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1.
Virology ; 570: 123-133, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35398776

RESUMO

The current outbreak of coronavirus disease-2019 (COVID-19) caused by SARS-CoV-2 poses unparalleled challenges to global public health. SARS-CoV-2 is a Betacoronavirus, one of four genera belonging to the Coronaviridae subfamily Orthocoronavirinae. Coronaviridae, in turn, are members of the order Nidovirales, a group of enveloped, positive-stranded RNA viruses. Here we present a systematic phylogenetic and evolutionary study based on protein domain architecture, encompassing the entire proteomes of all Orthocoronavirinae, as well as other Nidovirales. This analysis has revealed that the genomic evolution of Nidovirales is associated with extensive gains and losses of protein domains. In Orthocoronavirinae, the sections of the genomes that show the largest divergence in protein domains are found in the proteins encoded in the amino-terminal end of the polyprotein (PP1ab), the spike protein (S), and many of the accessory proteins. The diversity among the accessory proteins is particularly striking, as each subgenus possesses a set of accessory proteins that is almost entirely specific to that subgenus. The only notable exception to this is ORF3b, which is present and orthologous over all Alphacoronaviruses. In contrast, the membrane protein (M), envelope small membrane protein (E), nucleoprotein (N), as well as proteins encoded in the central and carboxy-terminal end of PP1ab (such as the 3C-like protease, RNA-dependent RNA polymerase, and Helicase) show stable domain architectures across all Orthocoronavirinae. This comprehensive analysis of the Coronaviridae domain architecture has important implication for efforts to develop broadly cross-protective coronavirus vaccines.


Assuntos
COVID-19 , Coronaviridae , Nidovirales , Coronaviridae/genética , Evolução Molecular , Humanos , Proteínas de Membrana/genética , Nidovirales/genética , Filogenia , SARS-CoV-2/genética
2.
Virus Res ; 313: 198727, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35257792

RESUMO

Research on the recently established Mesoniviridae family (Order Nidovirales), RNA genome insect-specific viruses, has been steadily growing in the last decade. However, after the last detailed phylogenetic characterization of mesoniviruses in 2014, numerous new sequences, even in organisms other than mosquitos, have been identified and characterized. In this study, we analyzed nucleotide and protein sequences of mesoniviruses with a wide range of molecular tools including genetic distance, Shannon entropy, selective pressure analysis, polymorphism identification, principal coordinate analysis, likelihood mapping and phylodynamic reconstruction. We also sought to revaluate new mesoniviruses sequence positions within the family, proposing a taxonomic revision. The different sub-lineages of mosquito mesoniviruses sequences presented low sequence diversity and entropy, with incongruences to the existing taxonomy being found after an extensive phylogenetic characterization. High sequence discrepancy and differences in genome organization were found between mosquito mesoniviruses and other mesoniviruses, so their future classification, as other meso-like viruses that are found in other organisms, should be approached with caution. No evidence of frequent recombination was found, and mesonivirus genomes seem to evolve under strong purifying selection. Insufficient data by root-to-tip analysis did not yet allow for an adequate phylogeographic reconstruction.


Assuntos
Culicidae , Nidovirales , Sequência de Aminoácidos , Animais , Variação Genética , Genoma Viral , Nidovirales/genética , Filogenia
3.
J Immunol ; 207(8): 2167-2178, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34535575

RESUMO

Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus-host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum-specific insertions, the 3-aa Ile52Glu53Arg54 and 1-aa Arg154 insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen-host interactions. Residue Arg154 insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.


Assuntos
Antígenos Virais/metabolismo , Quirópteros/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Infecções por Nidovirales/imunologia , Nidovirales/fisiologia , Peptídeos/metabolismo , Trichosurus/imunologia , Animais , Apresentação do Antígeno , Antígenos Virais/imunologia , Austrália , Evolução Biológica , Clonagem Molecular , Conservação dos Recursos Naturais , Antígenos de Histocompatibilidade Classe I/genética , Interações Hospedeiro-Patógeno , Mamíferos , Ligação Proteica , Conformação Proteica
4.
Viruses ; 13(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34452414

RESUMO

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.


Assuntos
Nucleotídeos/metabolismo , Vírus de RNA de Cadeia Positiva/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Caliciviridae/genética , Caliciviridae/metabolismo , Coronaviridae/genética , Coronaviridae/metabolismo , Genoma Viral , Nidovirales/genética , Nidovirales/metabolismo , Picornaviridae/genética , Picornaviridae/metabolismo , Vírus de RNA de Cadeia Positiva/genética , Potyviridae/genética , Potyviridae/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral
5.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
6.
PLoS One ; 16(3): e0246981, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33730017

RESUMO

Nidoviruses and arenaviruses are the only known RNA viruses encoding a 3'-5' exonuclease domain (ExoN). The proofreading activity of the ExoN domain has played a key role in the growth of nidoviral genomes, while in arenaviruses this domain partakes in the suppression of the host innate immune signaling. Sequence and structural homology analyses suggest that these proteins have been hijacked from cellular hosts many times. Analysis of the available nidoviral ExoN sequences reveals a high conservation level comparable to that of the viral RNA-dependent RNA polymerases (RdRp), which are the most conserved viral proteins. Two highly preserved zinc fingers are present in all nidoviral exonucleases, while in the arenaviral protein only one zinc finger can be identified. This is in sharp contrast with the reported lack of zinc fingers in cellular ExoNs, and opens the possibility of therapeutic strategies in the struggle against COVID-19.


Assuntos
Exonucleases/genética , Domínios Proteicos/genética , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Arenavirus/genética , COVID-19/virologia , Humanos , Imunidade Inata/genética , Nidovirales/genética , Vírus de RNA/genética , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/genética , Dedos de Zinco/genética
7.
Eur J Immunol ; 51(6): 1482-1493, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33788263

RESUMO

The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.


Assuntos
Anticorpos Monoclonais/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Rejeição de Enxerto/imunologia , Interleucina-12/metabolismo , Mastocitoma/imunologia , Esclerose Múltipla/imunologia , Infecções por Nidovirales/imunologia , Nidovirales/fisiologia , Subunidades Proteicas/metabolismo , Sepse/imunologia , Transplante de Pele , Animais , Anticorpos Monoclonais/isolamento & purificação , Modelos Animais de Doenças , Epitopos , Humanos , Hibridomas , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neoplasias Experimentais , Subunidades Proteicas/imunologia
8.
J Immunol ; 206(7): 1653-1667, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33637616

RESUMO

The reptile MHC class I (MCH-I) and MHC class II proteins are the key molecules in the immune system; however, their structure has not been investigated. The crystal structure of green anole lizard peptide-MHC-I-ß2m (pMHC-I or pAnca-UA*0101) was determined in the current study. Subsequently, the features of pAnca-UA*0101 were analyzed and compared with the characteristics of pMHC-I of four classes of vertebrates. The amino acid sequence identities between Anca-UA*0101 and MHC-I from other species are <50%; however, the differences between the species were reflected in the topological structure. Significant characteristics of pAnca-UA*0101 include a specific flip of ∼88° and an upward shift adjacent to the C terminus of the α1- and α2-helical regions, respectively. Additionally, the lizard MHC-I molecule has an insertion of 2 aa (VE) at positions 55 and 56. The pushing force from 55-56VE triggers the flip of the α1 helix. Mutagenesis experiments confirmed that the 55-56VE insertion in the α1 helix enhances the stability of pAnca-UA*0101. The peptide presentation profile and motif of pAnca-UA*0101 were confirmed. Based on these results, the proteins of three reptile lizard viruses were used for the screening and confirmation of the candidate epitopes. These data enhance our understanding of the systematic differences between five classes of vertebrates at the gene and protein levels, the formation of the pMHC-I complex, and the evolution of the MHC-I system.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Lagartos/imunologia , Infecções por Nidovirales/imunologia , Nidovirales/fisiologia , Proteínas de Répteis/química , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Cristalografia por Raios X , Epitopos/genética , Evolução Molecular , Antígenos de Histocompatibilidade Classe I/genética , Sistema Imunitário , Imunidade , Filogenia , Polimorfismo Genético , Conformação Proteica , Estabilidade Proteica , Proteínas de Répteis/genética
9.
Biochem Biophys Res Commun ; 538: 24-34, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33413979

RESUMO

Two pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st century raised unprecedented interest in coronavirus research and assigned it unseen urgency. The two viruses responsible for the outbreaks, SARS-CoV and SARS-CoV-2, respectively, are in the spotlight, and SARS-CoV-2 is the focus of the current fast-paced research. Its foundation was laid down by studies of many corona- and related viruses that collectively form the vast order Nidovirales. Comparative genomics of nidoviruses played a key role in this advancement over more than 30 years. It facilitated the transfer of knowledge from characterized to newly identified viruses, including SARS-CoV and SARS-CoV-2, as well as contributed to the dissection of the nidovirus proteome and identification of patterns of variations between different taxonomic groups, from species to families. This review revisits selected cases of protein conservation and variation that define nidoviruses, illustrates the remarkable plasticity of the proteome during nidovirus adaptation, and asks questions at the interface of the proteome and processes that are vital for nidovirus reproduction and could inform the ongoing research of SARS-CoV-2.


Assuntos
Infecções por Coronaviridae/virologia , Nidovirales/classificação , Nidovirales/genética , Sequência Conservada , Evolução Molecular , Variação Genética , Genômica , Humanos , Filogenia , Proteoma , Vírus da SARS/classificação , Vírus da SARS/genética , SARS-CoV-2/classificação , SARS-CoV-2/genética , Proteínas Virais
10.
Vet Q ; 41(1): 89-96, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33416037

RESUMO

BACKGROUND: Neutrophil extracellular traps (NETs) represent a novel cellular mechanism of antimicrobial defense activity. Intravascular neutrophils produce extracellular web-like structures composed of chromatin, histones, and cytoplasmic granule proteins to attack and kill microbes. They may impact both pathogen and host; NETs correlate strongly with disseminated intravascular coagulation and mortality in critically ill humans. The mechanism was first discovered in human neutrophils in 2004. Presumptive heterophil extracellular traps (HETs) in a non-avian reptile species were first described in blood films of a gopher tortoise with systemic inflammation. OBJECTIVE: While prior reports are limited to blood film review and in vitro studies, this descriptive case series highlights the cytological identification of presumptive HETs in nine reptile patients. METHODS: Subjects included six gopher tortoises, one blood python (Python curtus), one Burmese python (P. bivittatus), and one desert king snake (Lampropeltis getula splendida). All six gopher tortoises (Gopherus polyphemus) had upper respiratory disease with bacterial etiology (including Helicobacter sp. and/or Mycoplasma sp.), and snakes had upper respiratory tract infection confirmed with serpentovirus (n = 2) or bacterial dermatitis (n = 1). RESULTS: Cytology samples with identified HETs included tissue imprints (n = 4), nasal discharge (n = 3), an oral swab (n = 1), and a fine needle aspirate of a skin lesion (n = 1). The identification of specific bacterial (n = 6) and/or viral pathogens (n = 2) was notable. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of presumptive HETs recognized in reptile cytology specimens, suggesting an active cellular process in vivo in response to systemic inflammation in non-avian reptiles, and contributing to further understanding of extracellular traps in these species.


Assuntos
Armadilhas Extracelulares , Inflamação/veterinária , Neutrófilos , Animais , Boidae/virologia , Colubridae/virologia , Feminino , Helicobacter , Infecções por Helicobacter/veterinária , Masculino , Mycoplasma , Infecções por Mycoplasma/veterinária , Nidovirales , Infecções por Nidovirales/veterinária , Tartarugas/microbiologia
11.
J Zoo Wildl Med ; 52(4): 1167-1174, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34998286

RESUMO

Serpentoviruses (order Nidovirales) are an important cause of respiratory disease in snakes. Although transmission studies have shown that serpentoviruses can cause respiratory disease in pythons, the possible role of additional potential pathogens is not yet understood. Very little information is available on the role of mycoplasma and chlamydia infections in disease in pythons. Diagnostic samples from 271 pythons of different genera submitted to a laboratory for detection of serpentoviruses were also screened for mycoplasma and chlamydia infections by PCR. Most of the samples were oral swabs. Almost 30% of the samples were positive for serpentoviruses, and mycoplasmas were detected in more than 60% of the pythons. The occurrence of these two pathogens correlated significantly (P < 0.001). Additionally, about 3% of the snakes tested positive for Chlamydia. This study found a high prevalence of mycoplasmas in the tested pythons and a correlation between infections with these bacteria and serpentoviruses in python samples submitted for diagnostic testing. Because the role mycoplasmas play in respiratory diseases of snakes is still largely unknown, further investigations are necessary to evaluate the role of mixed infections in disease.


Assuntos
Boidae , Chlamydia , Infecções por Mycoplasma , Mycoplasma , Nidovirales , Doenças Respiratórias , Animais , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/veterinária , Doenças Respiratórias/veterinária
12.
PLoS Negl Trop Dis ; 14(11): e0008920, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253189

RESUMO

Advances in technology have greatly stimulated the understanding of insect-specific viruses (ISVs). Unfortunately, most of these findings are based on sequencing technology, and laboratory data are scarce on the transmission dynamics of ISVs in nature and the potential effects of these viruses on arboviruses. Mesonivirus is a class of ISVs with a wide geographical distribution. Recently, our laboratory reported the isolation of a novel strain of mesonivirus, Yichang virus (YCV), from Culex mosquitoes, China. In this study, the experimental infection of YCV by the oral route for adult and larvae mosquitoes, and the vertical transmission has been conducted, which suggests that YCV could adopt a mixed-mode transmission. Controlled experiments showed that the infectivity of YCV depends on the mosquito species, virus dose, and infection route. The proliferation curve and tissue distribution of YCV in Cx. quinquefasciatus and Ae. albopictus showed that YCV is more susceptible to Ae. albopictus and is located in the midgut. Furthermore, we also assessed the interference of YCV with flaviviruses both in vitro and in vivo. YCV significantly inhibited the proliferation of DENV-2 and ZIKV, in cell culture, and reduced transmission rate of DENV-2 in Ae. albopictus. Our work provides insights into the transmission of ISVs in different mosquito species during ontogeny and their potential ability to interact with mosquito-borne viruses.


Assuntos
Aedes/virologia , Culex/virologia , Infecções por Nidovirales/transmissão , Nidovirales/fisiologia , Aedes/crescimento & desenvolvimento , Animais , Culex/crescimento & desenvolvimento , Vírus da Dengue/crescimento & desenvolvimento , Cavalos , Transmissão Vertical de Doenças Infecciosas , Larva/virologia , Mosquitos Vetores/virologia , Replicação Viral , Microbiologia da Água , Zika virus/crescimento & desenvolvimento
13.
Viruses ; 12(11)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228135

RESUMO

Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (Chamaeleo calyptratus). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40-50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80-90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.


Assuntos
Coinfecção/veterinária , Lagartos/virologia , Doenças Pulmonares Intersticiais/veterinária , Nidovirales/patogenicidade , Orthoreovirus/patogenicidade , Infecções por Reoviridae/veterinária , Animais , Animais de Zoológico/virologia , Coinfecção/virologia , Surtos de Doenças/veterinária , Feminino , Doenças Pulmonares Intersticiais/virologia , Masculino , Metagenômica , Nidovirales/genética , Orthoreovirus/genética , Prevalência
14.
Viruses ; 12(10)2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33066222

RESUMO

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.


Assuntos
Antígenos Virais/imunologia , Culicidae/virologia , Transmissão de Doença Infecciosa , Nidovirales/genética , Nidovirales/imunologia , Animais , Austrália , Nidovirales/classificação , Filogenia , Análise de Sequência de DNA , Vietnã , Vírion
15.
Viruses ; 12(7)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32708079

RESUMO

Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5'-UTR and 3'-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification.


Assuntos
Arbovírus/genética , Surtos de Doenças , Perfilação da Expressão Gênica/métodos , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nidovirales/genética , Infecção por Zika virus/virologia , Zika virus/genética , Humanos , Mosquitos Vetores/virologia , Filogenia , Reação em Cadeia da Polimerase , Infecção por Zika virus/epidemiologia
16.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641481

RESUMO

In recent years, nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full postmortem examinations on 30 serpentovirus reverse transcription-PCR (RT-PCR)-positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus infects not only a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts, etc.), but also intravascular monocytes, intralesional macrophages, and endothelial cells. With next-generation sequencing we obtained a full-length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection-associated respiratory or systemic disease did not reveal sequence association to phenotypes; however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that comprises important zoonotic pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Serpentoviruses belong to the same order as the above-mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order Nidovirales.


Assuntos
Infecções por Nidovirales/virologia , Nidovirales/fisiologia , Infecções Respiratórias/virologia , Doenças dos Animais/diagnóstico , Doenças dos Animais/virologia , Animais , Biópsia , Boidae/virologia , Suscetibilidade a Doenças , Humanos , Imuno-Histoquímica , Nidovirales/isolamento & purificação , Infecções por Nidovirales/diagnóstico , Especificidade de Órgãos , Fenótipo , Filogenia , Recombinação Genética , Infecções Respiratórias/diagnóstico , Tropismo Viral , Eliminação de Partículas Virais
17.
J Vet Med Sci ; 82(6): 788-792, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32378646

RESUMO

Reptile-associated nidoviruses (serpentoviruses) have been reported to cause proliferative interstitial pneumonia in pythons and other reptile species. A captive, younger than 2 years old, intact female ball python (Python regius) showed increased oral mucus, wheezing, and audible breathing with weight loss. Gross and microscopic examination revealed large amounts of mucus in the esophagus and proliferative interstitial pneumonia. Serpentovirus genes were detected from the lung tissues by polymerase chain reaction. The current serpentoviruses was phylogenetically grouped with the serpentovirus previously identified in the US. No case of serpentovirus infection has been reported in Asia. The present report provides information of complete genome sequence and global distribution of serpentovirus.


Assuntos
Boidae/virologia , Infecções por Nidovirales/veterinária , Nidovirales/isolamento & purificação , Animais , Feminino , Genoma Viral , Doenças Pulmonares Intersticiais/veterinária , Doenças Pulmonares Intersticiais/virologia , Nidovirales/genética , Filogenia , Reação em Cadeia da Polimerase , Taiwan
18.
Viruses ; 12(5)2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32466150

RESUMO

This is the first record of a fish nidovirus isolated from a consignment of goldfish at the United Kingdom (UK) border. The full-length viral genome was 25,985 nt, sharing a 97.9% nucleotide identity with the Chinook salmon bafinivirus (CSBV) NIDO with two deletions of 537 and 480 nt on the ORF Ia protein. To assess the potential impact on UK fish species, Atlantic salmon, common carp and goldfish were exposed to the virus via an intraperitoneal (IP) injection and bath challenge. Moribundity was recorded in only 8% of IP-injected goldfish. A high viral load, ≈107 of the CSBV PpIa gene, was measured in the kidney of moribund goldfish. Mild histopathological changes were observed in the kidneys of challenged carps. Ultrastructural observations in renal tubule epithelial cells of goldfish showed cylindrical tubes (≈15 nm in diameter) and tubular structures budding spherical virions (≈200 nm in diameter) with external spike-like structures. Negative staining showed both circular and bacilliform virions. Seroconversion was measured in common carp and goldfish but not in Atlantic salmon. This study reinforces the potential risk of novel and emerging pathogens being introduced to recipient countries via the international ornamental fish trade and the importance of regular full health screens at the border inspection posts to reduce this risk.


Assuntos
Coronaviridae/isolamento & purificação , Doenças dos Peixes/virologia , Carpa Dourada/virologia , Salmão/virologia , Animais , Carpas/virologia , Coronaviridae/classificação , Coronaviridae/genética , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/patologia , Genes Virais/genética , Genoma Viral , Rim/patologia , Rim/virologia , Nidovirales , Filogenia , Reino Unido , Virulência
19.
J Insect Sci ; 20(2)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32282036

RESUMO

We report a new positive-sense single-stranded RNA (ss RNA+) virus from the brown citrus aphid Aphis citricidus. The 20,300 nucleotide (nt)-long viral genome contains five open-reading frames and encodes six conserved domains (TM2, 3CLpro, TM3, RdRp, Zm, and HEL1). Phylogenetic analysis and amino acid sequence analysis revealed this virus might belong to an unassigned genus in the family Mesoniviridae. The presence of the virus was also confirmed in the field population. Importantly, analysis of the virus-derived small RNAs showed a 22-nt peak, implying that viral infection triggers the small interfering RNA pathway as antiviral immunity in aphids. This is the first report of a mesonivirus in invertebrates other than mosquitoes.


Assuntos
Afídeos/virologia , Especificidade de Hospedeiro , Nidovirales/fisiologia , RNA Viral/análise , Animais , Afídeos/crescimento & desenvolvimento , Ninfa/crescimento & desenvolvimento , Ninfa/virologia , Vírus de Plantas/fisiologia , Análise de Sequência de RNA
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