. We found that none of them is likely to disrupt the interaction with the viral RBD of SARS-CoV-2.

Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: Similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism

SARS-CoV-2 orf1b Gene Sequence in the NTNG1 Gene on Human Chromosome 1.

Genetic Variant of SARS-CoV-2 Isolates in Indonesia: Spike Glycoprotein Gene

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel coronavirus and the primary causative agent of coronavirus disease 2019 (COVID-19), first occurred in China and rapidly spread worldwide The government of the Republic of Indonesia confirmed its first two cases of COVID-19 in March 2020 COVID-19 is a serious illness with no efficacious antiviral medication or approved vaccine currently available Therefore, there is a need to investigate the genome of SARS-CoV-2 In this study, we characterized SARS-CoV-2 spike glycoprotein genes from Indonesia to investigate their genetic composition and variability Overall, ten SARS-CoV-2 spike glycoprotein gene sequences retrieved from GenBank (National Center for Biotechnology Information, USA) and the GISAID EpiCoV database (Germany) were compared We analyzed nucleotide variants and amino acid changes using Molecular Evolutionary Genetics Analysis (MEGA) X and analyzed gene similarity using the LALIGN web server Interestingly, we revealed several specific mutation sites, however, there were no significant changes in the genetic composition of SARS-CoV-2 spike glycoprotein genes, when compared to the WuhanHu-1 isolate from China However, this is a preliminary study and we recommend that molecular epidemiology and surveillance programs against COVID-19 in Indonesia be improved

Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study

BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Jin Yin-tan Hospital of Wuhan, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 'SARS-related-conserved' regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 'SARS-CoV-2-conserved-structured' regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5' UTR, frame-shifting element, and 3' UTR. Last, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 'SARS-CoV-2-conserved-unstructured' genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

Importation of SARS-CoV-2 infection leads to major COVID-19 epidemic in Taiwan

OBJECTIVE: COVID-19 has recently become a pandemic affecting many countries worldwide. This study aims to evaluate the current status of COVID-19 in Taiwan and analyze the source of infection. METHODS: National data regarding SARS-CoV-2 infection were obtained from Taiwan. CDC at the end of April 2020. These data were subjected to analysis of the current status and correlation between indigenous and imported COVID-19 cases. A phylogenetic tree was created to analyze the phylogeny of Taiwanese SARS-CoV-2 isolates. RESULTS: The first case of SARS-CoV-2 infection in Taiwan was detected on January 21, 2020. Epidemiological data indicate that by April 30, there were a total of 429 COVID-19 confirmed cases with the death rate of 1.3%. Most cases were identified as imported (79.9%; 343/429), with the majority originating from the United States of America (22.1%) and the United Kingdom (17.6%). Results from phylogenetic tree analyses indicate that the Taiwanese SARS-CoV-2 isolates were clustered with the SARS-CoV-2 isolates from other countries (bootstrap value 98%) and sub-clustered with bat SARS-like coronaviruses (bootstrap value 99%). CONCLUSION: This study suggests that the importation of SARS-CoV-2 infection was the primary risk-factor resulting in the COVID-19 epidemic in Taiwan.

SARS-CoV Infection Was from at Least Two Origins in the Taiwan Area

OBJECTIVE: Severe acute respiratory syndrome (SARS) is caused by a new coronavirus. Genomic sequence analysis will provide the molecular epidemiology and help to develop vaccines. METHODS: We developed a rapid method to amplify and sequence the whole SARS-CoV genome from clinical specimens. The technique employed one-step multiplex RT-PCR to amplify the whole SARS-CoV genome, and then nested PCR was performed to amplify a 2-kb region separately. The PCR products were sequenced. RESULTS: We sequenced the genomes of SARS-CoV from 3 clinical specimens obtained in Taiwan. The sequences were similar to those reported by other groups, except that 17 single nucleotide variations and two 2-nucleotide deletions, and a 1-nucleotide deletion were found. All the variations in the clinical specimens did not alter the amino acid sequence. Of these 17 sequenced variants, two loci (positions 26203 and 27812) were segregated together as a specific genotype-T:T or C:C. Phylogenetic analysis showed two major clusters of SARS patients in Taiwan. CONCLUSION: We developed a very economical and rapid method to sequence the whole genome of SARS-CoV, which can avoid cultural influence. From our results, SARS patients in Taiwan may be infected from two different origins.

Whole-genome sequence analysis and homology modelling of the main protease and non-structural protein 3 of SARS-CoV-2 reveal an aza-peptide and a lead inhibitor with possible antiviral properties

Viruses belonging to the family Coronaviridae consist of virulent pathogens that have a zoonotic property. Severe acute respiratory syndrome coronaviruses (SARS-CoVs) and Middle East respiratory syndrome coronaviruses (MERS-CoVs) of this family have emerged before and SARS-CoV-2 has emerged now globally. The characterization of spike glycoproteins, polyproteins and other viral proteins from viruses is important for antiviral drug development. Homology modelling of these proteins with known templates offers the opportunity to discover ligand-binding sites and explore the possible antiviral properties of these protein-ligand complexes. In this study, we performed a complete bioinformatic analysis, sequence alignment, comparison of multiple sequences and modelling of the SARS-CoV-2 whole-genome sequences, the spike protein and the polyproteins for homology with known proteins. We also analysed binding sites in these models for possible binding with ligands that exhibit antiviral properties. Our results indicated that the sequence of the polyprotein isolate SARS-CoV-2_HKU-SZ-001_2020 showed 98.94 percent identity to SARS-coronavirus NSP12 bound to NSP7 and NSP8 co-factors. The results also indicated that a part of the viral genome (residues 3268-3573 in Frame 2 with 306 amino acids) of the SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank Accession Number MN908947.3) when modelled with templateof the PDB database showed 96 percent identity to a 3C-like peptidase of SARS-CoVs, which has the ability to bind with an aza-peptide epoxide (APE) known for the irreversible inhibition of SARS-CoV main peptidase. A docking profile with 9 different conformations of the ligand with the protein model using Autodock Vina showed an affinity of −7.1 kcal mol−1. This region was conserved in 831 genomes of SARS-CoV-2. The part of the genome (residues 1568-1882 in Frame 2 with 315 amino acids) when modelled with template of the PDB database showed 82 percent identity to a papain-like protease/deubiquitinase, which when complexed with ligand GRL0617 acts as an inhibitor and can block SARS-CoV replication. A docking profile with 9 different conformations of the ligand with the protein model using Autodock Vina showed an affinity of −7.9 kcal mol−1. This region was conserved in 831 genomes of SARS-CoV-2. It is possible that these ligands can be used as antivirals of SARS-CoV-2.

Genetic analysis of SARS-CoV-2 isolates collected from Bangladesh: insights into the origin, mutation spectrum, and possible pathomechanism

As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of 14 isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and the Middle East, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant interaction was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.

Comparing the Binding Interactions in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV

[Image: see text] SARS-CoV-2, since emerging in Wuhan, China, has been a major concern because of its high infection rate and has left more than six million infected people around the world. Many studies endeavored to reveal the structure of the SARS-CoV-2 compared to the SARS-CoV, in order to find solutions to suppress this high infection rate. Some of these studies showed that the mutations in the SARS-CoV spike (S) protein might be responsible for its higher affinity to the ACE2 human cell receptor. In this work, we used molecular dynamics simulations and Monte Carlo sampling to compare the binding affinities of the S proteins of SARS-CoV and SARS-CoV-2 to the ACE2. Our results show that the protein surface of the ACE2 at the receptor binding domain (RBD) exhibits negative electrostatic potential, while a positive potential is observed for the S proteins of SARS-CoV/SARS-CoV-2. In addition, the binding energies at the interface are slightly higher for SARS-CoV-2 because of enhanced electrostatic interactions. The major contributions to the electrostatic binding energies result from the salt bridges forming between R426 and ACE-2-E329 in the case of SARS-CoV and K417 and ACE2-D30 in the SARS-CoV-2. In addition, our results indicate that the enhancement in the binding energy is not due to a single mutant but rather because of the sophisticated structural changes induced by all these mutations together. This finding suggests that it is implausible for the SARS-CoV-2 to be a lab-engineered virus.

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look.

Endoplasmic reticulum as a potential therapeutic target for covid-19 infection management?

In December 2019, many pneumonia cases with unidentified sources appeared in Wuhan, Hubei, China, with clinical symptoms like viral pneumonia. Deep sequencing analysis of samples from lower respiratory tract revealed a novel coronavirus, called 2019 novel coronavirus (2019-nCoV). Currently there is a rapid global spread. World Health Organization declare the disease a pandemic condition. The pathologic source of this disease was a new RNA virus from Coronaviridae family, which was named COVID-19. SARS-CoV-2 entry starts with the binding of the spike glycoprotein expressed on the viral envelope to ACE2 on the alveolar surface followed by clathrin-dependent endocytosis of the SARS-CoV-2 and ACE2 complex. SARS-CoV-2 enters the cells through endocytosis process, which is possibly facilitated, via a pH dependent endosomal cysteine protease cathepsins. Once inside the cells, SARS-CoV-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate and spread through the entire lung. Endosomal acidic pH for SARS-CoV-2 processing and internalization is critical. After entering the cells, it possibly activates or hijack many intracellular pathways in favor of its replication. In the current opinion article, we will explain the possible involvement of unfolded protein response as a cellular stress response to the SARS-CoV-2 infection.

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2

Abstract SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 52,020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

SARS-CoV-2 host diversity: An update of natural infections and experimental evidences

Abstract Coronavirus disease-19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is now a pandemic threat. This virus is supposed to be spread by human to human transmission. Cellular angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2 which is identical or similar in different species of animals such as pigs, ferrets, cats, orangutans, monkeys, and humans. Moreover, a recent study predicted that dog might be secondary host during the evolution of SARS-CoV-2 from bat to human. Therefore, there is a possibility of spreading SARS-CoV-2 through domestic pets. There are now many reports of SARS-CoV-2 positive cases in dogs, cats, tigers, lion, and minks. Experimental data showed ferrets and cats are highly susceptible to SARS-CoV-2 as infected by virus inoculation and can transmit the virus directly or indirectly by droplets or airborne route. Based on these natural infection reports and experimental data, whether the pets are responsible for SARS-CoV-2 spread to human; needs to be deeply investigated. Humans showing clinical symptoms of respiratory infections have been undergoing for COVID-19 diagnostic test but many infected people and few pets confirmed with SARS-CoV-2 remained asymptomatic. In this review, we summarize the natural cases of SARS-CoV-2 in animals with the latest researches conducted in this field. This review will be helpful to think insights of SARS-CoV-2 transmissions, spread, and demand for sero-prevalence studies especially in companion animals.

Comparative genomics suggests limited variability and similar evolutionary patterns between major clades of SARS-CoV-2

Phylogenomic analysis of SARS-CoV-2 as available from publicly available repositories suggests the presence of 3 prevalent groups of viral episomes (super-clades), which are mostly associated with outbreaks in distinct geographic locations (China, USA and Europe). While levels of genomic variability between SARS-CoV-2 isolates are limited, to our knowledge, it is not clear whether the observed patterns of variability in viral super-clades reflect ongoing adaptation of SARS-CoV-2, or merely genetic drift and founder effects. Here, we analyze more than 1100 complete, high quality SARS-CoV-2 genome sequences, and provide evidence for the absence of distinct evolutionary patterns/signatures in the genomes of the currently known major clades of SARS-CoV-2. Our analyses suggest that the presence of distinct viral episomes at different geographic locations are consistent with founder effects, coupled with the rapid spread of this novel virus. We observe that while cross species adaptation of the virus is associated with hypervariability of specific protein coding regions (including the RDB domain of the spike protein), the more variable genomic regions between extant SARS-CoV-2 episomes correspond with the 3’ and 5’ UTRs, suggesting that at present viral protein coding genes should not be subjected to different adaptive evolutionary pressures in different viral strains. Although this study can not be conclusive, we believe that the evidence presented here is strongly consistent with the notion that the biased geographic distribution of SARS-CoV-2 isolates should not be associated with adaptive evolution of this novel pathogen.

Phylogenetic Analysis of SARS-CoV-2 Genomes in Turkey

COVID-19 has effectively spread worldwide. As of May 2020, Turkey is among the top ten countries with the most cases. A comprehensive genomic characterization of the virus isolates in Turkey is yet to be carried out. Here, we built a phylogenetic tree with globally obtained 15,277 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. We identified the subtypes based on the phylogenetic clustering in comparison with the previously annotated classifications. We performed a phylogenetic analysis of the first thirty SARS-CoV-2 genomes isolated and sequenced in Turkey. We suggest that the first introduction of the virus to the country is earlier than the first reported case of infection. Virus genomes isolated from Turkey are dispersed among most types in the phylogenetic tree. We find two of the seventeen sub-clusters enriched with the isolates of Turkey, which likely have spread expansively in the country. Finally, we traced virus genomes based on their phylogenetic placements. This analysis suggested multiple independent international introductions of the virus and revealed a hub for the inland transmission. We released a web application to track the global and interprovincial virus spread of the isolates from Turkey in comparison to thousands of genomes worldwide.

Evidence of the Recombinant Origin and Ongoing Mutations in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

The recent global outbreak of viral pneumonia designated as Coronavirus Disease 2019 (COVID-19) by coronavirus (SARS-CoV-2) has threatened global public health and urged to investigate its source. Whole genome analysis of SARS-CoV-2 revealed ~96% genomic similarity with bat CoV (RaTG13) and clustered together in phylogenetic tree. Furthermore, RaTGl3 also showed 97.43% spike protein similarity with SARS-CoV-2 suggesting that RaTGl3 is the closest strain. However, RBD and key amino acid residues supposed to be crucial for human-to-human and cross-species transmission are homologues between SARS-CoV-2 and pangolin CoVs. These results from our analysis suggest that SARS-CoV-2 is a recombinant virus of bat and pangolin CoVs. Moreover, this study also reports mutations in coding regions of 125 SARS-CoV-2 genomes signifying its aptitude for evolution. In short, our findings propose that homologous recombination has been occurred between bat and pangolin CoVs that triggered cross-species transmission and emergence of SARS-CoV-2, and, during the ongoing outbreak, SARS-CoV-2 is still evolving for its adaptability.

The establishment of reference sequence for SARS-CoV-2 and variation analysis

Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

Abstract SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.

A comprehensive analysis of genome composition and codon usage patterns of emerging coronaviruses

An outbreak of atypical pneumonia caused by a novel Betacoronavirus (ßCoV), named SARS-CoV-2 has been declared a public health emergency of international concern by the World Health Organization. In order to gain insight into the emergence, evolution and adaptation of SARS-CoV-2 viruses, a comprehensive analysis of genome composition and codon usage of ßCoV circulating in China was performed. A biased nucleotide composition was found for SARS-CoV-2 genome. This bias in genomic composition is reflected in its codon and amino acid usage patterns. The overall codon usage in SARS-CoV-2 is similar among themselves and slightly biased. Most of the highly frequent codons are A- and U-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. Significant differences in relative synonymous codon usage frequencies among SARS-CoV-2 and human cells were found. These differences are due to codon usage preferences.

A doubt of multiple introduction of SARS-CoV-2 in Italy: A preliminary overview

The emergence of the novel betacoronavirus, recently renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised serious concerns due to the virus's rapid dissemination worldwide. Nevertheless, there is limited information about the genomic epidemiology of SARS-CoV-2 circulating in Italy from surveillance studies. The shortage of complete genomic sequences available impairs our understanding of the SARS-CoV-2 introduction and establishment in the country. To better understand its dynamics in Italy, we analyzed complete genomes of SARS-CoV-2 isolates, obtained directly from clinical samples. Our phylogenetic reconstructions suggest possible multiple introduction of SARS-CoV-2. Continued genomic surveillance strategies are needed to improve monitoring and understanding of the current SARS-CoV-2 epidemics, which might help to attenuate public health impact of infectious diseases.

Analysis of the Hosts and Transmission Paths of SARS-CoV-2 in the COVID-19 Outbreak

The severe respiratory disease COVID-19 was initially reported in Wuhan, China, in December 2019, and spread into many provinces from Wuhan. The corresponding pathogen was soon identified as a novel coronavirus named SARS-CoV-2 (formerly, 2019-nCoV). As of 2 May, 2020, over 3 million COVID-19 cases had been confirmed, and 235,290 deaths had been reported globally, and the numbers are still increasing. It is important to understand the phylogenetic relationship between SARS-CoV-2 and known coronaviruses, and to identify its hosts for preventing the next round of emergency outbreak. In this study, we employ an effective alignment-free approach, the Natural Vector method, to analyze the phylogeny and classify the coronaviruses based on genomic and protein data. Our results show that SARS-CoV-2 is closely related to, but distinct from the SARS-CoV branch. By analyzing the genetic distances from the SARS-CoV-2 strain to the coronaviruses residing in animal hosts, we establish that the most possible transmission path originates from bats to pangolins to humans.

Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 site

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct 4 amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability and transmission.

Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective

SARS-CoV-2, a new coronavirus strain responsible for COVID-19 has emerged in Wuhan City, China and still continuing its worldwide pandemic nature. Considering the severity of the disease, a number of studies are underway, and full genomic sequences have already been released in the last few weeks to enable the understanding of the evolutionary origin and molecular characteristics of this virus. Bioinformatics analysis, satellite derived imaging data and epidemiological attributes were employed to investigate origin, immunogenic resemblance and global threat of newly pandemic SARS-CoV-2 including Bangladesh perspective. Based on currently available genomic information, a phylogeny study was employed focusing four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings clearly demonstrated that SARS-CoV-2 exhibited evolutionary convergent relation with previously reported SARS-CoV. It was also found that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed lower level of similarity and identical patterns. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. The study also prioritized the temperature comparison through satellite imaging alongside compiling and analyzing the epidemiological outbreak information on the 2019 novel coronavirus based on several open datasets on COVID-19 (SARS-CoV-2) and discussed possible threats to Bangladesh.

Genome-Wide Identification and Characterization of Point Mutations in the SARS-CoV-2 Genome

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and has been rapidly spreading worldwide. Although the causal relationship among mutations and the features of SARS-CoV-2 such as rapid transmission, pathogenicity, and tropism, remains unclear, our results of genomic mutations in SARS-CoV-2 may help to interpret the interaction between genomic characterization in SARS-CoV-2 and infectivity with the host. METHODS: A total of 4,254 genomic sequences of SARS-CoV-2 were collected from the Global Initiative on Sharing all Influenza Data (GISAID). Multiple sequence alignment for phylogenetic analysis and comparative genomic approach for mutation analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA), and an in-house program based on Perl language, respectively. RESULTS: Phylogenetic analysis of SARS-CoV-2 strains indicated that there were 3 major clades including S, V, and G, and 2 subclades (G.1 and G.2). There were 767 types of synonymous and 1,352 types of non-synonymous mutation. ORF1a, ORF1b, S, and N genes were detected at high frequency, whereas ORF7b and E genes exhibited low frequency. In the receptor-binding domain (RBD) of the S gene, 11 non-synonymous mutations were observed in the region adjacent to the angiotensin converting enzyme 2 (ACE2) binding site. CONCLUSION: It has been reported that the rapid infectivity and transmission of SARS-CoV-2 associated with host receptor affinity are derived from several mutations in its genes. Without these genetic mutations to enhance evolutionary adaptation, species recognition, host receptor affinity, and pathogenicity, it would not survive. It is expected that our results could provide an important clue in understanding the genomic characteristics of SARS-CoV-2.

The Architecture of SARS-CoV-2 Transcriptome

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

The proximal origin of SARS-CoV-2

Full-genome sequences of the first two SARS-CoV-2 viruses from India

BACKGROUND & OBJECTIVES: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. METHODS: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. RESULTS: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. INTERPRETATION & CONCLUSIONS: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

Phylogenetic analysis of SARS-CoV-2 genomes in Turkey

COVID-19 has effectively spread worldwide. As of May 2020, Turkey is among the top ten countries with the most cases. A comprehensive genomic characterization of the virus isolates in Turkey is yet to be carried out. Here, we built a phylogenetic tree with globally obtained 15,277 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. We identified the subtypes based on the phylogenetic clustering in comparison with the previously annotated classifications. We performed a phylogenetic analysis of the first 30 SARS-CoV-2 genomes isolated and sequenced in Turkey. We suggest that the first introduction of the virus to the country is earlier than the first reported case of infection. Virus genomes isolated from Turkey are dispersed among most types in the phylogenetic tree. We find 2 of the seventeen subclusters enriched with the isolates of Turkey, which likely have spread expansively in the country. Finally, we traced virus genomes based on their phylogenetic placements. This analysis suggested multiple independent international introductions of the virus and revealed a hub for the inland transmission. We released a web application to track the global and interprovincial virus spread of the isolates from Turkey in comparison to thousands of genomes worldwide.

[Variability analysis of S2 gene of SARS-CoV].

OBJECTIVE To determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene. METHOD S2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database. RESULT With the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope. CONCLUSION S2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.

Critical Differences between the Binding Features of the Spike Proteins of SARS-CoV-2 and SARS-CoV

The COVID-19 caused by SARS-CoV-2 has spread globally and caused tremendous loss of lives and properties, and it is of utmost urgency to understand its propagation process and to find ways to slow down the epidemic. In this work, we used a coarse-grained model to calculate the binding free energy of SARS-CoV-2 or SARS-CoV to their human receptor ACE2. The investigation of the free energy contribution of the interacting residues indicates that the residues located outside the receptor binding domain are the source of the stronger binding of the novel virus. Thus, the current results suggest that the essential evolution of SARS-CoV-2 happens remotely from the binding domain at the spike protein trimeric body. Such evolution may facilitate the conformational change and the infection process that occurs after the virus is bound to ACE2. By studying the binding pattern between SARS-CoV antibody m396 and SARS-CoV-2, it is found that the remote energetic contribution is missing, which might explain the absence of cross-reactivity of such antibodies.

A Novel Synonymous Mutation of SARS-CoV-2: Is This Possible to Affect Their Antigenicity and Immunogenicity?

The S glycoprotein of coronaviruses is important for viral entry and pathogenesis with most variable sequences. Therefore, we analyzed the S gene sequences of SARS-CoV-2 to better understand the antigenicity and immunogenicity of this virus in this study. In phylogenetic analysis, two subtypes (SARS-CoV-2a and -b) were confirmed within SARS-CoV-2 strains. These two subtypes were divided by a novel synonymous mutation of D614G. This may play a crucial role in the evolution of SARS-CoV-2 to evade the host immune system. The region containing this mutation point was confirmed as a B-cell epitope located in the S1 domain, and SARS-CoV-2b strains exhibited severe reduced antigenic indexes compared to SARS-CoV-2a in this area. This may allow these two subtypes to have different antigenicity. If the two subtypes have different serological characteristics, a vaccine for both subtypes will be more effective to prevent COVID-19. Thus, further study is urgently required to confirm the antigenicity of these two subtypes.

Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CL(pro) targeting repurposed drug candidates

BACKGROUND: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. METHODS: For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CL(pro)) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. RESULTS: Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CL(pro) enzyme, which regulates viral replication machinery. CONCLUSIONS: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

Isolation, sequence, infectivity and replication kinetics of SARS-CoV-2

SARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two cases of COVID-19 in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype using in vitro and in vivo infection models.

SARS-CoV-2 is well adapted for humans. What does this mean for re-emergence?

In a side-by-side comparison of evolutionary dynamics between the 2019/2020 SARS-CoV-2 and the 2003 SARS-CoV, we were surprised to find that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had developed several advantageous adaptations for human transmission. Our observations suggest that by the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV. However, no precursors or branches of evolution stemming from a less human-adapted SARS-CoV-2-like virus have been detected. The sudden appearance of a highly infectious SARS-CoV-2 presents a major cause for concern that should motivate stronger international efforts to identify the source and prevent near future re-emergence. Any existing pools of SARS-CoV-2 progenitors would be particularly dangerous if similarly well adapted for human transmission. To look for clues regarding intermediate hosts, we analyze recent key findings relating to how SARS-CoV-2 could have evolved and adapted for human transmission, and examine the environmental samples from the Wuhan Huanan seafood market. Importantly, the market samples are genetically identical to human SARS-CoV-2 isolates and were therefore most likely from human sources. We conclude by describing and advocating for measured and effective approaches implemented in the 2002-2004 SARS outbreaks to identify lingering population(s) of progenitor virus.