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1.
Nat Microbiol ; 9(9): 2383-2394, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39179693

RESUMO

Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization.


Assuntos
COVID-19 , Sistema Nervoso Central , Pulmão , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Tropismo Viral , Animais , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Camundongos , COVID-19/virologia , Pulmão/virologia , Pulmão/patologia , Sistema Nervoso Central/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Furina/metabolismo , Furina/genética , Virulência , Humanos , Mutação , Evolução Molecular , Feminino , Modelos Animais de Doenças
2.
J Immunol ; 213(5): 612-618, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39007643

RESUMO

Breast milk confers multiple benefits to the neonate, including passive immunity against multiple microorganisms via Abs. However, it remains unclear whether breast milk-derived Abs affect vaccine-induced immunity in the neonate. We evaluated in C57BL/6 and BALB/c mice whether breastfeeding from an mRNA-SARS-CoV-2-vaccinated dam affects vaccine-induced immunity in neonate mice. Using an experimental model that allows the distinction of maternal Abs and neonate Abs based on their allotype, we show that breastfeeding from an immune dam is associated with reduced vaccine immunity in the neonate. Importantly, mice that breastfed from an immune dam showed reduced numbers of plasma cells after vaccination, relative to mice that breastfed from a naive dam. Our subsequent studies using an mRNA-luciferase reporter system show that passive transfer of Abs through breastfeeding accelerates the clearance of vaccine Ag in suckling mice, resulting in reduced Ag availability. Altogether, maternal Abs transferred through breast milk can protect against infectious microorganisms, but they may also interfere with the neonate's response to vaccination by accelerating the clearance of vaccine Ag. These findings are important for understanding the effects of maternal Abs on the neonate's response to vaccines and may provide insights for improving neonatal vaccines.


Assuntos
Animais Recém-Nascidos , Imunidade Materno-Adquirida , Camundongos Endogâmicos BALB C , Leite Humano , Animais , Camundongos , Feminino , Imunidade Materno-Adquirida/imunologia , Leite Humano/imunologia , Animais Lactentes/imunologia , Camundongos Endogâmicos C57BL , Vacinação , Humanos , Aleitamento Materno
3.
J Clin Invest ; 134(15)2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861331

RESUMO

Viral vectors are being used for the treatment of cancer. Yet, their efficacy varies among tumors and their use poses challenges in immunosuppressed patients, underscoring the need for alternatives. We report striking antitumoral effects by a nonlytic viral vector based on attenuated lymphocytic choriomeningitis virus (r3LCMV). We show in multiple tumor models that injection of tumor-bearing mice with this vector results in improved tumor control and survival. Importantly, r3LCMV improved tumor control in immunodeficient Rag1-/- mice and MyD88-/- mice, suggesting that multiple pathways contributed to the antitumoral effects. The antitumoral effects of r3LCMV were also observed when this vector was administered several weeks before tumor challenges, suggesting the induction of trained immunity. Single-cell RNA sequencing analyses, antibody blockade experiments, and knockout models revealed a critical role for host-intrinsic IFN-I in the antitumoral efficacy of r3LCMV vectors. Collectively, these data demonstrate potent antitumoral effects by r3LCMV vectors and unveil multiple mechanisms underlying their antitumoral efficacy.


Assuntos
Vetores Genéticos , Interferon Tipo I , Vírus da Coriomeningite Linfocítica , Camundongos Knockout , Animais , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Interferon Tipo I/imunologia , Interferon Tipo I/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Humanos , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Neoplasias Experimentais/patologia , Proteínas de Homeodomínio
4.
bioRxiv ; 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38496467

RESUMO

mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.

5.
bioRxiv ; 2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38106001

RESUMO

Viral vectors are being used for the treatment of cancer. Yet their efficacy varies among tumors and their use poses challenges in immunosuppressed patients, underscoring the need for alternatives. We report striking antitumoral effects by a nonlytic viral vector based on attenuated lymphocytic choriomeningitis virus (r3LCMV). We show in multiple tumor models that injection of tumor-bearing mice with this novel vector results in improved tumor control and survival. Importantly, r3LCMV also improved tumor control in immunodeficient Rag1-/- mice. Single cell RNA-Seq analyses, antibody blockade experiments, and KO models revealed a critical role for host IFN-I in the antitumoral efficacy of r3LCMV vectors. Collectively, these data demonstrate potent antitumoral effects by a replication-attenuated LCMV vector and unveil mechanisms underlying its antitumoral efficacy.

6.
Res Sq ; 2023 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-37790412

RESUMO

Severe COVID-19 and post-acute sequelae of SARS-CoV-2 infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here, we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral diversity were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the Spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titers and decreased morbidity compared to the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but this required reversion of the FCS deletion. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization with possible implications for the treatment of neuroinvasive COVID-19.

7.
Cell Rep ; 42(3): 112167, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36857186

RESUMO

mRNA vaccines are effective in preventing severe COVID-19, but breakthrough infections, emerging variants, and waning immunity warrant the use of boosters. Although mRNA boosters are being implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we report that lower antibody levels before boost are associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the immunogenicity of mRNA vaccines. Our studies in mice show that pre-existing antibodies accelerate the clearance of vaccine antigen via Fc-dependent mechanisms, limiting the amount of antigen available to prime B cell responses after mRNA boosters. These data demonstrate a "tug of war" between pre-existing antibody responses and de novo B cell responses following mRNA vaccination, and they suggest that transient downmodulation of antibody effector function may improve the efficacy of mRNA boosters.


Assuntos
Vacina BNT162 , COVID-19 , Animais , Humanos , Camundongos , COVID-19/prevenção & controle , Imunização Secundária , Anticorpos , RNA Mensageiro/genética , Vacinas de mRNA , Anticorpos Antivirais , Anticorpos Neutralizantes
8.
J Clin Invest ; 132(23)2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36219482

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main antigen in all approved COVID-19 vaccines and is also the only target for monoclonal antibody (mAb) therapies. Immune responses to other viral antigens are generated after SARS-CoV-2 infection, but their contribution to the antiviral response remains unclear. Here, we interrogated whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine and then transferred sera from these mice into naive mice, followed by challenge with SARS-CoV-2. We show that mice that received nucleocapsid-specific sera or a nucleocapsid-specific mAb exhibited enhanced control of SARS-CoV-2. Nucleocapsid-specific antibodies elicited NK-mediated, antibody-dependent cellular cytotoxicity (ADCC) against infected cells. To our knowledge, these findings provide the first demonstration in the coronavirus literature that antibody responses specific to the nucleocapsid protein can improve viral clearance, providing a rationale for the clinical evaluation of nucleocapsid-based mAb therapies to treat COVID-19.


Assuntos
Anticorpos Monoclonais , COVID-19 , Nucleocapsídeo , Animais , Camundongos , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais , COVID-19/terapia , Vacinas contra COVID-19 , Nucleocapsídeo/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Front Immunol ; 13: 908707, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35958615

RESUMO

T cell-based therapies have been widely explored for the treatment of cancer and chronic infection, but B cell-based therapies have remained largely unexplored. To study the effect of B cell therapy, we adoptively transferred virus-specific B cells into mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). Adoptive transfer of virus-specific B cells resulted in increase in antibody titers and reduction of viral loads. Importantly, the efficacy of B cell therapy was partly dependent on antibody effector functions, and was improved by co-transferring virus-specific CD4 T cells. These findings provide a proof-of-concept that adoptive B cell therapy can be effective for the treatment of chronic infections, but provision of virus-specific CD4 T cells may be critical for optimal virus neutralization.


Assuntos
Coriomeningite Linfocítica , Animais , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia Adotiva , Coriomeningite Linfocítica/terapia , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL
10.
bioRxiv ; 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35794898

RESUMO

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.

11.
J Clin Invest ; 131(24)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34623973

RESUMO

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have shown efficacy against SARS-CoV-2, it is unknown if coronavirus vaccines can also protect against other coronaviruses that may infect humans in the future. Here, we show that coronavirus vaccines elicited cross-protective immune responses against heterologous coronaviruses. In particular, we show that a severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) vaccine developed in 2004 and known to protect against SARS-CoV-1 conferred robust heterologous protection against SARS-CoV-2 in mice. Similarly, prior coronavirus infections conferred heterologous protection against distinct coronaviruses. Cross-reactive immunity was also reported in patients with coronavirus disease 2019 (COVID-19) and in individuals who received SARS-CoV-2 vaccines, and transfer of plasma from these individuals into mice improved protection against coronavirus challenges. These findings provide the first demonstration to our knowledge that coronavirus vaccines (and prior coronavirus infections) can confer broad protection against heterologous coronaviruses and establish a rationale for universal coronavirus vaccines.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Animais , Linfócitos T CD8-Positivos/citologia , Reações Cruzadas , Mapeamento de Epitopos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , SARS-CoV-2 , Vacinação
12.
Sci Immunol ; 6(66): eabi8635, 2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34648369

RESUMO

SARS-CoV-2 has caused a global pandemic that has infected more than 250 million people worldwide. Although several vaccine candidates have received emergency use authorization, there is still limited knowledge on how vaccine dosing affects immune responses. We performed mechanistic studies in mice to understand how the priming dose of an adenovirus-based SARS-CoV-2 vaccine affects long-term immunity to SARS-CoV-2. We first primed C57BL/6 mice with an adenovirus serotype 5 vaccine encoding the SARS-CoV-2 spike protein, similar to that used in the CanSino and Sputnik V vaccines. The vaccine prime was administered at either a standard dose or 1000-fold lower dose, followed by a boost with the standard dose 4 weeks later. Initially, the low dose prime induced lower immune responses relative to the standard dose prime. However, the low dose prime elicited immune responses that were qualitatively superior and, upon boosting, exhibited substantially more potent recall and functional capacity. We also report similar effects with a simian immunodeficiency virus (SIV) vaccine. These findings show an unexpected advantage of fractionating vaccine prime doses, warranting a reevaluation of vaccine trial protocols for SARS-CoV-2 and other pathogens.


Assuntos
Vacinas contra COVID-19/imunologia , Imunogenicidade da Vacina , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas contra COVID-19/química , Relação Dose-Resposta Imunológica , Feminino , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Cell Rep ; 36(10): 109664, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34450033

RESUMO

SARS-CoV-2 infection causes respiratory insufficiency and neurological manifestations, including loss of smell and psychiatric disorders, and can be fatal. Most vaccines are based on the spike antigen alone, and although they have shown efficacy at preventing severe disease and death, they do not always confer sterilizing immunity. Here, we interrogate whether SARS-CoV-2 vaccines could be improved by incorporating nucleocapsid as an antigen. We show that, after 72 h of challenge, a spike-based vaccine confers acute protection in the lung, but not in the brain. However, combining a spike-based vaccine with a nucleocapsid-based vaccine confers acute protection in both the lung and brain. These findings suggest that nucleocapsid-specific immunity can improve the distal control of SARS-CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.


Assuntos
Vacinas contra COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/virologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/administração & dosagem , Humanos , Imunogenicidade da Vacina , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Fosfoproteínas/imunologia , Carga Viral/efeitos dos fármacos
14.
bioRxiv ; 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33821275

RESUMO

Since late 2019, SARS-CoV-2 has caused a global pandemic that has infected 128 million people worldwide. Although several vaccine candidates have received emergency use authorization (EUA), there are still a limited number of vaccine doses available. To increase the number of vaccinated individuals, there are ongoing discussions about administering partial vaccine doses, but there is still a paucity of data on how vaccine fractionation affects vaccine-elicited immunity. We performed studies in mice to understand how the priming dose of a SARS CoV-2 vaccine affects long-term immunity to SARS CoV-2. We first primed C57BL/6 mice with an adenovirus-based vaccine encoding SARS CoV-2 spike protein (Ad5-SARS-2 spike), similar to that used in the CanSino and Sputnik V vaccines. This prime was administered either at a low dose (LD) of 10 6 PFU or at a standard dose (SD) of 10 9 PFU, followed by a SD boost in all mice four weeks later. As expected, the LD prime induced lower immune responses relative to the SD prime. However, the LD prime elicited immune responses that were qualitatively superior, and upon boosting, mice that were initially primed with a LD exhibited significantly more potent immune responses. Overall, these data demonstrate that limiting the priming dose of a SARS CoV-2 vaccine may confer unexpected benefits. These findings may be useful for improving vaccine availability and for rational vaccine design.

15.
Curr Protoc Immunol ; 130(1): e99, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32940427

RESUMO

In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: LCMV infection routes in mice Support Protocol 1: Preparation of LCMV stocks ASSAYS TO MEASURE LCMV TITERS Support Protocol 2: Plaque assay Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC) Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody Support Protocol 4: Preparation of splenic lymphocytes Support Protocol 5: Making BHK21-LCMV lysate Basic Protocol 7: Challenge models TRANSGENIC MODELS Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge.


Assuntos
Imunidade Adaptativa , Interações Hospedeiro-Patógeno/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Memória Imunológica , Depleção Linfocítica , Coriomeningite Linfocítica/transmissão , Camundongos , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Viral/métodos , Ensaio de Placa Viral/métodos
16.
J Exp Med ; 217(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-32820330

RESUMO

Type I interferons (IFN-I) are a major antiviral defense and are critical for the activation of the adaptive immune system. However, early viral clearance by IFN-I could limit antigen availability, which could in turn impinge upon the priming of the adaptive immune system. In this study, we hypothesized that transient IFN-I blockade could increase antigen presentation after acute viral infection. To test this hypothesis, we infected mice with viruses coadministered with a single dose of IFN-I receptor-blocking antibody to induce a short-term blockade of the IFN-I pathway. This resulted in a transient "spike" in antigen levels, followed by rapid antigen clearance. Interestingly, short-term IFN-I blockade after coronavirus, flavivirus, rhabdovirus, or arenavirus infection induced a long-lasting enhancement of immunological memory that conferred improved protection upon subsequent reinfections. Short-term IFN-I blockade also improved the efficacy of viral vaccines. These findings demonstrate a novel mechanism by which IFN-I regulate immunological memory and provide insights for rational vaccine design.


Assuntos
Imunogenicidade da Vacina/imunologia , Interferon Tipo I/antagonistas & inibidores , Interferon-alfa/imunologia , Receptor de Interferon alfa e beta/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Antivirais/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Expressão Gênica/imunologia , Células HEK293 , Humanos , Memória Imunológica , Interferon-alfa/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Transfecção , Infecção por Zika virus/virologia
17.
Sci Rep ; 9(1): 16511, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712633

RESUMO

This study provides the first high-quality draft genome assembly (762.5 Mb) of Tenualosa ilisha that is highly contiguous and nearly complete. We observed a total of 2,864 contigs, with 96.4% completeness with N50 of 2.65 Mbp and the largest contig length of 17.4 Mbp, along with a complete mitochondrial genome of 16,745 bases. A total number of 33,042 protein coding genes were predicted, among these, 512 genes were classified under 61 Gene Ontology (GO) terms, associated with various homeostasis processes. Highest number of genes belongs to cellular calcium ion homeostasis, followed by tissue homeostasis. A total of 97 genes were identified, with 16 GO terms related to water homeostasis. Claudins, Aquaporins, Connexins/Gap junctions, Adenylate cyclase, Solute carriers and Voltage gated potassium channel genes were observed to be higher in number in T. ilisha, as compared to that in other teleost species. Seven novel gene variants, in addition to claudin gene (CLDZ), were found in T. ilisha. The present study also identified two putative novel genes, NKAIN3 and L4AM1, for the first time in fish, for which further studies are required for pinpointing their functions in fish. In addition, 1.6 million simple sequence repeats were mined from draft genome assembly. The study provides a valuable genomic resource for the anadromous Hilsa. It will form a basis for future studies, pertaining to its adaptation mechanisms to different salinity levels during migration, which in turn would facilitate in its domestication.


Assuntos
Peixes/fisiologia , Genoma , Genômica , Osmorregulação , Adaptação Biológica/genética , Animais , Biologia Computacional/métodos , Sequência Conservada , Perfilação da Expressão Gênica , Genoma Mitocondrial , Genômica/métodos , Homeostase , Anotação de Sequência Molecular , Família Multigênica , Sequências Repetitivas de Ácido Nucleico
18.
PLoS One ; 14(7): e0216144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31335900

RESUMO

The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.


Assuntos
Processamento Alternativo/fisiologia , Proteínas de Peixes , Peixes , alfa 2-Macroglobulinas Associadas à Gravidez , Animais , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Peixes/genética , Peixes/metabolismo , Humanos , Especificidade de Órgãos/fisiologia , alfa 2-Macroglobulinas Associadas à Gravidez/biossíntese , alfa 2-Macroglobulinas Associadas à Gravidez/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética
19.
Indian J Exp Biol ; 53(8): 514-21, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26349314

RESUMO

The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.


Assuntos
Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/genética , Influenza Humana/terapia , RNA Interferente Pequeno/uso terapêutico , Animais , Cães , Inativação Gênica , Humanos , Índia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/virologia , Células Madin Darby de Rim Canino , RNA Interferente Pequeno/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética
20.
J Med Virol ; 87(8): 1268-75, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25914198

RESUMO

Genetic variability in the hemagglutinin (HA1) and the neuraminidase (NA) genes of influenza viruses results in the emergence of new strains which differ in pathogenicity and severity. The present study was undertaken for genotypic characterization of the HA1 and NA genes of the influenza A(H3N2) strains, detected during the 2011-2013. A total of fifty five influenza A(H3N2) positive samples [2011 (n = 20), 2012 (n = 4) and 2013 (n = 31)] were studied. The 824 bp segment of HA1 gene and 931 bp segment of NA gene were amplified and sequenced by Big-Dye terminator kit on ABI3130, Genetic analyzer. Molecular and phylogenetic analysis was done by MEGA 5.05 software and PhyML program (v3.0). Mutations were determined by comparing the deduced amino acid sequences of study strains with that of 2009-2013 vaccine strains. The studied influenza A(H3N2) strains showed 98.1-99.6% similarity in HA1 and NA amino acid sequences with the influenza A/Victoria/361/2011 vaccine strain. Four mutations in the HA1 amino acid sequences (T128A, R142G, L157S and N278K) and three unique mutations in the NA amino acid sequences [D251V, S315G and V313A] were found. These mutations were observed only in strains from the year 2013 (cluster II). None of the strains showed the presence of mutations, N294S and R292K, markers of oseltamivir resistance. In conclusion, Lucknow strains have accumulated the significant number of mutations in the antigenic sites of the HA and the NA coding sequences and continue to be evolving from the 2013 vaccine strain [A/Victoria/361/2011], however, mutations specific for oseltamivir resistance were not detected.


Assuntos
Variação Genética , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Análise por Conglomerados , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Índia/epidemiologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Dados de Sequência Molecular , Mutação , Neuraminidase/genética , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Proteínas Virais/genética
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