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1.
Virulence ; 15(1): 2397512, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39282989

RESUMO

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.


Assuntos
Animais Recém-Nascidos , Infecções por Coronavirus , Diarreia , Filogenia , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Virulência , Diarreia/virologia , Diarreia/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Genoma Viral , Mutagênese Insercional , China , Células Vero
2.
Arch Virol ; 169(9): 180, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39150572

RESUMO

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Recombinação Genética , Glicoproteína da Espícula de Coronavírus , Doenças dos Suínos , Animais , Sequência de Aminoácidos , Substituição de Aminoácidos , China/epidemiologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/epidemiologia , Diarreia/virologia , Diarreia/veterinária , Diarreia/epidemiologia , Variação Genética , Genótipo , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia
3.
Microb Pathog ; 195: 106885, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39182857

RESUMO

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.


Assuntos
Infecções por Coronavirus , Deltacoronavirus , Fezes , Técnicas de Amplificação de Ácido Nucleico , Vírus da Diarreia Epidêmica Suína , RNA Viral , Sensibilidade e Especificidade , Doenças dos Suínos , Vírus da Gastroenterite Transmissível , Animais , Suínos , Vírus da Gastroenterite Transmissível/isolamento & purificação , Vírus da Gastroenterite Transmissível/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Fezes/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Diagnóstico Diferencial , Deltacoronavirus/isolamento & purificação , Deltacoronavirus/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/veterinária , Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Técnicas de Diagnóstico Molecular/métodos , Diarreia/virologia , Diarreia/veterinária , Diarreia/diagnóstico
4.
Talanta ; 280: 126712, 2024 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-39153256

RESUMO

Swine Enteric Coronaviruses (SECoVs), with high lethality and infectiousness, are the main pathogens causing fatal and watery diarrhea in piglets and spreading globally. Moreover, these SECoVs can cause similar clinical manifestations and are often co-infected, requiring an accurate assay suitable for rapid, in situ, and differential detection. Here, we developed a multiplexed fluorescent-based lateral flow immunoassay (mFB-LFIA) for the detection of three SECoVs, including porcine delta coronaviruses (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), in swine fecal samples. Thanks to the filter pad design and reasonable optimization, the mFB-LFIA was achieved within 15 min for three SECoVs detection simultaneously and improved the tolerance of the strips for feces samples. The limit of detection (LoD) of detecting PDCoV, TGEV, and PEDV were 2.1 × 104 TCID50 mL-1, 3.4 × 102 TCID50 mL-1, and 3.6 × 102 TCID50 mL-1, respectively. Additionally, the proposed assay was successfully applied to the detection of PDCoV, TGEV, and PEDV in swine feces with high accuracy. Compared with the gold standard nucleic acid testing, the total coincidence rate of the proposed assay was more than 90 %. Moreover, the mFB-LFIA performed excellent stability and repeatability. The proposed mFB-LFIA allows for rapid, in situ, more cost-effective and simultaneous detection of PDCoV, TGEV, and PEDV compared with nucleic acid testing. To the best of our knowledge, this is the first report to describe a multiplexed point-of-care assay capable of detecting PDCoV, TGEV, and PEDV in swine fecal samples. We believe our approach has a great potential for application to pig farm.


Assuntos
Fezes , Vírus da Diarreia Epidêmica Suína , Vírus da Gastroenterite Transmissível , Animais , Fezes/virologia , Fezes/química , Suínos , Vírus da Gastroenterite Transmissível/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Imunoensaio/métodos , Deltacoronavirus/isolamento & purificação , Limite de Detecção
5.
PLoS One ; 19(7): e0306532, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38968319

RESUMO

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Vírus da Gastroenterite Transmissível , Animais , Suínos , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Doenças dos Suínos/virologia , Doenças dos Suínos/diagnóstico , Estudos Retrospectivos , Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Gastroenterite Suína Transmissível/epidemiologia , Reação em Cadeia da Polimerase/métodos , Deltacoronavirus/genética , Deltacoronavirus/isolamento & purificação , Estados Unidos/epidemiologia
6.
Arch Virol ; 169(8): 158, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38970647

RESUMO

The highly pathogenic genotype 2b (HP-G2b) of porcine epidemic diarrhea virus (PEDV), which caused a pandemic in 2013-2014, evolved in South Korea and became endemic, affecting the domestic pig industry. This study describes the genotypic traits of novel HP-G2b PEDV strains identified on affected farms experiencing low disease severity with < 10% neonatal mortality. Nucleotide sequencing revealed common deletion patterns, termed S-DEL2, resulting in a two-amino-acid deletion at positions 60 and 61, 61 and 62, or 63 and 64 in the N-terminal domain of the spike (S) protein of all isolates. The S barcode profiles of S-DEL2 variants differed from each other and shared 96.0-99.4% and 98.5-99.6% nt sequence identity with other South Korean HP-G2b PEDV strains in the S gene and in the complete genome sequence, respectively. Genetic and phylogenetic analysis showed that the S-DEL2 strains belonged to diverse domestic clades: CK, CK.1, CK.2, or NC. The emergence of novel S-DEL2 strains suggests that continuous evolution of PEDV occurs under endemic circumstances, resulting in genetic diversity and distinct clinical presentations. This study advances our knowledge regarding the genetic and pathogenic heterogeneity of PEDV and emphasizes the importance of active monitoring and surveillance to identify novel variants and determine their genotypic and phenotypic characteristics.


Assuntos
Infecções por Coronavirus , Genótipo , Filogenia , Vírus da Diarreia Epidêmica Suína , Glicoproteína da Espícula de Coronavírus , Doenças dos Suínos , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , República da Coreia/epidemiologia , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Glicoproteína da Espícula de Coronavírus/genética , Variação Genética , Genoma Viral/genética , Deleção de Sequência
7.
Viruses ; 16(7)2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-39066270

RESUMO

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), emerges annually in several Asian countries. Its major symptoms include watery diarrhea, vomiting, anorexia, and dehydration. PED outbreaks incur significant economic losses. The efficacy of vaccines is limited by viral mutations and insufficient intestinal mucosal immunity. Therefore, new vaccines against these recent variants are urgently needed. Herein, we isolated and genetically characterized a novel Korean PEDV strain using NGS. Comparative genomic analysis demonstrated that the CKK1-1 strain belonged to genogroup 2. The isolated strain was cultured in sodium-glycochenodeoxycholic acid for 180 passages. Typically, PEDV isolation and passage require proteases, such as trypsin. However, the CKK1-1 strain adapted to this atypical culture condition, achieving a high titer of 8.83 ± 0.14 log TCID50/mL. In vitro biological analysis revealed no cell syncytium formation without trypsin; however, a cell-lysis-type cytopathic effect was noted. Notably, pathogenicity evaluation showed that CKK1-1 p0 exhibited naturally weakened virulence in five-day-old piglets, while piglets administered with CKK1-1 p180 exhibited 100% survival and reduced clinical symptoms. Collectively, our data demonstrate that this Korean PEDV strain, attenuated through atypical culture conditions with Na-glycochenodeoxycholic acid, has potential as a vaccine candidate, providing valuable insights into the genetic variation in and pathogenicity of PEDV.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , República da Coreia , Doenças dos Suínos/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Virulência , Filogenia , Genoma Viral , Chlorocebus aethiops , Células Vero
8.
Viruses ; 16(7)2024 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-39066288

RESUMO

The variant porcine epidemic diarrhea virus (PEDV) has caused considerable economic losses to the global pig industry since 2010. In this study, a total of 5859 diarrhea samples were collected from different pig farms in China's Guangxi province during January 2020 and March 2024 and tested for PEDV using RT-qPCR. The positivity rate of PEDV was 11.90% (697/5859). Ninety-two PEDV-positive samples were selected based on sampling time, and the sampling region for amplification, sequencing, and analysis of the S1, M, and N genes. Phylogenetic analysis of the S1 gene revealed that all strains from Guangxi province were distributed in three subgroups, i.e., 81.5% (75/92) in the G2a subgroup, 4.3% (4/92) in the G2b subgroup, and 14.1% (13/92) in the G2c subgroup. The sequence analysis revealed that the S1 gene sequences from Guangxi province had higher homology with the variant strains than with the classical strains, showing as high as 99.2% with the variant strain AJ1102 and only 94.3% with the classical strain CV777. Recombination analysis revealed that the GX-BS08-2023 strain (G2c) from Guangxi province originated from inter-lineage recombination between the GX-BS09-2023 (G2a) and CH-JN547228-2011 (G1a) strains. In addition, the S1 gene of the G2a and G2b subgroup strains shared many mutations and insertions. There were common mutations of N143D and P235L in the G2a subgroup. Evolutionary analysis revealed that all Guangxi strains belonged to the G2 genotype. These strains have spread rapidly since the PEDV variant strains that emerged in 2010, weakened until 2021, and then remained stable. In conclusion, the results revealed the latest genetic evolution of circulating PEDV strains in Guangxi province in recent years, providing important information for preventing and controlling PEDV infection. Currently, the G2a subgroup strains are the predominant strains circulating in pig herds in Guangxi province, southern China.


Assuntos
Infecções por Coronavirus , Evolução Molecular , Filogenia , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Suínos , China/epidemiologia , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Variação Genética , Diarreia/virologia , Diarreia/veterinária , Diarreia/epidemiologia , Genótipo , Glicoproteína da Espícula de Coronavírus/genética
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 320: 124670, 2024 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-38908108

RESUMO

Porcine epidemic diarrhea virus (PEDV) and rotavirus has posed a significant threat to the pig industry annually across different nations, resulting in huge economic losses. The frequent co-infection of these two viruses in clinical settings complicates the process of differential diagnoses. Rapid and accurate detection of PEDV and rotavirus is in great demand for timely diarrhea disease prevention and control. In this study, tris stabilized AuNPs were prepared and a sensitive lateral flow immunoassay (LFIA) sensor was developed for the simultaneous and rapid detection of PEDV and rotavirus on site. After the system optimization, the established LFIA can simultaneously identify PEDV and rotavirus with limits of detection (LOD) of 1.25 × 103 TCID50 mL-1 and 3.13 × 102 pg mL-1, respectively. When applying for clinical samples, the LFIA show a concordance of 95 % and 100 % to reverse transcript polymerase chain reaction (RT-PCR) for PEDV and rotavirus respectively. Therefore, this LFIA can qualitatively detect PEDV and rotavirus in 18 min with high sensitivity and accuracy without any sophisticated equipment and operation, making it a promising candidate for the early diagnosis of PEDV or/and rotavirus diarrhea on site.


Assuntos
Cromatografia de Afinidade , Ouro , Nanopartículas Metálicas , Vírus da Diarreia Epidêmica Suína , Rotavirus , Ouro/química , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Rotavirus/isolamento & purificação , Animais , Nanopartículas Metálicas/química , Suínos , Cromatografia de Afinidade/métodos , Limite de Detecção , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/veterinária , Infecções por Rotavirus/virologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Imunoensaio/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária
11.
Vet Microbiol ; 295: 110162, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38941767

RESUMO

Postweaning diarrhea (PWD) is a multifactorial disease caused by different aetiological agents, like viruses or bacteria and where the role of the microbiota remains unclear. The aim of this study was to assess differences between healthy and diarrheic weaned pigs concerning the prevalence of pathogens and changes in the intestinal microbiota. Eighteen farms with PWD were selected and 277 fecal samples were collected (152 diarrheic vs 125 healthy). Presence of Rotavirus A (RVA), B (RVB), C (RVC) and Porcine Epidemic Diarrhea Virus (PEDV), virulence factors of Escherichia coli and Clostridioides difficile were analyzed by PCR. Finally, the microbiota composition was also study by 16 S rRNA sequencing on 148 samples (102 diarrheic vs 46 healthy). RVA (53.95 % vs 36 %, p=0.04) and RVB (49.67 % vs 28.8 %, p<0.001) were more frequent in diarrheic animals. Furthermore, RVA viral load was higher in diseased animals. VT2 toxin was significantly associated with diarrhea, whereas other virulence factors were not. Presence of C. difficile and PEDV was almost negligible. Regarding microbiota changes, Fusobacteriota phylum was more frequent in diarrheic samples and Ruminococcaceae family in healthy penmates. During the first week postweaning, Enterobacteriace and Campylobacteria were enriched in animals presenting diarrhea. Furthermore, Lactobacillus was detected in those individuals with no RVA infection. In conclusion, RVA seems to play a primary role in PWD. Classic E. coli virulence factors were not associated with diarrhea, indicating the need for revising their implication in disease. Moreover, Lactobacillus was found frequently in animals negative for RVA, suggesting some protective effect.


Assuntos
Diarreia , Fezes , Microbioma Gastrointestinal , Doenças dos Suínos , Desmame , Animais , Suínos , Diarreia/veterinária , Diarreia/microbiologia , Diarreia/virologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Fezes/microbiologia , Fezes/virologia , Rotavirus/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Clostridioides difficile/isolamento & purificação , Clostridioides difficile/genética , Fatores de Virulência/genética , RNA Ribossômico 16S/genética , Escherichia coli/isolamento & purificação , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/genética
12.
J Virol Methods ; 329: 114986, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38914314

RESUMO

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China's implementation of a large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Reação em Cadeia da Polimerase em Tempo Real , Deleção de Sequência , Doenças dos Suínos , Vacinas Atenuadas , Vacinas Virais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , Suínos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Doenças dos Suínos/virologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/diagnóstico , Vacinas Virais/genética , Vacinas Virais/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , China , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Filogenia , Sensibilidade e Especificidade , Análise Custo-Benefício
13.
J Nanobiotechnology ; 22(1): 239, 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38735951

RESUMO

Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.


Assuntos
Técnicas Biossensoriais , Nanotubos de Carbono , Vírus da Diarreia Epidêmica Suína , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , Suínos , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Nanotubos de Carbono/química , Limite de Detecção , Imunoensaio/métodos , Imunoensaio/instrumentação , Anticorpos Monoclonais/imunologia , Transistores Eletrônicos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/análise , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Anticorpos Antivirais/imunologia , Desenho de Equipamento
14.
Microb Pathog ; 191: 106646, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38631414

RESUMO

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.


Assuntos
Vírus da Diarreia Epidêmica Suína , Reação em Cadeia da Polimerase em Tempo Real , Rotavirus , Sensibilidade e Especificidade , Doenças dos Suínos , Vírus da Gastroenterite Transmissível , Animais , Suínos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/classificação , Doenças dos Suínos/virologia , Doenças dos Suínos/diagnóstico , Rotavirus/genética , Rotavirus/isolamento & purificação , Rotavirus/classificação , Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Deltacoronavirus/genética , Deltacoronavirus/isolamento & purificação , Diarreia/virologia , Diarreia/veterinária , Diarreia/diagnóstico , Coronavirus/genética , Coronavirus/isolamento & purificação , Coronavirus/classificação , Fezes/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia
15.
J Hazard Mater ; 471: 134296, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38643574

RESUMO

The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only on disinfectants, whereas the capabilities of the treatment process have not been investigated. In this study, the performance and mechanism of an AnMBR in the removal of porcine hepatitis E virus (HEV), porcine kobuvirus (PKoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV) are systematically investigated. The results show that the AnMBR effectively removes the four viruses, with average removal efficiencies of 1.62, 3.05, 2.41, and 1.34 log for HEV, PKoV, PEDV and TGEV, respectively. Biomass adsorption contributes primarily to the total virus removal in the initial stage of reactor operation, with contributions to HEV and PKoV removal exceeding 71.7 % and 68.2 %, respectively. When the membrane is fouled, membrane rejection dominated virus removal. The membrane rejection contribution test shows the significant contribution of membrane pore foulants (23-76 %). Correlation analysis shows that the surface characteristics and size differences of the four viruses contribute primarily to their different effects on biomass adsorption and membrane rejection. This study provides technical guidance for viral removal during the treatment of high-concentration swine wastewater using an AnMBR.


Assuntos
Reatores Biológicos , Membranas Artificiais , Águas Residuárias , Animais , Águas Residuárias/virologia , Suínos , Anaerobiose , Vírus de RNA/isolamento & purificação , Purificação da Água/métodos , Adsorção , Biomassa , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Eliminação de Resíduos Líquidos/métodos
16.
Viruses ; 14(8)2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-36016441

RESUMO

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/µL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/µL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Rotavirus , Doenças dos Suínos , Animais , Infecções por Coronavirus/veterinária , Diarreia/diagnóstico , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Rotavirus/genética , Rotavirus/isolamento & purificação , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia
17.
Anal Bioanal Chem ; 413(30): 7521-7529, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34686895

RESUMO

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription-enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.


Assuntos
Sistemas CRISPR-Cas , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/diagnóstico , Vacinas Atenuadas/genética , Animais , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Endodesoxirribonucleases/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Recombinases/genética , Recombinases/metabolismo , Suínos , Doenças dos Suínos/virologia , Proteínas Virais/genética
18.
BMC Vet Res ; 17(1): 235, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225697

RESUMO

BACKGROUND: Porcine epidemic diarrhoea (PED) is a highly contagious infectious disease with negative economic impacts on the swine industry. PED outbreaks were reported from 2009 to 2015, but sporadic infection has been observed until now in Vietnam. However, the seroprevalence of PEDV infection has not yet been reported for commercial pig farms in Vietnam. The aim of this study was to assess the seroprevalence of PEDV infection in Vietnamese pig farms to reveal the endemic status of PEDV in northern Vietnam. RESULTS: A serological survey of PEDV infection was carried out using indirect ELISA in commercial pig farms in Hai Duong, Hung Yen and Thai Binh provinces in northern Vietnam in 2019. Twenty sera were randomly collected from each of 10 commercial pig farms, from each province; none of the farms had vaccinated for PEDV. Serological evidence of natural PEDV infection, expressed as a high antibody titre, was observed in the pig farms in all 3 provinces. The OD values were significantly higher (p < 0.001) for pig sera from Thai Binh than from Hai Duong and Hung Yen. No significant differences (p > 0.05) were detected for seropositivity to PEDV based on locality, age, pig breed and farm size. CONCLUSIONS: This study indicates serological evidence of natural PEDV infection with high antibody titre in commercial pig farms. PEDV infection was widespread among the pig population in these 3 provinces and that good management and strict biosecurity are needed at these pig farms.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Doenças dos Suínos/epidemiologia , Animais , Anticorpos Antivirais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Estudos Soroepidemiológicos , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Vietnã/epidemiologia
19.
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
20.
BMC Vet Res ; 17(1): 117, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712000

RESUMO

BACKGROUND: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation. RESULTS: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/µL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance. CONCLUSION: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.


Assuntos
Dispositivos Lab-On-A-Chip/veterinária , Reação em Cadeia da Polimerase Multiplex/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Doenças dos Suínos/virologia , Animais , Circovirus/genética , Circovirus/isolamento & purificação , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/isolamento & purificação , Ensaios de Triagem em Larga Escala , Dispositivos Lab-On-A-Chip/virologia , Microfluídica/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Suínos , Doenças dos Suínos/diagnóstico
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