RESUMO
Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-ß promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-ß mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.
Assuntos
Phlebovirus/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Humanos , Phlebovirus/genética , Vírus da Febre do Vale do Rift/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-ß gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.
Assuntos
Encéfalo/virologia , Doenças do Sistema Nervoso/virologia , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Flebótomo Napolitano/genética , Vírus da Febre do Flebótomo Napolitano/patogenicidade , Vacinas Atenuadas/efeitos adversos , Proteínas não Estruturais Virais/metabolismo , Vacinas Virais/efeitos adversos , Animais , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Flebótomo Napolitano/imunologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
An in vitro baculovirus cloning system has been developed for direct cloning of foreign DNA into baculovirus genomes. This system is called the "Homingbac system" because it uses homing endonucleases. The Homingbac system was engineered into the baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV and HzSNPV. All Homingbac viruses were designed to retain the polyhedra phenotype so that they could be inoculated per os to insects. This is the first time a common in vitro baculovirus cloning system has been made for multiple baculovirus species that include both groups I and II nucleopolyhedroviruses (NPVs). In this study, the Homingbac system was demonstrated by directly cloning a PCR-amplified beta-glucuronidase gene cassette into a parent Homingbac virus. This new collection of groups I and II NPV Homingbac viruses are a significant expansion of in vitro cloning technology and are new tools for making recombinant baculoviruses.
