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1.
Vet Res ; 52(1): 86, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127062

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly discovered swine enteropathogenic coronavirus with worldwide distribution. However, efficient strategies to prevent or treat the infection remain elusive. Our in vitro study revealed that ergosterol peroxide (EP) from the mushroom Cryptoporus volvatus has efficient anti-PDCoV properties. The aim of this study is to evaluate the potential of EP as a treatment for PDCoV in vivo and elucidate the possible mechanisms. Seven-day-old piglets were infected with PDCoV by oral administration in the presence or absence of EP. Piglets infected with PDCoV were most affected, whereas administration of EP reduced diarrhea incidence, alleviated intestinal lesion, and decreased viral load in feces and tissues. EP reduced PDCoV-induced apoptosis and enhanced tight junction protein expressions in the small intestine, maintaining the integrity of the intestinal barrier. EP showed immunomodulatory effect by suppressing PDCoV-induced pro-inflammatory cytokines and the activation of IκBα and NF-κB p65, and upregulating IFN-I expression. Knockdown of p38 inhibited PDCoV replication and alleviated PDCoV-induced apoptosis, implying that EP inhibited PDCoV replication and alleviated PDCoV-induced apoptosis via p38/MAPK signaling pathway. Collectively, ergosterol peroxide can protect piglets from PDCoV, revealing the potential of EP for development as a promising strategy for treating and controlling the infection of PDCoV.


Assuntos
Apoptose/efeitos dos fármacos , Infecções por Coronavirus/veterinária , Deltacoronavirus , Ergosterol/análogos & derivados , Doenças dos Suínos/virologia , Junções Íntimas/efeitos dos fármacos , Animais , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Deltacoronavirus/efeitos dos fármacos , Ergosterol/farmacologia , Ergosterol/uso terapêutico , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/virologia , Células LLC-PK1 , Masculino , Suínos , Doenças dos Suínos/tratamento farmacológico
2.
Vet Microbiol ; 257: 109097, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33933854

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes gastroenteritis in pigs and no vaccines or antiviral drugs are available. Bile acids are active factors in intestines and influence the replication of enteric viruses. Currently, the role of bile acids on PDCoV replication is unknown. In this study, we tested the effects of different types of bile acids on the replication of PDCoV in cell culture. We found that physiological concentrations of bile acids chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) had antiviral activity against PDCoV in porcine kidney cell line (LLC-PK1) and porcine small intestinal epithelial cell line (IPEC-J2). In IPEC-J2 cells, CDCA and LCA inhibited PDCoV replication at post-entry stages by inducing the production of interferon (IFN)-λ3 and IFN-stimulated gene 15 (ISG15) via G protein-coupled receptor (GPCR). In summary, bile acids CDCA and LCA restricted PDCoV infection and LCA functioned through a GPCR-IFN-λ3-ISG15 signaling axis in IPEC-J2 cells. Our results may open new avenues for the development of antiviral drugs to treat PDCoV infection in pigs.


Assuntos
Ácidos e Sais Biliares/farmacologia , Ácido Quenodesoxicólico/farmacologia , Deltacoronavirus/fisiologia , Ácido Litocólico/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Ácidos e Sais Biliares/química , Deltacoronavirus/efeitos dos fármacos , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Interferons/imunologia , Células LLC-PK1 , Suínos , Doenças dos Suínos/virologia
3.
Vet Microbiol ; 257: 109074, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940460

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg2+ and Mn2+ were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing antiviral drugs.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Vírus da Diarreia Epidêmica Suína/enzimologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , RNA Viral/genética , RNA Viral/metabolismo , Suínos , Doenças dos Suínos/virologia , Células Vero
4.
Arch Virol ; 166(7): 1951-1959, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33987752

RESUMO

A novel circovirus designated "porcine circovirus type 4" (PCV4) was recently reported in pigs with severe clinical disease in Hunan Province, China. Relatively little is known about the molecular epidemiology of this recently discovered virus. In order to assess the prevalence of PCV4 infection in pigs and to analyze its genomic characteristics, 1683 clinical samples were collected in Inner Mongolia, China, from 2016 to 2018. The overall infection rate of PCV4 was 1.6% (27/1683) at the sample level and 21.6% (11/51) at the farm level, with rates ranging from 3.2% (1/31) to 20.0% (6/30) on different PCV4-positive pig farms. In addition, the PCV4 infection rates at both the sample and farm level increased from 2016 to 2018. This also showed that PCV4 was present in pigs in 2016 in China and therefore did not arrive later than this date. Additionally, our findings showed that PCV4 infections had no association with PCV2 or PCV3 infections. We sequenced the complete genomes of three PCV4 strains and found that the PCV4 strains had a high degree of genetic stability but shared less than 80% sequence identity with other circoviruses. We identified six amino acid mutations in the Rep protein and seven in the Cap protein. Phylogenetic analysis based on Cap and Rep sequences confirmed that the PCV4 strains grouped in an independent branch. Our findings provide important information about the prevalence and genetic characteristics of PCV4 strains.


Assuntos
Infecções por Circoviridae/epidemiologia , Circovirus/genética , Doenças dos Suínos/epidemiologia , Animais , China/epidemiologia , Infecções por Circoviridae/virologia , Fazendas , Genoma Viral/genética , Genômica/métodos , Epidemiologia Molecular/métodos , Filogenia , Prevalência , Estudos Retrospectivos , Suínos , Doenças dos Suínos/virologia
5.
Viruses ; 13(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922604

RESUMO

Swine enteric viral infections are responsible for substantial economic losses in the pork industry worldwide. Porcine epidemic diarrhea (PEDV) is one of the main causative agents of diarrhea in lactating pigs, and reports of PEDV coinfection with other enteric viruses highlight the importance of viral interactions for disease presentation and outcomes. Using next-generation sequencing (NGS) and sequence analyses from samples taken from piglets with acute diarrhea, we explored the possible interactions between PEDV and other less reported pathogens. PEDV coinfection with porcine kobuvirus (PKV) was detected in 36.4% (27/74) of samples. Full genomes from porcine coronavirus and kobuvirus were obtained, as was a partial porcine sapovirus genome (PSaV). The phylogenetic results show the clustering of these strains corresponding to the geographical relationship. To our knowledge, this is the first full genome and isolation report for porcine kobuvirus in México, as well as the first phylogenetic analysis for porcine sapovirus in the country. The NGS approach provides a better perspective of circulating viruses and other pathogens in affected production units.


Assuntos
Coinfecção/virologia , Infecções por Coronavirus/virologia , Kobuvirus/genética , Kobuvirus/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , Coinfecção/epidemiologia , Infecções por Coronavirus/epidemiologia , Diarreia/virologia , Fezes/virologia , Genoma Viral , Kobuvirus/classificação , México/epidemiologia , Técnicas de Diagnóstico Molecular , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Sapovirus/genética , Análise de Sequência , Suínos , Doenças dos Suínos/virologia
6.
Vet Microbiol ; 257: 109068, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33894664

RESUMO

Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus (CoV) that continues to spread globally, placing strain on economic and public health. Currently, the pathogenic mechanism of PDCoV remains largely unclear, and effective strategies to prevent or treat PDCoV infection are still limited. In this study, the interaction between autophagy and PDCoV replication in LLC-PK1 cells was investigated. We demonstrated that PDCoV infection induced a complete autophagy process. Pharmacologically induced autophagy with rapamycin increased the expression of PDCoV N, while pharmacologically inhibited autophagy with wortmannin decreased the expression of PDCoV N, suggesting that PDCoV-induced autophagy facilitates virus replication. Further experiments showed that PDCoV infection activated p38 signaling pathway to trigger autophagy. Besides, ergosterol peroxide (EP) alleviated PDCoV-induced activation of p38 to suppress autophagy, thus exerting its antiviral effects. Finally, we employed a piglet model of PDCoV infection to demonstrate that EP prevented PDCoV infection by suppressing PDCoV-induced autophagy via p38 signaling pathway in vivo. Collectively, these findings accelerate the understanding of the pathogenesis of PDCoV infection and provide new insights for the development of EP as an effective therapeutic strategy for PDCoV.


Assuntos
Antivirais/farmacologia , Autofagia , Infecções por Coronavirus/veterinária , Deltacoronavirus/efeitos dos fármacos , Ergosterol/análogos & derivados , Sistema de Sinalização das MAP Quinases , Replicação Viral/efeitos dos fármacos , Animais , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Deltacoronavirus/fisiologia , Ergosterol/farmacologia , Células LLC-PK1 , Suínos , Doenças dos Suínos/virologia
7.
Vet Microbiol ; 257: 109081, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33901803

RESUMO

As the most abundant cell type in the blood, red blood cells (RBCs) are serving for transporting oxygen. However, the mechanism by which RBCs binding virus remains largely unknown. Here, we demonstrated that porcine epidemic diarrhea virus (PEDV), a kind of coronavirus, could hijack RBCs and cause typical diarrhea in neonatal piglets. In an epidemiology investigation of PEDV, the RBCs samples from diarrheic pigs in several pig farms were found to be PEDV-positive. PEDV could bind to neonatal RBCs through CD71 and clathrin-mediated endocytosis, and its viability was maintained for 12 h. PEDV-loaded RBCs could transfer the virus to CD3+ T cells by conjugation and reach the intestine mucosa, where it caused infection. Finally, a further animal challenge revealed that transfusing with PEDV-loaded RBCs could cause intestinal epithelial cells (IECs) infection and typical diarrhea symptom. Therefore, our studies illustrated the mechanism by which PEDV could cause intestinal infection through hijacking RBCs, further providing a novel insight into the role of RBCs as potential cells for viral transmission in coronavirus pathogenesis.


Assuntos
Transfusão de Sangue/veterinária , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Diarreia/veterinária , Eritrócitos/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/transmissão , Animais , Animais Recém-Nascidos , Chlorocebus aethiops , Diarreia/virologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Suínos , Doenças dos Suínos/virologia , Linfócitos T/virologia , Células Vero , Ligação Viral
8.
Arch Virol ; 166(7): 1859-1867, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33876315

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes emaciation and watery diarrhea in pigs. First identified in Europe in 1977, it eventually spread to Asia and North America, causing deadly outbreaks in neonatal piglets. In the Philippines, PEDV has caused several recorded outbreaks since 2005. However, DNA sequencing studies of local PEDV strains remain few and are limited to gene and gene fragment sequencing. Therefore, to provide updated sequence information about recent PEDV strains in the country, we performed reverse transcription PCR and sequencing of PEDV from swab samples collected from swine farms in the Philippines in 2017. Here, we report the first published whole genome sequence of PEDV from the Philippines as well as CO-26K equivalent (COE) domain sequences of strains from three provinces in Luzon where PEDV was detected in 2017. Sequence analysis suggested that PEDV from both the classical (genotype 1) and pandemic (genotype 2) groups are present in the Philippines, with possible East Asian and North American origins.


Assuntos
Infecções por Coronavirus/virologia , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/virologia , Animais , Ásia , Surtos de Doenças/veterinária , Europa (Continente) , Fazendas , Genoma Viral/genética , América do Norte , Filipinas , Filogenia , Análise de Sequência de DNA/métodos , Suínos
9.
Res Vet Sci ; 136: 535-539, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33882382

RESUMO

African swine fever (ASF) is one of the most devastating hemorrhagic infectious diseases that affect pigs and wild suids due to the lack of a vaccine or an effective treatment. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs that are predicted to encode proteins. Since its introduction to China in 2018, this genome has aroused the enthusiasm of researchers throughout the world. Here, we review the research progress on ASFV in recent years. Given the importance of this disease, this review will highlight recent discoveries in basic virology, focusing mainly on epidemiology, virulence, pathogenic mechanisms, diagnosis, vaccine development, and treatment; this will help in understanding virus-host interactions and disease prevention regarding ASFV.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana/epidemiologia , Doenças dos Suínos/epidemiologia , Animais , China/epidemiologia , Suínos , Doenças dos Suínos/virologia
10.
Viruses ; 13(4)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808275

RESUMO

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. Owing to the lack of effective vaccines and specific therapeutic options for PEDV, it is pertinent to develop new and available antivirals. This study identified, for the first time, a salinomycin that actively inhibited PEDV replication in Vero cells in a dose-dependent manner. Furthermore, salinomycin significantly inhibited PEDV infection by suppressing the entry and post-entry of PEDV in Vero cells. It did not directly interact with or inactivate PEDV particles, but it significantly ameliorated the activation of Erk1/2, JNK and p38MAPK signaling pathways that are associated with PEDV infection. This implied that salinomycin inhibits PEDV replication by altering MAPK pathway activation. Notably, the PEDV induced increase in reactive oxidative species (ROS) was not decreased, indicating that salinomycin suppresses PEDV replication through a pathway that is an independent pathway of viral-induced ROS. Therefore, salinomycin is a potential drug that can be used for treating PEDV infection.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Piranos/farmacologia , Doenças dos Suínos/virologia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Sistema de Sinalização das MAP Quinases , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
11.
Front Immunol ; 12: 584299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746943

RESUMO

Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus.


Assuntos
Quitosana/imunologia , Imunidade/imunologia , Vacinas contra Influenza/imunologia , Manose/imunologia , Infecções por Orthomyxoviridae/imunologia , Doenças dos Suínos/imunologia , Animais , Anticorpos Antivirais/imunologia , Células Cultivadas , Quitosana/metabolismo , Cães , Feminino , Imunidade/efeitos dos fármacos , Vacinas contra Influenza/administração & dosagem , Células Madin Darby de Rim Canino , Manose/metabolismo , Nanopartículas/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Gravidez , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinação/métodos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
12.
J Vet Diagn Invest ; 33(3): 457-468, 2021 May.
Artigo em Inglês | MEDLINE | ID: covidwho-1264088

RESUMO

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.


Assuntos
Infecções por Coronaviridae/veterinária , Coronaviridae/isolamento & purificação , Laboratórios/normas , Doenças dos Suínos/virologia , Animais , Teste para COVID-19/veterinária , Infecções por Coronaviridae/diagnóstico , Infecções por Coronaviridae/virologia , Surtos de Doenças , Fezes/virologia , Padrões de Referência , Estações do Ano , Suínos , Doenças dos Suínos/diagnóstico
13.
mBio ; 12(2)2021 03 30.
Artigo em Inglês | MEDLINE | ID: covidwho-1160040

RESUMO

Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection.IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.


Assuntos
Cavéolas/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Vírus da Diarreia Epidêmica Suína/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Membrana Celular/virologia , Chlorocebus aethiops , Infecções por Coronavirus , Suínos , Doenças dos Suínos/virologia , Células Vero
14.
Arch Virol ; 166(6): 1723-1728, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33721098

RESUMO

Porcine circovirus 2 (PCV-2) is the causative agent of porcine circovirus diseases (PCVD). A study was undertaken to determine whether PCV-2 was present in samples collected from commercial pigs (n = 46) and warthogs (n = 42) in Namibia between 2019 and 2020. Twenty-three of the collected samples were positive by PCR (13 from pigs and 10 from warthogs), and a phylogenetic analysis of ORF2 identified three genotypes (PCV-2b and PCV-2d in pigs and PCV-2c in warthogs). This is the first time that PCV-2 has been identified in warthogs and in Namibia. It is also the first report of PCV-2c in Africa.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Genótipo , Doenças dos Suínos/virologia , Animais , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Namíbia/epidemiologia , Filogenia , Suínos , Doenças dos Suínos/epidemiologia
15.
Mol Immunol ; 134: 86-99, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33740580

RESUMO

Porcine deltacoronavirus (PDCoV), an emerging porcine enteropathogenic coronavirus, causes acute watery diarrhea and vomiting in piglets. Here, we isolated a strain of PDCoV from intestinal content of a piglet with severe watery diarrhea on a farm located in Henan Province, named PDCoV strain HNZK-02. Subsequently, the complete genomes of cell-cultured PDCoV HNZK-02 passage 5 and 15 were sequenced and analyzed. There was a continuous 3-nucleotide deletion and 7 amino acid changes in S genes when compared with the other reported PDCoVs. RNA sequencing (RNA-seq)-based transcriptome analysis was used to quantitatively identify differentially expressed genes after PDCoV infection in ST cells. In total, 523 differentially expressed genes (DEGs) were identified, including 62 upregulated genes and 457 downregulated genes. The 62 upregulated genes were associated with TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, IL-17 signaling, chemokine signaling pathway and NF-κB signaling pathway. The significant expressing changed genes, including three antiviral genes (Mx1, OASL, OAS1) and three inflammatory chemokine related genes (CCL5, CXCL8, CXCL10) were further validated using quantitative real-time RT-PCR (qRT-PCR) assay. It showed the consistent expression patterns of the candidate genes with those from RNA-seq. Our results demonstrated that PDCoV infection activates NF-κB signaling pathway and leads to the expression of inflammatory factors, which may be related to TLRs but TLR2 is not a critical factor.In general, these results can help us to confirm the molecular regulation mechanism and also provide us a comprehensive resource of PDCoV infection.


Assuntos
Infecções por Coronavirus/veterinária , Deltacoronavirus/genética , Gastroenteropatias/veterinária , Gastroenteropatias/virologia , Genoma Viral/genética , Animais , China , Infecções por Coronavirus/virologia , Deltacoronavirus/isolamento & purificação , Gastroenteropatias/patologia , Perfilação da Expressão Gênica , Transdução de Sinais/genética , Suínos , Doenças dos Suínos/virologia , Transcriptoma/genética
16.
J Vet Diagn Invest ; 33(3): 457-468, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33739188

RESUMO

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.


Assuntos
Infecções por Coronaviridae/veterinária , Coronaviridae/isolamento & purificação , Laboratórios/normas , Doenças dos Suínos/virologia , Animais , Teste para COVID-19/veterinária , Infecções por Coronaviridae/diagnóstico , Infecções por Coronaviridae/virologia , Surtos de Doenças , Fezes/virologia , Padrões de Referência , Estações do Ano , Suínos , Doenças dos Suínos/diagnóstico
17.
Viruses ; 13(2)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33671997

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen's kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Infecções por Coronavirus/veterinária , Ensaio de Imunoadsorção Enzimática , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Baculoviridae/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Proteínas Recombinantes/imunologia , Spodoptera , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Células Vero
18.
mBio ; 12(2)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785615

RESUMO

Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection.IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.


Assuntos
Cavéolas/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Vírus da Diarreia Epidêmica Suína/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Membrana Celular/virologia , Chlorocebus aethiops , Infecções por Coronavirus , Suínos , Doenças dos Suínos/virologia , Células Vero
19.
Arch Virol ; 166(5): 1355-1370, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33709216

RESUMO

Porcine teschovirus (PTV) is a causative agent of reproductive disorders, encephalomyelitis, respiratory diseases, and diarrhea in swine, with a worldwide distribution. In this work, we identified PTV-associated nonsuppurative encephalitis as a potential cause of posterior paralysis in neonatal pigs in northeast China. Using indirect immunofluorescence assay, western blot, electron microscopy, and genome sequencing, we identified a neurotropic PTV strain, named CHN-NP1-2016, in the supernatants of pooled cerebrum and cerebellum samples from an affected piglet. Nucleotide sequence alignment revealed that the whole genome of CHN-NP1-2016 shared the highest sequence similarity (86.76% identity) with PTV 1 strain Talfan. A combination of phylogenetic and genetic divergence analysis was applied based on the deduced amino acid sequence of the P1 gene with a cutoff value of the genetic distance (0.102 ± 0.008) for defining PTV genotypes, and this showed that CHN-NP1-2016 is a variant of genotype 1. In total, 16 unique mutations and five mutant clusters were detected in the capsid proteins VP1 and VP2 of CHN-NP1-2016 when compared to other PTV1 isolates. Importantly, we detected three mutant clusters located in the exposed surface loops of the capsid protein, potentially indicating significant differences in major neutralization epitopes. Moreover, a potential recombination event in the P1 region of PTV CHN-NP1-2016 was detected. These findings provide valuable insights into the role of recombination in the evolution of teschoviruses. To our knowledge, this is the first case report of PTV-1-associated encephalitis in northeast China. Future investigations will narrow on the serology and pathogenicity of this novel isolate.


Assuntos
Encefalite Viral/veterinária , Infecções por Picornaviridae/veterinária , Doenças dos Suínos/virologia , Teschovirus/genética , Teschovirus/isolamento & purificação , Animais , Encéfalo/virologia , China/epidemiologia , Encefalite Viral/patologia , Encefalite Viral/virologia , Genoma Viral/genética , Genótipo , Mutação , Filogenia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , RNA Viral/genética , Recombinação Genética , Suínos , Teschovirus/classificação , Proteínas Virais/genética
20.
Arch Virol ; 166(5): 1463-1468, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33718993

RESUMO

Porcine circovirus 3 (PCV3) is a recently emerged circovirus discovered in 2016 that has drawn the attention of the swine industry worldwide. In this study, we evaluated the genetic diversity of PCV3 strains on pig farms. A total of 261 samples from sows, weaning pigs, growing pigs, and stillborn/mummified fetuses were analyzed by quantitative real-time PCR. The results revealed that at least two main lineages of PCV3 are circulating in Brazil. For the first time, it was possible to detect the presence of two different PCV3 strains in the same host.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Coinfecção/veterinária , Doenças dos Suínos/virologia , Animais , Brasil/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/isolamento & purificação , Coinfecção/virologia , DNA Viral/genética , Fazendas , Variação Genética , Genótipo , Fases de Leitura Aberta/genética , Filogenia , Suínos , Carga Viral
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