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1.
BMC Vet Res ; 19(1): 2, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36597091

RESUMO

BACKGROUND: Porcine Teschovirus (PTV), also named Teschovirus A, is prevalent in pig populations, mainly causing neurological symptoms, diarrhea, pneumonia, and reproductive failure, however the morbidity and mortality are usually low in pig farms. CASE PRESENTATION: In this study, we reported a PTV outbreak investigation in one large-scale pig farm in China with severe symptoms including diarrhea, lethargy, locomotor ataxia, nystagmus, paralysis of the hind limbs, and coma in piglets. More importantly, the mortality reached 38% in suckling pigs, which is remarkably high in PTV history. A novel PTV strain, named HeNZ1, was isolated from cerebral samples of one suckling pig and the genome sequence was obtained by NGS sequencing. Phylogenetic and evolutionary divergence analyses revealed that HeNZ1 belongs to PTV genotype 2. Surprisingly, the VP1 coding region of HeNZ1 shares the highest sequence similarity with European PTV-2 strains, instead of China domestic PTV-2 strains, implying it may not derive from China local PTV-2 strains. Multiple sequence alignment and B cell epitope prediction of PTV VP1 and VP2 protein revealed 10 B cell epitopes, 5 mutant clusters and 36 unique mutation sites, of which 19 unique mutation sites are located in B cell epitopes and exposed on the surface of VP1 or VP2, implying significant antigenic drift potential of HeNZ1. CONCLUSION: These results indicate that HeNZ1 is a highly virulent PTV-2 strain, which capable of causing severe neurological symptoms and high mortality in piglets. Bioinformatic analysis suggest that HeNZ1 is genetically and antigenically different from other Chinese PTV-2 strains. Overall, current case expanded our understanding of PTV-2 clinical spectrum and revealed the emergence of a highly virulent PTV-2 strain with substantial genetic diversity and antigenic drift potential in VP1 and VP2.


Assuntos
Encefalomielite , Infecções por Picornaviridae , Doenças dos Suínos , Teschovirus , Suínos , Animais , Filogenia , Epitopos de Linfócito B , Diarreia/veterinária , China/epidemiologia , Encefalomielite/veterinária , Infecções por Picornaviridae/veterinária
2.
Pathog Dis ; 80(1)2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36044999

RESUMO

Porcine teschovirus (PTV) is a causative agent of polioencephalomyelitis, encephalomyelitis, reproductive disorders and gastrointestinal and respiratory diseases in swine. In the present study, the PTV2 GX/2020 strain was isolated from pig intestinal tissue through the use of ST cells. Phylogenetic analysis of VP1 nucleotide sequences indicated that the GX/2020 isolate is closely related to PTV2. Furthermore, the full-length cDNA of an infectious GX/2020 clone was constructed using seamless ligation technology. The genome sequence of the rescued virus is largely consistent with the sequence of the parental virus, and it exhibits viral growth properties. The PTV2 virus was successfully isolated in the present study, and the reverse-genetic platform provides a foundation for studies of the pathogenic mechanisms of porcine teschovirus.


Assuntos
Doenças Transmissíveis , Infecções por Picornaviridae , Doenças dos Suínos , Teschovirus , Animais , Células Clonais , DNA Complementar/genética , Filogenia , Infecções por Picornaviridae/veterinária , Suínos
3.
Viruses ; 14(3)2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35336920

RESUMO

Porcine sapeloviruses, teschoviruses of family Picornaviridae and type 3 porcine astroviruses of family Astroviridae are (re-)emerging enteric pathogens that could be associated with severe, disseminated infections in swine, affecting multiple organs including the central nervous system (CNS). Furthermore, small-scale pioneer studies indicate the presence of these viruses in porcine nasal samples to various extents. The laboratory diagnostics are predominantly based on the detection of the viral RNA from faecal and tissue samples using different nucleic-acid-based techniques such as RT-qPCR. In this study, a novel highly sensitive one-step triplex RT-qPCR assay was introduced which can detect all known types of neurotropic sapelo-, tescho- and type 3 astroviruses in multiple types of samples of swine. The assay was evaluated using in vitro synthesized RNA standards and a total of 142 archived RNA samples including known sapelo-, tescho- and type 3 astrovirus positive and negative CNS, enteric and nasal specimens. The results of a large-scale epidemiological investigation of these viruses on n = 473 nasal swab samples from n = 28 industrial-type swine farms in Hungary indicate that all three neurotropic viruses, especially type 3 astroviruses, are widespread and endemically present on most of the investigated farms.


Assuntos
Infecções por Astroviridae , Astroviridae , Picornaviridae , Doenças dos Suínos , Teschovirus , Animais , Astroviridae/genética , Infecções por Astroviridae/diagnóstico , Infecções por Astroviridae/veterinária , Fezes , Mamastrovirus , Filogenia , Picornaviridae/genética , RNA Viral/genética , Suínos , Doenças dos Suínos/diagnóstico , Teschovirus/genética
4.
Artigo em Alemão | MEDLINE | ID: mdl-35235983

RESUMO

In a fattening farm in southern Germany, paralysis of the hind limbs was observed in 2 age groups (50 kg as well as 60 kg) during a 4 week period. Despite a low morbidity of 3.3 % the majority of the affected animals needed to be euthanized in consequence to the progression of their hind limb paralysis. During pathomorphological examinations of 2 affected fattening pigs severe lymphohistiocytic meningoencephalomyelitis and vasculitis were detected. Immunhistochemistry revealed the presence of Porcine Teschovirus antigen in all parts of the central nervous system as well as in several cell types (neurons, glia cells, endothelial cells, mononuclear cells). Porcine Teschovirus was detected by PCR in spinal cord samples. The subsequently performed phylogenetic analysis PCR revealed a close relation (88 % full genome sequence) to porcine Teschovirus A11 strain "Dresden". Other swine relevant pathogens were excluded by PCR, bacteriologic examination and sequencing. Following a period of 4 weeks no additional cases of hind limb paralysis were observed in the fattening farm.


Assuntos
Infecções por Picornaviridae , Doenças dos Suínos , Teschovirus , Animais , Células Endoteliais , Paralisia/veterinária , Filogenia , Infecções por Picornaviridae/veterinária , Suínos , Teschovirus/genética
5.
J Vet Diagn Invest ; 33(5): 864-874, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34151653

RESUMO

Porcine teschovirus (PTV), sapelovirus (PSV-A), and enterovirus (EV-G) are enteric viruses that can infect pigs and wild boars worldwide. The viruses have been associated with several diseases, primarily gastrointestinal, neurologic, reproductive, and respiratory disorders, but also with subclinical infections. However, for most serotypes, proof of a causal relationship between viral infection and clinical signs is still lacking. In Switzerland, there has been limited investigation of the occurrence of the 3 viruses. We used a modified multiplex reverse-transcription PCR protocol to study the distribution of the viruses in Swiss pigs by testing 363 fecal, brain, and placental or abortion samples from 282 healthy and diseased animals. We did not detect the 3 viruses in 94 placental or abortion samples or in 31 brain samples from healthy pigs. In brain tissue of 81 diseased pigs, we detected 5 PSV-A and 4 EV-G positive samples. In contrast, all 3 viruses were detected at high frequencies in fecal samples of both healthy and diseased pigs. In healthy animals, PTV was detected in 47%, PSV-A in 51%, and EV-G in 70% of the 76 samples; in diseased animals, frequencies in the 81 samples were 54%, 64%, and 68%, respectively. The viruses were detected more frequently in fecal samples from weaned and fattening pigs compared to suckling piglets and sows. Co-detections of all 3 viruses were the most common finding. Based on clinical and pathology data, statistical analysis yielded no evidence for an association of virus detection and disease. Further research is required to determine if pathogenicity is linked to specific serotypes of these viruses.


Assuntos
Enterovirus , Infecções por Picornaviridae , Picornaviridae , Doenças dos Suínos , Teschovirus , Animais , Enterovirus/genética , Feminino , Picornaviridae/genética , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/veterinária , Placenta , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia , Teschovirus/genética
6.
Braz J Microbiol ; 52(3): 1617-1622, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34024036

RESUMO

Porcine encephalomyelitis can be associated with many etiologies, including viral agents, such as Porcine teschovirus (PTV), Porcine sapelovirus (PSV), and Porcine astrovirus (PoAstV). In this study, we investigated the presence of these viruses in a neurological disease outbreak in a swine farm in Southern Brazil. The piglet production farm unity had 1200 weaning piglets, and 40 piglets with neurological signs such as motor incoordination, paresis, and paralysis of hind limbs, with an evolution time of approximately 4 days. Among these, 10 piglets were submitted to postmortem examination. Gross lesions were restricted to a mild enlargement of the nerve roots and ganglia of spinal cord segments. The microscopic lesions were characterized by nonsuppurative encephalomyelitis and ganglioneuritis with evident neuronal degeneration and necrosis. Samples of the central nervous system (CNS), cerebrospinal fluid, and feces were collected and submitted to molecular analysis. PTV was identified in all samples of the CNS, while eight of the piglets were also positive for PSV, and seven were positive for Porcine enterovirus (EV-G). PoAstV was identified in a pool of feces of healthy animals used as controls. This study demonstrates the occurrence of encephalomyelitis associated with PTV on a swine farm in Southern Brazil, as well as the presence of other viruses such as PSV, EV-G, and PoAstV in the swineherd. Sequences of the fragments that were previously amplified by PCR showed a high similarity to PTV 6. Herein, we describe the first case report of severe swine polioencephalomyelitis associated with PTV in South America.


Assuntos
Encefalomielite , Enterovirus Suínos , Infecções por Picornaviridae , Picornaviridae , Doenças dos Suínos , Teschovirus , Animais , Brasil/epidemiologia , Encefalomielite/epidemiologia , Encefalomielite/veterinária , Enterovirus Suínos/genética , Fazendas , Filogenia , Picornaviridae/genética , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/veterinária , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Teschovirus/genética
7.
Arch Virol ; 166(5): 1355-1370, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33709216

RESUMO

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


Assuntos
Encefalite Viral/veterinária , Infecções por Picornaviridae/veterinária , Doenças dos Suínos/virologia , Teschovirus/genética , Teschovirus/isolamento & purificação , Animais , Encéfalo/virologia , China/epidemiologia , Encefalite Viral/patologia , Encefalite Viral/virologia , Genoma Viral/genética , Genótipo , Mutação , Filogenia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , RNA Viral/genética , Recombinação Genética , Suínos , Teschovirus/classificação , Proteínas Virais/genética
8.
Virulence ; 12(1): 852-867, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33724149

RESUMO

Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.


Assuntos
Catalase/genética , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/patogenicidade , Estágios do Ciclo de Vida/genética , Proteínas de Protozoários/genética , Fatores de Virulência/genética , Animais , Catalase/metabolismo , Células Cultivadas , Feminino , Leishmania mexicana/genética , Camundongos , Camundongos Endogâmicos BALB C , Psychodidae/parasitologia , Teschovirus/genética , Virulência , Fatores de Virulência/metabolismo
9.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34561300

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.


Assuntos
COVID-19 , Regulação Viral da Expressão Gênica , Genes Reporter , SARS-CoV-2 , Proteínas Virais , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/biossíntese , Proteínas do Nucleocapsídeo de Coronavírus/genética , Feminino , Humanos , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Teschovirus/genética , Células Vero , Proteínas Virais/biossíntese , Proteínas Virais/genética
10.
Viruses ; 12(11)2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33138189

RESUMO

Porcine teschovirus (PTV) is an OIE-listed pathogen with 13 known PTV serotypes. Heterologous PTV serotypes frequently co-circulate and co-infect with another swine pathogen, causing various symptoms in all age groups, thus highlighting the need for a pan-PTV diagnostic tool. Here, a recombinant protein composed of a highly conserved "RNNQIPQDF" epitope on the GH loop of VP1, predicted in silico, and a tandem repeat of this epitope carrying the pan DR (PADRE) and Toxin B epitopes was constructed to serve as a PTV detection tool. This recombinant GST-PADRE-(RNNQIPQDF)n-Toxin B protein was used as an immunogen, which effectively raised non-neutralizing or undetectable neutralizing antibodies against PTV in mice. The raised antiserum was reactive against all the PTV serotypes (PTV-1-7) tested, but not against members of the closely related genera Sapelovirus and Cardiovirus, and the unrelated virus controls. This potential pan-PTV diagnostic reagent may be used to differentiate naturally infected animals from vaccinated animals that have antibodies against a subunit vaccine that does not contain this epitope or to screen for PTV before further subtyping. To our knowledge, this is the first report that utilized in silico PTV epitope prediction to find a reagent broadly reactive to various PTV serotypes.


Assuntos
Biologia Computacional , Mapeamento de Epitopos/métodos , Epitopos/genética , Epitopos/imunologia , Soros Imunes , Teschovirus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Simulação por Computador , Feminino , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sorogrupo , Suínos , Doenças dos Suínos/virologia , Teschovirus/patogenicidade
11.
Pathog Dis ; 78(5)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32691821

RESUMO

Porcine enterovirus G (EV-G) and teschovirus (PTV) generally cause asymptomatic infections. Although both viruses have been reported from various countries, they are rarely detected from India. To detect these viruses in Western India, fecal samples (n = 26) of diarrheic piglets aged below three months from private pig farms near Pune (Maharashtra) were collected. The samples were screened by reverse transcription-polymerase chain reaction using conserved enterovirus specific primers from 5' untranslated region. For genetic characterization of detected EV-G strain, nearly complete genome, and for PTV, partial VP1 gene were sequenced. EV-G strain showed the highest identity in a VP1 gene at nucleotide (78.61%) and amino acid (88.65%) level with EV-G15, prototype strain. However, its complete genome was homologous with the nucleotide (78.38% identity) and amino acid (91.24% identity) level to Ishi-Ka2 strain (LC316832), unassigned EV-G genotype detected from Japan. The nearly complete genome of EV-G15 consisted of 7398 nucleotides excluding the poly(A) tail and has an open reading frame that encodes a 2170 amino acid polyprotein. Genetic analysis of the partial VP1 gene of teschovirus identified porcine teschovirus 4 (PTV-4) and putative PTV-17 genotype. To the best of our knowledge, this is the first report on nearly full genome characterization of EV-G15, and detection of PTV-4 and putative PTV-17 genotypes from India. Further, detection and characterization of porcine enteroviruses are needed for a comprehensive understanding of their genetic diversity and their association with symptomatic infections from other geographical regions of India.


Assuntos
Enterovirus Suínos/classificação , Enterovirus Suínos/genética , Teschovirus/classificação , Teschovirus/genética , Regiões 5' não Traduzidas , Animais , Infecções Assintomáticas/epidemiologia , DNA Viral , Infecções por Enterovirus/veterinária , Infecções por Enterovirus/virologia , Enterovirus Suínos/isolamento & purificação , Fezes/virologia , Variação Genética , Genótipo , Índia/epidemiologia , Tipagem Molecular , Fases de Leitura Aberta , Filogenia , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos/virologia , Doenças dos Suínos/virologia , Teschovirus/isolamento & purificação , Sequenciamento Completo do Genoma
12.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611753

RESUMO

The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors.IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.


Assuntos
Células Epiteliais/virologia , Genoma Viral , RNA Viral/genética , Proteínas Recombinantes de Fusão/genética , Rotavirus/genética , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular , Cricetulus , Células Epiteliais/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Haplorrinos , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Genética Reversa/métodos , Rotavirus/metabolismo , Teschovirus/genética , Teschovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
13.
J Biosci Bioeng ; 130(2): 205-211, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32284303

RESUMO

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.


Assuntos
Fragmentos Fab das Imunoglobulinas/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Insetos/citologia , Peptídeos/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/genética , Teschovirus/genética , Transfecção
14.
BMC Vet Res ; 16(1): 51, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046722

RESUMO

BACKGROUND: Porcine teschovirus (PTV) circulates among wild and domesticated pig populations without causing clinical disease, however neuroinvasive strains have caused high morbidity and mortality in the past. In recent years, several reports appeared with viral agents as a cause for neurologic signs in weanling and growing pigs among which PTV and new strains of PTV were described. CASE PRESENTATION: On two unrelated pig farms in the Netherlands the weanling pig population showed a staggering gate, which developed progressively to paresis or paralysis of the hind legs with a morbidity up to 5%. After necropsy we diagnosed a non-suppurative encephalomyelitis on both farms, which was most consistent with a viral infection. PTV was detected within the central nervous system by qPCR. From both farms PTV full-length genomes were sequenced, which clustered closely with PTV-3 (98%) or PTV-11 (85%). Other common swine viruses were excluded by qPCR and sequencing of the virus. CONCLUSION: Our results show that new neuroinvasive PTV strains still emerge in pigs in the Netherlands. Further research is needed to investigate the impact of PTV and other viral agents causing encephalomyelitis within wild and domestic pig populations supported by the awareness of veterinarians.


Assuntos
Encefalomielite/veterinária , Infecções por Picornaviridae/veterinária , Doenças dos Suínos/virologia , Teschovirus/classificação , Animais , Encefalomielite/epidemiologia , Encefalomielite/virologia , Países Baixos/epidemiologia , Filogenia , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/virologia , Suínos , Doenças dos Suínos/epidemiologia , Teschovirus/genética , Teschovirus/isolamento & purificação
15.
Arch Virol ; 165(4): 993-1001, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32037488

RESUMO

Teschovirus A is currently the sole species in the genus Teschovirus, whose members are divided into 13 subtypes: porcine teschovirus (PTV) 1-13. However, recent discoveries of novel PTV genotypes have suggested that a new species, "Teschovirus B", should be established. Here, we have identified six of the 19 known genotypes and two novel genotypes (PTV 17-18), revealing the high genetic diversity of the PTV subpopulation in indigenous pigs of western Jiangxi, China. Moreover, we determined the nearly complete genome sequences of PTV 17-SG9 and PTV 18-SG10. Together with PTV 1-13, these novel genotypes were confirmed to be members of the species Teschovirus A based on phylogenetic and genetic divergence analysis. Consequently, the species Teschovirus A now includes at least 15 PTV genotypes.


Assuntos
Infecções por Picornaviridae/veterinária , Doenças dos Suínos/virologia , Teschovirus/genética , Teschovirus/isolamento & purificação , Animais , Sequência de Bases , China , Genoma Viral , Genótipo , Filogenia , Infecções por Picornaviridae/virologia , Suínos , Teschovirus/classificação
16.
Trop Anim Health Prod ; 52(3): 1161-1166, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31820308

RESUMO

Porcine teschovirus (PTV) previously classified as porcine enteroviruses in the family Picornaviridae are associated with a wide range of illnesses in swine ranging from asymptomatic infection to acute fatal encephalomyelitis, diarrhea, and pneumonia. This study was planned to investigate whether porcine teschovirus is prevalent among pigs in India and to characterize the PTV identified in the study population. The study conducted in certain farms of North India revealed that 13 of 190 (6.84%) fecal samples were PTV positive by RT-PCR. Three viruses were successfully isolated from fecal samples using IB-RS-2 cell lines which were confirmed by RT-PCR and sequencing. Molecular characterization based on the VP1 region of the viral genome identified the isolated viruses as serotype 5 and serotype 8 of PTV. A new variant of teschovirus was also identified which showed significant nucleotide diversity from the known serotypes of the teschoviruses. This is the first report of isolation, identification, and characterization of porcine teschoviruses in India.


Assuntos
Variação Genética , Infecções por Picornaviridae/veterinária , Doenças dos Suínos/epidemiologia , Teschovirus/genética , Animais , Fezes/virologia , Índia/epidemiologia , Filogenia , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/virologia , Prevalência , Sorogrupo , Suínos , Doenças dos Suínos/virologia , Teschovirus/classificação , Teschovirus/isolamento & purificação
17.
Transbound Emerg Dis ; 67(2): 1015-1018, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31657526

RESUMO

Conventionally, Porcine teschovirus (PTV) consists of 13 genotypes (PTV 1-13, which belong to Teschovirus A); however, several novel members including PTV 14-22 have been discovered recently. PTV 21 was identified as a novel Teschovirus species named Teschovirus B. In this study, almost all 22 reported PTV genotypes except PTV 6, 7, 12 and 20 were identified in the pig populations of western Jiangxi, China, which reflects the high genotype diversity. Moreover, to the best of our knowledge, the nearly complete genome of PTV 22-JiangX1 was first sequenced in the present study. The homology comparison of the polyprotein genes showed that PTV 22-JiangX1 shared a relatively high nucleotide and deduced amino acid sequence identities ranging from 78.3% to 82.0% and 84.6% to 89.3%, respectively, with PTV 19 and 21. Additionally, the PTV strain of JiangX1 represents genotype 22 with the PTV 19, and 21 strains proved to be members of Teschovirus B based on the phylogenetic and evolutionary divergence analyses. Finally, we determined that the novel Teschovirus B species comprises at least three PTV genotypes.


Assuntos
Infecções por Picornaviridae/veterinária , Doenças dos Suínos/virologia , Teschovirus/genética , Animais , Evolução Biológica , China/epidemiologia , Genótipo , Filogenia , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/virologia , Suínos , Teschovirus/isolamento & purificação
18.
Braz J Microbiol ; 51(2): 711-717, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31784949

RESUMO

Several emerging viral agents related to gastroenteritis are distributed in human and animal populations and may contaminate the environment due to anthropic activities. The objective of this study was to analyze the seasonal contamination by enteric virus and coliforms in water from streams in the Vale do Taquari, draining a large number of pig farms. Microbiological contamination was evidenced by the detection of total and thermotolerant coliforms, reaching their peak in December. Hepatitis E virus (HEV), Enterovirus-G (EV-G) genome, and Sapelovirus-A (SV-A) genome were not detected. On the other hand, Rotavirus (RV) was detected in 3% (1/32) of the samples, whereas Teschovirus-A (PTV) was detected in 6% (2/32). This is the first detection of PTV in environmental samples in Brazil, pointing that the virus is being shedded from swine herds to watersheds. Human mastadenovirus (HAdV) was the most frequent detected viral agent in 9.3% (3/32) with values of 2.54 × 105, 7.13 × 104, and 3.09 × 105 genome copies/liter (gc/L). The circulation of coliforms and viral pathogens is noticeable due to anthropic activities and to the management of animal waste from the pig farming. In this way, enteric viruses can assist in monitoring the quality of watersheds and in tracking sources of contamination.


Assuntos
Enterite/veterinária , Doenças dos Suínos/virologia , Teschovirus/isolamento & purificação , Eliminação de Partículas Virais , Vírus/isolamento & purificação , /virologia , Criação de Animais Domésticos , Animais , Brasil , Enterite/virologia , Fazendas , Fezes/virologia , Genoma Viral , Humanos , Suínos , Doenças dos Suínos/epidemiologia , Teschovirus/genética , Vírus/classificação , /microbiologia
19.
Mol Cells ; 42(5): 418-425, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31085809

RESUMO

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.


Assuntos
Vírus da Encefalomiocardite/genética , Sítios Internos de Entrada Ribossomal/genética , Teschovirus/genética , Animais , Vírus da Encefalomiocardite/metabolismo , Regulação Viral da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Proteínas Luminescentes , Camundongos , Modelos Moleculares , Teschovirus/metabolismo
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