Exploration of hosts and transmission traits for SARS-CoV-2 based on the k-mer natural vector.

A severe respiratory pneumonia COVID-19 has raged all over the world, and a coronavirus named SARS-CoV-2 is blamed for this global pandemic. Despite intensive research into the origins of the COVID-19 pandemic, the evolutionary history of its agent SARS-CoV-2 remains unclear, which is vital to control the pandemic and prevent another round of outbreak. Coronaviruses are highly recombinogenic, which are not well handled with alignment-based method. In addition, deletions have been found in the genomes of several SARS-CoV-2, which cannot be resolved with current phylogenetic methods. Therefore, the k-mer natural vector is proposed to explore hosts and transmission traits for SARS-CoV-2 using strict phylogenetic reconstruction. SARS-CoV-2 clustering with bat-origin coronaviruses strongly suggests bats to be the natural reservoir of SARS-CoV-2. By building bat-to-human transmission route, pangolin is identified as an intermediate host, and civet is predicted as a possible candidate. We speculate that SARS-CoV-2 undergoes cross-species recombination between bat and pangolin coronaviruses. This study also demonstrates transmission mode and features of SARS-CoV-2 in the COVID-19 pandemic when it broke out early around the world.

The 442th amino acid residue of the spike protein is critical for the adaptation to bat hosts for SARS-related coronaviruses.

Bats carry diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). The suspected interspecies transmission of SARSr-CoVs from bats to humans has caused two severe CoV pandemics, the SARS pandemic in 2003 and the recent COVID-19 pandemic. The receptor utilization of SARSr-CoV plays the key role in determining the host range and the interspecies transmission ability of the virus. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as their receptor. Previous studies showed that WIV1 strain, the first living coronavirus isolated from bat using ACE2 as its receptor, is the prototype of SARS-CoV. The receptor-binding domain (RBD) in the spike protein (S) of SARS-CoV and WIV1 is responsible for ACE2 binding and medicates the viral entry. Comparing to SARS-CoV, WIV1 has three distinct amino acid residues (442, 472, and 487) in its RBD. This study aimed at exploring whether these three residues could alter the receptor utilization of SARSr-CoVs. We replaced the three residues in SARS-CoV (BJ01 strain) S with their counterparts in WIV1 S, and then evaluated the change of their utilization of bat, civet, and human ACE2s using a lentivirus-based pseudovirus infection system. To further validate the S-ACE2 interactions, the binding affinity between the RBDs of these S proteins and the three ACE2s were verified by flow cytometry. The results showed that the single amino acid substitution Y442S in the RBD of BJ01 S enhanced its utilization of bat ACE2 and its binding affinity to bat ACE2. On the contrary, the reverse substitution in WIV1 S (S442Y) significantly attenuated the pseudovirus utilization of bat, civet and human ACE2s for cell entry, and reduced its binding affinity with the three ACE2s. These results suggest that the S442 is critical for WIV1 adapting to bats as its natural hosts. These findings will enhance our understanding of host adaptations and cross-species infections of coronaviruses, contributing to the prediction and prevention of coronavirus epidemics.

COVID-19, Your Pet and Other Animals: Are You at Risk?

Despite fast-tracked research, the precise origin, transmission and evolution of COVID-19 are still unknown. While the bat genus Rhinolophus is likely the primary source of the zoonotic-origin pathogen SARS-CoV-2 that causes COVID-19, its transmission route into the human population is still being studied.[1,2] Coronaviruses (CoV) affect humans and various animal species. Bats were the original hosts of the CoV that causes Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), for example, with masked palm civet cats and dromedaries, respectively, the intermediate hosts of those two viruses. Research is ongoing regarding intermediate species for SARS-CoV-2, but one possibility is the large stray cat and dog population around the live animal market in Wuhan, China, where the pandemic is thought to have started.

COVID19: an announced pandemic.

A severe upper respiratory tract syndrome caused by the new coronavirus has now spread to the entire world as a highly contagious pandemic. The large scale explosion of the disease is conventionally traced back to January of this year in the Chinese province of Hubei, the wet markets of the principal city of Wuhan being assumed to have been the specific causative locus of the sudden explosion of the infection. A number of findings that are now coming to light show that this interpretation of the origin and history of the pandemic is overly simplified. A number of variants of the coronavirus would in principle have had the ability to initiate the pandemic well before January of this year. However, even if the COVID-19 had become, so to say, ready, conditions in the local environment would have had to prevail to induce the loss of the biodiversity's "dilution effect" that kept the virus under control, favoring its spillover from its bat reservoir to the human target. In the absence of these appropriate conditions only abortive attempts to initiate the pandemic could possibly occur: a number of them did indeed occur in China, and probably elsewhere as well. These conditions were unfortunately present at the wet marked in Wuhan at the end of last year.

Carnivore Protoparvovirus-1 Associated With an Outbreak of Hemorrhagic Gastroenteritis in Small Indian Civets.

Carnivore protoparvovirus-1 (CPPV-1) infection has been reported frequently in both domestic and wildlife species including wild carnivores. Fifty-five captive small Indian civets (Viverricula indica), farmed for perfume production in Eastern Thailand, showed clinical signs of acute bloody diarrhea, anorexia, vomiting, circling, and seizures. The disease spread within the farm and resulted in the death of 38 of the 55 civets (69% mortality) within a month. Fecal swabs were collected from the 17 surviving civets, and necropsy was performed on 7 of the dead civets. Pathologic findings were severe hemorrhagic gastroenteritis with generalized lymphadenopathy. CPPV-1 was identified in both fecal swabs and postmortem samples by species-specific polymerase chain reaction. Further whole-gene sequencing and restriction fragment length polymorphism analysis suggested feline panleukopenia virus (FPV) as the causative agent. The viral tropism and tissue distribution were confirmed by immunohistochemistry, with immunolabeling in the cytoplasm and nucleus of small intestinal crypt epithelial cells, villous enterocytes, histiocytes in lymphoid tissues, myenteric nerve plexuses, and cerebral and cerebellar neurons. Phylogenetic analysis of civet-derived CPPV-1 indicated a genetic similarity close to the FPV HH-1/86 strain detected in a jaguar (Panthera onca) in China. To our knowledge, this mass die-off of civets is the first evidence of disease associated with CPPV-1 infection in the subfamily Viverrinae. These findings support the multi-host range of parvovirus infection and raises awareness for CPPV-1 disease outbreaks in wildlife species.

Susceptibility to SARS, MERS, and COVID-19 from animal health perspective.

Viruses are having great time as they seem to have bogged humans down. Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and novel coronavirus (COVID-19) are the three major coronaviruses of present-day global human and animal health concern. COVID-19 caused by SARS-CoV-2 is identified as the newest disease, presumably of bat origin. Different theories on the evolution of viruses are in circulation, yet there is no denying the fact that the animal source is the skeleton. The whole world is witnessing the terror of the COVID-19 pandemic that is following the same path of SARS and MERS, and seems to be more severe. In addition to humans, several species of animals are reported to have been infected with these life-threatening viruses. The possible routes of transmission and their zoonotic potentialities are the subjects of intense research. This review article aims to overview the link of all these three deadly coronaviruses among animals along with their phylogenic evolution and cross-species transmission. This is essential since animals as pets or food are said to pose some risk, and their better understanding is a must in order to prepare a possible plan for future havoc in both human and animal health. Although COVID-19 is causing a human health hazard globally, its reporting in animals are limited compared to SARS and MERS. Non-human primates and carnivores are most susceptible to SARS-coronavirus and SARS-CoV-2, respectively, whereas the dromedary camel is susceptible to MERS-coronavirus. Phylogenetically, the trio viruses are reported to have originated from bats and have special capacity to undergo mutation and genomic recombination in order to infect humans through its reservoir or replication host. However, it is difficult to analyze how the genomic pattern of coronaviruses occurs. Thus, increased possibility of new virus-variants infecting humans and animals in the upcoming days seems to be the biggest challenge for the future of the world. One health approach is portrayed as our best way ahead, and understanding the animal dimension will go a long way in formulating such preparedness plans.

[Etiology of epidemic outbreaks COVID-19 on Wuhan, Hubei province, Chinese People Republic associated with 2019-nCoV (Nidovirales, Coronaviridae, Coronavirinae, Betacoronavirus, Subgenus Sarbecovirus): lessons of SARS-CoV outbreak.]

Results of analysis of phylogenetic, virological, epidemiological, ecological, clinical data of COVID-19 outbreaks in Wuhan, China (PRC) in comparison with SARS-2002 and MERS-2012 outbreaks allow to conclude: - the etiological agent of COVID-19 is coronavirus (2019-CoV), phylogenetically close to the SARS-CoV, isolated from human, and SARS-related viruses isolated from bats (SARS-related bat CoV viruses). These viruses belong to the Sarbecovirus subgenus, Betacoronavirus genus, Orthocoronavirinae subfamily, Coronaviridae family (Cornidovirinea: Nidovirales). COVID-19 is a variant of SARS-2002 and is different from MERS-2012 outbreak, which were caused by coronavirus belonged to the subgenus Merbecovirus of the same genus; - according to the results of phylogenetic analysis of 35 different betacoronaviruses, isolated from human and from wild animals in 2002-2019, the natural source of COVID-19 and SARS-CoV (2002) is bats of Rhinolophus genus (Rhinolophidae) and, probably, some species of other genera. An additional reservoir of the virus could be an intermediate animal species (snakes, civet, hedgehogs, badgers, etc.) that are infected by eating of infected bats. SARS-like coronaviruses circulated in bats in the interepidemic period (2003-2019); - seasonal coronaviruses (subgenus Duvinacovirus, Alphacoronavirus) are currently circulating (November 2019 - January 2020) in the European part of Russia, Urals, Siberia and the Far East of Russia, along with the influenza viruses A(H1N1)pdm09, A(H3N2), and В, as well as six other respiratory viruses (HPIV, HAdV, HRSV, HRV, HBoV, and HMPV).

In silico studies on the comparative characterization of the interactions of SARS-CoV-2 spike glycoprotein with ACE-2 receptor homologs and human TLRs.

Coronavirus disease-2019 (COVID-19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has come out as a major threat for mankind in recent times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS-CoV-2), its mode of interaction with the angiotensin-converting enzyme-2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll-like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS-CoV-2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS-CoV-2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS-CoV-2 from a bat and also revealed that TLR4 may have a crucial role in the virus-induced inflammatory consequences associated with COVID-19. Therefore, selective targeting of TLR4-spike protein interaction by designing competitive TLR4-antagonists could pave a new way to treat COVID-19. Finally, this study is expected to improve our understanding on the immunobiology of SARS-CoV-2 and could be useful in adopting spike protein, ACE2, or TLR-guided intervention strategy against COVID-19 shortly.

Extended ORF8 Gene Region Is Valuable in the Epidemiological Investigation of Severe Acute Respiratory Syndrome-Similar Coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) was discovered as a novel pathogen in the 2002-2003 SARS epidemic. The emergence and disappearance of this pathogen have brought questions regarding its source and evolution. Within the genome sequences of 281 SARS-CoVs, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and SARS-related CoVs (SARSr-CoVs), a ~430 bp genomic region (from 27 701 bp to 28 131 bp in AY390556.1) with regular variations was investigated. This ~430 bp region overlaps with the ORF8 gene and is prone to deletions and nucleotide substitutions. Its complexity suggested the need for a new genotyping method for coronaviruses related to SARS-similar coronaviruses (SARS-CoV, SARSr-CoV, and SARS-CoV-2). Bat SARSr-CoV presented 3 genotypes, of which type 0 is only seen in bat SARSr-CoV, type I is present in SARS in the early phase, and type II is found in all SARS-CoV-2. This genotyping also shows potential usage in distinguishing the SARS-similar coronaviruses from different hosts and geographic areas. This genomic region has important implications for predicting the epidemic trend and studying the evolution of coronavirus.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Binding to ACE2 Receptors from Human, Pets, Farm Animals, and Putative Intermediate Hosts.

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a pandemic. Here, we used X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptors allows the definition of residues important for binding. From the 20 amino acids in ACE2 that contact S, up to 7 can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or acquisition of N-glycosylation sites located near the S interface. Of note, pigs and dogs, which are not infected or are not effectively infected and have only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with that from bat coronavirus strain RaTG13 (Bat-CoV-RaTG13) and pangolin coronavirus (Pangolin-CoV) strain hCoV-19/pangolin/Guangdong/1/2019 revealed that the latter contains only one substitution, whereas Bat-CoV-RaTG13 exhibits five. However, ACE2 of pangolin exhibits seven changes relative to human ACE2, and a similar number of substitutions is present in ACE2 of bats, raccoon dogs, and civets, suggesting that SARS-CoV-2 may not be especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-CoV-2.IMPORTANCE SARS-CoV-2 is threatening people worldwide, and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear, and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes, indicating that the species barrier might be low. Exceptions are dogs and especially pigs, which revealed relatively low ACE2 expression levels in the respiratory tract. Monitoring of animals is necessary to prevent the generation of a new coronavirus reservoir. Finally, our analysis also showed that SARS-CoV-2 may not be specifically adapted to any of its putative intermediate hosts.

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.

Genetic Adaptations, Biases, and Evolutionary Analysis of Canine Distemper Virus Asia-4 Lineage in a Fatal Outbreak of Wild-Caught Civets in Thailand.

Canine morbillivirus (CDV) is a serious pathogen that can cause fatal systemic disease in a wide range of domestic and wildlife carnivores. Outbreaks of CDV in wildlife species lead to questions regarding the dispersal of the CDV origin. In the present study, we identified a fatal CDV outbreak in caged wild-caught civets in Thailand. Full-length genetic analysis revealed that CDV from the Asia-4 lineage served as the likely causative agent, which was supported by the viral localization in tissues. Evolutionary analysis based on the CDV hemagglutinin (H) gene revealed that the present civet CDV has co-evolved with CDV strains in dogs in Thailand since about 2014. The codon usage pattern of the CDV H gene revealed that the CDV genome has a selective bias of an A/U-ended codon preference. Furthermore, the codon usage pattern of the CDV Asia-4 strain from potential hosts revealed that the usage pattern was related more to the codon usage of civets than of dogs. This finding may indicate the possibility that the discovered CDV had initially adapted its virulence to infect civets. Therefore, the CDV Asia-4 strain might pose a potential risk to civets. Further epidemiological, evolutionary, and codon usage pattern analyses of other CDV-susceptible hosts are required.

Rabies in the African Civet: An Incidental Host for Lyssaviruses?

In South Africa, canid rabies virus (RABV) infection is maintained in domestic and wildlife species. The identification of rabies in African civets raised the question of whether this wildlife carnivore is a potential reservoir host of RABVs of direct and ancestral dog origin (dog-maintained and dog-derived origins) with an independent cycle of transmission. Genetic analyses of African civet nucleoprotein sequences for 23 African civet RABVs and historically published sequences demonstrated that RABVs from African civets have two origins related to dog and mongoose rabies enzootics. The data support observations of the interaction of civets with domestic dogs and wildlife mongooses, mostly in Northern South Africa and North-East Zimbabwe. Within each host species clade, African civet RABVs group exclusively together, implying intra-species virus transfer occurs readily. The canid RABV clade appears to support virus transfer more readily between hosts than mongoose RABVs. Furthermore, these data probably indicate short transmission chains with conspecifics that may be related to transient rabies maintenance in African civets. Hence, it is important to continue monitoring the emergence of lyssaviruses in this host. Observations from this study are supported by ongoing and independent similar cases, in which bat-eared foxes and black-backed jackal species maintain independent rabies cycles of what were once dog-maintained RABVs.

Origins of SARS-CoV-1 and SARS-CoV-2 are often poorly explored in leading publications.

In the rush to understand the coronaviruses that threaten human health, authors of many prominent papers have not performed phylogenetic analyses to the standard of the field today. Errors include faulty placement of the root of the phylogeny, outdated methods of reconstruction, poor taxon sampling, inappropriate emphasis on selected functional elements, and inadequate consideration of ambiguity. As a result, certain conclusions regarding the origin of human infections are not supported soundly or are wrong.

Molecular epidemiology, evolution and phylogeny of SARS coronavirus.

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.

Canine Parvovirus-2c (CPV-2c) Infection in Wild Asian Palm Civets (Paradoxurus hermaphroditus) in Singapore.

We report pathogenic feline parvovirus and canine parvovirus-2c infection in wild Asian palm civets (Paradoxurus hermaphroditus), as demonstrated by histopathology and immunohistochemistry findings of parvoviral enteropathy. We performed molecular characterization and phylogeny studies to obtain an improved understanding of disease transmission dynamics between domestic and wild carnivores.

Identification and full-genome characterization of novel circoviruses in masked palm civets (Paguma larvata).

The family Circoviridae comprises a large group of small, circular, single-stranded DNA viruses and is classified into two genera: Circovirus and Cyclovirus. They have marked genetic diversity and a broad host range. In this study, three novel circovirus genomes were identified from wild-caught masked palm civets (Paguma larvata) in Japan and classified as a new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses. Of note, the presence of two predicted introns at the 5'-terminus of the rep gene was suggested in the Paguma larvata circovirus genomes.

Identification and whole genome characterization of novel anelloviruses in masked palm civets (Paguma larvata): Segregation into four distinct clades.

The members of the family Anelloviridae are small and single-stranded DNA viruses with marked diversity in sequence and length, which ubiquitously infect many vertebrates, including mammals, birds and reptiles. The anelloviruses isolated from mammals are currently classified into 11 assigned and four proposed genera; some anelloviruses remain unassigned. The present study was conducted to identify anelloviruses in wild-caught masked palm civets (Paguma larvata) in Japan using a rolling-circle amplification method. Thirteen novel anellovirus strains were identified from 8 of 10 masked palm civets and their entire genomic sequences (2039-2535 nucleotides) were determined; they were classifiable into four distinct clades. Comparative analyses of all reported anelloviruses for which the entire or near-entire genomic sequences have been determined, including the 13 strains obtained in the present study, revealed that anelloviruses can provisionally be classified into 20 clades, which may correspond to 20 genera (including 11 assigned and four proposed genera) by a >70% amino acid sequence difference in open reading frame 1 (ORF1). This study suggested that novel anelloviruses of marked diversity are circulating in animals worldwide, and that the rolling-circle amplification method would be useful for identifying novel anelloviruses and other viruses with a circular DNA genome.