RESUMO
Porcine epidemic diarrhea virus (PEDV) causes the third most important disease in the pig industry, after African swine fever and porcine reproductive and respiratory syndrome, and leads to illness or death of the entire litter, causing significant economic losses. In this study, three PEDV strains (HN-1, HN-2, and SC2023) were isolated from swine farms with suspected PEDV infections in Sichuan and Henan provinces. Phylogenetic analysis based on complete S gene sequences showed that all three strains belonged to the G2c subgroup. HN-1 adapted readily to cell culture, grew to a viral titer as high as 2 × 108 TCID50/mL in Vero cells, and caused the formation of large syncytia. We analyzed the amino acid sequence of the HN-1 isolate and found that its S1 subunit contained a three-amino-acid insertion (355KRL358). A seven-amino-acid-deletion (1377FEKVHVQ1383) in the S2 subunit resulted in the partial deletion of the endocytosis signal YxxΦ and the complete deletion of the endoplasmic reticulum retrieval signal (ERRS) KVHVQ in the cytoplasmic tail of the S protein. Consequently, HN-1 is predicted to be less pathogenic than its parent strain, an attribute that facilitates rapid cell-to-cell spread by enhancing syncytium formation. In addition, strain HN-1 was found to have the mutation 884-885SGâRR, which may favor adaptation to cell culture by providing new trypsin cleavage sites. These results suggest that HN-1 is a G2c subtype variant that adapts well to cell culture and can be used to study the adaptive mechanisms of PEDV and develop attenuated vaccines.
Assuntos
Infecções por Coronavirus , Filogenia , 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/classificação , Suínos , Células Vero , Chlorocebus aethiops , Doenças dos Suínos/virologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , China , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de AminoácidosRESUMO
Porcine epidemic diarrhea (PED) is an acute, highly contagious, and infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can affect pigs of all ages, with 50~100% mortality in neonatal piglets and substantial economic losses in the swine industry. In the present study, 347 fecal and intestinal samples were collected from seven regions in China during 2020-2022. A comprehensive molecular investigation of the spike (S) gene of PEDV strains was carried out, which included phylogenetic analysis of the obtained PEDV sequences. Epidemiological surveillance data indicate that the GIIc subgroup strains are widely distributed among pigs. A PEDV strain was successfully isolated from positive small intestine samples and identified through RT-PCR detection using specific N gene primers of PEDV, indirect immunofluorescence assay (IFA), TEM analysis, genome sequencing, and full-length S gene analysis, named PEDV/SC/2022. RDP and SimPlot analysis showed that the isolate originated from the recombination of PEDV/AH2012 and PEDV/AJ1102. In conclusion, our findings contribute to the current understanding of PEDV epidemiology and provide valuable information for the control of PED outbreaks in China.
Assuntos
Infecções por Coronavirus , Filogenia , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/classificação , Animais , Suínos , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/epidemiologia , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , China/epidemiologia , Glicoproteína da Espícula de Coronavírus/genética , Recombinação Genética , Fezes/virologiaRESUMO
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 VeroRESUMO
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/epidemiologiaRESUMO
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 VeroRESUMO
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/genéticaRESUMO
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 SequênciaRESUMO
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/virologiaRESUMO
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/genéticaRESUMO
Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172-554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5'UTR, ORF1a/1b, S, E, M, N and 3'UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.
Assuntos
Genótipo , Fases de Leitura Aberta/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Animais , Chlorocebus aethiops , Coinfecção/virologia , Infecções por Coronavirus/virologia , Diarreia/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/virologia , Células Vero , VirulênciaRESUMO
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/virologiaRESUMO
In recent years, a novel, highly virulent variant of porcine epidemic diarrhea virus (PEDV) has emerged, causing substantial economic losses to the pork industry worldwide. In this study, a PEDV strain named LNsy was successfully isolated in China. Phylogenetic analysis based on the whole genome revealed that PEDV LNsy belonged to the G2 subtype. For the first time, a unique four amino acids (4-aa) insertion was identified in the COE region of the spike (S) protein (residues 499-640), resulting in an extra alpha helix in the spatial structure of the COE region. To determine changes in virus-neutralization (VN) antibody reactivity of the virus, polyclonal antibodies (PAbs) against the S protein of different subtypes were used in a VN test. Both PAbs against the S protein of the G1 and G2 subtype showed reduced VN reactivity to PEDV LNsy. Further, recombination analyses revealed that PEDV LNsy was the result of recombination between PEDV GDS13 and GDS46 strains at the genomic breakpoints (nt 17,959-20,594 in the alignment) in the ORF1b gene of the genomes. Pathological examination showed gross morphological pathological changes in the gut, including significant villus atrophy and shedding of the infected piglets. These results indicated that a 4-aa insertion in the COE region of the S protein may have partly altered the profiles of VN antibodies and thus it will be important to develop vaccine candidates to resist wild virus infection and to monitor the genetic diversity of PEDV.
Assuntos
Aminoácidos/genética , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , China , Chlorocebus aethiops , Variação Genética , Genoma Viral , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Organismos Livres de Patógenos Específicos , Suínos/virologia , Doenças dos Suínos/virologia , Células VeroRESUMO
Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and a high rate of mortality in suckling pigs. The epidemic of PEDV that occurred after 2013 was caused by non-insertion and deletion of S gene (S-INDEL) PEDV strains. During this epidemic, a variant of the non-S-INDEL PEDV strain with a large deletion of 205 amino acids on the spike gene (5-17-V) was also found to co-exist with a non-S-INDEL PEDV without deletion (5-17-O). Herein, we describe the differences in the complete genome, distribution, virulence, and antigenicity between strain 5-17-O and variant strain 5-17-V. The deletion of 205 amino acids was primarily located in the S1O domain and was associated with milder clinical signs and lower mortality in suckling pigs than those of the 5-17-O strain. The 5-17-V strain-induced antibody did not completely cross-neutralize the 5-17-O strain. In conclusion, the deletion in the S1 region reduces the virulence of PEDV and influences the virus-neutralizing activities of the antibody it induces.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Infecções por Coronavirus/veterinária , Interações Hospedeiro-Patógeno/imunologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Diarreia/veterinária , Genoma Viral , Genômica/métodos , Testes de Neutralização , Variantes Farmacogenômicos , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Taiwan , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Porcine epidemic diarrhea (PED) is a viral enteric disease of pigs. It affects all age classes of animals but lethality is mainly seen in suckling piglets. After its first appearance in England in 1971, Porcine epidemic diarrhea virus (PEDV) has spread worldwide. While sporadic outbreaks prevailed in Europe, the disease had high impact in Asia. Following particularly severe outbreaks in 2011, high impact cases were also reported in the United States and neighboring countries in 2013. Subsequently, outbreaks were also reported in several European countries including Germany. These outbreaks were less severe. This case report describes a recent case of PED re-emergence in Germany and the sequence analyses of the causative PEDV. CASE PRESENTATION: In spring 2019 5 years after re-introduction of PED into Central Europe, a piglet-producer in northwestern Germany experienced an outbreak that affected sows, their suckling piglets, and weaners. After initial confirmation of PEDV by real-time RT-PCR, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. Phylogenetic analyses showed high identities among the PEDV sequences obtained from samples of different animals and a close relation to recent strains from Hungary and France. Compared to the PEDV strains analyzed in 2014, genetic drift could be confirmed. Changes were mainly observed in the spike protein encoding S gene segment. In addition, metagenomic analyses showed multiple Picobirnavirus reads in all investigated samples. CONCLUSION: This case report shows that PEDV is still circulating in Europe. The causative strains are moderately virulent and are still closely related to the so-called INDEL strains reported previously in Europe, including Germany. However, a genetic drift has taken place that can be seen in a novel cluster comprising strains from Germany, Hungary and France in 2019. Relevance and impact of the detected Picobirna sequences need further investigations.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Animais Recém-Nascidos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Surtos de Doenças/veterinária , Fezes/virologia , Feminino , Deriva Genética , Genoma Viral , Alemanha , Filogenia , Picobirnavirus/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/classificação , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Porcine epidemic diarrhea virus (PEDV) is a fatal epizootic swine coronavirus that presents a financial threat to the global swine industry. Since the discovery of the low-pathogenic genotype 1b (G1b) in 2014, it has been responsible for sporadic outbreaks in South Korea. In this study, we identified novel G1b variants arising from the natural recombination of a major pandemic-like G2b virus and a minor G1b virus currently circulating in the domestic field. The whole-genome sequences of two 2018-19 G1b recombinants, KNU-1808 and KNU-1909, were determined. A genomic comparison showed that these two viruses share the highest nucleotide sequence similarity with the 2017 G1b strain but share less similarity with the 2014 G1b emergent strain KNU-1406. However, the putative recombination breakpoints spanning the first 1,170 nucleotides of the spike (S) gene were almost identical among the emergent and contemporary G1b strains. Recombination detection indicated that the inter-subgroup G1b recombinant first emerged in 2017 by introducing the N-terminal domain of S from KNU-1406 into the backbone of KNU-1703, possibly leading to antigenic shift. It then evolved into KNU-1808 and KNU-1909 through genetic drift, moving toward a more G2b-like genotype. Phylogenetic analysis revealed that the 2018-2019 G1b recombinants belong to a cluster containing other G1b strains but form a new branch. This study provides an important advance warning in regard to the emergence and prevalence of new genotypes or variants that can result from genetic recombination between two different PEDV genotypes circulating in endemic areas and continuous non-lethal mutations essential for viral fitness in the host environment.
Assuntos
Infecções por Coronavirus/veterinária , Surtos de Doenças , Genoma Viral , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/epidemiologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Evolução Molecular , Variação Genética , Genótipo , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/patogenicidade , Recombinação Genética , República da Coreia/epidemiologia , Suínos , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Sequenciamento Completo do GenomaRESUMO
Porcine epidemic diarrhea virus (PEDV) causes continuous, significant damage to the swine industry worldwide. By RT-PCR-based methods, this study demonstrated the ongoing presence of PEDV in pigs of all ages in Korea at the average detection rate of 9.92%. By the application of Bayesian phylogenetic analysis, it was found that the nucleocapsid (N) gene of PEDV could evolve at similar rates to the spike (S) gene at the order of 10-4 substitutions/site/year. Based on branching patterns of PEDV strains, three main N gene-base genogroups (N1, N2, and N3) and two sub-genogroups (N3a, N3b) were proposed in this study. By analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. The antigenic indexes of genogroup N3 strains were significantly lower compared with those of genogroups N1 and N2 strains in the B-cell epitope of the nucleocapsid protein. Similarly, significantly lower antigenic indexes in some parts of the B-cell epitope sequences of the spike protein (COE, S1D, and 2C10) were also identified. PEDV mutants derived from genetic mutations of the S and N genes may cause severe damage to swine farms by evading established host immunities.
Assuntos
Infecções por Coronavirus/veterinária , Variação Genética , Proteínas do Nucleocapsídeo/genética , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Animais , Teorema de Bayes , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/genética , Fazendas , Fezes/virologia , Feminino , Genótipo , Mutação , Filogenia , República da Coreia , Suínos , Doenças dos Suínos/virologia , Sequenciamento Completo do GenomaRESUMO
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/virologiaRESUMO
A retrospective evaluation of PEDV-positive samples recovered in Spain before and after the re-emergence of this coronavirus in several European countries was carried out. We described for the first time recombinant SeCoV circulating in Spain between 1993 and 2014 and its misidentification as PEDV when diagnostic assays based on the S-protein or S-gene of the PEDV were used. The complete S-gene sequence of 7 Spanish SeCoV and 30 PEDV Spanish isolates was phylogenetically analysed including the S-gene sequences of the three SeCoV and a representative selection of the PEDV strains with complete genome sequences available in the GenBank. The tree showed a common ancestor for the S-gene of the PEDV and SeCoV, but no evolution from any known PEDV clade was shown for the SeCoV strains. Moreover, complete genome sequences were obtained from 23 PEDV strains recovered in Spanish swine farms since 2014. The phylogenetic tree showed the INDEL type genogroup of these Spanish strains, supporting the lower pathogenicity of this genogroup since no significant economic losses were reported in the affected Spanish swine farms. Four subgroups were detected among PEDV strains in Spain, closely related to the recent European strains. Moreover, eight of the most recent Spanish PEDV isolates formed a subclade together with three European strains from 2015, showing a new evolution branch with a recombinant virus.
Assuntos
Alphacoronavirus 1/isolamento & purificação , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Doenças dos Suínos/virologia , Alphacoronavirus 1/classificação , Animais , Infecções por Coronavirus/virologia , Fezes/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Estudos Retrospectivos , Espanha , Sus scrofa , SuínosRESUMO
A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1ß, IL6, IFN-ß, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/virologia , Carga Viral , Animais , Biomarcadores , Chlorocebus aethiops , Citocinas , Imuno-Histoquímica , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/patologia , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , VirulênciaRESUMO
Outbreaks of porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) infection have caused high mortality of piglets and significant economic losses to the Chinese swine industry. In the current study, 184 specimens from pigs with or without signs of diarrhea were collected from 39 farms across eight provinces, mainly around Hunan, People's Republic of China, in 2017 to 2018 in order to obtain epidemiological information on PEDV infections in these regions. The results indicated an average PEDV-positive rate of 38.04% (70/184) and more-pronounced disease severity in diarrheic pigs (48.76%; 59/121) than in non-diarrheic pigs (17.46%; 11/63). Phylogenetic and sequence analysis demonstrated that 14 representative PEDV strains from 14 swine farms belonged to the G2 group (G2-a and G2-b subgroups) and displayed a high degree of genetic variation. In particular, two out of the 14 PEDV strains were found to have unique indels in the S1 gene. The strain HN-SY-2017-Oct had a 9-nucleotide (T1152GAAGCCAAT1160T) insertion, and the strain ZJ-2018-May had a 3-nucleotide (AAA) deletion at position 1126 in the S1 gene. A three-dimensional structural prediction revealed that these unique insertions might lengthen the loop on the surface or increase the likelihood of the surface protein being phosphorylated at 388Y, thereby affecting the virulence or pathogenicity of PEDV. Collectively, the data show that PED remains a severe threat to the pig industry and that variant PEDV stains are circulating in China. The updated PEDV epidemiological data will facilitate the design of PEDV vaccines and the application of effective measures for PED prevention.