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Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5′ and 3′ ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5′ end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5′ end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates. Author summary: Filoviruses, such as Ebola virus and Marburg virus (EBOV and MARV, respectively), cause severe hemorrhagic fever with a high mortality rate in humans. Monoclonal antibodies (mAbs) are effective treatments for filovirus infection, but current therapies have limited breadth. Furthermore, a single mAb is susceptible to development of resistance. Here, we used protein engineering to create "bispecific" antibodies in which activities of two different mAbs were combined into one. These bispecific antibodies had broad activity, in one case providing protection against lethal challenge by two distant clades of filoviruses (EBOV and MARV). The bispecific antibodies were also less susceptible to resistance mutations. This work provides a roadmap for development of new bispecific antibody therapies for filoviruses. [ABSTRACT FROM AUTHOR]

Platinum and ruthenium nanoparticles stabilised by an amine modified polymer immobilised ionic liquid (MNP@NH2-PEGPIILS, M = Pt, Ru) catalyse the hydrolytic liberation of hydrogen from dimethylamine borane (DMAB), ammonia borane (AB) and NaBH4 under mild conditions. While RuNP@NH2-PEGPIILS and PtNP@NH2-PEGPIILS catalyse the hydrolytic evolution of hydrogen from NaBH4 with comparable initial TOFs of 6,250 molesH2.molcat−1.h−1 and 5,900 molesH2.molcat−1.h−1, respectively, based on the total metal content, RuNP@NH2-PEGPIILS is a markedly more efficient catalyst for the dehydrogenation of DMAB and AB than its platinum counterpart, as RuNP@NH2-PEGPIILS gave initial TOFs of 8,300 molesH2.molcat−1.h−1 and 21,200 molesH2.molcat−1.h−1, respectively, compared with 3,050 molesH2.molcat−1.h−1 and 8,500 molesH2.molcat−1.h−1, respectively, for PtNP@NH2-PEGPIILS. Gratifyingly, for each substrate tested RuNP@NH2-PEGPIILS and PtNP@NH2-PEGPIILS were markedly more active than commercial 5wt % Ru/C and 5wt% Pt/C, respectively. The apparent activation energies of 55.7 kJ mol−1 and 27.9 kJ mol−1 for the catalytic hydrolysis of DMAB and AB, respectively, with RuNP@NH2-PEGPIILS are significantly lower than the respective activation energies of 74.6 kJ mol−1 and 35.7 kJ mol−1 for its platinum counterpart, commensurate with the markedly higher initial rates obtained with the RuNPs. In comparison, the apparent activation energies of 44.1 kJ mol−1 and 46.5 kJ mol−1, for the hydrolysis NaBH4 reflect the similar initial TOFs obtained for both catalysts. The difference in apparent activation energies for the hydrolysis of DMAB compared with AB also reflect the higher rates of hydrolysis for the latter. Stability and reuse studies revealed that RuNP@NH2-PEGPIILS recycled efficiently as high conversions for the hydrolysis of DMAB were maintained across five runs with the catalyst retaining 97% of its activity.Graphical Abstract: Platinum and ruthenium nanoparticles stabilised by an amine modified polymer immobilised ionic liquid (MNP@NH2-PEGPIILS, M = Pt, Ru) catalyse the hydrolytic liberation of hydrogen from dimethylamine borane (DMAB), ammonia borane (AB) and NaBH4 under mild conditions. While RuNP@NH2-PEGPIILS and PtNP@NH2-PEGPIILS catalyse the hydrolytic evolution of hydrogen from NaBH4 with comparable initial TOFs of 6,250 molesH2.molcat−1.h−1 and 5,900 molesH2.molcat−1.h−1, respectively, based on the total metal content, RuNP@NH2-PEGPIILS is a markedly more efficient catalyst for the dehydrogenation of DMAB and AB than its platinum counterpart, as RuNP@NH2-PEGPIILS gave initial TOFs of 8,300 molesH2.molcat−1.h−1 and 21,200 molesH2.molcat−1.h−1, respectively, compared with 3,050 molesH2.molcat−1.h−1 and 8,500 molesH2.molcat−1.h−1, respectively, for PtNP@NH2-PEGPIILS. Gratifyingly, for each substrate tested RuNP@NH2-PEGPIILS and PtNP@NH2-PEGPIILS were markedly more active than commercial 5wt % Ru/C and 5wt% Pt/C, respectively. The apparent activation energies of 55.7 kJ mol−1 and 27.9 kJ mol−1 for the catalytic hydrolysis of DMAB and AB, respectively, with RuNP@NH2-PEGPIILS are significantly lower than the respective activation energies of 74.6 kJ mol−1 and 35.7 kJ mol−1 for its platinum counterpart, commensurate with the markedly higher initial rates obtained with the RuNPs. In comparison, the apparent activation energies of 44.1 kJ mol−1 and 46.5 kJ mol−1, for the hydrolysis NaBH4 reflect the similar initial TOFs obtained for both catalysts. The difference in apparent activation energies for the hydrolysis of DMAB compared with AB also reflect the higher rates of hydrolysis for the latter. Stability and reuse studies revealed that RuNP@NH2-PEGPIILS recycled efficiently as high conversions for the hydrolysis of DMAB were maintained across five runs with the catalyst retaining 97% of its activity. [ABSTRACT FROM AUTHOR]