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.

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...

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.

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34°C/35°C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.negative-strand RNA virus | respiratory syncytial virus | live attenuated vaccine | codon pair deoptimization | genetic stability T he availability and affordability of large-scale custom DNA synthesis opened the new field of synthetic biology (1). The combined approach of sequence design and synthetic biology allows the generation of DNA molecules with extensive targeted modifications. Synonymous genome recoding, in which one or more ORFs of a microbial pathogen are modified at the nucleotide level without affecting amino acid coding, currently is being widely evaluated to reduce pathogen fitness and create potential live attenuated vaccines, particularly for RNA viruses (reviewed in ref.2) (3-7). The main strategies for attenuation by synonymous genome recoding are codon deoptimization (CD), codon pair deoptimization (CPD), and increasing the dinucleotide CpG and UpA content (which is usually the result of CD and CPD) (2).The mechanisms of attenuation by these strategies are currently under intensive research. It has been suggested that the primary effect of CD and CPD is to reduce translation efficiency of pathogen mRNAs, thereby providing attenuation (8). Effects on mRNA stability also can be a factor (9). In addition, recent studies suggested that codon pa...

Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

SummarySARS-CoV-2 has become a major problem across the globe, with approximately 50 million cases and more than 1 million deaths and currently no approved treatment or vaccine. Chronic obstructive pulmonary disease (COPD) is one of the underlying conditions in adults of any age that place them at risk for developing severe illness associated with COVID-19. We established an airway epithelium model to study SARS-CoV-2 infection in healthy and COPD lung cells. We found that both the entry receptor ACE2 and the co-factor transmembrane protease TMPRSS2 are expressed at higher levels on nonciliated goblet cell, a novel target for SARS-CoV-2 infection. We observed that SARS-CoV-2 infected goblet cells and induced syncytium formation and cell sloughing. We also found that SARS-CoV-2 replication was increased in the COPD airway epithelium likely due to COPD associated goblet cell hyperplasia. Our results reveal goblet cells play a critical role in SARS-CoV-2 infection in the lung.

Patients with chronic lung disease are vulnerable to getting severe diseases associated with SARS-CoV-2 infection. Here, we describe protocols for subculturing and differentiating primary normal human bronchial epithelial (NHBE) cells of patients with chronic obstructive lung disease. The differentiation of NHBE cells in air-liquid interface mimics an in vivo airway and provides an in vitro model for studying SARS-CoV-2 infection. We also describe a protocol for detecting proteins in the sectioned epithelium for detailing SARS-CoV-2 infection-induced pathobiology with a vertical view.