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1.
Vaccines (Basel) ; 10(11)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36423034

RESUMO

Chikungunya virus (CHIKV) re-emergence in the last decade has resulted in explosive epidemics. Along with the classical symptoms of fever and debilitating arthralgia, there were occurrences of unusual clinical presentations such as neurovirulence and mortality. These generated a renewed global interest to develop prophylactic vaccines. Here, using the classical approach of virus attenuation, we developed an attenuated CHIKV strain (RGCB355/KL08-p75) for the purpose. Repeated passaging (75 times) of a local clinical isolate of ECSA lineage virus in U-87 MG human astrocytoma cells, an interferon-response-deficient cell line, resulted in efficient adaptation and attenuation. While experimental infection of 3-day old CHIKV-susceptible BALB/c pups with the parent strain RGCB355/KL08-p4 resulted in death of all the animals, there was 100% survival in mice infected with the attenuated p75. In adult, immunocompetent, CHIKV-non-susceptible C57BL/6 mice, inoculation with p75 induced high antibody response without any signs of disease. Both p4 and p75 strains are uniformly lethal to interferon-response-deficient AG129 mice. Passive protection studies in AG129 mice using immune serum against p75 resulted in complete survival. Whole-genome sequencing identified novel mutations that might be responsible for virus attenuation. Our results establish the usefulness of RGCB355/KL08-p75 as a strain for vaccine development against chikungunya.

2.
Mol Ther ; 30(5): 2058-2077, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34999210

RESUMO

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.


Assuntos
COVID-19 , Vacinas contra Dengue , Vírus da Dengue , Dengue , Vacinas de DNA , Anticorpos Neutralizantes , Anticorpos Antivirais , Dengue/prevenção & controle , Vacinas contra Dengue/genética , Vírus da Dengue/genética , Humanos , Pandemias , Proteínas do Envelope Viral/genética
3.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34328830

RESUMO

The 5' capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.


Assuntos
Autofagia , Vírus Chikungunya/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imidazóis/farmacologia , Biossíntese de Proteínas , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Apoptose , Linhagem Celular Tumoral , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Capuzes de RNA , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 114(7): 1666-1671, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28143925

RESUMO

Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Vírus Chikungunya/metabolismo , N-Glicosil Hidrolases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Aedes/virologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Linhagem Celular , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Chlorocebus aethiops , Insetos Vetores/virologia , N-Glicosil Hidrolases/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Células Vero , Proteínas não Estruturais Virais/genética , Virulência/genética , Replicação Viral/genética
5.
Infect Genet Evol ; 37: 174-84, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26611825

RESUMO

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Mutação , Proteínas do Envelope Viral/genética , Linhagem Celular , Vírus Chikungunya/isolamento & purificação , Evolução Molecular , Genótipo , Humanos , Técnicas In Vitro , Filogenia , RNA Viral/análise , Seleção Genética
6.
Infect Genet Evol ; 34: 402-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26160542

RESUMO

The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come.


Assuntos
Vírus da Dengue/genética , Dengue/virologia , Aedes/virologia , Animais , Linhagem Celular , Cricetinae , Dengue/epidemiologia , Vírus da Dengue/fisiologia , Surtos de Doenças , Genótipo , Insetos Vetores/virologia , Nepal/epidemiologia , Filogenia , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Replicação Viral
7.
Nat Commun ; 5: 4084, 2014 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-24933611

RESUMO

Host species-specific fitness landscapes largely determine the outcome of host switching during pathogen emergence. Using chikungunya virus (CHIKV) to study adaptation to a mosquito vector, we evaluated mutations associated with recently evolved sub-lineages. Multiple Aedes albopictus-adaptive fitness peaks became available after CHIKV acquired an initial adaptive (E1-A226V) substitution, permitting rapid lineage diversification observed in nature. All second-step mutations involved replacements by glutamine or glutamic acid of E2 glycoprotein amino acids in the acid-sensitive region, providing a framework to anticipate additional A. albopictus-adaptive mutations. The combination of second-step adaptive mutations into a single, 'super-adaptive' fitness peak also predicted the future emergence of CHIKV strains with even greater transmission efficiency in some current regions of endemic circulation, followed by their likely global spread.


Assuntos
Aedes/virologia , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Evolução Molecular , Insetos Vetores/virologia , Animais , Febre de Chikungunya/transmissão , Vírus Chikungunya/classificação , Vírus Chikungunya/fisiologia , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas do Envelope Viral/genética
8.
PLoS One ; 8(9): e75854, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086645

RESUMO

Chikungunya virus (CHIKV), an arthritogenic old-world alphavirus, has been implicated in the central nervous system (CNS) infection in infants and elderly patients. Astrocytes are the major immune cells of the brain parenchyma that mediate inflammation. In the present study we found that a local isolate of CHIKV infect and activate U-87 MG cells, a glioblastoma cell line of human astrocyte origin. The infection kinetics were similar in infected U-87 MG cells and the human embryo kidney (HEK293) cells as indicated by immunofluorescence and plaque assays, 24h post-infection (p.i.). In infected U-87 MG cells, apoptosis was detectable from 48h p.i. evidenced by DNA fragmentation, PARP cleavage, loss of mitochondrial membrane potential, nuclear condensation and visible cytopathic effects in a dose and time-dependent manner. XBP1 mRNA splicing and eIF2α phosphorylation studies indicated the occurrence of endoplasmic reticulum stress in infected cells. In U-87 MG cells stably expressing a green fluorescent protein-tagged light chain-3 (GFP-LC3) protein, CHIKV infection showed increased autophagy response. The infection led to an enhanced expression of the mRNA transcripts of the pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 and CXCL9 within 24h p.i. Significant up-regulation of the proteins of RIG-I like receptor (RLR) pathway, such as RIG-I and TRAF-6, was observed indicating the activation of the cytoplasmic-cellular innate immune response. The overall results show that the U-87 MG cell line is a potential in vitro model for in depth study of these molecular pathways in response to CHIKV infection. The responses in these cells of CNS origin, which are inherently defective in Type I interferon response, could be analogous to that occurring in infants and very old patients who also have a compromised interferon-response. The results also point to the intriguing possibility of using this virus for studies to develop oncolytic virus therapy approaches against glioblastoma, a highly aggressive malignancy.


Assuntos
Apoptose/fisiologia , Astrócitos/virologia , Vírus Chikungunya/metabolismo , Glioblastoma/virologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Fragmentação do DNA , Estresse do Retículo Endoplasmático/fisiologia , Imunofluorescência , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Terapia Viral Oncolítica/métodos , Ensaio de Placa Viral
9.
Virol J ; 10: 37, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23360780

RESUMO

BACKGROUND: Local epidemiology of Dengue is defined by the genetic diversity of the circulating Dengue virus (DENV) strains. This important information is not available for the virus strains from most parts of the Indian subcontinent. The present study focused on the genetic diversity of the serotype 3 DENV strains (DENV-3) from India. RESULTS: A total of 22 DENV-3 strains identified by reverse-transcription PCR analysis of serum samples from 709 patients were studied. These samples were collected over a period of 4 years (2008-2011) from dengue fever suspected patients from Kerala, a dengue endemic state in South India. Comparison of a 1740bp nucleotide sequence of the viral Capsid-Pre-membrane-Envelope coding region of our strains and previously reported DENV-3 strains from India, South Asia and South America revealed non-synonymous substitutions that were genotype III-specific as well as sporadic. Evidence of positive selection was detected in the I81 amino acid residue of the envelope protein. Out of the 22 samples, three had I81A and 18 had I81V substitutions. In the phylogenetic analysis by maximum likelihood method the strains from Kerala clustered in two different lineages (lineage III and IV) within genotype III clade of DENV-3 strains. The ten strains that belonged to lineage IV had a signature amino acid substitution T219A in the envelope protein. Interestingly, all these strains were found to be closely related to a Singapore strain GU370053 isolated in 2007. CONCLUSIONS: Our study identifies for the first time the presence of lineage IV strains in the Indian subcontinent. Results indicate the possibility of a recent exotic introduction and also a shift from the existing lineage III strains to lineage IV. Lineage shifts in DENV-3 strains have been attributed to dramatic increase in disease severity in many parts of the world. Hence the present observation could be significant in terms of the clinical severity of future dengue cases in the region.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Dengue/virologia , Variação Genética , Análise por Conglomerados , Dengue/epidemiologia , Vírus da Dengue/isolamento & purificação , Genótipo , Humanos , Índia/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Soro/virologia , Proteínas Estruturais Virais/genética
10.
Virol J ; 8: 363, 2011 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-21781334

RESUMO

BACKGROUND: The human hepatitis B virus (HBV), a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC). The duck hepatitis B virus (DHBV) infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference. FINDINGS: A subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH) identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD), Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study. CONCLUSIONS: The present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.


Assuntos
Perfilação da Expressão Gênica , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Vírus da Hepatite B do Pato/patogenicidade , Hepatócitos/virologia , Animais , Células Cultivadas , Patos , Biblioteca Gênica , Hibridização de Ácido Nucleico
11.
Virol J ; 7: 189, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20704755

RESUMO

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by infected Aedes (Ae.) aegypti and Ae.albopictus mosquitoes. In the study, reverse-transcription PCR (RT PCR) and virus isolation detected CHIKV in patient samples and also in adult Ae.albopictus mosquitoes that was derived from larvae collected during a chikungunya (CHIK) outbreak in Kerala in 2009. The CHIKV strains involved in the outbreak were the East, Central and South African (ECSA) genotype that had the E1 A226V mutation. The viral strains from the mosquitoes and CHIK patients from the same area showed a close relationship based on phylogenetic analysis. Genetic characterization by partial sequencing of non-structural protein 2 (nsP2; 378 bp), envelope E1 (505 bp) and E2 (428 bp) identified one critical mutation in the E2 protein coding region of these CHIKV strains. This novel, non-conservative mutation, L210Q, consistently present in both human and mosquito-derived samples studied, was within the region of the E2 protein (amino acids E2 200-220) that determines mosquito cell infectivity in many alpha viruses. Our results show the involvement of Ae. albopictus in this outbreak in Kerala and appearance of CHIKV with novel genetic changes. Detection of virus in adult mosquitoes, emerged in the laboratory from larvae, also points to the possibility of transovarial transmission (TOT) of mutant CHIKV strains in mosquitoes.


Assuntos
Aedes/virologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/isolamento & purificação , Surtos de Doenças , Substituição de Aminoácidos/genética , Animais , Vírus Chikungunya/genética , Análise por Conglomerados , Humanos , Índia/epidemiologia , Mutação de Sentido Incorreto , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Cultura de Vírus
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