RESUMEN

The coronavirus nonstructural protein (nsp) 13 encodes an RNA helicase (nsp13-HEL) with multiple enzymatic functions, including unwinding and nucleoside phosphatase (NTPase) activities. Attempts for enzymatic inactivation have defined the nsp13-HEL as a critical enzyme for viral replication and a high-priority target for antiviral development. Helicases have been shown to play numerous roles beyond their canonical ATPase and unwinding activities, though these functions are just beginning to be explored in coronavirus biology. Recent genetic and biochemical studies, as well as work in structurally-related helicases, have provided evidence that supports new hypotheses for the helicase's potential role in coronavirus replication. Here, we review several aspects of the coronavirus nsp13-HEL, including its reported and proposed functions in viral replication and highlight fundamental areas of research that may aid the development of helicase inhibitors.

Asunto(s)

ARN Helicasas , Proteínas no Estructurales Virales , Replicación Viral , ARN Helicasas/metabolismo , ARN Helicasas/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Humanos , Coronavirus/enzimología , Coronavirus/genética , Coronavirus/fisiología , Animales , Antivirales/farmacología , Metiltransferasas

RESUMEN

Nidovirales is one order of RNA virus, with the largest single-stranded positive sense RNA genome enwrapped with membrane envelope. It comprises four families (Arterividae, Mesoniviridae, Roniviridae, and Coronaviridae) and has been circulating in humans and animals for almost one century, posing great threat to livestock and poultry，as well as to public health. Nidovirales shares similar life cycle: attachment to cell surface, entry, primary translation of replicases, viral RNA replication in cytoplasm, translation of viral proteins, virion assembly, budding, and release. The viral RNA synthesis is the critical step during infection, including genomic RNA (gRNA) replication and subgenomic mRNAs (sg mRNAs) transcription. gRNA replication requires the synthesis of a negative sense full-length RNA intermediate, while the sg mRNAs transcription involves the synthesis of a nested set of negative sense subgenomic intermediates by a discontinuous strategy. This RNA synthesis process is mediated by the viral replication/transcription complex (RTC), which consists of several enzymatic replicases derived from the polyprotein 1a and polyprotein 1ab and several cellular proteins. These replicases and host factors represent the optimal potential therapeutic targets. Hereby, we summarize the Nidovirales classification, associated diseases, "replication organelle," replication and transcription mechanisms, as well as related regulatory factors.

RESUMEN

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.

Asunto(s)

COVID-19 , Coronaviridae , Nidovirales , Coronaviridae/genética , Evolución Molecular , Humanos , Proteínas de la Membrana/genética , Nidovirales/genética , Filogenia , SARS-CoV-2/genética

RESUMEN

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.

Asunto(s)

Culicidae , Nidovirales , Secuencia de Aminoácidos , Animales , Variación Genética , Genoma Viral , Nidovirales/genética , Filogenia

RESUMEN

The frequent emergence of coronavirus (CoV) epidemics has seriously threatened public health and stock farming. The major hosts for CoVs are birds and mammals. Although most CoVs inhabit their specific natural hosts, some may occasionally cross the host barrier to infect livestock and even people, causing a variety of diseases. Since the beginning of the new century, increasing attention has been given to research on CoVs due to the emergence of highly pathogenic and genetically diverse CoVs that have caused several epidemics, including the recent COVID-19 pandemic. CoVs belong to the Coronaviridae family of the Nidovirales order. Recently, advanced techniques for viral detection and viral genome analyses have enabled characterization of many new nidoviruses than ever and have greatly expanded the Nidovirales order with new classification and nomenclature. Here, we first provide an overview of the latest research progress in the classification of the Nidovirales order and then introduce the host range, genetic variation, genomic pattern and pathogenic features of epidemic CoVs and other epidemic viruses. This information will promote understanding of the phylogenetic relationship and infectious transmission of various pathogenic nidoviruses, including epidemic CoVs, which will benefit virological research and viral disease control.

RESUMEN

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b', is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

Asunto(s)

Arterivirus/genética , ADN Complementario/genética , Proteínas Fluorescentes Verdes/genética , Sistemas de Lectura Abierta , ARN Viral/genética , Recombinación Genética , Replicación Viral/genética , Animales , Arterivirus/fisiología , Línea Celular , Quirópteros , Hominidae , Humanos , Plásmidos/genética , Prueba de Estudio Conceptual , Roedores

RESUMEN

In a meta-transcriptome study of the giant freshwater prawn Macrobrachium rosenbergii sampled in 2018 from a hatchery, we identified a variant of Macrobrachium rosenbergii golda virus (MrGV) in postlarvae without clinical signs. The virus belongs to the family Roniviridae, and the genome of this MrGV variant, Mr-18, consisted of 28,957 nucleotides, including 4 open reading frames (ORFs): (1) ORF1a, encoding a 3C-like protein (3CLP) (4933 aa); (2) ORF1b, encoding a replicase polyprotein (2877 aa); (3) ORF2, encoding a hypothetical nucleocapsid protein (125 aa); and (4) ORF3, encoding a glycoprotein (1503 aa). ORF1a overlaps with ORF1b with 40 nucleotides, where a -1 ribosomal frameshift with slippage sequence 5'-G14925GGUUUU14931-3' produces the pp1ab polyprotein. The genomic sequence of Mr-18 shared 97.80% identity with MrGV LH1-2018 discovered in Bangladesh. The amino acid sequence identities between them were 99.30% (ORF1a), 99.60% (ORF1b), 100.00% (ORF2), and 99.80% (ORF3), respectively. Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) proteins revealed that they clustered together and formed a separate cluster from the genus Okavirus. The finding of MrGV in China warrants further studies to determine its pathogenicity and prevalence within the region.

RESUMEN

Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single­stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

Asunto(s)

Agua Dulce/virología , Larva/virología , Palaemonidae/virología , Infecciones por Virus ARN/mortalidad , Infecciones por Virus ARN/veterinaria , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , Animales , Acuicultura , Bangladesh/epidemiología , Nodaviridae/genética , Nodaviridae/aislamiento & purificación , Sistemas de Lectura Abierta , Filogenia , Virus ARN/genética , Alineación de Secuencia

RESUMEN

Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in December 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here we provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.

Asunto(s)

Enfermedades de los Animales/virología , Betacoronavirus/fisiología , Pollos/virología , Infecciones por Coronavirus/virología , Coronavirus/fisiología , Neumonía Viral/virología , Síndrome Respiratorio Agudo Grave/virología , Enfermedades de los Animales/diagnóstico , Enfermedades de los Animales/epidemiología , Enfermedades de los Animales/prevención & control , Animales , Betacoronavirus/genética , Betacoronavirus/patogenicidad , COVID-19 , Bovinos , Coronavirus/genética , Coronavirus/patogenicidad , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/prevención & control , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Pandemias/prevención & control , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Neumonía Viral/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/prevención & control , Glicoproteína de la Espiga del Coronavirus/genética , Porcinos , Virión , Replicación Viral

RESUMEN

Interest in coronaviruses because of the 2019 novel coronavirus (SARS-CoV-2) pandemic has generated concern about their occurrence and persistence in aquatic habitats. Coronaviruses are not quantitatively significant constituents of marine virioplankton. Members of the Nidovirales (to which human coronaviruses belong) infect marine mammals, teleosts and possibly invertebrates, and human coronaviruses may persist in marine plankton receiving wastewater effluent. However, virions likely experience significant particle and infectivity decay rates in surface seawater, similar to other enveloped RNA viruses.

RESUMEN

As an evergreen ecosystem, citrus orchards have specialized pest species and stable ecological homeostasis; thus, they provide an ideal model for investigating RNA viromes in diverse but ecologically related species. For this purpose, we collected specialized citrus pests from three classes of invertebrates, Insecta, Arachnida, and Gastropoda and we constructed two kinds of libraries (RNA and small RNA) for the pests by deep sequencing. In total, six virus-derived sequences were identified, including four Picornavirales, one Jingchuvirales and one Nidovirales. The picornavirus-derived small RNAs showed significant small RNA peaks and symmetric distribution patterns along the genome, which suggests these viruses infected the hosts and triggered host antiviral immunity RNA interference. Screening of virus-derived sequences in multiple species of citrus pests (n = 10 per species) showed that Eotetranychus kankitus picorna-like virus and Tetranychus urticae mivirus may be present in multiple pests. Our investigation in citrus pests confirmed that RNA viruses revealed by metagenomics could impact host immunity (e.g. RNAi). An approach with parallel deep sequencing of RNAs and small RNAs is useful not only for viral discoveries but also for understanding virus-host interactions of ecologically related but divergent pest species.

Asunto(s)

Interacciones Huésped-Patógeno , Insectos/virología , Ácaros/virología , ARN Viral/análisis , Caracoles/virología , Viroma , Animales , Biblioteca de Genes

RESUMEN

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

Asunto(s)

Coronaviridae/clasificación , Genoma Viral/genética , Infecciones por Nidovirales/virología , Nidovirales/clasificación , Transcriptoma , California , Biología Computacional , Coronaviridae/genética , Coronaviridae/aislamiento & purificación , Evolución Molecular , Nidovirales/genética , Nidovirales/aislamiento & purificación , Terminación de la Cadena Péptídica Traduccional/genética , Péptido Hidrolasas/genética , Filogenia , Proteínas Virales/genética , Virión

Asunto(s)

Infecciones por Coronavirus/epidemiología , Aislamiento de Pacientes/métodos , Virus de la Diarrea Epidémica Porcina/patogenicidad , Porcinos/genética , Animales , Infecciones por Coronavirus/virología , Brotes de Enfermedades/prevención & control , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , República de Corea/epidemiología , Porcinos/virología , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/virología

RESUMEN

The recent emergence of Middle East respiratory syndrome (MERS) highlights the need to engineer new methods for expediting vaccine development against emerging diseases. However, several obstacles prevent pursuit of a licensable MERS vaccine. First, the lack of a suitable animal model for MERS complicates the in vivo testing of candidate vaccines. Second, due to the low number of MERS cases, pharmaceutical companies have little incentive to pursue MERS vaccine production as the costs of clinical trials are high. In addition, the timeline from bench research to approved vaccine use is 10 years or longer. Using novel methods and cost-saving strategies, genetically engineered vaccines can be produced quickly and cost-effectively. Along with progress in MERS animal model development, these obstacles can be circumvented or at least mitigated.

Asunto(s)

Infecciones por Coronavirus/prevención & control , Vacunas Virales/inmunología , Vacunas Virales/aislamiento & purificación , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Descubrimiento de Drogas/economía , Descubrimiento de Drogas/tendencias , Evaluación Preclínica de Medicamentos/economía , Evaluación Preclínica de Medicamentos/métodos , Humanos

RESUMEN

In late 2013, outbreaks of porcine epidemic diarrhea virus (PEDV) infection recurred in South Korea. Genetic and phylogenetic analyses showed that isolates from the outbreaks were most closely related to emergent US strains of PEDV. These US strain-like PEDV variants are prevalent in South Korea and responsible for recent outbreaks in the country.

Asunto(s)

Infecciones por Coronavirus/epidemiología , Virus de la Diarrea Epidémica Porcina/genética , Enfermedades de los Porcinos/epidemiología , Animales , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Brotes de Enfermedades , Filogenia , República de Corea/epidemiología , Porcinos , Enfermedades de los Porcinos/virología

RESUMEN

Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2',5'-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2',5'-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L-mediated degradation. Thus, VP3-CTD is a 2',5'-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2',5'-PDEs that antagonize activation of innate immunity.

Asunto(s)

Inmunidad Innata/inmunología , Hidrolasas Diéster Fosfóricas/inmunología , Virus ARN/inmunología , Proteínas no Estructurales Virales/inmunología , 2',5'-Oligoadenilato Sintetasa/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Línea Celular , Células Cultivadas , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Immunoblotting , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/metabolismo , Virus de la Hepatitis Murina/fisiología , Mutación , Oligorribonucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Virus ARN/metabolismo , Virus ARN/fisiología , Rotavirus/inmunología , Rotavirus/metabolismo , Rotavirus/fisiología , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo

RESUMEN

The yellow head virus (YHV) has been reported to be one of most pathogenic viruses for cultivated shrimp; however, serious problems have only been reported in farms in south and southeastern Asian. Recently, a YHV strain was detected in Litopenaeus vannamei cultivated in Mexican farms that lacked virus-associated mortalities or epizooties, and the animals were apparently healthy. The identity of the virus was confirmed by sequencing replicative and structural protein-encoding regions and comparing with homologous virus sequences. Phylogenic relationships and genetic distances were also determined and, although some differences were observed, an influence on virulence was uncertain. In addition, the expression levels of several transcripts (3CLPRO, POL, GP64 and GP116) were evaluated by quantitative real-time polymerase chain reaction during an experimental infection. Although the transcript showed varying kinetics, viral genes were expressed in infected L. vannamei, demonstrating the replicative capability of this YHV strain.