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1.
Virulence ; 11(1): 707-718, 2020 01 01.
Article in English | MEDLINE | ID: mdl-32490723

ABSTRACT

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/µL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.


Subject(s)
Communicable Diseases, Emerging/veterinary , Coronaviridae Infections/veterinary , Coronaviridae/isolation & purification , Polymerase Chain Reaction , Swine Diseases/diagnosis , Animals , Coinfection/diagnosis , Coinfection/veterinary , Communicable Diseases, Emerging/diagnosis , Coronaviridae/genetics , Coronaviridae Infections/diagnosis , Diarrhea/diagnosis , Diarrhea/veterinary , Open Reading Frames/genetics , Polymerase Chain Reaction/standards , RNA, Viral/genetics , Reproducibility of Results , Sensitivity and Specificity , Swine
2.
Int J Mol Sci ; 21(11)2020 May 28.
Article in English | MEDLINE | ID: mdl-32481719

ABSTRACT

Mucociliary clearance, mediated by a coordinated function of cilia bathing in the airway surface liquid (ASL) on the surface of airway epithelium, protects the host from inhaled pathogens and is an essential component of the innate immunity. ASL is composed of the superficial mucus layer and the deeper periciliary liquid. Ion channels, transporters, and pumps coordinate the transcellular and paracellular movement of ions and water to maintain the ASL volume and mucus hydration. microRNA (miRNA) is a class of non-coding, short single-stranded RNA regulating gene expression by post-transcriptional mechanisms. miRNAs have been increasingly recognized as essential regulators of ion channels and transporters responsible for ASL homeostasis. miRNAs also influence the airway host defense. We summarize the most up-to-date information on the role of miRNAs in ASL homeostasis and host-pathogen interactions in the airway and discuss concepts for miRNA-directed therapy.


Subject(s)
Coronaviridae Infections/metabolism , Host-Pathogen Interactions , MicroRNAs/genetics , Respiratory Mucosa/metabolism , Respiratory Tract Absorption , Animals , Coronaviridae Infections/genetics , Coronaviridae Infections/virology , Homeostasis , Humans , MicroRNAs/metabolism , Respiratory Mucosa/virology
3.
Euro Surveill ; 25(23)2020 06.
Article in English | MEDLINE | ID: mdl-32553061

ABSTRACT

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.


Subject(s)
Antibodies, Viral/blood , Clinical Laboratory Techniques/methods , Coronaviridae Infections/diagnosis , Coronavirus Infections/diagnosis , Coronavirus/immunology , Pneumonia, Viral/diagnosis , Serologic Tests/standards , Severe Acute Respiratory Syndrome/immunology , Betacoronavirus , Clinical Laboratory Techniques/standards , Coronavirus/isolation & purification , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Predictive Value of Tests , Sensitivity and Specificity , Serologic Tests/methods , Severe Acute Respiratory Syndrome/blood
4.
s.l; s.n; 4 maio 2020. ilus.
Non-conventional in Portuguese | LILACS, BRISA/RedTESA | ID: biblio-1099490

ABSTRACT

CONTEXTO: Conforme classificação da OMS, a COVID-19 é uma pandemia de risco muito alto a nível global. Até o momento não existem terapias específicas para a doença, embora diferentes alternativas, incluindo o uso de plasma de pacientes recuperados, estejam em investigação. OBJETIVOS: Identificar, avaliar sistematicamente e sumarizar as melhores evidências científicas disponíveis sobre a eficácia e a segurança do uso de plasma de pacientes recuperados para COVID-19. MÉTODOS: Revisão sistemática rápida (rapid review methodology). RESULTADOS: Após o processo de seleção, 55 estudos foram incluídos: 15 estudos com resultados e 40 estudos clínicos em andamento. Os estudos evidenciaram que o plasma de paciente recuperado para tratamento de SARS-CoV-2 resultou em redução de mortalidade, carga viral e tempo de internação hospitalar. Entretanto, estes estudos têm risco de viés moderado a alto e os estudos clínicos têm amostra pequena. Estes fatores contribuíram para que a certeza na evidência fosse muito baixa. CONCLUSÃO: Esta revisão sistemática rápida identificou 15 estudos com qualidade metodológica baixa a moderada, que avaliaram os efeitos da terapia com plasma de pacientes recuperados para COVID-19. Com base nos achados destes estudos, a eficácia e a segurança do plasma de pacientes recuperados em pacientes com COVID-19 ainda são incertas e seu uso de rotina, para esta situação, não pode ser recomendado, até que resultados de ensaios clínicos em andamento possam ser avaliados.(AU)


Subject(s)
Humans , Pneumonia, Viral/drug therapy , Plasmapheresis/instrumentation , Coronaviridae Infections/drug therapy , Betacoronavirus/drug effects , Technology Assessment, Biomedical , Cost-Benefit Analysis
5.
Emerg Microbes Infect ; 9(1): 949-961, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32378471

ABSTRACT

The emergences of coronaviruses have caused a serious global public health problem because their infection in humans caused the severe acute respiratory disease and deaths. The outbreaks of lethal coronaviruses have taken place for three times within recent two decades (SARS-CoV in 2002, MERS-CoV in 2012 and SARS-CoV-2 in 2019). Much more serious than SARS-CoV in 2002, the current SARS-CoV-2 infection has been spreading to more than 213 countries, areas or territories and causing more than two million cases up to date (17 April 2020). Unfortunately, no vaccine and specific anti-coronavirus drugs are available at present time. Current clinical treatment at hand is inadequate to suppress viral replication and inflammation, and reverse organ failure. Intensive research efforts have focused on increasing our understanding of viral biology of SARS-CoV-2, improving antiviral therapy and vaccination strategies. The animal models are important for both the fundamental research and drug discovery of coronavirus. This review aims to summarize the animal models currently available for SARS-CoV and MERS-CoV, and their potential use for the study of SARS-CoV-2. We will discuss the benefits and caveats of these animal models and present critical findings that might guide the fundamental studies and urgent treatment of SARS-CoV-2-caused diseases.


Subject(s)
Betacoronavirus/physiology , Coronaviridae Infections/pathology , Coronaviridae Infections/prevention & control , Coronavirus Infections/pathology , Coronavirus Infections/prevention & control , Disease Models, Animal , Pandemics/prevention & control , Pneumonia, Viral/pathology , Pneumonia, Viral/prevention & control , Research/trends , Animals , Humans , Middle East Respiratory Syndrome Coronavirus/physiology , SARS Virus/physiology
6.
Sao paulo; APH; abr. 2020. 46 p.
Monography in Portuguese | LILACS, HomeoIndex Homeopathy | ID: biblio-1088074

ABSTRACT

A infecção humana causada pelo vírus SARS-CoV-2 (COVID-19), diagnosticada como pneumonia de causa desconhecida originalmente na cidade de Wuhan (China), foi considerada como pandemia pela Organização Mundial da Saúde. Sua transmissibilidade parece ser bastante elevada, tendo afetado quase dois milhões de pessoas em todo o mundo e provocado mais de 130 mil mortes. Surgiu no Brasil em fevereiro de 2020, inicialmente na cidade de São Paulo. Afeta de forma mais grave os idosos e portadores de algumas comorbidades (tais como doenças cardiovasculares, hipertensão arterial, diabetes, câncer, DPOC e doenças cerebrovasculares, entre outras), tendo uma sintomatologia variável e tratamentos empíricos que estão sendo testados de forma mais rigorosa desde o seu aparecimento. Na ausência de vacina para proteção dos sadios, tem sido adotada a estratégia de isolamento social e tratamento com medidas de suporte geral e/ou avançado. Neste contexto, cabe investigar a contribuição da terapêutica homeopática no enfrentamento da doença, notadamente no alívio dos sintomas desconfortáveis por ela provocados em sua fase inicial, com acompanhamento e registro dos resultados obtidos pelos médicos homeopatas. Este estudo nacional pretende coletar, durante o período em que durar a pandemia da COVID-19 no Brasil, informações de pacientes diagnosticados com a doença, tratados com medicamentos homeopáticos escolhidos de acordo com a sintomatologia apresentada pelo paciente, por experientes médicos homeopatas. Todos os medicamentos homeopáticos estão descritos na Farmacopéia Homeopática Brasileira, não envolvendo qualquer medicamento que não tenha sido anteriormente aprovado para uso homeopático. Serão avaliados os efeitos no estado de saúde do paciente, por meio de escores e escalas clínicas, bem como aspectos relacionados à segurança do medicamento, variação na duração da doença e medicamentos mais associados a eventuais sucessos terapêuticos. Questionário padronizado e específico para a COVID-19 foi elaborado e disponibilizado em formulários google para preenchimento dos médicos colaboradores do estudo durante o acompanhamento dos pacientes. Os dados serão armazenados em planilhas eletrônicas e serão analisados com técnicas estatísticas descritivas e inferenciais. Todos os dados dos pacientes serão coletados de forma totalmente anonimizada para proteger a privacidade dos pacientes, que serão identificados no formulário eletrônico, única e exclusivamente, por um código alfanumérico, escolhido pelo seu médico homeopata e registrado no seu prontuário médico. Dada a situação pandêmica, nos casos em que não for possível o atendimento presencial será enviado uma folha de informações sobre o estudo e TCLE para preenchimento pelo paciente, bem como feitas as teleconsultas de seguimento para acompanhamento do caso. Além de gerar o desenvolvimento de novas aplicações da telemedicina na área homeopática, o projeto visa também a coleta de informações úteis que poderão ser utilizadas em futuros estudos multicêntricos randomizados e controlados para tratamento com medicamentos homeopáticos de quadros epidêmicos, podendo servir também de modelo para novos estudos clínicos de avaliação dos benefícios do tratamento homeopático em outras doenças ou agravos à saúde. (AU)


Infection in humans caused by the SARS-CoV-2 virus (COVID-19), diagnosed as pneumonia of unknown cause originally in the city of Wuhan (China) in December 2019, was considered a pandemic by the World Health Organization. Its transmissibility seems to be quite high, affecting almost two million people worldwide and causing more than 130 thousand deaths. It appeared in Brazil in February 2020, initially in the city of São Paulo. It affects more severely the elderly and people with some comorbidities (such as cardiovascular diseases, high blood pressure, diabetes, malignancy, chronic obstructive pulmonary disease and cerebrovascular disease, among others), with a rich clinical symptomatology. Empirical treatments are being tested in more rigorous clinical trials. In the absence of a vaccine to protect the healthy, the strategy of social isolation and treatment with general and / or advanced support measures has been adopted. In this context, it is worth investigating the potential contribution of homeopathy for relieving the distressing symptoms caused by coronavirus in its initial phase, together with monitoring and recording outcomes collected by homeopathic doctors. This national study intends to collect information from suspected or confirmed cases of COVID-19 patients, during the current pandemic in Brazil. The patients will be attended by experienced homeopathic doctors with the prescription of remedies according to the symptoms presented by the patient in the pandemic. All homeopathic medicines are described in the Brazilian Homeopathic Pharmacopoeia. The effects on patients' health status will be evaluated by means of scores and clinical scales, together with measures on safety, duration of the disease and medicines better related with good results. A standardized and specific questionnaire for COVID-19 had been designed and will be available on google forms to be filled out by doctors during the study. The data will be stored in electronic spreadsheets and will be analyzed using descriptive and inferential statistical techniques. All patient data will be collected in a completely anonymous way to protect patients' privacy. Patients will be identified exclusively by an alphanumeric code, to be registered in doctors' medical records. Given the pandemic situation, in cases in which face-to-face health care is not obligatory, an information sheet about the study will be sent to the patient in order to get the informed consent. Teleconsultations will take place in some cases for follow-up of patients. In addition to enlarge telemedicine applications for homeopathy, this project also aims to collect useful information that could be used in future randomized and controlled multicenter trials to evaluate the role of homeopathy in epidemic or transmissible diseases. It could also be helpful for designing clinical studies using homeopathic medicines in other diseases or health problems. (AU)


La infección en humanos causada por el virus SARS-CoV-2 (COVID-19), diagnosticada como neumonía de causa desconocida originalmente en la ciudad de Wuhan (China) en diciembre de 2019, fue considerada una pandemia por la Organización Mundial de la Salud. Su transmisibilidad parece ser bastante alta, afectando a casi dos millones de personas en todo el mundo y causando más de 130 mil muertes. Apareció en Brasil en febrero de 2020, inicialmente en la ciudad de São Paulo. Afecta más severamente a los ancianos y personas con algunas comorbilidades (como enfermedades cardiovasculares, presión arterial alta, diabetes, malignidad, enfermedad pulmonar obstructiva crónica y enfermedad cerebrovascular, entre otros), con una rica sintomatología clínica. Los tratamientos empíricos se están probando en ensayos clínicos más rigurosos. En ausencia de una vacuna para proteger a los sanos, se ha adoptado la estrategia de aislamiento social y tratamiento con medidas de apoyo generales y / o avanzadas. En este contexto, se puede investigar la contribución potencial de la homeopatía para aliviar los síntomas causados por el coronavirus en su fase inicial, junto con el monitoreo y registro de los resultados recopilados por los médicos homeópatas. Este estudio nacional tiene la intención de recopilar información de casos sospechosos o confirmados de pacientes con COVID-19, durante la pandemia actual en Brasil. Los pacientes serán atendidos por médicos homeópatas experimentados con la prescripción de remedios de acuerdo con los síntomas presentados por el paciente en la pandemia. Todos los medicamentos homeopáticos se describen en la Farmacopea Homeopática Brasileña. Los efectos sobre el estado de salud de los pacientes se evaluarán mediante puntajes de síntomas y escalas clínicas, junto con medidas de seguridad, duración de la enfermedad y revelación de medicamentos más asociados con buenas respuestas clínicas. Se diseñó un cuestionario estandarizado y específico para COVID-19, que estará disponible en los formularios de Google para que los médicos lo completen durante el estudio. Los datos se almacenarán en hojas de cálculo electrónicas y se analizarán mediante técnicas estadísticas descriptivas e inferenciales. Todos los datos del paciente se recopilarán de forma completamente anónima para proteger la privacidad de los pacientes. Los pacientes serán identificados exclusivamente por un código alfanumérico, que se registrará en los registros médicos de los médicos. Dada la situación de pandemia, en los casos en que la atención médica presencial no es obligatoria, se enviará una hoja de información sobre el estudio al paciente para obtener el consentimiento informado. Se realizarán teleconsultas en algunos casos para el seguimiento de los pacientes. Además de ampliar las aplicaciones de telemedicina para la homeopatía, este protocolo también tiene como objetivo recopilar información útil que podría utilizarse en futuros ensayos multicéntricos controlados y aleatorizados para evaluar el papel de la homeopatía en enfermedades epidémicas o transmisibles. También podría ser útil para diseñar estudios clínicos con medicamentos homeopáticos en otras enfermedades o problemas de salud.(AU)


Subject(s)
Humans , Data Collection , Outcome Assessment, Health Care , Coronaviridae Infections/therapy , SARS Virus , Homeopathy , Brazil/epidemiology , Coronavirus Infections/epidemiology
7.
São Paulo; AMHB;APH; mar. 2020. 62 p.
Monography in Portuguese | LILACS, HomeoIndex Homeopathy, MOSAICO - Integrative health | ID: biblio-1087238

ABSTRACT

Além da reconhecida aplicação nas doenças crônicas, a homeopatia individualizada também pode atuar de forma resolutiva ou complementar nos casos agudos, incluindo as doenças epidêmicas. No entanto, para atingir esse intento, apresenta uma metodologia semiológica e terapêutica específica que deve ser seguida e respeitada, com o risco de não apresentar a eficácia e a segurança desejada. No caso das doenças epidêmicas, que pela virulência dos seus agentes provoca um quadro sintomatológico comum na maioria dos indivíduos suscetíveis, o medicamento homeopático individualizado (medicamento homeopático do gênio epidêmico) deve apresentar semelhança com o conjunto de sinais e sintomas característicos dos pacientes acometidos nos diferentes estágios de cada surto epidêmico. Estudos evidenciam a eficácia e a segurança desta prática profilática e/ou terapêutica em diversas epidemias do passado. Assim sendo, após o levantamento dos possíveis medicamentos homeopáticos individualizados do gênio epidêmico de cada epidemia, sua aplicação profilática e/ou terapêutica em larga escala deve ser sustentada por ensaios clínicos prévios que demonstrem sua eficácia e segurança, em consonância com os aspectos éticos e bioéticos da pesquisa envolvendo seres humanos. Cumprindo essas premissas da boa prática clínica, elaboramos o atual protocolo com o objetivo de investigar, em ensaio clínico randomizado, duplo-cego e placebo-controlado, a eficácia e a segurança de possíveis medicamentos homeopáticos individualizados do gênio epidêmico da COVID-19, em tratamento adjuvante e complementar de pacientes acometidos pela doença. Caso a a eficácia e a segurança se confirme, e tão somente, o(s) medicamento(s) poderão ser utilizado de forma generalizada e coletiva no tratamento e na prevenção da atual epidemia. (AU)


In addition to the recognized application in chronic diseases, individualized homeopathy can also act in a resolutive or complementary way in acute cases, including epidemic diseases. However, to achieve this intent, it presents a specific semiological and therapeutic methodology that must be followed and respected, with the risk of not presenting the desired efficacy and safety. In the case of epidemic diseases, which due to the virulence of their agents causes a common symptomatological picture in most susceptible individuals, the individualized homeopathic medicine (homeopathic medicine of the epidemic genius) should present similarity with the set of characteristic symptoms and signs of the patients affected in the different stages of each epidemic outbreak. Studies show the efficacy and safety of this prophylactic and/or therapeutic practice in several epidemics of the past. Therefore, after the survey of possible homeopathic drugs individualized from the epidemic genius of each epidemic, its prophylactic and/or large-scale therapeutic application should be supported by previous clinical trials that demonstrate its efficacy and safety, in line with the ethical and bioethical aspects of research involving human beings. Fulfilling these premises of good clinical practice, we developed the current protocol with the objective of investigating, in a randomized, double-blind and placebo-controlled clinical trial, the efficacy and safety of possible individualized homeopathic drugs of epidemic genius of COVID-19, in adjuvant and complementary treatment of patients affected by the disease. If efficacy and safety are confirmed, and only in this condition, the medicine may be used in a generalized and collective manner in the treatment and prevention of the current epidemic. (AU)


Subject(s)
Humans , Epidemic Genius , Clinical Protocols , Coronavirus , Coronaviridae Infections/prevention & control , Coronaviridae Infections/therapy , Ethics, Research , SARS Virus , Epidemics , Homeopathy , Brazil/epidemiology
8.
São Paulo; AMHB; mar. 3, 2020. 21 p.
Monography in Portuguese | LILACS, HomeoIndex Homeopathy, MOSAICO - Integrative health | ID: biblio-1087382

ABSTRACT

Estamos no meio da pandemia do Covid-19, em franco desenvolvimento no Brasil. No momento há perspectivas terapêuticas medicamentosa sem fases iniciais de teste, especialmente para o tratamento dos casos graves, aqueles que implicam em internação ou encaminhamento para Unidades de Terapia Intensiva. Pouco tem sido abordado sobre tentativas de tratamento de pacientes com síndrome respiratória leve. Não há escolha terapêutica efetiva para o início do quadro e, desta forma, a doença cursa de acordo com a resposta imune ou suscetibilidade individual do acometido, e há muito pouco de efetivo e específico que altere a história natural dessa enfermidade. (AU)


Subject(s)
Arsenicum Album/therapeutic use , China officinalis/therapeutic use , Chininum Arsenicosum/therapeutic use , Epidemic Genius , Coronaviridae , Coronaviridae Infections , Bryonia , Pandemics , Homeopathy , Brazil/epidemiology
9.
Vet Microbiol ; 237: 108398, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31585653

ABSTRACT

Feline infectious peritonitis (FIP) is a highly fatal disease caused by a virulent feline coronavirus in domestic and wild cats. We have previously reported the synthesis of potent coronavirus 3C-like protease (3CLpro) inhibitors and the efficacy of a protease inhibitor, GC376, in client-owned cats with FIP. In this study, we studied the effect of the amino acid changes in 3CLpro of feline coronavirus from a feline patient who received antiviral treatment for prolonged duration. We generated recombinant 3CLpro containing the identified amino acid changes (N25S, A252S or K260 N) and determined their susceptibility to protease inhibitors in the fluorescence resonance energy transfer assay. The assay showed that N25S in 3CLpro confers a small change (up to 1.68-fold increase in the 50% inhibitory concentration) in susceptibility to GC376, but other amino acid changes do not affect susceptibility. Modelling of 3CLpro carrying the amino acid changes was conducted to probe the structural basis for these findings. The results of this study may explain the observed absence of clinical resistance to the long-term antiviral treatment in the patients.


Subject(s)
Cat Diseases/virology , Coronaviridae Infections/veterinary , Coronavirus, Feline/enzymology , Feline Infectious Peritonitis/complications , Protease Inhibitors/therapeutic use , Pyrrolidines/therapeutic use , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cats , Coronaviridae Infections/drug therapy , Coronaviridae Infections/virology , Male , Models, Molecular , Protease Inhibitors/pharmacology , Protein Conformation , Pyrrolidines/pharmacology , RNA, Viral , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/metabolism
10.
J Microbiol Biotechnol ; 29(11): 1817-1829, 2019 Nov 28.
Article in English | MEDLINE | ID: mdl-31546302

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. Highresolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two upregulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.


Subject(s)
Coronaviridae Infections/veterinary , Coronaviridae/physiology , Swine Diseases/virology , Viral Regulatory and Accessory Proteins/metabolism , Actinin/metabolism , Animals , Cell Line , Coronaviridae/genetics , Coronaviridae/metabolism , Coronaviridae Infections/virology , Host-Pathogen Interactions , Mitochondria/metabolism , Proteomics , Swine , Viral Regulatory and Accessory Proteins/genetics
11.
J Virol ; 92(15)2018 08 01.
Article in English | MEDLINE | ID: mdl-29769348

ABSTRACT

The emergence of Middle East respiratory syndrome showed once again that coronaviruses (CoVs) in animals are potential source for epidemics in humans. To explore the diversity of deltacoronaviruses in animals in the Middle East, we tested fecal samples from 1,356 mammals and birds in Dubai, The United Arab Emirates. Four novel deltacoronaviruses were detected from eight birds of four species by reverse transcription-PCR (RT-PCR): FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon, and QuaCoV UAE-HKU30 from five quails. Complete genome sequencing showed that FalCoV UAE-HKU27, HouCoV UAE-HKU28, and PiCoV UAE-HKU29 belong to the same CoV species, suggesting recent interspecies transmission between falcons and their prey, houbara bustards and pigeons, possibly along the food chain. Western blotting detected specific anti-FalCoV UAE-HKU27 antibodies in 33 (75%) of 44 falcon serum samples, supporting genuine infection in falcons after virus acquisition. QuaCoV UAE-HKU30 belongs to the same CoV species as porcine coronavirus HKU15 (PorCoV HKU15) and sparrow coronavirus HKU17 (SpCoV HKU17), discovered previously from swine and tree sparrows, respectively, supporting avian-to-swine transmission. Recombination involving the spike protein is common among deltacoronaviruses, which may facilitate cross-species transmission. FalCoV UAE-HKU27, HouCoV UAE-HKU28, and PiCoV UAE-HKU29 originated from recombination between white-eye coronavirus HKU16 (WECoV HKU16) and magpie robin coronavirus HKU18 (MRCoV HKU18), QuaCoV UAE-HKU30 from recombination between PorCoV HKU15/SpCoV HKU17 and munia coronavirus HKU13 (MunCoV HKU13), and PorCoV HKU15 from recombination between SpCoV HKU17 and bulbul coronavirus HKU11 (BuCoV HKU11). Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.IMPORTANCE During an attempt to explore the diversity of deltacoronaviruses among mammals and birds in Dubai, four novel deltacoronaviruses were detected in fecal samples from eight birds of four different species: FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon, and QuaCoV UAE-HKU30 from five quails. Genome analysis revealed evidence of recent interspecies transmission between falcons and their prey, houbara bustards and pigeons, possibly along the food chain, as well as avian-to-swine transmission. Recombination, which is known to occur frequently in some coronaviruses, was also common among these deltacoronaviruses and occurred predominantly at the spike region. Such recombination, involving the receptor binding protein, may contribute to the emergence of new viruses capable of infecting new hosts. Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.


Subject(s)
Bird Diseases , Birds/virology , Coronaviridae Infections , Coronavirus , High-Throughput Nucleotide Sequencing , Swine/virology , Animals , Bird Diseases/genetics , Bird Diseases/transmission , Coronaviridae Infections/genetics , Coronaviridae Infections/transmission , Coronaviridae Infections/veterinary , Coronavirus/classification , Coronavirus/genetics , Coronavirus/isolation & purification , Coronavirus/pathogenicity , Saudi Arabia
12.
J Virol ; 92(4)2018 02 15.
Article in English | MEDLINE | ID: mdl-29093093

ABSTRACT

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5-Å-resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified Deltacoronavirus genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved compared with the human respiratory coronavirus NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates in rendering the spike largely protease resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.IMPORTANCE Coronaviruses use transmembrane S glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic-resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic PDCoV, which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease resistant, which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.


Subject(s)
Coronaviridae Infections/virology , Coronaviridae/physiology , Membrane Fusion , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment , Animals , Coronaviridae/classification , Coronaviridae Infections/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/virology , Humans , Polysaccharides/chemistry , Protein Conformation , Swine , Swine Diseases/pathology , Swine Diseases/virology
13.
Virology ; 502: 33-38, 2017 02.
Article in English | MEDLINE | ID: mdl-27984784

ABSTRACT

Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-ß), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-ß production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses.


Subject(s)
Coronaviridae Infections/veterinary , Coronaviridae/enzymology , Cysteine Endopeptidases/metabolism , I-kappa B Kinase/metabolism , Interferon-beta/metabolism , Swine Diseases/enzymology , Viral Nonstructural Proteins/metabolism , Animals , Coronaviridae/genetics , Coronaviridae Infections/enzymology , Coronaviridae Infections/metabolism , Coronaviridae Infections/virology , Cysteine Endopeptidases/genetics , Host-Pathogen Interactions , I-kappa B Kinase/genetics , Interferon-beta/genetics , Protein Processing, Post-Translational , Swine , Swine Diseases/genetics , Swine Diseases/metabolism , Swine Diseases/virology , Viral Nonstructural Proteins/genetics
14.
Transbound Emerg Dis ; 64(1): 3-10, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27718337

ABSTRACT

Porcine deltacoronavirus (PDCoV) was detected by RT-PCR in 12 of 97 (12.4%) intestinal samples collected during 2015 from piglets with diarrhoea in Thailand, Vietnam and Lao PDR. Spike, membrane and nucleocapsid genes were characterized, and phylogenetic analyses demonstrated that PDCoV isolates from Thai and Lao PDR form a novel cluster, separated from US and China isolates, but relatively were more closely related to China PDCoV than US isolates. Vietnam PDCoVs, however, were grouped together with US PDCoV. The analyses of amino acid changes suggested that they were from different lineage.


Subject(s)
Coronaviridae/genetics , DNA, Viral/genetics , Swine Diseases/virology , Animals , Coronaviridae Infections/genetics , Intestines/virology , Laos , Phylogeny , Polymerase Chain Reaction , Swine , Thailand , Vietnam
15.
Prev Vet Med ; 135: 87-94, 2016 Dec 01.
Article in English | MEDLINE | ID: mdl-27931933

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme-linked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or non-infected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ=0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1-74.4%) and 97.5% (95.2-99.0%) for ELISA 1, 73.7% (71.5-79.6%) and 98.4% (96.6-99.5%) for ELISA 2, 86.2% (81.1-90.6%) and 91.6% (87.7-94.8%) for ELISA 3, 78.3% (72.8-83.5%) and 99.7% (98.2-100%) for ELISA 4, and 93.5% (90.3-96.0%) and 91.2% (83.8-97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used.


Subject(s)
Antibodies, Viral/blood , Coronaviridae Infections/veterinary , Diagnostic Techniques and Procedures/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Porcine epidemic diarrhea virus/isolation & purification , Swine Diseases/diagnosis , Animals , Coronaviridae Infections/diagnosis , Coronaviridae Infections/virology , Denmark , Italy , Sensitivity and Specificity , Swine , Swine Diseases/virology , United States
16.
Vet Microbiol ; 193: 28-35, 2016 Sep 25.
Article in English | MEDLINE | ID: mdl-27599927

ABSTRACT

Population of wild boar is increasing in the whole Europe, the animals migrate close to human habitats which greatly increases the possibility of natural transmission between domestic animals or humans and wild boars. The aim of the study was to estimate in population of free-living wild boar in the Czech Republic the prevalence of enteric viral pathogens, namely rotavirus groups A and C (RVA and RVC), porcine reproductive and respiratory syndrome virus (PRRSV), and members of family Coronaviridae (transmissible gastroenteritis virus - TGEV, porcine epidemic diarrhea virus - PEDV, porcine respiratory coronavirus - PRCV, and porcine hemagglutination encephalomyelitis virus - PHEV) and Picornaviridae,(teschovirus A - PTV, sapelovirus A - PSV, and enterovirus G - EV-G). In our study, stool samples from 203 wild boars culled during hunting season 2014-2015 (from October to January) were examined by RT-PCR. RVA was detected in 2.5% of tested samples. Nucleotide analysis of VP7, VP4, and VP6 genes revealed that four RVA strains belong to G4P[25]I1, G4P[6]I5, G11P[13]I5, and G5P[13]I5 genotypes and phylogenetic analysis suggested close relation to porcine and human RVAs. The prevalence of RVC in wild boar population reached 12.8%, PTV was detected in 20.2%, PSV in 8.9%, and EV-G in 2.5% of samples. During our study no PRRSV or coronaviruses were detected. Our study provides the first evidence of RVC prevalence in wild boars and indicates that wild boars might contribute to the genetic variability of RVA and also serve as an important reservoir of other enteric viruses.


Subject(s)
Coronaviridae Infections/veterinary , Picornaviridae Infections/veterinary , Rotavirus Infections/veterinary , Rotavirus/isolation & purification , Swine Diseases/virology , Animals , Antigens, Viral/genetics , Capsid Proteins/genetics , Coronaviridae/genetics , Coronaviridae/isolation & purification , Coronaviridae Infections/epidemiology , Coronaviridae Infections/virology , Czech Republic/epidemiology , Disease Reservoirs , Feces/virology , Female , Genotype , Humans , Male , Phylogeny , Picornaviridae/genetics , Picornaviridae/isolation & purification , Picornaviridae Infections/epidemiology , Picornaviridae Infections/virology , Rotavirus/genetics , Rotavirus Infections/epidemiology , Rotavirus Infections/virology , Sus scrofa , Swine , Swine Diseases/epidemiology
17.
Virol J ; 13: 136, 2016 08 05.
Article in English | MEDLINE | ID: mdl-27496131

ABSTRACT

BACKGROUND: Following the initial isolation of porcine deltacoronavirus (PDCoV) from pigs with diarrheal disease in the United States in 2014, the virus has been detected on swine farms in some provinces of China. To date, little is known about the molecular epidemiology of PDCoV in southern China where major swine production is operated. RESULTS: To investigate the prevalence of PDCoV in this region and compare its activity to other enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis coronavirus (TGEV), and porcine rotavirus group C (Rota C), 390 fecal samples were collected from swine of various ages from 15 swine farms with reported diarrhea. Fecal samples were tested by reverse transcription-PCR (RT-PCR) that targeted PDCoV, PEDV, TGEV, and Rota C, respectively. PDCoV was detected exclusively from nursing piglets with an overall prevalence of approximate 1.28 % (5/390), not in suckling and fattening piglets. Interestingly, all of PDCoV-positive samples were from 2015 rather than 2012-2014. Despite a low detection rate, PDCoV emerged in each province/region of southern China. In addition, compared to TGEV (1.54 %, 5/390) or Rota C (1.28 %, 6/390), there were highly detection rates of PEDV (22.6 %, 88/390) in those samples. Notably, all five PDCoV-positive piglets were co-infected by PEDV. Furthermore, phylogenetic analysis of spike (S) and nucleocapsid (N) gene sequences of PDCoVs revealed that currently circulating PDCoVs in southern China were more closely related to other Chinese strains of PDCoVs than to those reported in United States, South Korea and Thailand. CONCLUSIONS: This study demonstrated that PDCoV was present in southern China despite the low prevalence, and supported an evolutionary theory of geographical clustering of PDCoVs.


Subject(s)
Coronaviridae Infections/veterinary , Coronaviridae/isolation & purification , Feces/virology , Swine Diseases/virology , Animals , China/epidemiology , Coronaviridae/classification , Coronaviridae/genetics , Coronaviridae Infections/epidemiology , Coronaviridae Infections/virology , Phylogeny , Sequence Analysis, DNA , Swine , Viral Proteins/genetics
18.
Virology ; 497: 185-197, 2016 10.
Article in English | MEDLINE | ID: mdl-27479465

ABSTRACT

Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell ß-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles.


Subject(s)
Coronaviridae Infections/metabolism , Coronaviridae Infections/virology , Coronaviridae/physiology , Spike Glycoprotein, Coronavirus/metabolism , Tubulin/metabolism , Virus Assembly , Virus Replication , Animals , Cell Line , Coronaviridae/drug effects , Gastroenteritis, Transmissible, of Swine/metabolism , Gastroenteritis, Transmissible, of Swine/virology , Humans , Intracellular Space/metabolism , Intracellular Space/virology , Nocodazole/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , Spike Glycoprotein, Coronavirus/chemistry , Swine , Virus Assembly/drug effects , Virus Release , Virus Replication/drug effects
19.
J Virol ; 90(18): 8281-92, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27384656

ABSTRACT

UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of the IFN response. However, the molecular mechanisms by which PEDV antagonizes the antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts IFN signaling components has yet to be elucidated. In this study, we observed that PEDV was relatively resistant to treatment with type I IFN. Western blot analysis showed that STAT1 expression was markedly reduced in PEDV-infected cells and that this reduction was not due to inhibition of STAT1 transcription. STAT1 downregulation was blocked by a proteasome inhibitor but not by an autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is independent of the IFN signaling pathway. Furthermore, we report that PEDV-induced STAT1 degradation inhibits IFN-α signal transduction pathways. Pharmacological inhibition of STAT1 degradation rescued the ability of the host to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation. IMPORTANCE: In this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is the degradation of STAT1 by PEDV infection in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to PEDV replication. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response.


Subject(s)
Antiviral Agents/antagonists & inhibitors , Host-Pathogen Interactions , Immune Evasion , Interferon-alpha/antagonists & inhibitors , Porcine epidemic diarrhea virus/pathogenicity , STAT1 Transcription Factor/antagonists & inhibitors , Animals , Blotting, Western , Cell Line , Chlorocebus aethiops , Coronaviridae Infections , Down-Regulation , Proteolysis , STAT1 Transcription Factor/metabolism , Signal Transduction , Swine
20.
Virus Res ; 226: 142-151, 2016 12 02.
Article in English | MEDLINE | ID: mdl-27397100

ABSTRACT

Porcine enteric coronaviruses (CoVs) cause severe disease in the porcine herds worldwide, leading to important economic losses. Despite the knowledge of these viruses since the 1970s, vaccination strategies have not been implemented, leading to continuous re-emergence of novel virulent strains. Live attenuated vaccines historically have been the most efficient. We consider that the new trend is the development of recombinant vaccines by using reverse genetics systems to engineer attenuated viruses, which could be used as effective and safe modified live vaccine candidates. To this end, host cell signaling pathways influencing porcine CoV virulence should be identified. Similarly, the identity of viral proteins involved in the modulation of host cell pathways influencing CoV pathogenesis should be analyzed. With this information, and using reverse genetics systems, it is possible to design viruses with modifications in the viral proteins acting as virulence factors, which may lead to attenuated viruses and, therefore, vaccine candidates. In addition, novel antiviral drugs may be developed once the host cell pathways and the molecular mechanism affecting porcine CoV replication and virulence are known. This review is focused in the host cell responses to enteric porcine CoV infection and the viral proteins involved in pathogenesis.


Subject(s)
Coronaviridae Infections/veterinary , Coronavirus/immunology , Coronavirus/pathogenicity , Swine Diseases/immunology , Swine Diseases/virology , Viral Vaccines/immunology , Virulence Factors/immunology , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Coronavirus/genetics , Host-Pathogen Interactions/immunology , Immunity, Innate , Swine , Swine Diseases/metabolism , Swine Diseases/prevention & control , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/genetics , Virulence Factors/genetics
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