RESUMO
The outbreak of novel coronavirus (nCoV) or severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in December 2019 in Wuhan, China, has posed an international public health emergency worldwide and forced people to be confined in their homes. This virus is of high-risk category and is declared a pandemic by the World Health Organization (WHO). The worldwide researchers and various health professionals are working together to determine the best way to stop its spread or halt this virus's spread and circumvent this pandemic condition threatening millions of human lives. The absence of definitive treatment is possible to explore to reduce virus infection and enhance patient recovery. Along with off-label medicines, plasma therapy, vaccines, the researchers exploit the various plants/herbs and their constituents to effectively treat nCoV infection. The present study aimed to present brief and most informative salient features of the numerous facts regarding the SARS-CoV-2, including the structure, genomic sequence, recent mutation, targeting possibility, and various hurdles in research progress, and off-labeled drugs, convalescent plasma therapy, vaccine and plants/herbs for the treatment of coronavirus disease-2019 (COVID-19). Results showed that off-labeled drugs such as hydroxychloroquine, dexamethasone, tocilizumab, antiviral drug (remdesivir, favipiravir), etc., give positive results and approved for use or approved for restricted use in some countries like India. Future research should focus on these possibilities that may allow the development of an effective treatment for COVID-19.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Vacinas contra COVID-19/administração & dosagem , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/uso terapêutico , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/virologia , Ensaios Clínicos como Assunto , Quimioterapia Combinada/métodos , Humanos , Terapia de Alvo Molecular/métodos , Mutação , Uso Off-Label , Pandemias/prevenção & controle , Extratos Vegetais/uso terapêutico , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Resultado do Tratamento , Proteínas Estruturais Virais/antagonistas & inibidores , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.
Assuntos
Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Fibroblastos/virologia , Expressão Gênica/efeitos dos fármacos , Fármacos Neuroprotetores , Extratos Vegetais/farmacologia , Tussilago , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Depressão Química , Relação Dose-Resposta a Droga , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/tratamento farmacológico , Prepúcio do Pênis/citologia , Células Hep G2 , Humanos , Masculino , Extratos Vegetais/uso terapêuticoRESUMO
Enterovirus 71 (EV71) can cause central nervous system infections with mortality and neurologic sequelae. At present, there is no effective therapeutic modality for EV71 infection. The infection is more common in families with poor socioeconomic status. Therefore, finding a readily available, cost-effective therapeutic modality would be very helpful to these socioeconomically disadvantaged families. Yakammaoto is a cheap and readily available traditional prescription that is proven to have antiviral activity against coxsackievirus B4 (CVB4). CVB4 and EV71 are enteroviruses. In this study, we evaluated the antiviral activity of hot water extract of yakammaoto against EV71. The results of plaque reduction assay and flow cytometry demonstrated that yakammaoto dose dependently inhibited EV71 infection. In addition, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR results showed that yakammaoto reduced viral replication. Western blotting analysis showed that yakammaoto can inhibit viral protein production. Thus, our results suggest that yakammaoto should be considered to manage EV71 infection in the future.
Assuntos
Antivirais/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Enterovirus Humano A/fisiologia , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/efeitos dos fármacos , Genes Virais , Células Hep G2 , Humanos , Biossíntese de Proteínas , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Ligação Viral , Internalização do Vírus , Replicação Viral/efeitos dos fármacosRESUMO
Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368-607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1-198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection.
Assuntos
Vírus da Hepatite A/imunologia , Hepatite E/imunologia , Imunidade nas Mucosas , Mucosa/imunologia , Tuftsina/imunologia , Vacinas Combinadas/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Feminino , Vírus da Hepatite A/genética , Anticorpos Anti-Hepatite/imunologia , Hepatite E/genética , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina A Secretora/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Tuftsina/genética , Proteínas Estruturais Virais/genéticaRESUMO
Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.
Assuntos
Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Enterovirus Humano A/genética , Proteínas de Fluorescência Verde/análise , Coloração e Rotulagem/métodos , Regiões 5' não Traduzidas , Animais , Chlorocebus aethiops , Enterovirus Humano A/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Recombinação Genética , Células Vero , Proteínas Estruturais Virais/genéticaRESUMO
The production of plant-derived pharmaceuticals essentially requires stable concentrations of plant constituents, especially recombinant proteins; nonetheless, soil and seasonal variations might drastically interfere with this stability. In addition, variability might depend on the plant organ used for production. Therefore, we investigated the variability in plant constituents and antigen expression in potato plants under greenhouse and field growth conditions and in leaves compared to tubers. Using potatoes expressing VP60, the only structural capsid protein of the rabbit haemorrhagic disease virus (RHDV), CTB, the non-toxic B subunit (CTB) of the cholera toxin (CTA-CTB(5)) and the marker protein NPTII (neomycinphosphotransferase) as a model, we compare greenhouse and field production of potato-derived antigens. The influence of the production organ turned out to be transgene specific. In general, yield, plant quality and transgene expression levels in the field were higher than or similar to those observed in the greenhouse. The variation (CV) of major plant constituents and the amount of transgene-encoded protein was not influenced by the higher variation of soil properties observed in the field. Amazingly, for specific events, the variability in the model protein concentrations was often lower under field than under greenhouse conditions. The changes in gene expression under environmental stress conditions in the field observed in another event do not reduce the positive influence on variability since events like these should excluded from production. Hence, it can be concluded that for specific applications, field production of transgenic plants producing pharmaceuticals is superior to greenhouse production, even concerning the stability of transgene expression over different years. On the basis of our results, we expect equal or even higher expression levels with lower variability of recombinant pharmaceuticals in the field compared to greenhouse production combined with approximately 10 times higher tuber yield in the field.
Assuntos
Antígenos Virais/metabolismo , Toxina da Cólera/metabolismo , Solanum tuberosum/metabolismo , Proteínas Estruturais Virais/metabolismo , Agricultura/métodos , Animais , Antígenos Virais/genética , Toxina da Cólera/genética , Ecossistema , Ambiente Controlado , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solo/análise , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Fatores de Tempo , Transgenes/genética , Proteínas Estruturais Virais/genéticaRESUMO
Despite several optimizations, the production of CTB::VP60 antigen fusion proteins in tobacco is still very low. This might be due to the size of the fusion partner VP60 (579 aa). Hence, two different N-terminal truncations of VP60 were fused to CTB, either with or without an ER retention signal. CTB::VP60 expression levels, in vitro and in vivo antigenicity and immunogenicity were analyzed in plants carrying one of four different transgenes. Only one of the truncated CTB::VP60 fusions (365 aa) directed to the endoplasmic reticulum led to similar but not enhanced expression levels as compared to the complete protein in tobacco and possessed similar in vitro antigenicity. In contrast to the complete protein, no anti-VP60-specific antibodies were induced in rabbits after the intramuscular application of plant extracts containing the truncated protein.
Assuntos
Antígenos Virais/imunologia , Toxina da Cólera/imunologia , Nicotiana/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Toxina da Cólera/genética , DNA de Plantas/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Técnicas de Transferência de Genes , Vírus da Doença Hemorrágica de Coelhos/imunologia , Plantas Geneticamente Modificadas/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Nicotiana/imunologia , Transgenes , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
The use of attenuated classical swine fever virus (CSFV) strains as live vaccines is no longer allowed for the control of classical swine fever in Europe, due to the inability to differentiate between infected and vaccinated animals (Differentiating Infected from Vaccinated Animals; DIVA), except as emergency vaccines or as bait vaccines for wild boars. Thus, the establishment of a DIVA vaccine(s) is of pivotal importance for the control of this infectious disease. In this study, recombinant versions of the live-attenuated vaccine strain CSFV Riems were generated by replacing parts of the E2 gene with the corresponding sequence of border disease virus strain Gifhorn. Three cDNA clones were constructed: pRiems-ABC-Gif, pRiems-A-Gif and pRiems-BC-Gif. Infectious particles were obtained from clones pRiems-ABC-Gif and pRiems-BC-Gif only, whereas transfected RNA from clone pRiems-A-Gif behaved like a replicon. Based on its ability to be differentiated in vitro from wild-type CSFV by mAbs, vRiems-ABC-Gif was assessed for immunogenicity and protection against challenge infection in pigs. Before challenge, no CSFV-specific anti-E2 antibodies could be detected with commercial E2-blocking ELISAs in vRiems-ABC-Gif-vaccinated animals, whereas vRiems-vaccinated pigs developed high titres of anti-E2 antibodies, confirming the marker properties of this vaccine candidate. After oral vaccination, only partial protection against challenge infection was observed in the vRiems-ABC-Gif vaccinees, whereas all intramuscularly vaccinated animals and all vRiems-vaccinated animals were fully protected. These experiments suggest that the strategy of exchanging specific antigenic epitopes among pestiviruses is a promising tool for the development of new CSFV marker vaccines.
Assuntos
Vírus da Doença da Fronteira/imunologia , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Genes Virais/genética , Glicoproteínas/biossíntese , Vírus Reordenados/imunologia , Vacinação , Proteínas Estruturais Virais/biossíntese , Vacinas Virais/administração & dosagem , Administração Oral , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Vírus da Doença da Fronteira/química , Vírus da Doença da Fronteira/metabolismo , Linhagem Celular , Peste Suína Clássica/imunologia , Vírus da Febre Suína Clássica/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Injeções Intramusculares , Vírus Reordenados/metabolismo , Suínos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.
Assuntos
Agrobacterium tumefaciens/genética , Antígenos Virais , Imunização , Vacinas/uso terapêutico , Proteínas Estruturais Virais , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/uso terapêutico , Sequência de Bases , Clonagem Molecular , Técnicas de Transferência de Genes , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , RNA/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Vacinas/genética , Vacinas/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/uso terapêuticoRESUMO
New vaccine candidates exploiting the rational combination of regulatory and structural HIV gene products are being developed within the program of the AIDS Vaccine Integrated Project (AVIP) and will be tested in comparative preclinical and clinical trials with the ultimate goal of selecting proper candidates for advanced clinical testing in developing countries.
Assuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells. METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD), Fufang Huangqi (FFHQ) and Bingganling (BGL) were respectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresis after screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After co-culture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCV gene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQ and BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61 respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene in Hela D cells.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Hepacivirus/genética , Transcrição Gênica , Proteínas Estruturais Virais/genética , Sequência de Bases , Sobrevivência Celular , Primers do DNA , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , TransfecçãoRESUMO
The genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26.
Assuntos
Hemípteros/virologia , Insetos Vetores/virologia , Proteínas Virais/metabolismo , Waikavirus/metabolismo , Zea mays/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Dados de Sequência Molecular , Doenças das Plantas/virologia , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Waikavirus/genética , Waikavirus/patogenicidadeRESUMO
Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.
Assuntos
Antígenos Virais/imunologia , Infecções por Caliciviridae/prevenção & controle , Vírus da Doença Hemorrágica de Coelhos/imunologia , Solanum tuberosum , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/genética , Infecções por Caliciviridae/imunologia , Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Vírus da Doença Hemorrágica de Coelhos/genética , Imunização , Plantas Geneticamente Modificadas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/genética , Proteínas Estruturais Virais/genética , Vacinas Virais/genéticaRESUMO
Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient in vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate in tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles.
Assuntos
Mapeamento Cromossômico/métodos , Genes Virais/genética , Genes Virais/fisiologia , Genoma Viral , Genômica/métodos , Potyvirus/genética , Solanum tuberosum/virologia , Proteínas Estruturais Virais/genética , Replicação Viral/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Pegada de DNA , Ordem dos Genes , Mutagênese Insercional/genética , Proteínas Virais/genética , Integração Viral/genética , Replicação Viral/fisiologiaRESUMO
A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.
Assuntos
Cisteína/genética , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Proteínas Virais , Animais , Sequência de Bases , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral , Humanos , Dados de Sequência Molecular , Plasmídeos , Edição de RNA , Transfecção , Células Vero , Proteínas Estruturais Virais/genéticaRESUMO
Two types of strains of serotype I of infectious bursal disease virus (IBDV) have been described, on the basis of their ability (IBDV-TC) or inability (IBDV-BU) to infect chicken embryonic cells in culture. However, both types infect B lymphocytes in the bursa of Fabricius of young chickens. To determine the molecular basis for tissue culture infectivity, virus recombinants with chimeric segments A were constructed from IBDV-TC and IBDV-BU by reverse genetics. The region responsible for the different phenotypes was located in VP2. Site-directed mutagenesis identified single amino acids that are responsible for the restriction in infectivity. However, the appropriate amino acid exchanges are strain-specific.
Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/fisiologia , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Embrião de Galinha , Técnicas de Cultura , DNA Viral , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , RNA Complementar , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.
Assuntos
Antígenos Virais/imunologia , Infecções por Caliciviridae/prevenção & controle , Capsídeo/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Solanum tuberosum , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/genética , Infecções por Caliciviridae/imunologia , Capsídeo/genética , Vírus da Doença Hemorrágica de Coelhos/genética , Plantas Geneticamente Modificadas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/genética , Proteínas Estruturais Virais/genética , Vacinas Virais/genéticaRESUMO
The sequence of the 20 N-terminal amino acids of the viral protein (VPg) which is covalently attached to the genomic RNA of the bean strain of Southern bean mosaic virus (SBMV-B) has been determined. The obtained VPg sequence mapped to position 327 to 346 of the SBMV-B ORF2 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Thus indicating that the sobemovirus genomic arrangement is similar to that of subgroup II luteoviruses. Comparison with other viral sequences revealed a high similarity with the sequence of the ORF2-product of the cowpea strain of SBMV (SBMV-C). No significant similarities were detected with amino acid sequences derived of other sobemoviruses or non-related viruses.
Assuntos
Fabaceae/virologia , Vírus do Mosaico/genética , Fases de Leitura Aberta/genética , Plantas Medicinais , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Genes Virais/genética , Genoma Viral , Dados de Sequência Molecular , Vírus do Mosaico/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Core Viral/química , Proteínas do Core Viral/isolamento & purificação , Proteínas Estruturais Virais/genéticaRESUMO
Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.
Assuntos
Genes Virais , Vírus do Mosaico/genética , Potyvirus/genética , Solanum tuberosum/virologia , Tobamovirus/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Brassica/virologia , Bromovirus/genética , Bromovirus/fisiologia , Clonagem Molecular , Teste de Complementação Genética , Genoma Viral , Solanum lycopersicum/virologia , Vírus do Mosaico/fisiologia , Folhas de Planta , Proteínas do Movimento Viral em Plantas , Poaceae/virologia , Potyvirus/fisiologia , RNA Viral/genética , Proteínas Recombinantes/metabolismo , Tobamovirus/fisiologia , Proteínas Virais/metabolismoRESUMO
Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.