scholarly journals Subtyping Cryptosporidium xiaoi, a Common Pathogen in Sheep and Goats

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 800
Author(s):  
Yingying Fan ◽  
Xitong Huang ◽  
Sheng Guo ◽  
Fang Yang ◽  
Xin Yang ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Furin Cleavage ◽  
Sheep And Goats ◽  
Gp60 Gene ◽  
Furin Cleavage Site ◽  
Glycosylation Sites ◽  
Sequence Patterns ◽  
Established Species ◽  
Sequence Types ◽  
Subtyping Tool

Cryptosporidiosis is a significant cause of diarrhea in sheep and goats. Among the over 40 established species of Cryptosporidium, Cryptosporidium xiaoi is one of the dominant species infecting ovine and caprine animals. The lack of subtyping tools makes it impossible to examine the transmission of this pathogen. In the present study, we identified and characterized the 60-kDa glycoprotein (gp60) gene by sequencing the genome of C. xiaoi. The GP60 protein of C. xiaoi had a signal peptide, a furin cleavage site of RSRR, a glycosylphosphatidylinositol anchor, and over 100 O-glycosylation sites. Based on the gp60 sequence, a subtyping tool was developed and used in characterizing C. xiaoi in 355 positive samples from sheep and goats in China. A high sequence heterogeneity was observed in the gp60 gene, with 94 sequence types in 12 subtype families, namely XXIIIa to XXIIIl. Co-infections with multiple subtypes were common in these animals, suggesting that genetic recombination might be responsible for the high diversity within C. xiaoi. This was supported by the mosaic sequence patterns among the subtype families. In addition, a potential host adaptation was identified within this species, reflected by the exclusive occurrence of XXIIIa, XXIIIc, XXIIIg, and XXIIIj in goats. This subtyping tool should be useful in studies of the genetic diversity and transmission dynamics of C. xiaoi.

scholarly journals Development and Application of a gp60-Based Subtyping Tool for Cryptosporidium bovis

2021 ◽  
Vol 9 (10) ◽  
pp. 2067
Author(s):  
Weijian Wang ◽  
Muchun Wan ◽  
Fang Yang ◽  
Na Li ◽  
Lihua Xiao ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Genetic Recombination ◽  
Transmission Characteristics ◽  
Typical Structure ◽  
Sequence Patterns ◽  
Geographic Differences ◽  
Sequence Types ◽  
Sequence Characteristics ◽  
Multiple Episodes ◽  
Subtyping Tool

Cryptosporidium bovis is a common enteric pathogen in bovine animals. The research on transmission characteristics of the pathogen is hampered by the lack of subtyping tools. In this study, we retrieve the nucleotide sequence of the 60 kDa glycoprotein (GP60) from the whole genome sequences of C. bovis we obtained previously and analyze its sequence characteristics. Despite a typical structure of the GP60 protein, the GP60 of C. bovis had only 19.3–45.3% sequence identity to those of other Cryptosporidium species. On the basis of the gene sequence, a subtype typing tool was developed for C. bovis and used in the analysis of 486 C. bovis samples from dairy cattle, yaks, beef cattle, and water buffalos from China. Sixty-eight sequence types were identified from 260 subtyped samples, forming six subtype families, namely XXVIa to XXVIf. The mosaic sequence patterns among subtype families and the 121 potential recombination events identified among the sequences both suggest the occurrence of genetic recombination at the locus. No obvious host adaptation and geographic differences in the distribution of subtype families were observed. Most farms with more extensive sampling had more than one subtype family, and the dominant subtype families on a farm appeared to differ between pre- and post-weaned calves, indicating the likely occurrence of multiple episodes of C. bovis infections. There was an association between XXVId infection and occurrence of moderate diarrhea in dairy cattle. The subtyping tool developed and the data generated in the study might improve our knowledge of the genetic diversity and transmission of C. bovis.

scholarly journals Subtyping Cryptosporidium ryanae: A Common Pathogen in Bovine Animals

2020 ◽  
Vol 8 (8) ◽  
pp. 1107 ◽  
Author(s):  
Xin Yang ◽  
Ni Huang ◽  
Wen Jiang ◽  
Xinrui Wang ◽  
Na Li ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Sequence Data ◽  
Southern China ◽  
Eastern China ◽  
Whole Genome Sequence ◽  
Furin Cleavage ◽  
Genetic Characteristics ◽  
Water Buffaloes ◽  
Furin Cleavage Site ◽  
Subtyping Tool

Cryptosporidium ryanae is one of the most common species for cryptosporidiosis in cattle. However, little is known of the genetic characteristics of C. ryanae due to the lack of subtyping tools. In the present study, the 60-kDa glycoprotein (gp60) gene of C. ryanae was identified in whole genome sequence data and analyzed for sequence characteristics using bioinformatics tools. The protein it encodes had some of the typical characteristics of GP60 proteins, with a signal peptide, a furin cleavage site, and a glycosylphosphatidylinositol anchor at the C terminus of the protein, and numerous O-glycosylation sites. The gene sequence was used in the development of a subtyping tool, which was used in characterizing C. ryanae from 110 specimens from dairy cattle, 2 from beef cattle, 6 from yaks, and 4 from water buffaloes in China. Altogether, 17 subtypes from 8 subtype families were recognized, namely XXIa to XXIh. Possible host adaption was identified within this species, reflected by the unique occurrence of XXIa, XXIc, and XXIh in dairy cattle, yaks, and water buffaloes, respectively. Some geographical differences were detected in the distribution of subtype families in dairy cattle; specimens from southern China showed higher genetic diversity than from northern China, and the XXIa subtype family was only seen in dairy cattle in southern and eastern China. The gp60-based subtyping tool should be useful in molecular epidemiological studies of the transmission of C. ryanae.

scholarly journals SARS-CoV-2 and the Secret of the Furin Site

2021 ◽  
Author(s):  
Antonio R. Romeu ◽  
Enric Ollé
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Cleavage Site ◽  
Genetic Recombination ◽  
Evolutionary Model ◽  
Viral Proteins ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Bat Coronavirus ◽  
High Infectivity

The SARS-CoV-2 high infectivity is due to the functional polybasic furin cleavage site in the S protein. How it was acquired is unknown. There are two challenges to face: (i) an evolutionary model, to fit the origin of the coronavirus; and (ii) a molecular mechanism for the site acquisition. Here we show genomic fingerprints which are specific of Pangolin-CoVs, Bat-SARS-like (CoVZC45, CoVZXC21), bat RatG13 and human SARS-CoV-2 coronaviruses. This, along with phylogenetic analysis, we found that these species have the same evolutionary origin in the bat, including a genetic recombination of S gene between Pangolin-CoV (2017) and RatG13 ancestors. However, this does not explain why SARS-CoV-2 is the only of them with the furin site, which consists in four amino acid (PRRA) motif. The Arginine doublet is encoded by CGGCGG codons. Surprisingly, none of the Arginine doublet of other furin site of viral proteins from several type of viruses, are encoded by the CGGCGG codons. This makes it difficult to consider a virus recombination as mechanism for the PRRA acquisition. The origin of SARS-CoV-2, is the origin of the recognition cleavage site. The bat coronavirus RaTG13 appears to be the closest relative of the SARS-CoV-2, but was isolated in 2013. So, new RatG13 samples would provide insights into the acquisition of the polybasic motif.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Subtype Characterization and Zoonotic Potential of Cryptosporidium felis in Cats in Guangdong and Shanghai, China

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 89
Author(s):  
Jiayu Li ◽  
Fuxian Yang ◽  
Ruobing Liang ◽  
Sheng Guo ◽  
Yaqiong Guo ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
Genetic Characterization ◽  
Zoonotic Potential ◽  
Common Occurrence ◽  
High Genetic Diversity ◽  
Gp60 Gene ◽  
The Common ◽  
Subtyping Tool

Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis.

Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio

2021 ◽  
Author(s):  
Huolin Tu ◽  
Matthew R Avenarius ◽  
Laura Kubatko ◽  
Matthew Hunt ◽  
Xiaokang Pan ◽  
...  
Keyword(s):  
Clinical Impact ◽  
Clinical Samples ◽  
Membrane Glycoprotein ◽  
Receptor Binding Domain ◽  
Furin Cleavage ◽  
S Gene ◽  
The United Kingdom ◽  
Furin Cleavage Site ◽  
Two Samples ◽  
The Uk

AbstractFollowing the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

2021 ◽  
Author(s):  
Alison E Stout ◽  
Qinghua Guo ◽  
Jean K Millet ◽  
Gary R Whittaker
Keyword(s):  
Viral Pathogenesis ◽  
Spike Protein ◽  
Future Research ◽  
Receptor Binding Domain ◽  
Viral Reservoirs ◽  
Furin Cleavage ◽  
Proteolytic Activation ◽  
Furin Cleavage Site ◽  
Unique Aspect ◽  
Genome Level

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

scholarly journals Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

2021 ◽  
Vol 118 (47) ◽  
pp. e2109905118
Author(s):  
Liping Zhang ◽  
Matthew Mann ◽  
Zulfeqhar A. Syed ◽  
Hayley M. Reynolds ◽  
E. Tian ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Protein S ◽  
Viral Infectivity ◽  
The Novel ◽  
Furin Cleavage ◽  
Global Pandemic ◽  
Furin Cleavage Site ◽  
Respiratory Cells ◽  
Syncytia Formation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

scholarly journals SARS-CoV-2 convergent evolution as a guide to explore adaptive advantage

2021 ◽  
Author(s):  
Jiri Zahradnik ◽  
Jaroslav Nunvar ◽  
Gideon Schreiber
Keyword(s):  
Receptor Binding ◽  
Convergent Evolution ◽  
Great Majority ◽  
Viral Population ◽  
Binding Motif ◽  
Furin Cleavage ◽  
S Protein ◽  
Adaptive Mutations ◽  
Furin Cleavage Site ◽  
Protein Receptor

Much can be learned from 1.2 million sequences of SARS-CoV-2 generated during the last 15 months. Out of the overwhelming number of mutations sampled so far, only few rose to prominence in the viral population. Many of these emerged recently and independently in multiple lineages. Such a textbook example of convergent evolution at the molecular level is not only curiosity but a guide to uncover the basis for adaptive advantage behind these events. Focusing on the extent of the convergent evolution in the spike (S) protein, our report confirms that the most concerning SARS-CoV-2 lineages carry the heaviest burden of convergent S-protein mutations, suggesting their fundamental adaptive advantage. The great majority (21/25) of S-protein sites under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. We further show that among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favored. While the probed mutation space in the S protein covered all amino-acids reachable by single nucleotide changes, substitutions requiring two nucleotide changes or epistatic mutations of multiple-residues have only recently started to emerge. Unfortunately, despite their convergent emergence and physical association, most of these adaptive mutations and their combinations remain understudied. We aim to promote research of current variants which are currently understudied but may become important in the future.

scholarly journals Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

2020 ◽  
Author(s):  
Alexandra C. Walls ◽  
Young-Jun Park ◽  
M. Alexandra Tortorici ◽  
Abigail Wall ◽  
Andrew T. McGuire ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Furin Cleavage ◽  
Humoral Immune ◽  
Binding Domains ◽  
Furin Cleavage Site ◽  
Recent Emergence ◽  
Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

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