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1.
J Med Microbiol ; 69(9): 1183-1196, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32812860

RESUMO

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms.Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization.Methodology. Based on the non-structural Rep' protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets.Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep' antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep' antibody-positive. The combined detection results for the Rep' iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep' antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep' antibody-positive.Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Circoviridae/sangue , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Suínos/sangue , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Infecções por Circoviridae/imunologia , Circovirus/genética , Imunização , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
2.
Methods Mol Biol ; 2203: 77-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833205

RESUMO

Porcine deltacoronavirus (PDCoV) has emerged as a novel, contagious swine enteric coronavirus that causes watery diarrhea and/or vomiting and intestinal villous atrophy in nursing piglets. PDCoV-related diarrhea first occurred in the USA in 2014 and was subsequently reported in South Korea, China, Thailand, Vietnam, and Lao People's Democratic Republic, leading to massive economic losses and posing a threat to the swine industry worldwide. Currently, no treatments or vaccines for PDCoV are available. The critical step in the development of potential vaccines against PDCoV infection is the isolation and propagation of PDCoV in cell culture. This chapter provides a detailed protocol for isolation and propagation of PDCoV in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures supplemented with pancreatin and trypsin, respectively. Filtered clinical samples (swine intestinal contents or feces) applied to ST or LLC-PK cells produce cytopathic effects characterized by rounding, clumping, and detachment of cells. PDCoV replication in cells can be quantifiably monitored by qRT-PCR, immunofluorescence assays, and immune-electron microscopy. Infectious viral titers can be evaluated by using plaque assays or 50% tissue culture infectious dose (TCID50) assays. The ST or LLC-PK cells efficiently supported serial passage and propagation of PDCoV. After serial passage of PDCoV in either ST or LLC-PK cells, the virus can be purified further in ST cells by plaque assays.


Assuntos
Coronavirus/isolamento & purificação , Doenças dos Suínos/virologia , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Fezes/virologia , Inoculações Seriadas , Suínos
3.
Virology ; 548: 59-72, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838947

RESUMO

Methylation of the N6 position of adenosine (m6A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m6A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m6A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m6A in host RNA and decrease the expression of FTO. The m6A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m6A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m6A modification in PEDV replication and interactions with the host.


Assuntos
Adenosina/análogos & derivados , Infecções por Coronavirus/veterinária , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , Adenosina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral , Metilação , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Ligação Proteica , RNA Viral , Proteínas de Ligação a RNA/metabolismo , Suínos , Células Vero
4.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
5.
Vet Microbiol ; 247: 108785, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32768229

RESUMO

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Interferons/antagonistas & inibidores , Animais , Linhagem Celular , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/imunologia , Interferons/imunologia , Intestinos/citologia , Intestinos/virologia , Rim/citologia , Rim/virologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia
6.
PLoS Pathog ; 16(7): e1008682, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730327

RESUMO

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of newborn piglets causing acute, severe atrophic enteritis. The underlying mechanisms of PEDV infection and the reasons why newborn piglets are more susceptible than older pigs remain incompletely understood. Iron deficiency is common in newborn piglets. Here we found that high levels of transferrin receptor 1 (TfR1) distributed in the apical tissue of the intestinal villi of newborns, and intracellular iron levels influence the susceptibility of newborn piglets to PEDV. We show that iron deficiency induced by deferoxamine (DFO, an iron chelating agent) promotes PEDV infection while iron accumulation induced by ferric ammonium citrate (FAC, an iron supplement) impairs PEDV infection in vitro and in vivo. Besides, PEDV infection was inhibited by occluding TfR1 with antibodies or decreasing TfR1 expression. Additionally, PEDV infection was increased in PEDV-resistant Caco-2 and HEK 293T cells over-expressed porcine TfR1. Mechanistically, the PEDV S1 protein interacts with the extracellular region of TfR1 during PEDV entry, promotes TfR1 re-localization and clustering, then activates TfR1 tyrosine phosphorylation mediated by Src kinase, and heightens the internalization of TfR1, thereby promoting PEDV entry. Taken together, these data suggest that the higher expression of TfR1 in the apical tissue of the intestinal villi caused by iron deficiency, accounts for newborn piglets being acutely susceptible to PEDV.


Assuntos
Infecções por Coronavirus/veterinária , Suscetibilidade a Doenças/metabolismo , Mucosa Intestinal/metabolismo , Vírus da Diarreia Epidêmica Suína , Receptores da Transferrina/metabolismo , Doenças dos Suínos/metabolismo , Animais , Animais Recém-Nascidos , Suscetibilidade a Doenças/virologia , Ferro/deficiência , Suínos , Doenças dos Suínos/virologia
7.
Proc Natl Acad Sci U S A ; 117(29): 17204-17210, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32601207

RESUMO

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.


Assuntos
Genes Virais , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Animais , China , Reações Cruzadas , Células Epiteliais/virologia , Variação Genética , Genótipo , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Influenza Humana/imunologia , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/transmissão , Pandemias , Filogenia , Prevalência , Vírus Reordenados/genética , Estudos Soroepidemiológicos , Suínos
8.
Arch Virol ; 165(10): 2323-2333, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32715325

RESUMO

To investigate the epidemic characteristics of porcine epidemic diarrhea virus (PEDV), 135 clinical samples (including intestinal tissues and feces) were collected from diseased piglets during outbreaks of diarrhea from 2015 to 2019 on farms in Henan and Shanxi provinces of China where swine had been immunized with attenuated PEDV (CV777). A total of 86 clinical samples (86/135, 63.7%) were positive for PEDV by RT-PCR, and subsequently, the complete spike (S) and ORF3 genes of 32 PEDV samples were sequenced. Phylogenetic analysis showed that the 32 PEDV strains obtained in this study belonged to group 2 (pandemic variant strains) and had a close relationship to 17 Chinese strains after 2010, two South Korean strains (KNU-1305 and KNU-1807), three American strains (PC22A-P140.BI, USA/Colorado/2013, and USA/OK10240-6/2017) and a Mexican strain (PEDV/MEX/QRO/02/2017), but differed genetically from a South Korean strain (SM98), a European strain (Br1/87), a Chinese strain (LZC), and a vaccine strain (CV777). G2-a subgroup strains were the dominant pandemic variant strains circulating in Henan and Shanxi provinces of China. Furthermore, a cross-recombination event was identified in the S region of the SX/TY2/2017 strain, and the putative parental strains were the epidemic strains CH/GDGZ/2012 and CH/YZ1/2015, identified in China in 2012 and 2015, respectively. These results provide further information about PEDV evolution, which could improve our understanding of the circulation of PEDV in Henan and Shanxi provinces. This information will also be helpful for developing new strategies for prevention and control of variant strains.


Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Surtos de Doenças , Genoma Viral , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/epidemiologia , Proteínas Virais/genética , Animais , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Diarreia/epidemiologia , Diarreia/virologia , Fazendas , Fezes/virologia , Variação Genética , Intestinos/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Recombinação Genética , Suínos/virologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia
9.
PLoS One ; 15(7): e0235660, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667952

RESUMO

Transmission network modelling to infer 'who infected whom' in infectious disease outbreaks is a highly active area of research. Outbreaks of foot-and-mouth disease have been a key focus of transmission network models that integrate genomic and epidemiological data. The aim of this study was to extend Lau's systematic Bayesian inference framework to incorporate additional parameters representing predominant species and numbers of animals held on a farm. Lau's Bayesian Markov chain Monte Carlo algorithm was reformulated, verified and pseudo-validated on 100 simulated outbreaks populated with demographic data Japan and Australia. The modified model was then implemented on genomic and epidemiological data from the 2010 outbreak of foot-and-mouth disease in Japan, and outputs compared to those from the SCOTTI model implemented in BEAST2. The modified model achieved improvements in overall accuracy when tested on the simulated outbreaks. When implemented on the actual outbreak data from Japan, infected farms that held predominantly pigs were estimated to have five times the transmissibility of infected cattle farms and be 49% less susceptible. The farm-level incubation period was 1 day shorter than the latent period, the timing of the seeding of the outbreak in Japan was inferred, as were key linkages between clusters and features of farms involved in widespread dissemination of this outbreak. To improve accessibility the modified model has been implemented as the R package 'BORIS' for use in future outbreaks.


Assuntos
Doenças dos Bovinos/transmissão , Febre Aftosa/transmissão , Doenças dos Suínos/transmissão , Animais , Austrália/epidemiologia , Teorema de Bayes , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/virologia , Surtos de Doenças , Fazendas , Febre Aftosa/epidemiologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/isolamento & purificação , Japão/epidemiologia , Cadeias de Markov , Método de Monte Carlo , Filogenia , Quarentena/veterinária , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia
10.
PLoS One ; 15(7): e0235832, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706778

RESUMO

Porcine circovirus type 3 (PCV3) is a newly emerging virus in the swine industry, first reported recently in 2016. PCV3 assembles into a 2000 bp circular genome; slightly larger than PCV1 (1758-1760 bp), PCV2 (1766-1769 bp) and PCV4 (1770 bp). Apart from being associated with porcine dermatitis and nephropathy syndrome (PDNS), PCV3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (PRDC) and neurologic disease. Given that PCV3 is increasingly reported in countries including Thailand and U.S. with whom Malaysia shares trade and geographical relationship; and that PCV3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of PCV3 in Malaysian swine farms. Twenty-four commercial swine farms, three abattoirs and retail shops in Peninsular Malaysia were sampled using convenience sampling method. A total of 281 samples from 141 pigs, including 49 lung archive samples were tested for PCV3 by conventional PCR. Twenty-eight lung samples from wild boar population in Peninsular Malaysia were also included. Nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. Results revealed that PCV3 is present in Peninsular Malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. Despite wide reports of PCV3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. PCV3 strain A1 and A2 were present in Malaysia, and Malaysian PCV3 strains were found to be phylogenetically related to Spanish, U.S. and Mexico strains.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Genoma Viral , Doenças dos Suínos/virologia , Suínos/virologia , Animais , Sequência de Bases , Infecções por Circoviridae/virologia , Circovirus/isolamento & purificação , DNA Viral/genética , Dermatite/veterinária , Dermatite/virologia , Nefropatias/veterinária , Nefropatias/virologia
11.
PLoS One ; 15(7): e0230257, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658910

RESUMO

Hepatitis E virus is a zoonotic pathogen for which pigs are recognized as the major reservoir in industrialised countries. A multiscale model was developed to assess the HEV transmission and persistence pattern in the pig production sector through an integrative approach taking into account within-farm dynamics and animal movements based on actual data. Within-farm dynamics included both demographic and epidemiological processes. Direct contact and environmental transmission routes were considered along with the possible co-infection with immunomodulating viruses (IMVs) known to modify HEV infection dynamics. Movements were limited to 3,017 herds forming the largest community on the swine commercial network in France and data from the national pig movement database were used to build the contact matrix. Between-herd transmission was modelled by coupling within-herd and network dynamics using the SimInf package. Different introduction scenarios were tested as well as a decrease in the prevalence of IMV-infected farms. After introduction of a single infected gilt, the model showed that the transmission pathway as well as the prevalence of HEV-infected pigs at slaughter age were affected by the type of the index farm, the health status of the population and the type of the infected farms. These outcomes could help design HEV control strategies at a territorial scale based on the assessment of the farms' and network's risk.


Assuntos
Hepatite E/patologia , Doenças dos Suínos/patologia , Animais , Cruzamento , Bases de Dados Factuais , França/epidemiologia , Nível de Saúde , Hepatite E/epidemiologia , Hepatite E/transmissão , Modelos Logísticos , Dinâmica Populacional , Prevalência , Modelos de Riscos Proporcionais , Análise Espaço-Temporal , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia
12.
Virus Res ; 286: 198045, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502552

RESUMO

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. Two different genogroups of PEDV, S INDEL [PEDV variant containing multiple deletions and insertions in the S1 subunit of the spike (S) protein, G1b] and non-S INDEL (G2b) strains were detected during the diarrheal disease outbreak in US swine in 2013-2014. Similar viruses are also circulating globally. Continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent PEDV infections worldwide. Although the non-S INDEL PEDV was highly virulent and the S INDEL PEDV caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. The main PEDV transmission route is fecal-oral, but airborne transmission via the fecal-nasal route may play a role in pig-to-pig and farm-to-farm spread. PEDV infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of PEDV RNA in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. PEDV-specific IgA effector and memory B cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. This review focuses on the etiology, transmission, pathogenesis, and prevention and control of PEDV infection.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/transmissão , Mucosa Intestinal/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/transmissão , Aerossóis , Animais , Infecções por Coronavirus/prevenção & controle , Progressão da Doença , Imunidade Humoral/imunologia , Mucosa Intestinal/patologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Receptores Virais/metabolismo , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Tropismo Viral/fisiologia , Viremia/sangue
13.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32554695

RESUMO

Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.


Assuntos
Infecções por Coronavirus/veterinária , Exocitose , Interações Hospedeiro-Patógeno , Fases de Leitura Aberta , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Engenharia Genética , Redes e Vias Metabólicas , Plasmídeos/genética , Transporte Proteico , Proteômica , Suínos , Proteínas Virais/química , Proteínas Virais/genética
14.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32554697

RESUMO

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.


Assuntos
Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Interferons/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Vacinas Atenuadas/imunologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Betacoronavirus/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/prevenção & controle , Diarreia/patologia , Diarreia/virologia , Modelos Animais de Doenças , Endorribonucleases/antagonistas & inibidores , Fezes/virologia , Íleo/patologia , Imunidade Inata , Jejuno/patologia , Pandemias , Pneumonia Viral/imunologia , Vírus da Diarreia Epidêmica Suína/genética , RNA Replicase , RNA Viral , Suínos , Doenças dos Suínos/virologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
15.
Arch Virol ; 165(9): 1969-1977, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32529459

RESUMO

Porcine epidemic diarrhea (PED) is an acute enteric disease caused by porcine epidemic diarrhea virus (PEDV). In China, variant PEDV causes severe watery diarrhea, vomiting, and dehydration in piglets, leading to very high morbidity and mortality. However, the pathogenesis of PEDV is still not fully understood. In our study, we analyzed the long noncoding RNA (lncRNA) and mRNA expression profiles of PEDV GDgh16 in infected Vero cells at 60 h postinfection. A total of 61,790 annotated mRNAs, 14,247 annotated lncRNAs and 1290 novel lncRNAs were identified. A total of 227 annotated lncRNAs and 13 novel lncRNAs were significantly and differentially expressed after viral infection. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used to identify genes adjacent to the lncRNAs, and it was found that these lncRNAs were enriched in pathways related to immune and antiviral responses. Next, we selected candidate lncRNAs and their predicted target genes for study. RT-qPCR demonstrated that these lncRNAs and genes were differentially expressed after PEDV infection. Our study investigated the function of lncRNAs involved in PEDV infection, providing new insight into the pathogenic mechanisms of PEDV.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , RNA Longo não Codificante/genética , Doenças dos Suínos/genética , Animais , China , Chlorocebus aethiops , Infecções por Coronavirus/genética , Genoma , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Doenças dos Suínos/virologia , Células Vero
16.
Arch Virol ; 165(9): 2003-2011, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32594321

RESUMO

Porcine circovirus 3 (PCV3) is a recently identified virus that is associated with reproductive failure, porcine dermatitis and nephropathy syndrome, and multi-systemic inflammation. To investigate the molecular epidemic characteristics and genetic evolution of PCV3 in northern China, a commercial TaqMan-based real-time quantitative PCR kit was used to detect PCV3 in 435 tissue specimens collected from pigs with various clinical signs from 105 different swine farms in northern China. The results showed that 48 out of 105 (45.7%) farms and 97 out of 435 (22.3%) samples tested positive for PCV3. Of the 97 PCV3-positive samples, 80 (82.5%) tested positive for other pathogens. PCV3 was found more frequently in pigs with reproductive failure than in those with other clinical signs. This study is the first to detect PCV3 in Tianjin. The complete genome sequences of six PCV3 isolates and the capsid (Cap) protein gene sequences of 11 isolates were determined. Based on the predicted amino acids at positions 24 and 27 of the Cap protein and their evolutionary relationships, the 17 PCV3 strains obtained from northern China and 49 reference strains downloaded from the GenBank database were divided into four major groups (3a-3d). An analysis of selection pressure and polymorphism indicated that the PCV3 Cap protein seems to be evolving under balancing selection, that the population is in dynamic equilibrium, and that no population expansion occurred during the study period. Our results provide new information about the molecular epidemiology and evolution of PCV3.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/classificação , Circovirus/isolamento & purificação , Filogenia , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , China/epidemiologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/genética , Epidemiologia Molecular , Suínos , Doenças dos Suínos/epidemiologia
17.
J Clin Microbiol ; 58(8)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522830

RESUMO

Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Doenças dos Bovinos/fisiopatologia , Infecções por Coronavirus/veterinária , Coronavirus Bovino/crescimento & desenvolvimento , Pneumonia Viral/fisiopatologia , Infecções Respiratórias/veterinária , Doenças dos Suínos/fisiopatologia , Animais , Betacoronavirus/patogenicidade , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Coronavirus Bovino/patogenicidade , Especificidade de Hospedeiro , Humanos , Pandemias , Pneumonia Viral/patologia , Coronavirus Respiratório Porcino/crescimento & desenvolvimento , Coronavirus Respiratório Porcino/patogenicidade , Infecções Respiratórias/patologia , Infecções Respiratórias/fisiopatologia , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Tropismo Viral
18.
Virus Res ; 285: 198024, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32482591

RESUMO

Discovered in 2017, swine enteric alphacoronavirus (SeACoV), also known as swine acute diarrhea syndrome coronavirus (SADS-CoV) or porcine enteric alphacoronavirus (PEAV), is the fifth porcine CoV identified in diarrheal piglets. The presumed name "SADS-CoV" may not be appropriate since current studies have not provided strong evidence for high pathogenicity of the virus. SeACoV was the most recently recognized CoV of potential bat origin prior to the novel human severe acute respiratory syndrome CoV 2 (SARS-CoV-2), associated with the pandemic CoV disease 2019 (COVID-19). Although SeACoV is recognized as a regional epizootic virus currently, it possesses the most extensive cell species tropism in vitro among known CoVs. This review summarizes the emergence of SeACoV and updates the research progress made from 2017 to early 2020, mainly focusing on the etiology, epidemiology, evolutionary perspective, potential for interspecies transmission, pathogenesis and diagnosis.


Assuntos
Alphacoronavirus , Infecções por Coronavirus/veterinária , Doenças dos Suínos/virologia , Alphacoronavirus/genética , Alphacoronavirus/patogenicidade , Alphacoronavirus/ultraestrutura , Animais , Linhagem Celular , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Genoma Viral , Humanos , Epidemiologia Molecular , Especificidade da Espécie , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Tropismo Viral
19.
Arch Virol ; 165(7): 1653-1658, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: covidwho-245259

RESUMO

Although porcine deltacoronavirus (PDCoV) is a significant pandemic threat in the swine population and has caused significant economic losses, information regarding the immune response in conventionally weaned pigs infected with PDCoV is scarce. Hence, the immune response in conventionally weaned pigs infected with PDCoV was assessed after challenge and rechallenge. After the first challenge, obvious diarrhea and viral shedding developed successively in all pigs in the four inoculation dose groups from 3 to 14 days postinfection (dpi), and all pigs recovered (no clinical symptoms or viral shedding) by 21 dpi. All pigs in the four groups exhibited significantly increased PDCoV-specific IgG, IgA and virus-neutralizing (VN) antibody (Ab) titers and IFN-γ levels in the serum after the first challenge. All pigs were completely protected against rechallenge at 21 dpi. The serum levels of PDCoV-specific IgG, IgA, and VN Abs increased further after rechallenge. Notably, the IFN-γ level declined continuously after 7 dpi. In addition, the levels of PDCoV-specific IgG, IgA and VN Abs in saliva increased significantly after rechallenge and correlated well with the serum Ab titers. Furthermore, the appearance of clinical symptoms of PDCoV infection in conventionally weaned pigs was delayed with reduced inoculation doses. In summary, the data presented here offer important reference information for future PDCoV animal infection and vaccine-induced immunoprotection experiments.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/fisiologia , Doenças dos Suínos/imunologia , Animais , Anticorpos Antivirais/imunologia , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Diarreia/imunologia , Diarreia/virologia , Interferon gama/imunologia , Suínos , Doenças dos Suínos/virologia , Eliminação de Partículas Virais
20.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376622

RESUMO

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.


Assuntos
Antígenos CD13/genética , Infecções por Coronavirus/veterinária , Coronavirus/fisiologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Tropismo Viral , Animais , Enterocolite/metabolismo , Enterocolite/patologia , Enterocolite/virologia , Interferons/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Suínos , Doenças dos Suínos/patologia , Replicação Viral
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