Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Virology ; 540: 45-56, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31756532

RESUMO

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

2.
Emerg Microbes Infect ; 9(1): 20-31, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31859605

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea, resulting in high mortality in neonatal piglets. Despite widespread outbreaks in many countries, no effective PDCoV vaccines are currently available. Here, we generated, for the first time, a full-length infectious cDNA clone of PDCoV. We further manipulated the infectious clone by replacing the NS6 gene with a green fluorescent protein (GFP) to generate rPDCoV-ΔNS6-GFP; likewise, rPDCoV-ΔNS7 was constructed by removing the ATG start codons of the NS7 gene. Growth kinetics studies suggest that rPDCoV-ΔNS7 could replicate similarly to that of the wild-type PDCoV, whereas rPDCoV-ΔNS6-GFP exhibited a substantial reduction of viral titer in vitro and in vivo. Piglets inoculated with rPDCoV-ΔNS7 or wild-type PDCoV showed similar diarrheic scores and pathological injury. In contrast, rPDCoV-ΔNS6-GFP-infected piglets did not show any clinical signs, indicating that the NS6 protein is an important virulence factor of PDCoV and that the NS6-deficient mutant virus might be a promising live-attenuated vaccine candidate. Taken together, the reverse genetics platform described here not only provides more insights into the role of PDCoV accessory proteins in viral replication and pathogenesis, but also allows the development of novel vaccines against PDCoV infection.

3.
Viruses ; 11(4)2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022991

RESUMO

The accessory protein ORF3 of porcine epidemic diarrhea virus (PEDV) has been proposed to play a key role in virus replication. However, our understanding of its function regarding virus and host interaction is still limited. In this study, we employed immunoprecipitation and mass spectrometry to screen for cellular interacting partners of ORF3. Gene ontology analysis of the host interactome highlighted the involvement of ORF3 in endosomal and immune signaling pathways. Among the identified ORF3-interacting proteins, the vacuolar protein-sorting-associated protein 36 (VPS36) was assessed for its role in PEDV replication. VPS36 was found to interact with ORF3 regardless of its GLUE domain. As a result of VPS36-ORF3 interaction, PEDV replication was substantially suppressed in cells overexpressing VPS36. Interestingly, the ORF3 protein expression was diminished in VPS36-overexpressing cells, an effect that could not be restored by treatment of lysosomal inhibitors. In addition, disruption of endogenously-expressed VPS36 by siRNA could partially augment PEDV replication. Taken together, our study provides mechanistic insights into the contribution of ORF3 in PEDV replication.

4.
Viruses ; 11(3)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897856

RESUMO

While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells.


Assuntos
Mutação , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Fusão Celular , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Suínos , Doenças dos Suínos/virologia , Células Vero
5.
PLoS One ; 14(3): e0212632, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840701

RESUMO

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.


Assuntos
Proteínas do Nucleocapsídeo/biossíntese , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Animais , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/veterinária , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Células Vero
6.
Front Vet Sci ; 6: 34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30854373

RESUMO

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host's first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution.

7.
Arch Virol ; 164(2): 413-425, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30377826

RESUMO

Porcine deltacoronavirus (PDCoV) was first detected in Hong Kong and has recently spread to many countries around the world. PDCoV causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. A nucleotide sequence alignment showed that the whole genome of CHN-HG-2017 is 97.6%-99.1% identical to other PDCoV strains. Analysis of potential recombination sites showed that CHN-HG-2017 is a possible recombinant originating from the strains CH/SXD1/2015 and Vietnam/HaNoi6/2015. Furthermore, the pathogenicity of this recombinant PDCoV strain was investigated in 5-day-old piglets by oral inoculation. The challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from 1 to 7 days post-inoculation (DPI). Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets. Interestingly, high titers of virus-neutralizing antibodies in sera were detected at 21 DPI. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. Our analysis of the full genome sequence of a recombinant PDCoV and its virulence in suckling piglets might provide new insights into the pathogenesis of PDCoV and facilitate further investigation of this newly emerged pathogen.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/isolamento & purificação , Coronavirus/patogenicidade , Doenças dos Suínos/virologia , Animais , China , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/virologia , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Genoma Viral , Genômica , Filogenia , Suínos , Vietnã , Virulência
8.
Virology ; 525: 161-169, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30290311

RESUMO

BST-2 is an antiviral protein described as a powerful cross-species transmission barrier for simian immunodeficiency viruses. Influenza viruses appear to interact with BST-2, raising the possibility that BST-2 may be a barrier for cross-species transmission. An MDCK-based cell line expressing human BST-2 was generated to study human-derived A/Puerto Rico/8/36 (H1N1; PR8) as well as two low pathogenic avian influenza viruses (subtypes H4N6 and H6N1). The H4N6 and H6N1 viruses were less affected by BST-2 expression than PR8, due to their ability to decrease BST-2 levels, a function localized to the PA segment of both avian viruses. Experiments with PA-mutant and -chimeric viruses confirmed that the avian PA segment conferred BST-2 downregulation and antagonism. These results indicate a species-specific ability of PA from low pathogenic avian viruses to mitigate human BST-2 antiviral activity, suggesting that BST-2 is unlikely to be a general cross-species barrier to transmission of such viruses to humans.


Assuntos
Antígeno 2 do Estroma da Médula Óssea/antagonistas & inibidores , Vírus da Influenza A/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Cães , Regulação da Expressão Gênica , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Fases de Leitura Aberta
9.
Arch Virol ; 163(12): 3255-3264, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30136251

RESUMO

The coronavirus spike protein and the influenza virus hemagglutinin are class I viral membrane fusion proteins. While the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. Whether they are functionally interchangeable is currently unknown. In this study, we constructed scIAV-S, a single-cycle influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (PEDV), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. Treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. Moreover, the infectivity of scIAV-S was associated with the degree of cell adaptation of PEDV-S. Intriguingly, scIAV-S lacking functional neuraminidase (NA) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of PEDV. Together, scIAV-S offers a robust platform for the investigation of the entry mechanism of PEDV or, possibly, of other coronaviruses.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Influenza A/genética , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Infecções por Coronavirus/virologia , Vírus da Influenza A/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos
10.
Viruses ; 10(8)2018 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-30060558

RESUMO

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.


Assuntos
Vírus da Diarreia Epidêmica Suína/fisiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Replicação Viral , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Citoplasma/química , Citoplasma/virologia , Replicação do DNA , Diarreia/veterinária , Diarreia/virologia , Células HEK293 , Humanos , Microscopia Confocal , Plasmídeos/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/virologia , Transfecção , Células Vero , Proteínas Virais Reguladoras e Acessórias/genética
11.
Mol Biotechnol ; 59(9-10): 385-393, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28791613

RESUMO

Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optimal protective profile. Virus-like particles (VLPs) offer an attractive alternate vaccination strategy due to the effectively native presentation of epitopes in the absence of any infectious genetic material. However, the production of amounts of VLP in a platform that can support commercial development remains a major obstacle. This study generated two DENV 2 VLPs [codon-optimized and chimeric DENV/Japanese encephalitis virus (JEV)] and directly compared yields of these constructs by western blotting and dot blot hybridization. The effect of oleic acid supplementation, a process known to increase DENV production in natural infection, was also investigated. Results showed that the chimeric construct gave a two- to threefold higher yield than the codon-optimized construct and that while oleic acid increased DENV virion production in natural infection, it inhibited VLP production. These results suggest that further optimization of DENV VLP expression is possible, but it will require more understanding of how native DENV infection remodels the host cell machinery.


Assuntos
Vírus da Dengue/genética , Dengue/prevenção & controle , Ácido Oleico/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Anticorpos Antivirais/imunologia , Culicidae/virologia , Dengue/imunologia , Dengue/transmissão , Dengue/virologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Vírus da Encefalite Japonesa (Espécie)/genética , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Transfecção , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/uso terapêutico , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
12.
Arch Virol ; 162(9): 2553-2563, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28474223

RESUMO

The ORF3 accessory protein has been shown to impede reverse genetics of cell-culture-adapted porcine epidemic diarrhea virus (PEDV). Its absence or truncated variants are also associated with viral attenuation in vivo. Here, three ORF3 variants (ORF3NP12, ORF3NP14 and ORF3RB14) and their truncated counterparts were investigated for their regulatory role in recovery of cell-adapted PEDV in vitro. We demonstrate that ORF3NP12, but not the truncated form, can inhibit recovery of reverse-genetics-derived PEDV when expressed in trans. When testing with other RNA viruses, ORF3 was found to inhibit rescue of porcine respiratory and reproductive syndrome virus (PRRSV), but not of influenza virus. Interestingly, results from mutagenesis of ORF3NP12 suggest that F81 and M167 are responsible for impairing PEDV rescue in vitro. By changing specific residues of ORF3, the recombinant PEDV bearing the modified ORF3NP12 can be productively propagated in VeroE6-APN cells. These results may provide mechanistic insights into ORF3-mediated inhibition of PEDV replication in new host cells.


Assuntos
Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Sequência de Aminoácidos , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Regulação Viral da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Mutação Puntual , Suínos , Doenças dos Suínos/virologia , Células Vero , Proteínas Virais/genética
13.
Virology ; 506: 99-109, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28371631

RESUMO

Several mechanisms underlying intertypic interference between co-infecting influenza types A and B viruses (IAV and IBV) have been proposed. We have recently described one in which IBV's nucleoprotein (BNP) sequestered IAV's nucleoprotein (ANP) and suppressed IAV polymerase and growth. However, its anti-IAV capacity and limitations have not been fully explored. Here, we showed that BNP's inhibitory effect was more potent toward a wide array of avian IAVs, whereas human IAVs revealed moderate resistance. BNP sensitivity was largely determined by ANP's residue 343 at the NP oligomerization interface. An avian IAV polymerase carrying an NP-V343L mutation switched from being highly BNP-sensitive to moderately BNP-resistant, and vice versa for a human IAV polymerase carrying a reverse mutation. To highlight its capacity, we demonstrated that the polymerases of highly-pathogenic H5N1 and the pandemic 2009 (H1N1) strains are strongly inhibited by BNP. Our work provides insights into lineage-specific sensitivity to BNP-mediated intertypic interference.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Aviária/psicologia , Influenza Aviária/virologia , Influenza Humana/virologia , Nucleoproteínas/genética , Animais , Aves , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza B/fisiologia , Mutação , Nucleoproteínas/metabolismo , Polimorfismo de Nucleotídeo Único
14.
Asian Pac J Allergy Immunol ; 35(1): 20-26, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27543728

RESUMO

BACKGROUD: Avian influenza H5N1 and H7N9 viruses have jumped across species from avian to humans and become a threat to public health. Not much is known about pre-existing cross-reactive antibodies against these avian viruses in human population. OBJECTIVE: To determine the prevalence of cross-reactive anti-HA and anti-NA antibodies to avian influenza H5N1 and H7N9 viruses in Thai population. METHOD: Archival serum samples from 100 blood donors and 21 patients infected with 2009 pandemic influenza A (H1N1) (pdmH1N1) virus were investigated by hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays for anti-HA and anti-NA antibodies, respectively. The test antigens comprised 2 human viruses (pdmH1N1 and H3N2 viruses), and 6 reassortant viruses carrying HA and NA genes of avian H5N1 or H7N9 virus generated by reverse genetics. RESULTS: HAI antibody titers ≥ 10 were found in 58, 89, 0 and 15% of blood donors as tested against pdmH1N1, H3N2, H5N1 and H7N9 viruses, respectively. On the other hand, NAI antibodies were detected in 98, 94, 73 and 53% of blood donors when reverse genetic-derived viruses harboring NA gene from pdmH1N1, H3N2, H5N1 or H7N9 virus were used as the test antigens. Moreover, 66.7% of pdmH1N1 patients who had > 4 fold increase in HAI antibody titers in paired sera developed > 4 fold increase in NAI antibody titers. CONCLUSIONS: Anti-NA antibody has broader reactivity than anti-HA antibody, therefore, it can be a supplement to anti-HA antibody in the prevention against novel influenza viruses.


Assuntos
Anticorpos Antivirais/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Reações Cruzadas , Testes de Inibição da Hemaglutinação , Humanos , Tailândia
15.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807240

RESUMO

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE: Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.


Assuntos
Cisteína Endopeptidases/metabolismo , Nucleocapsídeo/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Infecções por Coronavirus/virologia , Genoma Viral , Nucleocapsídeo/química , Proteólise , Suínos , Doenças dos Suínos/virologia , Células Vero
16.
Virology ; 498: 99-108, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27567258

RESUMO

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein.


Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Genes Reporter , Teste de Complementação Genética , Humanos , Fenótipo , Recombinação Genética , Proteínas da Matriz Viral/genética
17.
Arch Virol ; 161(10): 2693-704, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27406044

RESUMO

Recombinant vaccinia viruses harboring the complete hemagglutinin (HA) or neuraminidase (NA) genes from the influenza A/Anhui/1/2013 (H7N9) virus were constructed (rVac-H7 HA and rVac-N9 NA viruses). The HA and NA proteins were expressed in the cytoplasm and on the plasma membrane of thymidine-kinase-negative (TK(-)) cells infected with these recombinant viruses. Only one form of the HA protein was expressed in infected TK(-) cells, with a molecular weight (MW) of 75 kDa, but three forms were found when the culture medium was supplemented with trypsin (MWs of 75, 50 and 27 kDa), which was similar to what was found in Madin-Darby canine kidney (MDCK) cells infected with reverse genetic (rg) influenza viruses carrying HA genes of H7N9 virus origin. One form of hyperglycosylated NA protein with a MW of 75 kDa was produced in rVac-N9-NA-virus-infected TK(-) or MDCK cells. The MW decreased to 55 kDa after deglycosylation. The hyperglycosylated recombinant NA protein demonstrated sialidase activity in a fetuin-based neuraminidase assay. The rVac-H7 HA and rVac-N9 NA viruses elicited significantly higher anti-HA and anti-NA antibody titers in BALB/c mice that were immunized once than in ICR mice. The anti-HA and anti-NA antibodies showed activity against homosubtypic HA or NA, but not against heterosubtypic HA or NA, as determined by hemagglutination-inhibition and microneutralization assays for anti-HA antibodies and neuraminidase-inhibition and replication-inhibition assays for anti-NA antibodies. Taken together, our data demonstrated immunobiological properties of recombinant HA and NA proteins that might be useful for vaccine development.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Subtipo H7N9 do Vírus da Influenza A/imunologia , Neuraminidase/imunologia , Neuraminidase/metabolismo , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Peso Molecular , Neuraminidase/química , Neuraminidase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vírus Vaccinia/genética
18.
PLoS One ; 11(6): e0157287, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27315286

RESUMO

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication.


Assuntos
Vetores Genéticos/genética , Vesiculovirus/genética , Vacinas Virais/uso terapêutico , Replicação Viral/genética , Animais , Regulação Viral da Expressão Gênica/genética , Vetores Genéticos/efeitos adversos , Vetores Genéticos/uso terapêutico , Hemaglutininas/genética , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Camundongos , Vírus da Doença de Newcastle/genética , Fosfoproteínas/genética , Deleção de Sequência/genética , Vesiculovirus/patogenicidade , Vacinas Virais/genética
19.
Virus Res ; 226: 152-171, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27212685

RESUMO

Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/fisiologia , Genética Reversa , Doenças dos Suínos/virologia , Adaptação Biológica/genética , Adaptação Biológica/imunologia , Animais , Linhagem Celular , Genoma Viral , Fases de Leitura Aberta , Receptores Virais/metabolismo , Genética Reversa/métodos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/prevenção & controle , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vacinas Virais/genética , Vacinas Virais/imunologia , Ligação Viral , Cultura de Vírus/métodos
20.
J Virol ; 90(9): 4637-4646, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26912622

RESUMO

UNLABELLED: Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE: Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Resistência à Doença/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/imunologia , Influenza Humana/virologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Farmacorresistência Viral , Feminino , Furões , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Oseltamivir/farmacologia , Carga Viral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA