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1.
J Virol ; 98(2): e0197523, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38294249

RESUMO

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.


Assuntos
Caspases , Citoplasma , Febre Hemorrágica Americana , Interações Hospedeiro-Patógeno , Imunidade Inata , Vírus Junin , Nucleoproteínas , Biossíntese de Proteínas , Humanos , Apoptose , Inibidores de Caspase/metabolismo , Caspases/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Ativação Enzimática , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Interferons/genética , Interferons/imunologia , Vírus Junin/genética , Vírus Junin/metabolismo , Vírus Junin/patogenicidade , Nucleoproteínas/biossíntese , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Replicação Viral
2.
Front Immunol ; 14: 1172792, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37334351

RESUMO

Junin virus (JUNV) is a member of the Arenaviridae family of viruses and is the pathogen responsible for causing Argentine hemorrhagic fever, a potentially lethal disease endemic to Argentina. A live attenuated vaccine for human use, called Candid#1, is approved only in Argentina. Candid#1 vaccine strain of Junin virus was obtained through serial passage in mouse brain tissues followed by passage in Fetal Rhesus macaque lung fibroblast (FRhL) cells. Previously, the mutations responsible for attenuation of this virus in Guinea pigs were mapped in the gene encoding for glycoprotein precursor (GPC) protein. The resulting Candid#1 glycoprotein complex has been shown to cause endoplasmic reticulum (ER) stress in vitro resulting in the degradation of the GPC. To evaluate the attenuating properties of specific mutations within GPC, we created recombinant viruses expressing GPC mutations specific to key Candid#1 passages and evaluated their pathogenicity in our outbred Hartley guinea pig model of Argentine hemorrhagic fever. Here, we provide evidence that early mutations in GPC obtained through serial passaging attenuate the visceral disease and increase immunogenicity in guinea pigs. Specific mutations acquired prior to the 13th mouse brain passage (XJ13) are responsible for attenuation of the visceral disease while having no impact on the neurovirulence of Junin virus. Additionally, our findings demonstrate that the mutation within an N-linked glycosylation motif, acquired prior to the 44th mouse brain passage (XJ44), is unstable but necessary for complete attenuation and enhanced immunogenicity of Candid#1 vaccine strain. The highly conserved N-linked glycosylation profiles of arenavirus glycoproteins could therefore be viable targets for designing attenuating viruses for vaccine development against other arenavirus-associated illnesses.


Assuntos
Febre Hemorrágica Americana , Vírus Junin , Humanos , Animais , Cobaias , Camundongos , Vírus Junin/genética , Macaca mulatta/metabolismo , Glicoproteínas/metabolismo , Mutação
3.
Medicina (B Aires) ; 83(1): 129-132, 2023.
Artigo em Espanhol | MEDLINE | ID: mdl-36774608

RESUMO

We present the case of a 38-year-old woman with no relevant medical history, resident of the City of Buenos Aires, who was admitted in hospital for presenting fever, retroocular headache, myalgia, arthralgia, and maculopapular pruritic rash on the back of the hands and feet of 6 days of evolution. Laboratory tests revealed lymphopenia, severe thrombocytopenia, and anicteric hepatitis. Her husband had been hospitalized three weeks earlier for a condition of similar characteristics without etiological diagnosis. Subsequently, it evolved with metrorrhagia and axillary petechiae associated with photophobia, drowsiness, and fine tremor of the tongue with normal cerebrospinal fluid, treated with intravenous ceftriaxone 2 g/day for 7 days. Computed tomography of abdomen and pelvis showed a left abdominal wall hematoma. Serological samples were sent to the National Institute of Human Viral Diseases Dr. Julio I. Maiztegui for dengue virus, leptospirosis and hantavirus with non-reactive results, and RT-PCR of Junín virus that was positive. Retrospectively, the spouse was diagnosed by detection of IgG antibodies to Junin virus by ELISA and neutralization tests. Neither of the two cases had a clear epidemiological link. Our aim is to highlight the importance of clinical suspicion outside of endemic areas.


Presentamos el caso de una mujer de 38 años sin antecedentes personales relevantes, residente de Ciudad Autónoma de Buenos Aires, que consultó por fiebre, cefalea retroocular mialgias, artralgias y exantema maculopapular pruriginoso en dorso de manos y pies de 6 días de evolución. El laboratorio presentaba linfopenia, trombocitopenia grave y hepatitis anictérica. El cónyuge había cursado internación tres semanas antes por un cuadro de similares características sin diagnóstico etiológico. Posteriormente, la paciente evolucionó con metrorragia y petequias axilares asociados a fotofobia, somnolencia y temblor fino de la lengua, con líquido cefalorraquídeo normal, cumpliendo tratamiento con ceftriaxona 2 g/día intravenoso por 7 días. La tomografía computarizada de abdomen y pelvis evidenciaba un hematoma de pared abdominal izquierdo. Se derivaron muestras serológicas al Instituto Nacional de Enfermedades Virales Humanas Dr. Julio I. Maiztegui para virus dengue, leptospirosis y hantavirus con resultados no reactivos y RT-PCR de virus Junín que resultó positiva. Retrospectivamente se realizó el diagnóstico del cónyuge por detección de anticuerpos IgG para virus Junín por ELISA y prueba de neutralización. Ninguno de los dos casos presentaba un nexo epidemiológico claro. Nuestro objetivo es remarcar la importancia de la sospecha clínica fuera de áreas endémicas.


Assuntos
Febre Hemorrágica Americana , Humanos , Feminino , Adulto , Estudos Retrospectivos , Febre
4.
Rev Med Virol ; 33(2): e2419, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36635519

RESUMO

Junin virus consists of ribonucleic acid as the genome and is responsible for a rapidly changing tendency of the virus. The virus is accountable for ailments in the human body and causes Argentine Haemorrhagic Fever (AHF). The infection is may be transmitted through contact between an infected animal/host and a person, and later between person to person. Prevention of outbreaks of AHF in humans can be a tough practice, as their occurrence is infrequent and unpredictable. In this review, recent information from the past 5 years available on the Junin virus including the risk of its emergence, infectious agents, its pathogenesis in humans, available diagnostic and therapeutic approaches, and disease management has been summarised. Altogether, this article would be highly significant in understanding the mechanistic basis behind virus interaction and other processes during the life cycle. Currently, no specific therapeutic options are available to treat the Junin virus infection. The information covered in this review could be important for finding possible treatment options for Junin virus infections.


Assuntos
Febre Hemorrágica Americana , Vírus Junin , Animais , Humanos , Vírus Junin/genética , Febre Hemorrágica Americana/diagnóstico , Febre Hemorrágica Americana/patologia
5.
Antiviral Res ; 208: 105444, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36243175

RESUMO

Infections by pathogenic New World mammarenaviruses (NWM)s, including Junín virus (JUNV), can result in a severe life-threatening viral hemorrhagic fever syndrome. In the absence of FDA-licensed vaccines or antivirals, these viruses are considered high priority pathogens. The mammarenavirus envelope glycoprotein complex (GPC) mediates pH-dependent fusion between viral and cellular membranes, which is essential to viral entry and may be vulnerable to small-molecule inhibitors that disrupt this process. ARN-75039 is a potent fusion inhibitor of a broad spectrum of pseudotyped and native mammarenaviruses in cell culture and Tacaribe virus infection in mice. In the present study, we evaluated ARN-75039 against pathogenic JUNV in the rigorous guinea pig infection model. The compound was well-tolerated and had favorable pharmacokinetics supporting once-per-day oral dosing in guinea pigs. Importantly, significant protection against JUNV challenge was observed even when ARN-75039 was withheld until 6 days after the viral challenge when clinical signs of disease are starting to develop. We also show that ARN-75039 combination treatment with favipiravir, a viral polymerase inhibitor, results in synergistic activity in vitro and improves survival outcomes in JUNV-challenged guinea pigs. Our findings support the continued development of ARN-75039 as an attractive therapeutic candidate for treating mammarenaviral hemorrhagic fevers, including those associated with NWM infection.


Assuntos
Arenaviridae , Febre Hemorrágica Americana , Febres Hemorrágicas Virais , Vírus Junin , Cobaias , Camundongos , Animais , Febre Hemorrágica Americana/tratamento farmacológico , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Amidas/farmacologia , Amidas/uso terapêutico , Antirretrovirais/farmacologia
6.
PLoS Pathog ; 18(7): e1010689, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35816544

RESUMO

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.


Assuntos
Arenavirus , Febre Hemorrágica Americana , Vírus Junin , Amidas , Antivirais/farmacologia , Antivirais/uso terapêutico , Febre Hemorrágica Americana/tratamento farmacológico , Humanos , Vírus Junin/genética , Pirazinas , RNA Polimerase Dependente de RNA/genética , Replicação Viral
7.
N Engl J Med ; 386(24): 2283-2294, 2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35704480

RESUMO

BACKGROUND: In June 2019, the Bolivian Ministry of Health reported a cluster of cases of hemorrhagic fever that started in the municipality of Caranavi and expanded to La Paz. The cause of these cases was unknown. METHODS: We obtained samples for next-generation sequencing and virus isolation. Human and rodent specimens were tested by means of virus-specific real-time quantitative reverse-transcriptase-polymerase-chain-reaction assays, next-generation sequencing, and virus isolation. RESULTS: Nine cases of hemorrhagic fever were identified; four of the patients with this illness died. The etiologic agent was identified as Mammarenavirus Chapare mammarenavirus, or Chapare virus (CHAPV), which causes Chapare hemorrhagic fever (CHHF). Probable nosocomial transmission among health care workers was identified. Some patients with CHHF had neurologic manifestations, and those who survived had a prolonged recovery period. CHAPV RNA was detected in a variety of human body fluids (including blood; urine; nasopharyngeal, oropharyngeal, and bronchoalveolar-lavage fluid; conjunctiva; and semen) and in specimens obtained from captured small-eared pygmy rice rats (Oligoryzomys microtis). In survivors of CHHF, viral RNA was detected up to 170 days after symptom onset; CHAPV was isolated from a semen sample obtained 86 days after symptom onset. CONCLUSIONS: M. Chapare mammarenavirus was identified as the etiologic agent of CHHF. Both spillover from a zoonotic reservoir and possible person-to-person transmission were identified. This virus was detected in a rodent species, O. microtis. (Funded by the Bolivian Ministry of Health and others.).


Assuntos
Arenavirus do Novo Mundo , Febre Hemorrágica Americana , RNA Viral , Roedores , Animais , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/isolamento & purificação , Bolívia/epidemiologia , Infecção Hospitalar/transmissão , Infecção Hospitalar/virologia , Transmissão de Doença Infecciosa , Febre Hemorrágica Americana/complicações , Febre Hemorrágica Americana/genética , Febre Hemorrágica Americana/transmissão , Febre Hemorrágica Americana/virologia , Febres Hemorrágicas Virais/genética , Febres Hemorrágicas Virais/transmissão , Febres Hemorrágicas Virais/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/isolamento & purificação , Ratos/virologia , Roedores/virologia , Zoonoses Virais/transmissão , Zoonoses Virais/virologia
8.
Viruses ; 14(6)2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35746604

RESUMO

Junín virus (JUNV) belongs to the Arenaviridae family and is the causative agent of Argentine hemorrhagic fever (AHF), a severe human disease endemic to agricultural areas in Argentina. At this moment, there are no effective antiviral therapeutics to battle pathogenic arenaviruses. Cumulative reports from recent years have widely provided information on cellular factors playing key roles during JUNV infection. In this review, we summarize research on host molecular determinants that intervene in the different stages of the viral life cycle: viral entry, replication, assembly and budding. Alongside, we describe JUNV tight interplay with the innate immune system. We also review the development of different reverse genetics systems and their use as tools to study JUNV biology and its close teamwork with the host. Elucidating relevant interactions of the virus with the host cell machinery is highly necessary to better understand the mechanistic basis beyond virus multiplication, disease pathogenesis and viral subversion of the immune response. Altogether, this knowledge becomes essential for identifying potential targets for the rational design of novel antiviral treatments to combat JUNV as well as other pathogenic arenaviruses.


Assuntos
Arenaviridae , Arenavirus , Febre Hemorrágica Americana , Vírus Junin , Antivirais , Arenaviridae/genética , Humanos , Vírus Junin/fisiologia , Replicação Viral
9.
Buenos Aires; GCBA. Gerencia Operativa de Epidemiología; 24 jun. 2022. f:11 l:13 p. tab, graf.(Boletín Epidemiológico Semanal: Ciudad Autónoma de Buenos Aires, 7, 305).
Monografia em Espanhol | LILACS, InstitutionalDB, BINACIS, UNISALUD | ID: biblio-1391661

RESUMO

Características generales de la Fiebre Hemorrágica Argentina (FHA): Transmisión, presentación clínica, definición de caso sospechoso, tratamiento, medidas de prevención, y situación regional. Se describe el caso confirmado en la Ciudad de Buenos Aires.


Assuntos
Humanos , Feminino , Adulto , Febre Hemorrágica Americana/fisiopatologia , Febre Hemorrágica Americana/patologia , Febre Hemorrágica Americana/transmissão , Febre Hemorrágica Americana/epidemiologia , Notificação de Doenças , Monitoramento Epidemiológico
10.
Multimedia | Recursos Multimídia | ID: multimedia-9683

RESUMO

Destinado a todo el personal de salud, Alejandra Rodríguez aborda aspectos epidemiológicos de la Fiebre Hemorrágica Argentina, la Dra. Andrea Uboldi diserta sobre la vacuna y la Dra. Alejandra Gaiano sobre los desafíos futuros


Assuntos
Febre Hemorrágica Americana/complicações , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/epidemiologia , Argentina , Controle de Doenças Transmissíveis , Vacinas
11.
Medicina (B Aires) ; 82(3): 344-350, 2022.
Artigo em Espanhol | MEDLINE | ID: mdl-35639054

RESUMO

Since the identification of Junin virus in the 1950s, many studies were carried out in wild rodents within the endemic area of the Argentine Hemorrhagic Fever (AHF) that recorded also the activity of the lymphocytic choriomeningitis virus (LCMV) and the Latino virus (LATV). The absence of confirmed cases of AHF since the 1990s in the department of Rio Cuarto, Córdoba province, promoted ecoepidemiological surveillance of infection of Calomys musculinus (Junin virus reservoir) and the search of reservoirs of the other mammarenaviruses. During two years of seasonal sampling, with a capture, mark and release system, 857 rodents were captured, corresponding 57.3% to the rodent reservoirs: C. musculinus, C. venustus and Mus musculus, being the first the most abundant species. Antibodies were detected and the three viral agents were molecularly characterized, showing a prevalence of infection of 3.5% (9/254) for Junin virus, 100% (3/3) for LCMV and 24.1% (21/87) for LATV. In conclusion, we demonstrated Junin virus circulation in its rodent reservoir in a region considered historic for AHF with potential risk for the population and the spatio-temporal co-circulation of the three mammarenaviruses in the central region of Argentina.


Desde la identificación del virus Junin en la década del 50, se realizaron numerosos estudios en roedores silvestres dentro del área endémica de la Fiebre Hemorrágica Argentina (FHA) que permitieron registrar, además, actividad del virus de la coriomeningitis linfocitaria (LCMV) y del virus Latino (LATV). La ausencia de casos confirmados de FHA desde la década del 90 en el departamento Río Cuarto, provincia de Córdoba, promovió la vigilancia ecoepidemiológica y de infección del Calomys musculinus (reservorio del virus Junin) y la búsqueda de reservorios e infección de los otros mammarenavirus. Durante dos años de muestreo estacional, con un sistema de captura, marcación y liberación capturamos 857 roedores, que correspondieron 57.3% a los reservorios: C. musculinus (especie más abundante), C. venustus y Mus musculus. Detectamos anticuerpos y caracterizamos molecularmente los tres agentes virales. Observamos una prevalencia de infección de 3.5% (9/254) para virus Junin, 100% (3/3) para LCMV y 24.1% (21/87) para LATV. En conclusión, demostramos circulación de virus Junin en su roedor reservorio, en una región considerada histórica para FHA con riesgo potencial para la población y cocirculación espacio-temporal de los tres mammarenavirus en la región central de Argentina.


Assuntos
Arenaviridae , Arenavirus do Novo Mundo , Febre Hemorrágica Americana , Vírus Junin , Animais , Reservatórios de Doenças , Febre Hemorrágica Americana/epidemiologia , Humanos , Camundongos , Roedores
12.
Front Cell Infect Microbiol ; 12: 798978, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35463647

RESUMO

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.


Assuntos
Febre Hemorrágica Americana , Vírus Junin , Proteínas de Choque Térmico HSP27 , Humanos , Vírus Junin/genética , Proteômica , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno
13.
Biochem Biophys Res Commun ; 606: 168-173, 2022 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-35364325

RESUMO

The New World (NW) mammarenavirus Junín (JUNV) is the etiological agent of Argentine hemorrhagic fever, a human endemic disease of Argentina. Promyelocytic leukemia protein (PML) has been reported as a restriction factor for several viruses although the mechanism/s behind PML-mediated antiviral effect may be diverse and are a matter of debate. Previous studies have reported a nuclear to cytoplasm translocation of PML during the murine Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) infection. This translocation was found to be mediated by the viral Z protein. Here, we show that PML restricts JUNV infection in human A549 cells. However, in contrast to LCVM, JUNV infection enhances PML expression and PML is not translocated to the cytoplasm neither it colocalizes with JUNV Z protein. Our study demonstrates that a NW mammarenavirus as JUNV interacts differently with the antiviral protein PML than LCMV.


Assuntos
Febre Hemorrágica Americana , Vírus Junin , Proteína da Leucemia Promielocítica , Células A549 , Febre Hemorrágica Americana/metabolismo , Humanos , Proteína da Leucemia Promielocítica/genética , Proteínas Virais , Replicação Viral
14.
J Virol ; 96(8): e0020922, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35343792

RESUMO

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.


Assuntos
Arenavirus do Novo Mundo , Febre Hemorrágica Americana , Vacinas Virais , Animais , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/imunologia , Modelos Animais de Doenças , Glicoproteínas/metabolismo , Glicosilação , Cobaias , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Vírus Junin/genética , Vírus Junin/imunologia , Mutação , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia
15.
Nat Commun ; 13(1): 558, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35091550

RESUMO

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.


Assuntos
Antígenos CD/metabolismo , Arenaviridae/metabolismo , Febre Hemorrágica Americana/metabolismo , Receptores da Transferrina/metabolismo , Proteínas do Envelope Viral/metabolismo , Células A549 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Arenaviridae/efeitos dos fármacos , Arenaviridae/fisiologia , Chlorocebus aethiops , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus Junin/efeitos dos fármacos , Vírus Junin/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Receptores da Transferrina/antagonistas & inibidores , Receptores da Transferrina/imunologia , Células Vero
16.
Nat Commun ; 12(1): 4134, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226547

RESUMO

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.


Assuntos
Arenavirus/enzimologia , Arenavirus/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Febre Hemorrágica Americana/virologia , Interações Hospedeiro-Patógeno , Humanos , Vírus Junin/enzimologia , Vírus Junin/genética , Modelos Moleculares , Conformação Proteica , RNA Viral
17.
J Virol ; 95(17): e0186820, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34132574

RESUMO

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Arenavirus do Novo Mundo/fisiologia , Glicoproteínas/imunologia , Febre Hemorrágica Americana/prevenção & controle , Receptores da Transferrina/imunologia , Células A549 , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Humanos , Estrutura Terciária de Proteína , Receptores da Transferrina/química , Receptores da Transferrina/genética
18.
J Virol ; 95(14): e0039721, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33952638

RESUMO

Live-attenuated virus vaccines are highly effective in preventing viral disease but carry intrinsic risks of residual virulence and reversion to pathogenicity. The classically derived Candid#1 virus protects seasonal field workers in Argentina against zoonotic infection by Junín virus (JUNV) but is not approved in the United States, in part due to the potential for reversion at the attenuating locus, a phenylalanine-to-isoleucine substitution at position 427 in the GP2 subunit of the GPC envelope glycoprotein. Previously, we demonstrated facile reversion of recombinant Candid#1 (rCan) in cell culture and identified an epistatic interaction between the attenuating I427 and a secondary K33S mutation in the stable signal peptide (SSP) subunit of GPC that imposes an evolutionary barrier to reversion. The magnitude of this genetic barrier is manifest in our repeated failures to rescue the hypothetical revertant virus. In this study, we show that K33S rCan is safe and attenuated in guinea pigs and capable of eliciting potent virus-neutralizing antibodies. Immunized animals are fully protected against lethal challenge with virulent JUNV. In addition, we employed a more permissive model of infection in neonatal mice to investigate genetic reversion. RNA sequence analysis of the recovered virus identified revertant viruses in pups inoculated with the parental rCan virus and none in mice receiving K33S rCan (P < 0.0001). Taken together, our findings support the further development of K33S rCan as a safe second-generation JUNV vaccine. IMPORTANCE Our most successful vaccines comprise weakened strains of virus that initiate a limited and benign infection in immunized persons. The live-attenuated Candid#1 strain of Junín virus (JUNV) was developed to protect field workers in Argentina from rodent-borne hemorrhagic fever but is not licensed in the United States, in part due to the likelihood of genetic reversion to virulence. A single-amino-acid change in the GPC envelope glycoprotein of the virus is responsible for attenuation, and a single nucleotide change may regenerate the pathogenic virus. Here, we take advantage of a unique genetic interaction between GPC subunits to design a mutant Candid#1 virus that establishes an evolutionary barrier to reversion. The mutant virus (K33S rCan) is fully attenuated and protects immunized guinea pigs against lethal JUNV infection. We find no instances of reversion in mice inoculated with K33S rCan. This work supports the further development of K33S rCan as a second-generation JUNV vaccine.


Assuntos
Febre Hemorrágica Americana/prevenção & controle , Vírus Junin/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Cobaias , Febre Hemorrágica Americana/imunologia , Imunogenicidade da Vacina , Vírus Junin/genética , Vírus Junin/patogenicidade , Masculino , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Células Vero , Vacinas Virais/genética , Virulência
19.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836604

RESUMO

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Antivirais/farmacologia , Febre Hemorrágica Americana/prevenção & controle , Vírus Junin/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Cobaias , Febre Hemorrágica Americana/sangue , Humanos , Macaca fascicularis
20.
PLoS Pathog ; 17(3): e1009356, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33647064

RESUMO

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.


Assuntos
Anticorpos Antivirais/imunologia , Arenavirus/imunologia , Febre Hemorrágica Americana/virologia , Vírus Junin/patogenicidade , Animais , Anticorpos Neutralizantes/imunologia , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/imunologia , Arenavirus do Novo Mundo/patogenicidade , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/prevenção & controle , Humanos , Vírus Junin/imunologia , Vacinas Virais/imunologia
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