Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32°C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negativestrand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.negative strand RNA virus | pneumovirus | live attenuated vaccine H uman respiratory syncytial virus (RSV) is a negative-strand RNA virus of genus Pneumovirus, family Paramyxoviridae. RSV is the most important viral agent of serious respiratory tract illness in infants and children worldwide (1-3). Worldwide, nearly all children are infected by RSV at least once by the age of 2 y. RSV disease ranges from rhinitis to bronchiolitis or pneumonia. The RSV genome consists of a single-stranded negative-sense 15.2-kb RNA and has 10 genes in the order 3′ NS1-NS2-N-P-M-SH-F-G-M2-L 5′ (for a review, see ref. 4). The M2 gene encodes two separate proteins, M2-1 and M2-2, from overlapping ORFs.RSV vaccines and new antiviral drugs are in preclinical and clinical development; however, no RSV vaccines or antiviral drugs suitable for routine use are commercially available. The goal of the present study was to design and generate new vaccine candidates for RSV using the recently described strategy of codon-pair deoptimization (CPD) (5). By this strategy, one or more ORFs in a virus or other pathogen are recoded by rearranging existing synonymous codons so as to increase the presence of underrepresented codon pairs within the ORF. CPD can be done without changing codon use although, in the present study, codon use was occasionally changed slightly when we manually edited the sequence to remove features such as long homooligomers. Amino acid coding was completely unaffected, and nontranslated genome regions were unchanged. A major effect of CPD is...

Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP 1,2 ) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP 1,2 , and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

Marburg virus belongs to the genus Marburgvirus in the family Filoviridae and causes a severe hemorrhagic fever, known as Marburg hemorrhagic fever (MHF), in both humans and nonhuman primates. Similar to the more widely known Ebola hemorrhagic fever, MHF is characterized by systemic viral replication, immunosuppression and abnormal inflammatory responses. These pathological features of the disease contribute to a number of systemic dysfunctions including hemorrhages, edema, coagulation abnormalities and, ultimately, multiorgan failure and shock, often resulting in death. A detailed understanding of the pathological processes that lead to this devastating disease remains elusive, a fact that contributes to the lack of licensed vaccines or effective therapeutics. This article will review the clinical aspects of MHF and discuss the pathogenesis and possible options for diagnosis, treatment and prevention.