cis-Acting Element at the 5' Noncoding Region of Tacaribe Virus S RNA Modulates Genome Replication.

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that includes several members that cause hemorrhagic fevers in humans. The TCRV genome comprises two RNA segments, named S (small) and L (large). Both genomic segments contain noncoding regions (NCRs) at their 5' and 3' ends. While the 5'- and 3'-terminal 19-nucleotide sequences are known to be essential for promoter function, the role of their neighboring internal noncoding region (iNCR) sequences remains poorly understood. To analyze the relevance of the 5' and 3' iNCRs in TCRV S RNA synthesis, mutant S-like minigenomes and miniantigenomes were generated. Using a minireplicon assay, Northern blotting, and reverse transcription-quantitative PCR, we demonstrated that the genomic 5' iNCR is specifically engaged in minigenome replication yet is not directly involved in minigenome transcription, and we showed that the S genome 3' iNCR is barely engaged in this process. Analysis of partial deletions and point mutations, as well as total or partial substitution of the 5' iNCR sequence, led us to conclude that the integrity of the whole genomic 5' iNCR is essential and that a local predicted secondary structure or RNA-RNA interactions between the 5' and 3' iNCRs are not strictly required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic approach to ask whether manipulation of the S genomic 5' iNCR sequence may be suitable for viral attenuation. We found that mutagenesis of the 5' promoter-proximal subregion slightly impacted recombinant TCRV virulence in vivo. IMPORTANCE The Mammarenavirus genus of the Arenaviridae family includes several members that cause severe hemorrhagic fevers associated with high morbidity and mortality rates, for which no FDA-approved vaccines and limited therapeutic resources are available. We provide evidence demonstrating the specific involvement of the TCRV S 5' noncoding sequence adjacent to the viral promoter in replication. In addition, we examined the relevance of this region in the context of an in vivo infection. Our findings provide insight into the mechanism through which this 5' viral RNA noncoding region assists the L polymerase for efficient viral S RNA synthesis. Also, these findings expand our understanding of the effect of genetic manipulation of New World mammarenavirus sequences aimed at the rational design of attenuated recombinant virus vaccine platforms.

Protocol for the Production of a Vaccine Against Argentinian Hemorrhagic Fever.

Argentinian hemorrhagic Fever (AHF) is a febrile, acute disease caused by Junín virus (JUNV), a member of the Arenaviridae. Different approaches to obtain an effective antigen to prevent AHF using complete live or inactivated virus, as well as molecular constructs, have reached diverse development stages. This chapter refers to JUNV live attenuated vaccine strain Candid #1, currently used in Argentina to prevent AHF. A general standardized protocol used at Instituto Nacional de Enfermedades Virales Humanas (Pergamino, Pcia. Buenos Aires, Argentina) to manufacture the tissue culture derived Candid #1 vaccine is described. Intermediate stages like viral seeds and cell culture bank management, bulk vaccine manufacture, and finished product processing are also separately presented in terms of Production and Quality Control/Quality Assurance requirements, under the Adminitracion Nacional de Medicamentos, Alimentos y Tecnología Medica (ANMAT), the Argentine national regulatory authority.

[Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain]. / Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.(AU)

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.(AU)

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1. / [Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain].

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Argentine hemorrhagic fever vaccines.

Argentine hemorrhagic fever (AHF), an acute disease caused by Junin virus (JUNV, Arenaviridae), has been an important issue to public health in Argentina since the early 1950s. The field rodent Calomys musculinus is JUNV natural reservoir and human disease is a consequence of contact with infected rodents. A steady extention of AHF endemic area is being observed since the first reports of the disease. Important achievements have been made in: (a) improvement of methods for the etiological diagnosis; (b) implementation and validation of therapeutical measures; (c) development of vaccines to protect against AHF. Reference is made to different research strategies used to obtain anti-AHF vaccines in the past and anti-arenaviral diseases in the present. Information is updated on features and field performance of Candid #1 vaccine, a live attenuted vaccine currently used to prevent AHF. This vaccine was developed through a joint international effort that envisioned it as an orphan drug. With transferred technology, Argentine government was committed to be Candid #1 manufacturer and to register this vaccine as a novel medical product under the Argentine regulatory authority. Candid #1 vaccine is the first one used to control an arenaviral hemorrhagic fever, the first live viral vaccine to be manufactured and registered in Argentina, reaching its target population through governmental effort.

Estudio de marcadores fenotípicos de atenuación en cepas de virus Junin recuperadas de individuos recientemente vacunados con la cepa Junin Candid #1: índices de letalidad y protección en animales de laboratorio / Study of phenotypic markers of attenuation in Junin virus strains recovered from individuals vaccinated with strain recently Junin Candid # 1: lethality rates and protection of laboratory animals

La fiebre hemorrágica argentica (FHA), es una enfermedad aguda producida por el vírus Junin (VJUN) siendo el roedor Calomys musculinus el principal reservorio del VJUN.Para la prevención de la FHA, tanto el control de los roedores como el control del contacto humano com la problación de roedores infectados sin impraticables. Por esto, desde el aislamiento de VJUN, todos los esfuerzos han estado dirigidos a la obtenciõn de una vacuna efectiva. La cepa atenuada de VJUN Candid#1 há sido resultado de estas investigaciones. Durante el ensayo clínico puente realizado em el Instituto Nacional de Enfermedades Virales Humans (INEVH), em el año 2005, para comparar la vacuna Candid#1 producida em EE.UU. com la vacuna producida em la Argentina, se logro aislar por cocultivo de células mononucleares de sangre periférica (CMNs) dos cepas de VJUN (65604 y 65727) de 10 voluntarios inoculados com Candid#1 que concurrieron entre los dias 9 y 10 post vacunación com sintomas de cefalea, mialgia y decaimiento. Dichas cepas fueron identificadas como VJUN por netralización de placas bajo agar enfrentadas com um suero espercífico anti-Candid obtenido em conejo. El objetivo de este trabajo fue comparar lãs características fenotípicas de atenuación de las dos cepas recuperadias con la vacuna Candid#. Para ello se utilizaron cobayos y ratones de diferentes edades como modelo de este estúdio. Se midieron os índices de letalidad, infección y proteccíón. El ratón recién nacido (rrn) fue el único que mostro letalidad com las tres cepas estudiadas, sin embargo para ratónde 10 a 12 días, ratón adulto (rad) y cobayo, ninguna de las cepas fue letal, aún para la más baja dilución inoculada. Los cobayos inoculados com las cepas recuperadas65604 y 65727 com la cepa Candid#1 no mostraron sintomas de enfermedad y resultaron protegidos cuando fueron desafiados com la cepa patógena P3790. Se observo que los valores obtenidos de tanto de los índices de infección como de protección fueron muy bajos, lo que indica que estas cepas tienen un alto poder infectante y que son altamente protectoras. En base a los resultados obtenidos podemos concluir que las cepas de VJUN recuperadas de indivíduos vacunados com Candid#1 mantienen las características de atenuaciõn de la vacuna, no detectándose ninguma evidencia de reversión de Candid#1 luego de um pasaje em humanos.