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1.
J Virol ; 96(3): e0156121, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-34817201

RESUMO

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.


Assuntos
DNA Complementar , Genoma Viral , Genética Reversa , Torovirus/genética , Animais , Bovinos , Doenças dos Bovinos/virologia , Linhagem Celular , Células Cultivadas , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Genes Reporter , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Mutação , Plasmídeos/genética , Torovirus/isolamento & purificação , Infecções por Torovirus , Transfecção
2.
Transbound Emerg Dis ; 69(2): 598-608, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33555108

RESUMO

Toroviruses (ToVs), closely related but genetically distinct from coronaviruses, are known to infect horses, cows, pigs, goats and humans, mainly causing enteritic disorders. However, due to the lack of an adaptive culture system, porcine ToV (PToV) has received less attention. In this study, we developed a novel serological detection method based on the PToV envelope spike subunit 1 (S1) protein for the first time, and compared it to an existing indirect enzyme-linked immunosorbent assay (ELISA) based on the nucleocapsid protein. By using the S1-based ELISA, we carried out the first seroepidemiological survey of PToV in China, assaying both specific IgG and IgA responses in 1,037 serum samples collected from diarrheic pigs in eastern China. There was a relatively high incidence of seropositivity in pigs of different ages, especially one-week-old piglets and sows (78% and 43%), the former probably reflecting maternal antibodies. Furthermore, 3/20 (15%) of faecal samples collected from one PToV-seropositive swine herd in Zhejiang province tested positive by RT-PCR. The complete PToV genome was sequenced from one of these samples, and its phylogenetic relationship with other full-length PToV sequences available in GenBank was determined. Our data provide the first serological evidence for PToV infection in pigs from China, which will help elucidate the potential pathogenicity of PToV in pigs.


Assuntos
Doenças dos Bovinos , Doenças dos Cavalos , Doenças dos Suínos , Infecções por Torovirus , Torovirus , Animais , Anticorpos Antivirais , Bovinos , China/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Cavalos , Filogenia , Suínos , Torovirus/genética , Infecções por Torovirus/epidemiologia , Infecções por Torovirus/veterinária
3.
Mol Microbiol ; 117(4): 837-850, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34967475

RESUMO

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes. We previously identified double membrane vesicles (DMVs) in the cytoplasm of cells infected with Berne virus (BEV), the prototype member of the Torovirus genus (Nidovirales Order). Our previous analysis by transmission electron microscopy suggested that the DMVs form a reticulovesicular network (RVN) analogous those described for the related severe acute respiratory syndrome coronavirus (SARS-CoV-1). Here, we used serial sectioning and electron tomography to characterize the architecture of torovirus replication organelles, and to learn about their biogenesis and dynamics during the infection. The formation of a RVN in BEV infected cells was confirmed, where the outer membranes of the DMVs are interconnected with each other and with the ER. Paired or zippered ER membranes connected with the DMVs were also observed, and likely represent early structures that evolve to give rise to DMVs. Also, paired membranes forming small spherule-like invaginations were observed at late time post-infection. Although resembling in size, the tomographic analysis show that these structures are clearly different from the true spherules described previously for coronaviruses. Hence, BEV shows important similarities, but also some differences, in the architecture of the replication organelles with other nidoviruses.


Assuntos
Torovirus , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático , Replicação Viral
4.
Viruses ; 13(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800523

RESUMO

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.


Assuntos
Infecções por Torovirus/virologia , Torovirus/fisiologia , Animais , Coronavirus/genética , Coronavirus/fisiologia , Infecções por Coronavirus/virologia , Humanos , Torovirus/genética
5.
Arch Virol ; 166(7): 2017-2025, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33881617

RESUMO

Bovine torovirus (BToV) is an important diarrhea-causing pathogen affecting bovines. To facilitate BToV detection, a reverse transcription insulated isothermal PCR (RT-iiPCR) assay was developed that targets the BToV M gene with high specificity and reproducibility. The assay has a limit of detection of 23 copies/µL. Out of 69 diarrheic fecal samples from yaks collected on six farms in Tibet and Sichuan provinces in China, 11.59% (8/69) tested positive for BToV using this assay. The full-length spike (S) and hemagglutinin-esterase (HE) genes of three positive samples were subsequently sequenced. Notably, an identical recombination event was identified in the S1 subunit of the S protein of three isolates. All of the HE genes were found to belong to genotype III and shared the same unique aa variation (P44S) in the esterase domain. This study is the first confirmation of BToV in yaks and the first report of an S gene recombination event in BToV. Our findings will enhance the current understanding of the molecular characteristics and genetic evolution of BToV.


Assuntos
Doenças dos Bovinos/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Reversa/genética , Infecções por Torovirus/virologia , Torovirus/genética , Animais , Bovinos , China , Fezes/virologia , Genes Virais/genética , Genótipo , Filogenia , RNA Viral/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Tibet , Infecções por Torovirus/veterinária , Proteínas Virais/genética
6.
ACS Chem Biol ; 16(10): 1951-1960, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33769035

RESUMO

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.


Assuntos
Hemaglutininas Virais/metabolismo , Polissacarídeos/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virais de Fusão/metabolismo , Acetilação , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Hemaglutininas Virais/química , Estrutura Molecular , Oxirredução , Ácido Periódico/química , Fenilenodiaminas/química , Polissacarídeos/análise , Polissacarídeos/química , Ligação Proteica , Ácidos Siálicos/análise , Ácidos Siálicos/química , Torovirus/enzimologia , Proteínas Virais de Fusão/química
7.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33177195

RESUMO

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.


Assuntos
Núcleo Celular/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Torovirus/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Mutação , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Proteínas do Nucleocapsídeo/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Torovirus/crescimento & desenvolvimento , Torovirus/metabolismo , Replicação Viral/genética
8.
BMC Vet Res ; 16(1): 272, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32758221

RESUMO

BACKGROUND: Coronaviruses are notorious pathogens that cause diarrheic and respiratory diseases in humans and animals. Although the epidemiology and pathogenicity of coronaviruses have gained substantial attention, little is known about bovine coronavirus in cattle, which possesses a close relationship with human coronavirus. Bovine torovirus (BToV) is a newly identified relevant pathogen associated with cattle diarrhoea and respiratory diseases, and its epidemiology in the Chinese cattle industry remains unknown. RESULTS: In this study, a total of 461 diarrhoeic faecal samples were collected from 38 different farms in three intensive cattle farming regions and analysed. Our results demonstrated that BToV is present in China, with a low prevalence rate of 1.74% (8/461). The full-length spike genes were further cloned from eight clinical samples (five farms in Henan Province). Phylogenetic analysis showed that two different subclades of BToV strains are circulating in China. Meanwhile, the three BToV strains identified from dairy calves, 18,307, 2YY and 5YY, all contained the amino acid variants R614Q, I801T, N841S and Q885E. CONCLUSIONS: This is the first report to confirm the presence of BToV in beef and dairy calves in China with diarrhea, which extend our understanding of the epidemiology of BToVs worldwide.


Assuntos
Doenças dos Bovinos/virologia , Infecções por Torovirus/veterinária , Torovirus/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , China/epidemiologia , Diarreia/epidemiologia , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Filogenia , RNA Viral , Análise de Sequência de DNA , Torovirus/genética , Infecções por Torovirus/epidemiologia , Infecções por Torovirus/virologia , Proteínas Virais/genética
9.
Int J Mol Sci ; 21(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604730

RESUMO

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Evolução Molecular , Interações entre Hospedeiro e Microrganismos/fisiologia , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Internalização do Vírus , Acetilesterase/metabolismo , Animais , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Linhagem Celular , Coronavirus/genética , Esterases , Transferência Genética Horizontal , Glicosaminoglicanos/metabolismo , Hemaglutininas Virais/genética , Humanos , Lectinas/metabolismo , Pandemias , Polissacarídeos , Receptores Virais/química , SARS-CoV-2 , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Torovirus , Proteínas Virais de Fusão/genética
10.
J Virol ; 94(20)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727876

RESUMO

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.


Assuntos
Proteases 3C de Coronavírus/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Infecções por Torovirus/embriologia , Torovirus/enzimologia , Animais , Proteases 3C de Coronavírus/genética , Células HEK293 , Humanos , Especificidade por Substrato , Suínos , Torovirus/genética , Infecções por Torovirus/genética
11.
Arch Virol ; 165(7): 1577-1583, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32388597

RESUMO

Bovine torovirus (BToV) is a diarrhea-causing pathogen. In this study, 92 diarrheic fecal samples from five farms in four provinces in China were collected and tested for BToV using a RT-PCR assay, and 21.73% samples were found to be BToV positive. Moreover, two complete BToV genome sequences (MN073058 and MN073059) were obtained from the clinical samples, which were 28,297 and 28,301 nucleotides in length, respectively. Sequence analysis showed that the two isolates shared 10 identical amino acid mutations in the S protein compared to the complete S sequences of BToV available in the GenBank database. In addition, seven consecutive amino acid mutations were found from aa 1,486 to 1,492 in the S protein of isolate MN073058. Moreover, the two isolates shared one identical amino acid mutation in the receptor binding sites of the HE protein. To the best of our knowledge, this is the first report on the epidemic and genomic characterization of BToV in China, which is helpful for further understanding the genetic evolution of BToV.


Assuntos
Doenças dos Bovinos/virologia , Diarreia/veterinária , Infecções por Torovirus/veterinária , Torovirus/isolamento & purificação , Animais , Bovinos , Diarreia/virologia , Fezes/virologia , Genoma Viral , Genômica , Filogenia , Torovirus/classificação , Torovirus/genética , Infecções por Torovirus/virologia , Proteínas Virais/genética
12.
J Vet Med Sci ; 82(3): 314-319, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31941845

RESUMO

It can be judged that if the detection frequency of prevalent pathogenic viruses decreases, biosecurity has been enhanced. To monitor bovine farm biosecurity levels, one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of group A rotavirus (RVA), bovine torovirus (BToV), bovine enterovirus (BEV), and bovine coronavirus (BCV) was designed, with the aim of configuring candidates for "viral pathogen indicators". A total of 322 bovine fecal samples were collected from calves aged less than three months at 48 bovine farms in Ibaraki and Chiba prefectures. At farm A, 20 calves were selected and sampled weekly for 12 weeks (184 samples); at farm B, 10 calves were selected and sampled for five weeks (50 samples); and at the rest of the 46 farms, 88 calves were sampled once. The screening on the 358 field samples proved positive for 27 RVA, 4 BToV, 55 BEV, and 52 BCV. In the successive sampling, RVA was detected once but not continuously, whereas BEV and BCV were detected in succession for up to five weeks. The results revealed that RVA was the primary agent among the positive samples obtained from calves aged three weeks or less, while BEV was the primary among those from the older than three weeks old. They can be employed as useful viral pathogen indicators for soundly evaluating biosecurity at bovine farms.


Assuntos
Doenças dos Bovinos/virologia , Coronavirus Bovino/isolamento & purificação , Enterovirus Bovino/isolamento & purificação , Rotavirus/isolamento & purificação , Torovirus/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/prevenção & controle , Fezes/virologia , Japão/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária
13.
Arch Virol ; 165(2): 471-477, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31863265

RESUMO

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.


Assuntos
Fezes/virologia , Genoma Viral , Infecções por Torovirus/veterinária , Torovirus/genética , Torovirus/isolamento & purificação , Sequenciamento Completo do Genoma , Animais , Biologia Computacional , Evolução Molecular , Humanos , Japão , Recombinação Genética , Suínos , Infecções por Torovirus/virologia
14.
PLoS One ; 14(7): e0219428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31306441

RESUMO

Autophagy is a conserved eukaryotic process that mediates lysosomal degradation of cytoplasmic macromolecules and damaged organelles, also exerting an important role in the elimination of intracellular pathogens. Despite the antiviral role of autophagy, many studies suggest that some positive-stranded RNA viruses exploit this pathway to facilitate their own replication. In this study, we demonstrate that the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae Family, Nidovirales Order), induces autophagy at late times post-infection. Conversion of microtubule associated protein 1B light chain 3 (LC3) from cytosolic (LC3 I) to the membrane associated form (LC3 II), a canonical marker of autophagosome formation, is enhanced in BEV infected cells. However, neither autophagy induction, via starvation, nor pharmacological blockade significantly affect BEV replication. Similarly, BEV infection is not altered in autophagy deficient cells lacking either Beclin 1 or LC3B protein expression. Unexpectedly, the cargo receptor p62, a selective autophagy receptor, aggregates within the region where the BEV main protease (Mpro) localizes. This finding, coupled with observation that BEV replication also induces ER stress at the time when selective autophagy is taking place, suggests that the autophagy pathway is activated in response to the hefty accumulation of virus-encoded polypeptides during the late phase of BEV infection.


Assuntos
Autofagia , Infecções por Torovirus/virologia , Torovirus/fisiologia , Replicação Viral , Animais , Autofagossomos , Proteína Beclina-1/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Cavalos , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Transdução de Sinais , Infecções por Torovirus/fisiopatologia
15.
Transbound Emerg Dis ; 66(2): 1023-1028, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30431236

RESUMO

Enterovirus species G (EV-G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV-G strain (KOR/KNU-1811/2018) that resulted from cross-order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV-G genome possessed an exogenous 594-nucleotide (198-amino acid) sequence, flanked by two viral 3Cpro cleavage sites at the 5' and 3' ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain-like cysteine protease (PLCP), which was recently found in the EV-G1, -G2, and -G17 genomes. The complete KNU-1811 genome shared 73.7% nucleotide identity with a prototype EV-G1 strain, but had 83.9%-86.7% sequence homology with the global EV-G1-PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV-G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1-PLCP and G17-PLCP strains respectively. These results implied that the torovirus-derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV-G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV-G driven by infrequent viral recombination events, by which EV-G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.


Assuntos
Cisteína Proteases/genética , Diarreia/veterinária , Infecções por Enterovirus/veterinária , Enterovirus Suínos/genética , Vírus Reordenados/genética , Recombinação Genética , Torovirus/genética , Sequência de Aminoácidos , Animais , Diarreia/epidemiologia , Diarreia/virologia , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Enterovirus Suínos/isolamento & purificação , Evolução Molecular , Fezes/virologia , Genoma Viral , Genótipo , Filogenia , República da Coreia/epidemiologia , Homologia de Sequência , Suínos , Doenças dos Suínos
16.
J Virol ; 92(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950409

RESUMO

The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5' untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames "hidden" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.


Assuntos
Perfilação da Expressão Gênica , Biossíntese de Proteínas , Torovirus/crescimento & desenvolvimento , Torovirus/genética , Transcrição Genética , Proteínas Virais/biossíntese , Animais , Células Cultivadas , Cavalos , Interações Hospedeiro-Patógeno , Análise de Sequência de RNA , Proteínas Virais/genética , Cultura de Vírus
17.
Arch Virol ; 163(9): 2471-2476, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29786119

RESUMO

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.


Assuntos
Infecções por Enterovirus/veterinária , Enterovirus Suínos/genética , Genoma Viral , Vírus Reordenados/genética , Doenças dos Suínos/epidemiologia , Infecções por Torovirus/veterinária , Torovirus/genética , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , China/epidemiologia , Endopeptidases/genética , Endopeptidases/metabolismo , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Enterovirus Suínos/classificação , Enterovirus Suínos/metabolismo , Fazendas , Fezes/virologia , Deleção de Genes , Variação Genética , Metagenômica/métodos , Filogenia , Prevalência , Vírus Reordenados/classificação , Vírus Reordenados/metabolismo , Recombinação Genética , Suínos , Doenças dos Suínos/virologia , Torovirus/classificação , Torovirus/metabolismo , Infecções por Torovirus/epidemiologia , Infecções por Torovirus/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
Virus Res ; 246: 28-34, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29337162

RESUMO

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.


Assuntos
Antivirais/farmacologia , Arterivirus/efeitos dos fármacos , Benzamidas/farmacologia , Coronaviridae/efeitos dos fármacos , Equartevirus/efeitos dos fármacos , Piperidinas/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Torovirus/efeitos dos fármacos , Animais , Arterivirus/genética , Arterivirus/crescimento & desenvolvimento , Arterivirus/metabolismo , Carpas , Linhagem Celular , Chlorocebus aethiops , Coronaviridae/genética , Coronaviridae/crescimento & desenvolvimento , Coronaviridae/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Equartevirus/genética , Equartevirus/crescimento & desenvolvimento , Equartevirus/metabolismo , Mesocricetus , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , RNA de Cadeia Dupla/antagonistas & inibidores , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , RNA Viral/genética , Torovirus/genética , Torovirus/crescimento & desenvolvimento , Torovirus/metabolismo , Replicação Viral/efeitos dos fármacos
19.
J Virol ; 91(14)2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28490584

RESUMO

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-ß expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.


Assuntos
Enzimas Desubiquitinantes/genética , Enterovirus/enzimologia , Enterovirus/genética , Fezes/virologia , Mutagênese Insercional , Torovirus/enzimologia , Torovirus/genética , Animais , Animais Recém-Nascidos , Diarreia/veterinária , Enterovirus/isolamento & purificação , Metagenômica , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Suínos , Doenças dos Suínos/virologia , Estados Unidos
20.
ACS Chem Biol ; 12(1): 214-224, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-27936566

RESUMO

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac2, with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.


Assuntos
Ácidos Siálicos/metabolismo , Acetilação , Antígenos CD/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Glicosilação , Hemaglutininas Virais/metabolismo , Humanos , Ligantes , Análise em Microsséries , Conformação Molecular , Simulação de Dinâmica Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Ácidos Siálicos/síntese química , Ácidos Siálicos/química , Torovirus/enzimologia , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/metabolismo
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