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1.
J Virol Methods ; 286: 113972, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32941977

RESUMO

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , COVID-19 , Teste para COVID-19 , Simulação por Computador , Proteínas do Envelope de Coronavírus , Humanos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , DNA Polimerase Dirigida por RNA , SARS-CoV-2 , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/genética
2.
J Vet Intern Med ; 34(1): 307-321, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31793692

RESUMO

BACKGROUND: Prolonged exposure to environmental antigens or allergens elicits an immune response in both healthy horses and those with mild asthma. Corticosteroids often are used to treat lower airway inflammation. OBJECTIVE: To investigate the changes in equine herpesvirus (EHV)-1,2,4,5 glycoprotein B gene expression and changes in respiratory bacterial and fungal communities after nebulized dexamethasone treatment of horses with asthma. ANIMALS: Horses with naturally occurring mild asthma (n = 16) and healthy control horses (n = 4). METHODS: Prospective, randomized, controlled, blinded clinical trial. Polymerase chain reaction amplification of EHV-1,2,4,5 in bronchoalveolar lavage fluid, and 16S (microbiome) and ITS2 (mycobiome) genes with subsequent sequencing was performed on DNA extracted from nasal swabs and transendoscopic tracheal aspirates before and after 13 days treatment with nebulized dexamethasone (15 mg q24h) and saline (control). RESULTS: Nebulized dexamethasone treatment decreased microbial diversity; relative abundance of 8 genera in the upper respiratory tract were altered. For both the microbiota and the mycobiota, environment had a dominant effect over treatment. Alternaria, an opportunistic pathogen and allergen in humans recognized as a risk factor for asthma, asthma severity, and exacerbations, was increased with treatment. Treatment affected relative quantification of the equine gamma herpesviruses (EHV-2 and -5); EHV-2 DNA levels increased and those of EHV-5 decreased. CONCLUSIONS: Nebulized dexamethasone treatment affected the upper respiratory tract microbiota, but not the mycobiota, which was overwhelmed by the effect of a sustained dusty environment.


Assuntos
Asma/veterinária , Bactérias/efeitos dos fármacos , Dexametasona/uso terapêutico , Fungos/efeitos dos fármacos , Herpesviridae/efeitos dos fármacos , Sistema Respiratório/microbiologia , Animais , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Herpesviridae/classificação , Doenças dos Cavalos , Cavalos , Nebulizadores e Vaporizadores , Terapia Respiratória
3.
BMC Vet Res ; 15(1): 397, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694631

RESUMO

BACKGROUND: Mild equine asthma is a common inflammatory airway disease of the horse. The primary treatment of mild equine asthma is corticosteroids. The purpose of this study was to investigate the effects of injected dexamethasone on relative IL-1ß, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-17, IL-23, IFN-γ, Eotaxin-2 and TNF-α mRNA expression in bronchoalveolar lavage (BAL) fluid in healthy Thoroughbred horses (n = 6), and those with mild equine asthma (n = 7). RESULTS: Horses with mild equine asthma had a significantly greater bronchoalveolar lavage mast cell percentage than healthy horses both before and after treatment. Mild equine asthma was associated with a 4.95-fold up-regulation of IL-17 (p = 0.026) and a 2.54-fold down-regulation of IL-10 (p = 0.049) compared to healthy horses. TNF-α was down-regulated in response to dexamethasone treatment in both healthy horses (3.03-fold, p = 0.023) and those with mild equine asthma (1.75-fold, p = 0.023). IL-5 was also down-regulated in horses with mild asthma (2.17-fold, p = 0.048). CONCLUSIONS: Horses with mild equine asthma have a lower concentration of IL-10 in BAL fluid than healthy controls which concurs with human asthmatics. The marked up-regulation of IL-17 in horses with mild asthma suggests these horses had a true tendency of "allergic" airway inflammation in response to environmental allergens. Dexamethasone administration exerted anti-inflammatory effects associated with down-regulation of TNF-α in all horses, and decreased levels of IL-5 mRNA expression in horses with mild equine asthma. The inhibition of the Th-2 response, without any alterations to the airway cytology, indicates that maintained exposure to environmental allergens perpetuates airway inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Asma/veterinária , Líquido da Lavagem Broncoalveolar/química , Citocinas/metabolismo , Dexametasona/farmacologia , Doenças dos Cavalos/tratamento farmacológico , Animais , Asma/tratamento farmacológico , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/genética , Cavalos , Masculino , Mastócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
PLoS One ; 10(2): e0117514, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25658101

RESUMO

Hepatitis C virus (HCV) is a small positive-sense single-stranded RNA virus that causes severe liver diseases. Current anti-HCV therapies involving direct-acting antivirals have significantly enhanced efficacy in comparison to traditional interferon and ribavirin combination. However, further improvement is needed to eradicate HCV. Anacardic acid (AnA) is a phytochemical compound that can inhibit the activity of various cellular enzymes including histone acetyltransferases (HATs). In this study, we investigated the effects of AnA on different phases of HCV life cycle. Our data showed that AnA can inhibit HCV entry, replication, translation, and virion secretion in a dose-dependent manner with no measurable effects on cell viability. In addition, we showed that two HAT inhibitors and knocking down HAT (PCAF) by RNAi can reduce HCV replication, suggesting a mechanism of AnA's inhibitory effects on HCV. Elucidation of the AnA-mediated inhibitory mechanism should facilitate the development of new drug candidates for HCV infection.


Assuntos
Ácidos Anacárdicos/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Hepacivirus/fisiologia , Histona Acetiltransferases/metabolismo , Humanos
5.
PLoS One ; 8(11): e79165, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24265752

RESUMO

Reassortment of influenza A virus genes enables antigenic shift resulting in the emergence of pandemic viruses with novel hemagglutinins (HA) acquired from avian strains. Here, we investigated whether historic and contemporary avian strains with different replication capacity in human cells can donate their hemagglutinin to a pandemic human virus. We performed double-infections with two avian H3 strains as HA donors and a human acceptor strain, and determined gene compositions and replication of HA reassortants in mammalian cells. To enforce selection for the avian virus HA, we generated a strictly elastase-dependent HA cleavage site mutant from A/Hong Kong/1/68 (H3N2) (Hk68-Ela). This mutant was used for co-infections of human cells with A/Duck/Ukraine/1/63 (H3N8) (DkUkr63) or the more recent A/Mallard/Germany/Wv64-67/05 (H3N2) (MallGer05) in the absence of elastase but presence of trypsin. Among 21 plaques analyzed from each assay, we found 12 HA reassortants with DkUkr63 (4 genotypes) and 14 with MallGer05 (10 genotypes) that replicated in human cells comparable to the parental human virus. Although DkUkr63 replicated in mammalian cells at a reduced level compared to MallGer05 and Hk68, it transmitted its HA to the human virus, indicating that lower replication efficiency of an avian virus in a mammalian host may not constrain the emergence of viable HA reassortants. The finding that HA and HA/NA reassortants replicated efficiently like the human virus suggests that further HA adaptation remains a relevant barrier for emergence of novel HA reassortants.


Assuntos
Aptidão Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus Reordenados/genética , Vírus Reordenados/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Cães , Humanos , Vírus da Influenza A Subtipo H3N2/metabolismo , Mutação , Elastase Pancreática/metabolismo , Proteólise , Vírus Reordenados/metabolismo , Replicação Viral
6.
World J Gastroenterol ; 19(47): 8929-39, 2013 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-24379618

RESUMO

Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.


Assuntos
Hepacivirus/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Animais , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/patogenicidade , Humanos , Proteínas Virais/genética , Replicação Viral
7.
Proc Natl Acad Sci U S A ; 109(7): 2579-84, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22308331

RESUMO

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Animais , Aves , Vírus da Influenza A/patogenicidade
8.
J Virol ; 85(15): 7730-41, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21613402

RESUMO

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.


Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A Subtipo H7N7/fisiologia , Influenza Aviária/metabolismo , Interferons/biossíntese , Animais , Sequência de Bases , Galinhas , Primers do DNA , Imuno-Histoquímica , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/fisiologia
9.
J Gen Virol ; 92(Pt 8): 1843-1853, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21525207

RESUMO

In the field, highly pathogenic avian influenza viruses (HPAIV) originate from low-pathogenic strains of the haemagglutinin (HA) serotypes H5 and H7 that have acquired a polybasic HA cleavage site. This observation suggests the presence of a cryptic virulence potential of H5 and H7 low-pathogenic avian influenza viruses (LPAIV). Among all other LPAIV, the H9N2 strains are of particular relevance as they have become widespread across many countries in several avian species and have been transmitted to humans. To assess the potential of these strains to transform into an HPAIV, we introduced a polybasic cleavage site into the HA of a contemporary H9N2 isolate. Whereas the engineered polybasic HA cleavage site mutant remained a low-pathogenic strain like its parent virus, a reassortant expressing the modified H9 HA with engineered polybasic cleavage site and all the other genes from an H5N1 HPAIV became highly pathogenic in chicken with an intravenous pathogenicity index of 1.23. These results suggest that an HPAIV with a subtype other than H5 or H7 would only emerge under conditions where the HA gene could acquire a polybasic cleavage site and the other viral genes carry additional virulence determinants.


Assuntos
Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/virologia , Vírus Reordenados/patogenicidade , Motivos de Aminoácidos , Animais , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H9N2/metabolismo , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Vírus Reordenados/genética , Vírus Reordenados/metabolismo , Organismos Livres de Patógenos Específicos , Virulência
10.
J Gen Virol ; 92(Pt 1): 51-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20881092

RESUMO

The prime virulence determinant of highly pathogenic avian influenza viruses (HPAIVs) is the polybasic haemagglutinin (HA) cleavage site. However, engineering of a polybasic cleavage site into an avian influenza virus of low pathogenicity does not result in transformation into an HPAIV, indicating the importance of other adaptations. Here, the influence of amino acids adjacent to the HA cleavage site on virulence was studied. Most HPAIVs of subtype H5 carry serine or threonine at position 346 (corresponding to position 323 according to H3 numbering), whereas almost all low-pathogenic H5 viruses have valine. Moreover, all H5 low-pathogenic strains carry threonine at position 351 (corresponding to position 328 according to H3 numbering), suggesting that acquisition of a polybasic cleavage site involves several steps. This study generated a virus mutant derived from HPAIV A/Swan/Germany/R65/06 H5N1 (R65) with a monobasic cleavage site, R65(mono)-S-ER, and the following additional mutants: R65(mono)-V-ER with serine changed to valine at position 346, and R65(mono)-S-ETR and R65(mono)-V-ETR with threonine inserted at position 351. Moreover, in the R65 HA, serine was replaced with valine at position 346 (R65-V). Infection of chickens with R65(mono)-S-ETR or R65(mono)-S-ER led to slight transient respiratory symptoms, whereas R65-infected animals died within 2 days. However, chickens infected with R65-V survived longer than R65-infected animals, indicating that serine 346 in R65 HA contributes to virulence. These data suggest that evolution of H5 HPAIVs from low-pathogenic precursors, besides acquisition of a polybasic cleavage site, involves adaptation of neighbouring regions.


Assuntos
Motivos de Aminoácidos/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/patologia , Influenza Aviária/virologia , Substituição de Aminoácidos/genética , Animais , Galinhas , Alemanha , Influenza Aviária/mortalidade , Mutagênese Insercional , Mutação de Sentido Incorreto , Sistema Respiratório/patologia , Sistema Respiratório/virologia , Análise de Sobrevida , Virulência
11.
PLoS One ; 5(7): e11826, 2010 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-20676399

RESUMO

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05(poly)). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HA(R65)) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HA(TG05poly)). In vitro, TG05(poly) and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05(poly), TG05-HA(R65), and R65-HA(TG05poly) are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05(poly) led to temporary non-lethal disease in all animals, the reassortant TG05-HA(R65) caused death in 3 of 10 animals. Furthermore, the reassortant R65-HA(TG05poly) displayed the highest lethality as 8 of 10 chickens died, resembling "natural" HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas do Envelope Viral/metabolismo , Virulência/genética , Animais , Western Blotting , Linhagem Celular , Galinhas , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imuno-Histoquímica , Virus da Influenza A Subtipo H5N1/genética , Proteínas do Envelope Viral/genética
12.
FASEB J ; 23(6): 1643-54, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19144697

RESUMO

Late-onset Alzheimer's disease is often connected with nutritional misbalance, such as enhanced cholesterol intake, deficiency in polyunsaturated fatty acids, or hypovitaminosis. The alpha-secretase ADAM10 has been found to be regulated by retinoic acid, the bioreactive metabolite of vitamin A. Here we show that retinoids induce gene expression of ADAM10 and alpha-secretase activity by nonpermissive retinoid acid receptor/retinoid X receptor (RAR/RXR) heterodimers, whereby alpha- and beta-isotypes of RAR play a major role. However, ligands of other RXR binding partners, such as the vitamin D receptor, do not stimulate alpha-secretase activity. On the basis of these findings, we examined the effect of synthetic retinoids and found a strong enhancement of nonamyloidogenic processing of the amyloid precursor protein by the vitamin A analog acitretin: it stimulated ADAM10 promoter activity with an EC(50) of 1.5 microM and led to an increase of mature ADAM10 protein that resulted in a two- to three-fold increase of the ratio between alpha- and beta-secretase activity in neuroblastoma cells. The alpha-secretase stimulation by acitretin was completely inhibited by the ADAM10-specific inhibitor GI254023X. Intracerebral injection of acitretin in APP/PS1-21 transgenic mice led to a reduction of Abeta(40) and Abeta(42). The results of this study may have clinical relevance because acitretin has been approved for the treatment of psoriasis since 1997 and found generally safe for long-term use in humans.


Assuntos
Proteínas ADAM/metabolismo , Acitretina/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ceratolíticos/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Acitretina/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Receptores X do Fígado , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Receptores Nucleares Órfãos , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptores X de Retinoides/genética , Tretinoína/química , Tretinoína/metabolismo , Regulação para Cima
13.
Nucleic Acids Res ; 36(21): e139, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18832366

RESUMO

Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.


Assuntos
Clonagem Molecular/métodos , Genes Virais , Vírus da Influenza A/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Primers do DNA , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Fatores de Tempo
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